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Sample records for extremely abundant sirnas

  1. LITHIUM ABUNDANCES OF EXTREMELY METAL-POOR TURNOFF STARS

    SciTech Connect

    Aoki, Wako; Inoue, Susumu; Barklem, Paul S.; Beers, Timothy C.; Christlieb, Norbert; Perez, Ana E. GarcIa; Norris, John E.; Carollo, Daniela E-mail: Paul.Barklem@physics.uu.se E-mail: N.Christlieb@lsw.uni-heidelberg.de E-mail: jen@mso.anu.edu.au E-mail: inoue@tap.scphys.kyoto-u.ac.jp

    2009-06-20

    We have determined Li abundances for eleven metal-poor turnoff stars, among which eight have [Fe/H] <-3, based on LTE analyses of high-resolution spectra obtained with the High Dispersion Spectrograph on the Subaru Telescope. The Li abundances for four of these eight stars are determined for the first time by this study. Effective temperatures are determined by a profile analysis of H{alpha} and H{beta}. While seven stars have Li abundances as high as the Spite Plateau value, the remaining four objects with [Fe/H] <-3 have A(Li) =log (Li/H)+ 12 {approx}< 2.0, confirming the existence of extremely metal-poor (EMP) turnoff stars having low Li abundances, as reported by previous work. The average of the Li abundances for stars with [Fe/H]<-3 is lower by 0.2 dex than that of the stars with higher metallicity. No clear constraint on the metallicity dependence or scatter of the Li abundances is derived from our measurements for the stars with [Fe/H]<-3. Correlations of the Li abundance with effective temperatures, with abundances of Na, Mg, and Sr, and with the kinematical properties are investigated, but no clear correlation is seen in the EMP star sample.

  2. Extreme possible variations of the deuterium abundance within the Galaxy

    NASA Astrophysics Data System (ADS)

    Delbourgo-Salvador, P.; Audouze, J.; Vidal-Madjar, A.

    1987-03-01

    In order to reconcile the present baryonic densities deduced respectively from the primordial abundances of D and 4He, some recent chemical evolution models imply that D could have been destroyed more thoroughly during the Galaxy evolution than what was previously predicted. Under the conditions outlined by these models, the present abundance of D may vary by factors as large as 50 in different parts of the Galaxy. If such variations are not observed, this implies that the ratio X(D)prim/X(D)present is not large (2 - 3): the simplest Big Bang models may then be unable to reconcile the baryonic densities predicted by D and 4He respectively.

  3. Abundance analysis of extremely metal-poor stars

    NASA Astrophysics Data System (ADS)

    Hansen, T.; Hansen, C. J.; Christlieb, N.; Andersen, J.

    2016-01-01

    The outer atmosphere of the first generations of low-mass (M < 0.8 M⊙) stars retain to a great extent the original chemical composition of the interstellar medium (ISM) at the time and place of their birth. The composition of this pristine gas represents the nucleosynthesis of the very first massive stars, that produced and ejected the first heavy elements into the early ISM. Thus a detailed abundance analysis of low-mass, metal-poor stars can help us track these gasses and provide insight into the formation processes that took place in the very early stages of our Galaxy. Preliminary result of a 25-star homogeneously analysed sample of metal- poor candidates from the Hamburg/ESO survey is presented. The main focus is on the most metal-poor stars of the sample; stars with [Fe/H] < -4. The abundance pattern of these ultra metal-poor (UMP) stars is used to extract key information of the earliest ongoing formation processes (ranging from hydrostatic burning to neutron-capture processes).

  4. Lithium Abundances in Extremely Metal-Poor Turn-Off Stars

    SciTech Connect

    Aoki, W.; Barklem, P.; Christlieb, N.; Beers, T. C.; Inoue, S.

    2008-05-21

    The Lithium (Li) abundances measured for very metal-poor turn-off (unevolved) stars have been interpreted as the result of Big Bang nucleosynthesis. However, the value is lower by a factor of two or three than the prediction of standard Big Bang nucleosynthesis models, adopting the cosmological parameters determined by the measurements of cosmic microwave background radiation with the WMAP satellite. Moreover, the recent measurements for extremely metal-poor stars (objects having iron abundances less than 1/1000th solar) suggest a scatter of the Li abundance, or a possible decreasing trend with decreasing metallicity. In order to further investigate the Li production and destruction processes in the very early universe, we have determined Li abundances for extremely metal-poor stars based on high-resolution spectra for the resonance line of neutral Li. The result of our analysis, combined with previous measurements, indicates that the Li abundances of extremely metal-poor stars are, on average, lower than those of stars with higher metallicity, while the scatter or trend of the Li abundance remains unclear. We discuss possible reasons for the lower Li abundances in extremely metal-poor stars, such as depletion of Li in low-mass unevolved stars, or destruction of Li by the first generations of massive progenitors.

  5. Lithium abundance in a turnoff halo star on an extreme orbit

    NASA Astrophysics Data System (ADS)

    Spite, M.; Spite, F.; Caffau, E.; Bonifacio, P.

    2015-10-01

    Context. The lithium abundance in turnoff stars of the old population of our Galaxy is remarkably constant in the metallicity interval -2.8 < [Fe/H] < -2.0, defining a plateau. The Li abundance of these turnoff stars is clearly lower than the abundance predicted by the primordial nucleosynthesis in the frame of the standard Big Bang nucleosynthesis. Different scenarios have been proposed for explaining this discrepancy, along with the very low scatter of the lithium abundance around the plateau. Aims: The recently identified very high velocity star, WISE J0725-2351 appears to belong to the old Galactic population, and appears to be an extreme halo star on a bound, retrograde Galactic orbit. In this paper, we study the abundance ratios and, in particular the lithium abundance, in this star. Methods: The available spectra (ESO-Very Large Telescope) are analyzed and the abundances of Li, C, Na, Mg, Al, Si, Ca, Sc, Ti, Cr, Mn, Fe, Co, Ni, Sr and Ba are determined. Results: The abundance ratios in WISE J0725-2351 are those typical of old turnoff stars. The lithium abundance in this star is in close agreement with the lithium abundance found in the metal-poor turnoff stars located at moderate distance from the Sun. This high velocity star confirms, in an extreme case, that the very small scatter of the lithium plateau persists independent of the dynamic and kinematic properties of the stars. Based on observations obtained at the ESO Paranal Observatory, Chile Programmes 093.D-0127, PI: S. Geier and 189.B-0925, PI: S. Trager.Table 2 (line by line abundances of the elements) is only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (ftp://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/582/A74

  6. Distribution, abundance and diversity of the extremely halophilic bacterium Salinibacter ruber

    PubMed Central

    Antón, Josefa; Peña, Arantxa; Santos, Fernando; Martínez-García, Manuel; Schmitt-Kopplin, Philippe; Rosselló-Mora, Ramon

    2008-01-01

    Since its discovery in 1998, representatives of the extremely halophilic bacterium Salinibacter ruber have been found in many hypersaline environments across the world, including coastal and solar salterns and solar lakes. Here, we review the available information about the distribution, abundance and diversity of this member of the Bacteroidetes. PMID:18957079

  7. First high-precision differential abundance analysis of extremely metal-poor stars

    NASA Astrophysics Data System (ADS)

    Reggiani, Henrique; Meléndez, Jorge; Yong, David; Ramírez, Ivan; Asplund, Martin

    2016-02-01

    Context. Studies of extremely metal-poor stars indicate that chemical abundance ratios [X/Fe] have a root mean square scatter as low as 0.05 dex (12%). It remains unclear whether this reflects observational uncertainties or intrinsic astrophysical scatter arising from physical conditions in the interstellar medium at early times. Aims: We measure differential chemical abundance ratios in extremely metal-poor stars to investigate the limits of precision and to understand whether cosmic scatter or observational errors are dominant. Methods: We used high-resolution (R ~ 95 000) and high signal-to-noise (S/N = 700 at 5000 Å) HIRES/Keck spectra to determine high-precision differential abundances between two extremely metal-poor stars through a line-by-line differential approach. We determined stellar parameters for the star G64-37 with respect to the standard star G64-12. We performed EW measurements for the two stars for the lines recognized in both stars and performed spectral synthesis to study the carbon abundances. Results: The differential approach allowed us to obtain errors of σ(Teff) = 27 K, σ(log g) = 0.06 dex, σ( [Fe/H] ) = 0.02 dex and σ(vt) = 0.06 km s-1. We estimated relative chemical abundances with a precision as low as σ([X/Fe]) ≈ 0.01 dex. The small uncertainties demonstrate that there are genuine abundance differences larger than the measurement errors. The observed Li difference cannot be explained by the difference in mass because the less massive star has more Li. Conclusions: It is possible to achieve an abundance precision around ≈ 0.01-0.05 dex for extremely metal-poor stars, which opens new windows on the study of the early chemical evolution of the Galaxy. Table A.1 is also available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (ftp://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/586/A67

  8. NEON AND CNO ABUNDANCES FOR EXTREME HELIUM STARS-A NON-LTE ANALYSIS

    SciTech Connect

    Pandey, Gajendra; Lambert, David L. E-mail: dll@astro.as.utexas.edu

    2011-02-01

    A non-LTE (NLTE) abundance analysis was carried out for three extreme helium stars (EHes): BD+10{sup 0} 2179, BD-9{sup 0} 4395, and LS IV+6{sup 0} 002, from their optical spectra with NLTE model atmospheres. NLTE TLUSTY model atmospheres were computed with H, He, C, N, O, and Ne treated in NLTE. Model atmosphere parameters were chosen from consideration of fits to observed He I line profiles and ionization equilibria of C and N ions. The program SYNSPEC was then used to determine the NLTE abundances for Ne as well as H, He, C, N, and O. LTE neon abundances from Ne I lines in the EHes: LSE 78, V1920 Cyg, HD 124448, and PV Tel, are derived from published models and an estimate of the NLTE correction applied to obtain the NLTE Ne abundance. We show that the derived abundances of these key elements, including Ne, are well matched with semi-quantitative predictions for the EHe resulting from a cold merger (i.e., no nucleosynthesis during the merger) of an He white dwarf with a C-O white dwarf.

  9. Abundance profiling of extremely metal-poor stars and supernova properties in the early universe

    SciTech Connect

    Tominaga, Nozomu; Iwamoto, Nobuyuki; Nomoto, Ken'ichi E-mail: iwamoto.nobuyuki@jaea.go.jp

    2014-04-20

    After the big bang nucleosynthesis, the first heavy element enrichment in the universe was made by a supernova (SN) explosion of a population (Pop) III star (Pop III SN). The abundance ratios of elements produced from Pop III SNe are recorded in abundance patterns of extremely metal-poor (EMP) stars. The observations of the increasing number of EMP stars have made it possible to statistically constrain the explosion properties of Pop III SNe. We present Pop III SN models whose nucleosynthesis yields well reproduce, individually, the abundance patterns of 48 such metal-poor stars as [Fe/H] ≲ – 3.5. We then derive relations between the abundance ratios of EMP stars and certain explosion properties of Pop III SNe: the higher [(C + N)/Fe] and [(C + N)/Mg] ratios correspond to the smaller ejected Fe mass and the larger compact remnant mass, respectively. Using these relations, the distributions of the abundance ratios of EMP stars are converted to those of the explosion properties of Pop III SNe. Such distributions are compared with those of the explosion properties of present day SNe: the distribution of the ejected Fe mass of Pop III SNe has the same peak as that of the present day SNe but shows an extended tail down to ∼10{sup –2}-10{sup –5} M {sub ☉}, and the distribution of the mass of the compact remnant of Pop III SNe is as wide as that of the present-day, stellar-mass black holes. Our results demonstrate the importance of large samples of EMP stars obtained by ongoing and future EMP star surveys and subsequent high-dispersion spectroscopic observations in clarifying the nature of Pop III SNe in the early universe.

  10. Extreme abundance ratios in the polluted atmosphere of the cool white dwarf NLTT 19868

    NASA Astrophysics Data System (ADS)

    Kawka, Adela; Vennes, Stéphane

    2016-05-01

    We present an analysis of intermediate-dispersion spectra and photometric data of the newly identified cool, polluted white dwarf NLTT 19868. The spectra obtained with X-shooter on the Very Large Telescope-Melipal show strong lines of calcium, and several lines of magnesium, aluminium and iron. We use these spectra and the optical-to-near-infrared spectral energy distribution to constrain the atmospheric parameters of NLTT 19868. Our analysis shows that NLTT 19868 is iron poor with respect to aluminium and calcium. A comparison with other cool, polluted white dwarfs shows that the Fe to Ca abundance ratio (Fe/Ca) varies by up to approximately two orders of magnitudes over a narrow temperature range with NLTT 19868 at one extremum in the Fe/Ca ratio and, in contrast, NLTT 888 at the other extremum. The sample shows evidence of extreme diversity in the composition of the accreted material: in the case of NLTT 888, the inferred composition of the accreted matter is akin to iron-rich planetary core composition, while in the case of NLTT 19868 it is close to mantle composition depleted by subsequent chemical separation at the bottom of the convection zone.

  11. Preliminary determination of the Non-LTE Calcium abundance in a sample of extremely metal-poor stars*

    NASA Astrophysics Data System (ADS)

    Spite, M.; Spite, F.; Bonifacio, P.; Caffau, E.; Andrievsky, S.; Korotin, S.; Cayrel, R.; François, P.

    2011-12-01

    The abundance ratios of the elements found in the extremely metal-poor stars (EMP) are a test of the yields predicted by the models of supernovae. For precise comparisons, it is of course preferable to avoid the approximation of LTE. The difference of LTE and NLTE profiles is displayed for three strong lines. The NLTE abundances of Ca are derived from the profiles of about 15 Ca I lines in the EMP giants and about 10 lines in the turnoff stars. The improved abundance trends are consistent with a [Ca/Fe] ratio constant vs. [Fe/H], and with a [Ca/Mg] ratio slightly declining when [Mg/H] increases. Also [Ca/Mg] presents a scatter larger than [Ca/Fe]. As far as the comparison with sulfur (another alpha elment) is concerned we find that [S/Ca] presents a scatter smaller than [S/Mg].

  12. Chemical abundances in the extremely carbon-rich and xenon-rich halo planetary nebula H4-1

    SciTech Connect

    Otsuka, Masaaki; Tajitsu, Akito E-mail: tajitsu@subaru.naoj.org

    2013-12-01

    We performed detailed chemical abundance analysis of the extremely metal-poor ([Ar/H] ∼ –2) halo planetary nebula (PN) H4-1 based on the multi-wavelength spectra from Subaru/HDS, GALEX, SDSS, and Spitzer/IRS and determined the abundances of 10 elements. The C and O abundances were derived from collisionally excited lines (CELs) and are almost consistent with abundances from recombination lines (RLs). We demonstrated that the large discrepancy in the C abundance between CEL and RL in H4-1 can be solved using the temperature fluctuation model. We reported the first detection of the [Xe III] λ5846 line in H4-1 and determination of its elemental abundance ([Xe/H] > +0.48). H4-1 is the most Xe-rich PN among the Xe-detected PNe. The observed abundances are close to the theoretical prediction by a 2.0 M {sub ☉} single star model with an initially element rich ([r/Fe] = +2.0 dex) rapid neutron-capture process (r-process). The observed Xe abundance would be a product of the r-process in primordial supernovae. The [C/O]-[Ba/(Eu or Xe)] diagram suggests that the progenitor of H4-1 shares the evolution with carbon-enhanced metal-poor (CEMP)-r/s and CEMP-no stars. The progenitor of H4-1 is presumably a binary formed in an r-process-rich environment.

  13. Solar Abundances of Rock Forming Elements, Extreme Oxygen and Hydrogen in a Young Polluted White Dwarf

    NASA Astrophysics Data System (ADS)

    Farihi, J.; Koester, D.; Zuckerman, B.; Vican, L.; Gänsicke, B. T.; Smith, N.; Walth, G.; Breedt, E.

    2016-09-01

    The Teff = 20 800 K white dwarf WD 1536+520 is shown to have broadly solar abundances of the major rock forming elements O, Mg, Al, Si, Ca, and Fe, together with a strong relative depletion in the volatile elements C and S. In addition to the highest metal abundances observed to date, including log (O/He) =-3.4, the helium-dominated atmosphere has an exceptional hydrogen abundance at log (H/He) =-1.7. Within the uncertainties, the metal-to-metal ratios are consistent with the accretion of an H2O-rich and rocky parent body, an interpretation supported by the anomalously high trace hydrogen. The mixed atmosphere yields unusually short diffusion timescales for a helium atmosphere white dwarf, of no more than a few hundred yr, and equivalent to those in a much cooler, hydrogen-rich star. The overall heavy element abundances of the disrupted parent body deviate modestly from a bulk Earth pattern, and suggest the deposition of some core-like material. The total inferred accretion rate is 4.2 × 109 g s-1, and at least 4 times higher than any white dwarf with a comparable diffusion timescale. Notably, when accretion is exhausted in this system, both metals and hydrogen will become undetectable within roughly 300 Myr, thus supporting a scenario where the trace hydrogen is related to the ongoing accretion of planetary debris.

  14. Abundances of Extremely Metal-Poor Stars, aNnew HIRES Sample

    NASA Astrophysics Data System (ADS)

    Lai, David K.; Bolte, M.; Johnson, J. A.; Lucatello, S.

    2006-12-01

    We present the results of an abundance analysis for a sample of stars with -2>[Fe/H]> -4. The set includes 29 stars, with effective temperature ranging from 4800 K to 6300 K. The data were obtained with the HIRES spectrograph at Keck Observatory. For most objects our wavelength range reaches from about 3100 angstroms to 5800 angstroms. Our spectra allow us to further constrain the abundance scatter at low metallicities for the light elements including carbon and nitrogen, up through the iron group, and for many neutron-capture elements. Most of our objects have come from the Beers et al. HK survey (1992, AJ, 103, 1987) for metal-poor stars, and for many of them this is the first high-resolution study. This research is based on work supported by the National Science Foundation under the grant AST-0607770.

  15. Snow cover and extreme winter warming events control flower abundance of some, but not all species in high arctic Svalbard

    PubMed Central

    Semenchuk, Philipp R; Elberling, Bo; Cooper, Elisabeth J

    2013-01-01

    Abstract The High Arctic winter is expected to be altered through ongoing and future climate change. Winter precipitation and snow depth are projected to increase and melt out dates change accordingly. Also, snow cover and depth will play an important role in protecting plant canopy from increasingly more frequent extreme winter warming events. Flower production of many Arctic plants is dependent on melt out timing, since season length determines resource availability for flower preformation. We erected snow fences to increase snow depth and shorten growing season, and counted flowers of six species over 5 years, during which we experienced two extreme winter warming events. Most species were resistant to snow cover increase, but two species reduced flower abundance due to shortened growing seasons. Cassiope tetragona responded strongly with fewer flowers in deep snow regimes during years without extreme events, while Stellaria crassipes responded partly. Snow pack thickness determined whether winter warming events had an effect on flower abundance of some species. Warming events clearly reduced flower abundance in shallow but not in deep snow regimes of Cassiope tetragona, but only marginally for Dryas octopetala. However, the affected species were resilient and individuals did not experience any long term effects. In the case of short or cold summers, a subset of species suffered reduced reproductive success, which may affect future plant composition through possible cascading competition effects. Extreme winter warming events were shown to expose the canopy to cold winter air. The following summer most of the overwintering flower buds could not produce flowers. Thus reproductive success is reduced if this occurs in subsequent years. We conclude that snow depth influences flower abundance by altering season length and by protecting or exposing flower buds to cold winter air, but most species studied are resistant to changes. Winter warming events, often

  16. The Abundance of Distant and Extremely Red Galaxies: The Role of AGN Feedback in Hierarchical Models

    NASA Astrophysics Data System (ADS)

    Menci, N.; Fontana, A.; Giallongo, E.; Grazian, A.; Salimbeni, S.

    2006-08-01

    We investigate the effect of AGN feedback associated with the bright QSO phase on the color distribution of galaxies from z=0 up to z=4. To this aim, we insert a blast-wave model of AGN feedback in our semianalytic model of galaxy formation, which includes the growth of supermassive black holes and the AGN activity triggered by interactions of the host galaxies. The AGN feedback is directly related to the impulsive, luminous quasar phase. We test our model by checking the consistency of its results against (1) the QSO luminosity functions from z=0 to 4, and (2) the observed local relation between the black hole mass mBH and the mass of the host galaxy. At low redshift the inclusion of AGN feedback enhances the number of red bright galaxies so that the color distribution of Mr<-22 objects is entirely dominated by red (u-r>1.5) galaxies; at 0.5extremely red object (ERO) population with mK<20 (Vega system); at such a magnitude, the model yields an ERO surface density of 6.3×103 deg2, matching existing data. Extending our analysis to z=4, the model matches the observed surface density 1.5×103 deg2 of distant red galaxies (DRGs) at mK=20; such a population is predicted to be dominated by galaxies with old stellar populations for z>2.5.

  17. Oxygen in the Early Galaxy: OH Lines as Tracers of Oxygen Abundance in Extremely Metal-Poor Giant Stars

    NASA Astrophysics Data System (ADS)

    Kucinskas, A.; Dobrovolskas, V.; Bonifacio, P.; Caffau, E.; Ludwig, H.-G.; Steffen, M.; Spite, M.

    2015-01-01

    Oxygen is a powerful tracer element of Galactic chemical evolution. Unfortunately, only a few oxygen lines are available in the ultraviolet-infrared stellar spectra for the reliable determination of its abundance. Moreover, oxygen abundances obtained using different spectral lines often disagree significantly. In this contribution we therefore investigate whether the inadequate treatment of convection in 1D hydrostatic model atmospheres used in the abundance determinations may be responsible for this disagreement. For this purpose, we used VLT CRIRES spectra of three EMP giants, as well as 3D hydrodynamical COBOLD and 1D hydrostatic LHD model atmospheres, to investigate the role of convection in the formation of infrared (IR) OH lines. Our results show that the presence of convection leads to significantly stronger IR OH lines. As a result, the difference in the oxygen abundance determined from IR OH lines with 3D hydrodynamical and classical 1D hydrostatic model atmospheres may reach -0.2 dots -0.3 dex. In case of the three EMP giants studied here, we obtain a good agrement between the 3D LTE oxygen abundances determined by us using vibrational-rotational IR OH lines in the spectral range of 1514-1626 nm, and oxygen abundances determined from forbidden [O I] 630 nm line in previous studies.

  18. THE CHEMICAL ABUNDANCES OF STARS IN THE HALO (CASH) PROJECT. II. A SAMPLE OF 14 EXTREMELY METAL-POOR STARS ,

    SciTech Connect

    Hollek, Julie K.; Sneden, Christopher; Shetrone, Matthew; Frebel, Anna; Roederer, Ian U.; Beers, Timothy C.; Kang, Sung-ju; Thom, Christopher E-mail: chris@astro.as.utexas.edu E-mail: afrebel@cfa.harvard.edu E-mail: beers@pa.msu.edu E-mail: cthom@stsci.edu

    2011-11-20

    We present a comprehensive abundance analysis of 20 elements for 16 new low-metallicity stars from the Chemical Abundances of Stars in the Halo (CASH) project. The abundances have been derived from both Hobby-Eberly Telescope High Resolution Spectrograph snapshot spectra (R {approx}15, 000) and corresponding high-resolution (R {approx}35, 000) Magellan Inamori Kyocera Echelle spectra. The stars span a metallicity range from [Fe/H] from -2.9 to -3.9, including four new stars with [Fe/H] < -3.7. We find four stars to be carbon-enhanced metal-poor (CEMP) stars, confirming the trend of increasing [C/Fe] abundance ratios with decreasing metallicity. Two of these objects can be classified as CEMP-no stars, adding to the growing number of these objects at [Fe/H]< - 3. We also find four neutron-capture-enhanced stars in the sample, one of which has [Eu/Fe] of 0.8 with clear r-process signatures. These pilot sample stars are the most metal-poor ([Fe/H] {approx}< -3.0) of the brightest stars included in CASH and are used to calibrate a newly developed, automated stellar parameter and abundance determination pipeline. This code will be used for the entire {approx}500 star CASH snapshot sample. We find that the pipeline results are statistically identical for snapshot spectra when compared to a traditional, manual analysis from a high-resolution spectrum.

  19. Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing

    PubMed Central

    Rajeswaran, Rajendran; Gubaeva, Ekaterina G.; Zvereva, Anna S.; Windels, David; Vazquez, Franck; Blevins, Todd; Farinelli, Laurent; Pooggin, Mikhail M.

    2012-01-01

    In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR). However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a GFP transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the GFP stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the GFP 3′-untranslated region (UTR) induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust GFP silencing, except when viral siRNAs targeted GFP 5′-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause GFP silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs. PMID:23028332

  20. Abundance analysis of SDSS J134338.67+484426.6; an extremely metal-poor star from the MARVELS pre-survey

    NASA Astrophysics Data System (ADS)

    Susmitha Rani, A.; Sivarani, T.; Beers, T. C.; Fleming, S.; Mahadevan, S.; Ge, J.

    2016-05-01

    We present an elemental-abundance analysis of an extremely metal-poor (EMP; [Fe/H] <-3.0) star, SDSS J134338.67+484426.6, identified during the course of the Multi-object Apache Point Observatory Radial Velocity Exoplanet Large-area Survey spectroscopic pre-survey of some 20 000 stars to identify suitable candidates for exoplanet searches. This star, with an apparent magnitude V = 12.14, is the lowest metallicity star found in the pre-survey, and is one of only ˜20 known EMP stars that are this bright or brighter. Our high-resolution spectroscopic analysis shows that this star is a subgiant with [Fe/H] = -3.42, having `normal' carbon and no enhancement of neutron-capture abundances. Strontium is underabundant, [Sr/Fe] = -0.47, but the derived lower limit on [Sr/Ba] indicates that Sr is likely enhanced relative to Ba. This star belongs to the sparsely populated class of α-poor EMP stars that exhibit low ratios of [Mg/Fe], [Si/Fe], and [Ca/Fe] compared to typical halo stars at similar metallicity. The observed variations in radial velocity from several epochs of (low- and high-resolution) spectroscopic follow-up indicate that SDSS J134338.67+484426.6 is a possible long-period binary. We also discuss the abundance trends in EMP stars for r-process elements, and compare with other magnesium-poor stars.

  1. Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer

    PubMed Central

    Vargas, Diana Y.; Kramer, Fred Russell; Tyagi, Sanjay; Marras, Salvatore A. E.

    2016-01-01

    We describe the use of “SuperSelective” primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long “5' anchor sequence” that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, “3' foot sequence” that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation’s abundance by comparing its threshold value to the threshold value of a reference gene present in the sample. PMID:27244445

  2. Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer.

    PubMed

    Vargas, Diana Y; Kramer, Fred Russell; Tyagi, Sanjay; Marras, Salvatore A E

    2016-01-01

    We describe the use of "SuperSelective" primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long "5' anchor sequence" that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, "3' foot sequence" that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation's abundance by comparing its threshold value to the threshold value of a reference gene present in the sample. PMID:27244445

  3. Definition of an Abrupt Transition Between Regions of Abundant and Extremely Low Magma Supplies Along the Mid-Atlantic Between 14o and 16oN

    NASA Astrophysics Data System (ADS)

    Casey, J. F.; Fujiwara, T.

    2001-12-01

    anomaly patterns to the north accompany this change in magma supply. The data suggests that a highly focused mantle upwelling zone, roughly 1o-2o in length and focused at ~14oN leads to abundant magma supply that quickly dissipates near this sharp transition zone, which has persisted in the area for tens of millions of years based on off-axes traces of discontinuous seamount chains. The stark contrast with this zone is due to an extreme magma drought to the north over the last several million years.

  4. siRNA Design Software for a Target Gene-Specific RNA Interference

    PubMed Central

    Naito, Yuki; Ui-Tei, Kumiko

    2012-01-01

    RNA interference (RNAi) is a mechanism through which small interfering RNA (siRNA) induces sequence-specific posttranscriptional gene silencing. RNAi is commonly recognized as a powerful tool not only for functional genomics but also for therapeutic applications. Twenty-one-nucleotide-long siRNA suppresses the expression of the intended gene whose transcript possesses perfect complementarity to the siRNA guide strand. Hence, its silencing effect has been assumed to be extremely specific. However, accumulated evidences revealed that siRNA could downregulate unintended genes with partial complementarities mainly to the seven-nucleotide seed region of siRNA. This phenomenon is referred to as off-target effect. We have revealed that the capability to induce off-target effect is strongly correlated to the thermodynamic stability in siRNA seed-target duplex. For understanding accurate target gene function and successful therapeutic application, it may be critical to select a target gene-specific siRNA with minimized off-target effect. Here we present our siRNA design software for a target-specific RNAi. In addition, we also introduce the software programs open to the public for designing functional siRNAs. PMID:22701467

  5. siRNA Design Software for a Target Gene-Specific RNA Interference.

    PubMed

    Naito, Yuki; Ui-Tei, Kumiko

    2012-01-01

    RNA interference (RNAi) is a mechanism through which small interfering RNA (siRNA) induces sequence-specific posttranscriptional gene silencing. RNAi is commonly recognized as a powerful tool not only for functional genomics but also for therapeutic applications. Twenty-one-nucleotide-long siRNA suppresses the expression of the intended gene whose transcript possesses perfect complementarity to the siRNA guide strand. Hence, its silencing effect has been assumed to be extremely specific. However, accumulated evidences revealed that siRNA could downregulate unintended genes with partial complementarities mainly to the seven-nucleotide seed region of siRNA. This phenomenon is referred to as off-target effect. We have revealed that the capability to induce off-target effect is strongly correlated to the thermodynamic stability in siRNA seed-target duplex. For understanding accurate target gene function and successful therapeutic application, it may be critical to select a target gene-specific siRNA with minimized off-target effect. Here we present our siRNA design software for a target-specific RNAi. In addition, we also introduce the software programs open to the public for designing functional siRNAs. PMID:22701467

  6. Hyaluronic acid-siRNA conjugate/reducible polyethylenimine complexes for targeted siRNA delivery.

    PubMed

    Jang, Yeon Lim; Ku, Sook Hee; Jin, So; Park, Jae Hyung; Kim, Won Jong; Kwon, Ick Chan; Kim, Sun Hwa; Jeong, Ji Hoon

    2014-10-01

    The clinical applications of therapeutic siRNA remain as a challenge due to the lack of efficient delivery system. In the present study, hyaluronic acid-siRNA conjugate (HA-SS-siRNA)/reducible polyethylenimine (BPEI1.2k-SS) complexes were developed to efficiently deliver the siRNA to HA receptor abundant region with the improved siRNA stability. HA and siRNA were conjugated with disulfide bonds, which are cleavable in cytoplasm. The synthesized HA-SS-siRNA was further complexed with BPEI1.2k-SS, resulting in the formation of spherical nanostructures with approximately 190 nm of size and neutral surface charge. HA-SS-siRNA/BPEI1.2k-SS complexes exhibited the improved stability against serum proteins or polyanions. These complexes were successfully translocated into intracellular region via HA receptor-mediated endocytosis, and silenced target gene expression. PMID:25942799

  7. SiRNAs conjugated with aromatic compounds induce RISC-mediated antisense strand selection and strong gene-silencing activity

    SciTech Connect

    Kubo, Takanori; Yanagihara, Kazuyoshi; Takei, Yoshifumi; Mihara, Keichiro; Sato, Yuichiro; Seyama, Toshio

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer SiRNAs conjugated with aromatic compounds (Ar-siRNAs) at 5 Prime -sense strand were synthesized. Black-Right-Pointing-Pointer Ar-siRNAs increased resistance against nuclease degradation. Black-Right-Pointing-Pointer Ar-siRNAs were thermodynamically stable compared with the unmodified siRNA. Black-Right-Pointing-Pointer High levels of cellular uptake and cytoplasmic localization were found. Black-Right-Pointing-Pointer Strong gene-silencing efficacy was exhibited in the Ar-siRNAs. -- Abstract: Short interference RNA (siRNA) is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with aromatic compounds in order to improve the potency of RNAi and thus to overcome several problems with siRNAs, such as cellular delivery and nuclease stability. The siRNAs conjugated with phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives showed strong resistance to nuclease degradation, and were thermodynamically stable compared with unmodified siRNA. A high level of membrane permeability in HeLa cells was also observed. Moreover, these siRNAs exhibited enhanced RNAi efficacy, which exceeded that of locked nucleic acid (LNA)-modified siRNAs, against exogenous Renilla luciferase in HeLa cells. In particular, abundant cytoplasmic localization and strong gene-silencing efficacy were found in the siRNAs conjugated with phenyl and hydroxyphenyl derivatives. The novel siRNAs conjugated with aromatic compounds are promising candidates for a new generation of modified siRNAs that can solve many of the problems associated with RNAi technology.

  8. Advances in Systemic siRNA Delivery

    PubMed Central

    Leng, Qixin; Woodle, Martin C; Lu, Patrick Y; Mixson, A James

    2009-01-01

    Sequence-specific gene silencing with small interfering RNA (siRNA) has transformed basic science research, and the efficacy of siRNA therapeutics toward a variety of diseases is now being evaluated in pre-clinical and clinical trials. Despite its potential value, the highly negatively charged siRNA has the classic delivery problem of requiring transport across cell membranes to the cytosol. Consequently, carrier development for siRNA delivery is one of the most important problems to solve before siRNA can achieve widespread clinical use. An assortment of non-viral carriers including liposomes, peptides, polymers, and aptamers are being evaluated for their ability to shepherd siRNA to the target tissue and cross the plasma membrane barrier into the cell. Several promising carriers with low toxicity and increased specificity for disease targets have emerged for siRNA-based therapeutics. This review will discuss non-viral approaches for siRNA therapeutics, with particular focus on synthetic carriers for in vivo systemic delivery of siRNA. PMID:20161621

  9. Delivery materials for siRNA therapeutics

    NASA Astrophysics Data System (ADS)

    Kanasty, Rosemary; Dorkin, Joseph Robert; Vegas, Arturo; Anderson, Daniel

    2013-11-01

    RNA interference (RNAi) has broad potential as a therapeutic to reversibly silence any gene. To achieve the clinical potential of RNAi, delivery materials are required to transport short interfering RNA (siRNA) to the site of action in the cells of target tissues. This Review provides an introduction to the biological challenges that siRNA delivery materials aim to overcome, as well as a discussion of the way that the most effective and clinically advanced classes of siRNA delivery systems, including lipid nanoparticles and siRNA conjugates, are designed to surmount these challenges. The systems that we discuss are diverse in their approaches to the delivery problem, and provide valuable insight to guide the design of future siRNA delivery materials.

  10. siRNA and RNAi optimization.

    PubMed

    Alagia, Adele; Eritja, Ramon

    2016-05-01

    The discovery and examination of the posttranscriptional gene regulatory mechanism known as RNA interference (RNAi) contributed to the identification of small interfering RNA (siRNA) and the comprehension of its enormous potential for clinical purposes. Theoretically, the ability of specific target gene downregulation makes the RNAi pathway an appealing solution for several diseases. Despite numerous hurdles resulting from the inherent properties of siRNA molecule and proper delivery to the target tissue, more than 50 RNA-based drugs are currently under clinical testing. In this work, we analyze the recent literature in the optimization of siRNA molecules. In detail, we focused on describing the most recent advances of siRNA field aimed at optimize siRNA pharmacokinetic properties. Special attention has been given in describing the impact of RNA modifications in the potential off-target effects (OTEs) such as saturation of the RNAi machinery, passenger strand-mediated silencing, immunostimulation, and miRNA-like OTEs as well as to recent developments on the delivery issue. The novel delivery systems and modified siRNA provide significant steps toward the development of reliable siRNA molecules for therapeutic use. WIREs RNA 2016, 7:316-329. doi: 10.1002/wrna.1337 For further resources related to this article, please visit the WIREs website. PMID:26840434

  11. Label-free Quantitative Proteomics for the Extremely Thermophilic Bacterium Caldicellulosiruptor obsidiansis Reveal Distinct Abundance Patterns upon Growth on Cellobiose, Crystalline Cellulose, and Switchgrass

    SciTech Connect

    Giannone, Richard J; Lochner, Adriane; Keller, Martin; Antranikian, Garabed; Graham, David E; Hettich, Robert {Bob} L

    2011-01-01

    Mass spectrometric analysis of Caldicellulosiruptor obsidiansis cultures grown on four different carbon sources identified 65% of the cells predicted proteins in cell lysates and supernatants. Biological and technical replication together with sophisticated statistical analysis were used to reliably quantify protein abundances and their changes as a function of carbon source. Extracellular, multifunctional glycosidases were significantly more abundant on cellobiose than on the crystalline cellulose substrates Avicel and filter paper, indicating either disaccharide induction or constitutive protein expression. Highly abundant flagellar, chemotaxis, and pilus proteins were detected during growth on insoluble substrates, suggesting motility or specific substrate attachment. The highly abundant extracellular binding protein COB47-0549 together with the COB47-1616 ATPase might comprise the primary ABC-transport system for cellooligosaccharides, while COB47-0096 and COB47-0097 could facilitate monosaccharide uptake. Oligosaccharide degradation can occur either via extracellular hydrolysis by a GH1 {beta}-glycosidase or by intracellular phosphorolysis using two GH94 enzymes. When C. obsidiansis was grown on switchgrass, the abundance of hemicellulases (including GH3, GH5, GH51, and GH67 enzymes) and certain sugar transporters increased significantly. Cultivation on biomass also caused a concerted increase in cytosolic enzymes for xylose and arabinose fermentation.

  12. Rational Design of Immunostimulatory siRNAs

    PubMed Central

    Gantier, Michael P; Tong, Stephen; Behlke, Mark A; Irving, Aaron T; Lappas, Martha; Nilsson, Ulrika W; Latz, Eicke; McMillan, Nigel AJ; Williams, Bryan RG

    2010-01-01

    Short-interfering RNAs (siRNAs) have engendered much enthusiasm for their ability to silence the expression of specific genes. However, it is now well established that siRNAs, depending on their sequence, can be variably sensed by the innate immune system through recruitment of toll-like receptors 7 and 8 (TLR7/8). Here, we aimed to identify sequence-based modifications allowing for the design of bifunctional siRNAs with both proinflammatory and specific silencing activities, and with potentially increased therapeutic benefits. We found that the introduction of a micro-RNA (miRNA)-like nonpairing uridine-bulge in the passenger strand robustly increased immunostimulatory activity on human immune cells. This sequence modification had no effect on the silencing efficiency of the siRNA. Increased immunostimulation with the uridine-bulge design was specific to human cells, and conserved silencing efficiency required a Dicer-substrate scaffold. The increased cytokine production with the uridine-bulge design resulted in enhanced protection against Semliki Forest virus (SFV) infection, in viral assays. Thus, we characterize a design scaffold applicable to any given siRNA sequence, that results in increased innate immune activation without affecting gene silencing. Our data suggest that this sequence modification coupled with structural modification differentially recruits human TLR8 over TLR7, and could have potential application in antiviral therapies. PMID:20125126

  13. De novo reconstruction of consensus master genomes of plant RNA and DNA viruses from siRNAs.

    PubMed

    Seguin, Jonathan; Rajeswaran, Rajendran; Malpica-López, Nachelli; Martin, Robert R; Kasschau, Kristin; Dolja, Valerian V; Otten, Patricia; Farinelli, Laurent; Pooggin, Mikhail M

    2014-01-01

    Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense. PMID:24523907

  14. Albumin pre-coating enhances intracellular siRNA delivery of multifunctional amphiphile/siRNA nanoparticles

    PubMed Central

    Kummitha, China M; Malamas, Anthony S; Lu, Zheng-Rong

    2012-01-01

    Nonspecific association of serum molecules with short-interfering RNA (siRNA) nanoparticles can change their physiochemical characteristics, and results in reduced cellular uptake in the target tissue during the systemic siRNA delivery process. Serum albumin is the most abundant protein in the body and has been used to modify the surface of nanoparticles, to inhibit association of other serum molecules. Here, we hypothesized that surface modification of lipid-based nanoparticular siRNA delivery systems with albumin could prevent their interaction with serum proteins, and improve intracellular uptake. In this study, we investigated the influence of albumin on the stability and intracellular siRNA delivery of the targeted siRNA nanoparticles of a polymerizable and pH-sensitive multifunctional surfactant N-(1-aminoethyl) iminobis[N-(oleoylcysteinylhistinyl-1-aminoethyl)propionamide] (EHCO) in serum. Serum resulted in a significant increase in the size of targeted EHCO/siRNA nanoparticles and inhibited cellular uptake of the nanoparticles. Coating of targeted EHCO/siRNA nanoparticles with bovine serum albumin at 9.4 μM prior to cell transfection improved cellular uptake and gene silencing efficacy of EHCO/siRNA targeted nanoparticles in serum-containing media, as compared with the uncoated nanoparticles. At a proper concentration, albumin has the potential to minimize interactions of serum proteins with siRNA nanoparticles for effective systemic in vivo siRNA delivery. PMID:23055731

  15. Recent advances in siRNA delivery.

    PubMed

    Sarisozen, Can; Salzano, Giuseppina; Torchilin, Vladimir P

    2015-12-01

    In the 1990s an unexpected gene-silencing phenomena in plants, the later called RNA interference (RNAi), perplexed scientists. Following the proof of activity in mammalian cells, small interfering RNAs (siRNAs) have quickly crept into biomedical research as a new powerful tool for the potential treatment of different human diseases based on altered gene expression. In the past decades, several promising data from ongoing clinical trials have been reported. However, despite surprising successes in many pre-clinical studies, concrete obstacles still need to be overcome to translate therapeutic siRNAs into clinical reality. Here, we provide an update on the recent advances of RNAi-based therapeutics and highlight novel synthetic platforms for the intracellular delivery of siRNAs. PMID:26609865

  16. Development of a simple, biocompatible and cost-effective Inulin-Diethylenetriamine based siRNA delivery system.

    PubMed

    Sardo, C; Farra, R; Licciardi, M; Dapas, B; Scialabba, C; Giammona, G; Grassi, M; Grassi, G; Cavallaro, G

    2015-07-30

    Small interfering RNAs (siRNAs) have the potential to be of therapeutic value for many human diseases. So far, however, a serious obstacle to their therapeutic use is represented by the absence of appropriate delivery systems able to protect them from degradation and to allow an efficient cellular uptake. In this work we developed a siRNA delivery system based on inulin (Inu), an abundant and natural polysaccharide. Inu was functionalized via the conjugation with diethylenetriamine (DETA) residues to form the complex Inu-DETA. We studied the size, surface charge and the shape of the Inu-DETA/siRNA complexes; additionally, the cytotoxicity, the silencing efficacy and the cell uptake-mechanisms were studied in the human bronchial epithelial cells (16HBE) and in the hepatocellular carcinoma derived cells (JHH6). The results presented here indicate that Inu-DETA copolymers can effectively bind siRNAs, are highly cytocompatible and, in JHH6, can effectively deliver functional siRNAs. Optimal delivery is observed using a weight ratio Inu-DETA/siRNA of 4 that corresponds to polyplexes with an average size of 600nm and a slightly negative surface charge. Moreover, the uptake and trafficking mechanisms, mainly based on micropinocytosis and clatrin mediated endocytosis, allow the homogeneous diffusion of siRNA within the cytoplasm of JHH6. Notably, in 16 HBE where the trafficking mechanism (caveolae mediated endocytosis) does not allow an even distribution of siRNA within the cell cytoplasm, no significant siRNA activity is observed. In conclusion, we developed a novel inulin-based siRNA delivery system able to efficiently release siRNA in JHH6 with negligible cytotoxicity thus opening the way for further testing in more complex in vivo models. PMID:25845631

  17. Bioengineered Nanoparticles for siRNA delivery

    PubMed Central

    Kozielski, Kristen L.; Tzeng, Stephany Y.; Green, Jordan J.

    2014-01-01

    Short interfering RNA (siRNA) has been an important laboratory tool in the last two decades and has allowed researchers to better understand the functions of non-protein-coding genes through RNA interference (RNAi). Although RNAi holds great promise for this purpose as well as for treatment of many diseases, efforts at using siRNA have been hampered by the difficulty of safely and effectively introducing it into cells of interest, both in vitro and in vivo. To overcome this challenge, many biomaterials and nanoparticles (NPs) have been developed and optimized for siRNA delivery, often taking cues from the DNA delivery field, although different barriers exist for these two types of molecules. In this review, we discuss general properties of biomaterials and nanoparticles that are necessary for effective nucleic acid delivery. We also discuss specific examples of bioengineered materials, including lipid-based NPs, polymeric NPs, inorganic NPs, and RNA-based NPs, which clearly illustrate the problems and successes in siRNA delivery. PMID:23821336

  18. Cuboplexes: Topologically Active siRNA Delivery.

    PubMed

    Kim, Hojun; Leal, Cecilia

    2015-10-27

    RNAi technology is currently experiencing a revival due to remarkable improvements in efficacy and viability through oligonucleotide chemical manipulations and/or via their packaging into nanoscale carriers. At present, there is no FDA-approved system for siRNA technology in humans. The design of the next generation of siRNA carriers requires a deep understanding of how a nanoparticle's physicochemical properties truly impart biological stability and efficiency. For example, we now know that nanoparticles need to be sterically stabilized in order to meet adequate biodistribution profiles. At present, targeting, uptake, and, in particular, endosomal escape are among the most critical challenges impairing RNAi technologies. The disruption of endosomes encompasses membrane transformations (for example, pore formation) that cost significant elastic energy. Nanoparticle size and shape have been identified as relevant parameters impacting tissue accumulation and cellular uptake. In this paper, we demonstrate that the internal structure of lipid-based particles offers a different handle to promote endosomal membrane topological disruptions that enhance siRNA delivery. Specifically, we designed sterically stabilized lipid-based particles that differ from traditional liposomal systems by displaying highly ordered bicontinuous cubic internal structures that can be loaded with large amounts of siRNA. This system differs from traditional siRNA-containing liposomes (lipoplexes) as the particle-endosomal membrane interactions are controlled by elasticity energetics and not by electrostatics. The resulting "PEGylated cuboplex" has the ability to deliver siRNA and specifically knockdown genes with efficiencies that surpass those achieved by traditional lipoplex systems. PMID:26390340

  19. De Novo Reconstruction of Consensus Master Genomes of Plant RNA and DNA Viruses from siRNAs

    PubMed Central

    Seguin, Jonathan; Rajeswaran, Rajendran; Malpica-López, Nachelli; Martin, Robert R.; Kasschau, Kristin; Dolja, Valerian V.; Otten, Patricia; Farinelli, Laurent; Pooggin, Mikhail M.

    2014-01-01

    Virus-infected plants accumulate abundant, 21–24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this ‘siRNA omics’ approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense. PMID:24523907

  20. Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs in Arabidopsis

    PubMed Central

    Cao, Mengji; Du, Peng; Wang, Xianbing; Yu, Yun-Qi; Qiu, Yan-Hong; Li, Wanxiang; Gal-On, Amit; Zhou, Changyong; Li, Yi; Ding, Shou-Wei

    2014-01-01

    Antiviral immunity controlled by RNA interference (RNAi) in plants and animals is thought to specifically target only viral RNAs by the virus-derived small interfering RNAs (siRNAs). Here we show that activation of antiviral RNAi in Arabidopsis plants is accompanied by the production of an abundant class of endogenous siRNAs mapped to the exon regions of more than 1,000 host genes and rRNA. These virus-activated siRNAs (vasiRNAs) are predominantly 21 nucleotides long with an approximately equal ratio of sense and antisense strands. Genetically, vasiRNAs are distinct from the known plant endogenous siRNAs characterized to date and instead resemble viral siRNAs by requiring Dicer-like 4 and RNA-dependent RNA polymerase 1 (RDR1) for biogenesis. However, loss of EXORIBONUCLEASE4/THYLENE-INSENSITIVE5 enhances vasiRNA biogenesis and virus resistance without altering the biogenesis of viral siRNAs. We show that vasiRNAs are active in directing widespread silencing of the target host genes and that Argonaute-2 binds to and is essential for the silencing activity of vasiRNAs. Production of vasiRNAs is readily detectable in Arabidopsis after infection by viruses from two distinct supergroups of plant RNA virus families and is targeted for inhibition by the silencing suppressor protein 2b of Cucumber mosaic virus. These findings reveal RDR1 production of Arabidopsis endogenous siRNAs and identify production of vasiRNAs to direct widespread silencing of host genes as a conserved response of plants to infection by diverse viruses. A possible function for vasiRNAs to confer broad-spectrum antiviral activity distinct to the virus-specific antiviral RNAi by viral siRNAs is discussed. PMID:25201959

  1. Dendrimers for siRNA Delivery

    PubMed Central

    Biswas, Swati; Torchilin, Vladimir P.

    2013-01-01

    Since the discovery of the “starburst polymer”, later renamed as dendrimer, this class of polymers has gained considerable attention for numerous biomedical applications, due mainly to the unique characteristics of this macromolecule, including its monodispersity, uniformity, and the presence of numerous functionalizable terminal groups. In recent years, dendrimers have been studied extensively for their potential application as carriers for nucleic acid therapeutics, which utilize the cationic charge of the dendrimers for effective dendrimer-nucleic acid condensation. siRNA is considered a promising, versatile tool among various RNAi-based therapeutics, which can effectively regulate gene expression if delivered successfully inside the cells. This review reports on the advancements in the development of dendrimers as siRNA carriers. PMID:24275946

  2. Simple gene silencing using the trans-acting siRNA pathway.

    PubMed

    Jacobs, Thomas B; Lawler, Noah J; LaFayette, Peter R; Vodkin, Lila O; Parrott, Wayne A

    2016-01-01

    In plants, particular micro-RNAs (miRNAs) induce the production of a class of small interfering RNAs (siRNA) called trans-acting siRNA (ta-siRNA) that lead to gene silencing. A single miRNA target is sufficient for the production of ta-siRNAs, which target can be incorporated into a vector to induce the production of siRNAs, and ultimately gene silencing. The term miRNA-induced gene silencing (MIGS) has been used to describe such vector systems in Arabidopsis. Several ta-siRNA loci have been identified in soybean, but, prior to this work, few of the inducing miRNAs have been experimentally validated, much less used to silence genes. Nine ta-siRNA loci and their respective miRNA targets were identified, and the abundance of the inducing miRNAs varies dramatically in different tissues. The miRNA targets were experimentally verified by silencing a transgenic GFP gene and two endogenous genes in hairy roots and transgenic plants. Small RNAs were produced in patterns consistent with the utilization of the ta-siRNA pathway. A side-by-side experiment demonstrated that MIGS is as effective at inducing gene silencing as traditional hairpin vectors in soybean hairy roots. Soybean plants transformed with MIGS vectors produced siRNAs and silencing was observed in the T1 generation. These results complement previous reports in Arabidopsis by demonstrating that MIGS is an efficient way to produce siRNAs and induce gene silencing in other species, as shown with soybean. The miRNA targets identified here are simple to incorporate into silencing vectors and offer an effective and efficient alternative to other gene silencing strategies. PMID:25816689

  3. Highly efficient siRNA delivery from core-shell mesoporous silica nanoparticles with multifunctional polymer caps

    NASA Astrophysics Data System (ADS)

    Möller, Karin; Müller, Katharina; Engelke, Hanna; Bräuchle, Christoph; Wagner, Ernst; Bein, Thomas

    2016-02-01

    A new general route for siRNA delivery is presented combining porous core-shell silica nanocarriers with a modularly designed multifunctional block copolymer. Specifically, the internal storage and release of siRNA from mesoporous silica nanoparticles (MSN) with orthogonal core-shell surface chemistry was investigated as a function of pore-size, pore morphology, surface properties and pH. Very high siRNA loading capacities of up to 380 μg per mg MSN were obtained with charge-matched amino-functionalized mesoporous cores, and release profiles show up to 80% siRNA elution after 24 h. We demonstrate that adsorption and desorption of siRNA is mainly driven by electrostatic interactions, which allow for high loading capacities even in medium-sized mesopores with pore diameters down to 4 nm in a stellate pore morphology. The negatively charged MSN shell enabled the association with a block copolymer containing positively charged artificial amino acids and oleic acid blocks, which acts simultaneously as capping and endosomal release agent. The potential of this multifunctional delivery platform is demonstrated by highly effective cell transfection and siRNA delivery into KB-cells. A luciferase reporter gene knock-down of up to 80-90% was possible using extremely low cell exposures with only 2.5 μg MSN containing 0.5 μg siRNA per 100 μL well.A new general route for siRNA delivery is presented combining porous core-shell silica nanocarriers with a modularly designed multifunctional block copolymer. Specifically, the internal storage and release of siRNA from mesoporous silica nanoparticles (MSN) with orthogonal core-shell surface chemistry was investigated as a function of pore-size, pore morphology, surface properties and pH. Very high siRNA loading capacities of up to 380 μg per mg MSN were obtained with charge-matched amino-functionalized mesoporous cores, and release profiles show up to 80% siRNA elution after 24 h. We demonstrate that adsorption and desorption of

  4. siRNA Against KIR3DL1 as a Potential Gene Therapeutic Agent in Controlling HIV-1 Infection

    PubMed Central

    Fu, Geng-Feng; Pan, Ji-Cheng; Lin, Nan; Hu, Hai-Yang; Tang, Wei-Ming; Xu, Jin-Shui; Wang, Xiao-Liang; Xu, Xiao-Qin; Qiu, Tao; Liu, Xiao-Yan; Chen, Guo-Hong; Mahapatra, Tanmay; Huan, Xi-Ping

    2014-01-01

    Abstract Objectives: The aim of this study was to develop a small interfering RNA (siRNA) against the expression of KIR3DL1 receptor on natural killer (NK) cells, in order to promote the ability of NK cells to destroy human immunodeficiency virus (HIV)-infected cells and thus prevent failure of siRNA therapy targeting human immunodeficiency virus type 1 (HIV-1) virus among HIV-1 infected patients in vitro. Methods: A siRNA targeting KIR3DL1 was synthesized and then modified with cholesterol, methylene, and sulfate. The inhibitory action of the siRNAs on primary cultured NK cells was detected. The amount of IFN-γ and TNF-α secretions in NK cells was measured. The intended functions of NK cells in vitro were analyzed by CFSE and PI methods. Results: There were no significant differences in inhibiting the expression of KIR3DL1 on NK cells between the modified and unmodified siRNAs, while inhibition by each of them differed significantly from controls. The amount of IFN-γ and TNF-α secretions in the NK cells was abundant due to unsuccessful expression of KIR3DL1 on NK cells, which further promoted function of the NK cells. Conclusion: The siRNA against KIR3DL1 could enhance the ability of the NK cells to kill the HIV-1 infected cells in vitro and successfully prevented the failure of siRNA therapy targeting the HIV-1 virus. Therefore, it can act as a potential gene therapeutic agent among HIV-1 infected people. PMID:24834927

  5. siRNA Delivery to the Glomerular Mesangium Using Polycationic Cyclodextrin Nanoparticles Containing siRNA

    PubMed Central

    Gale, Aaron; Wu, Peiwen; Ma, Rong; Davis, Mark E.

    2015-01-01

    There is an urgent need for new therapies that can halt or reverse the course of chronic kidney disease with minimal side-effect burden on the patient. Small interfering RNA (siRNA) nanoparticles are new therapeutic entities in clinical development that could be useful for chronic kidney disease treatment because they combine the tissue-specific targeting properties of nanoparticles with the gene-specific silencing effects of siRNA. Recent reports have emerged demonstrating that the kidney, specifically the glomerulus, is a readily accessible site for nanoparticle targeting. Here, we explore the hypothesis that intravenously administered polycationic cyclodextrin nanoparticles containing siRNA (siRNA/CDP-NPs) can be used for delivery of siRNA to the glomerular mesangium. We demonstrate that siRNA/CDP-NPs localize to the glomerular mesangium with limited deposition in other areas of the kidney after intravenous injection. Additionally, we report that both mouse and human mesangial cells rapidly internalize siRNA/CDP-NPs in vitro and that nanoparticle uptake can be enhanced by attaching the targeting ligands mannose or transferrin to the nanoparticle surface. Lastly, we show knockdown of mesangial enhanced green fluorescent protein expression in a reporter mouse strain following iv treatment with siRNA/CDP-NPs. Altogether, these data demonstrate the feasibility of mesangial targeting using intravenously administered siRNA/CDP-NPs. PMID:25734248

  6. How extreme are extremes?

    NASA Astrophysics Data System (ADS)

    Cucchi, Marco; Petitta, Marcello; Calmanti, Sandro

    2016-04-01

    High temperatures have an impact on the energy balance of any living organism and on the operational capabilities of critical infrastructures. Heat-wave indicators have been mainly developed with the aim of capturing the potential impacts on specific sectors (agriculture, health, wildfires, transport, power generation and distribution). However, the ability to capture the occurrence of extreme temperature events is an essential property of a multi-hazard extreme climate indicator. Aim of this study is to develop a standardized heat-wave indicator, that can be combined with other indices in order to describe multiple hazards in a single indicator. The proposed approach can be used in order to have a quantified indicator of the strenght of a certain extreme. As a matter of fact, extremes are usually distributed in exponential or exponential-exponential functions and it is difficult to quickly asses how strong was an extreme events considering only its magnitude. The proposed approach simplify the quantitative and qualitative communication of extreme magnitude

  7. Structural and Functional Analysis of Viral siRNAs

    PubMed Central

    Szittya, Gyorgy; Moxon, Simon; Pantaleo, Vitantonio; Toth, Gabor; Rusholme Pilcher, Rachel L.; Moulton, Vincent; Burgyan, Jozsef; Dalmay, Tamas

    2010-01-01

    A large amount of short interfering RNA (vsiRNA) is generated from plant viruses during infection, but the function, structure and biogenesis of these is not understood. We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs was the same in different plant species and in the absence of RDR6. We used the Terminator 5′-Phosphate-Dependent Exonuclease to study the 5′ end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5′ monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5′ end of short RNAs or after replacing any potential 5′ ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were complementary to non-abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double stranded RNA, and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution

  8. Coordinative Amphiphiles as Tunable siRNA Transporters.

    PubMed

    Kim, Jin Bum; Lee, Yeong Mi; Ryu, Jooyeon; Lee, Eunji; Kim, Won Jong; Keum, Gyochang; Bang, Eun-Kyoung

    2016-08-17

    In this study, we developed coordinative amphiphiles for use as novel siRNA transporters. As a modification of a conventional cationic lipid structure, we replaced the cationic head with zinc(II)-dipicolylamine complex (Zn/DPA) as a phosphate-directing group, and used various membrane-directing groups in the place of the hydrophobic tails. These simple amphiphiles are readily synthesized and easy to modify. The Zn/DPA head groups bind to the phosphate backbones of siRNAs, and to our surprise, they prevented the enzymatic degradation of siRNAs by RNase A. Interestingly, the Zn/DPA head itself exhibited moderate transfection efficiency, and its combination with a membrane-directing group-oleoyl (CA1), pyrenebutyryl (CA2), or biotin (CA3)-enhanced the delivery efficiency without imparting significant cytotoxicity. Notably, the uptake pathway was tunable depending on the nature of the membrane-directing group. CA1 delivered siRNAs mainly through caveolae-mediated endocytosis, and CA2 through clathrin- and caveolin-independent endocytosis; CA3 recruited siRNAs specifically into biotin receptor-positive HepG2 cells through receptor-mediated endocytosis. Thus, it appears possible to develop tunable siRNA transporters simply by changing the membrane-directing parts. These are the first examples of amphiphilic siRNA transporters accompanying coordinative interactions between the amphiphiles and siRNAs. PMID:27364494

  9. Bone site-specific delivery of siRNA

    PubMed Central

    Liu, Xinli

    2016-01-01

    Abstract Small interfering RNAs (siRNA) have enormous potential as therapeutics to target and treat various bone disorders such as osteoporosis and cancer bone metastases. However, effective and specific delivery of siRNA therapeutics to bone and bone-specific cells in vivo is very challenging. To realize the full therapeutic potential of siRNA in treating bone disorders, a safe and efficient, tissue- and cell-specific delivery system must be developed. This review focuses on recent advances in bone site-specific delivery of siRNA at the tissue or cellular level. Bone-targeted nanoparticulate siRNA carriers and various bone-targeted moieties such as bisphosphonates, oligopeptides (Asp)8 and (AspSerSer)6, and aptamers are highlighted. Incorporation of these bone-seeking targeting moieties into siRNA carriers allows for recognition of different sub-tissue functional domains of bone and also specific cell types residing in bone tissue. It also provides a means for bone-formation surface-, bone-resorption surface-, or osteoblast-specific targeting and transportation of siRNA therapeutics. The discussion mainly focuses on systemic and local bone-specific delivery of siRNA in osteoporosis and bone metastasis preclinical models. PMID:26642236

  10. Translocation and encapsulation of siRNA inside carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Mogurampelly, Santosh; Maiti, Prabal K.

    2013-01-01

    We report spontaneous translocation of small interfering RNA (siRNA) inside carbon nanotubes (CNTs) of various diameters and chirality using all atom molecular dynamics simulations with explicit solvent. We use umbrella sampling method to calculate the free energy landscape of the siRNA entry and translocation event. Free energy profiles show that siRNA gains free energy while translocating inside CNT, and barrier for siRNA exit from CNT ranges from 40 to 110 kcal/mol depending on CNT chirality and salt concentration. The translocation time τ decreases with the increase of CNT diameter with a critical diameter of 24 Å for the translocation. In contrast, double strand DNA of the same sequence does not translocate inside CNT due to large free energy barrier for the translocation. This study helps in understanding the nucleic acid transport through nanopores at microscopic level and may help designing carbon nanotube based sensor for siRNA.

  11. Effect of surface properties on liposomal siRNA delivery.

    PubMed

    Xia, Yuqiong; Tian, Jie; Chen, Xiaoyuan

    2016-02-01

    Liposomes are one of the most widely investigated carriers for siRNA delivery. The surface properties of liposomal carriers, including the surface charge, PEGylation, and ligand modification can significantly affect the gene silencing efficiency. Three barriers of systemic siRNA delivery (long blood circulation, efficient tumor penetration and efficient cellular uptake/endosomal escape) are analyzed on liposomal carriers with different surface charges, PEGylations and ligand modifications. Cationic formulations dominate siRNA delivery and neutral formulations also have good performance while anionic formulations are generally not proper for siRNA delivery. The PEG dilemma (prolonged blood circulation vs. reduced cellular uptake/endosomal escape) and the side effect of repeated PEGylated formulation (accelerated blood clearance) were discussed. Effects of ligand modification on cationic and neutral formulations were analyzed. Finally, we summarized the achievements in liposomal siRNA delivery, outlined existing problems and provided some future perspectives. PMID:26695117

  12. Small-interfering RNAs (siRNAs) as a promising tool for ocular therapy.

    PubMed

    Guzman-Aranguez, A; Loma, P; Pintor, J

    2013-10-01

    RNA interference (RNAi) can be used to inhibit the expression of specific genes in vitro and in vivo, thereby providing an extremely useful tool for investigating gene function. Progress in the understanding of RNAi-based mechanisms has opened up new perspectives in therapeutics for the treatment of several diseases including ocular disorders. The eye is currently considered a good target for RNAi therapy mainly because it is a confined compartment and, therefore, enables local delivery of small-interfering RNAs (siRNAs) by topical instillation or direct injection. However, delivery strategies that protect the siRNAs from degradation and are suitable for long-term treatment would be help to improve the efficacy of RNAi-based therapies for ocular pathologies. siRNAs targeting critical molecules involved in the pathogenesis of glaucoma, retinitis pigmentosa and neovascular eye diseases (age-related macular degeneration, diabetic retinopathy and corneal neovascularization) have been tested in experimental animal models, and clinical trials have been conducted with some of them. This review provides an update on the progress of RNAi in ocular therapeutics, discussing the advantages and drawbacks of RNAi-based therapeutics compared to previous treatments. PMID:23937539

  13. Repression of multiple CYP2D genes in mouse primary hepatocytes with a single siRNA construct.

    PubMed

    Elraghy, Omaima; Baldwin, William S

    2015-01-01

    The Cyp2d subfamily is the second most abun-dant subfamily of hepatic drug-metabolizing CYPs. In mice, there are nine Cyp2d members that are believed to have redundant catalytic activity. We are testing and optimizing the ability of one short interfering RNA (siRNA) construct to knockdown the expression of multiple mouse Cyp2ds in primary hepatocytes. Expression of Cyp2d10, Cyp2d11, Cyp2d22, and Cyp2d26 was observed in the primary male mouse hepatocytes. Cyp2d9, which is male-specific and growth hormone-dependent, was not expressed in male primary hepatocytes, potentially because of its dependence on pulsatile growth hormone release from the anterior pituitary. Several different siRNAs at different concentrations and with different reagents were used to knockdown Cyp2d expression. siRNA constructs designed to repress only one construct often mildly repressed several Cyp2d isoforms. A construct designed to knockdown every Cyp2d isoform provided the best results, especially when incubated with transfection reagents designed specifically for primary cell culture. Interestingly, a construct designed to knockdown all Cyp2d isoforms, except Cyp2d10, caused a 2.5× increase in Cyp2d10 expression, presumably because of a compensatory response. However, while RNA expression is repressed 24 h after siRNA treatment, associated changes in Cyp2d-mediated metabolism are tenuous. Overall, this study provides data on the expression of murine Cyp2ds in primary cell lines, valuable information on designing siRNAs for silencing multiple murine CYPs, and potential pros and cons of using siRNA as a tool for repressing Cyp2d and estimating Cyp2d's role in murine xenobiotic metabolism. PMID:25124873

  14. Efficient prediction methods for selecting effective siRNA sequences.

    PubMed

    Takasaki, Shigeru

    2010-02-01

    Although short interfering RNA (siRNA) has been widely used for studying gene functions in mammalian cells, its gene silencing efficacy varies markedly and there are only a few consistencies among the recently reported design rules/guidelines for selecting siRNA sequences effective for mammalian genes. Another shortcoming of the previously reported methods is that they cannot estimate the probability that a candidate sequence will silence the target gene. This paper first reviewed the recently reported siRNA design guidelines and clarified the problems concerning the guidelines. It then proposed two prediction methods-Radial Basis Function (RBF) network and decision tree learning-and their combined method for selecting effective siRNA target sequences from many possible candidate sequences. They are quite different from the previous score-based siRNA design techniques and can predict the probability that a candidate siRNA sequence will be effective. The methods imply high estimation accuracy for selecting candidate siRNA sequences. PMID:20022002

  15. Exosomes: Natural Carriers for siRNA Delivery.

    PubMed

    Kumar, Lalit; Verma, Shivani; Vaidya, Bhuvaneshwar; Gupta, Vivek

    2015-01-01

    Various cells of the human physiological system have the capability to release extracellular vesicles (EVs) involved in intercellular transport of proteins and nucleic acids. Exosomes are a subtype of extracellular vesicles having their origin through endocytic pathway. While being involved in intercellular transport of macromolecules, exosomes, due to their presence in several body fluids, can also be utilized as a system to commute RNA molecules and proteins in the body. Recent advances in gene therapy have provided a new outlook in disease therapeutics by modulation of gene expression using oligonucleotide based approach and exosomes have been reported a potential carrier for nucleic acid based therapeutic moieties. In recent years, small interfering RNA (siRNA) has emerged as promising therapeutic alternative for diseases with gene-based pathophysiology, however poor bioavailability limits its therapeutic potential. For effective delivery and enhancement of bioavailability of siRNA, several carriers including dendrimers, liposomes, siRNA conjugates, and siRNA aptamer chimeras, to name a few, have been explored. Exosomes can be considered a promising carrier for effective delivery of siRNA due to their existence in body's endogenous system and high tolerance. The present review focuses on delivering knowledge about exosomes, siRNA, and capability of exosomes to act as natural carriers for siRNA delivery. PMID:26486142

  16. Porous silicon microparticles for delivery of siRNA therapeutics.

    PubMed

    Shen, Jianliang; Wu, Xiaoyan; Lee, Yeonju; Wolfram, Joy; Yang, Zhizhou; Mao, Zong-Wan; Ferrari, Mauro; Shen, Haifa

    2015-01-01

    Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described. PMID:25651103

  17. SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

    PubMed Central

    Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

    2015-01-01

    Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments. PMID:26464441

  18. Optimization of Transfection Conditions for siRNA Screening.

    PubMed

    Montoya, Justin J; Azorsa, David O

    2016-01-01

    RNAi screening of mammalian cells is often performed using siRNAs and cationic lipids as transfection reagents. Efficiency of transfection depends on growth characteristics of the cells and the cationic lipid used. With a large selection of cationic lipids available, it can often be difficult to select the optimal lipid and lipid:siRNA (vol:wt) ratio. Here, we describe the process of optimizing siRNA transfection conditions for efficient reverse transfection of mammalian cells using specific positive and negative siRNA controls. PMID:27581281

  19. Characterization of viral siRNA populations in honey bee colony collapse disorder.

    PubMed

    Chejanovsky, Nor; Ophir, Ron; Schwager, Michal Sharabi; Slabezki, Yossi; Grossman, Smadar; Cox-Foster, Diana

    2014-04-01

    Colony Collapse Disorder (CCD), a special case of collapse of honey bee colonies, has resulted in significant losses for beekeepers. CCD-colonies show abundance of pathogens which suggests that they have a weakened immune system. Since honey bee viruses are major players in colony collapse and given the important role of viral RNA interference (RNAi) in combating viral infections we investigated if CCD-colonies elicit an RNAi response. Deep-sequencing analysis of samples from CCD-colonies from US and Israel revealed abundant small interfering RNAs (siRNA) of 21-22 nucleotides perfectly matching the Israeli acute paralysis virus (IAPV), Kashmir virus and Deformed wing virus genomes. Israeli colonies showed high titers of IAPV and a conserved RNAi-pattern of matching the viral genome. That was also observed in sample analysis from colonies experimentally infected with IAPV. Our results suggest that CCD-colonies set out a siRNA response that is specific against predominant viruses associated with colony losses. PMID:24725944

  20. Generation of siRNA Nanosheets for Efficient RNA Interference

    PubMed Central

    Kim, Hyejin; Lee, Jae Sung; Lee, Jong Bum

    2016-01-01

    After the discovery of small interference RNA (siRNA), nanostructured siRNA delivery systems have been introduced to achieve an efficient regulation of the target gene expression. Here we report a new siRNA-generating two dimensional nanostructure in a formation of nanosized sheet. Inspired by tunable mechanical and functional properties of the previously reported RNA membrane, siRNA nanosized sheets (siRNA-NS) with multiple Dicer cleavage sites were prepared. The siRNA-NS has two dimensional structure, providing a large surface area for Dicer to cleave the siRNA-NS for the generation of functional siRNAs. Furthermore, downregulation of the cellular target gene expression was achieved by delivery of siRNA-NS without chemical modification of RNA strands or conjugation to other substances. PMID:27120975

  1. Generation of siRNA Nanosheets for Efficient RNA Interference

    NASA Astrophysics Data System (ADS)

    Kim, Hyejin; Lee, Jae Sung; Lee, Jong Bum

    2016-04-01

    After the discovery of small interference RNA (siRNA), nanostructured siRNA delivery systems have been introduced to achieve an efficient regulation of the target gene expression. Here we report a new siRNA-generating two dimensional nanostructure in a formation of nanosized sheet. Inspired by tunable mechanical and functional properties of the previously reported RNA membrane, siRNA nanosized sheets (siRNA-NS) with multiple Dicer cleavage sites were prepared. The siRNA-NS has two dimensional structure, providing a large surface area for Dicer to cleave the siRNA-NS for the generation of functional siRNAs. Furthermore, downregulation of the cellular target gene expression was achieved by delivery of siRNA-NS without chemical modification of RNA strands or conjugation to other substances.

  2. Chitosan Nanoparticles for SiRNA Delivery In Vitro.

    PubMed

    Ragelle, Héloïse; Vanvarenberg, Kevin; Vandermeulen, Gaëlle; Préat, Véronique

    2016-01-01

    RNA interference, the process in which small interfering RNAs (SiRNAs) silence a specific gene and thus inhibit the associated protein, has opened new doors for the treatment of a wide range of diseases. However, efficient delivery of SiRNAs remains a challenge, especially due to their instability in biological environments and their inability to cross cell membranes. To protect and deliver SiRNAs to mammalian cells, a variety of polymeric nanocarriers have been developed. Among them, the polysaccharide chitosan has generated great interests. This derivative of natural chitin is biodegradable and biocompatible, and can complex SiRNAs into nanoparticles on account of its positive charges. However, chitosan presents some limitations that need to be taken into account when designing chitosan/SiRNA nanoparticles. Here, we describe a method to prepare SiRNA/chitosan nanoparticles with high gene silencing efficiency and low cytotoxicity by using the ionic gelation technique. PMID:26472448

  3. Tertiary siRNAs Mediate Paramutation in C. elegans

    PubMed Central

    Miska, Eric A.

    2015-01-01

    In the nematode Caenorhabditis elegans, different small RNA-dependent gene silencing mechanisms act in the germline to initiate transgenerational gene silencing. Piwi-interacting RNAs (piRNAs) can initiate transposon and gene silencing by acting upstream of endogenous short interfering RNAs (siRNAs), which engage a nuclear RNA interference (RNAi) pathway to trigger transcriptional gene silencing. Once gene silencing has been established, it can be stably maintained over multiple generations without the requirement of the initial trigger and is also referred to as RNAe or paramutation. This heritable silencing depends on the integrity of the nuclear RNAi pathway. However, the exact mechanism by which silencing is maintained across generations is not understood. Here we demonstrate that silencing of piRNA targets involves the production of two distinct classes of small RNAs with different genetic requirements. The first class, secondary siRNAs, are localized close to the direct target site for piRNAs. Nuclear import of the secondary siRNAs by the Argonaute HRDE-1 leads to the production of a distinct class of small RNAs that map throughout the transcript, which we term tertiary siRNAs. Both classes of small RNAs are necessary for full repression of the target gene and can be maintained independently of the initial piRNA trigger. Consistently, we observed a form of paramutation associated with tertiary siRNAs. Once paramutated, a tertiary siRNA generating allele confers dominant silencing in the progeny regardless of its own transmission, suggesting germline-transmitted siRNAs are sufficient for multigenerational silencing. This work uncovers a multi-step siRNA amplification pathway that promotes germline integrity via epigenetic silencing of endogenous and invading genetic elements. In addition, the same pathway can be engaged in environmentally induced heritable gene silencing and could therefore promote the inheritance of acquired traits. PMID:25811365

  4. Enhancing endosomal escape for nanoparticle mediated siRNA delivery

    NASA Astrophysics Data System (ADS)

    Ma, Da

    2014-05-01

    Gene therapy with siRNA is a promising biotechnology to treat cancer and other diseases. To realize siRNA-based gene therapy, a safe and efficient delivery method is essential. Nanoparticle mediated siRNA delivery is of great importance to overcome biological barriers for systemic delivery in vivo. Based on recent discoveries, endosomal escape is a critical biological barrier to be overcome for siRNA delivery. This feature article focuses on endosomal escape strategies used for nanoparticle mediated siRNA delivery, including cationic polymers, pH sensitive polymers, calcium phosphate, and cell penetrating peptides. Work has been done to develop different endosomal escape strategies based on nanoparticle types, administration routes, and target organ/cell types. Also, enhancement of endosomal escape has been considered along with other aspects of siRNA delivery to ensure target specific accumulation, high cell uptake, and low toxicity. By enhancing endosomal escape and overcoming other biological barriers, great progress has been achieved in nanoparticle mediated siRNA delivery.

  5. New paradigms on siRNA local application

    PubMed Central

    Pan, Meng; Ni, Jinwen; He, Huiming; Gao, Shan; Duan, Xiaohong

    2015-01-01

    Small interfering RNA (siRNA) functions through pairing with specific mRNA sequences and results in the mRNA’s degradation. It is a potential therapeutic approach for many diseases caused by altered gene expression. The delivery of siRNA is still a major problem due to its rapid degradation in the circulation. Various strategies have been proposed to help with the cellular uptake of siRNA and short or small hairpin RNA (shRNA). Here, we reviewed recently published data regarding local applications of siRNA. Compared with systemic delivery methods, local delivery of siRNA/shRNA has many advantages, such as targeting the specific tissues or organs, mimicking a gene knockout effect, or developing certain diseases models. The eye, brain, and tumor tissues are ‘hot’ target tissues/organs for local siRNA delivery. The siRNA can be delivered locally, in naked form, with chemical modifications, or in formulations with viral or non-viral vectors, such as liposomes and nanoparticles. This review provides a comprehensive overview of RNAi local administration and potential future applications in clinical treatment. [BMB Reports 2015; 48(3): 147-152] PMID:25081998

  6. Transdermal Delivery of siRNA through Microneedle Array.

    PubMed

    Deng, Yan; Chen, Jiao; Zhao, Yi; Yan, Xiaohui; Zhang, Li; Choy, Kwongwai; Hu, Jun; Sant, Himanshu J; Gale, Bruce K; Tang, Tao

    2016-01-01

    Successful development of siRNA therapies has significant potential for the treatment of skin conditions (alopecia, allergic skin diseases, hyperpigmentation, psoriasis, skin cancer, pachyonychia congenital) caused by aberrant gene expression. Although hypodermic needles can be used to effectively deliver siRNA through the stratum corneum, the major challenge is that this approach is painful and the effects are restricted to the injection site. Microneedle arrays may represent a better way to deliver siRNAs across the stratum corneum. In this study, we evaluated for the first time the ability of the solid silicon microneedle array for punching holes to deliver cholesterol-modified housekeeping gene (Gapdh) siRNA to the mouse ear skin. Treating the ear with microneedles showed permeation of siRNA in the skin and could reduce Gapdh gene expression up to 66% in the skin without accumulation in the major organs. The results showed that microneedle arrays could effectively deliver siRNA to relevant regions of the skin noninvasively. PMID:26888011

  7. Transdermal Delivery of siRNA through Microneedle Array

    PubMed Central

    Deng, Yan; Chen, Jiao; Zhao, Yi; Yan, Xiaohui; Zhang, Li; Choy, Kwongwai; Hu, Jun; Sant, Himanshu J.; Gale, Bruce K.; Tang, Tao

    2016-01-01

    Successful development of siRNA therapies has significant potential for the treatment of skin conditions (alopecia, allergic skin diseases, hyperpigmentation, psoriasis, skin cancer, pachyonychia congenital) caused by aberrant gene expression. Although hypodermic needles can be used to effectively deliver siRNA through the stratum corneum, the major challenge is that this approach is painful and the effects are restricted to the injection site. Microneedle arrays may represent a better way to deliver siRNAs across the stratum corneum. In this study, we evaluated for the first time the ability of the solid silicon microneedle array for punching holes to deliver cholesterol-modified housekeeping gene (Gapdh) siRNA to the mouse ear skin. Treating the ear with microneedles showed permeation of siRNA in the skin and could reduce Gapdh gene expression up to 66% in the skin without accumulation in the major organs. The results showed that microneedle arrays could effectively deliver siRNA to relevant regions of the skin noninvasively. PMID:26888011

  8. Transdermal Delivery of siRNA through Microneedle Array

    NASA Astrophysics Data System (ADS)

    Deng, Yan; Chen, Jiao; Zhao, Yi; Yan, Xiaohui; Zhang, Li; Choy, Kwongwai; Hu, Jun; Sant, Himanshu J.; Gale, Bruce K.; Tang, Tao

    2016-02-01

    Successful development of siRNA therapies has significant potential for the treatment of skin conditions (alopecia, allergic skin diseases, hyperpigmentation, psoriasis, skin cancer, pachyonychia congenital) caused by aberrant gene expression. Although hypodermic needles can be used to effectively deliver siRNA through the stratum corneum, the major challenge is that this approach is painful and the effects are restricted to the injection site. Microneedle arrays may represent a better way to deliver siRNAs across the stratum corneum. In this study, we evaluated for the first time the ability of the solid silicon microneedle array for punching holes to deliver cholesterol-modified housekeeping gene (Gapdh) siRNA to the mouse ear skin. Treating the ear with microneedles showed permeation of siRNA in the skin and could reduce Gapdh gene expression up to 66% in the skin without accumulation in the major organs. The results showed that microneedle arrays could effectively deliver siRNA to relevant regions of the skin noninvasively.

  9. Nanocarriers for delivery of siRNA and co-delivery of siRNA and other therapeutic agents.

    PubMed

    Zhao, Jing; Feng, Si-Shen

    2015-07-01

    A major problem in cancer treatment is the multidrug resistance. siRNA inhibitors have great advantages to solve the problem, if the bottleneck of their delivery could be well addressed by the various nanocarriers. Moreover, co-delivery of siRNA together with the various anticancer agents in one nanocarrier may maximize their additive or synergistic effect. This review provides a comprehensive summary on the state-of-the-art of the nanocarriers, which may include prodrugs, micelles, liposomes, dendrimers, nanohydrogels, solid lipid nanoparticles, nanoparticles of biodegradable polymers and nucleic acid nanocarriers for delivery of siRNA and co-delivery of siRNA together with anticancer agents with focus on synthesis of the nanocarrier materials, design and characterization, in vitro and in vivo evaluation, and prospect and challenges of nanocarriers. PMID:26214357

  10. Detection of Pol IV/RDR2-dependent transcripts at the genomic scale in Arabidopsis reveals features and regulation of siRNA biogenesis

    PubMed Central

    Li, Shaofang; Vandivier, Lee E.; Tu, Bin; Gao, Lei; Won, So Youn; Li, Shengben; Zheng, Binglian; Gregory, Brian D.

    2015-01-01

    Twenty-four-nucleotide small interfering (si)RNAs are central players in RNA-directed DNA methylation (RdDM), a process that establishes and maintains DNA methylation at transposable elements to ensure genome stability in plants. The plant-specific RNA polymerase IV (Pol IV) is required for siRNA biogenesis and is believed to transcribe RdDM loci to produce primary transcripts that are converted to double-stranded RNAs (dsRNAs) by RDR2 to serve as siRNA precursors. Yet, no such siRNA precursor transcripts have ever been reported. Here, through genome-wide profiling of RNAs in genotypes that compromise the processing of siRNA precursors, we were able to identify Pol IV/RDR2-dependent transcripts from tens of thousands of loci. We show that Pol IV/RDR2-dependent transcripts correspond to both DNA strands, whereas the RNA polymerase II (Pol II)-dependent transcripts produced upon derepression of the loci are derived primarily from one strand. We also show that Pol IV/RDR2-dependent transcripts have a 5′ monophosphate, lack a poly(A) tail at the 3′ end, and contain no introns; these features distinguish them from Pol II-dependent transcripts. Like Pol II-transcribed genic regions, Pol IV-transcribed regions are flanked by A/T-rich sequences depleted in nucleosomes, which highlights similarities in Pol II- and Pol IV-mediated transcription. Computational analysis of siRNA abundance from various mutants reveals differences in the regulation of siRNA biogenesis at two types of loci that undergo CHH methylation via two different DNA methyltransferases. These findings begin to reveal features of Pol IV/RDR2-mediated transcription at the heart of genome stability in plants. PMID:25414514

  11. Lipid-based vectors for siRNA delivery

    PubMed Central

    Zhang, Shubiao; Zhi, Defu; Huang, Leaf

    2016-01-01

    siRNA therapeutics has developed rapidly and already there are clinical trials ongoing or planned; however, the delivery of siRNA into cells, tissues or organs remains to be a major obstacle. Lipid-based vectors hold the most promising position among non-viral vectors, as they have a similar structure to cell or organelle membranes. But when used in the form of liposomes, these vectors have shown some problems. Therefore, either the nature of lipids themselves or forms used should be improved. As a novel class of lipid like materials, lipidoids have the advantages of easy synthesis and the ability for delivering siRNA to obtain excellent silencing activity. However, the toxicities of lipidoids have not been thoroughly studied. pH responsive lipids have also gained great attention recently, though some of the amine-based lipids are not novel in terms of chemical structures. More complex self-assembly structures, such as LPD (LPH) and LCP, may provide a good solution to siRNA delivery. They have demonstrated controlled particle morphology and size and siRNA delivery activity for both in vitro and in vivo. PMID:22994300

  12. Advances in Lipid Nanoparticles for siRNA Delivery

    PubMed Central

    Tam, Yuen Yi C.; Chen, Sam; Cullis, Pieter R.

    2013-01-01

    Technological advances in both siRNA (small interfering RNA) and whole genome sequencing have demonstrated great potential in translating genetic information into siRNA-based drugs to halt the synthesis of most disease-causing proteins. Despite its powerful promises as a drug, siRNA requires a sophisticated delivery vehicle because of its rapid degradation in the circulation, inefficient accumulation in target tissues and inability to cross cell membranes to access the cytoplasm where it functions. Lipid nanoparticle (LNP) containing ionizable amino lipids is the leading delivery technology for siRNA, with five products in clinical trials and more in the pipeline. Here, we focus on the technological advances behind these potent systems for siRNA-mediated gene silencing. PMID:24300520

  13. Nanovector Delivery of siRNA for Cancer Therapy

    PubMed Central

    Ferrari, Mauro; Sun, Tong; Shen, Haifa

    2013-01-01

    RNA interference holds the promise to knock down expression of every cancer gene. Both academic laboratories and pharmaceutical companies have committed heavily on manpower and financial resources to develop siRNA cancer therapeutics over the last decade. While significant advances have been made in the design of siRNA therapeutics and mechanism of action on cancer cell killing, there are still many hurdles to overcome including effective delivery of therapeutics in vivo. Nanotechnology has played an important role in the development of delivery vectors so far. This article summarizes current nanovectors for siRNA delivery, discusses technical challenges in overcoming biological barriers, and introduces the multistage vector system for tumor-specific delivery. PMID:22555511

  14. Prediction of siRNA potency using sparse logistic regression.

    PubMed

    Hu, Wei; Hu, John

    2014-06-01

    RNA interference (RNAi) can modulate gene expression at post-transcriptional as well as transcriptional levels. Short interfering RNA (siRNA) serves as a trigger for the RNAi gene inhibition mechanism, and therefore is a crucial intermediate step in RNAi. There have been extensive studies to identify the sequence characteristics of potent siRNAs. One such study built a linear model using LASSO (Least Absolute Shrinkage and Selection Operator) to measure the contribution of each siRNA sequence feature. This model is simple and interpretable, but it requires a large number of nonzero weights. We have introduced a novel technique, sparse logistic regression, to build a linear model using single-position specific nucleotide compositions which has the same prediction accuracy of the linear model based on LASSO. The weights in our new model share the same general trend as those in the previous model, but have only 25 nonzero weights out of a total 84 weights, a 54% reduction compared to the previous model. Contrary to the linear model based on LASSO, our model suggests that only a few positions are influential on the efficacy of the siRNA, which are the 5' and 3' ends and the seed region of siRNA sequences. We also employed sparse logistic regression to build a linear model using dual-position specific nucleotide compositions, a task LASSO is not able to accomplish well due to its high dimensional nature. Our results demonstrate the superiority of sparse logistic regression as a technique for both feature selection and regression over LASSO in the context of siRNA design. PMID:21091052

  15. An atlas of soybean small RNAs identifies phased siRNAs from hundreds of coding genes.

    PubMed

    Arikit, Siwaret; Xia, Rui; Kakrana, Atul; Huang, Kun; Zhai, Jixian; Yan, Zhe; Valdés-López, Oswaldo; Prince, Silvas; Musket, Theresa A; Nguyen, Henry T; Stacey, Gary; Meyers, Blake C

    2014-12-01

    Small RNAs are ubiquitous, versatile repressors and include (1) microRNAs (miRNAs), processed from mRNA forming stem-loops; and (2) small interfering RNAs (siRNAs), the latter derived in plants by a process typically requiring an RNA-dependent RNA polymerase. We constructed and analyzed an expression atlas of soybean (Glycine max) small RNAs, identifying over 500 loci generating 21-nucleotide phased siRNAs (phasiRNAs; from PHAS loci), of which 483 overlapped annotated protein-coding genes. Via the integration of miRNAs with parallel analysis of RNA end (PARE) data, 20 miRNA triggers of 127 PHAS loci were detected. The primary class of PHAS loci (208 or 41% of the total) corresponded to NB-LRR genes; some of these small RNAs preferentially accumulate in nodules. Among the PHAS loci, novel representatives of TAS3 and noncanonical phasing patterns were also observed. A noncoding PHAS locus, triggered by miR4392, accumulated preferentially in anthers; the phasiRNAs are predicted to target transposable elements, with their peak abundance during soybean reproductive development. Thus, phasiRNAs show tremendous diversity in dicots. We identified novel miRNAs and assessed the veracity of soybean miRNAs registered in miRBase, substantially improving the soybean miRNA annotation, facilitating an improvement of miRBase annotations and identifying at high stringency novel miRNAs and their targets. PMID:25465409

  16. Innovative Delivery of siRNA to Solid Tumors by Super Carbonate Apatite

    PubMed Central

    Wu, Xin; Yamamoto, Hirofumi; Nakanishi, Hiroyuki; Yamamoto, Yuki; Inoue, Akira; Tei, Mitsuyoshi; Hirose, Hajime; Uemura, Mamoru; Nishimura, Junichi; Hata, Taishi; Takemasa, Ichiro; Mizushima, Tsunekazu; Hossain, Sharif; Akaike, Toshihiro; Matsuura, Nariaki; Doki, Yuichiro; Mori, Masaki

    2015-01-01

    RNA interference (RNAi) technology is currently being tested in clinical trials for a limited number of diseases. However, systemic delivery of small interfering RNA (siRNA) to solid tumors has not yet been achieved in clinics. Here, we introduce an in vivo pH-sensitive delivery system for siRNA using super carbonate apatite (sCA) nanoparticles, which is the smallest class of nanocarrier. These carriers consist simply of inorganic ions and accumulate specifically in tumors, yet they cause no serious adverse events in mice and monkeys. Intravenously administered sCA-siRNA abundantly accumulated in the cytoplasm of tumor cells at 4 h, indicating quick achievement of endosomal escape. sCA-survivin-siRNA induced apoptosis in HT29 tumors and significantly inhibited in vivo tumor growth of HCT116, to a greater extent than two other in vivo delivery reagents. With innovative in vivo delivery efficiency, sCA could be a useful nanoparticle for the therapy of solid tumors. PMID:25738937

  17. Abundance of field galaxies

    NASA Astrophysics Data System (ADS)

    Klypin, Anatoly; Karachentsev, Igor; Makarov, Dmitry; Nasonova, Olga

    2015-12-01

    We present new measurements of the abundance of galaxies with a given circular velocity in the Local Volume: a region centred on the Milky Way Galaxy and extending to distance ˜10 Mpc. The sample of ˜750 mostly dwarf galaxies provides a unique opportunity to study the abundance and properties of galaxies down to absolute magnitudes MB ≈ -10 and virial masses M_vir= 109{ M_{⊙}}. We find that the standard Λ cold dark matter (ΛCDM) model gives remarkably accurate estimates for the velocity function of galaxies with circular velocities V ≳ 70 kms-1 and corresponding virial masses M_vir≳ 5× 10^{10}{ M_{⊙}}, but it badly fails by overpredicting ˜5 times the abundance of large dwarfs with velocities V = 30-40 kms-1. The warm dark matter (WDM) models cannot explain the data either, regardless of mass of WDM particle. Just as in previous observational studies, we find a shallow asymptotic slope dN/dlog V ∝ Vα, α ≈ -1 of the velocity function, which is inconsistent with the standard ΛCDM model that predicts the slope α = -3. Though reminiscent to the known overabundance of satellite problem, the overabundance of field galaxies is a much more difficult problem. For the standard ΛCDM model to survive, in the 10 Mpc radius of the Milky Way there should be 1000 not yet detected galaxies with virial mass M_vir≈ 10^{10}{ M_{⊙}}, extremely low surface brightness and no detectable H I gas. So far none of this type of galaxies have been discovered.

  18. siRNA Delivery by Stimuli-Sensitive Nanocarriers

    PubMed Central

    Salzano, Giuseppina; Costa, Daniel F.; Torchilin, Vladimir P.

    2016-01-01

    Since its discovery in late 1990s, small interfering RNA (siRNA) has become a significant biopharmaceutical research tool and a powerful option for the treatment of different human diseases based on altered gene-expression. Despite promising data from many pre-clinical studies, concrete hurdles still need to be overcome to bring therapeutic siRNAs in clinic. The design of stimuli-sensitive nanopreparations for gene therapy is a lively area of the current research. Compared to conventional systems for siRNA delivery, this type of platform can respond to local stimuli that are characteristics of the pathological area of interest, allowing the release of nucleic acids at the desired site. Acidic pH, abnormal levels of enzymes, altered redox potential and magnetic field are examples of stimuli exploited in the design of stimuli-sensitive nanoparticles. In this review, we discuss on recent stimuli-sensitive strategies for siRNA delivery and we highlight on the potential of combining multiple stimuli-sensitive strategies in the same nano-platform for a better therapeutic outcome. PMID:26486143

  19. Acoustic Liquid Handling for Rapid siRNA Transfection Optimization.

    PubMed

    Xiao, Andrew S; Lightcap, Eric S; Bouck, David C

    2015-09-01

    Gene knockdown by small interfering RNA (siRNA) has been used extensively to investigate the function of genes in targeted and genome-wide studies. One of the primary challenges of siRNA studies of any scale is to achieve sufficient gene knockdown to produce the biological changes that lead to measurable phenotypes. Reverse, lipid-based transfection efficiency minimally requires the optimization of the following parameters: cell number, knockdown duration, siRNA oligonucleotide concentration, type/brand of transfection lipid, and transfection lipid concentration. In this study, we describe a methodology to utilize the flexibility and low-volume range of the Echo acoustic liquid handler to rapidly screen a matrix of transfection conditions. The matrix includes six different transfection lipids from three separate vendors across a broad range of concentrations. Our results validate acoustic liquid transfer for the delivery of siRNAs and transfection reagents. Finally, this methodology is applied to rapidly optimize transfection conditions across many tissue culture cell lines derived from various originating tissues. PMID:25924619

  20. Acoustically Propelled Nanomotors for Intracellular siRNA Delivery.

    PubMed

    Esteban-Fernández de Ávila, Berta; Angell, Chava; Soto, Fernando; Lopez-Ramirez, Miguel Angel; Báez, Daniela F; Xie, Sibai; Wang, Joseph; Chen, Yi

    2016-05-24

    An effective intracellular gene silencing strategy based on acoustically propelled nanowires modified with an interfering RNA's (siRNA) payload is described. The gold nanowires (AuNW) are wrapped with a Rolling Circle Amplification (RCA) DNA strand, which serves to anchor the siRNA therapy. The ultrasound (US)-powered propulsion of the AuNW leads to fast internalization and rapid intracellular movement and hence to an accelerated siRNA delivery and silencing response. To optimize the micromotor gene silencing procedure, the influence of motion, time, and siRNA dosage was investigated, leading up to a 94% silencing after few minutes treatment with US-propelled siRNA-AuNWs, and to a dramatic (∼13-fold) improvement in the silencing response compared to the static modified nanowires. The ability of the nanomotor-based method for gene silencing has been demonstrated by measuring the GFP silencing response in two different cell lines (HEK-293 and MCF-7) and using detailed control experiments. The viability of the cells after the nanomotors treatment was examined using the MCF-7 cancer cell line. The use of DNA structures carried by the US-propelled nanomotors for gene silencing represents an efficient tool that addresses the challenges associated with RNA transportation and intracellular delivery. Future implementation of nanomachines in gene therapy applications can be expanded into a co-delivery platform for therapeutics. PMID:27022755

  1. Argonaute Proteins Affect siRNA Levels and Accumulation of a Novel Extrachromosomal DNA from the Dictyostelium Retrotransposon DIRS-1*

    PubMed Central

    Boesler, Benjamin; Meier, Doreen; Förstner, Konrad U.; Friedrich, Michael; Hammann, Christian; Sharma, Cynthia M.; Nellen, Wolfgang

    2014-01-01

    The retrotransposon DIRS-1 is the most abundant retroelement in Dictyostelium discoideum and constitutes the pericentromeric heterochromatin of the six chromosomes in D. discoideum. The vast majority of cellular siRNAs is derived from DIRS-1, suggesting that the element is controlled by RNAi-related mechanisms. We investigated the role of two of the five Argonaute proteins of D. discoideum, AgnA and AgnB, in DIRS-1 silencing. Deletion of agnA resulted in the accumulation of DIRS-1 transcripts, the expression of DIRS-1-encoded proteins, and the loss of most DIRS-1-derived secondary siRNAs. Simultaneously, extrachromosomal single-stranded DIRS-1 DNA accumulated in the cytoplasm of agnA− strains. These DNA molecules appear to be products of reverse transcription and thus could represent intermediate structures before transposition. We further show that transitivity of endogenous siRNAs is impaired in agnA− strains. The deletion of agnB alone had no strong effect on DIRS-1 transposon regulation. However, in agnA−/agnB− double mutant strains strongly reduced accumulation of extrachromosomal DNA compared with the single agnA− strains was observed. PMID:25352599

  2. Docosahexaenoic Acid Conjugation Enhances Distribution and Safety of siRNA upon Local Administration in Mouse Brain.

    PubMed

    Nikan, Mehran; Osborn, Maire F; Coles, Andrew H; Godinho, Bruno Mdc; Hall, Lauren M; Haraszti, Reka A; Hassler, Matthew R; Echeverria, Dimas; Aronin, Neil; Khvorova, Anastasia

    2016-01-01

    The use of siRNA-based therapies for the treatment of neurodegenerative disease requires efficient, nontoxic distribution to the affected brain parenchyma, notably the striatum and cortex. Here, we describe the synthesis and activity of a fully chemically modified siRNA that is directly conjugated to docosahexaenoic acid (DHA), the most abundant polyunsaturated fatty acid in the mammalian brain. DHA conjugation enables enhanced siRNA retention throughout both the ipsilateral striatum and cortex following a single, intrastriatal injection (ranging from 6-60 μg). Within these tissues, DHA conjugation promotes internalization by both neurons and astrocytes. We demonstrate efficient and specific silencing of Huntingtin mRNA expression in both the ipsilateral striatum (up to 73%) and cortex (up to 51%) after 1 week. Moreover, following a bilateral intrastriatal injection (60 μg), we achieve up to 80% silencing of a secondary target, Cyclophilin B, at both the mRNA and protein level. Importantly, DHA-hsiRNAs do not induce neural cell death or measurable innate immune activation following administration of concentrations over 20 times above the efficacious dose. Thus, DHA conjugation is a novel strategy for improving siRNA activity in mouse brain, with potential to act as a new therapeutic platform for the treatment of neurodegenerative disorders. PMID:27504598

  3. Mucus barrier-triggered disassembly of siRNA nanocarriers

    NASA Astrophysics Data System (ADS)

    Thomsen, Troels B.; Li, Leon; Howard, Kenneth A.

    2014-10-01

    The mucus overlying mucosal epithelial surfaces presents not only a biological barrier to the penetration of potential pathogens, but also therapeutic modalities including RNAi-based nanocarriers. Movement of nanomedicines across the mucus barriers of the gastrointestinal mucosa is modulated by interactions of the nanomedicine carriers with mucin glycoproteins inside the mucus, potentiated by the large surface area of the nanocarrier. We have developed a fluorescence activation-based reporter system showing that the interaction between polyanionic mucins and the cationic chitosan/small interfering RNA (siRNA) nanocarriers (polyplexes) results in the disassembly and consequent triggered release of fluorescent siRNA. The quantity of release was found to be dependent on the molar ratio between chitosan amino groups and siRNA phosphate groups (NP ratio) of the polyplexes with a maximal estimated 48.6% release of siRNA over 30 min at NP 60. Furthermore, a microfluidic in vitro model of the gastrointestinal mucus barrier was used to visualize the dynamic interaction between chitosan/siRNA nanocarriers and native purified porcine stomach mucins. We observed strong interactions and aggregations at the mucin-liquid interface, followed by an NP ratio dependent release and consequent diffusion of siRNA across the mucin barrier. This work describes a new model of interaction at the nanocarrier-mucin interface and has important implications for the design and development of nucleic acid-based nanocarrier therapeutics for mucosal disease treatments and also provides insights into nanoscale pathogenic processes.The mucus overlying mucosal epithelial surfaces presents not only a biological barrier to the penetration of potential pathogens, but also therapeutic modalities including RNAi-based nanocarriers. Movement of nanomedicines across the mucus barriers of the gastrointestinal mucosa is modulated by interactions of the nanomedicine carriers with mucin glycoproteins inside the

  4. Beryllium and Boron abundances in population II stars

    NASA Technical Reports Server (NTRS)

    1995-01-01

    The scientific focus of this program was to undertake UV spectroscopic abundance analyses of extremely metal poor stars with attention to determining abundances of light elements such as beryllium and boron. The abundances are likely to reflect primordial abundances within the early galaxy and help to constrain models for early galactic nucleosynthesis. The general metal abundances of these stars are also important for understanding stellar evolution.

  5. The Transition from Primary siRNAs to Amplified Secondary siRNAs That Regulate Chalcone Synthase During Development of Glycine max Seed Coats

    PubMed Central

    Cho, Young B.; Jones, Sarah I.; Vodkin, Lila

    2013-01-01

    The I locus is a 27-kb inverted repeat cluster of chalcone synthase genes CHS1-3-4 that mediates siRNA down-regulation of CHS7 and CHS8 target mRNAs during seed development leading to yellow seed coats lacking anthocyanin pigments. Here, we report small RNA sequencing of ten stages of seed development from a few days post fertilization through maturity, revealing the amplification from primary to secondary short interfering RNAs (siRNAs) occurring during development. The young seed populations had a higher proportion of siRNAs representing the CHS1-3-4 gene family members, consistent with this region as the origin of the primary siRNAs. More intriguingly, the very young seed had a higher proportion of 22-nt CHS siRNAs than did the mid-maturation seed. We infer that the primary CHS siRNAs increase during development to levels sufficient to trigger amplification of secondary CHS siRNAs from the CHS7/8 target mRNAs, enabling the total levels of 21-nt CHS siRNAs to rise dramatically. Further, we demonstrate that the soybean system exhibits tissue-specific CHS siRNA production because primary CHS siRNA levels are not sufficient to trigger secondary amplification in tissues other than the seed coat. PMID:24204712

  6. EGFP-Based Protein Nanoparticles with Cell-Penetrating Peptide for Efficient siRNA Delivery.

    PubMed

    Guan, Xingang; Hu, Xiuli; Cui, Fengchao; Li, Yunqi; Jing, Xiabing; Xie, Zhigang

    2015-11-01

    Development of an innovative nucleic acid nanocarriers still represents a challenge. In this study, we develop a protein nanoparticle (H6-TatEGFP) and examine its siRNA condensing activity. Gel retardation assay show that protein nanoparticle can condense siRNA into stable nanoparticle/siRNA complexes. UsingCy3-labelled siRNA, we also evaluate siRNA transport characteristic of protein nanoparticles in tumor cells, the results indicate that H6-TatEGFP nanoparticle may be a potential nanocarrier for siRNA in tumor cells. PMID:26109167

  7. A peptidomimetic siRNA transfection reagent forhighly effectivegene silencing

    SciTech Connect

    Utku, Yeliz; Dehan, Elinor; Ouerfelli, Ouathek; Piano, Fabio; Zuckermann, Ronald N.; Pagano, Michele; Kirshenbaum, Kent

    2006-05-17

    RNA interference (RNAi) techniques hold forth great promisefor therapeutic silencing of deleterious genes. However, clinicalapplications of RNAi require the development of safe and efficientmethods for intracellular delivery of small interfering RNA (siRNA)oligonucleotides specific to targeted genes. We describe the use of alipitoid, a cationic oligopeptoid phospholipid conjugate, for non-viraltransfection of synthetic siRNA oligos in cell culture. Thispeptidomimetic delivery vehicle allows for efficient siRNA transfectionin a variety of human cell lines with negligible toxicity and promotesextensive downregulation of the targeted genes at both the protein andthe mRNA level. We compare the lipitoid reagent to a standard commercialtransfection reagent. The lipitoid is highly efficient even in primaryIMR-90 human lung fibroblasts in which other commercial reagents aretypically ineffective.

  8. High Throughput siRNA Screening Using Reverse Transfection.

    PubMed

    von Schantz, Carina; Saarela, Jani

    2016-01-01

    RNA interference (RNAi) is a commonly used technique to knockdown gene function. Here, we describe a high throughput screening method for siRNA mediated gene silencing of the breast cancer cell line MDA-MB-231 using reverse transfection. Furthermore, we describe the setup for two separate methods for detecting viable and dead cells using either homogenous assays or image-based analysis. PMID:27581282

  9. Ocular neuroprotection by siRNA targeting caspase-2

    PubMed Central

    Ahmed, Z; Kalinski, H; Berry, M; Almasieh, M; Ashush, H; Slager, N; Brafman, A; Spivak, I; Prasad, N; Mett, I; Shalom, E; Alpert, E; Di Polo, A; Feinstein, E; Logan, A

    2011-01-01

    Retinal ganglion cell (RGC) loss after optic nerve damage is a hallmark of certain human ophthalmic diseases including ischemic optic neuropathy (ION) and glaucoma. In a rat model of optic nerve transection, in which 80% of RGCs are eliminated within 14 days, caspase-2 was found to be expressed and cleaved (activated) predominantly in RGC. Inhibition of caspase-2 expression by a chemically modified synthetic short interfering ribonucleic acid (siRNA) delivered by intravitreal administration significantly enhanced RGC survival over a period of at least 30 days. This exogenously delivered siRNA could be found in RGC and other types of retinal cells, persisted inside the retina for at least 1 month and mediated sequence-specific RNA interference without inducing an interferon response. Our results indicate that RGC apoptosis induced by optic nerve injury involves activation of caspase-2, and that synthetic siRNAs designed to inhibit expression of caspase-2 represent potential neuroprotective agents for intervention in human diseases involving RGC loss. PMID:21677688

  10. Intracellular Delivery of siRNA by Polycationic Superparamagnetic Nanoparticles

    PubMed Central

    Castillo, Betzaida; Bromberg, Lev; López, Xaira; Badillo, Valerie; González Feliciano, Jose A.; González, Carlos I.; Hatton, T. Alan; Barletta, Gabriel

    2012-01-01

    The siRNA transfection efficiency of nanoparticles (NPs), composed of a superparamagnetic iron oxide core modified with polycationic polymers (poly(hexamethylene biguanide) or branched polyethyleneimine), were studied in CHO-K1 and HeLa cell lines. Both NPs demonstrated to be good siRNA transfection vehicles, but unmodified branched polyethyleneimine (25 kD) was superior on both cell lines. However, application of an external magnetic field during transfection (magnetofection) increased the efficiency of the superparamagnetic NPs. Furthermore, our results reveal that these NPs are less toxic towards CHO-K1 cell lines than the unmodified polycationic-branched polyethyleneimine (PEI). In general, the external magnetic field did not alter the cell's viability nor it disrupted the cell membranes, except for the poly(hexamethylene biguanide)-modified NP, where it was observed that in CHO-K1 cells application of the external magnetic field promoted membrane damage. This paper presents new polycationic superparamagnetic NPs as promising transfection vehicles for siRNA and demonstrates the advantages of magnetofection. PMID:22970377

  11. Local administration of siRNA through Microneedle: Optimization, Bio-distribution, Tumor Suppression and Toxicity

    PubMed Central

    Tang, Tao; Deng, Yan; Chen, Jiao; Zhao, Yi; Yue, Ruifeng; Choy, Kwong Wai; Wang, Chi Chiu; Du, Quan; Xu, Yan; Han, Linxiao; Chung, Tony Kwok Hung

    2016-01-01

    Although RNA interference may become a novel therapeutic approach for cancer treatment, target-site accumulation of siRNA to achieve therapeutic dosage will be a major problem. Microneedle represents a better way to deliver siRNAs and we have evaluated for the first time the capability of a silicon microneedle array for delivery of Gapdh siRNA to the skin in vivo and the results showed that the microneedle arrays could effectively deliver siRNA to relevant regions of the skin noninvasively. For the further study in this field, we evaluated the efficacy of the injectable microneedle device for local delivery of siRNA to the mouse xenograft. The results presented here indicate that local administration of siRNA through injectable microneedle could effectively deliver siRNA into the tumor region, and inhibit tumor progression without major adverse effects. PMID:27457182

  12. Recent Developments in Nanoparticle-Based siRNA Delivery for Cancer Therapy

    PubMed Central

    Lee, Jong-Min; Yoon, Tae-Jong; Cho, Young-Seok

    2013-01-01

    RNA interference (RNAi) is a gene regulation mechanism initiated by RNA molecules that enables sequence-specific gene silencing by promoting degradation of specific mRNAs. Molecular therapy using small interfering RNA (siRNA) has shown great therapeutic potential for diseases caused by abnormal gene overexpression or mutation. The major challenges to application of siRNA therapeutics include the stability and effective delivery of siRNA in vivo. Important progress in nanotechnology has led to the development of efficient siRNA delivery systems. In this review, the authors discuss recent advances in nanoparticle-mediated siRNA delivery and the application of siRNA in clinical trials for cancer therapy. This review will also offer perspectives on future applications of siRNA therapeutics. PMID:23844368

  13. Local administration of siRNA through Microneedle: Optimization, Bio-distribution, Tumor Suppression and Toxicity

    NASA Astrophysics Data System (ADS)

    Tang, Tao; Deng, Yan; Chen, Jiao; Zhao, Yi; Yue, Ruifeng; Choy, Kwong Wai; Wang, Chi Chiu; Du, Quan; Xu, Yan; Han, Linxiao; Chung, Tony Kwok Hung

    2016-07-01

    Although RNA interference may become a novel therapeutic approach for cancer treatment, target-site accumulation of siRNA to achieve therapeutic dosage will be a major problem. Microneedle represents a better way to deliver siRNAs and we have evaluated for the first time the capability of a silicon microneedle array for delivery of Gapdh siRNA to the skin in vivo and the results showed that the microneedle arrays could effectively deliver siRNA to relevant regions of the skin noninvasively. For the further study in this field, we evaluated the efficacy of the injectable microneedle device for local delivery of siRNA to the mouse xenograft. The results presented here indicate that local administration of siRNA through injectable microneedle could effectively deliver siRNA into the tumor region, and inhibit tumor progression without major adverse effects.

  14. Local administration of siRNA through Microneedle: Optimization, Bio-distribution, Tumor Suppression and Toxicity.

    PubMed

    Tang, Tao; Deng, Yan; Chen, Jiao; Zhao, Yi; Yue, Ruifeng; Choy, Kwong Wai; Wang, Chi Chiu; Du, Quan; Xu, Yan; Han, Linxiao; Chung, Tony Kwok Hung

    2016-01-01

    Although RNA interference may become a novel therapeutic approach for cancer treatment, target-site accumulation of siRNA to achieve therapeutic dosage will be a major problem. Microneedle represents a better way to deliver siRNAs and we have evaluated for the first time the capability of a silicon microneedle array for delivery of Gapdh siRNA to the skin in vivo and the results showed that the microneedle arrays could effectively deliver siRNA to relevant regions of the skin noninvasively. For the further study in this field, we evaluated the efficacy of the injectable microneedle device for local delivery of siRNA to the mouse xenograft. The results presented here indicate that local administration of siRNA through injectable microneedle could effectively deliver siRNA into the tumor region, and inhibit tumor progression without major adverse effects. PMID:27457182

  15. Essential Role for Endogenous siRNAs during Meiosis in Mouse Oocytes

    PubMed Central

    Stein, Paula; Rozhkov, Nikolay V.; Li, Fan; Cárdenas, Fabián L.; Davydenk, Olga; Vandivier, Lee E.; Gregory, Brian D.; Hannon, Gregory J.; Schultz, Richard M.

    2015-01-01

    The RNase III enzyme DICER generates both microRNAs (miRNAs) and endogenous short interfering RNAs (endo-siRNAs). Both small RNA species silence gene expression post-transcriptionally in association with the ARGONAUTE (AGO) family of proteins. In mammals, there are four AGO proteins (AGO1-4), of which only AGO2 possesses endonucleolytic activity. siRNAs trigger endonucleolytic cleavage of target mRNAs, mediated by AGO2, whereas miRNAs cause translational repression and mRNA decay through association with any of the four AGO proteins. Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because mouse oocytes express both miRNAs and endo-siRNAs, this phenotype could be due to the absence of either class of small RNA, or both. However, we and others demonstrated that miRNA function is suppressed in mouse oocytes, which suggested that endo-siRNAs, not miRNAs, are essential for female meiosis. To determine if this was the case we generated mice that express a catalytically inactive knock-in allele of Ago2 (Ago2ADH) exclusively in oocytes and thereby disrupted the function of siRNAs. Oogenesis and hormonal response are normal in Ago2ADH oocytes, but meiotic maturation is impaired, with severe defects in spindle formation and chromosome alignment that lead to meiotic catastrophe. The transcriptome of these oocytes is widely perturbed and shows a highly significant correlation with the transcriptome of Dicer null and Ago2 null oocytes. Expression of the mouse transcript (MT), the most abundant transposable element in mouse oocytes, is increased. This study reveals that endo-siRNAs are essential during meiosis I in mouse females, demonstrating a role for endo-siRNAs in mammals. PMID:25695507

  16. Modified gold nanoparticles for intracellular delivery of anti-liver cancer siRNA.

    PubMed

    Shaat, Hanan; Mostafa, Amany; Moustafa, Moustafa; Gamal-Eldeen, Amira; Emam, Ahmed; El-Hussieny, Enas; Elhefnawi, Mahmoud

    2016-05-17

    To overcome the rapid enzymatic degradation and low transfection efficiency of siRNA, the delivery carriers for siRNA is a therapeutic demand to increase its stability. Gold nanoparticles (AuNPs) modified by branched polyethyleneimine (bPEI) were developed as an efficient and safe intracellular delivery carriers for siRNA. The current study implied that siRNA designed against an oncogene c-Myc could be delivered by a modified AuNPs complex without significant cytotoxicity. The comparative semi-quantitative and quantitative real time PCR were used to measure the c-Myc gene expression after transfection with naked siRNA and siRNA/bPEI/AuNPs, but AuNPs interfered with PCR. However, the c-Myc protein translation was successfully detected in the transfected HuH7 cells with naked siRNA and siRNA/bPEI/AuNPs and it was found to be inhibited by siRNA/bPEI/AuNPs more than naked siRNA. The results validate the successful silencing of c-Myc gene. Accordingly, it may confirm the promising and effective delivery of siRNA by bPEI/AuNPs. The complex enhances the cellular uptake of siRNA without significant cytotoxicity and confirms that bPEI modified AuNPs could be used as a good candidate for safe cellular delivery of siRNA. PMID:27036397

  17. Extreme Physics

    NASA Astrophysics Data System (ADS)

    Colvin, Jeff; Larsen, Jon

    2013-11-01

    Acknowledgements; 1. Extreme environments: what, where, how; 2. Properties of dense and classical plasmas; 3. Laser energy absorption in matter; 4. Hydrodynamic motion; 5. Shocks; 6. Equation of state; 7. Ionization; 8. Thermal energy transport; 9. Radiation energy transport; 10. Magnetohydrodynamics; 11. Considerations for constructing radiation-hydrodynamics computer codes; 12. Numerical simulations; Appendix: units and constants, glossary of symbols; References; Bibliography; Index.

  18. Current siRNA targets in atherosclerosis and aortic aneurysm.

    PubMed

    Pradhan-Nabzdyk, Leena; Huang, Chenyu; LoGerfo, Frank W; Nabzdyk, Christoph S

    2014-05-01

    Atherosclerosis (ATH) and aortic aneurysms (AA) remain challenging chronic diseases that confer high morbidity and mortality despite advances in medical, interventional, and surgical care. RNA interference represents a promising technology that may be utilized to silence genes contributing to ATH and AA. Despite positive results in preclinical and some clinical feasibility studies, challenges such as target/sequence validation, tissue specificity, transfection efficiency, and mitigation of unwanted off-target effects remain to be addressed. In this review the most current targets and some novel approaches in siRNA delivery are being discussed. Due to the plethora of investigated targets, only studies published between 2010 and 2014 were included. PMID:24882715

  19. Calcium phosphate nanoparticles-based systems for siRNA delivery.

    PubMed

    Xu, Xiaochun; Li, Zehao; Zhao, Xueqin; Keen, Lawrence; Kong, Xiangdong

    2016-09-01

    Despite the enormous therapeutic potential of siRNA as a treatment strategy, the delivery is still a problem due to unfavorable biodistribution profiles and poor intracellular bioavailability. Calcium phosphate (CaP) co-precipitate has been used for nearly 40 years for in vitro transfection due to its non-toxic nature and simplicity of preparation. The surface charge of CaP will be tuned into positive by surface modification, which is important for siRNA loading and crossing cell membrane without enzymatic degradation. The new siRNA carrier system will also promote the siRNA escape from lysosome to achieve siRNA sustained delivery and high-efficiency silence. In this review, we focus on the current research activity in the development of CaP nanoparticles for siRNA delivery. These nanoparticles are mainly classified into lipid coated, polymer coated and various other types for discussion. PMID:27252888

  20. Pharmacokinetics and biodistribution of recently-developed siRNA nanomedicines.

    PubMed

    Park, Jinho; Park, Joonyoung; Pei, Yihua; Xu, Jun; Yeo, Yoon

    2016-09-01

    Small interfering RNA (siRNA) is a promising drug candidate, expected to have broad therapeutic potentials toward various diseases including viral infections and cancer. With recent advances in bioconjugate chemistry and carrier technology, several siRNA-based drugs have advanced to clinical trials. However, most cases address local applications or diseases in the filtering organs, reflecting remaining challenges in systemic delivery of siRNA. The difficulty in siRNA delivery is in large part due to poor circulation stability and unfavorable pharmacokinetics and biodistribution profiles of siRNA. This review describes the pharmacokinetics and biodistribution of siRNA nanomedicines, focusing on those reported in the past 5years, and their pharmacological effects in selected disease models such as hepatocellular carcinoma, liver infections, and respiratory diseases. The examples discussed here will provide an insight into the current status of the art and unmet needs in siRNA delivery. PMID:26686832

  1. Calcium phosphate nanoparticles-based systems for siRNA delivery

    PubMed Central

    Xu, Xiaochun; Li, Zehao; Zhao, Xueqin; Keen, Lawrence; Kong, Xiangdong

    2016-01-01

    Despite the enormous therapeutic potential of siRNA as a treatment strategy, the delivery is still a problem due to unfavorable biodistribution profiles and poor intracellular bioavailability. Calcium phosphate (CaP) co-precipitate has been used for nearly 40 years for in vitro transfection due to its non-toxic nature and simplicity of preparation. The surface charge of CaP will be tuned into positive by surface modification, which is important for siRNA loading and crossing cell membrane without enzymatic degradation. The new siRNA carrier system will also promote the siRNA escape from lysosome to achieve siRNA sustained delivery and high-efficiency silence. In this review, we focus on the current research activity in the development of CaP nanoparticles for siRNA delivery. These nanoparticles are mainly classified into lipid coated, polymer coated and various other types for discussion. PMID:27252888

  2. Hydroxychloroquine-conjugated gold nanoparticles for improved siRNA activity.

    PubMed

    Perche, F; Yi, Y; Hespel, L; Mi, P; Dirisala, A; Cabral, H; Miyata, K; Kataoka, K

    2016-06-01

    Current technology of siRNA delivery relies on pharmaceutical dosage forms to route maximal doses of siRNA to the tumor. However, this rationale does not address intracellular bottlenecks governing silencing activity. Here, we tested the impact of hydroxychloroquine conjugation on the intracellular fate and silencing activity of siRNA conjugated PEGylated gold nanoparticles. Addition of hydroxychloroquine improved endosomal escape and increased siRNA guide strand distribution to the RNA induced silencing complex (RISC), both crucial obstacles to the potency of siRNA. This modification significantly improved gene downregulation in cellulo. Altogether, our data suggest the benefit of this modification for the design of improved siRNA delivery systems. PMID:26986857

  3. RNase non-sensitive and endocytosis independent siRNA delivery system: delivery of siRNA into tumor cells and high efficiency induction of apoptosis

    NASA Astrophysics Data System (ADS)

    Jiang, Xinglu; Wang, Guobao; Liu, Ru; Wang, Yaling; Wang, Yongkui; Qiu, Xiaozhong; Gao, Xueyun

    2013-07-01

    To date, RNase degradation and endosome/lysosome trapping are still serious problems for siRNA-based molecular therapy, although different kinds of delivery formulations have been tried. In this report, a cell penetrating peptide (CPP, including a positively charged segment, a linear segment, and a hydrophobic segment) and a single wall carbon nanotube (SWCNT) are applied together by a simple method to act as a siRNA delivery system. The siRNAs first form a complex with the positively charged segment of CPP via electrostatic forces, and the siRNA-CPP further coats the surface of the SWCNT via hydrophobic interactions. This siRNA delivery system is non-sensitive to RNase and can avoid endosome/lysosome trapping in vitro. When this siRNA delivery system is studied in Hela cells, siRNA uptake was observed in 98% Hela cells, and over 70% mRNA of mammalian target of rapamycin (mTOR) is knocked down, triggering cell apoptosis on a significant scale. Our siRNA delivery system is easy to handle and benign to cultured cells, providing a very efficient approach for the delivery of siRNA into the cell cytosol and cleaving the target mRNA therein.

  4. Intracellular trafficking and exocytosis of a multi-component siRNA nanocomplex.

    PubMed

    Shukla, Ravi S; Jain, Akshay; Zhao, Zhen; Cheng, Kun

    2016-07-01

    Despite the importance of siRNA delivery systems, understanding of their intracellular fate remains elusive. We recently developed a multi-component siRNA nanocomplex to deliver siRNA to hepatic stellate cells (HSCs). The objective of this study is to study post-internalization trafficking of this siRNA nanocomplex and its multiple components like siRNA, protamine, and streptavidin, in HSCs. After internalization, the nanocomplex entrapped in early endosomes undergoes three possible routes including endosomal escape, exocytosis, and entrapment in lysosomes. Significant amount of siRNA dissociates from the nanocomplex to exert silencing activity. After escaping from endosomes, protamine dissociates from the nanocomplex and stays inside the cytoplasm. Golgi complex plays an important role in exocytosis of the nanocomplex. We also demonstrate that exocytosis is one of the major reasons accounting for the transient silencing activity of nonviral siRNA delivery. Incorporation of exocytosis inhibitors in nonviral siRNA delivery systems may extend the silencing activity of siRNA. PMID:26970028

  5. Pharmacokinetic Behaviors of Intravenously Administered siRNA in Glandular Tissues.

    PubMed

    Huang, Yuanyu; Cheng, Qiang; Ji, Jia-Li; Zheng, Shuquan; Du, Lili; Meng, Lingwei; Wu, Yidi; Zhao, Deyao; Wang, Xiaoxia; Lai, Li; Cao, Huiqing; Xiao, Kai; Gao, Shan; Liang, Zicai

    2016-01-01

    The pharmacokinetics of small interfering RNAs (siRNAs) is a pivotal issue for siRNA-based drug development. In this study, we comprehensively investigated the behavior of siRNAs in vivo in various tissues and demonstrated that intravenously-injected naked siRNA accumulated remarkably in the submandibular gland, bulbourethral gland, and pancreas, with a respective half-life of ~22.7, ~45.6, and ~30.3 h. This was further confirmed by gel separation of tissue homogenates and/or supernatants. In vivo imaging and cryosectioning suggested that delivery carriers significantly influence the distribution and elimination profiles of siRNA. Gene-silencing assays revealed that neither naked nor liposome-formulated siRNA resulted in gene knockdown in the submandibular and bulbourethral glands after systemic administration, suggesting that these glands function as drug reservoirs that enable slow siRNA release into the circulation. But robust gene-silencing was achieved by local injection of liposome-encapsulated siRNA into the submandibular gland. Our results enhance understanding of the pharmacokinetic properties of siRNAs and we believe that they will facilitate the development of siRNA therapy, especially for the submandibular gland. PMID:27446488

  6. Turning Squalene into Cationic Lipid Allows a Delivery of siRNA in Cultured Cells

    PubMed Central

    Bertrand, Jean-Rémi; Lucas, Claire; Pham, Ngoc Minh; Durieu, Catherine; Couvreur, Patrick; Desmaële, Didier

    2015-01-01

    Covalent binding of squalene to siRNA has already been shown to be an interesting way of delivering siRNA in vivo. Whether squalene derivatives could also be used to deliver siRNA in cells without covalent binding similar to usual transfection with cationic lipids is the question addressed in this article. Accordingly, we investigated the activity of two squalene derivatives bearing a quaternary ammonium head group and a guanidinium group, respectively. The second derivative displayed interesting properties for delivering siRNA into cells in vitro. PMID:25894614

  7. Precise and efficient siRNA design: a key point in competent gene silencing.

    PubMed

    Fakhr, E; Zare, F; Teimoori-Toolabi, L

    2016-04-01

    RNA interference-related strategies have become appealing methods in various fields of research. Exact sequence design of these small molecules is an essential step in the silencing procedure. Numerous researchers have tried to define some algorithms in order to increase the chance of short interfering RNA's (siRNA's) success. In recent decades, online designing software has aimed at promoting the quality of siRNA designing based on the most cited algorithms. According to our previous experiments, a combination of different criteria would be helpful. That is, siRNAs suggested by a combination of tools seem to be more efficient. Furthermore, different factors such as distance of target region to transcription start site, nucleotide composition, absence of off-target effects and secondary structures in the target site and siRNA and the presence of asymmetry and energy valley within the siRNA will increase the efficiency of siRNAs. Despite application of different online tools and fulfilling the criteria, there is no guarantee for designing an effective siRNA. However, meticulous designing of siRNAs according to the suggested algorithms and scoring systems and using different siRNAs for targeting the same gene would lead to improved silencing outcome. In this review, we focus on common algorithms and online software, and introduce a new scoring system used in our experiments. PMID:26987292

  8. Pharmacokinetic Behaviors of Intravenously Administered siRNA in Glandular Tissues

    PubMed Central

    Huang, Yuanyu; Cheng, Qiang; Ji, Jia-Li; Zheng, Shuquan; Du, Lili; Meng, Lingwei; Wu, Yidi; Zhao, Deyao; Wang, Xiaoxia; Lai, Li; Cao, Huiqing; Xiao, Kai; Gao, Shan; Liang, Zicai

    2016-01-01

    The pharmacokinetics of small interfering RNAs (siRNAs) is a pivotal issue for siRNA-based drug development. In this study, we comprehensively investigated the behavior of siRNAs in vivo in various tissues and demonstrated that intravenously-injected naked siRNA accumulated remarkably in the submandibular gland, bulbourethral gland, and pancreas, with a respective half-life of ~22.7, ~45.6, and ~30.3 h. This was further confirmed by gel separation of tissue homogenates and/or supernatants. In vivo imaging and cryosectioning suggested that delivery carriers significantly influence the distribution and elimination profiles of siRNA. Gene-silencing assays revealed that neither naked nor liposome-formulated siRNA resulted in gene knockdown in the submandibular and bulbourethral glands after systemic administration, suggesting that these glands function as drug reservoirs that enable slow siRNA release into the circulation. But robust gene-silencing was achieved by local injection of liposome-encapsulated siRNA into the submandibular gland. Our results enhance understanding of the pharmacokinetic properties of siRNAs and we believe that they will facilitate the development of siRNA therapy, especially for the submandibular gland. PMID:27446488

  9. Development of siRNA payloads to target KRAS-mutant cancer

    PubMed Central

    Ritchie, Cayde D.; Thapar, Vishal; Lee, Liam C.; Hsu, Dennis J.; Grace, Danielle; Carver, Joseph O.; Zuber, Johannes; Luo, Ji; McCormick, Frank; Lowe, Scott W.

    2014-01-01

    RNA interference (RNAi) is a powerful tool for target identification and can lead to novel therapies for pharmacologically intractable targets such as KRAS. RNAi therapy must combine potent siRNA payloads with reliable in vivo delivery for efficient target inhibition. We employed a functional “Sensor” assay to establish a library of potent siRNAs against RAS pathway genes and show they efficiently suppress their targets at low dose. This reduces off-target effects and enables combination gene knockdown. We administered Sensor siRNAs in vitro and in vivo and validated the delivery of KRAS siRNA alone and siRNA targeting the complete RAF effector node (A/B/C-RAF) as promising strategies to treat KRAS-mutant colorectal cancer. We further demonstrate that improved therapeutic efficacy is achieved by formulating siRNA payloads that combine both single-gene siRNA and node-targeted siRNAs (KRAS+PIK3C-A/B). The customizable nature of Sensor siRNA payloads offers a universal platform for combination target identification and development of RNAi therapeutics. PMID:25100204

  10. Cationic lipid nanodisks as an siRNA delivery vehicle.

    PubMed

    Ghosh, Mistuni; Ren, Gang; Simonsen, Jens B; Ryan, Robert O

    2014-06-01

    The term nanodisk (ND) describes reconstituted high-density lipoprotein particles that contain one or more exogenous bioactive agents. In the present study, ND were assembled from apolipoprotein A-I, the zwitterionic glycerophospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the synthetic cationic lipid 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP). ND formulated at a DMPC:DMTAP ratio of 70:30 (by weight) were soluble in aqueous media. The particles generated were polydisperse, with diameters ranging from ∼20 to <50 nm. In nucleic acid binding studies, agarose gel retardation assays revealed that a synthetic 23-mer double-stranded oligonucleotide (dsOligo) bound to DMTAP containing ND but not to ND formulated with DMPC alone. Sucrose density gradient ultracentrifugation studies provided additional evidence for stable dsOligo binding to DMTAP-ND. Incubation of cultured hepatoma cells with DMTAP-ND complexed with a siRNA directed against glyceraldehyde 3-phosphate dehydrogenase showed 60% knockdown efficiency. Thus, incorporation of synthetic cationic lipid (i.e., DMTAP) to ND confers an ability to bind siRNA and the resulting complexes possess target gene knockdown activity in a cultured cell model. PMID:24840721

  11. Interfering cancer with polymeric siRNA nanomedicines.

    PubMed

    Tiram, Galia; Scomparin, Anna; Ofek, Paula; Satchi-Fainaro, Ronit

    2014-01-01

    The ability to specifically silence genes using RNA interference (RNAi) has wide therapeutic applications for the treatment of disease. Numerous studies have demonstrated global gene and protein signatures distinguishing malignant and nonmalignant tissues. This worldwide pursuit of optimal cancer targets has so far provided a wide list of potential targets for each cancer type and for each patient, for which RNAi-based therapies can be applied. Nevertheless, due to poor stability of RNAi molecules in physiological conditions and their inability to cross cellular membranes, the delivery of siRNA and microRNA (miRNA) in vivo holds a great challenge and remains a crucial issue for their therapeutic success. Supramolecular carriers are often used in order to improve the physicochemical and biopharmaceutical properties of RNAi. Nano-sized delivery systems enable the accumulation of drugs and oligonucleotides (ONTs) in angiogenesis-dependent areas due to the enhanced permeability and retention (EPR) effect, and are able to cross cellular membranes and release the siRNA/miRNA only inside the target cell. In addition, a targeting moiety can increase the selectivity and specific uptake in the target tissue. Several vehicles (dendrimers, nanoparticles, liposomes, polyplex, lipoplex, polymeric nanoconjugates) are being developed for siRNA/miRNA delivery. These vehicles provide an important tool for exploiting the full potential of ONTs as therapeutic agents. In this review we will focus on the polymer-based approaches to deliver siRNA to cancer in vivo. PMID:24724498

  12. Nanolayered siRNA dressing for sustained localized knockdown.

    PubMed

    Castleberry, Steven; Wang, Mary; Hammond, Paula T

    2013-06-25

    The success of RNA interference (RNAi) in medicine relies on the development of technology capable of successfully delivering it to tissues of interest. Significant research has focused on the difficult task of systemic delivery of RNAi; however its local delivery could be a more easily realized approach. Localized delivery is of particular interest for many medical applications, including the treatment of localized diseases, the modulation of cellular response to implants or tissue engineering constructs, and the management of wound healing and regenerative medicine. In this work we present an ultrathin electrostatically assembled coating for localized and sustained delivery of short interfering RNA (siRNA). This film was applied to a commercially available woven nylon dressing commonly used for surgical applications and was demonstrated to sustain significant knockdown of protein expression in multiple cell types for more than one week in vitro. Significantly, this coating can be easily applied to a medically relevant device and requires no externally delivered transfection agents for effective delivery of siRNA. These results present promising opportunities for the localized administration of RNAi. PMID:23672676

  13. siRNA as a tool for investigating organogenesis

    PubMed Central

    Lee, Wen-Chin; Berry, Rachel; Davies, Jamie

    2008-01-01

    Removing the function of a specific gene from a developing organ, by making a ‘knockout’ mouse, is a powerful method for analyzing the molecular pathways that control organogenesis. The technique is expensive, though, in terms of time and money, and complex strategies for producing conditional knockouts are needed for genes that are essential for early development of the embryo, for which an unconditional knockout would be lethal before the organ of interest begins to form. Small interfering RNAs (siRNAs) offer a method of knocking down the expression of specific genes with no need for genomic manipulation. Almost as soon as they had been discovered, siRNAs began to be used to explore the molecular biology of mammalian cells in conventional, two-dimensional culture. They have now also been applied successfully, by several groups, to knock down specific genes in various organ rudiments developing in organ culture. This article reviews the basic technique of siRNA-mediated gene knockdown and how it is being applied to organ culture. It also reviews some of the current problems and challenges in the field, and the ways in which these problems are likely to be overcome. PMID:19279730

  14. Advancements in the field of intravaginal siRNA delivery.

    PubMed

    Yang, Sidi; Chen, Yufei; Ahmadie, Roien; Ho, Emmanuel A

    2013-04-10

    The vaginal tract is a suitable site for the administration of both local and systemic acting drugs. There are numerous vaginal products on the market such as those approved for contraception, treatment of yeast infection, hormonal replacement therapy, and feminine hygiene. Despite the potential in drug delivery, the vagina is a complex and dynamic organ that requires greater understanding. The recent discovery that injections of double stranded RNA (dsRNA) in Caenorhabditis elegans (C. elegans) results in potent gene specific silencing, was a major scientific revolution. This phenomenon known as RNA interference (RNAi), is believed to protect host genome against invasion by mobile genetic elements such as transposons and viruses. Gene silencing or RNAi has opened new potential opportunities to study the function of a gene in an organism. Furthermore, its therapeutic potential is being investigated in the field of sexually transmitted infections such as human immunodeficiency virus (HIV) and other diseases such as age-related macular degeneration (AMD), diabetes, hypercholesterolemia, respiratory disease, and cancer. This review will focus on the therapeutic potential of siRNA for the treatment and/or prevention of infectious diseases such as HIV, HPV, and HSV within the vaginal tract. Specifically, formulation design parameters to improve siRNA stability and therapeutic efficacy in the vaginal tract will be discussed along with challenges, advancements, and future directions of the field. PMID:23298612

  15. Hybrid PET/CT for noninvasive pharmacokinetic evaluation of dynamic PolyConjugates, a synthetic siRNA delivery system.

    PubMed

    Mudd, Sarah R; Trubetskoy, Vladimir S; Blokhin, Andrei V; Weichert, Jamey P; Wolff, Jon A

    2010-07-21

    Positron emission tomography/computed tomography (PET/CT) hybrid imaging can be used to gain insights into a synthetic siRNA delivery system targeted to the liver. Either siRNA or the delivery vehicle was labeled with (64)Cu via 1, 4, 7, 10- tetraazacyclododecane- 1, 4, 7, 10- tetraacetic acid (DOTA) chelation. This study confirmed that the siRNA delivery system was successfully targeted to the liver. Incorporation of the siRNA into the delivery system protected the siRNA from renal filtration long enough so that the siRNA could be delivered to the liver. PET/CT imaging was important for confirming biodistribution and for determining differences in the distribution of labeled siRNA, siRNA incorporated into the delivery system, and the delivery system without siRNA. PMID:20552976

  16. An Atlas of Soybean Small RNAs Identifies Phased siRNAs from Hundreds of Coding Genes[W

    PubMed Central

    Kakrana, Atul; Huang, Kun; Zhai, Jixian; Yan, Zhe; Valdés-López, Oswaldo; Prince, Silvas; Musket, Theresa A.; Stacey, Gary

    2014-01-01

    Small RNAs are ubiquitous, versatile repressors and include (1) microRNAs (miRNAs), processed from mRNA forming stem-loops; and (2) small interfering RNAs (siRNAs), the latter derived in plants by a process typically requiring an RNA-dependent RNA polymerase. We constructed and analyzed an expression atlas of soybean (Glycine max) small RNAs, identifying over 500 loci generating 21-nucleotide phased siRNAs (phasiRNAs; from PHAS loci), of which 483 overlapped annotated protein-coding genes. Via the integration of miRNAs with parallel analysis of RNA end (PARE) data, 20 miRNA triggers of 127 PHAS loci were detected. The primary class of PHAS loci (208 or 41% of the total) corresponded to NB-LRR genes; some of these small RNAs preferentially accumulate in nodules. Among the PHAS loci, novel representatives of TAS3 and noncanonical phasing patterns were also observed. A noncoding PHAS locus, triggered by miR4392, accumulated preferentially in anthers; the phasiRNAs are predicted to target transposable elements, with their peak abundance during soybean reproductive development. Thus, phasiRNAs show tremendous diversity in dicots. We identified novel miRNAs and assessed the veracity of soybean miRNAs registered in miRBase, substantially improving the soybean miRNA annotation, facilitating an improvement of miRBase annotations and identifying at high stringency novel miRNAs and their targets. PMID:25465409

  17. Solar abundance of osmium

    PubMed Central

    Jacoby, George; Aller, Lawrence H.

    1976-01-01

    The abundance parameter, log gfA, where g is the statistical weight of the lower level, f is the oscillator strength, and A is the abundance (by numbers of atoms with respect to hydrogen), has been derived for three lines of osmium by a method of spectrum synthesis. An apparent discordance of the derived abundance with that found from the carbonaceous chondrites is probably to be attributed primarily to errors in the f-values, and blending with unknown contributors. PMID:16592314

  18. An efficient algorithm for systematic analysis of nucleotide strings suitable for siRNA design

    PubMed Central

    2011-01-01

    Background The "off-target" silencing effect hinders the development of siRNA-based therapeutic and research applications. Existing solutions for finding possible locations of siRNA seats within a large database of genes are either too slow, miss a portion of the targets, or are simply not designed to handle a very large number of queries. We propose a new approach that reduces the computational time as compared to existing techniques. Findings The proposed method employs tree-based storage in a form of a modified truncated suffix tree to sort all possible short string substrings within given set of strings (i.e. transcriptome). Using the new algorithm, we pre-computed a list of the best siRNA locations within each human gene ("siRNA seats"). siRNAs designed to reside within siRNA seats are less likely to hybridize off-target. These siRNA seats could be used as an input for the traditional "set-of-rules" type of siRNA designing software. The list of siRNA seats is available through a publicly available database located at http://web.cos.gmu.edu/~gmanyam/siRNA_db/search.php Conclusions In attempt to perform top-down prediction of the human siRNA with minimized off-target hybridization, we developed an efficient algorithm that employs suffix tree based storage of the substrings. Applications of this approach are not limited to optimal siRNA design, but can also be useful for other tasks involving selection of the characteristic strings specific to individual genes. These strings could then be used as siRNA seats, as specific probes for gene expression studies by oligonucleotide-based microarrays, for the design of molecular beacon probes for Real-Time PCR and, generally, any type of PCR primers. PMID:21619643

  19. Extreme Events

    NASA Astrophysics Data System (ADS)

    Nott, Jonathan

    2006-04-01

    The assessment of risks posed by natural hazards such as floods, droughts, earthquakes, tsunamis or cyclones, is often based on short-term historical records that may not reflect the full range or magnitude of events possible. As human populations grow, especially in hazard-prone areas, methods for accurately assessing natural hazard risks are becoming increasingly important. In Extreme Events Jonathan Nott describes the many methods used to reconstruct such hazards from natural long-term records. He demonstrates how long-term (multi-century to millennial) records are essential in gaining a realistic understanding of the variability of natural hazards, and how short-term historical records can often misrepresent the likely risks associated with natural hazards. This book will form a useful resource for students taking courses covering natural hazards and risk assessment. It will also be valuable for urban planners, policy makers and non-specialists as a guide to understanding and reconstructing long-term records of natural hazards. Explains mechanisms that cause extreme events and discusses their prehistoric records Describes how to reconstruct long-term records of natural hazards in order to make accurate risk assessments Demonstrates that natural hazards can follow cycles over time and do not occur randomly

  20. siRNA Delivery: Mastering Dendrimer Self-Assembly for Efficient siRNA Delivery: From Conceptual Design to In Vivo Efficient Gene Silencing (Small 27/2016).

    PubMed

    Chen, Chao; Posocco, Paola; Liu, Xiaoxuan; Cheng, Qiang; Laurini, Erik; Zhou, Jiehua; Liu, Cheng; Wang, Yang; Tang, Jingjie; Col, Valentina Dal; Yu, Tianzhu; Giorgio, Suzanne; Fermeglia, Maurizio; Qu, Fanqi; Liang, Zicai; Rossi, John J; Liu, Minghua; Rocchi, Palma; Pricl, Sabrina; Peng, Ling

    2016-07-01

    Supramolecular dendrimers created from small amphiphilic dendrons are able to mimic covalently constructed high-generation dendrimers for siRNA delivery, as presented by S. Pricl, L. Peng, and co-workers on page 3667. An optimal balance between the hydrophobic alkyl chain length and the hydrophilic dendritic portion is crucial for self-assembly of these amphiphilic dendrons into micellar nanostructures, and critically impacts the effectiveness of siRNA delivery and functional gene silencing. PMID:27412303

  1. In Situ Functionalized Polymers for siRNA Delivery.

    PubMed

    Priegue, Juan M; Crisan, Daniel N; Martínez-Costas, José; Granja, Juan R; Fernandez-Trillo, Francisco; Montenegro, Javier

    2016-06-20

    A new method is reported herein for screening the biological activity of functional polymers across a consistent degree of polymerization and in situ, that is, under aqueous conditions and without purification/isolation of candidate polymers. In brief, the chemical functionality of a poly(acryloyl hydrazide) scaffold was activated under aqueous conditions using readily available aldehydes to obtain amphiphilic polymers. The transport activity of the resulting polymers can be evaluated in situ using model membranes and living cells without the need for tedious isolation and purification steps. This technology allowed the rapid identification of a supramolecular polymeric vector with excellent efficiency and reproducibility for the delivery of siRNA into human cells (HeLa-EGFP). The reported method constitutes a blueprint for the high-throughput screening and future discovery of new polymeric functional materials with important biological applications. PMID:27100572

  2. siRNAmod: A database of experimentally validated chemically modified siRNAs.

    PubMed

    Dar, Showkat Ahmad; Thakur, Anamika; Qureshi, Abid; Kumar, Manoj

    2016-01-01

    Small interfering RNA (siRNA) technology has vast potential for functional genomics and development of therapeutics. However, it faces many obstacles predominantly instability of siRNAs due to nuclease digestion and subsequently biologically short half-life. Chemical modifications in siRNAs provide means to overcome these shortcomings and improve their stability and potency. Despite enormous utility bioinformatics resource of these chemically modified siRNAs (cm-siRNAs) is lacking. Therefore, we have developed siRNAmod, a specialized databank for chemically modified siRNAs. Currently, our repository contains a total of 4894 chemically modified-siRNA sequences, comprising 128 unique chemical modifications on different positions with various permutations and combinations. It incorporates important information on siRNA sequence, chemical modification, their number and respective position, structure, simplified molecular input line entry system canonical (SMILES), efficacy of modified siRNA, target gene, cell line, experimental methods, reference etc. It is developed and hosted using Linux Apache MySQL PHP (LAMP) software bundle. Standard user-friendly browse, search facility and analysis tools are also integrated. It would assist in understanding the effect of chemical modifications and further development of stable and efficacious siRNAs for research as well as therapeutics. siRNAmod is freely available at: http://crdd.osdd.net/servers/sirnamod. PMID:26818131

  3. Lipid-based siRNA Delivery Systems: Challenges, Promises and Solutions Along the Long Journey.

    PubMed

    Sarisozen, Can; Salzano, Giuseppina; Torchilin, Vladimir P

    2016-01-01

    RNA interference (RNAi) is an evolutionary conserved highly specific gene-silencing mechanism initiated by small interfering RNA (siRNA) molecules. Fast-paced preclinical and clinical studies helped the siRNA technology become an efficient tool for undruggable targets in different diseases including genetic diseases, viral diseases and cancer. Despite great feature of siRNAs that can down-regulate any protein in the cells, the full potential and the success of the preclinical studies could not be translated into largely successful clinical outcomes. It has become clear that the possibility of overcoming the pitfalls for in vivo siRNA therapy fully depends on delivery systems. In this review, we start with the challenges and barriers for in vivo siRNA delivery. Then we briefly discuss the recent developments in siRNA modification technology. We specifically focused on siRNA lipidation and delivery approaches with special emphasis on the lipid based hybrid systems. Here we summarize the journey of lipid-based micelle-like nanoparticle systems that combine longevity in blood, effective cellular uptake and endosomal escape for successful siRNA delivery and discuss the multifunctional stimuli-sensitive systems based on lipids as the next generation smart systems. PMID:27033509

  4. De novo reconstruction of plant RNA and DNA virus genomes from viral siRNAs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In antiviral defense, plants produce massive quantities of 21-24 nucleotide siRNAs. Here we demonstrate that the complete genomes of DNA and RNA viruses and viroids can be reconstructed by deep sequencing and de novo assembly of viral/viroid siRNAs from experimentally- and naturally-infected plants....

  5. Targeted delivery of anti-coxsackievirus siRNAs using ligand-conjugated packaging RNAs.

    PubMed

    Zhang, Huifang M; Su, Yue; Guo, Songchuan; Yuan, Ji; Lim, Travis; Liu, Jing; Guo, Peixuan; Yang, Decheng

    2009-09-01

    Coxsackievirus B3 (CVB3) is a common pathogen of myocarditis. We previously synthesized a siRNA targeting the CVB3 protease 2A (siRNA/2A) gene and achieved reduction of CVB3 replication by 92% in vitro. However, like other drugs under development, CVB3 siRNA faces a major challenge of targeted delivery. In this study, we investigated a novel approach to deliver CVB3 siRNAs to a specific cell population (e.g. HeLa cells containing folate receptor) using receptor ligand (folate)-linked packaging RNA (pRNA) from bacterial phage phi29. pRNA monomers can spontaneously form dimers and multimers under optimal conditions by base-pairing between their stem loops. By covalently linking a fluorescence-tag to folate, we delivered the conjugate specifically to HeLa cells without the need of transfection. We further demonstrated that pRNA covalently conjugated to siRNA/2A achieved an equivalent antiviral effect to that of the siRNA/2A alone. Finally, the drug targeted delivery was further evaluated by using pRNA monomers or dimers, which carried both the siRNA/2A and folate ligand and demonstrated that both of them strongly inhibited CVB3 replication. These data indicate that pRNA as a siRNA carrier can specifically deliver the drug to target cells via its ligand and specific receptor interaction and inhibit virus replication effectively. PMID:19616030

  6. Inhibition of Hepatitis C Virus Replication by Intracellular Delivery of Multiple siRNAs by Nanosomes

    PubMed Central

    Chandra, Partha K; Kundu, Anup K; Hazari, Sidhartha; Chandra, Sruti; Bao, Lili; Ooms, Tara; Morris, Gilbert F; Wu, Tong; Mandal, Tarun K; Dash, Srikanta

    2012-01-01

    Sustained antiviral responses of chronic hepatitis C virus (HCV) infection have improved recently by the use of direct-acting antiviral agents along with interferon (IFN)-α and ribavirin. However, the emergence of drug-resistant variants is expected to be a major problem. We describe here a novel combinatorial small interfering RNA (siRNA) nanosome-based antiviral approach to clear HCV infection. Multiple siRNAs targeted to the highly conserved 5′-untranslated region (UTR) of the HCV genome were synthesized and encapsulated into lipid nanoparticles called nanosomes. We show that siRNA can be repeatedly delivered to 100% of cells in culture using nanosomes without toxicity. Six siRNAs dramatically reduced HCV replication in both the replicon and infectious cell culture model. Repeated treatments with two siRNAs were better than a single siRNA treatment in minimizing the development of an escape mutant, resulting in rapid inhibition of viral replication. Systemic administration of combinatorial siRNA-nanosomes is well tolerated in BALB/c mice without liver injury or histological toxicity. As a proof-of-principle, we showed that systemic injections of siRNA nanosomes significantly reduced HCV replication in a liver tumor-xenotransplant mouse model of HCV. Our results indicate that systemic delivery of combinatorial siRNA nanosomes can be used to minimize the development of escape mutants and inhibition of HCV infection. PMID:22617108

  7. Fast degrading polyesters as siRNA nano-carriers for pulmonary gene therapy.

    PubMed

    Nguyen, Juliane; Steele, Terry W J; Merkel, Olivia; Reul, Regina; Kissel, Thomas

    2008-12-18

    A potential siRNA carrier for pulmonary gene delivery was assessed by encapsulating siRNA into biodegradable polyester nanoparticles consisting of tertiary-amine-modified polyvinyl alcohol (PVA) backbones grafted to poly(d,l-lactide-co-glycolide) (PLGA). The resulting siRNA nanoparticles were prepared using a solvent displacement method that offers the advantage of forming small nanoparticles without using shear forces. The nanoparticles were characterized with regard to particle size, zeta-potential, and degradation at pH 7.4 using dynamic and static light scattering. SiRNA release studies were performed and correlated to the nanoparticle degradation. In vitro knockdown of firefly luciferase reporter gene was used to assess the potential of the nanoparticles as siRNA carriers in a human lung epithelial cell line, H1299 luc. The amine-modified-PVA-PLGA/siRNA nanoparticles form 150-200 nm particles with zeta-potentials of +15-+20 mV in phosphate buffered saline (PBS). Break down of the nanoparticles was seen within 4 h in PBS with sustained release of siRNA. These nanoparticles have achieved 80-90% knockdown of a luciferase reporter gene with only 5 pmol anti-luc siRNA, even after nebulization. Hence we conclude that amine-modified-PVA-PLGA/siRNA nanoparticles could be a promising siRNA carrier for pulmonary gene delivery due to their fast degradation and potent gene knockdown profile. PMID:18619502

  8. Anti-angiogenic therapy via cationic liposome-mediated systemic siRNA delivery.

    PubMed

    Tagami, Tatsuaki; Suzuki, Takuya; Matsunaga, Mariko; Nakamura, Kazuya; Moriyoshi, Naoto; Ishida, Tatsuhiro; Kiwada, Hiroshi

    2012-01-17

    siRNA has been touted as a therapeutic molecule against genetic diseases, which include cancers. But several challenging issues remain in order to achieve efficient systemic siRNA delivery and a sufficient therapeutic effect for siRNA in vivo. Cationic liposome shows promise as a carrier for nucleic acids, as it can selectively bind to angiogenic tumor blood vessels. In this way, anti-angiogenic therapy via cationic liposome-mediated systemic siRNA delivery could be achieved in cancer therapy. In the present study, we proved our assumption by preparing various kinds of polyethylene glycol (PEG)-coated siRNA/cationic liposome complexes (siRNA-lipoplexes) and screening the avidity of these siRNA-lipoplexes upon angiogenic tumor blood vessels by means of a murine dorsal air sac (DAS) model. The lipoplex, having a lipid composition of DC-6-14/POPC/CHOL/DOPE/mPEG(2000)-DSPE=20/30/30/20/5 (molar ratio) and a charge ratio of cationic liposome and siRNA=3.81 (+/-), showed a higher binding index to newly formed blood vessels. Systemic injection with the lipoplex containing siRNA for the Argonaute2 gene (apoptosis-inducible siRNA) resulted in significant anti-tumor effect without severe side effects in mice with Lewis lung carcinoma. Our results indicate that the PEGylated cationic liposome-mediated systemic delivery of cytotoxic siRNA achieves anti-angiogenesis, resulting in the suppression of tumor growth. PMID:22101286

  9. siRNAmod: A database of experimentally validated chemically modified siRNAs

    PubMed Central

    Dar, Showkat Ahmad; Thakur, Anamika; Qureshi, Abid; Kumar, Manoj

    2016-01-01

    Small interfering RNA (siRNA) technology has vast potential for functional genomics and development of therapeutics. However, it faces many obstacles predominantly instability of siRNAs due to nuclease digestion and subsequently biologically short half-life. Chemical modifications in siRNAs provide means to overcome these shortcomings and improve their stability and potency. Despite enormous utility bioinformatics resource of these chemically modified siRNAs (cm-siRNAs) is lacking. Therefore, we have developed siRNAmod, a specialized databank for chemically modified siRNAs. Currently, our repository contains a total of 4894 chemically modified-siRNA sequences, comprising 128 unique chemical modifications on different positions with various permutations and combinations. It incorporates important information on siRNA sequence, chemical modification, their number and respective position, structure, simplified molecular input line entry system canonical (SMILES), efficacy of modified siRNA, target gene, cell line, experimental methods, reference etc. It is developed and hosted using Linux Apache MySQL PHP (LAMP) software bundle. Standard user-friendly browse, search facility and analysis tools are also integrated. It would assist in understanding the effect of chemical modifications and further development of stable and efficacious siRNAs for research as well as therapeutics. siRNAmod is freely available at: http://crdd.osdd.net/servers/sirnamod. PMID:26818131

  10. Use of guanidinopropyl-modified siRNAs to silence gene expression.

    PubMed

    Buff, Maximilian C R; Bernhardt, Stefan; Marimani, Musa D; Ely, Abdullah; Engels, Joachim W; Arbuthnot, Patrick

    2015-01-01

    Silencing gene expression by harnessing the RNA interference (RNAi) pathway with short interfering RNAs (siRNAs) has useful analytical and potentially therapeutic application. To augment silencing efficacy of siRNAs, chemical modification has been employed to improve stability, target specificity, and delivery to target tissues. siRNAs incorporating guanidinopropyl (GP) moieties have demonstrated enhanced target gene silencing in cell culture and in vivo models of hepatitis B virus replication. Here we describe the synthesis of GP-modified siRNAs and use of 5' rapid amplification of cDNA ends (5' RACE) to verify an RNAi-mediated mechanism of action of these novel chemically modified siRNAs. PMID:25319654

  11. Delivery strategies and potential targets for siRNA in major cancer types.

    PubMed

    Lee, So Jin; Kim, Min Ju; Kwon, Ick Chan; Roberts, Thomas M

    2016-09-01

    Small interfering RNA (siRNA) has gained attention as a potential therapeutic reagent due to its ability to inhibit specific genes in many genetic diseases. For many years, studies of siRNA have progressively advanced toward novel treatment strategies against cancer. Cancer is caused by various mutations in hundreds of genes including both proto-oncogenes and tumor suppressor genes. In order to develop siRNAs as therapeutic agents for cancer treatment, delivery strategies for siRNA must be carefully designed and potential gene targets carefully selected for optimal anti-cancer effects. In this review, various modifications and delivery strategies for siRNA delivery are discussed. In addition, we present current thinking on target gene selection in major tumor types. PMID:27259398

  12. Biological effects of hexitol and altritol-modified siRNAs targeting B-Raf

    PubMed Central

    Fisher, Michael; Abramov, Mikhail; Van Aerschot, Arthur; Rozenski, Jef; Dixit, Vidula; Juliano, Rudy L.; Herdewijn, Piet

    2009-01-01

    Increasing the effectiveness of siRNAs through chemical modification is an important task. Here we describe altritol and hexitol modified oligonucleotides targeting the B-Raf oncogene that is critical for the growth and survival of melanoma cells. Using assays for apoptosis, DNA synthesis, colony formation and B-Raf protein and message levels, we demonstrate that certain hexitol modifications can improve the effectiveness of B-Raf siRNAs and also increase duration of action. Altritol modified siRNAs were similar to or slightly less effective than unmodified B-Raf siRNA. Modifications at the 3′ or 5′ end of the sense strand, at the 3′ end of the antisense strand, or within either strand were well tolerated. The basis for the increased effectiveness of the hexitol-modified siRNAs is not fully understood but may be partly due to increased stability to nucleases. PMID:19374843

  13. Delivery of siRNA to ovarian cancer cells using laser-activated carbon nanoparticles

    PubMed Central

    Sengupta, Aritra; Mezencev, Roman; McDonald, John F; Prausnitz, Mark R

    2015-01-01

    Aim The RNAi-mediated knockdown of gene expression is an attractive tool for research and therapeutic purposes but its implementation is challenging. Here we report on a new method based on photoacoustic delivery of siRNA developed to address some of these challenges. Materials & methods Physical properties and photoacoustic emission of carbon black (CB) particles upon near-infrared laser irradiation were characterized. Next, ovarian cancer cells Hey A8-F8 were exposed to near-infrared nanosecond laser pulses in the presence of siRNA targeting EGFR gene and CB particles. The intracellular delivery of siRNA and silencing of the target gene were determined by specific qPCR assays. Results & conclusion Laser-activated CB nanoparticles generated photoacoustic emission and enabled intracellular delivery of siRNA and significant knockdown of its target EGFR mRNA. This physical method represents a new promising approach to targeted therapeutic delivery of siRNA. PMID:26080699

  14. Smart polymeric micelles as nanocarriers for oligonucleotides and siRNA delivery.

    PubMed

    Kataoka, Kazunori; Itaka, Keiji; Nishiyama, Nobuhiro; Yamasaki, Yuichi; Oishi, Motoi; Nagasaki, Yukio

    2005-01-01

    The development of in vivo delivery systems for oligonucleotides and siRNA is strongly desired to achieve their clinical applications. Recently, polyplex micelles, which are formed through an electrostatic interaction between nucleic acid compounds (DNA and RNA) and poly(ethylene glycol) (PEG)-polycation block copolymers, have received much attention due to their nanometric-scaled size and excellent biocompatibility. Here, three types of newly engineered block copolymers were developed to construct polyplex micelles useful for oligonucleotides and siRNA delivery: (1) PEG-polycation diblock copolymers possessing diamine side-chain with distinctive pKa for siRNA encapsulation into polyplex micelles with high endosomal escaping ability, (2) Lactosylated PEG-(oligonucleotide or siRNA) conjugate through acid-labile beta-thiopropionate linkage to construct pH-sensitive PIC micelles, and (3) PEG-poly(methacrylic acid) block copolymer for the construction of organic/inorganic hybrid nanoparticles encapsulating siRNA. PMID:17150611

  15. Cyclodextrin and Polyethylenimine Functionalized Mesoporous Silica Nanoparticles for Delivery of siRNA Cancer Therapeutics

    PubMed Central

    Shen, Jianliang; Kim, Han-Cheon; Su, Hua; Wang, Feng; Wolfram, Joy; Kirui, Dickson; Mai, Junhua; Mu, Chaofeng; Ji, Liang-Nian; Mao, Zong-Wan; Shen, Haifa

    2014-01-01

    Effective delivery holds the key to successful in vivo application of therapeutic small interfering RNA (siRNA). In this work, we have developed a universal siRNA carrier consisting of a mesoporous silica nanoparticle (MSNP) functionalized with cyclodextrin-grafted polyethylenimine (CP). CP provides positive charge for loading of siRNA through electrostatic interaction and enables effective endosomal escape of siRNA. Using intravital microscopy we were able to monitor tumor enrichment of CP-MSNP/siRNA particles in live mice bearing orthotopic MDA-MB-231 xenograft tumors. CP-MSNP delivery of siRNA targeting the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2) resulted in effective knockdown of gene expression in vitro and in vivo. Suppression of PKM2 led to inhibition of tumor cell growth, invasion, and migration. PMID:24672582

  16. Multi-target siRNA: Therapeutic Strategy for Hepatocellular Carcinoma

    PubMed Central

    Li, Tiejun; Xue, Yuwen; Wang, Guilan; Gu, Tingting; Li, Yunlong; Zhu, York Yuanyuan; Chen, Li

    2016-01-01

    Multiple targets RNAi strategy is a preferred way to treat multigenic diseases, especially cancers. In the study, multi-target siRNAs were designed to inhibit NET-1, EMS1 and VEGF genes in hepatocellular carcinoma (HCC) cells. And multi-target siRNAs showed better silencing effects on NET-1, EMS1 and VEGF, compared with single target siRNA. Moreover, multi-target siRNA showed greater suppression effects on proliferation, migration, invasion, angiogenesis and induced apoptosis in HCC cells. The results suggested that multi-target siRNA might be a preferred strategy for cancer therapy and NET-1, EMS1 and VEGF could be effective targets for HCC treatments. PMID:27390607

  17. Identification of miniature inverted-repeat transposable elements (MITEs) and biogenesis of their siRNAs in the Solanaceae: New functional implications for MITEs

    PubMed Central

    Kuang, Hanhui; Padmanabhan, Chellappan; Li, Feng; Kamei, Ayako; Bhaskar, Pudota B.; Ouyang, Shu; Jiang, Jiming; Buell, C. Robin; Baker, Barbara

    2009-01-01

    Small RNAs regulate the genome by guiding transcriptional and post-transcriptional silencing machinery to specific target sequences, including genes and transposable elements (TEs). Although miniature inverted-repeat transposable elements (MITEs) are closely associated with euchromatic genes, the broader functional impact of these short TE insertions in genes is largely unknown. We identified 22 families of MITEs in the Solanaceae (MiS1–MiS22) and found abundant MiS insertions in Solanaceae genomic DNA and expressed sequence tags (EST). Several Solanaceae MITEs generate genome changes that potentially affect gene function and regulation, most notably, a MiS insertion that provides a functionally indispensable alternative exon in the tobacco mosaic virus N resistance gene. We show that MITEs generate small RNAs that are primarily 24 nt in length, as detected by Northern blot hybridization and by sequencing small RNAs of Solanum demissum, Nicotiana glutinosa, and Nicotiana benthamiana. Additionally, we show that stable RNAi lines silencing DICER-LIKE3 (DCL3) in tobacco and RNA-dependent RNA polymerase 2 (RDR2) in potato cause a reduction in 24-nt MITE siRNAs, suggesting that, as in Arabidopsis, TE-derived siRNA biogenesis is DCL3 and RDR2 dependent. We provide evidence that DICER-LIKE4 (DCL4) may also play a role in MITE siRNA generation in the Solanaceae. PMID:19037014

  18. Inhibition of hepatitis C virus in mouse models by lipidoid nanoparticle-mediated systemic delivery of siRNA against PRK2.

    PubMed

    Moon, Jae-Su; Lee, Seung-Hoon; Han, Song-Hee; Kim, Eun-Jung; Cho, Hee; Lee, Wooseong; Kim, Mi-Kyung; Kim, Tae-Eun; Park, Hyun-Ji; Rhee, Jin-Kyu; Kim, Seong-Jun; Cho, Seung-Woo; Han, Seung Hyun; Oh, Jong-Won

    2016-08-01

    Host-targeting antivirals have an advantage over direct-acting antivirals in that they have a high genetic barrier to resistance. Here, we describe in vivo anti-hepatitis C virus (HCV) efficacy of a potent siRNA targeting the protein kinase C-related kinase 2 (PRK2), which phosphorylates HCV NS5B RNA-dependent RNA polymerase and promotes HCV replication. PRK2-silencing reduced the phosphorylated NS5B level and resulted in inhibition of NS5B RdRp activity to decrease HCV genome abundance. Systemic administration of lipidoid nanoparticle-formulated PRK2 siRNA (once every three days for a total of three injections at a dose of 3mgkg(-1)) resulted in a 3.72 and 1.96 log10 reduction in serum HCV RNA titer, in mouse subcutaneous and orthotopic xenograft models for HCV replication, respectively. Our results verify the essential role of PRK2 in HCV replication and offer a host-targeting anti-HCV siRNA therapy that might be beneficial for non-responders to current treatment regimens. PMID:27013134

  19. Controlling mesenchymal stem cell gene expression using polymer-mediated delivery of siRNA

    PubMed Central

    Benoit, Danielle S.W.; Boutin, Molly E.

    2012-01-01

    siRNA treatment has great promise to specifically control gene expression and select cell behaviors but have delivery challenges limiting their use. Particularly for applications in regenerative medicine, uniform and consistent delivery of siRNA to control gene expression and subsequent stem cell functions, such as differentiation, is paramount. Therefore, a diblock copolymer was examined for its ability to effective delivery siRNA to mesenchymal stem cells (MSCs). The diblock copolymers, which are composed of cationic blocks for siRNA complexation, protection, and uptake and pH-responsive blocks for endosomal escape, were shown to facilitate nearly 100% MSC uptake of siRNA, which is vastly superior to a commercially-available control, DharmaFECT, which resulted in only ~60% siRNA positive MSCs. Moreover, the diblock copolymer, at conditions that result in excellent knockdown (down to ~10% of control gene expression), is cytocompatible, causing no negative effects on MSC survivability. In contrast, DharmaFECT:siRNA treatment results in only ~60% survivability of MSCs. Longitudinal knockdown after siRNA treatment was examined and protein knockdown persists for ~6 days regardless of delivery system (diblock copolymer or DharmaFECT). Finally, MSC phenotype and differentiation capacity was examined after treatment with control siRNA. There is no statistically significant differences on cell surface markers of diblock copolymer:siRNA or DharmaFECT:siRNA treated or cells measured 2 weeks after siRNA delivery compared to untreated cells. Upon differentiation with typical media/culture conditions to adipogenic, chondrogenic, and osteogenic lineages and examination of histological staining markers, there is no discernable differences between treated and untreated cells, regardless of delivery mechanism. Thus, diblock copolymers examined herein facilitate uniform siRNA treatment of MSCs, inducing siRNA-specific gene and protein knockdown without adversely affecting MSC

  20. Dicetyl phosphate-tetraethylenepentamine-based liposomes for systemic siRNA delivery.

    PubMed

    Asai, Tomohiro; Matsushita, Saori; Kenjo, Eriya; Tsuzuku, Takuma; Yonenaga, Norihito; Koide, Hiroyuki; Hatanaka, Kentaro; Dewa, Takehisa; Nango, Mamoru; Maeda, Noriyuki; Kikuchi, Hiroshi; Oku, Naoto

    2011-03-16

    Dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) conjugate was newly synthesized and formed into liposomes for efficient siRNA delivery. Formulation of DCP-TEPA-based polycation liposomes (TEPA-PCL) complexed with siRNA was examined by performing knockdown experiments using stable EGFP-transfected HT1080 human fibrosarcoma cells and siRNA for GFP. An adequate amount of DCP-TEPA in TEPA-PCL and N/P ratio of TEPA-PCL/siRNA complexes were determined based on the knockdown efficiency. Then, the biodistribution of TEPA-PCL modified with poly(ethylene glycol) (PEG) was examined in BALB/c mice. As a result, TEPA-PCL modified with PEG6000 avoided reticuloendothelial system uptake and showed long circulation in the bloodstream. On the other hand, PEGylation of TEPA-PCL/siRNA complexes caused dissociation of a portion of the siRNA from the liposomes. However, we found that the use of cholesterol-conjugated siRNA improved the interaction between TEPA-PCL and siRNA, which allowed PEGylation of TEPA-PCL/siRNA complexes without siRNA dissociation. In addition, TEPA-PCL complexed with cholesterol-conjugated siRNA showed potent knockdown efficiency in stable luciferase-transfected B16-F10 murine melanoma cells. Finally, the biodistribution of cholesterol-conjugated siRNA formulated in PEGylated TEPA-PCL was examined by performing near-infrared fluorescence imaging in Colon26 NL-17 murine carcinoma-bearing mice. Our results showed that tumor targeting with siRNA via systemic administration was achieved by using PEGylated TEPA-PCL combined with active targeting with Ala-Pro-Arg-Pro-Gly, a peptide used for targeting angiogenic endothelium. PMID:21361311

  1. siRNA liposome-gold nanorod vectors for multispectral optoacoustic tomography theranostics

    NASA Astrophysics Data System (ADS)

    Taruttis, Adrian; Lozano, Neus; Nunes, Antonio; Jasim, Dhifaf A.; Beziere, Nicolas; Herzog, Eva; Kostarelos, Kostas; Ntziachristos, Vasilis

    2014-10-01

    Therapeutic applications of gene silencing using siRNA have seen increasing interest over the past decade. The optimization of the delivery and biodistribution of siRNA using liposome-gold nanorod (AuNRs) nanoscale carriers can greatly benefit from adept imaging methods that can visualize the time-resolved delivery performance of such vectors. In this work, we describe the effect of AuNR length incorporated with liposomes and show their complexation with siRNA as a novel gene delivery vehicle. We demonstrate the application of multispectral optoacoustic tomography (MSOT) to longitudinally visualize the localisation of siRNA carrying liposome-AuNR hybrids within tumors. Combination of in vivo MSOT with ex vivo fluorescence cryo-slice imaging offers further insight into the siRNA transport and activity obtained.Therapeutic applications of gene silencing using siRNA have seen increasing interest over the past decade. The optimization of the delivery and biodistribution of siRNA using liposome-gold nanorod (AuNRs) nanoscale carriers can greatly benefit from adept imaging methods that can visualize the time-resolved delivery performance of such vectors. In this work, we describe the effect of AuNR length incorporated with liposomes and show their complexation with siRNA as a novel gene delivery vehicle. We demonstrate the application of multispectral optoacoustic tomography (MSOT) to longitudinally visualize the localisation of siRNA carrying liposome-AuNR hybrids within tumors. Combination of in vivo MSOT with ex vivo fluorescence cryo-slice imaging offers further insight into the siRNA transport and activity obtained. Electronic supplementary information (ESI) available: Experimental section and dark-field microscopy in both tumors 24 h after injection of the complex have been included. See DOI: 10.1039/c4nr04164j

  2. Effective cytoplasmic release of siRNA from liposomal carriers by controlling the electrostatic interaction of siRNA with a charge-invertible peptide, in response to cytoplasmic pH.

    PubMed

    Itakura, Shoko; Hama, Susumu; Matsui, Ryo; Kogure, Kentaro

    2016-05-19

    Condensing siRNA with cationic polymers is a major strategy used in the development of siRNA carriers that can avoid degradation by nucleases and achieve effective delivery of siRNA into the cytoplasm. However, ineffective release of siRNA from such condensed forms into the cytoplasm is a limiting step for induction of RNAi effects, and can be attributed to tight condensation of siRNA with the cationic polymers, due to potent electrostatic interactions. Here, we report that siRNA condensed with a slightly acidic pH-sensitive peptide (SAPSP), whose total charge is inverted from positive to negative in response to cytoplasmic pH, is effectively released via electrostatic repulsion of siRNA with negatively charged SAPSP at cytoplasmic pH (7.4). The condensed complex of siRNA and positively-charged SAPSP at acidic pH (siRNA/SAPSP) was found to result in almost complete release of siRNA upon charge inversion of SAPSP at pH 7.4, with the resultant negatively-charged SAPSP having no undesirable interactions with endogenous mRNA. Moreover, liposomes encapsulating siRNA/SAPSP demonstrated knockdown efficiencies comparable to those of commercially available siRNA carriers. Taken together, SAPSP may be very useful as a siRNA condenser, as it facilitates effective cytoplasmic release of siRNA, and subsequent induction of specific RNAi effects. PMID:27145993

  3. MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs.

    PubMed

    Zhai, Jixian; Jeong, Dong-Hoon; De Paoli, Emanuele; Park, Sunhee; Rosen, Benjamin D; Li, Yupeng; González, Alvaro J; Yan, Zhe; Kitto, Sherry L; Grusak, Michael A; Jackson, Scott A; Stacey, Gary; Cook, Douglas R; Green, Pamela J; Sherrier, D Janine; Meyers, Blake C

    2011-12-01

    Legumes and many nonleguminous plants enter symbiotic interactions with microbes, and it is poorly understood how host plants respond to promote beneficial, symbiotic microbial interactions while suppressing those that are deleterious or pathogenic. Trans-acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide (nt) "phased" intervals, a pattern formed by DICER-LIKE 4 (DCL4) processing. A search for phased siRNAs (phasiRNAs) found at least 114 Medicago loci, the majority of which were defense-related NB-LRR-encoding genes. We identified three highly abundant 22-nt microRNA (miRNA) families that target conserved domains in these NB-LRRs and trigger the production of trans-acting siRNAs. High levels of small RNAs were matched to >60% of all ∼540 encoded Medicago NB-LRRs; in the potato, a model for mycorrhizal interactions, phasiRNAs were also produced from NB-LRRs. DCL2 and SGS3 transcripts were also cleaved by these 22-nt miRNAs, generating phasiRNAs, suggesting synchronization between silencing and pathogen defense pathways. In addition, a new example of apparent "two-hit" phasiRNA processing was identified. Our data reveal complex tasiRNA-based regulation of NB-LRRs that potentially evolved to facilitate symbiotic interactions and demonstrate miRNAs as master regulators of a large gene family via the targeting of highly conserved, protein-coding motifs, a new paradigm for miRNA function. PMID:22156213

  4. Elemental Abundances in NGC 3516

    NASA Technical Reports Server (NTRS)

    Turner, T. J.; Kraemer, S. B.; Mushotzky, R. F.; George, I. M.; Gabel, J. R.

    2003-01-01

    We present Reflection Grating Spectrometer data from an XMM-Newton observation of the Seyfert 1 galaxy NGC 3516, taken while the continuum source was in an extremely low flux state. This observation offers a rare opportunity for a detailed study of emission from a Seyfert 1 galaxy as these are usually dominated by high nuclear continuum levels and heavy absorption. The spectrum shows numerous narrow emission lines (FWHM approximately less than 1300 kilometers per second) in the 0.3 - 2 keV range, including the H-like lines of C, N, and O and the He-like lines of N, O and Ne. The emission-line ratios and the narrow width of the radiative recombination continuum of CVI indicate that the gas is photoionized and of fairly low temperature (kT approximately less than 0.01 keV). The availability of emission lines from different elements for two iso-electronic sequences allows us to constrain the element abundances. These data show that the N lines are far stronger than would be expected from gas of solar abundances. Based on our photoionization models we find that nitrogen is overabundant in the central regions of the galaxy, compared to carbon, oxygen and neon by at least a factor of 2.5. We suggest that this is the result of secondary production of nitrogen in intermediate mass stars, and indicative of the history of star formation in NGC 3516.

  5. OXYGEN ABUNDANCES IN CEPHEIDS

    SciTech Connect

    Luck, R. E.; Andrievsky, S. M.; Korotin, S. N.; Kovtyukh, V. V. E-mail: serkor@skyline.od.ua E-mail: scan@deneb1.odessa.ua

    2013-07-01

    Oxygen abundances in later-type stars, and intermediate-mass stars in particular, are usually determined from the [O I] line at 630.0 nm, and to a lesser extent, from the O I triplet at 615.7 nm. The near-IR triplets at 777.4 nm and 844.6 nm are strong in these stars and generally do not suffer from severe blending with other species. However, these latter two triplets suffer from strong non-local thermodynamic equilibrium (NLTE) effects and thus see limited use in abundance analyses. In this paper, we derive oxygen abundances in a large sample of Cepheids using the near-IR triplets from an NLTE analysis, and compare those abundances to values derived from a local thermodynamic equilibrium (LTE) analysis of the [O I] 630.0 nm line and the O I 615.7 nm triplet as well as LTE abundances for the 777.4 nm triplet. All of these lines suffer from line strength problems making them sensitive to either measurement complications (weak lines) or to line saturation difficulties (strong lines). Upon this realization, the LTE results for the [O I] lines and the O I 615.7 nm triplet are in adequate agreement with the abundance from the NLTE analysis of the near-IR triplets.

  6. siRNA Knock-Down of RANK Signaling to Control Osteoclast-Mediated Bone Resorption

    PubMed Central

    Wang, Yuwei; Grainger, David W.

    2010-01-01

    Purpose To demonstrate the ability of small interfering (si)RNA targeting the cell receptor, RANK, to control osteoclast function in cultures of both primary and secondary osteoclasts and their precursor cells. Methods siRNA targeting RANK was transfected into both RAW264.7 and primary bone marrow cell cultures. RANK knock-down by siRNA and functional inhibition were assessed in both mature osteoclast and their precursor cell cultures. RANK mRNA message and protein expression after the transfections were analyzed by PCR and Western blot, respectively. Off-target effects were assessed. The inhibition of osteoclast formation was evaluated using tartrate-resistant acid phosphatase (TRAP) assay, and subsequent bone resorption was determined by resorption pit assay. Results Both osteoclasts and osteoclast precursors can be targeted by siRNA in serum-containing media. Delivery of siRNA targeting RANK to both RAW 264.7 and primary bone marrow cell cultures produces short term repression of RANK expression without off-targeting effects, and significantly inhibits both osteoclast formation and bone resorption. Moreover, data support successful RANK knock-down by siRNA specifically in mature osteoclast cultures. Conclusions RANK is demonstrated to be an attractive target for siRNA control of osteoclast activity, with utility for development of new therapeutics for low bone mass pathologies or osteoporosis. PMID:20333451

  7. PepFect6 Mediated SiRNA Delivery into Organotypic Cultures.

    PubMed

    Dash-Wagh, Suvarna; Langel, Ülo; Ulfendahl, Mats

    2016-01-01

    Gene silencing by small interfering RNA (SiRNA) is an attractive therapeutic approach for pathological disorders that targets a specific gene. However, its applications are limited, as naked RNA is rapidly degraded by RNases and is inadequately internalized by the target cells in the body. Several viral and nonviral vectors have been described to improve the delivery of SiRNAs both in cultured cells as well as in vivo. Increasing evidence suggests that cell-penetrating peptides (CPPs) are an efficient, non-cytotoxic tool for intracellular delivery of SiRNA. Recently, a new peptide, PepFect6 (PF6), based system has been described for efficient SiRNA delivery in various cell types. PF6 is an amphipathic stearyl-TP10 peptide carrying a pH titratable trifluoromethylquinoline moiety that facilitate endosomal release. PF6 forms stable non-covalent complexes with SiRNA. Upon internalization, the complexes rapidly escape the endosomal compartment, resulting in robust RNA interference (RNAi) responses. This chapter describes a protocol to use the PF6-nanoparticle technology for SiRNA delivery into organotypic cultures of the inner ear i.e., cochlea. We also highlight different critical points in the peptide/SiRNA complex preparation, transfection and in analyzing the efficacy of PF6-SiRNA associated RNAi response. PMID:26472439

  8. Fabrication of electrospun zein nanofibers for the sustained delivery of siRNA.

    PubMed

    Karthikeyan, K; Krishnaswamy, Venkat Raghavan; Lakra, Rachita; Kiran, M S; Korrapati, Purna Sai

    2015-02-01

    In this study, zein nanofibers based siRNA delivery system has been attempted for the first time. Here, the amphiphilic property of zein and the size advantage of nanofibers have been brought together in developing an ideal delivery system for siRNA. The morphological analysis of the GAPDH-siRNA loaded zein nanofibers revealed the proper encapsulation of the siRNA in the polymeric matrix. The loading efficiency of this delivery system was found to be 58.57±2.4% (w/w). The agarose gel analysis revealed that the zein nanofibers preserved the integrity of siRNA for a longer period even at the room temperature. The in vitro release studies not only depicted the sustaining potential of the zein nanofibers but also ensured the release of sufficient quantity of siRNA required to induce the gene silencing effect. The amphiphilic property of zein supported the cell attachment and thereby facilitated the transfection of siRNA into the cells. qRT-PCR analysis confirmed the potential of the developed system in inducing the desired gene silencing effect. Thus, electrospun zein nanofibers have been successfully employed for the delivery of siRNA which has a great therapeutic potential. PMID:25655500

  9. More complete gene silencing by fewer siRNAs: transparent optimized design and biophysical signature.

    PubMed

    Ladunga, Istvan

    2007-01-01

    Highly accurate knockdown functional analyses based on RNA interference (RNAi) require the possible most complete hydrolysis of the targeted mRNA while avoiding the degradation of untargeted genes (off-target effects). This in turn requires significant improvements to target selection for two reasons. First, the average silencing activity of randomly selected siRNAs is as low as 62%. Second, applying more than five different siRNAs may lead to saturation of the RNA-induced silencing complex (RISC) and to the degradation of untargeted genes. Therefore, selecting a small number of highly active siRNAs is critical for maximizing knockdown and minimizing off-target effects. To satisfy these needs, a publicly available and transparent machine learning tool is presented that ranks all possible siRNAs for each targeted gene. Support vector machines (SVMs) with polynomial kernels and constrained optimization models select and utilize the most predictive effective combinations from 572 sequence, thermodynamic, accessibility and self-hairpin features over 2200 published siRNAs. This tool reaches an accuracy of 92.3% in cross-validation experiments. We fully present the underlying biophysical signature that involves free energy, accessibility and dinucleotide characteristics. We show that while complete silencing is possible at certain structured target sites, accessibility information improves the prediction of the 90% active siRNA target sites. Fast siRNA activity predictions can be performed on our web server at http://optirna.unl.edu/. PMID:17169992

  10. OligoWalk: an online siRNA design tool utilizing hybridization thermodynamics.

    PubMed

    Lu, Zhi John; Mathews, David H

    2008-07-01

    Given an mRNA sequence as input, the OligoWalk web server generates a list of small interfering RNA (siRNA) candidate sequences, ranked by the probability of being efficient siRNA (silencing efficacy greater than 70%). To accomplish this, the server predicts the free energy changes of the hybridization of an siRNA to a target mRNA, considering both siRNA and mRNA self-structure. The free energy changes of the structures are rigorously calculated using a partition function calculation. By changing advanced options, the free energy changes can also be calculated using less rigorous lowest free energy structure or suboptimal structure prediction methods for the purpose of comparison. Considering the predicted free energy changes and local siRNA sequence features, the server selects efficient siRNA with high accuracy using a support vector machine. On average, the fraction of efficient siRNAs selected by the server that will be efficient at silencing is 78.6%. The OligoWalk web server is freely accessible through internet at http://rna.urmc.rochester.edu/servers/oligowalk. PMID:18490376

  11. Analysis of siRNA specificity on targets with double-nucleotide mismatches

    PubMed Central

    Dahlgren, Cecilia; Zhang, Hong-Yan; Du, Quan; Grahn, Maria; Norstedt, Gunnar; Wahlestedt, Claes

    2008-01-01

    Although RNA interference as a tool for gene knockdown is a great promise for future applications, the specificity of small interfering RNA (siRNA)-mediated gene silencing needs to be thoroughly investigated. Most research regarding siRNA specificity has involved analysis of affected off-target genes instead of exploring the specificity of the siRNA itself. In this study we have developed an efficient method for generating a siRNA target library by combining a siRNA target validation vector with a nucleotide oligomix. We have used this library to perform an analysis of the silencing effects of a functional siRNA towards its target site with double-nucleotide mismatches. The results indicated that not only the positions of the mismatched base pair have an impact on silencing efficiency but also the identity of the mismatched nucleotide. Our data strengthen earlier observations of widespread siRNA off-target effects and shows that ∼35% of the double-mutated target sites still causes knockdown efficiency of >50%. We also provide evidence that there may be substantial differences in knockdown efficiency depending on whether the mutations are positioned within the siRNA itself or in the corresponding target site. PMID:18420656

  12. Prediction of siRNA knockdown efficiency using artificial neural network models

    SciTech Connect

    Ge Guangtao . E-mail: guge@eecs.tufts.edu; Wong, G.William . E-mail: wong@wi.mit.edu; Luo Biao . E-mail: bluo@broad.mit.edu

    2005-10-21

    Selective knockdown of gene expression by short interference RNAs (siRNAs) has allowed rapid validation of gene functions and made possible a high throughput, genome scale approach to interrogate gene function. However, randomly designed siRNAs display different knockdown efficiencies of target genes. Hence, various prediction algorithms based on siRNA functionality have recently been constructed to increase the likelihood of selecting effective siRNAs, thereby reducing the experimental cost. Toward this end, we have trained three Back-propagation and Bayesian neural network models, previously not used in this context, to predict the knockdown efficiencies of 180 experimentally verified siRNAs on their corresponding target genes. Using our input coding based primarily on RNA structure thermodynamic parameters and cross-validation method, we showed that our neural network models outperformed most other methods and are comparable to the best predicting algorithm thus far published. Furthermore, our neural network models correctly classified 74% of all siRNAs into different efficiency categories; with a correlation coefficient of 0.43 and receiver operating characteristic curve score of 0.78, thus highlighting the potential utility of this method to complement other existing siRNA classification and prediction schemes.

  13. Small Players Ruling the Hard Game: siRNA in Bone Regeneration.

    PubMed

    Ghadakzadeh, Saber; Mekhail, Mina; Aoude, Ahmed; Hamdy, Reggie; Tabrizian, Maryam

    2016-03-01

    Silencing gene expression through a sequence-specific manner can be achieved by small interfering RNAs (siRNAs). The discovery of this process has opened the doors to the development of siRNA therapeutics. Although several preclinical and clinical studies have shown great promise in the treatment of neurological disorders, cancers, dominant disorders, and viral infections with siRNA, siRNA therapy is still gaining ground in musculoskeletal tissue repair and bone regeneration. Here we present a comprehensive review of the literature to summarize different siRNA delivery strategies utilized to enhance bone regeneration. With advancement in understanding the targetable biological pathways involved in bone regeneration and also the rapid progress in siRNA technologies, application of siRNA for bone regeneration has great therapeutic potential. High rates of musculoskeletal injuries and diseases, and their inevitable consequences, impose a huge financial burden on individuals and healthcare systems worldwide. © 2016 American Society for Bone and Mineral Research. PMID:26890411

  14. Effect of Inducible Co-Stimulatory Molecule siRNA in Cerebral Infarction Rat Models

    PubMed Central

    Luo, Yingquan; Yang, Yu; Zhang, Hui; Zhang, Ting; Wang, Yina; Tan, Shengyu; Xu, Yan; Li, Dan; Ye, Ling; Chen, Ping

    2015-01-01

    Background T cell-induced inflammatory response and related cytokine secretion at the injury site may participate in the pathogenesis of cerebral infarction. Recent studies established inducible co-stimulatory molecule (ICOS) as a novel T cell-related factor for its activation and functions. We thus investigate the role of ICOS in cerebral infarction. Material/Methods The siRNA of ICOS was first used to suppress the gene expression in cultured lymphocytes. An in vivo study was then performed by intravenous application of ICOS siRNA in cerebral infarction rats. Survival rates, neurological scores, serum tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-17 levels were observed. Results The expression of ICOS in cultured lymphocytes was significantly suppressed by siRNA. In the in vivo study, the application of siRNA effectively lowered mortality rates of rats, in addition to the improvement of neurological behaviors and amelioration of cerebral tissue damage. Serum levels of TNF-α, IL-1 and IL-17 were all significantly suppressed after siRNA injection. Conclusions ICOS siRNA can protect brain tissues from ischemia injuries after cerebral infarction, improve limb movement and coordination, lower the mortality rate of rats, and inhibit T cell-induced cytokines. These results collectively suggest the potential treatment efficacy of ICOS siRNA against cerebral infarction. PMID:26436531

  15. Structure-Guided Control of siRNA Off-Target Effects.

    PubMed

    Suter, Scott R; Sheu-Gruttadauria, Jessica; Schirle, Nicole T; Valenzuela, Rachel; Ball-Jones, Alexi A; Onizuka, Kazumitsu; MacRae, Ian J; Beal, Peter A

    2016-07-20

    Short interfering RNAs (siRNAs) are promising therapeutics that make use of the RNA interference (RNAi) pathway, but liabilities arising from the native RNA structure necessitate chemical modification for drug development. Advances in the structural characterization of components of the human RNAi pathway have enabled structure-guided optimization of siRNA properties. Here we report the 2.3 Å resolution crystal structure of human Argonaute 2 (hAgo2), a key nuclease in the RNAi pathway, bound to an siRNA guide strand bearing an unnatural triazolyl nucleotide at position 1 (g1). Unlike natural nucleotides, this analogue inserts deeply into hAgo2's central RNA binding cleft and thus is able to modulate pairing between guide and target RNAs. The affinity of the hAgo2-siRNA complex for a seed-only matched target was significantly reduced by the triazolyl modification, while the affinity for a fully matched target was unchanged. In addition, siRNA potency for off-target repression was reduced (4-fold increase in IC50) by the modification, while on-target knockdown was improved (2-fold reduction in IC50). Controlling siRNA on-target versus microRNA (miRNA)-like off-target potency by projection of substituent groups into the hAgo2 central cleft from g1 is a new approach to enhance siRNA selectivity with a strong structural rationale. PMID:27387838

  16. Dendrimers as Carriers for siRNA Delivery and Gene Silencing: A Review

    PubMed Central

    Huang, Weizhe; He, Ziying

    2013-01-01

    RNA interference (RNAi) was first literaturally reported in 1998 and has become rapidly a promising tool for therapeutic applications in gene therapy. In a typical RNAi process, small interfering RNAs (siRNA) are used to specifically downregulate the expression of the targeted gene, known as the term “gene silencing.” One key point for successful gene silencing is to employ a safe and efficient siRNA delivery system. In this context, dendrimers are emerging as potential nonviral vectors to deliver siRNA for RNAi purpose. Dendrimers have attracted intense interest since their emanating research in the 1980s and are extensively studied as efficient DNA delivery vectors in gene transfer applications, due to their unique features based on the well-defined and multivalent structures. Knowing that DNA and RNA possess a similar structure in terms of nucleic acid framework and the electronegative nature, one can also use the excellent DNA delivery properties of dendrimers to develop effective siRNA delivery systems. In this review, the development of dendrimer-based siRNA delivery vectors is summarized, focusing on the vector features (siRNA delivery efficiency, cytotoxicity, etc.) of different types of dendrimers and the related investigations on structure-activity relationship to promote safe and efficient siRNA delivery system. PMID:24288498

  17. Antineoplastic Effects of siRNA against TMPRSS2-ERG Junction Oncogene in Prostate Cancer.

    PubMed

    Urbinati, Giorgia; Ali, Hafiz Muhammad; Rousseau, Quentin; Chapuis, Hubert; Desmaële, Didier; Couvreur, Patrick; Massaad-Massade, Liliane

    2015-01-01

    TMPRSS2-ERG junction oncogene is present in more than 50% of patients with prostate cancer and its expression is frequently associated with poor prognosis. Our aim is to achieve gene knockdown by siRNA TMPRSS2-ERG and then to assess the biological consequences of this inhibition. First, we designed siRNAs against the two TMPRSS2-ERG fusion variants (III and IV), most frequently identified in patients' biopsies. Two of the five siRNAs tested were found to efficiently inhibit mRNA of both TMPRSS2-ERG variants and to decrease ERG protein expression. Microarray analysis further confirmed ERG inhibition by both siRNAs TMPRSS2-ERG and revealed one common down-regulated gene, ADRA2A, involved in cell proliferation and migration. The siRNA against TMPRSS2-ERG fusion variant IV showed the highest anti-proliferative effects: Significantly decreased cell viability, increased cleaved caspase-3 and inhibited a cluster of anti-apoptotic proteins. To propose a concrete therapeutic approach, siRNA TMPRSS2-ERG IV was conjugated to squalene, which can self-organize as nanoparticles in water. The nanoparticles of siRNA TMPRSS2-ERG-squalene injected intravenously in SCID mice reduced growth of VCaP xenografted tumours, inhibited oncoprotein expression and partially restored differentiation (decrease in Ki67). In conclusion, this study offers a new prospect of treatment for prostate cancer based on siRNA-squalene nanoparticles targeting TMPRSS2-ERG junction oncogene. PMID:25933120

  18. DNA as Tunable Adaptor for siRNA Polyplex Stabilization and Functionalization.

    PubMed

    Heissig, Philipp; Klein, Philipp M; Hadwiger, Philipp; Wagner, Ernst

    2016-01-01

    siRNA and microRNA are promising therapeutic agents, which are engaged in a natural mechanism called RNA interference that modulates gene expression posttranscriptionally. For intracellular delivery of such nucleic acid triggers, we use sequence-defined cationic polymers manufactured through solid phase chemistry. They consist of an oligoethanamino amide core for siRNA complexation and optional domains for nanoparticle shielding and cell targeting. Due to the small size of siRNA, electrostatic complexes with polycations are less stable, and consequently intracellular delivery is less efficient. Here we use DNA oligomers as adaptors to increase size and charge of cargo siRNA, resulting in increased polyplex stability, which in turn boosts transfection efficiency. Extending a single siRNA with a 181-nucleotide DNA adaptor is sufficient to provide maximum gene silencing aided by cationic polymers. Interestingly, this simple strategy was far more effective than merging defined numbers (4-10) of siRNA units into one DNA scaffolded construct. For DNA attachment, the 3' end of the siRNA passenger strand was beneficial over the 5' end. The impact of the attachment site however was resolved by introducing bioreducible disulfides at the connection point. We also show that DNA adaptors provide the opportunity to readily link additional functional domains to siRNA. Exemplified by the covalent conjugation of the endosomolytic influenza peptide INF-7 to siRNA via a DNA backbone strand and complexing this construct with a targeting polymer, we could form a highly functional polyethylene glycol-shielded polyplex to downregulate a luciferase gene in folate receptor-positive cells. PMID:26928236

  19. N-Alkyl-PEI Functional Iron Oxide Nanocluster for Efficient siRNA Delivery**

    PubMed Central

    Liu, Gang; Xie, Jin; Zhang, Fan; Wang, Zhi-Yong; Luo, Kui; Zhu, Lei; Quan, Qi-Meng; Niu, Gang; Lee, Seulki

    2013-01-01

    Small interfering RNA (siRNA) is an emerging class of therapeutics, working by regulating the expression of a specific gene involved in disease progression. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. In this study, a non-viral nanoparticle gene carrier has been developed and its efficiency for siRNA delivery and transfection has been validated at both in vitro and in vivo levels. Such a nanocarrier, abbreviated as Alkyl-PEI2k-IO, was constructed with a core of iron oxide (IO) and a shell of alkylated PEI2000 (Alkyl-PEI2k). It was found to be able to bind with siRNA, resulting in well-dispersed nanoparticles with a controlled clustering structure and narrow size distribution. Electrophoresis studies showed that the Alkyl-PEI2k-IOs could retard siRNA completely at N/P ratios above 10, protect siRNA from enzymatic degradation in serum and release complexed siRNA efficiently in the presence of polyanionic heparin. The knockdown efficiency of the siRNA loaded nanocarriers was assessed with 4T1 cells stably expressing luciferase (fluc-4T1) and further, with a fluc-4T1 xenograft model. Significant downregulation of luciferase was observed, and unlike the high molecular weight analogs, the Alkyl-PEI2k coated IOs showed a good biocompatibility. In conclusion, Alkyl-PEI2k-IOs demonstrate highly efficient delivery of siRNA and an innocuous toxic profile, making it a potential carrier for gene therapy. PMID:21861295

  20. Simultaneous cytosolic delivery of a chemotherapeutic and siRNA using nanoparticle-stabilized nanocapsules

    NASA Astrophysics Data System (ADS)

    Hardie, Joseph; Jiang, Ying; Tetrault, Emily R.; Ghazi, Phaedra C.; Yesilbag Tonga, Gulen; Farkas, Michelle E.; Rotello, Vincent M.

    2016-09-01

    We report on nanoparticle-stabilized capsules (NPSCs) as a platform for the co-delivery of survivin-targeted siRNA and tamoxifen. These capsules feature an inner oil core that provides a carrier for tamoxifen, and is coated on the surface with positively charged nanoparticles self-assembled with siRNA. The multifaceted chemical nature of the NPSC system enables the simultaneous delivery of both payloads directly into the cytosol in vitro. The NPSC co-delivery of tamoxifen and survivin-targeted siRNA into breast cancer cells disables the pathways that inhibit apoptosis, resulting in enhanced breast cell death.

  1. Delivery of siRNA Using Cationic Liposomes Incorporating Stearic Acid-modified Octa-Arginine.

    PubMed

    Yang, Dongsheng; Li, Yuhuan; Qi, Yuhang; Chen, Yongzhen; Yang, Xuewei; Li, Yujing; Liu, Songcai; Lee, Robert J

    2016-07-01

    Cationic liposomes incorporating stearic acid-modified octa-arginine (StA-R8) were evaluated for survivin small interfering RNA (siRNA) delivery. StA-R8 was synthesized and incorporated into liposomes. The composition of liposomes was optimized. Physicochemical properties, cytotoxicity, cellular uptake and gene silencing activity of the liposomes complexed to survivin siRNA were investigated. The results showed that StA-R8-containing liposomes had reduced cytotoxicity and improved delivery efficiency of siRNA into cancer cells compared with StA-R8 by itself. PMID:27354583

  2. Simultaneous cytosolic delivery of a chemotherapeutic and siRNA using nanoparticle-stabilized nanocapsules.

    PubMed

    Hardie, Joseph; Jiang, Ying; Tetrault, Emily R; Ghazi, Phaedra C; Tonga, Gulen Yesilbag; Farkas, Michelle E; Rotello, Vincent M

    2016-09-16

    We report on nanoparticle-stabilized capsules (NPSCs) as a platform for the co-delivery of survivin-targeted siRNA and tamoxifen. These capsules feature an inner oil core that provides a carrier for tamoxifen, and is coated on the surface with positively charged nanoparticles self-assembled with siRNA. The multifaceted chemical nature of the NPSC system enables the simultaneous delivery of both payloads directly into the cytosol in vitro. The NPSC co-delivery of tamoxifen and survivin-targeted siRNA into breast cancer cells disables the pathways that inhibit apoptosis, resulting in enhanced breast cell death. PMID:27505356

  3. The Abundance of Interstellar Fluorine

    NASA Technical Reports Server (NTRS)

    Lauroesch, James T.

    2005-01-01

    The primary objective of this program was to obtain FUSE observations of the interstellar absorption lines of F I at 951 and 954 Angstroms to derive the abundance of fluorine toward the star HD 164816. The nucleosynthetic source(s) of fluorine are still a matter of debate - the present day abundance of fluorine can potentially constrain models for pulsationally driven dredge-up in asymptotic giant branch stars. An accurate measure for the depletion behavior of fluorine will determine whether it may be detectable in QSO absorption line systems - an unambiguous detection of fluorine at suitably high redshifts would provide the best evidence to date for the neutrino process in massive stars. Furthermore, due to its extreme reactivity, measurement of the gas-phase interstellar fluorine abundance is important for models of grain chemistry. Despite the importance of measuring the interstellar fluorine abundance, at the time of our proposal only one previous detection has been made due to the low relative abundance of fluorine, the lack of lines outside the far-UV, and the blending of the available F I transitions with lines of Hz. The star HD 164816 is associated with the Lagoon nebula (M8), and at a distance of approximately 1.5 kpc probes both distant and local gas. Beginning April 8th, 2004 FUSE FP-Split observations of the star HD 164816 were obtained for this program. This data became available in the FUSE data archive May 21, 2004, and these observations were then downloaded and we began our analysis. Our analysis procedure has involved (1) fitting stellar models to the FUSE spectra, (2) using the multiple lines of Hz and N I at other wavelengths in the FUSE bandpass to derive column densities for the lines of H2 and N I which are blended with the F I features at 951 and 954 angstroms (3) the measurement of the column densities of F I and the species O I and C1 I which are important species for the dis-entangling of dust and nucleosynthetic effects. As discussed in

  4. Assorted Processing of Synthetic Trans-Acting siRNAs and Its Activity in Antiviral Resistance

    PubMed Central

    Zhao, Mingmin; San León, David; Mesel, Frida; García, Juan Antonio; Simón-Mateo, Carmen

    2015-01-01

    The use of syn-tasiRNAs has been proposed as an RNA interference technique alternative to those previously described: hairpin based, virus induced gene silencing or artificial miRNAs. In this study we engineered the TAS1c locus to impair Plum pox virus (PPV) infection by replacing the five native siRNAs with two 210-bp fragments from the CP and the 3´NCR regions of the PPV genome. Deep sequencing analysis of the small RNA species produced by both constructs in planta has shown that phased processing of the syn-tasiRNAs is construct-specific. While in syn-tasiR-CP construct the processing was as predicted 21-nt phased in register with miR173-guided cleavage, the processing of syn-tasiR-3NCR is far from what was expected. A 22-nt species from the miR173-guided cleavage was a guide of two series of phased small RNAs, one of them in an exact 21-nt register, and the other one in a mixed of 21-/22-nt frame. In addition, both constructs produced abundant PPV-derived small RNAs in the absence of miR173 as a consequence of a strong sense post-transcriptional gene silencing induction. The antiviral effect of both constructs was also evaluated in the presence or absence of miR173 and showed that the impairment of PPV infection was not significantly higher when miR173 was present. The results show that syn-tasiRNAs processing depends on construct-specific factors that should be further studied before the so-called MIGS (miRNA-induced gene silencing) technology can be used reliably. PMID:26147769

  5. Oral delivery of double-stranded RNAs and siRNAs induces RNAi effects in the potato/tomato psyllid, Bactericerca cockerelli.

    PubMed

    Wuriyanghan, Hada; Rosa, Cristina; Falk, Bryce W

    2011-01-01

    The potato/tomato psyllid, Bactericerca cockerelli (B. cockerelli), and the Asian citrus psyllid, Diaphorina citri (D. citri), are very important plant pests, but they are also vectors of phloem-limited bacteria that are associated with two devastating plant diseases. B. cockerelli is the vector of Candidatus Liberibacter psyllaurous (solanacearum), which is associated with zebra chip disease of potatoes, and D. citri is the vector of Ca. Liberibacter asiaticus, which is associated with the Huanglongbing (citrus greening) disease that currently threatens the entire Florida citrus industry. Here we used EST sequence information from D. citri to identify potential targets for RNA interference in B. cockerelli. We targeted ubiquitously expressed and gut-abundant mRNAs via injection and oral acquisition of double-stranded RNAs and siRNAs and were able to induce mortality in recipient psyllids. We also showed knockdown of target mRNAs, and that oral acquisition resulted primarily in mRNA knockdown in the psyllid gut. Concurrent with gene knockdown was the accumulation of target specific ∼ 21 nucleotide siRNAs for an abundant mRNA for BC-Actin. These results showed that RNAi can be a powerful tool for gene function studies in psyllids, and give support for continued efforts for investigating RNAi approaches as possible tools for psyllid and plant disease control. PMID:22110747

  6. Solar abundance of platinum

    PubMed Central

    Burger, Harry; Aller, Lawrence H.

    1975-01-01

    Three lines of neutral platinum, located at λ 2997.98 Å, λ 3064.71 Å, and λ 3301.86 Å have been used to determine the solar platinum abundance by the method of spectral synthesis. On the scale, log A(H) = 12.00, the thus-derived solar platinum abundance is 1.75 ± 0.10, in fair accord with Cameron's value of log A(Pt) = 1.69 derived by Mason from carbonaceous chondrites and calculated on the assumption that log A(Si) = 7.55 in the sun. PMID:16592278

  7. Epigenome Editing of Potato by Grafting Using Transgenic Tobacco as siRNA Donor.

    PubMed

    Kasai, Atsushi; Bai, Songling; Hojo, Hatsune; Harada, Takeo

    2016-01-01

    In plants, it is possible to induce heritable transcriptional gene silencing (TGS) via RNA-directed DNA methylation (RdDM) using artificially synthesized small RNA (siRNA) homologous to the 5'-flanking region of the target gene. As the siRNA signal with a specific RNA determinant moves through plasmodesmata and sieve elements, we attempted to induce TGS of a transgene and an endogenous gene of potato (Solanum tuberosum) rootstock by grafting using siRNA produced in a tobacco (Nicotiana benthamiana) scion. Our results provide evidence that this system can induce TGS of target genes in tubers formed on potato rootstock. The TGS is maintained in the progeny tubers lacking the transported siRNAs. Our findings reveal that epigenome editing using mobile RNA has the potential to allow breeding of artificial sport cultivars in vegetative propagation crops. PMID:27564864

  8. Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells.

    PubMed

    Ghildiyal, Megha; Seitz, Hervé; Horwich, Michael D; Li, Chengjian; Du, Tingting; Lee, Soohyun; Xu, Jia; Kittler, Ellen L W; Zapp, Maria L; Weng, Zhiping; Zamore, Phillip D

    2008-05-23

    Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA (dsRNA) as a defense against viral infection. We identified endogenous siRNAs (endo-siRNAs), 21 nucleotides in length, that correspond to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to messenger RNAs (mRNAs); these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form double-stranded RNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease Dicer-2 and the RNAi effector protein Argonaute2 (Ago2). We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma, much as Piwi-interacting RNAs do in the germ line. PMID:18403677

  9. Reductively Cleavable Nanocaplets for siRNA Delivery by Template-Assisted Oxidative Polymerization.

    PubMed

    Hashim, P K; Okuro, Kou; Sasaki, Shigekazu; Hoashi, Yasutaka; Aida, Takuzo

    2015-12-23

    A series of water-soluble telechelic dithiol monomers bearing multiple guanidinium ion (Gu(+)) units in their main chains were synthesized for packaging siRNA by template-assisted oxidative polymerization at their thiol termini. In the presence of siRNA, oxidative polymerization of (TEG)Gu4 affords a uniform-sized (7 ± 2 nm) nanocaplet containing siRNA (P(TEG)Gu4⊃siRNA; P(TEG)Gu4 = polymerized (TEG)Gu4). When this small conjugate is incubated with live cells, cellular uptake occurs, and the nanocaplet undergoes depolymerization in the reductive cytosolic environment to liberate the packaged siRNA. Consequently, gene expression in the live cells is suppressed. PMID:26648391

  10. Epigenome Editing of Potato by Grafting Using Transgenic Tobacco as siRNA Donor

    PubMed Central

    Hojo, Hatsune; Harada, Takeo

    2016-01-01

    In plants, it is possible to induce heritable transcriptional gene silencing (TGS) via RNA-directed DNA methylation (RdDM) using artificially synthesized small RNA (siRNA) homologous to the 5'-flanking region of the target gene. As the siRNA signal with a specific RNA determinant moves through plasmodesmata and sieve elements, we attempted to induce TGS of a transgene and an endogenous gene of potato (Solanum tuberosum) rootstock by grafting using siRNA produced in a tobacco (Nicotiana benthamiana) scion. Our results provide evidence that this system can induce TGS of target genes in tubers formed on potato rootstock. The TGS is maintained in the progeny tubers lacking the transported siRNAs. Our findings reveal that epigenome editing using mobile RNA has the potential to allow breeding of artificial sport cultivars in vegetative propagation crops. PMID:27564864

  11. In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight

    NASA Astrophysics Data System (ADS)

    Dahlman, James E.; Barnes, Carmen; Khan, Omar F.; Thiriot, Aude; Jhunjunwala, Siddharth; Shaw, Taylor E.; Xing, Yiping; Sager, Hendrik B.; Sahay, Gaurav; Speciner, Lauren; Bader, Andrew; Bogorad, Roman L.; Yin, Hao; Racie, Tim; Dong, Yizhou; Jiang, Shan; Seedorf, Danielle; Dave, Apeksha; Singh Sandhu, Kamaljeet; Webber, Matthew J.; Novobrantseva, Tatiana; Ruda, Vera M.; Lytton-Jean, Abigail K. R.; Levins, Christopher G.; Kalish, Brian; Mudge, Dayna K.; Perez, Mario; Abezgauz, Ludmila; Dutta, Partha; Smith, Lynelle; Charisse, Klaus; Kieran, Mark W.; Fitzgerald, Kevin; Nahrendorf, Matthias; Danino, Dganit; Tuder, Rubin M.; von Andrian, Ulrich H.; Akinc, Akin; Panigrahy, Dipak; Schroeder, Avi; Koteliansky, Victor; Langer, Robert; Anderson, Daniel G.

    2014-08-01

    Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.

  12. Systematic Comparisons of Formulations of Linear Oligolysine Peptides with siRNA and Plasmid DNA.

    PubMed

    Kwok, Albert; McCarthy, David; Hart, Stephen L; Tagalakis, Aristides D

    2016-05-01

    The effects of lysine peptide lengths on DNA and siRNA packaging and delivery were studied using four linear oligolysine peptides with 8 (K8), 16 (K16), 24 (K24) and 32 (K32) lysines. Oligolysine peptides with 16 lysines or longer were effective for stable monodisperse particle formation and optimal transfection efficiency with plasmid DNA (pDNA), but K8 formulations were less stable under anionic heparin challenge and consequently displayed poor transfection efficiency. However, here we show that the oligolysines were not able to package siRNA to form stable complexes, and consequently, siRNA transfection was unsuccessful. These results indicate that the physical structure and length of cationic peptides and their charge ratios are critical parameters for stable particle formation with pDNA and siRNA and that without packaging, delivery and transfection cannot be achieved. PMID:26684657

  13. Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages

    PubMed Central

    Perez, Ana Paula; Cosaka, Maria Luz; Romero, Eder Lilia; Morilla, Maria Jose

    2011-01-01

    Background Gene silencing using small interfering RNA (siRNA) is a promising new therapeutic approach for glioblastoma. The endocytic uptake and delivery of siRNA to intracellular compartments could be enhanced by complexation with polyamidoamine dendrimers. In the present work, the uptake mechanisms and intracellular traffic of siRNA/generation 7 dendrimer complexes (siRNA dendriplexes) were screened in T98G glioblastoma and J774 macrophages. Methods The effect of a set of chemical inhibitors of endocytosis on the uptake and silencing capacity of dendriplexes was determined by flow cytometry. Colocalization of fluorescent dendriplexes with endocytic markers and occurrence of intracellular dissociation were assessed by confocal laser scanning microscopy. Results Uptake of siRNA dendriplexes by T98G cells was reduced by methyl-β-cyclodextrin, and genistein, and cytochalasine D, silencing activity was reduced by genistein; dendriplexes colocalized with cholera toxin subunit B. Therefore, caveolin-dependent endocytosis was involved both in the uptake and silencing activity of siRNA dendriplexes. On the other hand, uptake of siRNA dendriplexes by J774 cells was reduced by methyl-β-cyclodextrin, genistein, chlorpromazine, chloroquine, cytochalasine D, and nocodazole, the silencing activity was not affected by chlorpromazine, genistein or chloroquine, and dendriplexes colocalized with transferrin and cholera toxin subunit B. Thus, both clathrin-dependent and caveolin-dependent endocytosis mediated the uptake and silencing activity of the siRNA dendriplexes. SiRNA dendriplexes were internalized at higher rates by T98G but induced lower silencing than in J774 cells. SiRNA dendriplexes showed relatively slow dissociation kinetics, and their escape towards the cytosol was not mediated by acidification independently of the uptake pathway. Conclusion The extent of cellular uptake of siRNA dendriplexes was inversely related to their silencing activity. The higher silencing

  14. Therapeutic antidepressant potential of a conjugated siRNA silencing the serotonin transporter after intranasal administration

    PubMed Central

    Ferrés-Coy, A; Galofré, M; Pilar-Cuéllar, F; Vidal, R; Paz, V; Ruiz-Bronchal, E; Campa, L; Pazos, Á; Caso, J R; Leza, J C; Alvarado, G; Montefeltro, A; Valdizán, E M; Artigas, F; Bortolozzi, A

    2016-01-01

    Major depression brings about a heavy socio-economic burden worldwide due to its high prevalence and the low efficacy of antidepressant drugs, mostly inhibiting the serotonin transporter (SERT). As a result, ~80% of patients show recurrent or chronic depression, resulting in a poor quality of life and increased suicide risk. RNA interference (RNAi) strategies have been preliminarily used to evoke antidepressant-like responses in experimental animals. However, the main limitation for the medical use of RNAi is the extreme difficulty to deliver oligonucleotides to selected neurons/systems in the mammalian brain. Here we show that the intranasal administration of a sertraline-conjugated small interfering RNA (C-SERT-siRNA) silenced SERT expression/function and evoked fast antidepressant-like responses in mice. After crossing the permeable olfactory epithelium, the sertraline-conjugated-siRNA was internalized and transported to serotonin cell bodies by deep Rab-7-associated endomembrane vesicles. Seven-day C-SERT-siRNA evoked similar or more marked responses than 28-day fluoxetine treatment. Hence, C-SERT-siRNA (i) downregulated 5-HT1A-autoreceptors and facilitated forebrain serotonin neurotransmission, (ii) accelerated the proliferation of neuronal precursors and (iii) increased hippocampal complexity and plasticity. Further, short-term C-SERT-siRNA reversed depressive-like behaviors in corticosterone-treated mice. The present results show the feasibility of evoking antidepressant-like responses by selectively targeting neuronal populations with appropriate siRNA strategies, opening a way for further translational studies. PMID:26100539

  15. Therapeutic antidepressant potential of a conjugated siRNA silencing the serotonin transporter after intranasal administration.

    PubMed

    Ferrés-Coy, A; Galofré, M; Pilar-Cuéllar, F; Vidal, R; Paz, V; Ruiz-Bronchal, E; Campa, L; Pazos, Á; Caso, J R; Leza, J C; Alvarado, G; Montefeltro, A; Valdizán, E M; Artigas, F; Bortolozzi, A

    2016-03-01

    Major depression brings about a heavy socio-economic burden worldwide due to its high prevalence and the low efficacy of antidepressant drugs, mostly inhibiting the serotonin transporter (SERT). As a result, ~80% of patients show recurrent or chronic depression, resulting in a poor quality of life and increased suicide risk. RNA interference (RNAi) strategies have been preliminarily used to evoke antidepressant-like responses in experimental animals. However, the main limitation for the medical use of RNAi is the extreme difficulty to deliver oligonucleotides to selected neurons/systems in the mammalian brain. Here we show that the intranasal administration of a sertraline-conjugated small interfering RNA (C-SERT-siRNA) silenced SERT expression/function and evoked fast antidepressant-like responses in mice. After crossing the permeable olfactory epithelium, the sertraline-conjugated-siRNA was internalized and transported to serotonin cell bodies by deep Rab-7-associated endomembrane vesicles. Seven-day C-SERT-siRNA evoked similar or more marked responses than 28-day fluoxetine treatment. Hence, C-SERT-siRNA (i) downregulated 5-HT1A-autoreceptors and facilitated forebrain serotonin neurotransmission, (ii) accelerated the proliferation of neuronal precursors and (iii) increased hippocampal complexity and plasticity. Further, short-term C-SERT-siRNA reversed depressive-like behaviors in corticosterone-treated mice. The present results show the feasibility of evoking antidepressant-like responses by selectively targeting neuronal populations with appropriate siRNA strategies, opening a way for further translational studies. PMID:26100539

  16. Topical Delivery of siRNA into Skin using SPACE-peptide Carriers

    PubMed Central

    Chen, Ming; Zakrewsky, Michael; Gupta, Vivek; Anselmo, Aaron C.; Slee, Deborah H.; Muraski, John A.; Mitragotri, Samir

    2014-01-01

    Short-interfering RNAs (siRNAs) offer a potential tool for the treatment of skin disorders. However, applications of siRNA for dermatological conditions are limited by their poor permeation across the stratum corneum of the skin and low penetration into skin’s viable cells. In this study, we report the use of SPACE-peptide in combination with a DOTAP-based ethosomal carrier system to enhance skin delivery of siRNA. A DOTAP-based SPACE Ethosomal System significantly enhanced siRNA penetration into porcine skin in vitro by 6.3±1.7-fold (p<0.01) with an approximately 10-fold (p<0.01) increase in epidermis accumulation of siRNA compared to that from an aqueous solution. Penetration of siRNA was also enhanced at the cellular level. Internalization of SPACE-peptide occurred in a concentration dependent manner marked by a shift in intracellular distribution from punctate spots to diffused cytoplasmic staining at a peptide concentration of 10 mg/mL. In vitro delivery of GAPDH siRNA by SPACE peptide led to 83.3±3.0% knockdown relative to the control. In vivo experiments performed using female BALB/C mice also confirmed the efficacy of DOTAP-SES in delivering GAPDH-siRNA into skin. Topical application of DOTAP-SES on mice skin resulted in 63.2%±7.7% of GAPDH knockdown, which was significantly higher than that from GAPDH-siRNA PBS (p<0.05). DOTAP-SES formulation reported here may open new opportunities for cutaneous siRNA delivery. PMID:24434423

  17. siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro.

    PubMed

    Liu, Haibing; Qin, Yanyan; Kong, Zhenzhen; Shao, Qixiang; Su, Zhaoliang; Wang, Shengjun; Chen, Jianguo

    2016-01-01

    Enterovirus 71 (EV71) is the most important etiological agent of hand, foot, and mouth disease (HFMD) in young children, which is associated with severe neurological complications and has caused significant mortalities in recent HFMD outbreaks in Asia. However, there is no effective antiviral therapy against EV71. In this study, RNA interference (RNAi) was used as an antiviral strategy to inhibit EV71 replication. Three small interfering RNAs (siRNAs) targeting the 2Apro region of the EV71 genome were designed and synthesized. All the siRNAs were transfected individually into rhabdomyosarcoma (RD) cells, which were then infected with strain EV71-2006-52-9. The cytopathic effects (CPEs) in the infected RD cells, cell viability, viral titer, and viral RNA and protein expression were examined to evaluate the specific viral inhibition by the siRNAs. The results of cytopathogenicity and MTT tests indicated that the RD cells transfected with the three siRNAs showed slight CPEs and significantly high viability. The 50% tissue culture infective dose (TCID50) values demonstrated that the viral titer of the groups treated with three siRNAs were lower than those of the control groups. qRT-PCR and western blotting revealed that the levels of viral RNA and protein in the RD cells treated with the three siRNAs were lower than those in the controls. When RD cells transfected with siRNAs were also infected with strain EV71-2008-43-16, the expression of the VP1 protein was significantly inhibited. The levels of interferon α (IFN-α) and IFN-β did not differ significantly in any group. These results suggest that siRNAs targeting the 2Apro region of the EV71 genome exerted antiviral effects in vitro. PMID:26886455

  18. Off-target effects of siRNA specific for GFP

    PubMed Central

    Tschuch, Cordula; Schulz, Angela; Pscherer, Armin; Werft, Wiebke; Benner, Axel; Hotz-Wagenblatt, Agnes; Barrionuevo, Leticia Serra; Lichter, Peter; Mertens, Daniel

    2008-01-01

    Background Gene knock down by RNAi is a highly effective approach to silence gene expression in experimental as well as therapeutic settings. However, this widely used methodology entails serious pitfalls, especially concerning specificity of the RNAi molecules. Results We tested the most widely used control siRNA directed against GFP for off-target effects and found that it deregulates in addition to GFP a set of endogenous target genes. The off-target effects were dependent on the amount of GFP siRNA transfected and were detected in a variety of cell lines. Since the respective siRNA molecule specific for GFP is widely used as negative control for RNAi experiments, we studied the complete set of off-target genes of this molecule by genome-wide expression profiling. The detected modulated mRNAs had target sequences homologous to the siRNA as small as 8 basepairs in size. However, we found no restriction of sequence homology to 3'UTR of target genes. Conclusion We can show that even siRNAs without a physiological target have sequence-specific off-target effects in mammalian cells. Furthermore, our analysis defines the off-target genes affected by the siRNA that is commonly used as negative control and directed against GFP. Since off-target effects can hardly be avoided, the best strategy is to identify false positives and exclude them from the results. To this end, we provide the set of false positive genes deregulated by the commonly used GFP siRNA as a reference resource for future siRNA experiments. PMID:18577207

  19. Enhancing potency of siRNA targeting fusion genes by optimization outside of target sequence.

    PubMed

    Gavrilov, Kseniya; Seo, Young-Eun; Tietjen, Gregory T; Cui, Jiajia; Cheng, Christopher J; Saltzman, W Mark

    2015-12-01

    Canonical siRNA design algorithms have become remarkably effective at predicting favorable binding regions within a target mRNA, but in some cases (e.g., a fusion junction site) region choice is restricted. In these instances, alternative approaches are necessary to obtain a highly potent silencing molecule. Here we focus on strategies for rational optimization of two siRNAs that target the junction sites of fusion oncogenes BCR-ABL and TMPRSS2-ERG. We demonstrate that modifying the termini of these siRNAs with a terminal G-U wobble pair or a carefully selected pair of terminal asymmetry-enhancing mismatches can result in an increase in potency at low doses. Importantly, we observed that improvements in silencing at the mRNA level do not necessarily translate to reductions in protein level and/or cell death. Decline in protein level is also heavily influenced by targeted protein half-life, and delivery vehicle toxicity can confound measures of cell death due to silencing. Therefore, for BCR-ABL, which has a long protein half-life that is difficult to overcome using siRNA, we also developed a nontoxic transfection vector: poly(lactic-coglycolic acid) nanoparticles that release siRNA over many days. We show that this system can achieve effective killing of leukemic cells. These findings provide insights into the implications of siRNA sequence for potency and suggest strategies for the design of more effective therapeutic siRNA molecules. Furthermore, this work points to the importance of integrating studies of siRNA design and delivery, while heeding and addressing potential limitations such as restricted targetable mRNA regions, long protein half-lives, and nonspecific toxicities. PMID:26627251

  20. Peptide- and Amine-Modified Glucan Particles for the Delivery of Therapeutic siRNA.

    PubMed

    Cohen, Jessica L; Shen, Yuefei; Aouadi, Myriam; Vangala, Pranitha; Tencerova, Michaela; Amano, Shinya U; Nicoloro, Sarah M; Yawe, Joseph C; Czech, Michael P

    2016-03-01

    Translation of siRNA technology into the clinic is limited by the need for improved delivery systems that target specific cell types. Macrophages are particularly attractive targets for RNAi therapy because they promote pathogenic inflammatory responses in a number of important human diseases. We previously demonstrated that a multicomponent formulation of β-1,3-d-glucan-encapsulated siRNA particles (GeRPs) can specifically and potently silence genes in mouse macrophages. A major advance would be to simplify the GeRP system by reducing the number of delivery components, thus enabling more facile manufacturing and future commercialization. Here we report the synthesis and evaluation of a simplified glucan-based particle (GP) capable of delivering siRNA in vivo to selectively silence macrophage genes. Covalent attachment of small-molecule amines and short peptides containing weak bases to GPs facilitated electrostatic interaction of the particles with siRNA and aided in the endosomal release of siRNA by the proton-sponge effect. Modified GPs were nontoxic and were efficiently internalized by macrophages in vitro. When injected intraperitoneally (i.p.), several of the new peptide-modified GPs were found to efficiently deliver siRNA to peritoneal macrophages in lean, healthy mice. In an animal model of obesity-induced inflammation, i.p. administration of one of the peptide-modified GPs (GP-EP14) bound to siRNA selectively reduced the expression of target inflammatory cytokines in the visceral adipose tissue macrophages. Decreasing adipose tissue inflammation resulted in an improvement of glucose metabolism in these metabolically challenged animals. Thus, modified GPs represent a promising new simplified system for the efficient delivery of therapeutic siRNAs specifically to phagocytic cells in vivo for modulation of inflammation responses. PMID:26815386

  1. Evaluation of Exogenous siRNA Addition as a Metabolic Engineering Tool for Modifying Biopharmaceuticals

    PubMed Central

    Tummala, Seshu; Titus, Michael; Wilson, Lee; Wang, Chunhua; Ciatto, Carlo; Foster, Donald; Szabo, Zoltan; Guttman, Andras; Li, Chen; Bettencourt, Brian; Jayaraman, Muthuswamy; Deroot, Jack; Thill, Greg; Kocisko, David; Pollard, Stuart; Charisse, Klaus; Kuchimanchi, Satya; Hinkle, Greg; Milstein, Stuart; Myers, Rachel; Wu, Shiaw-Lin; Karger, Barry; Rossomando, Anthony

    2012-01-01

    Traditional metabolic engineering approaches, including homologous recombination, zinc finger nucleases, and short hairpin RNA (shRNA), have previously been employed to generate biologics with specific characteristics that improve efficacy, potency, and safety. An alternative approach is to exogenously add soluble small interfering RNA (siRNA) duplexes, formulated with a cationic lipid, directly to cells grown in shake flasks or bioreactors, This approach has the following potential advantages : no cell line development required, ability to tailor mRNA silencing by adjusting siRNA concentration, simultaneous silencing of multiple target genes, and potential temporal control of down regulation of target gene expression. In this study, we demonstrate proof of concept of the siRNA feeding approach as a metabolic engineering tool in the context of increasing monoclonal antibody afucosylation. First, potent siRNA duplexes targeting fut8 and gmds were dosed into shake flasks with cells that express an anti-CD20 monoclonal antibody. Dose response studies demonstrated the ability to titrate the silencing effect. Furthermore, siRNA addition resulted in no deleterious effects on cell growth, final protein titer, or specific productivity. In bioreactors, antibodies produced by cells following siRNA treatment exhibited improved functional characteristics compared to antibodies from untreated cells, including increased levels of afucosylation (63%), a 17-fold improvement in FCgRIIIa binding, and an increase in specific cell lysis by up to 30%, as determined in an ADCC assay. In addition, standard purification procedures effectively cleared the exogenously added siRNA and transfection agent. Moreover, no differences were observed when other key product quality structural attributes were compared to untreated controls. These results establish that exogenous addition of siRNA represents a potentially novel metabolic engineering tool to improve biopharmaceutical function and

  2. Systemic Administration of siRNA via cRGD-containing Peptide

    PubMed Central

    Huang, Yuanyu; Wang, Xiaoxia; Huang, Weiyan; Cheng, Qiang; Zheng, Shuquan; Guo, Shutao; Cao, Huiqing; Liang, Xing-Jie; Du, Quan; Liang, Zicai

    2015-01-01

    Although small interfering RNAs (siRNAs) have been demonstrated to specifically silence their target genes in disease models and clinical trials, in vivo siRNA delivery is still the technical bottleneck that limits their use in therapeutic applications. In this study, a bifunctional peptide named RGD10-10R was designed and tested for its ability to deliver siRNA in vitro and in vivo. Because of their electrostatic interactions with polyarginine (10R), negatively charged siRNAs were readily complexed with RGD10-10R peptides, forming spherical RGD10-10R/siRNA nanoparticles. In addition to enhancing their serum stability by preventing RNase from attacking siRNA through steric hindrance, peptide binding facilitated siRNA transfection into MDA-MB-231 cells, as demonstrated by FACS and confocal microscopy assays and by the repressed expression of target genes. When RGD10 peptide, a receptor competitor of RGD10-10R, was added to the transfection system, the cellular internalization of RGD10-10R/siRNA was significantly compromised, suggesting a mechanism of ligand/receptor interaction. Tissue distribution assays indicated that the peptide/siRNA complex preferentially accumulated in the liver and in several exocrine/endocrine glands. Furthermore, tumor-targeted delivery of siRNA was also demonstrated by in vivo imaging and cryosection assays. In summary, RGD10-10R might constitute a novel siRNA delivery tool that could potentially be applied in tumor treatment. PMID:26300278

  3. [Efficacy of siRNA on feline leukemia virus replication in vitro].

    PubMed

    Lehmann, Melanie; Weber, Karin; Rauch, Gisep; Hofmann-Lehmann, Regina; Hosie, Margaret J; Meli, Marina L; Hartmann, Katrin

    2015-01-01

    Feline leukemia virus (FeLV) can lead to severe clinical signs in cats. Until now, there is no effective therapy for FeLV-infected cats. RNA interference-based antiviral therapy is a new concept. Specific small interfering RNA (siRNA) are designed complementary to the mRNA of a target region, and thus inhibit replication. Several studies have proven efficacy of siRNAs in inhibiting virus replication. The aim of this study was to evaluate the inhibitory potential of siRNAs against FeLV replication in vitro. siRNAs against the FeLV env gene and the host cell surface receptor (feTHTR1) which is used by FeLV-A for entry as well as siRNA that were not complementary to the FeLV or cat genome, were tested. Crandell feline kidney cells (CrFK cells) were transfected with FeLV-A/Glasgow-1. On day 13, infected cells were transfected with siRNAs. As control, cells were mock-transfected or treated with azidothymidine (AZT) (5 μg/ml). Culture supernatants were analyzed for FeLV RNA using quantitative real-time RT-PCR and for FeLV p27 by ELISA every 24 hours for five days. All siRNAs significantly reduced viral RNA and p27 production, starting after 48 hours. The fact that non-complementary siRNAs also inhibited virus replication may lead to the conclusion that unspecific mechanisms rather than specific binding lead to inhibition. PMID:26054227

  4. Effects of hydrophobic core components in amphiphilic PDMAEMA nanoparticles on siRNA delivery.

    PubMed

    Han, Shangcong; Cheng, Qiang; Wu, Yidi; Zhou, Junhui; Long, Xingwen; Wei, Tuo; Huang, Yuanyu; Zheng, Shuquan; Zhang, Jianhua; Deng, Liandong; Wang, Xiaoxia; Liang, Xing-Jie; Cao, Huiqing; Liang, Zicai; Dong, Anjie

    2015-04-01

    Due to their biodegradable character, polyesters such as polycaprolactone (PCL), poly(D,L-lactide) (PDLLA), and polylactic-co-glycolic acid (PLGA) were widely used as the hydrophobic cores of amphiphilic cationic nanoparticles (NPs) for siRNA delivery. However, fewer researches focused on facilitating siRNA delivery by adjusting the polyester composition of these nanoparticles. Herein, we investigated the contribution of polyester segments in siRNA delivery in vitro by introducing different ratio of DLLA moieties in PCL segments of mPEG-block-PCL-graft-poly(dimethylamino ethyl methacrylate)(PEG-b-PCL-g-PDMAEMA). It was noticed that compared with the other ratios of DLLA moieties, a certain molar ratio (about 70%) of the NPs, named mPEG45-P(CL21-co-DLLA48)-g-(PDMAEMA29)2 (PECLD-70), showed the highest gene knockdown efficiency but poorest cellular uptake ability in vitro. Further research revealed that NPs with various compositions of the polyester cores showed different physicochemical properties including particle size, zeta potential and stiffness, leading to different endocytosis mechanisms thus influencing the cellular uptake efficiency. Subsequently, we observed that the cells treated by PECLD-70 NPs/Cy5 siRNA complexes exhibited more diffuse Cy5 signal distribution than other NPs by confocal laser scanning microscope, which suggested that siRNA delivered by PECLD-70 NPs/Cy5 siRNA complexes possessed of stronger capabilities in escaping from endosome/lysosome, entering the RNA-induced silencing complex (RISC) and cutting the target mRNA efficiently. The different siRNA release profile was dominated by the degradation rate of polyester segments. Therefore, it could be concluded that the adjustment of hydrophobic core of cationic nanoparticles could significantly affect their transfection behavior and appropriate polyester composition should be concerned in designing of analogous siRNA vectors. PMID:25701031

  5. siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro

    PubMed Central

    Kong, Zhenzhen; Shao, Qixiang; Su, Zhaoliang; Wang, Shengjun; Chen, Jianguo

    2016-01-01

    Enterovirus 71 (EV71) is the most important etiological agent of hand, foot, and mouth disease (HFMD) in young children, which is associated with severe neurological complications and has caused significant mortalities in recent HFMD outbreaks in Asia. However, there is no effective antiviral therapy against EV71. In this study, RNA interference (RNAi) was used as an antiviral strategy to inhibit EV71 replication. Three small interfering RNAs (siRNAs) targeting the 2Apro region of the EV71 genome were designed and synthesized. All the siRNAs were transfected individually into rhabdomyosarcoma (RD) cells, which were then infected with strain EV71-2006-52-9. The cytopathic effects (CPEs) in the infected RD cells, cell viability, viral titer, and viral RNA and protein expression were examined to evaluate the specific viral inhibition by the siRNAs. The results of cytopathogenicity and MTT tests indicated that the RD cells transfected with the three siRNAs showed slight CPEs and significantly high viability. The 50% tissue culture infective dose (TCID50) values demonstrated that the viral titer of the groups treated with three siRNAs were lower than those of the control groups. qRT–PCR and western blotting revealed that the levels of viral RNA and protein in the RD cells treated with the three siRNAs were lower than those in the controls. When RD cells transfected with siRNAs were also infected with strain EV71-2008-43-16, the expression of the VP1 protein was significantly inhibited. The levels of interferon α (IFN-α) and IFN-β did not differ significantly in any group. These results suggest that siRNAs targeting the 2Apro region of the EV71 genome exerted antiviral effects in vitro. PMID:26886455

  6. Enhancing potency of siRNA targeting fusion genes by optimization outside of target sequence

    PubMed Central

    Gavrilov, Kseniya; Seo, Young-Eun; Tietjen, Gregory T.; Cui, Jiajia; Cheng, Christopher J.; Saltzman, W. Mark

    2015-01-01

    Canonical siRNA design algorithms have become remarkably effective at predicting favorable binding regions within a target mRNA, but in some cases (e.g., a fusion junction site) region choice is restricted. In these instances, alternative approaches are necessary to obtain a highly potent silencing molecule. Here we focus on strategies for rational optimization of two siRNAs that target the junction sites of fusion oncogenes BCR-ABL and TMPRSS2-ERG. We demonstrate that modifying the termini of these siRNAs with a terminal G-U wobble pair or a carefully selected pair of terminal asymmetry-enhancing mismatches can result in an increase in potency at low doses. Importantly, we observed that improvements in silencing at the mRNA level do not necessarily translate to reductions in protein level and/or cell death. Decline in protein level is also heavily influenced by targeted protein half-life, and delivery vehicle toxicity can confound measures of cell death due to silencing. Therefore, for BCR-ABL, which has a long protein half-life that is difficult to overcome using siRNA, we also developed a nontoxic transfection vector: poly(lactic-coglycolic acid) nanoparticles that release siRNA over many days. We show that this system can achieve effective killing of leukemic cells. These findings provide insights into the implications of siRNA sequence for potency and suggest strategies for the design of more effective therapeutic siRNA molecules. Furthermore, this work points to the importance of integrating studies of siRNA design and delivery, while heeding and addressing potential limitations such as restricted targetable mRNA regions, long protein half-lives, and nonspecific toxicities. PMID:26627251

  7. pH-responsive hybrid quantum dots for targeting hypoxic tumor siRNA delivery.

    PubMed

    Zhu, HongYan; Zhang, ShengYu; Ling, Yong; Meng, GuoLiang; Yang, Yu; Zhang, Wei

    2015-12-28

    Hypoxia is a characteristic of cancer and plays a key role in tumorigenesis, angiogenesis and resistance to cancer therapies. SiRNA treatment is effective against hypoxic tumors by gene silencing. However, siRNA delivery to the hypoxic regions of solid tumors still presents a challenge due to the distance from blood vessels and the increased presence of efflux transporters. Therefore, tumor therapies would be improved through the immediate development of an effective siRNA delivery system to hypoxic regions. To this end, we synthesized a system to deliver HIF-1α siRNA into hypoxic tumor cells. The system consists of a functional shell composed of 2-deoxyglucose (DG)-polyethylene glycol (PEG) connected with the compound of lipoic acid, lysine and 9-poly-d-arginine (LA-Lys-9R) by a hydrazone bond and a core of CdTe quantum dots (QDs). The molecular structure of DG-PEG-LA-Lys-9R was confirmed by liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared spectroscopy (FTIR), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The multifunctional CdTe QDs measured approximately 200 nm and showed excellent biocompatibility, perfect siRNA binding capability and enhanced hypoxic tumor targeting. Importantly, the system described here is pH-responsive with a hydrazone bond; therefore, it avoids GLUT1 receptor-mediated endocytic recycling, resulting in irreversible delivery of the siRNA. We used Western blots to confirm the superior gene silencing efficiency induced by the DG-PEG-LA-Lys-9R with hydrazone modified CdTe QDs. Here, we demonstrate high efficacy of the siRNA tumor delivery system using in vitro and in vivo experiments. In addition, these studies demonstrate that pH-responsive hybrid quantum dots show improved antitumor efficacy with decreased organ toxicity, indicating a promising siRNA delivery system for hypoxic cancer therapy. PMID:26590349

  8. Effective cytoplasmic release of siRNA from liposomal carriers by controlling the electrostatic interaction of siRNA with a charge-invertible peptide, in response to cytoplasmic pH

    NASA Astrophysics Data System (ADS)

    Itakura, Shoko; Hama, Susumu; Matsui, Ryo; Kogure, Kentaro

    2016-05-01

    Condensing siRNA with cationic polymers is a major strategy used in the development of siRNA carriers that can avoid degradation by nucleases and achieve effective delivery of siRNA into the cytoplasm. However, ineffective release of siRNA from such condensed forms into the cytoplasm is a limiting step for induction of RNAi effects, and can be attributed to tight condensation of siRNA with the cationic polymers, due to potent electrostatic interactions. Here, we report that siRNA condensed with a slightly acidic pH-sensitive peptide (SAPSP), whose total charge is inverted from positive to negative in response to cytoplasmic pH, is effectively released via electrostatic repulsion of siRNA with negatively charged SAPSP at cytoplasmic pH (7.4). The condensed complex of siRNA and positively-charged SAPSP at acidic pH (siRNA/SAPSP) was found to result in almost complete release of siRNA upon charge inversion of SAPSP at pH 7.4, with the resultant negatively-charged SAPSP having no undesirable interactions with endogenous mRNA. Moreover, liposomes encapsulating siRNA/SAPSP demonstrated knockdown efficiencies comparable to those of commercially available siRNA carriers. Taken together, SAPSP may be very useful as a siRNA condenser, as it facilitates effective cytoplasmic release of siRNA, and subsequent induction of specific RNAi effects.Condensing siRNA with cationic polymers is a major strategy used in the development of siRNA carriers that can avoid degradation by nucleases and achieve effective delivery of siRNA into the cytoplasm. However, ineffective release of siRNA from such condensed forms into the cytoplasm is a limiting step for induction of RNAi effects, and can be attributed to tight condensation of siRNA with the cationic polymers, due to potent electrostatic interactions. Here, we report that siRNA condensed with a slightly acidic pH-sensitive peptide (SAPSP), whose total charge is inverted from positive to negative in response to cytoplasmic pH, is

  9. Stellar Oxygen Abundances

    NASA Astrophysics Data System (ADS)

    King, Jeremy

    1994-04-01

    This dissertation addresses several issues concerning stellar oxygen abundances. The 7774 {\\AA} O I triplet equivalent widths of Abia & Rebolo [1989, AJ, 347, 186] for metal-poor dwarfs are found to be systematically too high. I also argue that current effective temperatures used in halo star abundance studies may be ~150 K too low. New color-Teff relations are derived for metal-poor stars. Using the revised Teff values and improved equivalent widths for the 7774A O I triplet, the mean [O/Fe] ratio for a handful of halo stars is found to be +0.52 with no dependence on Teff or [Fe/H]. Possible cosmological implications of the hotter Teff scale are discussed along with additional evidence supporting the need for a higher temperature scale for metal-poor stars. Our Teff scale leads to a Spite Li plateau value of N(Li)=2.28 +/- 0.09. A conservative minimal primordial value of N(Li)=2.35 is inferred. If errors in the observations and models are considered, consistency with standard models of Big Bang nucleosynthesis is still achieved with this larger Li abundance. The revised Teff scale raises the observed B/Be ratio of HD 140283 from 10 to 12, making its value more comfortably consistent with the production of the observed B and Be by ordinary spallation. Our Teff values are found to be in good agreement with values predicted from both the Victoria and Yale isochrone color-Teff relations. Thus, it appears likely that no changes in globular cluster ages would result. Next, we examine the location of the break in the [O/Fe] versus [Fe/H] plane in a quantitative fashion. Analysis of a relatively homogeneous data set does not favor any unique break point in the range -1.7 /= -3), in agreement with the new results for halo dwarfs. We find that the gap in the observed [O/H] distribution, noted by Wheeler et al

  10. Abundances of light elements.

    PubMed Central

    Pagel, B E

    1993-01-01

    Recent developments in the study of abundances of light elements and their relevance to cosmological nucleosynthesis are briefly reviewed. The simplest model, based on standard cosmology and particle physics and assuming homogeneous baryon density at the relevant times, continues to stand up well. PMID:11607388

  11. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection.

    PubMed

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  12. siRNA Versus miRNA as Therapeutics for Gene Silencing

    PubMed Central

    Lam, Jenny K W; Chow, Michael Y T; Zhang, Yu; Leung, Susan W S

    2015-01-01

    Discovered a little over two decades ago, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are noncoding RNAs with important roles in gene regulation. They have recently been investigated as novel classes of therapeutic agents for the treatment of a wide range of disorders including cancers and infections. Clinical trials of siRNA- and miRNA-based drugs have already been initiated. siRNAs and miRNAs share many similarities, both are short duplex RNA molecules that exert gene silencing effects at the post-transcriptional level by targeting messenger RNA (mRNA), yet their mechanisms of action and clinical applications are distinct. The major difference between siRNAs and miRNAs is that the former are highly specific with only one mRNA target, whereas the latter have multiple targets. The therapeutic approaches of siRNAs and miRNAs are therefore very different. Hence, this review provides a comparison between therapeutic siRNAs and miRNAs in terms of their mechanisms of action, physicochemical properties, delivery, and clinical applications. Moreover, the challenges in developing both classes of RNA as therapeutics are also discussed. PMID:26372022

  13. Rigid nanoparticle-baseddelivery of anti-cancer siRNA: challenges and opportunities

    PubMed Central

    Wang, Zhiyong; Liu, Gang; Zheng, Hairong; Chen, Xiaoyuan

    2013-01-01

    Gene therapy is a promising strategy to treat various genetic and acquired diseases. Small interfering RNA (siRNA) is a revolutionary tool for gene therapy and the analysis of gene function. However, the development of a safe, efficient, and targetable non-viral siRNA delivery system remains a major challenge in gene therapy. An ideal delivery system should be able to encapsulate and protect the siRNA cargo from serum proteins, exhibit target tissue and cell specificity, penetrate the cell membrane, and release its cargo in the desired intracellular compartment. Nanomedicine has the potential to deal with these challenges faced by siRNA delivery. The unique characteristics of rigid nanoparticles mostly inorganic nanoparticles and allotropes of carbon nanomaterials, including high surface area, facile surface modification, controllable size, and excellent magnetic/optical/electrical properties, make them promising candidates for targeted siRNA delivery. In this review, recent progresses on rigid nanoparticle-based siRNA delivery systems will be summarized. PMID:24013011

  14. Self-assembled RNA interference microsponges for efficient siRNA delivery

    NASA Astrophysics Data System (ADS)

    Lee, Jong Bum; Hong, Jinkee; Bonner, Daniel K.; Poon, Zhiyong; Hammond, Paula T.

    2012-04-01

    The encapsulation and delivery of short interfering RNA (siRNA) has been realized using lipid nanoparticles, cationic complexes, inorganic nanoparticles, RNA nanoparticles and dendrimers. Still, the instability of RNA and the relatively ineffectual encapsulation process of siRNA remain critical issues towards the clinical translation of RNA as a therapeutic. Here we report the synthesis of a delivery vehicle that combines carrier and cargo: RNA interference (RNAi) polymers that self-assemble into nanoscale pleated sheets of hairpin RNA, which in turn form sponge-like microspheres. The RNAi-microsponges consist entirely of cleavable RNA strands, and are processed by the cell’s RNA machinery to convert the stable hairpin RNA to siRNA only after cellular uptake, thus inherently providing protection for siRNA during delivery and transport to the cytoplasm. More than half a million copies of siRNA can be delivered to a cell with the uptake of a single RNAi-microsponge. The approach could lead to novel therapeutic routes for siRNA delivery.

  15. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection

    PubMed Central

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  16. Targeted delivery of siRNA to cell death proteins in sepsis

    PubMed Central

    Brahmamdam, Pavan; Watanabe, Eizo; Unsinger, Jacqueline; Chang, Katherine C.; Schierding, William; Hoekzema, Andrew S.; Zhou, Tony T.; McDonough, Jacquelyn S.; Holemon, Heather; Heidel, Jeremy D.; Coopersmith, Craig M.; McDunn, Jonathan E.; Hotchkiss, Richard S.

    2010-01-01

    Immune suppression is a major cause of morbidity and mortality in the septic patient. Apoptotic loss of immune effector cells such as CD4 T and B cells is a key component in the loss immune competence in sepsis. Inhibition of lymphocyte apoptosis has led to improved survival in animal models of sepsis. Using qRT-PCR of isolated splenic CD4 T and B cells, we determined that Bim and PUMA, two key cell death proteins, are markedly up-regulated during sepsis. Lymphocytes have been notoriously difficult to transfect with siRNA. Consequently a novel, cyclodextrin polymer-based, transferrin receptor-targeted, delivery vehicle was employed to co-administer siRNA to Bim and PUMA to mice immediately after cecal ligation and puncture. Anti-apoptotic siRNA based therapy markedly decreased lymphocyte apoptosis and prevented the loss of splenic CD4 T and B cells. Flow cytometry confirmed in vivo delivery of siRNA to CD4 T and B cells and also demonstrated decreases in intracellular Bim and PUMA protein. In conclusion, Bim and PUMA are two critical mediators of immune cell death in sepsis. Use of a novel cyclodextrin polymer-based, transferrin receptor-targeted siRNA delivery vehicle enables effective administration of anti-apoptotic siRNAs to lymphocytes and reverses the immune cell depletion that is a hallmark of this highly lethal disorder. PMID:19033888

  17. Design of a Multicomponent Peptide-Woven Nanocomplex for Delivery of siRNA

    PubMed Central

    Jun, Eunsung; Kim, Soyoun; Kim, Jong-Ho; Cha, Kiweon; So, In-Seop; Son, Hye-Nam; Lee, Byung-Heon; Kim, Kwangmeyung; Kwon, Ick Chan; Kim, Sang Yoon; Kim, In-San

    2015-01-01

    We developed and tested a multicomponent peptide-woven siRNA nanocomplex (PwSN) comprising different peptides designed for efficient cellular targeting, endosomal escape, and release of siRNA. To enhance tumor-specific cellular uptake, we connected an interleukin-4 receptor-targeting peptide (I4R) to a nine-arginine peptide (9r), yielding I4R-9r. To facilitate endosomal escape, we blended endosomolytic peptides into the I4R-9r to form a multicomponent nanocomplex. Lastly, we modified 9r peptides by varying the number and positions of positive charges to obtain efficient release of siRNA from the nanocomplex in the cytosol. Using this step-wise approach for overcoming the biological challenges of siRNA delivery, we obtained an optimized PwSN with significant biological activity in vitro and in vivo. Interestingly, surface plasmon resonance analyses and three-dimensional peptide models demonstrated that our designed peptide adopted a unique structure that was correlated with faster complex disassembly and a better gene-silencing effect. These studies further elucidate the siRNA nanocomplex delivery pathway and demonstrate the applicability of our stepwise strategy to the design of siRNA carriers capable of overcoming multiple challenges and achieving efficient delivery. PMID:25705892

  18. Low-Molecular-Weight Polyethyleneimine Grafted Polythiophene for Efficient siRNA Delivery

    PubMed Central

    He, Pan; Hagiwara, Kyoji; Chong, Hui; Yu, Hsiao-hua; Ito, Yoshihiro

    2015-01-01

    Owing to its hydrophilicity, negative charge, small size, and labile degradation by endogenous nucleases, small interfering RNA (siRNA) delivery must be achieved by a carrier system. In this study, cationic copolymers composed of low-molecular-weight polyethylenimine and polythiophenes were synthesized and evaluated as novel self-tracking siRNA delivery vectors. The concept underlying the design of these copolymers is that hydrophobicity and rigidity of polythiophenes should enhance the transport of siRNA across the cell membrane and endosomal membrane. A gel retardation assay showed that the nanosized complexes formed between the copolymers and siRNA were stable even at a molar ratio of 1 : 2. The high cellular uptake (>80%) and localization of the copolymer vectors inside the cells were easily analyzed by tracking the fluorescence of polythiophene using fluorescent microscopy and cytometry. An in vitro luciferase knockdown (KD) assay in A549-luc cells demonstrated that the siRNA complexes with more hydrophobic copolymers achieved a higher KD efficiency of 52.8% without notable cytotoxicity, indicating protein-specific KD activity rather than solely the cytotoxicity of the materials. Our polythiophene copolymers should serve as novel, efficient, low cell toxicity, and label-free siRNA delivery systems. PMID:26539490

  19. Preparation of Novel Curdlan Nanoparticles for Intracellular siRNA Delivery

    PubMed Central

    Han, Jingfen; Caia, Jia; Borjihan, Wuyinga; Ganbolda, Tsogzolmaa; Rana, Tariq M.; Baigude, Huricha

    2014-01-01

    RNA interference (RNAi) down-regulates gene expression post-transcriptionally, which is a therapeutically significant phenomenon that could potentially reduce the level of disease related proteins that are undruggable by conventional small molecular approaches. However, clinical application of small interference RNAs (siRNAs) requires design of potent siRNA sequences and development of safe and efficient delivery systems. To create a biocompatible siRNA delivery agent, we chemically modified natural polysaccharide curdlan in a regioselective manner to introduce amino group in the glucose units. The resulting 6-amino-curdlan (6AC) is water soluble and forms nanoparticles upon complexing with siRNAs. The novel curdlan-based nanoparticles efficiently delivered siRNAs to human cancer cells and mouse primary cells, and reduced 70–90% of target mRNA level. Moreover, 6AC nanoparticles delivered siRNA targeting eGFP to mouse embryonic stem (mES) cells stably expressing eGFP, and produced substantial reductions of eGFP protein level. The novel curdlan-based nanoparticle is a promising vehicle for delivery of short RNAs to knock down endogenous mRNAs. PMID:25498642

  20. New Type of BACE1 siRNA Delivery to Cells

    PubMed Central

    Jabłkowski, Maciej; Szemraj, Maciej; Oszajca, Katarzyna; Janiszewska, Grażyna; Bartkowiak, Jacek; Szemraj, Janusz

    2014-01-01

    Background Small interfering RNA (siRNA) gene therapy is a new molecular approach in the search for an efficient therapy for Alzheimer disease (AD), based on the principle of RNA interference. Reducing BACE activity can have great therapeutic potential for the treatment of AD. In this study, receptor-mediated delivery was used to deliver opioid peptide-conjugated BACE 1 to INR-32 human neuroblastoma cells. Material/Methods An INR-32 human neuroblastoma cell line was stably transfected to express the APP cDNA coding fragment containing the predicted sites for cleavage by α, β, or γ-secretase. This was then treated with BACE 1 siRNA to silence BACE gene expression. BACE gene transcription and translation was determined using BACE-1 siRNA cross-linked with opioid peptide, together with RT-PCR, Western blot analysis, and ELISA. Results Receptor-mediated delivery was used to introduce BACE1 siRNA to the APP – INR 32 human neuroblastoma cells. Decreased BACE mRNA and protein expression were observed after the cells were transfected with BACE1 siRNA. Conclusions Delivery of BACE1 siRNA appears to specifically reduce the cleavage of APP by inhibiting BACE1 activity. PMID:25491230

  1. Accurate abundance determinations in S stars

    NASA Astrophysics Data System (ADS)

    Neyskens, P.; Van Eck, S.; Plez, B.; Goriely, S.; Siess, L.; Jorissen, A.

    2011-12-01

    S-type stars are thought to be the first objects, during their evolution on the asymptotic giant branch (AGB), to experience s-process nucleosynthesis and third dredge-ups, and therefore to exhibit s-process signatures in their atmospheres. Until present, the modeling of these processes is subject to large uncertainties. Precise abundance determinations in S stars are of extreme importance for constraining e.g., the depth and the formation of the 13C pocket. In this paper a large grid of MARCS model atmospheres for S stars is used to derive precise abundances of key s-process elements and iron. A first estimation of the atmospheric parameters is obtained using a set of well-chosen photometric and spectroscopic indices for selecting the best model atmosphere of each S star. Abundances are derived from spectral line synthesis, using the selected model atmosphere. Special interest is paid to technetium, an element without stable isotopes. Its detection in stars is considered as the best possible signature that the star effectively populates the thermally-pulsing AGB (TP-AGB) phase of evolution. The derived Tc/Zr abundances are compared, as a function of the derived [Zr/Fe] overabundances, with AGB stellar model predictions. The computed [Zr/Fe] overabundances are in good agreement with the AGB stellar evolution model predictions, while the Tc/Zr abundances are slightly over-predicted. This discrepancy can help to set stronger constraints on nucleosynthesis and mixing mechanisms in AGB stars.

  2. Relative Abundance Measurements in Plumes and Interplumes

    NASA Astrophysics Data System (ADS)

    Guennou, C.; Hahn, M.; Savin, D. W.

    2015-07-01

    We present measurements of relative elemental abundances in plumes and interplumes. Plumes are bright, narrow structures in coronal holes that extend along open magnetic field lines far out into the corona. Previous work has found that in some coronal structures the abundances of elements with a low first ionization potential (FIP) <10 eV are enhanced relative to their photospheric abundances. This coronal-to-photospheric abundance ratio, commonly called the FIP bias, is typically 1 for elements with a high-FIP (>10 eV). We have used Extreme Ultraviolet Imaging Spectrometer observations made on 2007 March 13 and 14 over a ≈24 hr period to characterize abundance variations in plumes and interplumes. To assess their elemental composition, we used a differential emission measure analysis, which accounts for the thermal structure of the observed plasma. We used lines from ions of iron, silicon, and sulfur. From these we estimated the ratio of the iron and silicon FIP bias relative to that for sulfur. From the results, we have created FIP-bias-ratio maps. We find that the FIP-bias ratio is sometimes higher in plumes than in interplumes and that this enhancement can be time dependent. These results may help to identify whether plumes or interplumes contribute to the fast solar wind observed in situ and may also provide constraints on the formation and heating mechanisms of plumes.

  3. Surface abundances of ON stars

    NASA Astrophysics Data System (ADS)

    Martins, F.; Simón-Díaz, S.; Palacios, A.; Howarth, I.; Georgy, C.; Walborn, N. R.; Bouret, J.-C.; Barbá, R.

    2015-06-01

    Context. Massive stars burn hydrogen through the CNO cycle during most of their evolution. When mixing is efficient or when mass transfer in binary systems occurs, chemically processed material is observed at the surface of O and B stars. Aims: ON stars show stronger lines of nitrogen than morphologically normal counterparts. Whether this corresponds to the presence of material processed through the CNO cycle is not known. Our goal is to answer this question. Methods: We performed a spectroscopic analysis of a sample of ON stars with atmosphere models. We determined the fundamental parameters as well as the He, C, N, and O surface abundances. We also measured the projected rotational velocities. We compared the properties of the ON stars to those of normal O stars. Results: We show that ON stars are usually rich in helium. Their CNO surface abundances are fully consistent with predictions of nucleosynthesis. ON stars are more chemically evolved and rotate - on average - faster than normal O stars. Evolutionary models including rotation cannot account for the extreme enrichment observed among ON main sequence stars. Some ON stars are members of binary systems, but others are single stars as indicated by stable radial velocities. Mass transfer is therefore not a simple explanation for the observed chemical properties. Conclusions: We conclude that ON stars show extreme chemical enrichment at their surface, consistent with nucleosynthesis through the CNO cycle. Its origin is not clear at present. Based on observations obtained 1) at the Anglo-Australian Telescope; 2) at the Canada-France-Hawaii Telescope (CFHT), which is operated by the National Research Council (NRC) of Canada, the Institut National des Science de l'Univers of the Centre National de la Recherche Scientifique (CNRS) of France, and the University of Hawaii; 3) at the ESO/La Silla Observatory under programs 081.D-2008, 083.D-0589, 086.D-0997; 4) the Nordic Optical Telescope, operated on the island of La

  4. Lipid-based nanoparticles for siRNA delivery in cancer therapy: paradigms and challenges.

    PubMed

    Gomes-da-Silva, Lígia C; Fonseca, Nuno A; Moura, Vera; Pedroso de Lima, Maria C; Simões, Sérgio; Moreira, João N

    2012-07-17

    RNA interference (RNAi) is a specific gene-silencing mechanism that can be mediated by the delivery of chemical synthesized small-interfering RNA (siRNA). RNAi might constitute a novel therapeutic approach for cancer treatment because researchers can easily design siRNA molecules to inhibit, specifically and potently, the expression of any protein involved in tumor initiation and progression. Despite all the potential of siRNA as a novel class of drugs, the limited cellular uptake, low biological stability, and unfavorable pharmacokinetics of siRNAs have limited their application in the clinic. Indeed, blood nucleases easily degrade naked siRNAs, and the kidneys rapidly eliminate these molecules. Furthermore, at the level of target cells, the negative charge and hydrophilicity of siRNAs strongly impair their cellular internalization. Therefore, the translation of siRNA to the clinical setting is highly dependent on the development of an appropriate delivery system, able to ameliorate siRNA pharmacokinetic and biodistribution properties. In this regard, major advances have been achieved with lipid-based nanocarriers sterically stabilized by poly(ethylene glycol) (PEG), such as the stabilized nucleic acid lipid particles (SNALP). However, PEG has not solved all the major problems associated with siRNA delivery. In this Account, the major problems associated with PEGylated lipid-based nanoparticles, and the different strategies to overcome them are discussed. Although PEG has revolutionized the field of nanocarriers, cumulative experience has revealed that upon repeated administration, PEGylated liposomes lose their ability to circulate over long periods in the bloodstream, a phenomenon known as accelerated blood clearance. In addition, PEGylation impairs the internalization of the siRNA into the target cell and its subsequent escape from the endocytic pathway, which reduces biological activity. An interesting approach to overcome such limitations relies on the design

  5. Structural Dynamics of Human Argonaute2 and Its Interaction with siRNAs Designed to Target Mutant tdp43

    PubMed Central

    Bhandare, Vishwambhar

    2016-01-01

    The human Argonaute2 protein (Ago2) is a key player in RNA interference pathway and small RNA recognition by Ago2 is the crucial step in siRNA mediated gene silencing mechanism. The present study highlights the structural and functional dynamics of human Ago2 and the interaction mechanism of Ago2 with a set of seven siRNAs for the first time. The human Ago2 protein adopts two conformations such as “open” and “close” during the simulation of 25 ns. One of the domains named as PAZ, which is responsible for anchoring the 3′-end of siRNA guide strand, is observed as a highly flexible region. The interaction between Ago2 and siRNA, analyzed using a set of siRNAs (targeting at positions 128, 251, 341, 383, 537, 1113, and 1115 of mRNA) designed to target tdp43 mutants causing Amyotrophic Lateral Sclerosis (ALS) disease, revealed the stable and strong recognition of siRNA by the Ago2 protein during dynamics. Among the studied siRNAs, the siRNA341 is identified as a potent siRNA to recognize Ago2 and hence could be used further as a possible siRNA candidate to target the mutant tdp43 protein for the treatment of ALS patients. PMID:27110240

  6. Solar abundance of iridium

    PubMed Central

    Drake, Stephen; Aller, Lawrence H.

    1976-01-01

    By a method of spectrum synthesis, which yields log gfA, where g is the statistical weight of the lower level, f is the oscillator strength, and A is the abundance, an attempt is made to deduce the solar iridium abundance from one relatively unblended, but fairly weak IrI line, λ 3220.78 Å. If the Corliss-Bozman f-value for this line is adopted, we find log A(Ir) = 0.82 on the scale log A(H) = 12.00. The discordance with the value found from carbonaceous chondrites may arise from faulty f-values or from difficulties arising from line blending in this far ultraviolet domain of the solar spectrum. PMID:16578735

  7. Potential application of injectable chitosan hydrogel treated with siRNA in chronic rhinosinusitis therapy

    PubMed Central

    CAO, CHENG; YAN, CHUNHONG; HU, ZHIQIANG; ZHOU, SHAO

    2015-01-01

    Chronic rhinosinusitis is a condition with severe clinical symptoms and limited therapeutic solutions. It has been reported that vascular endothelial growth factor (VEGF) can promote nasal epithelial cell growth and result in hyperplasia of the sinuses. Therefore, the downregulation of VEGF may inhibit the process of hyperplasia. In the present study, small interfering RNA (siRNA) targeting VEGF was used to silence the expression of VEGF, and injectable chitosan based hydrogel, which is suitable for sinus injection and exhibits long-term retention, was prepared as the siRNA carrier. Human bronchial epithelial cells were cultured directly on the hydrogel to observe the biological performance in vitro. Further in vivo effects were investigated by the injection of the hydrogel into the sinus cavity. Following the introduction of siRNA introducing, the expression of VEGF in the bronchial epithelial cells was significantly suppressed at mRNA and protein levels. The number of living cells on the gel was significantly decreased, thus resulting in the inhibition of proliferation. However, the cytoskeletal arrangement of the remaining cells were not affected substantially. The hydrogel was able to retain the siRNA for an extended duration, which enabled a sustained supply of siRNA. The in vivo sinus mucosa analysis revealed that the siRNA was able to collocate with cells and the mucosa thickness was substantially decreased. In conclusion, the results of the present study suggested that injectable chitosan based hydrogel, treated with siRNA targeting VEGF, may be used as a convenient therapeutic option for chronic rhinosinusitis. PMID:26299569

  8. Magnetic nanoparticle and magnetic field assisted siRNA delivery in vitro.

    PubMed

    Mykhaylyk, Olga; Sanchez-Antequera, Yolanda; Vlaskou, Dialechti; Cerda, Maria Belen; Bokharaei, Mehrdad; Hammerschmid, Edelburga; Anton, Martina; Plank, Christian

    2015-01-01

    This chapter describes how to design and conduct experiments to deliver siRNA to adherent cell cultures in vitro by magnetic force-assisted transfection using self-assembled complexes of small interfering RNA (siRNA) and cationic lipids or polymers that are associated with magnetic nanoparticles (MNPs). These magnetic complexes are targeted to the cell surface by the application of a gradient magnetic field. A further development of the magnetic drug-targeting concept is combining it with an ultrasound-triggered delivery using magnetic microbubbles as a carrier for gene or drug delivery. For this purpose, selected MNPs, phospholipids, and siRNAs are assembled in the presence of perfluorocarbon gas into flexible formulations of magnetic lipospheres (microbubbles). Methods are described how to accomplish the synthesis of magnetic nanoparticles for magnetofection and how to test the association of siRNA with the magnetic components of the transfection vector. A simple method is described to evaluate magnetic responsiveness of the magnetic siRNA transfection complexes and estimate the complex loading with magnetic nanoparticles. Procedures are provided for the preparation of magnetic lipoplexes and polyplexes of siRNA as well as magnetic microbubbles for magnetofection and downregulation of the target gene expression analysis with account for the toxicity determined using an MTT-based respiration activity test. A modification of the magnetic transfection triplexes with INF-7, fusogenic peptide, is described resulting in reporter gene silencing improvement in HeLa, Caco-2, and ARPE-19 cells. The methods described can also be useful for screening vector compositions and novel magnetic nanoparticle preparations for optimized siRNA transfection by magnetofection in any cell type. PMID:25319646

  9. Fatty acid modified octa-arginine for delivery of siRNA.

    PubMed

    Li, Yuhuan; Li, Yujing; Wang, Xinmei; Lee, Robert J; Teng, Lesheng

    2015-11-10

    Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Four fatty acids derivatives of octa-arginine (R8) were synthesized and evaluated for the delivery of siRNA into hepatocellular carcinoma Hep G2 and human lung adenocarcinoma A549 cells. The results showed that the long chain acid oleic acid or stearic acid derivatives of R8, OA-R8 and StA-R8, were more efficient in siRNA complexation and form nanoparticles with greater stability compared to the native R8. Cellular uptake of fluorescence-labeled siRNA delivered by OA-R8 and StA-R8 in Hep G2 and A549 cells was substantially 40-50 times higher than unmodified R8. A significant reduction in siRNA cellular uptake was observed in the presence of sucrose and cytochalasin D, indicating endocytosis as a primary mechanism of cellular entry. A survivin siRNA was used to prepare nanoparticles with OA-R8 or StA-R8 and evaluated for silencing of survivin mRNA and protein in A549 cells, and the inhibition efficiencies of survivin protein reached to 50.3% and 54.6%, respectively. The results showed greater effectiveness with the derivatized R8. Taken together, these findings showed that long chain fatty acid derivatives of R8 are efficient delivery agents for siRNA and may facilitate its therapeutic application. PMID:26386137

  10. Tau silencing by siRNA in the P301S mouse model of tauopathy.

    PubMed

    Xu, Hong; Rösler, Thomas W; Carlsson, Thomas; de Andrade, Anderson; Fiala, Ondrej; Hollerhage, Matthias; Oertel, Wolfgang H; Goedert, Michel; Aigner, Achim; Höglinger, Gunter U

    2014-01-01

    Suppression of tau protein expression has been shown to improve behavioral deficits in mouse models of tauopathies, offering an attractive therapeutic approach. Experimentally this had been achieved by switching off the promoters controlling the transgenic human tau gene (MAPT), which is not possible in human patients. The aim of the present study was therefore to evaluate the effectiveness of small interfering RNAs (siRNAs) and their cerebral delivery to suppress human tau expression in vivo, which might be a therapeutic option for human tauopathies. We used primary cortical neurons of transgenic mice expressing P301S-mutated human tau and Lund human mesencephalic (LUHMES) cells to validate the suppressive effect of siRNA in vitro. For measuring the effect in vivo, we stereotactically injected siRNA into the brains of P301S mice to reveal the suppression of tau by immunochemistry (AT180, MC1, and CP13 antibodies). We found that the Accell™ SMART pool siRNA against MAPT can effectively suppress tau expression in vitro and in vivo without a specific delivery agent. The siRNA showed a moderate distribution in the hippocampus of mice after single injection. NeuN, GFAP, Iba-1, MHC II immunoreactivities and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed neither signs of neurotoxicity or neuroinflammation nor apoptosis when MAPT siRNA is present in the hippocampus. Our data suggest that siRNA against MAPT can serve as a potential tool for gene therapy in tauopathies. PMID:25687501

  11. Plant siRNAs from introns mediate DNA methylation of host genes.

    PubMed

    Chen, Dijun; Meng, Yijun; Yuan, Chunhui; Bai, Lin; Huang, Donglin; Lv, Shaolei; Wu, Ping; Chen, Ling-Ling; Chen, Ming

    2011-06-01

    Small RNAs (sRNAs), largely known as microRNAs (miRNAs) and short interfering RNAs (siRNAs), emerged as the critical components of genetic and epigenetic regulation in eukaryotic genomes. In animals, a sizable portion of miRNAs reside within the introns of protein-coding genes, designated as mirtron genes. Recently, high-throughput sequencing (HTS) revealed a huge amount of sRNAs that derived from introns in plants, such as the monocot rice (Oryza sativa). However, the biogenesis and the biological functions of this kind of sRNAs remain elusive. Here, we performed a genome-scale survey of intron-derived sRNAs in rice based on HTS data. Several introns were found to have great potential to form internal hairpin structures, and the short hairpins could generate miRNAs while the larger ones could produce siRNAs. Furthermore, 22 introns, termed "sirtrons," were identified from the rice protein-coding genes. The single-stranded sirtrons produced a diverse set of siRNAs from long hairpin structures. These sirtron-derived siRNAs are dominantly 21 nt, 22 nt, and 24 nt in length, whose production relied on DCL4, DCL2, and DCL3, respectively. We also observed a strong tendency for the sirtron-derived siRNAs to be coexpressed with their host genes. Finally, the 24-nt siRNAs incorporated with Argonaute 4 (AGO4) could direct DNA methylation on their host genes. In this regard, homeostatic self-regulation between intron-derived siRNAs and their host genes was proposed. PMID:21518803

  12. Expression data of HeLa cells treated with CENP-E siRNA or Eg5 siRNA in the presence of BubR1 siRNA.

    PubMed

    Nakayama, Yusuke; Ohashi, Akihiro

    2015-12-01

    The molecular mechanism responsible for cell fate after mitotic slippage is unclear. We investigated the postmitotic effects of different mitotic aberrations (Ohashi et al. [1]), misaligned chromosomes produced by CENP-E siRNA (siCENP-E), and monopolar spindles resulting from Eg5 siRNA (siEg5) (Miki et al. [2]). To determine which signaling pathways contribute to the postmitotic effect of siCENP-E in the presence of siBubR1 (siCENP-E + siBubR1) compared with siEg5 + siBubR1, we performed comprehensive gene expression analysis using microarray comparisons [1]. The microarray data have been deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE67905. PMID:26697328

  13. Highly efficient siRNA delivery system into human and murine cells using single-wall carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Ladeira, M. S.; Andrade, V. A.; Gomes, E. R. M.; Aguiar, C. J.; Moraes, E. R.; Soares, J. S.; Silva, E. E.; Lacerda, R. G.; Ladeira, L. O.; Jorio, A.; Lima, P.; Leite, M. Fatima; Resende, R. R.; Guatimosim, S.

    2010-09-01

    Development of RNA interference (RNAi) technology utilizing short interfering RNA sequences (siRNA) has focused on creating methods for delivering siRNAs to cells and for enhancing siRNA stability in vitro and in vivo. Here, we describe a novel approach for siRNA cellular delivery using siRNA coiling into carboxyl-functionalized single-wall carbon nanotubes (SWCNTs). The CNT-siRNA delivery system successfully demonstrates nonspecific toxicity and transfection efficiency greater than 95%. This approach offers the potential for siRNA delivery into different types of cells, including hard-to-transfect cells, such as neuronal cells and cardiomyocytes. We also tested the CNT-siRNA system in a non-metastatic human hepatocellular carcinoma cell line (SKHep1). In all types of cells used in this work the CNT-siRNA delivery system showed high efficiency and apparent no side effects for various in vitro applications.

  14. The Origin of Element Abundance Variations in Solar Energetic Particles

    NASA Astrophysics Data System (ADS)

    Reames, Donald V.

    2016-08-01

    Abundance enhancements, during acceleration and transport in both gradual and impulsive solar energetic particle (SEP) events, vary approximately as power laws in the mass-to-charge ratio [A/Q] of the ions. Since the Q-values depend upon the electron temperature of the source plasma, this has allowed a determination of this temperature from the pattern of element-abundance enhancements and a verification of the expected inverse-time dependence of the power of A/Q for diffusive transport of ions from the SEP events, with scattering mean free paths found to be between 0.2 and 1 AU. SEP events derived from plasma of different temperatures map into different regions in typical cross-plots of abundances, spreading the distributions. In comparisons of SEP events with temperatures above 2 MK, impulsive events show much broader non-thermal variation of abundances than do gradual events. The extensive shock waves accelerating ions in gradual events may average over much of an active region where numerous but smaller magnetic reconnections, "nanojets", produce suprathermal seed ions, thus averaging over varying abundances, while an impulsive SEP event only samples one local region of abundance variations. Evidence for a reference He/O-abundance ratio of 91, rather than 57, is also found for the hotter plasma. However, while this is similar to the solar-wind abundance of He/O, the solar-wind abundances otherwise provide an unacceptably poor reference for the SEP-abundance enhancements, generating extremely large errors.

  15. The Origin of Element Abundance Variations in Solar Energetic Particles

    NASA Astrophysics Data System (ADS)

    Reames, Donald V.

    2016-07-01

    Abundance enhancements, during acceleration and transport in both gradual and impulsive solar energetic particle (SEP) events, vary approximately as power laws in the mass-to-charge ratio [ A/Q] of the ions. Since the Q-values depend upon the electron temperature of the source plasma, this has allowed a determination of this temperature from the pattern of element-abundance enhancements and a verification of the expected inverse-time dependence of the power of A/Q for diffusive transport of ions from the SEP events, with scattering mean free paths found to be between 0.2 and 1 AU. SEP events derived from plasma of different temperatures map into different regions in typical cross-plots of abundances, spreading the distributions. In comparisons of SEP events with temperatures above 2 MK, impulsive events show much broader non-thermal variation of abundances than do gradual events. The extensive shock waves accelerating ions in gradual events may average over much of an active region where numerous but smaller magnetic reconnections, "nanojets", produce suprathermal seed ions, thus averaging over varying abundances, while an impulsive SEP event only samples one local region of abundance variations. Evidence for a reference He/O-abundance ratio of 91, rather than 57, is also found for the hotter plasma. However, while this is similar to the solar-wind abundance of He/O, the solar-wind abundances otherwise provide an unacceptably poor reference for the SEP-abundance enhancements, generating extremely large errors.

  16. Unique Gene-Silencing and Structural Properties of 2;#8242;-Fluoro-Modified siRNAs

    SciTech Connect

    Manoharan, Muthiah; Akinc, Akin; Pandey, Rajendra K.; Qin, June; Hadwiger, Philipp; John, Matthias; Mills, Kathy; Charisse, Klaus; Maier, Martin A.; Nechev, Lubomir; Greene, Emily M.; Pallan, Pradeep S.; Rozners, Eriks; Rajeev, Kallanthottathil G.; Egli, Martin

    2015-10-15

    With little or no negative impact on the activity of small interfering RNAs (siRNAs), regardless of the number of modifications or the positions within the strand, the 2'-deoxy-2'-fluoro (2'-F) modification is unique. Furthermore, the 2'-F-modified siRNA (see crystal structure) was thermodynamically more stable and more nuclease-resistant than the parent siRNA, and produced no immunostimulatory response.

  17. Hapten-Binding Bispecific Antibodies for the Targeted Delivery of SiRNA and SiRNA-Containing Nanoparticles.

    PubMed

    Thorey, Irmgard S; Grote, Michael; Mayer, Klaus; Brinkmann, Ulrich

    2016-01-01

    Hapten-binding bispecific antibodies (bsAbs) are effective and versatile tools for targeting diverse payloads, including siRNAs, to specific cells and tissues. In this chapter, we provide examples for successful SiRNA delivery using this powerful targeting platform. We further provide protocols for designing and producing bsAbs, for combining bsAbs with SiRNA into functional complexes, and achieving specific mRNA knockdown in cells by using these functional complexes. PMID:26472454

  18. Antibody-Mediated Targeting of siRNA Via the Human Insulin Receptor Using Avidin-Biotin Technology

    PubMed Central

    Xia, Chun-Fang; Boado, Ruben J.; Pardridge, William M.

    2013-01-01

    Delivery of short interfering RNA (siRNA) to cells in culture, and in vivo, is possible with combined use of a receptor-specific monoclonal antibody (MAb) and avidin-biotin technology. In the present studies, the luciferase gene is transiently expressed in human 293 epithelial cells. The siRNA delivery system is comprised of the siRNA, mono-biotinylated on the 3′-terminus of the sense strand, and a conjugate of streptavidin (SA) and a MAb to the human insulin receptor (HIR). Exposure of cells to 3′-biotinyl-siRNA bound to the HIRMAb/SA conjugate, but not to unconjugated SA, avidin, or the HIRMAb, causes a >90% reduction in luciferase gene expression. The receptor-targeted siRNA effect is maximal at 48 hours after delivery of the siRNA to the cells, and the effect is lost by 7 days after a single application of the targeted siRNA in culture. The KI of the receptor-targeted siRNA inhibition of gene expresssion is 30.5 ± 11.7 nM, and significant inhibition is observed with siRNA concentrations as low as 3 nM. In conclusion, the combination of a receptor-specific targeting ligand, such as the HIRMAb, and avidin-biotin technology, allows for high affinity capture of the mono-biotinylated siRNA by the targeting MAb. The siRNA is effectively delivered to the cytosol of cells and knockdown of gene expression with the HIRMAb/SA delivery system is comparable to RNA interference effects obtained with cationic polyplexes. Whereas the use of cationic polyplexes in vivo is problematic, the bond between the targeting MAb and the siRNA is stable with avidin-biotin technology, and RNAi effects at distant sites such as brain are observed in vivo following an intravenous administration of the targeted siRNA. PMID:19093871

  19. Efficient inhibition of fibroblast proliferation and collagen expression by ERK2 siRNAs

    SciTech Connect

    Li, Fengfeng; Fan, Cunyi; Cheng, Tao; Jiang, Chaoyin; Zeng, Bingfang

    2009-05-01

    Transforming growth factor-{beta}1 and fibroblast growth factor-2 play very important roles in fibroblast proliferation and collagen expression. These processes lead to the formation of joint adhesions through the SMAD and MAPK pathways, in which ERK2 is supposed to be crucial. Based on these assumptions, lentivirus (LV)-mediated small interfering RNAs (siRNAs) targeting ERK2 were used to suppress the proliferation and collagen expression of rat joint adhesion tissue fibroblasts (RJATFs). Among four siRNAs examined, siRNA1 caused an 84% reduction in ERK2 expression (p < 0.01) and was selected as the most efficient siRNA for use in this study. In subsequent experiments, significant downregulation of types I and III collagen were observed by quantitative RT-PCR and Western blot analyses. MTT assays and flow cytometry revealed marked inhibition of RJATF proliferation, but no apoptosis. In conclusion, LV-mediated ERK2 siRNAs may represent novel therapies or drug targets for preventing joint adhesion formation.

  20. Glycerol monooleate-based nanocarriers for siRNA delivery in vitro.

    PubMed

    Zhen, Guoliang; Hinton, Tracey M; Muir, Benjamin W; Shi, Shuning; Tizard, Mark; McLean, Keith M; Hartley, Patrick G; Gunatillake, Pathiraja

    2012-09-01

    We present studies of the delivery of short interfering ribonucleic acid (siRNA) into a green fluorescent protein (GFP) expressing cell line, using lipid nanocarriers in cubic lyotropic liquid crystal form. These carriers are based on glycerol monooleate (GMO) and employ the use of varying concentrations of cationic siRNA binding lipids. The essential physicochemical parameters of the cationic lipid/GMO/siRNA complexes such as particle size, ζ otential, siRNA uptake stability, lyotropic mesophase behavior, cytotoxicity,and gene silencing efficiency were systematically assessed. We find that the lipid nanocarriers were effectively taken up by mammalian cells and that their siRNA payload was able to induce gene silencing in vitro. More importantly, it was found that the nonlamellar structure of some of the lipid nanocarrier formulations were more effective at gene silencing than their lamellar structured counterparts. The development of cationic lipid functionalized nonlamellar GMO-based nanostructured nanoparticles may lead to improved siRNA delivery vehicles. PMID:22794355

  1. PEGylation of 6-amino-6-deoxy-curdlan for efficient in vivo siRNA delivery.

    PubMed

    Altangerel, Altanzul; Cai, Jia; Liu, Lixia; Wu, Yinga; Baigude, Huricha; Han, Jingfen

    2016-05-01

    RNA interference (RNAi) is an evolutionarily conserved gene-silencing phenomenon that shows great promise for developing new therapies. However, the development of small interfering RNA (siRNA)-based therapies need to establish efficient delivery system that silences target genes with siRNA doses that is clinically feasible in humans. Here we report synthesis and in vivo study of a novel PEGylated curdlan-based nanoparticle, designated as 6AC-100PEG, obtained by conjugation of mPEG 2000 to 6-amino-6-deoxy-curdlan. The complex of siRNA/6AC-100PEG showed homogenous nanoparticles with an average diameter of 200nm. MTT assay indicated that 6AC-100PEG does not have apparent cytotoxicity. Systemic administration of a complex of siapoB/6AC-100PEG significantly reduced the level of apoB mRNA in mouse liver, indicating that 6AC-100PEG can efficiently deliver siRNA to mouse liver and induce RNAi. Administration of siRNA/6AC-100PEG to mouse did not elevate liver enzyme level in the serum, indicating that 6AC-100PEG nanoparticle is a promising in vivo siRNA delivery agent. PMID:26877000

  2. Topical and Targeted Delivery of siRNAs to Melanoma Cells Using a Fusion Peptide Carrier

    PubMed Central

    Ruan, Renquan; Chen, Ming; Sun, Sijie; Wei, Pengfei; Zou, Lili; Liu, Jing; Gao, Dayong; Wen, Longping; Ding, Weiping

    2016-01-01

    Topical application of siRNAs through the skin is a potentially effective strategy for the treatment of melanoma tumors. In this study, we designed a new and safe fusion peptide carrier SPACE-EGF to improve the skin and cell penetration function of the siRNAs and their targeting ability to B16 cells, such that the apoptosis of B16 cells can be induced. The results show that the carrier is stable and less toxic. The EGF motif does not affect the skin and cell penetration function of the SPACE. Because EGF can strongly bind EGFR, which is overexpressed in cancer cells, the targeting ability of the SPACE-EGF-siRNA complex is increased. In vitro experiments indicate that GAPDH siRNAs conjugated with SPACE-EGF can significantly reduce the GAPDH concentration in B16 cells, and c-Myc siRNAs can cause the gene silencing of c-Myc and thus the apoptosis of cells. In vivo experiments show that the topical application of c-Myc siRNAs delivered by SPACE-EGF through the skin can significantly inhibit the growth of melanoma tumors. This work may provide insight into the development of new transdermal drug carriers to treat a variety of skin disorders. PMID:27374619

  3. Fluorescence imaging of siRNA delivery by peptide nucleic acid-based probe.

    PubMed

    Sato, Takaya; Sato, Yusuke; Iwai, Kenta; Kuge, Shusuke; Teramae, Norio; Nishizawa, Seiichi

    2015-01-01

    We report on the use of a peptide nucleic acid (PNA)-based fluorescent probe for the analysis of siRNA delivery to living cells. The probe, Py-AA-TO, possesses thiazole orange (TO) and pyrene moieties in the C- and N-termini of PNA, and can function as a light-up probe capable of selective binding to 3'-overhanging nucleotides of target siRNAs. The affinity-labeling of the siRNAs with Py-AA-TO facilitates fluorescence imaging of cellular uptake of polymer-based carriers encapsulating the siRNAs (polyplexes) through endocytosis and subsequent sequestration into lysosome. In addition, flow cytometric measurements reveal that the monitoring of Py-AA-TO fluorescence inside the cells is successfully applicable to the analysis of the polyplex disassembly. These promising functions of Py-AA-TO are presented and discussed as a basis for the design of molecular probes for fluorescent imaging and quantitative analysis of the siRNA delivery process. PMID:25864675

  4. Promoting siRNA delivery via enhanced cellular uptake using an arginine-decorated amphiphilic dendrimer

    NASA Astrophysics Data System (ADS)

    Liu, Xiaoxuan; Liu, Cheng; Zhou, Jiehua; Chen, Chao; Qu, Fanqi; Rossi, John J.; Rocchi, Palma; Peng, Ling

    2015-02-01

    RNA interference (RNAi) with small interfering RNA (siRNA) is expected to offer an attractive means to specifically and efficiently silence disease-associated genes for treating various diseases provided that safe and efficient delivery systems are available. In this study, we have established an arginine-decorated amphiphilic dendrimer composed of a hydrophobic alkyl chain and a hydrophilic PAMAM dendron bearing arginine terminals as nonviral vector for siRNA delivery. Indeed, this dendrimer proved to be very effective at delivering siRNAs in human prostate cancer PC-3 cells and in human hematopoietic CD34+ stem cells, leading to improved gene silencing compared to the corresponding nonarginine decorated dendrimer. Further investigation confirmed that this dendrimer was granted with the capacity to form stable nanoparticles with siRNA and significantly enhance cellular uptake of siRNA. In addition, this dendrimer revealed no discernible cytotoxicity. All these findings demonstrate that decoration of the dendrimer surface with arginine residues is indeed a useful strategy to improve the delivery ability of dendrimers.

  5. Synthesis of folate-functionalized RAFT polymers for targeted siRNA delivery

    PubMed Central

    Srinivasan, Selvi; Shubin, Andrew D.; Stayton, Patrick S.

    2011-01-01

    Receptor-mediated, cell-specific delivery of siRNA enables silencing of target genes in specific tissues, opening the door to powerful therapeutic options for a multitude of diseases. However, development of delivery systems capable of targeted and effective siRNA delivery typically requires multiple steps and use of sophisticated, orthogonal chemistries. Previously, we developed diblock copolymers consisting of dimethaminoethyl methacrylate-b-dimethylaminoethyl methacrylate-co-butyl methacrylate-copropylacrylic acid as potent siRNA delivery systems that protect siRNA from enzymatic degradation and enable its cytosolic delivery through pH-responsive, endosomolytic behavior.1,2 These architectures were polymerized using a living radical polymerization method, specifically reversible addition-fragmentation chain transfer (RAFT) polymerization, which employs a chain transfer agent (CTA) to modulate the rate of reaction, resulting in polymers with low polydispersity and telechelic chain ends reflecting the chemistry of the CTA. Here, we describe the straightforward, facile synthesis of a folate receptor-targeted diblock copolymer siRNA delivery system, as the folate receptor is an attractive target for tumor-selective therapies due to its overexpression in a number of cancers. Specifically, we detail the de novo synthesis of a folate-functionalized CTA, use the folate-CTA for controlled polymerizations of diblock copolymers, and demonstrate efficient, specific cellular folate receptor interaction and in vitro gene knockdown using the folate-functionalized polymer. PMID:21634800

  6. Antibody targeting facilitates effective intratumoral siRNA nanoparticle delivery to HER2-overexpressing cancer cells

    PubMed Central

    Palanca-Wessels, Maria C.; Booth, Garrett C.; Convertine, Anthony J.; Lundy, Brittany B.; Berguig, Geoffrey Y.; Press, Michael F.; Stayton, Patrick S.; Press, Oliver W.

    2016-01-01

    The therapeutic potential of RNA interference (RNAi) has been limited by inefficient delivery of short interfering RNA (siRNA). Tumor-specific recognition can be effectively achieved by antibodies directed against highly expressed cancer cell surface receptors. We investigated the utility of linking an internalizing streptavidin-conjugated HER2 antibody to an endosome-disruptive biotinylated polymeric nanocarrier to improve the functional cytoplasmic delivery of siRNA in breast and ovarian cancer cells in vitro and in an intraperitoneal ovarian cancer xenograft model in vivo, yielding an 80% reduction of target mRNA and protein levels with sustained repression for at least 96 hours. RNAi-mediated site specific cleavage of target mRNA was demonstrated using the 5′ RLM-RACE (RNA ligase mediated-rapid amplification of cDNA ends) assay. Mice bearing intraperitoneal human ovarian tumor xenografts demonstrated increased tumor accumulation of Cy5.5 fluorescently labeled siRNA and 70% target gene suppression after treatment with HER2 antibody-directed siRNA nanocarriers. Detection of the expected mRNA cleavage product by 5′ RLM-RACE assay confirmed that suppression occurs via the expected RNAi pathway. Delivery of siRNA via antibody-directed endosomolytic nanoparticles may be a promising strategy for cancer therapy. PMID:26840082

  7. Targeted delivery of siRNA to cell death proteins in sepsis.

    PubMed

    Brahmamdam, Pavan; Watanabe, Eizo; Unsinger, Jacqueline; Chang, Katherine C; Schierding, William; Hoekzema, Andrew S; Zhou, Tony T; McDonough, Jacquelyn S; Holemon, Heather; Heidel, Jeremy D; Coopersmith, Craig M; McDunn, Jonathan E; Hotchkiss, Richard S

    2009-08-01

    Immune suppression is a major cause of morbidity and mortality in the patients with sepsis. Apoptotic loss of immune effector cells such as CD4 T and B cells is a key component in the loss of immune competence in sepsis. Inhibition of lymphocyte apoptosis has led to improved survival in animal models of sepsis. Using quantitative real-time polymerase chain reaction of isolated splenic CD4 T and B cells, we determined that Bim and PUMA, two key cell death proteins, are markedly upregulated during sepsis. Lymphocytes have been notoriously difficult to transfect with small interfering RNA (siRNA). Consequently a novel, cyclodextrin polymer-based, transferrin receptor-targeted, delivery vehicle was used to coadminister siRNA to Bim and PUMA to mice immediately after cecal ligation and puncture. Antiapoptotic siRNA-based therapy markedly decreased lymphocyte apoptosis and prevented the loss of splenic CD4 T and B cells. Flow cytometry confirmed in vivo delivery of siRNA to CD4 T and B cells and also demonstrated decreases in intracellular Bim and PUMA protein. In conclusion, Bim and PUMA are two critical mediators of immune cell death in sepsis. Use of a novel cyclodextrin polymer-based, transferrin receptor-targeted siRNA delivery vehicle enables effective administration of antiapoptotic siRNAs to lymphocytes and reverses the immune cell depletion that is a hallmark of this highly lethal disorder. PMID:19033888

  8. Silencing Myostatin Using Cholesterol-conjugated siRNAs Induces Muscle Growth.

    PubMed

    Khan, Tayeba; Weber, Hans; DiMuzio, Jillian; Matter, Andrea; Dogdas, Belma; Shah, Tosha; Thankappan, Anil; Disa, Jyoti; Jadhav, Vasant; Lubbers, Laura; Sepp-Lorenzino, Laura; Strapps, Walter R; Tadin-Strapps, Marija

    2016-01-01

    Short interfering RNAs (siRNAs) are a valuable tool for gene silencing with applications in both target validation and therapeutics. Many advances have recently been made to improve potency and specificity, and reduce toxicity and immunostimulation. However, siRNA delivery to a variety of tissues remains an obstacle for this technology. To date, siRNA delivery to muscle has only been achieved by local administration or by methods with limited potential use in the clinic. We report systemic delivery of a highly chemically modified cholesterol-conjugated siRNA targeting muscle-specific gene myostatin (Mstn) to a full range of muscles in mice. Following a single intravenous injection, we observe 85-95% knockdown of Mstn mRNA in skeletal muscle and >65% reduction in circulating Mstn protein sustained for >21 days. This level of Mstn knockdown is also accompanied by a functional effect on skeletal muscle, with animals showing an increase in muscle mass, size, and strength. The cholesterol-conjugated siRNA platform described here could have major implications for treatment of a variety of muscle disorders, including muscular atrophic diseases, muscular dystrophy, and type II diabetes. PMID:27483025

  9. Kinetic analysis of the effects of target structure on siRNA efficiency

    NASA Astrophysics Data System (ADS)

    Chen, Jiawen; Zhang, Wenbing

    2012-12-01

    RNAi efficiency for target cleavage and protein expression is related to the target structure. Considering the RNA-induced silencing complex (RISC) as a multiple turnover enzyme, we investigated the effect of target mRNA structure on siRNA efficiency with kinetic analysis. The 4-step model was used to study the target cleavage kinetic process: hybridization nucleation at an accessible target site, RISC-mRNA hybrid elongation along with mRNA target structure melting, target cleavage, and enzyme reactivation. At this model, the terms accounting for the target accessibility, stability, and the seed and the nucleation site effects are all included. The results are in good agreement with that of experiments which show different arguments about the structure effects on siRNA efficiency. It shows that the siRNA efficiency is influenced by the integrated factors of target's accessibility, stability, and the seed effects. To study the off-target effects, a simple model of one siRNA binding to two mRNA targets was designed. By using this model, the possibility for diminishing the off-target effects by the concentration of siRNA was discussed.

  10. Co-delivery of siRNA and therapeutic agents using nanocarriers to overcome cancer resistance

    PubMed Central

    Creixell, Mar; Peppas, Nicholas A.

    2015-01-01

    Summary There are two main mechanisms by which cells become multidrug resistant (MDR): by increasing drug efflux pumps on the cell membrane and by increasing anti-apoptotic pathways. The use of nanotechnology to develop nanodelivery systems has allowed researchers to overcome limitations of antineoplastic drugs by increasing the solubility of the drug and decreasing the toxicity to healthy tissues. By encapsulating drugs into nanoparticles that bypass the efflux pumps, drug efflux is reduced, hence increasing the intracellular concentration of the drug. siRNA has the ability to disrupt cellular pathways by knocking down genes, opening the door to down regulating anti-apoptotic pathways. The use of nanocarriers to deliver siRNA, prevents both renal clearance and RNase degradation by protecting siRNA chains, increasing their half life in blood. It has been suggested that co-delivering drugs and siRNA together in the same delivery system would be more effective in overcoming resistance of cancer cells than co-treatment of cancer cells with delivery systems carrying either siRNA or drugs. In this study we discuss the progress of nanoscale co-delivery systems in overcoming multidrug cancer resistance. PMID:26257819

  11. Target-specific intracellular delivery of siRNA using degradable hyaluronic acid nanogels.

    PubMed

    Lee, Hyukjin; Mok, Hyejung; Lee, Soohyeon; Oh, Yu-Kyoung; Park, Tae Gwan

    2007-06-01

    Novel hyaluronic acid (HA) nanogels physically encapsulating small interfering RNA (siRNA) were fabricated by an inverse water-in-oil emulsion method. Thiol-conjugated HA dissolved in aqueous emulsion droplets was ultrasonically crosslinked via the formation of disulfide linkages to produce HA nanogels with a size distribution from 200 to 500 nm. Green fluorescence protein (GFP) siRNA was physically entrapped within the HA nanogels during the emulsion/crosslinking process. The HA/siRNA nanogels were readily taken up by HA receptor positive cells (HCT-116 cells) having HA-specific CD44 receptors on the surface. Release rates of siRNA from the HA nanogels could be modulated by changing the concentration of glutathione (GSH) in the buffer solution, indicating that the degradation/erosion of disulfide crosslinked HA nanogels, triggered by an intracellular reductive agent, controlled the release pattern of siRNA. When HA nanogels containing GFP siRNA were co-transfected with GFP plasmid/Lipofectamine to HCT-116 cells, a significant extent of GFP gene silencing was observed in both serum and non-serum conditions. The gene silencing effect was reduced in the presence of free HA in the transfection medium, revealing that HA nanogels were selectively taken up by HCT-116 cells via receptor mediated endocytosis. PMID:17408798

  12. Late embryogenesis abundant proteins

    PubMed Central

    Olvera-Carrillo, Yadira; Reyes, José Luis

    2011-01-01

    Late Embryogenesis Abundant (LEA) proteins accumulate at the onset of seed desiccation and in response to water deficit in vegetative plant tissues. The typical LEA proteins are highly hydrophilic and intrinsically unstructured. They have been classified in different families, each one showing distinctive conserved motifs. In this manuscript we present and discuss some of the recent findings regarding their role in plant adaptation to water deficit, as well as those concerning to their possible function, and how it can be related to their intrinsic structural flexibility. PMID:21447997

  13. Genome-wide analysis of single non-templated nucleotides in plant endogenous siRNAs and miRNAs.

    PubMed

    Wang, Feng; Johnson, Nathan R; Coruh, Ceyda; Axtell, Michael J

    2016-09-01

    Plant small RNAs are subject to various modifications. Previous reports revealed widespread 3' modifications (truncations and non-templated tailing) of plant miRNAs when the 2'-O-methyltransferase HEN1 is absent. However, non-templated nucleotides in plant heterochromatic siRNAs have not been deeply studied, especially in wild-type plants. We systematically studied non-templated nucleotide patterns in plant small RNAs by analyzing small RNA sequencing libraries from Arabidopsis, tomato, Medicago, rice, maize and Physcomitrella Elevated rates of non-templated nucleotides were observed at the 3' ends of both miRNAs and endogenous siRNAs from wild-type specimens of all species. 'Off-sized' small RNAs, such as 25 and 23 nt siRNAs arising from loci dominated by 24 nt siRNAs, often had very high rates of 3'-non-templated nucleotides. The same pattern was observed in all species that we studied. Further analysis of 24 nt siRNA clusters in Arabidopsis revealed distinct patterns of 3'-non-templated nucleotides of 23 nt siRNAs arising from heterochromatic siRNA loci. This pattern of non-templated 3' nucleotides on 23 nt siRNAs is not affected by loss of known small RNA 3'-end modifying enzymes, and may result from modifications added to longer heterochromatic siRNA precursors. PMID:27207877

  14. Efficient in vitro gene therapy with PEG siRNA lipid nanocapsules for passive targeting strategy in melanoma.

    PubMed

    Resnier, Pauline; LeQuinio, Pierre; Lautram, Nolwenn; André, Emilie; Gaillard, Cédric; Bastiat, Guillaume; Benoit, Jean-Pierre; Passirani, Catherine

    2014-11-01

    Small interfering RNA (siRNA)-mediated gene therapy is a promising strategy to temporarily inhibit the expression of proteins implicated in carcinogenesis or chemotherapy resistance. Although intra-tumoral administration can be envisaged, studies currently focus on formulating nanomedicines for intravenous injection to target tumor sites as well as metastases. The development of synthetic nanoparticles and liposomes has advanced greatly during the last decade. The objective of this work consists in formulating and optimizing the encapsulation of siRNA into lipid nanocapsules (LNCs) for efficient gene therapy to target melanoma cells. SiRNA LNCs were prepared from DOTAP/DOPE lipoplexes, and the siRNA amount and lipid/siRNA charge ratio were assayed to improve the stability and the encapsulation yield. Cryo-TEM imaging of the siRNA lipoplexes and LNC morphology revealed specific organization of the siRNA DOTAP/DOPE lipoplexes as well as specific lipid microstructures that can be eliminated by purification. No cytotoxicity of the siRNA LNCs against the melanoma SK-Mel28 cell line was observed at concentrations of up to 500 ng/mL siRNA. In vitro siRNA transfection experiments, compared to Oligofectamine™, demonstrated interesting targeted gene silencing effects. Finally, complement activation assays confirmed the feasibility of the PEGylation of siRNA LNCs as part of a passive targeting strategy for future in vivo melanoma- and metastasis-targeting experiments. PMID:25262914

  15. Genome-wide analysis of single non-templated nucleotides in plant endogenous siRNAs and miRNAs

    PubMed Central

    Wang, Feng; Johnson, Nathan R.; Coruh, Ceyda; Axtell, Michael J.

    2016-01-01

    Plant small RNAs are subject to various modifications. Previous reports revealed widespread 3′ modifications (truncations and non-templated tailing) of plant miRNAs when the 2′-O-methyltransferase HEN1 is absent. However, non-templated nucleotides in plant heterochromatic siRNAs have not been deeply studied, especially in wild-type plants. We systematically studied non-templated nucleotide patterns in plant small RNAs by analyzing small RNA sequencing libraries from Arabidopsis, tomato, Medicago, rice, maize and Physcomitrella. Elevated rates of non-templated nucleotides were observed at the 3′ ends of both miRNAs and endogenous siRNAs from wild-type specimens of all species. ‘Off-sized’ small RNAs, such as 25 and 23 nt siRNAs arising from loci dominated by 24 nt siRNAs, often had very high rates of 3′-non-templated nucleotides. The same pattern was observed in all species that we studied. Further analysis of 24 nt siRNA clusters in Arabidopsis revealed distinct patterns of 3′-non-templated nucleotides of 23 nt siRNAs arising from heterochromatic siRNA loci. This pattern of non-templated 3′ nucleotides on 23 nt siRNAs is not affected by loss of known small RNA 3′-end modifying enzymes, and may result from modifications added to longer heterochromatic siRNA precursors. PMID:27207877

  16. Synergistic effect of phosphorothioate, 5'-vinylphosphonate and GalNAc modifications for enhancing activity of synthetic siRNA.

    PubMed

    Prakash, Thazha P; Kinberger, Garth A; Murray, Heather M; Chappell, Alfred; Riney, Stan; Graham, Mark J; Lima, Walt F; Swayze, Eric E; Seth, Punit P

    2016-06-15

    Chemical modifications are essential to improve metabolic stability and pharmacokinetic properties of siRNA to enable their systemic delivery. We investigated the effect of combing the phosphorothioate (PS) modification with metabolically stable phosphate analog (E)-5'-vinylphosphonate and GalNAc cluster conjugation on the activity of fully 2'-modified siRNA in cell culture and mice. Our data suggest that integrating multiple chemical approaches in one siRNA molecule improved potency 5-10 fold and provide a roadmap for developing more efficient siRNA drugs. PMID:27161280

  17. Quantitative Silencing of EGFP Reporter Gene by Self-Assembled siRNA Lipoplexes of LinOS and Cholesterol

    PubMed Central

    2012-01-01

    Nonviral siRNA vectors prepared by the direct mixing of siRNA and mixtures of an asymmetric N4,N9-diacyl spermine conjugate, N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), with either cholesterol or DOPE, at various molar ratios of the neutral lipids, are reported. The effects of varying the lipid formulation and changing the N/P charge ratio on the intracellular delivery of siRNA to HeLa cells and on the siRNA-mediated gene silencing of a stably expressed reporter gene (EGFP) were evaluated. The presence of either cholesterol or DOPE in the mixture resulted in a marked increase in the delivery of the siRNA as well as enhanced EGFP silencing as evaluated by FACS. A LinOS/Chol 1:2 mixture resulted in the highest siRNA delivery and the most efficient EGFP silencing (reduced to 20%) at N/P = 3.0. Lowering the amount of siRNA from 15 pmol to 3.75 pmol, thus increasing the N/P charge ratio to 11.9, resulted in decreasing the amount of delivered siRNA, while the efficiency of gene silencing was comparable to that obtained with 15 pmol (N/P = 3.0) of siRNA. Mixtures of symmetrical N4,N9-dioleoyl spermine (DOS) with cholesterol at 1:2 molar ratio showed less siRNA delivery than with LinOS/Chol at N/P = 3.0 (15 pmol of siRNA), and comparable delivery at N/P = 11.9 (3.75 pmol of siRNA). The EGFP silencing was comparable with LinOS and with DOS when mixed with cholesterol 1:2 (lipoplexes prepared with 15 pmol of siRNA), but LinOS mixtures showed better EGFP silencing when the siRNA was reduced to 3.75 pmol. Lipoplex particle size determination by DLS of cholesterol mixtures was 106–118 nm, compared to 194–356 nm for lipoplexes prepared with the spermine conjugates only, and to 685 nm for the LinOS/DOPE 1:1 mixture. Confocal microscopy showed successful siRNA delivery of red tagged siRNA and quantitative EGFP knockdown in HeLa EGFP cells; Z-stack photomicrographs showed that the delivered siRNA is distributed intracellularly. Cryo-TEM of siRNA LinOS/Chol 1

  18. Flare Plasma Iron Abundance

    NASA Technical Reports Server (NTRS)

    Dennis, Brian R.; Dan, Chau; Jain, Rajmal; Schwartz, Richard A.; Tolbert, Anne K.

    2008-01-01

    The equivalent width of the iron-line complex at 6.7 keV seen in flare X-ray spectra suggests that the iron abundance of the hottest plasma at temperatures >approx.10 MK may sometimes be significantly lower than the nominal coronal abundance of four times the photospheric value that is commonly assumed. This conclusion is based on X-ray spectral observations of several flares seen in common with the Ramaty High Energy Solar Spectroscopic Imager (RHESSI) and the Solar X-ray Spectrometer (SOXS) on the second Indian geostationary satellite, GSAT-2. The implications of this will be discussed as it relates to the origin of the hot flare plasma - either plasma already in the corona that is directly heated during the flare energy release process or chromospheric plasma that is heated by flare-accelerated particles and driven up into the corona. Other possible explanations of lower-than-expected equivalent widths of the iron-line complex will also be discussed.

  19. Carbon nanotube-mediated siRNA delivery for gene silencing in cancer cells

    NASA Astrophysics Data System (ADS)

    Hong, Tu; Guo, Honglian; Xu, Yaqiong

    2011-10-01

    Small interfering RNA (siRNA) is potentially a promising tool in influencing gene expression with a high degree of target specificity. However, its poor intracellular uptake, instability in vivo, and non-specific immune stimulations impeded its effect in clinical applications. In this study, carbon nanotubes (CNTs) functionalized with two types of phospholipid-polyethylene glycol (PEG) have shown capabilities to stabilize siRNA in cell culture medium during the transfection and efficiently deliver siRNA into neuroblastoma and breast cancer cells. Moreover, the intrinsic optical properties of CNTs have been investigated through absorption and fluorescence measurements. We have found that the directly-functionalized groups play an important role on the fluorescence imaging of functionalized CNTs. The unique fluorescence imaging and high delivery efficiency make CNTs a promising material to deliver drugs and evaluate the treatment effect simultaneously.

  20. Ferritin-mediated siRNA delivery and gene silencing in human tumor and primary cells.

    PubMed

    Li, Le; Muñoz-Culla, Maider; Carmona, Unai; Lopez, Maria Paz; Yang, Fan; Trigueros, Cesar; Otaegui, David; Zhang, Lianbing; Knez, Mato

    2016-08-01

    We demonstrate a straightforward method to encapsulate siRNA into naturally available and unmodified human apoferritin. The encapsulation into apoferritin is independent of the sequence of the siRNA and provides superior protection for those sensitive molecules. High efficiency in transfection can be achieved in human tumorigenic cells, human primary mesenchymal stem cells (hMSC) and peripheral blood mononuclear cells (PBMCs). In contrast to Lipofectamine, highly effective gene silencing can be achieved with ferritin as the delivery agent in both tumor cells and PBMCs at low siRNA concentrations (10 nM). As an endogenous delivery agent, apoferritin does not induce immune activation of T- and B-cells in human PBMCs. Apoferritin shows intrinsic anti-inflammatory effects and apoferritin-mediated delivery shows a preference for immune-activated T- and B-cells, a natural selectivity which may turn useful for drug delivery in case of infections or inflammatory diseases. PMID:27187278

  1. N-Isopropylacrylamide Modified Polyethylenimines as Effective siRNA Carriers for Cancer Therapy.

    PubMed

    Chen, Jie; Lin, Lin; Guo, Zhaopei; Xu, Caina; Li, Yanhui; Tian, Huayu; Tang, Zhaohui; He, Chaoliang; Chen, Xuesi

    2016-06-01

    N-isopropylacrylamide modified PEI (PEN) was synthesized via Michael addition and was developed as an efficient siRNA delivery system both in vitro and in vivo. PEN showed significant enhanced cytocompatibility compared with commercial PEI-25k. The complexation of PEN with siRNA was studied by gel retardation, particle size and zeta potential measurement. The in vitro transfection ability of PEN was measured by qRT-PCR assay, and achieved obviously enhanced gene silencing efficiency compared with PEI-25k. The confocal imaging and flow cytometric analysis further validated its excellent intracellular trafficking ability. For antitumor treatment experiment, PEN mediated siVEGF showed obviously therapeutic effects for the treatment of CT26 tumor. Therefore, the present study demonstrated a useful strategy for constructing efficient siRNA delivery vehicles for antitumor therapy. PMID:27427585

  2. Liver-Targeted SiRNA Delivery Using Biodegradable Poly(amide) Polymer Conjugates.

    PubMed

    Barrett, Stephanie E; Guidry, Erin N

    2016-01-01

    The realization of polymer conjugate-based RNA delivery as a clinical modality requires the development and optimization of novel formulations. Although many literature examples of polymer conjugate-based SiRNA delivery systems exist, the protocols described herein represent a robust and facile way of screening any poly(amine)-based polymer system for SiRNA delivery. In this chapter, we describe the synthetic methods used to prepare poly(amide) polymers using a controlled polymerization method, as well as the preparation of the resulting targeted SiRNA polymer conjugates. In addition, detailed methods are provided for the characterization of the biodegradable poly(peptides) as well as the polymer conjugate that ensues. PMID:26472438

  3. Nanodiamond as a vector for siRNA delivery to Ewing sarcoma cells.

    PubMed

    Alhaddad, Anna; Adam, Marie-Pierre; Botsoa, Jacques; Dantelle, Géraldine; Perruchas, Sandrine; Gacoin, Thierry; Mansuy, Christelle; Lavielle, Solange; Malvy, Claude; Treussart, François; Bertrand, Jean-Rémi

    2011-11-01

    The ability of diamond nanoparticles (nanodiamonds, NDs) to deliver small interfering RNA (siRNA) into Ewing sarcoma cells is investigated with a view to the possibility of in-vivo anticancer nucleic-acid drug delivery. siRNA is adsorbed onto NDs that are coated with cationic polymer. Cell uptake of NDs is demonstrated by taking advantage of the NDs' intrinsic fluorescence from embedded color-center defects. Cell toxicity of these coated NDs is shown to be low. Consistent with the internalization efficacy, a specific inhibition of EWS/Fli-1 gene expression is shown at the mRNA and protein level by the ND-vectorized siRNA in a serum-containing medium. PMID:21913326

  4. Recent progress in development of siRNA delivery vehicles for cancer therapy.

    PubMed

    Kim, Hyun Jin; Kim, Ahram; Miyata, Kanjiro; Kataoka, Kazunori

    2016-09-01

    Recent progress in RNA biology has broadened the scope of therapeutic targets of RNA drugs for cancer therapy. However, RNA drugs, typically small interfering RNAs (siRNAs), are rapidly degraded by RNases and filtrated in the kidney, thereby requiring a delivery vehicle for efficient transport to the target cells. To date, various delivery formulations have been developed from cationic lipids, polymers, and/or inorganic nanoparticles for systemic delivery of siRNA to solid tumors. This review describes the current status of clinical trials related to siRNA-based cancer therapy, as well as the remaining issues that need to be overcome to establish a successful therapy. It, then introduces various promising design strategies of delivery vehicles for stable and targeted siRNA delivery, including the prospects for future design. PMID:27352638

  5. Bioinformatics prediction of siRNAs as potential antiviral agents against dengue viruses

    PubMed Central

    Villegas-Rosales, Paula M; Méndez-Tenorio, Alfonso; Ortega-Soto, Elizabeth; Barrón, Blanca L

    2012-01-01

    Dengue virus (DENV 1-4) represents the major emerging arthropod-borne viral infection in the world. Currently, there is neither an available vaccine nor a specific treatment. Hence, there is a need of antiviral drugs for these viral infections; we describe the prediction of short interfering RNA (siRNA) as potential therapeutic agents against the four DENV serotypes. Our strategy was to carry out a series of multiple alignments using ClustalX program to find conserved sequences among the four DENV serotype genomes to obtain a consensus sequence for siRNAs design. A highly conserved sequence among the four DENV serotypes, located in the encoding sequence for NS4B and NS5 proteins was found. A total of 2,893 complete DENV genomes were downloaded from the NCBI, and after a depuration procedure to identify identical sequences, 220 complete DENV genomes were left. They were edited to select the NS4B and NS5 sequences, which were aligned to obtain a consensus sequence. Three different servers were used for siRNA design, and the resulting siRNAs were aligned to identify the most prevalent sequences. Three siRNAs were chosen, one targeted the genome region that codifies for NS4B protein and the other two; the region for NS5 protein. Predicted secondary structure for DENV genomes was used to demonstrate that the siRNAs were able to target the viral genome forming double stranded structures, necessary to activate the RNA silencing machinery. PMID:22829722

  6. Surface engineering of gold nanoparticles for in vitro siRNA delivery

    NASA Astrophysics Data System (ADS)

    Zhao, Enyu; Zhao, Zhixia; Wang, Jiancheng; Yang, Chunhui; Chen, Chengjun; Gao, Lingyan; Feng, Qiang; Hou, Wenjie; Gao, Mingyuan; Zhang, Qiang

    2012-07-01

    Cellular uptake, endosomal/lysosomal escape, and the effective dissociation from the carrier are a series of hurdles for specific genes to be delivered both in vitro and in vivo. To construct siRNA delivery systems, poly(allylamine hydrochloride) (PAH) and siRNA were alternately assembled on the surface of 11.8 +/- 0.9 nm Au nanoparticles (GNP), stabilized by denatured bovine serum albumin, by the ionic layer-by-layer (LbL) self-assembly method. By manipulating the outmost PAH layer, GNP-PAH vectors with different surface electric potentials were prepared. Then, the surface potential-dependent cytotoxicity of the resultant GNP-PAH particles was evaluated via sulforhodamine B (SRB) assay, while the surface potential-dependent cellular uptake efficiency was quantitatively analyzed by using the flow cytometry method based on carboxyfluorescein (FAM)-labeled siRNA. It was revealed that the GNP-PAH particles with surface potential of +25 mV exhibited the optimal cellular uptake efficiency and cytotoxicity for human breast cancer MCF-7 cells. Following these results, two more positively charged polyelectrolytes with different protonating abilities in comparison with PAH, i.e., polyethylenimine (PEI), and poly(diallyl dimethyl ammonium chloride) (PDDA), were chosen to fabricate similarly structured vectors. Confocal fluorescence microscopy studies indicated that siRNA delivered by GNP-PAH and GNP-PEI systems was better released than that delivered by the GNP-PDDA system. Further flow cytometric assays based on immunofluorescence staining of the epidermal growth factor receptor (EGFR) revealed that EGFR siRNA delivered by GNP-PAH and GNP-PEI exhibited similar down-regulation effects on EGFR expression in MCF-7 cells. The following dual fluorescence flow cytometry assays by co-staining phosphatidylserine and DNA suggested the EGFR siRNA delivered by GNP-PAH exhibited an improved silencing effect in comparison with that delivered by the commercial transfection reagent

  7. Screening nylon-3 polymers, a new class of cationic amphiphiles, for siRNA delivery.

    PubMed

    Nadithe, Venkatareddy; Liu, Runhui; Killinger, Bryan A; Movassaghian, Sara; Kim, Na Hyung; Moszczynska, Anna B; Masters, Kristyn S; Gellman, Samuel H; Merkel, Olivia M

    2015-02-01

    Amphiphilic nucleic acid carriers have attracted strong interest. Three groups of nylon-3 copolymers (poly-β-peptides) possessing different cationic/hydrophobic content were evaluated as siRNA delivery agents in this study. Their ability to condense siRNA was determined in SYBR Gold assays. Their cytotoxicity was tested by MTT assays, their efficiency of delivering Alexa Fluor-488-labeled siRNA intracellularly in the presence and absence of uptake inhibitors was assessed by flow cytometry, and their transfection efficacies were studied by luciferase knockdown in a cell line stably expressing luciferase (H1299/Luc). Endosomal release was determined by confocal laser scanning microscopy and colocalization with lysotracker. All polymers efficiently condensed siRNA at nitrogen-to-phosphate (N/P) ratios of 5 or lower, as reflected in hydrodynamic diameters smaller than that at N/P 1. Although several formulations had negative zeta potentials at N/P 1, G2C and G2D polyplexes yielded >80% uptake in H1299/Luc cells, as determined by flow cytometry. Luciferase knockdown (20-65%) was observed after transfection with polyplexes made of the high molecular weight polymers that were the most hydrophobic. The ability of nylon-3 polymers to deliver siRNA intracellularly even at negative zeta potential implies that they mediate transport across cell membranes based on their amphiphilicity. The cellular uptake route was determined to strongly depend on the presence of cholesterol in the cell membrane. These polymers are, therefore, very promising for siRNA delivery at reduced surface charge and toxicity. Our study identified nylon-3 formulations at low N/P ratios for effective gene knockdown, indicating that nylon-3 polymers are a new, promising type of gene delivery agent. PMID:25437915

  8. Screening Nylon-3 Polymers, a New Class of Cationic Amphiphiles, for siRNA Delivery

    PubMed Central

    2015-01-01

    Amphiphilic nucleic acid carriers have attracted strong interest. Three groups of nylon-3 copolymers (poly-β-peptides) possessing different cationic/hydrophobic content were evaluated as siRNA delivery agents in this study. Their ability to condense siRNA was determined in SYBR Gold assays. Their cytotoxicity was tested by MTT assays, their efficiency of delivering Alexa Fluor-488-labeled siRNA intracellularly in the presence and absence of uptake inhibitors was assessed by flow cytometry, and their transfection efficacies were studied by luciferase knockdown in a cell line stably expressing luciferase (H1299/Luc). Endosomal release was determined by confocal laser scanning microscopy and colocalization with lysotracker. All polymers efficiently condensed siRNA at nitrogen-to-phosphate (N/P) ratios of 5 or lower, as reflected in hydrodynamic diameters smaller than that at N/P 1. Although several formulations had negative zeta potentials at N/P 1, G2C and G2D polyplexes yielded >80% uptake in H1299/Luc cells, as determined by flow cytometry. Luciferase knockdown (20–65%) was observed after transfection with polyplexes made of the high molecular weight polymers that were the most hydrophobic. The ability of nylon-3 polymers to deliver siRNA intracellularly even at negative zeta potential implies that they mediate transport across cell membranes based on their amphiphilicity. The cellular uptake route was determined to strongly depend on the presence of cholesterol in the cell membrane. These polymers are, therefore, very promising for siRNA delivery at reduced surface charge and toxicity. Our study identified nylon-3 formulations at low N/P ratios for effective gene knockdown, indicating that nylon-3 polymers are a new, promising type of gene delivery agent. PMID:25437915

  9. High Sensitivity RT-qPCR Assay of Nonlabeled siRNA in Small Blood Volume for Pharmacokinetic Studies: Application to Survivin siRNA.

    PubMed

    Yeung, Bertrand Z; Lu, Ze; Wientjes, Guillaume M; Au, Jessie L-S

    2015-11-01

    RNAi therapeutics provide an opportunity to correct faulty genes, and several RNAi have entered clinical evaluation. The existing quantification methods typically use radioactivity- or fluorescence-labeled RNAi, require large blood volumes, and/or have a limited dynamic detection range. We established a quantitative reverse transcriptase real-time polymerase chain reaction (RT-qPCR) assay to measure RNAi; the model analyte was survivin siRNA (siSurvivin). A second siRNA was used as the internal standard. The three major steps were (a) extraction of the two siRNAs from blood or water, (b) synthesis of their cDNA by poly-A extension, and (c) qPCR of cDNA. Standard curves were established. Utility of the assay was demonstrated in a pharmacokinetic study where all 12 samples for the blood concentration-time profile were obtained from a single mouse given an intravenous dose of 1 nmole siSurvivin (prepared as lipoplex with pegylated cationic liposomes). The RT-qPCR assay was sensitive (lower detection limit of 100 fM) and had a 5 × 107-fold dynamic range and low sample volume requirement (10 μL). The 16-point standard curves constructed using whole blood samples were linear (R (2) > 0.98). The intraday and interday variations for the slopes were ≤6%, although the variations for accuracy and precision at individual concentrations were substantially higher (58-145%). Standard curves prepared with water in place of blood showed similar results (<6% difference), indicating water may be used when blood is not available. The current RT-qPCR assay enabled the measurement of nonlabeled siRNA in small volume of blood samples. PMID:26286676

  10. A Bifurcated Proteoglycan Binding Small Molecule Carrier for siRNA Delivery

    PubMed Central

    Gooding, Matt; Adigbli, Derick; Edith Chan, A W; Melander, Roberta J; MacRobert, Alexander J; Selwood, David L

    2014-01-01

    A wider application of siRNA- and miRNA- based therapeutics is restricted by the currently available delivery systems. We have designed a new type of small molecule carrier (SMoC) system for siRNA modeled to interact with cell surface proteoglycans. This bifurcated SMoC has similar affinity for the model proteoglycan heparin to an equivalent polyarginine peptide and exhibits significant mRNA knockdown of protein levels comparable to lipofectamine and the previously reported linear SMoC. PMID:24472581

  11. Clustering siRNA conjugates for MMP-responsive therapeutics in chronic wounds of diabetic animals

    NASA Astrophysics Data System (ADS)

    Kim, Hye Sung; Son, Young Ju; Yoo, Hyuk Sang

    2016-07-01

    The MMP-responsive breakdown of siRNA clusters was translated to site-specific gene transfection and enhanced wound healing in diabetic ulcers. MMP-2 siRNA was chemically tethered to the end of multi-armed PEG via MMP-cleavable linkers (4PEG-siRNA) and subsequently clustered into submicron particles complexed with LPEI. 4PEG-siRNA was more tightly complexed with LPEI and the associated cluster showed higher resistance against RNase attack, in comparison to naked siRNA. Because the size of the clusters increased depending on the increase in charge ratio of LPEI to siRNA, cellular uptake of the 4PEG-siRNA/LPEI cluster was significantly attenuated due to the huge size of the cluster. However, upon MMP treatment, the cluster dissociated into smaller particles and was efficiently endocytosed by cells. An in vivo fluorescence resonance energy transfer (FRET) study also revealed that the clusters were effectively dissociated in MMP-rich environments of dorsal wounds in diabetic animals. In addition, diabetic ulcers treated with the clusters showed a faster wound closure rate and the recovered tissue expressed a larger amount of cytokeratin along with a lower expression level of MMP-2 compared to the other groups.The MMP-responsive breakdown of siRNA clusters was translated to site-specific gene transfection and enhanced wound healing in diabetic ulcers. MMP-2 siRNA was chemically tethered to the end of multi-armed PEG via MMP-cleavable linkers (4PEG-siRNA) and subsequently clustered into submicron particles complexed with LPEI. 4PEG-siRNA was more tightly complexed with LPEI and the associated cluster showed higher resistance against RNase attack, in comparison to naked siRNA. Because the size of the clusters increased depending on the increase in charge ratio of LPEI to siRNA, cellular uptake of the 4PEG-siRNA/LPEI cluster was significantly attenuated due to the huge size of the cluster. However, upon MMP treatment, the cluster dissociated into smaller particles and was

  12. Amylose-Based Cationic Star Polymers for siRNA Delivery

    PubMed Central

    Nishimura, Tomoki; Umezaki, Kaori; Mukai, Sada-atsu; Sawada, Shin-ichi; Akiyoshi, Kazunari

    2015-01-01

    A new siRNA delivery system using a cationic glyco-star polymer is described. Spermine-modified 8-arm amylose star polymer (with a degree of polymerization of approximately 60 per arm) was synthesized by chemoenzymatic methods. The cationic star polymer effectively bound to siRNA and formed spherical complexes with an average hydrodynamic diameter of 230 nm. The cationic 8-arm star polymer complexes showed superior cellular uptake characteristics and higher gene silencing effects than a cationic 1-arm polymer. These results suggest that amylose-based star polymers are a promising nanoplatform for glycobiomaterials. PMID:26539548

  13. siRNA targeting RBP2 inhibits expression, proliferation, tumorigenicity and invasion in thyroid carcinoma cells

    PubMed Central

    KONG, LING-LING; MAN, DONG-MEI; WANG, TIAN; ZHANG, GUO-AN; CUI, WEN

    2015-01-01

    In order to estimate the effects of small interfering RNA (siRNA) targeting retinoblastoma binding protein 2 (RBP2) on the proliferation, expression, invasion, migration and tumorigenicity abilities of papillary thyroid carcinoma K1 cells, siRNA targeting RBP2 (RBP2-siRNA) and negative control siRNA were transfected into K1 cells. The mRNA levels of RBP2 in the transfected cells were estimated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the protein levels of RBP2 in these cells were evaluated by western blot analysis and immunocytochemical (ICC) analyses. The growth, tumorigenicity, migration and invasion abilities of the transfected cells were measured by Cell Counting Kit-8 (CCK-8), soft agar colony formation and transwell chamber assay, respectively. The ICC results demonstrated that the protein expression levels of RBP2 were lower in the RBP2-siRNA-transfected cells than in the blank and control cells (analysis of variance, F=26.754, P<0.01). RBP2-siRNA downregulated RBP2 at the mRNA (t=8.869) and protein level (F=60.835) (P=0.000 vs. control cells). In addition, the transfection of RBP2-siRNA into K1 cells also suppressed cell proliferation at 24, 48 and 72 h post-transfection (t=7.650, P<0.01; t=2.606, P=0.016; and t=2.377, P=0.027, respectively). Compared with the control group, the number of invasive and migrated cells were significantly reduced in the RBP2-siRNA-transfected group (t=4.774 and t=6.366, respectively; P<0.01). Furthermore, the tumorigenic potential of the cells transfected with RBP2-siRNA was markedly reduced, as indicated by the soft agar formation assay (t=2.749, P=0.014 vs. control cells). In conclusion, the transfection of RBP2-siRNA into papillary thyroid carcinoma K1 cells suppressed the expression of RBP2 in these cells, and reduced their proliferation, invasion, migration and tumorigenic potential. Therefore, targeting RBP2 may be an efficient approach to control thyroid carcinoma. PMID:26788140

  14. siRNA Delivery to the Lung: What’s New?

    PubMed Central

    Merkel, Olivia M.; Rubinstein, Israel; Kissel, Thomas

    2014-01-01

    RNA interference (RNAi) has been thought of as the general answer to many unmet medical needs. After the first success stories, it soon became obvious that short interfering RNA (siRNA) is not suitable for systemic administration due to its poor pharmacokinetics. Therefore local administration routes have been adopted for more successful in vivo RNAi. This paper reviews nucleic acid modifications, nanocarrier chemistry, animal models used in successful pulmonary siRNA delivery, as well as clinical translation approaches. We summarize what has been published recently and conclude with the potential problems that may still hamper the efficient clinical application of RNAi in the lung. PMID:24907426

  15. Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes.

    PubMed

    Bender, Heather R; Kane, Sarah; Zabel, Mark D

    2016-01-01

    Prion diseases result from the misfolding of the normal, cellular prion protein (PrP(C)) to an abnormal protease resistant isomer called PrP(Res). The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrP(Res) isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrP(Res) formation in brain tissue, do not specifically target neuronal PrP(C), and are often too toxic to use in animal or human subjects. We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrP(C) is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to Ach

  16. Oxygen abundance and convection

    NASA Astrophysics Data System (ADS)

    Van't Veer, C.; Cayrel, R.

    The triplet IR lines of O I near 777 nm are computed with the Kurucz's code, modified to accept several convection models. The program has been run with the MLT algorithm, with l/H = 1.25 and 0.5, and with the Canuto-Mazzitelli and Canuto-Goldman-Mazzitelli approaches, on a metal-poor turnoff-star model atmosphere with Teff=6200 K, log g = 4.3, [Fe/H]= -1.5. The results show that the differences in equivalent widths for the 4 cases do not exceed 2 per cent (0.3 mA). The convection treatment is therefore not an issue for the oxygen abundance derived from the permitted lines.

  17. VIRsiRNApred: a web server for predicting inhibition efficacy of siRNAs targeting human viruses

    PubMed Central

    2013-01-01

    Background Selection of effective viral siRNA is an indispensable step in the development of siRNA based antiviral therapeutics. Despite immense potential, a viral siRNA efficacy prediction algorithm is still not available. Moreover, performances of the existing general mammalian siRNA efficacy predictors are not satisfactory for viral siRNAs. Therefore, we have developed “VIRsiRNApred” a support vector machine (SVM) based method for predicting the efficacy of viral siRNA. Methods In the present study, we have employed a new dataset of 1725 viral siRNAs with experimentally verified quantitative efficacies tested under heterogeneous experimental conditions and targeting as many as 37 important human viruses including HIV, Influenza, HCV, HBV, SARS etc. These siRNAs were divided into training (T1380) and validation (V345) datasets. Important siRNA sequence features including mono to penta nucleotide frequencies, binary pattern, thermodynamic properties and secondary structure were employed for model development. Results During 10-fold cross validation on T1380 using hybrid approach, we achieved a maximum Pearson Correlation Coefficient (PCC) of 0.55 between predicted and actual efficacy of viral siRNAs. On V345 independent dataset, our best model achieved a maximum correlation of 0.50 while existing general siRNA prediction methods showed PCC from 0.05 to 0.18. However, using leave one out cross validation PCC was improved to 0.58 and 0.55 on training and validation datasets respectively. SVM performed better than other machine learning techniques used like ANN, KNN and REP Tree. Conclusion VIRsiRNApred is the first algorithm for predicting inhibition efficacy of viral siRNAs which is developed using experimentally verified viral siRNAs. We hope this algorithm would be useful in predicting highly potent viral siRNA to aid siRNA based antiviral therapeutics development. The web server is freely available at http://crdd.osdd.net/servers/virsirnapred/. PMID:24330765

  18. Evaluation of carrier-mediated siRNA delivery: lessons for the design of a stem-loop qPCR-based approach for quantification of intracellular full-length siRNA.

    PubMed

    Colombo, Stefano; Nielsen, Hanne Mørck; Foged, Camilla

    2013-03-28

    Harnessing the RNA interference (RNAi) process with chemically synthesized small interfering RNA (siRNA) is dependent on the development of efficient delivery vehicles that can help overcome the numerous barriers existing for siRNA delivery. However, quantifying the intracellular amount of siRNA delivered by use of carriers remains an analytical challenge. The purpose of the present study was to optimize and validate an analytical protocol based on stem-loop reverse transcription quantitative polymerase chain reaction (RT qPCR) to quantitatively monitor the carrier-mediated intracellular siRNA delivery. An in vitro cell culture model system expressing enhanced green fluorescent protein (EGFP) was used to develop the assay, which was based on the intracellular quantification of a full-length double-stranded Dicer substrate siRNA by stem-loop RT qPCR. The result is a well-documented protocol for accurate and sensitive determination of the effective intracellular siRNA concentration upon transfection with different reagents. Specific guidelines for the customization of the protocol are provided and reported together with an example of its application for studying a specific siRNA delivery case. The outcome of the present study is a thoroughly discussed analytical protocol generally applicable to characterize carrier-mediated siRNA delivery processes. PMID:23313963

  19. Discovery of novel peptides targeting pro-atherogenic endothelium in disturbed flow regions -Targeted siRNA delivery to pro-atherogenic endothelium in vivo

    PubMed Central

    Chung, Jihwa; Shim, Hyunbo; Kim, Kwanchang; Lee, Duhwan; Kim, Won Jong; Kang, Dong Hoon; Kang, Sang Won; Jo, Hanjoong; Kwon, Kihwan

    2016-01-01

    Atherosclerosis occurs preferentially in arterial regions exposed to disturbed blood flow. Targeting these pro-atherogenic regions is a potential anti-atherogenic therapeutic approach, but it has been extremely challenging. Here, using in vivo phage display approach and the partial carotid ligation model of flow-induced atherosclerosis in mouse, we identified novel peptides that specifically bind to endothelial cells (ECs) exposed to disturbed flow condition in pro-atherogenic regions. Two peptides, CLIRRTSIC and CPRRSHPIC, selectively bound to arterial ECs exposed to disturbed flow not only in the partially ligated carotids but also in the lesser curvature and branching point of the aortic arch in mice as well as human pulmonary artery branches. Peptides were conjugated to branched polyethylenimine-polyethylene glycol polymer to generate polyplexes carrying siRNA targeting intercellular adhesion molecule-1 (siICAM-1). In mouse model, CLIRRTSIC polyplexes carrying si-ICAM-1 specifically bound to endothelium in disturbed flow regions, reducing endothelial ICAM-1 expression. Mass spectrometry analysis revealed that non-muscle myosin heavy chain II A (NMHC IIA) is a protein targeted by CLIRRTSIC peptide. Further studies showed that shear stress regulates NMHC IIA expression and localization in ECs. The CLIRRTSIC is a novel peptide that could be used for targeted delivery of therapeutics such as siRNAs to pro-atherogenic endothelium. PMID:27173134

  20. Actinide abundances in ordinary chondrites

    NASA Technical Reports Server (NTRS)

    Hagee, B.; Bernatowicz, T. J.; Podosek, F. A.; Johnson, M. L.; Burnett, D. S.

    1990-01-01

    Measurements of actinide and light REE (LREE) abundances and of phosphate abundances in equilibrated ordinary chondrites were obtained and were used to define the Pu abundance in the solar system and to determine the degree of variation of actinide and LREE abundances. The results were also used to compare directly the Pu/U ratio with the earlier obtained ratio determined indirectly, as (Pu/Nd)x(Nd/U), assuming that Pu behaves chemically as a LREE. The data, combined with high-accuracy isotope-dilution data from the literature, show that the degree of gram-scale variability of the Th, U, and LREE abundances for equilibrated ordinary chondrites is a factor of 2-3 for absolute abundances and up to 50 percent for relative abundances. The observed variations are interpreted as reflecting the differences in the compositions and/or proportions of solar nebula components accreted to ordinary chondrite parent bodies.

  1. De novo reconstruction of consensus master genomes of plant RNA and DNA viruses from siRNAs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In antiviral defense, plants produce massive quantities of 21-24 nucleotide siRNAs. Here we demonstrate that the complete genomes of DNA and RNA viruses and viroids can be reconstructed by deep sequencing and de novo assembly of viral/viroid siRNAs from experimentally- and naturally-infected plants....

  2. A bridge to silencing: Co-assembling anionic nanoparticles of siRNA and hyaluronan sulfate via calcium ion bridges.

    PubMed

    Forti, Efrat; Kryukov, Olga; Elovic, Edan; Goldshtein, Matan; Korin, Efrat; Margolis, Gal; Felder, Shani; Ruvinov, Emil; Cohen, Smadar

    2016-06-28

    Therapeutic implementation of RNA interference (RNAi) through delivery of short interfering RNA (siRNA) is still facing several critical hurdles, which mostly can be solved through the use of an efficient delivery system. We hereby introduce anionic siRNA nanoparticles (NPs) co-assembled by the electrostatic interactions of the semi-synthetic polysaccharide hyaluronan-sulfate (HAS), with siRNA, mediated by calcium ion bridges. The NPs have an average size of 130nm and a mild (-10mV) negative surface charge. Transmission electron microscopy (TEM) using gold-labeled components and X-ray photoelectron spectroscopy (XPS) demonstrated the spatial organization of siRNA molecules in the particle core, surrounded by a layer of HAS. The anionic NPs efficiently encapsulated siRNA, were stable in physiological-relevant environments and were cytocompatible, not affecting cell viability or homeostasis. Efficient cellular uptake of the anionic siRNA NPs, associated with potent gene silencing (>80%), was observed across multiple cell types, including murine primary peritoneal macrophages and human hepatocellular carcinoma cells. In a clinically-relevant model of acute inflammatory response in IL-6-stimulated human hepatocytes, STAT3 silencing induced by HAS-Ca(2+)-siRNA NPs resulted in marked decrease in the total and activated STAT3 protein levels, as well as in the expression levels of downstream acute phase response genes. Collectively, anionic NPs prove to be an efficient and cytocompatible delivery system for siRNA. PMID:27117458

  3. Anticancer siRNA cocktails as a novel tool to treat cancer cells. Part (A). Mechanisms of interaction.

    PubMed

    Ionov, Maksim; Lazniewska, Joanna; Dzmitruk, Volha; Halets, Inessa; Loznikova, Svetlana; Novopashina, Darya; Apartsin, Evgeny; Krasheninina, Olga; Venyaminova, Alya; Milowska, Katarzyna; Nowacka, Olga; Gomez-Ramirez, Rafael; de la Mata, Francisco Javier; Majoral, Jean-Pierre; Shcharbin, Dzmitry; Bryszewska, Maria

    2015-05-15

    This paper examines a perspective on the use of newly engineered nanomaterials as effective and safe carriers of genes for the therapy of cancer. Three different groups of cationic dendrimers (PAMAM, phosphorus and carbosilane) were complexed with anticancer siRNA and their biophysical properties of the dendriplexes analyzed. The potential of the dendrimers as nanocarriers for anticancer siBcl-xl, siBcl-2, siMcl-1 siRNAs and a siScrambled sequence was explored. Dendrimer/siRNA complexes were characterized by methods including fluorescence, zeta potential, dynamic light scattering, circular dichroism, gel electrophoresis and transmission electron microscopy. Some of the experiments were done with heparin to check if siRNA can be easily disassociated from the complexes, and whether released siRNA maintains its structure after interaction with the dendrimer. The results indicate that siRNAs form complexes with all the dendrimers tested. Oligoribonucleotide duplexes can be released from dendriplexes after heparin treatment and the structure of siRNA is maintained in the case of PAMAM or carbosilane dendrimers. The dendrimers were also effective in protecting siRNA from RNase A activity. The selection of the best siRNA carrier will be made based on cell culture studies (Part B). PMID:25791760

  4. Comparison of small interfering RNA (siRNA) delivery into bovine monocyte-derived macrophages by transfection and electroporation

    PubMed Central

    Jensen, Kirsty; Anderson, Jennifer A.; Glass, Elizabeth J.

    2014-01-01

    The manipulation of the RNA interference pathway using small interfering RNA (siRNA) has become the most frequently used gene silencing method. However, siRNA delivery into primary cells, especially primary macrophages, is often considered challenging. Here we report the investigation of the suitability of two methodologies: transient transfection and electroporation, to deliver siRNA targeted against the putative immunomodulatory gene Mediterranean fever (MEFV) into primary bovine monocyte-derived macrophages (bMDM). Eleven commercial transfection reagents were investigated with variable results with respect to siRNA uptake, target gene knock-down, cell toxicity and type I interferon (IFN) response induction. Three transfection reagents: Lipofectamine 2000, Lipofectamine RNAiMAX and DharmaFECT 3, were found to consistently give the best results. However, all the transfection reagents tested induced an IFN response in the absence of siRNA, which could be minimized by reducing the transfection reagent incubation period. In addition, optimized siRNA delivery into bMDM by electroporation achieved comparable levels of target gene knock-down as transient transfection, without a detectable IFN response, but with higher levels of cell toxicity. The optimized transient transfection and electroporation methodologies may provide a starting point for optimizing siRNA delivery into macrophages derived from other species or other cells considered difficult to investigate with siRNA. PMID:24598124

  5. Capella: Structure and Abundances

    NASA Technical Reports Server (NTRS)

    Brickhouse, Nancy S.

    1999-01-01

    This grant covers the analysis of EUVE spectra of the cool star binary system Capella. This project has also required the analysis of simultaneous Advanced Satellite for Cosmology and Astrophysics (ASCA) data. The ASCA spectrum of Capella could not be fit with standard models; by imposing models based on strong lines observed with EUVE, a problem wavelength region was identified. Correcting the problem required calculations of atomic collision strengths of higher principal quantum number than had ever been calculated. With these new models applied to the ASCA spectrum, better fits were obtained. Findings are that: (1) ASCA and EUVE spectra are both dominated by a region at 6 x 10(exp 6) K. (2) The high energy cut-off of the ASCA spectrum is consistent with emission from the highest ionization stages of EUVE, namely Fe XXIV. (3) EUVE requires a continuous emission measure distribution with more than two temperatures. (4) The ASCA spectra are of such high statistical significance that systematic uncertainties dominate, including atomic physics issues and calibration issues. (5) While the ASCA spectral fits achieve lower Chi(exp 2 with two-temperature fits, the EUVE-derived emission measure distribution models are also consistent with the spectra. (6) The Fe/H ratio obtained from the ASCA fit is within 20 % of the Fe/H abundance obtained from the summed spectra of Capella over 5 EUVE pointings, as well as the 1996 EUVE data. This result confirms our claims that quasi-continua composed of weak emission lines in the short wavelength spectrometer of EUVE are not major contributors to the measured Capella continuum. Other abundance ratios are also determined from the ASCA data, using models derived with EUVE. Si, Si, and Mg appear to be close to solar photospheric values, while the ratio of Ne/Fe is three to four times lower than solar photospheric values. Whether there is a general First Ionization Potential (FIP) effect or a specific neon anomaly cannot be determined

  6. Development of Pre-Clinical Models for Evaluating the Therapeutic Potential of Candidate siRNA Targeting STAT6

    PubMed Central

    Healey, Gareth D.; Lockridge, Jennifer A.; Zinnen, Shawn; Hopkin, Julian M.; Richards, Ivan; Walker, William

    2014-01-01

    Developing siRNA therapeutics poses technical challenges including appropriate molecular design and testing in suitable pre-clinical models. We previously detailed sequence-selection and modification strategies for siRNA candidates targeting STAT6. Here, we describe methodology that evaluates the suitability of candidate siRNA for respiratory administration. Chemically-modified siRNA exhibited similar inhibitory activity (IC50) against STAT6 in vitro compared to unmodified siRNA and apical exposure testing with Caco-2 cell monolayers showed modification was not associated with cellular toxicity. Use of a modified RNA extraction protocol improved the sensitivity of a PCR-based bio-analytical assay (lower limit of siRNA strand quantification  =  0.01 pg/µl) which was used to demonstrate that lung distribution profiles for both siRNAs were similar following intra-tracheal administration. However, after 6 hours, modified siRNA was detected in lung tissue at concentrations >1000-fold higher than unmodified siRNA. Evaluation in a rat model of allergic inflammation confirmed the persistence of modified siRNA in vivo, which was detectable in broncho-alveolar lavage (BAL) fluid, BAL cells and lung tissue samples, 72 hours after dosing. Based upon the concept of respiratory allergy as a single airway disease, we considered nasal delivery as a route for respiratory targeting, evaluating an intra-nasal exposure model that involved simple dosing followed by fine dissection of the nasal cavity. Notably, endogenous STAT6 expression was invariant throughout the nasal cavities and modified siRNA persisted for at least 3 days after administration. Coupled with our previous findings showing upregulated expression of inflammatory markers in nasal samples from asthmatics, these findings support the potential of intranasal siRNA delivery. In summary, we demonstrate the successful chemical modification of STAT6 targeting siRNA, which enhanced bio-availability without cellular

  7. Extreme Scale Visual Analytics

    SciTech Connect

    Wong, Pak C.; Shen, Han-Wei; Pascucci, Valerio

    2012-05-08

    Extreme-scale visual analytics (VA) is about applying VA to extreme-scale data. The articles in this special issue examine advances related to extreme-scale VA problems, their analytical and computational challenges, and their real-world applications.

  8. Structural studies of the formation of lipoplexes between siRNA and selected bis-imidazolium gemini surfactants.

    PubMed

    Andrzejewska, W; Pietralik, Z; Skupin, M; Kozak, M

    2016-10-01

    Dicationic (gemini) surfactants are agents that can be used for the preparation of stable complexes of nucleic acids, particularly siRNA for therapeutic purposes. In this study, we demonstrated that bis-imidazolium gemini surfactants with variable lengths of dioxyalkyl linker groups (from dioxyethyl to dioxydodecyl) and dodecyl side chains are excellent for the complexation of siRNA. All of these compounds effectively complexed siRNA in a charge ratio range (p/n) of 1.5-10. The low resolution structure of siRNA oligomers was characterised by small angle scattering of synchrotron radiation (SR-SAXS) and ab initio modelling. The structures of the formed complexes were also analysed using SR-SAXS, circular dichroism studies and electrophoretic mobility tests. The most promising agents for complexation with siRNA were the surfactants that contained dioxyethyl and dioxyhexyl spacer groups. PMID:27424091

  9. Gene regulation with carbon-based siRNA conjugates for cancer therapy.

    PubMed

    Zhang, Lingmin; Zheng, Wenfu; Tang, Rongbing; Wang, Nuoxin; Zhang, Wei; Jiang, Xingyu

    2016-10-01

    We report fluorescent carbon nanoparticle (FCN)-based small interfering RNA (siRNA) conjugates (C-siRNA) for gene regulation and cancer therapy. The C-siRNA has a core of chitosan-derived FCN and a shell of siRNA, and can down-regulate the expression of polo-like kinase-1 (Plk1), a master regulator of mitosis, via siRNA targeting Plk1 (siPlk1), for cancer therapy. The required amount of the FCNs is only ∼1/30 of that of the gold nanoparticles in delivering equal amount of siRNA. The C-siPlk1 led to ∼80% knockdown of cellular Plk1 mRNA in A375 cells, and induced apoptosis of the A375 cells (31.9%) and MCF-7 cells (20.33%), much higher than those by commercial nonviral gene delivery vectors, such as Lipofectamine 2000 in both cell lines (apoptosis rate < 10%). After the C-siPlk1 was administrated to A375 tumor-bearing mice intravenously, the tumor volume was less than 1/11 of the control groups. The C-siRNA can thus be powerful tools for gene delivery and gene therapy. PMID:27472164

  10. Clustering siRNA conjugates for MMP-responsive therapeutics in chronic wounds of diabetic animals.

    PubMed

    Kim, Hye Sung; Son, Young Ju; Yoo, Hyuk Sang

    2016-07-21

    The MMP-responsive breakdown of siRNA clusters was translated to site-specific gene transfection and enhanced wound healing in diabetic ulcers. MMP-2 siRNA was chemically tethered to the end of multi-armed PEG via MMP-cleavable linkers (4PEG-siRNA) and subsequently clustered into submicron particles complexed with LPEI. 4PEG-siRNA was more tightly complexed with LPEI and the associated cluster showed higher resistance against RNase attack, in comparison to naked siRNA. Because the size of the clusters increased depending on the increase in charge ratio of LPEI to siRNA, cellular uptake of the 4PEG-siRNA/LPEI cluster was significantly attenuated due to the huge size of the cluster. However, upon MMP treatment, the cluster dissociated into smaller particles and was efficiently endocytosed by cells. An in vivo fluorescence resonance energy transfer (FRET) study also revealed that the clusters were effectively dissociated in MMP-rich environments of dorsal wounds in diabetic animals. In addition, diabetic ulcers treated with the clusters showed a faster wound closure rate and the recovered tissue expressed a larger amount of cytokeratin along with a lower expression level of MMP-2 compared to the other groups. PMID:27251781

  11. siRNA knockdown validation 101: Incorporating negative controls in antibody research

    PubMed Central

    Olds, Will; Li, Jason

    2016-01-01

    More antibody validation protocols would identify non-specific reagents – a major source of irreproducible research – with the inclusion of negative controls. This article presents an overview of one such method: siRNA knockdown. The authors outline a general protocol, the knockdown mechanism, and tips for evaluating knockdown experiments. PMID:26998240

  12. Functionalized silicon quantum dots tailored for targeted siRNA delivery

    SciTech Connect

    Klein, S.; Zolk, O.; Fromm, M.F.; Schroedl, F.; Kryschi, C.

    2009-09-11

    For RNA interference (RNAi) mediated silencing of the ABCB1 gene in Caco-2 cells biocompatible luminescent silicon quantum dots (SiQDs) were developed to serve as self-tracking transfection tool for ABCB1 siRNA. While the 2-3 nm sized SiQD core exhibits green luminescence, the QD surfaces are completely saturated with covalently linked 2-vinylpyridine that may electrostatically bind siRNA. For down-regulating P-glycoprotein (Pgp) expression of the ABCB1 gene the SiQDs were complexed with siRNA. The cellular uptake and allocation of SiQD-siRNA complexes in Caco-2 cells were monitored using confocal laser scanning microscopy and transmission electron microscopy. The release of siRNA to the cytoplasm was verified through real-time PCR quantification of the reduced ABCB1 mRNA level. Additional evidence was obtained from time-resolved in situ fluorescence spectroscopic monitoring of the Pgp efflux dynamics in transfected Caco-2 cells which yielded significantly reduced transporter efficiencies for the Pgp substrate Rhodamine 123.

  13. Chitosan nanoparticles for siRNA delivery: optimizing formulation to increase stability and efficiency.

    PubMed

    Ragelle, H; Riva, R; Vandermeulen, G; Naeye, B; Pourcelle, V; Le Duff, C S; D'Haese, C; Nysten, B; Braeckmans, K; De Smedt, S C; Jérôme, C; Préat, V

    2014-02-28

    This study aims at developing chitosan-based nanoparticles suitable for an intravenous administration of small interfering RNA (siRNA) able to achieve (i) high gene silencing without cytotoxicity and (ii) stability in biological media including blood. Therefore, the influence of chitosan/tripolyphosphate ratio, chitosan physicochemical properties, PEGylation of chitosan as well as the addition of an endosomal disrupting agent and a negatively charged polymer was assessed. The gene silencing activity and cytotoxicity were evaluated on B16 melanoma cells expressing luciferase. We monitored the integrity and the size behavior of siRNA nanoparticles in human plasma using fluorescence fluctuation spectroscopy and single particle tracking respectively. The presence of PEGylated chitosan and poly(ethylene imine) was essential for high levels of gene silencing in vitro. Chitosan nanoparticles immediately released siRNA in plasma while the inclusion of hyaluronic acid and high amount of poly(ethylene glycol) in the formulation improved the stability of the particles. The developed formulations of PEGylated chitosan-based nanoparticles that achieve high gene silencing in vitro, low cytotoxicity and high stability in plasma could be promising for intravenous delivery of siRNA. PMID:24389132

  14. Dual-Functional Nanoparticles Targeting CXCR4 and Delivering Antiangiogenic siRNA Ameliorate Liver Fibrosis.

    PubMed

    Liu, Chun-Hung; Chan, Kun-Ming; Chiang, Tsaiyu; Liu, Jia-Yu; Chern, Guann-Gen; Hsu, Fu-Fei; Wu, Yu-Hsuan; Liu, Ya-Chi; Chen, Yunching

    2016-07-01

    The progression of liver fibrosis, an intrinsic response to chronic liver injury, is associated with hepatic hypoxia, angiogenesis, abnormal inflammation, and significant matrix deposition, leading to the development of cirrhosis and hepatocellular carcinoma (HCC). Due to the complex pathogenesis of liver fibrosis, antifibrotic drug development has faced the challenge of efficiently and specifically targeting multiple pathogenic mechanisms. Therefore, CXCR4-targeted nanoparticles (NPs) were formulated to deliver siRNAs against vascular endothelial growth factor (VEGF) into fibrotic livers to block angiogenesis during the progression of liver fibrosis. AMD3100, a CXCR4 antagonist that was incorporated into the NPs, served dual functions: it acted as a targeting moiety and suppressed the progression of fibrosis by inhibiting the proliferation and activation of hepatic stellate cells (HSCs). We demonstrated that CXCR4-targeted NPs could deliver VEGF siRNAs to fibrotic livers, decrease VEGF expression, suppress angiogenesis and normalize the distorted vessels in the fibrotic livers in the carbon tetrachloride (CCl4) induced mouse model. Moreover, blocking SDF-1α/CXCR4 by CXCR4-targeted NPs in combination with VEGF siRNA significantly prevented the progression of liver fibrosis in CCl4-treated mice. In conclusion, the multifunctional CXCR4-targeted NPs delivering VEGF siRNAs provide an effective antifibrotic therapeutic strategy. PMID:27224003

  15. Therapeutic siRNA for drug-resistant HER2-positive breast cancer

    PubMed Central

    Ngamcherdtrakul, Worapol; Castro, David J.; Morry, Jingga; Reda, Moataz M.; Gray, Joe W.; Yantasee, Wassana

    2016-01-01

    HER2 is overexpressed in about 20% of breast cancers and contributes to poor prognosis. Unfortunately, a large fraction of patients have primary or acquired resistance to the HER2-targeted therapy trastuzumab, thus a multi-drug combination is utilized in the clinic, putting significant burden on patients. We systematically identified an optimal HER2 siRNA from 76 potential sequences and demonstrated its utility in overcoming intrinsic and acquired resistance to trastuzumab and lapatinib in 18 HER2-positive cancer cell lines. We provided evidence that the drug-resistant cancer maintains dependence on HER2 for survival. Importantly, cell lines did not readily develop resistance following extended treatment with HER2 siRNA. Using our recently developed nanoparticle platform, systemic delivery of HER2 siRNA to trastuzumab-resistant tumors resulted in significant growth inhibition. Moreover, the optimal HER2 siRNA could also silence an exon 16 skipped HER2 splice variant reported to be highly oncogenic and linked to trastuzumab resistance. PMID:26894975

  16. Nanoparticle-Mediated Systemic Delivery of siRNA for Treatment of Cancers and Viral Infections

    PubMed Central

    Draz, Mohamed Shehata; Fang, Binbin Amanda; Zhang, Pengfei; Hu, Zhi; Gu, Shenda; Weng, Kevin C.; Gray, Joe W.; Chen, Fanqing Frank

    2014-01-01

    RNA interference (RNAi) is an endogenous post-transcriptional gene regulatory mechanism, where non-coding, double-stranded RNA molecules interfere with the expression of certain genes in order to silence it. Since its discovery, this phenomenon has evolved as powerful technology to diagnose and treat diseases at cellular and molecular levels. With a lot of attention, short interfering RNA (siRNA) therapeutics has brought a great hope for treatment of various undruggable diseases, including genetic diseases, cancer, and resistant viral infections. However, the challenge of their systemic delivery and on how they are integrated to exhibit the desired properties and functions remains a key bottleneck for realizing its full potential. Nanoparticles are currently well known to exhibit a number of unique properties that could be strategically tailored into new advanced siRNA delivery systems. This review summarizes the various nanoparticulate systems developed so far in the literature for systemic delivery of siRNA, which include silica and silicon-based nanoparticles, metal and metal oxides nanoparticles, carbon nanotubes, graphene, dendrimers, polymers, cyclodextrins, lipids, hydrogels, and semiconductor nanocrystals. Challenges and barriers to the delivery of siRNA and the role of different nanoparticles to surmount these challenges are also included in the review. PMID:25057313

  17. Ultrasound-responsive microbubbles for sonography-guided siRNA delivery.

    PubMed

    Wang, Ping; Yin, Tinghui; Li, Jingguo; Zheng, Bowen; Wang, Xiaoli; Wang, Yiru; Zheng, Jian; Zheng, Rongqin; Shuai, Xintao

    2016-05-01

    RNA interfering is a gene therapeutic approach of great potential for cancer. However, tumor-targeted delivery of small interfering RNA (siRNA) solely based on the enhanced permeability and retention effect of nanocarriers is often insufficient. To address this challenge, siRNA encapsulated ultrasound-responsive microbubble (MB) was developed from polymeric siRNA micelles and liposomal MBs using hetero-assembling strategy. 1MHz low-frequency ultrasound exposure of the tumor site after intratumoral injection of XIAP siRNA/MBs led to enhanced permeability for much more siRNA delivery into deep tumor regions. Significant improvement of XIAP gene silencing and cleaved caspase-3 activation was achieved, resulting in good therapeutic effect on human cervical cancer xenograft model in nude mice. Moreover, real-time US monitoring of the tumor was also possible using the siRNA/MBs as a contrast agent during the therapeutic process. These results show that the multi-functional siRNA/MBs are a promising theranostic system for cancer gene therapy. PMID:26733262

  18. Mechanistic profiling of the siRNA delivery dynamics of lipid-polymer hybrid nanoparticles.

    PubMed

    Colombo, Stefano; Cun, Dongmei; Remaut, Katrien; Bunker, Matt; Zhang, Jianxin; Martin-Bertelsen, Birte; Yaghmur, Anan; Braeckmans, Kevin; Nielsen, Hanne M; Foged, Camilla

    2015-03-10

    Understanding the delivery dynamics of nucleic acid nanocarriers is fundamental to improve their design for therapeutic applications. We investigated the carrier structure-function relationship of lipid-polymer hybrid nanoparticles (LPNs) consisting of poly(DL-lactic-co-glycolic acid) (PLGA) nanocarriers modified with the cationic lipid dioleoyltrimethyl-ammoniumpropane (DOTAP). A library of siRNA-loaded LPNs was prepared by systematically varying the nitrogen-to-phosphate (N/P) ratio. Atomic force microscopy (AFM) and cryo-transmission electron microscopy (cryo-TEM) combined with small angle X-ray scattering (SAXS) and confocal laser scanning microscopy (CLSM) studies suggested that the siRNA-loaded LPNs are characterized by a core-shell structure consisting of a PLGA matrix core coated with lamellar DOTAP structures with siRNA localized both in the core and in the shell. Release studies in buffer and serum-containing medium combined with in vitro gene silencing and quantification of intracellular siRNA suggested that this self-assembling core-shell structure influences the siRNA release kinetics and the delivery dynamics. A main delivery mechanism appears to be mediated via the release of transfection-competent siRNA-DOTAP lipoplexes from the LPNs. Based on these results, we suggest a model for the nanostructural characteristics of the LPNs, in which the siRNA is organized in lamellar superficial assemblies and/or as complexes entrapped in the polymeric matrix. PMID:25540904

  19. Clustered magnetite nanocrystals cross-linked with PEI for efficient siRNA delivery.

    PubMed

    Park, Ji Won; Bae, Ki Hyun; Kim, Chunsoo; Park, Tae Gwan

    2011-02-14

    Magnetofection has been utilized as a powerful tool to enhance gene transfection efficiency via magnetic field-enforced cellular transport processes. The accelerated accumulation of nucleic acid molecules by applying an external magnetic force enables the rapid and improved transduction efficiency. In this study, we developed magnetite nanocrystal clusters (PMNCs) cross-linked with polyethylenimine (PEI) to magnetically trigger intracellular delivery of small interfering RNA (siRNA). PMNCs were produced by cross-linked assembly of catechol-functionalized branched polyethylenimine (bPEI) around magnetite nanocrystals through an oil-in-water (O/W) emulsion and solvent evaporation method. The physical properties of PMNC were characterized by TEM, DLS, TSA, and FT-IR. Finely tuned formulation of clustered magnetite nanocrystals with controlled size and shape exhibited superior saturation of magnetization value. Magnetite nanocrystal clusters could form nanosized polyelectrolyte complexes with negatively charged siRNA molecules, enabling efficient delivery of siRNA into cells upon exposure to an external magnetic field within a short time. This study introduces a new class of magnetic nanomaterials that can be utilized for magnetically driven intracellular siRNA delivery. PMID:21190334

  20. siRNA transfection in larvae of the barnacle Amphibalanus amphitrite.

    PubMed

    Zhang, Gen; He, Li-Sheng; Wong, Yue Him; Yu, Li; Qian, Pei-Yuan

    2015-08-01

    RNA interference (RNAi) provides an efficient and specific technique for functional genomic studies. Yet, no successful application of RNAi has been reported in barnacles. In this study, siRNA against p38 MAPK was synthesized and then transfected into A. amphitrite larvae at either the nauplius or cyprid stage, or at both stages. Effects of siRNA transfection on the p38 MAPK level were hardly detectable in the cyprids when they were transfected at the nauplius stage. In contrast, larvae that were transfected at the cyprid stage showed lower levels of p38 MAPK than the blank and reagent controls. However, significantly decreased levels of phosphorylated p38 MAPK (pp38 MAPK) and reduced settlement rates were observed only in 'double transfections', in which larvae were exposed to siRNA solution at both the nauplius and cyprid stages. A relatively longer transfection time and more larval cells directly exposed to siRNA might explain the higher efficiency of double transfection experiments. PMID:26113139

  1. Identification of siRNA delivery enhancers by a chemical library screen

    PubMed Central

    Gilleron, Jerome; Paramasivam, Prasath; Zeigerer, Anja; Querbes, William; Marsico, Giovanni; Andree, Cordula; Seifert, Sarah; Amaya, Pablo; Stöter, Martin; Koteliansky, Victor; Waldmann, Herbert; Fitzgerald, Kevin; Kalaidzidis, Yannis; Akinc, Akin; Maier, Martin A.; Manoharan, Muthiah; Bickle, Marc; Zerial, Marino

    2015-01-01

    Most delivery systems for small interfering RNA therapeutics depend on endocytosis and release from endo-lysosomal compartments. One approach to improve delivery is to identify small molecules enhancing these steps. It is unclear to what extent such enhancers can be universally applied to different delivery systems and cell types. Here, we performed a compound library screen on two well-established siRNA delivery systems, lipid nanoparticles and cholesterol conjugated-siRNAs. We identified fifty-one enhancers improving gene silencing 2–5 fold. Strikingly, most enhancers displayed specificity for one delivery system only. By a combination of quantitative fluorescence and electron microscopy we found that the enhancers substantially differed in their mechanism of action, increasing either endocytic uptake or release of siRNAs from endosomes. Furthermore, they acted either on the delivery system itself or the cell, by modulating the endocytic system via distinct mechanisms. Interestingly, several compounds displayed activity on different cell types. As proof of principle, we showed that one compound enhanced siRNA delivery in primary endothelial cells in vitro and in the endocardium in the mouse heart. This study suggests that a pharmacological approach can improve the delivery of siRNAs in a system-specific fashion, by exploiting distinct mechanisms and acting upon multiple cell types. PMID:26220182

  2. Modified siRNA effectively silence inducible immunoproteasome subunits in NSO cells.

    PubMed

    Gvozdeva, Olga V; Belogurov, Alexey A; Kuzina, Ekaterina S; Gabibov, Alexander G; Meschaninova, Mariya I; Ven'yaminova, Alya G; Zenkova, Marina A; Vlassov, Valentin V; Chernolovskaya, Elena L

    2016-06-01

    The pathogenesis of autoimmune and neurodegenerative diseases involves overexpression of inducible subunits of the immunoproteasome. However, the clinical application of inhibitors to inducible subunits of the immunoproteasome has been limited due to systemic toxicity. Here, we designed siRNAs that efficiently silence LMP2, LMP7 and MECL-1 gene expression. Inducible subunits of the immunoproteasome are complex siRNA targets because they have a long half-life; therefore, we introduced 2'-O-methyl modifications into nuclease-sensitive sites. This led to 90-95% silencing efficiency and prolonged silencing, eliminating the need for multiple transfections. Furthermore, we showed that in the absence of transfection reagent, siRNAs with lipophilic residues were able to penetrate cells more effectively and decrease the expression of inducible immunoproteasome subunits by 35% after 5 days. These results show that siRNA targeted to inducible immunoproteasome subunits have great potential for the development of novel therapeutics for autoimmune and neurodegenerative diseases. PMID:26944796

  3. Combination of integrin siRNA and irradiation for breast cancer therapy

    SciTech Connect

    Cao Qizhen; Cai Weibo; Li Tianfang; Yang Yong; Chen Kai; Xing Lei; Chen Xiaoyuan . E-mail: shawchen@stanford.edu

    2006-12-22

    Up-regulation of integrin {alpha}{sub v}{beta}{sub 3} has been shown to play a key role in tumor angiogenesis and metastasis. In this study, we evaluated the role of integrin {alpha}{sub v}{beta}{sub 3} in breast cancer cell resistance to ionizing irradiation (IR) and tested the anti-tumor efficacy of combining integrin {alpha}{sub v} siRNA and IR. Colonogenic survival assay, cell proliferation, apoptosis, and cell cycle analysis were carried out to determine the treatment effect of siRNA, IR, or combination of both on MDA-MB-435 cells (integrin {alpha}{sub v}{beta}{sub 3}-positive). Integrin {alpha}{sub v}{beta}{sub 3}-negative MCF-7 cells exert more radiosensitivity than MDA-MB-435 cells. IR up-regulates integrin {alpha}{sub v}{beta}{sub 3} expression in MDA-MB-435 cells and integrin {alpha}{sub v} siRNA can effectively reduce both {alpha}{sub v} and {alpha}{sub v}{beta}{sub 3} integrin expression, leading to increased radiosensitivity. Integrin {alpha}{sub v} siRNA also promotes IR-induced apoptosis and enhances IR-induced G2/M arrest in cell cycle progression. This study, with further optimization, may provide a simple and highly efficient treatment strategy for breast cancer as well as other integrin {alpha}{sub v}{beta}{sub 3}-positive cancer types.

  4. Polymers modified with double-tailed fluorous compounds for efficient DNA and siRNA delivery.

    PubMed

    He, Bingwei; Wang, Yitong; Shao, Naimin; Chang, Hong; Cheng, Yiyun

    2015-08-01

    Cationic polymers are widely used as gene carriers, however, these polymers are usually associated with low transfection efficacy and non-negligible toxicity. Fluorination on polymers significantly improves their performances in gene delivery, but a high density of fluorous chains must be conjugated on a single polymer. Here we present a new strategy to construct fluorinated polymers with minimal fluorous chains for efficient DNA and siRNA delivery. A double-tailed fluorous compound 2-chloro-4,6-bis[(perfluorohexyl)propyloxy]-1,3,5-triazine (CBT) was conjugated on dendrimers of different generations and low molecular weight polyethylenimine via a facile synthesis. The yielding products with average numbers of 1-2 conjugated CBT moieties showed much improved EGFP and luciferase transfection efficacy compared to unmodified polymers. In addition, these polymers show high siRNA delivery efficacy on different cell lines. Among the synthesized polymers, generation 1 (G1) dendrimer modified with an average number of 1.9 CBT moieties (G1-CBT1.9) shows the highest efficacy when delivering both DNA and siRNA and its efficacy approaches that of Lipofectamine 2000. G1-CBT1.9 also shows efficient gene silencing in vivo. All of the CBT-modified polymers exhibit minimal toxicity on the cells at their optimal transfection conditions. This study provides a new strategy to design efficient fluorous polymers for DNA and siRNA delivery. PMID:25937003

  5. Delivery of small interfering RNA (siRNA) using the sleeping beauty transposon.

    PubMed

    Fletcher, Bradley S

    2010-11-01

    RNA interference (RNAi) is an evolutionarily conserved process that silences gene expression through double-stranded RNA species in a sequence-specific manner. Small interfering RNAs (siRNAs) can promote sequence-specific degradation and/or translational repression of target RNA by activation of the RNA-induced silencing complex (RISC). Traditionally, silencing in mammalian cells had been achieved by transfection of synthetically derived siRNA duplexes, resulting in transient gene suppression of the target sequence. As the technology was advanced, inhibitory short-hairpin-shaped RNAs (shRNAs) could be produced by transcription from RNA polymerase-III (pol-III)-driven promoters, such as H1, U6, or cytomegalovirus (CMV)-enhanced pol III promoters. Following transcription, the shRNAs are processed by the enzyme Dicer into active siRNA. This approach allows for the continuous production of siRNA within cells using a DNA template and offers increased options for delivery of the pol-III-driven transcriptional units. A number of different viral vectors, as well as plasmid DNAs, have been utilized to deliver shRNA to mammalian cells. Here, the Tc1/mariner DNA transposon Sleeping Beauty (SB) is used as a tool to deliver shRNA-encoding transcriptional units. The SB transposon system uses a "cut-and-paste" mechanism to insert the transposon into random TA dinucleotides within the target genome. The shRNAs are then processed and used for gene knockdown. PMID:21041394

  6. Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision

    PubMed Central

    Denise, Hubert; Moschos, Sterghios A.; Sidders, Benjamin; Burden, Frances; Perkins, Hannah; Carter, Nikki; Stroud, Tim; Kennedy, Michael; Fancy, Sally-Ann; Lapthorn, Cris; Lavender, Helen; Kinloch, Ross; Suhy, David; Corbau, Romu

    2014-01-01

    TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034–encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5′ RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC). PMID:24496437

  7. Polyethylenimine as a promising vector for targeted siRNA delivery.

    PubMed

    Nimesh, Surendra

    2012-05-01

    Recent discovery of RNA interference (RNAi) technology for gene therapy has triggered explosive research efforts towards development of small interfering RNA (siRNA) as therapeutic modality for gene silencing. Owing to its large molecular weight (~13 kDa), polyanionic nature (~40 negative phosphate groups) and rapid enzymatic degradation, delivery of siRNA remains an unresolved issue. Hence, there arises a need of an appropriate delivery vector to overcome the intrinsic, poor intracellular uptake and limited in vitro and in vivo stability. Amongst the various non-viral delivery vectors, the application of polymeric vectors such as polyethylenimine (PEI) or its derivatives has attracted much attention due to its high transfection efficiency and ease of manipulation. PEI has been extensively investigated for DNA delivery, only recently this polymer has been employed for siRNA delivery. This review will focus on studies done on PEI to deliver siRNA, with emphasis on the targeted, self-assembled polymeric nanoparticles with promising potential to evolve as therapeutic tool in gene therapy. PMID:22432843

  8. Engineering RNA for Targeted siRNA Delivery and Medical Application

    PubMed Central

    Guo, Peixuan; Coban, Oana; Snead, Nick; Trebley, Joe; Hoeprich, Steve; Guo, Songchuan; Shu, Yi

    2010-01-01

    RNA engineering for nanotechnology and medical applications is an exciting emerging research field. RNA has intrinsically defined features on the nanometer scale and is a particularly interesting candidate for such applications due to its amazing diversity, flexibility and versatility in structure and function. Specifically, the current use of siRNA to silence target genes involved in disease has generated much excitement in the scientific community. The intrinsic ability to sequence-specifically down-regulate gene expression in a temporally- and spatially-controlled fashion has led to heightened interest and rapid development of siRNA-based therapeutics. Though methods for gene silencing with high efficacy and specificity have been achieved in vitro, the effective delivery of nucleic acids to specific cells in vivo has been a hurdle for RNA therapeutics. This review covers different RNA-based approaches for diagnosis, prevention and treatment of human disease, with a focus on the latest developments of nonviral carriers of siRNA for delivery in vivo. The applications and challenges of siRNA therapy, as well as potential solutions to these problems, the approaches for using phi29 pRNA-based vectors as polyvalent vehicles for specific delivery of siRNA, ribozymes, drugs or other therapeutic agents to specific cells for therapy will also be addressed. PMID:20230868

  9. Chlorotoxin bound magnetic nanovector tailored for cancer cell targeting, imaging, and siRNA delivery.

    PubMed

    Veiseh, Omid; Kievit, Forrest M; Fang, Chen; Mu, Ni; Jana, Soumen; Leung, Matthew C; Mok, Hyejung; Ellenbogen, Richard G; Park, James O; Zhang, Miqin

    2010-11-01

    Ribonucleic acid interference (RNAi) is a powerful molecular tool that has potential to revolutionize the treatment of cancer. One major challenge of applying this technology for clinical application is the lack of site-specific carriers that can effectively deliver short interfering RNA (siRNA) to cancer cells. Here we report the development and assessment of a cancer-cell specific magnetic nanovector construct for efficient siRNA delivery and non-invasive monitoring through magnetic resonance imaging (MRI). The base of the nanovector construct is comprised of a superparamagnetic iron oxide nanoparticle core coated with polyethylene glycol (PEG)-grafted chitosan, and polyethylenimine (PEI). The construct was then further functionalized with siRNA and a tumor-targeting peptide, chlorotoxin (CTX), to improve tumor specificity and potency. Flow cytometry, quantitative RT-PCR, and fluorescence microscopy analyses confirmed receptor-mediated cellular internalization of nanovectors and enhanced gene knockdown through targeted siRNA delivery. The ability of this nanovector construct to generate specific contrast enhancement of glioblastoma cells was demonstrated through MR imaging. These findings suggest that this CTX enabled nanoparticle carrier may be well suited for delivery of RNAi therapeutics to brain cancer cells. PMID:20673683

  10. Octa-functional PLGA nanoparticles for targeted and efficient siRNA delivery to tumors

    PubMed Central

    Zhou, Jiangbing; Patel, Toral R.; Fu, Michael; Bertram, James P.; Saltzman, W. Mark

    2014-01-01

    Therapies based on RNA interference, using agents such as siRNA, are limited by the absence of safe, efficient vehicles for targeted delivery in vivo. The barriers to siRNA delivery are well known and can be individually overcome by addition of functional modules, such as conjugation of moieties for cell penetration or targeting. But, so far, it has been impossible to engineer multiple modules into a single unit. Here, we describe the synthesis of degradable nanoparticles that carry eight synergistic functions: 1) polymer matrix for stabilization/controlled release; 2) siRNA for gene knockdown; 3) agent to enhance endosomal escape; 4) agent to enhance siRNA potency; 5) surface-bound PEG for enhancing circulatory time; and surface-bound peptides for 6) cell penetration; 7) endosomal escape; and 8) tumor targeting. Further, we demonstrate that this approach can provide prolonged knockdown of PLK1 and control of tumor growth in vivo. Importantly, all elements in these octa-functional nanoparticles are known to be safe for human use and each function can be individually controlled, giving this approach to synthetic RNA-loaded nanoparticles potential in a variety of clinical applications. PMID:22014944

  11. Solid lipid and chitosan particulate systems for delivery of siRNA.

    PubMed

    Senel, G; Büyükköroğlu, G; Yazan, Y

    2015-11-01

    Due to the recent advances in molecular biology, there are promising gene therapy studies for prevention and treatment of cancer, genetic and infectious diseases. Many technologies in molecular biology and biotechnology were developed, and among those technologies, 'antisense technology' has become prominent in recent years. In this study, non-viral gene delivery systems such as solid lipid and chitosan nanoparticles were developed for improving intercellular delivery of siRNA. Commercially available Bcl-2 siRNA which is specific for Bcl-2 mRNA was used as a genetic material. Particle size, zeta potential, siRNA binding abilities and cytotoxic properties of the systems were evaluated and transfection assay was performed on among the prepared formulations. When the results of those studies were compared with Lipofectamine 2000, prepared formulations were found to show usable results. A novel method was developed in this study for producing solid lipid nanoparticles (SLNs) with highly efficient siRNA encapsulation. The results of this study showed that the genetic materials can be encapsulated in SLNs and SLNs have the potential to be used as a transfection agents. PMID:26790185

  12. Efficient delivery of siRNA to cortical neurons using layered double hydroxide nanoparticles.

    PubMed

    Wong, Yunyi; Markham, Kathryn; Xu, Zhi Ping; Chen, Min; Max Lu, Gao Qing; Bartlett, Perry F; Cooper, Helen M

    2010-11-01

    Small interfering RNAs (siRNAs) are capable of targeting and destroying specific mRNAs, making them particularly suited to the treatment of neurodegenerative conditions such as Huntington's Disease where the production of abnormal proteins results in a gain-of-function phenotype. Although a variety of nanoparticle formulations are currently under development as siRNA delivery systems, application of these technologies has been limited by their high cytotoxicity, low drug loading capacity and release, and inability to penetrate cell membranes. Layered double hydroxide (LDH) nanoparticles are now emerging as a potential new drug delivery system as they exhibit low cytotoxicity and are highly biocompatible. Here we present the first study investigating LDH delivery of siRNAs to primary cultured neurons. We show that internalization by neurons is rapid, dose-dependent and saturable, and markedly more efficient than in other cell types. We demonstrate that siRNA-LDH complexes are internalized by clathrin-dependent endocytosis at the cell body and in neurites, with subsequent retrograde transport to the cell body followed by efficient release into the cytoplasm. Finally we show that LDH mediated siRNA delivery effectively silences neuronal gene expression. This study therefore confirms the potential of LDH nanoparticles as a drug delivery system for patients suffering from neurodegenerative disease. PMID:20709387

  13. Chemically defined polyethylene glycol siRNA conjugates with enhanced gene silencing effect

    PubMed Central

    Gaziova, Zuzana; Baumann, Volker; Winkler, Anna-Maria; Winkler, Johannes

    2014-01-01

    The therapeutic application of siRNA suffers from poor bioavailability caused by rapid degradation and elimination. The covalent attachment of PEG is a universal concept to increase molecular size and enhance the pharmacokinetic properties of biomacromolecules. We devised a facile approach for attachment of PEG molecules with a defined molecular weight, and successful purification of the resulting conjugates. We directly conjugated structurally defined PEG chains with twelve ethylene glycol units to the 3′-terminal hydroxyl group of both sense and antisense strands via an aminoalkyl linker. The conjugates were easily purified by HPLC and successful PEGylation and molecule integrity were confirmed by ESI-MS. The evaluation of in vitro gene knockdown of two different targets in MCF-7 breast cancer cells showed stable pharmacologic activity when combined with a standard transfection reagent. Sense strand PEGylation even increased the silencing potency of a CRCX4-siRNA which had modest activity in its wild-type form. The results indicate that PEG chains at the 3′-terminus of both strands of siRNA are well tolerated by the RNAi effector. The attachment of short, chemically defined PEG chains is a feasible approach to improve the pharmacokinetic properties of siRNA, and can be combined with other targeted and untargeted delivery vehicles. PMID:24613624

  14. Polymer Nanoparticles Encapsulating siRNA for Treatment of HSV-2 Genital Infection

    PubMed Central

    Steinbach, Jill M.; Weller, Caroline E.; Booth, Carmen J.; Saltzman, W. Mark

    2012-01-01

    Effective, low-cost, and safe treatments for sexually transmitted viral infections are urgently needed. Here, we show for the first time that intravaginal administration with nanoparticles of poly(lactic acid-co-glycolic acid) (PLGA) encapsulating short interfering RNA (siRNA) molecules is effective for prevention of genital HSV-2 infections in mice. PLGA nanoparticles (NPs) were designed to interfere with HSV-2 infection by siRNA-mediated knockdown of nectin, a host cell protein. NPs were characterized in vitro to determine the optimal formulation based on siRNA loading, controlled release profile, and mRNA knockdown. Mice inoculated intravaginally with a lethal dose of HSV-2, and treated with PLGA NPs, showed increased survival from ~9 days (in untreated mice) to > 28 days (in PLGA NP treated mice) - the longest survival ever observed with siRNA treatment in this mouse model. This work provides proof-of-concept that topical administration of NPs containing siRNA against a pathologically relevant host cell target can knockdown the gene in tissue and improve survival after HSV-2 infection. Furthermore, this system provides a safe delivery platform that employs materials that are already approved by the FDA and can be modified to enhance delivery of other microbicides. PMID:22705461

  15. In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight

    PubMed Central

    Khan, Omar; Thiriot, Aude; Jhunjunwala, Siddharth; Shaw, Taylor E.; Xing, Yiping; Sager, Hendrik B.; Sahay, Gaurav; Speciner, Lauren; Bader, Andrew; Bogorad, Roman L.; Yin, Hao; Racie, Tim; Dong, Yizhou; Jiang, Shan; Seedorf, Danielle; Dave, Apeksha; Sandu, Kamaljeet S.; Webber, Matthew J.; Novobrantseva, Tatiana; Ruda, Vera M.; Lytton-Jean, Abigail K.R.; Levins, Christopher G.; Kalish, Brian; Mudge, Dayna K.; Perez, Mario; Abezgauz, Ludmila; Dutta, Partha; Smith, Lynelle; Charisse, Klaus; Kieran, Mark W.; Fitzgerald, Kevin; Nahrendorf, Matthias; Danino, Dganit; Tuder, Rubin M.; von Andrian, Ulrich H.; Akinc, Akin; Schroeder, Avi; Panigrahy, Dipak; Kotelianski, Victor; Langer, Robert; Anderson, Daniel G.

    2014-01-01

    Dysfunctional endothelium contributes to more disease than any other tissue in the body. Small interfering RNAs (siRNAs) have the potential to help study and treat endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here we show that polymeric nanoparticles made of low molecular weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. It mediates the most durable non-liver silencing reported to date, and facilitates the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth, and metastasis. We believe these nanoparticles improve the ability to study endothelial gene function in vivo, and may be used to treat diseases caused by vascular dysfunction. PMID:24813696

  16. A Cell-penetrating Helical Polymer For siRNA Delivery to Mammalian Cells

    PubMed Central

    Gabrielson, Nathan P; Lu, Hua; Yin, Lichen; Kim, Kyung Hoon; Cheng, Jianjun

    2012-01-01

    Cell-penetrating peptides (CPPs) are routinely used for intracellular delivery of a variety of cargo, including drugs, genes, and short interfering RNA (siRNA). Most CPPs are active only upon exposure to acidic environments inside of late endosomes, thereby facilitating the endosomal escape of internalized vectors. Here, we describe the generation of a synthetic polypeptide—PVBLGn-8—that is able to adopt a helical structure independent of pH. Like other CPPs, the helical structure of PVBLGn-8 allows the polypeptide to destabilize membranes. However, since the helix is stable at all physiologically relevant pH values between pH 2 and pH 7.4, the membrane permeation properties of PVBLGn-8 are irreversible. Given its pH-insensitive activity, our results suggest that PVBLGn-8 is able to facilitate efficient siRNA delivery by causing pore formation in the cell membranes through which either free or complexed siRNA is able to diffuse. This nonspecific form of entry into the cell cytosol may prove useful when trying to deliver siRNA to cells which have proven to be difficult to transfect. PMID:22643866

  17. Differential sensitivity of Arabidopsis siRNA biogenesis mutants to genotoxic stress.

    PubMed

    Yao, Youli; Bilichak, Andriy; Golubov, Andrey; Blevins, Todd; Kovalchuk, Igor

    2010-12-01

    Plant response to stress has been linked to different RNA-silencing processes and epigenetic mechanisms. Our recent results showed that Arabidopsis thaliana Dicer-like (DCL) mutants were impaired in transgenerational changes, recombination frequency and stress tolerance. We also found that transgenerational changes were dependent on changes in DNA methylation. Here, we hypothesized that plants deficient in the production of small RNAs would show an impaired abiotic stress response. To test this, we exposed A. thaliana dcl2, dcl3, dcl4, dcl2 dcl3 (d2d3), dcl2 dcl4 (d2d4), dcl2 dcl3 dcl4 (d2d3d4), nrpd1a, rdr2 and rdr6 mutants to methyl methane sulfonate (MMS). We found dcl4 and rdr6 to be more sensitive and dcl2, dcl3, d2d3 and rdr2 plants more resistant to MMS, as shown by fresh weight, root length and survival rate. The in vitro repair assay showed the lower ability of dcl2 and dcl3 to repair UV-damaged DNA. To summarize, we found that whereas mutants impaired in transactivating siRNA biogenesis were more sensitive to MMS, mutants impaired in natural antisense siRNA and heterochromatic siRNA biogeneses were more tolerant. Our data suggest that plant response to MMS is in part regulated through biogenesis of various siRNAs. PMID:20953786

  18. Synthesis and Gene Silencing Properties of siRNAs Containing Terminal Amide Linkages

    PubMed Central

    Gaglione, Maria; Mercurio, M. Emilia; Mosca, Nicola; Novellino, Ettore; Messere, Anna

    2014-01-01

    The active components of the RNAi are 21 nucleotides long dsRNAs containing a 2 nucleotide overhang at the 3′ end, carrying 5′-phosphate and 3′-hydroxyl groups (siRNAs). Structural analysis revealed that the siRNA is functionally bound at both ends to RISC. Terminal modifications are considered with interest as the introduction of chemical moieties interferes with the 3′ overhang recognition by the PAZ domain and the 5′-phosphate recognition by the MID and PIWI domains of RISC. Herein, we report the synthesis of modified siRNAs containing terminal amide linkages by introducing hydroxyethylglycine PNA (hegPNA) moieties at 5′, and at 3′ positions and on both terminals. Results of gene silencing studies highlight that some of these modifications are compatible with the RNAi machinery and markedly increase the resistance to serum-derived nucleases even after 24 h of incubation. Molecular docking simulations were attained to give at atomistic level a clearer picture of the effect of the most performing modifications on the interactions with the human Argonaute 2 PAZ, MID, and PIWI domains. This study adds another piece to the puzzle of the heterogeneous chemical modifications that can be attained to enhance the silencing efficiency of siRNAs. PMID:24791003

  19. DELIVERY OF siRNA INTO BREAST CANCER CELLS VIA PHAGE FUSION PROTEIN-TARGETED LIPOSOMES

    PubMed Central

    Bedi, Deepa; Musacchio, Tiziana; Fagbohun, Olusegun A.; Gillespie, James W.; Deinnocentes, Patricia; Bird, R. Curtis; Bookbinder, Lonnie; Torchilin, Vladimir P.; Petrenko, Valery A.

    2011-01-01

    Efficacy of siRNAs as potential anticancer therapeutics can be increased by their targeted delivery into cancer cells via tumor-specific ligands. Phage display offers an unique approach to identify highly specific and selective ligands that can deliver nanocarriers to the site of disease. In this study, we proved a novel approach for intracellular delivery of siRNAs into breast cancer cells through their encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins. The targeted siRNA liposomes were obtained by a fusion of two parental liposomes containing spontaneously inserted siRNA and fusion phage proteins. The presence of pVIII coat protein fused to a MCF-7 cell-targeting peptide DMPGTVLP in the liposomes was confirmed by Western blotting. The novel phage-targeted siRNA-nanopharmaceuticals demonstrate significant down-regulation of PRDM14 gene expression and PRDM14 protein synthesis in the target MCF- 7 cells. This approach offers the potential for development of new anticancer siRNA-based targeted nanomedicines. PMID:21050894

  20. Efficient intracellular siRNA delivery by ethyleneimine-modified amphiphilic macromolecules.

    PubMed

    Sparks, Sarah M; Waite, Carolyn L; Harmon, Alexander M; Nusblat, Leora M; Roth, Charles M; Uhrich, Kathryn E

    2011-09-01

    New materials that can bind and deliver oligonucleotides such as short interfering RNA (siRNA) without toxicity are greatly needed to fulfill the promise of therapeutic gene silencing. Amphiphilic macromolecules (AMs) were functionalized with linear ethyleneimines to create cationic AMs capable of complexing with siRNA. Structurally, the parent AM is formed from a mucic acid backbone whose tetra-hydroxy groups are alkylated with 12-carbon aliphatic chains to form the hydrophobic component of the macromolecule. This alkylated mucic acid is then mono-functionalized with poly(ethylene glycol) (PEG) as a hydrophilic component. The resulting AM contains a free carboxylic acid within the hydrophobic domain. In this work, linear ethyleneimines were conjugated to the free carboxylic acid to produce an AM with one primary amine (1N) or one primary amine and four secondary amines (5N). Further, an AM with amine substitution both to the free carboxylic acid in the hydrophobic domain and also to the adjacent PEG was synthesized to produce a polymer with one primary amine and eight secondary amines (9N), four located on each side of the AM hydrophobic domain. All amine-functionalized AMs formed nanoscale micelles but only the 5N and 9N AMs had cationic zeta potentials, which increased with increasing number of amines. All AMs exhibited less inherent cytotoxicity than linear polyethyleneimine (L-PEI) at concentrations of 10 µM and above. By increasing the length of the cationic ethyleneimine chain and the total number of amines, successful siRNA complexation and cellular siRNA delivery was achieved in a malignant glioma cell line. In addition, siRNA-induced silencing of firefly luciferase was observed using complexes of siRNA with the 9N AM and comparable to L-PEI, yet showed better cell viability at higher concentrations (above 10 µM). This work highlights the promise of cationic AMs as safe and efficient synthetic vectors for siRNA delivery. Specifically, a novel polymer (9N

  1. Characterization and comparison of two novel nanosystems associated with siRNA for cellular therapy.

    PubMed

    André, E M; Pensado, A; Resnier, P; Braz, L; Rosa da Costa, A M; Passirani, C; Sanchez, A; Montero-Menei, C N

    2016-01-30

    To direct stem cell fate, a delicate control of gene expression through small interference RNA (siRNA) is emerging as a new and safe promising strategy. In this way, the expression of proteins hindering neuronal commitment may be transiently inhibited thus driving differentiation. Mesenchymal stem cells (MSC), which secrete tissue repair factors, possess immunomodulatory properties and may differentiate towards the neuronal lineage, are a promising cell source for cell therapy studies in the central nervous system. To better drive their neuronal commitment the repressor Element-1 silencing transcription (REST) factor, may be inhibited by siRNA technology. The design of novel nanoparticles (NP) capable of safely delivering nucleic acids is crucial in order to successfully develop this strategy. In this study we developed and characterized two different siRNA NP. On one hand, sorbitan monooleate (Span(®)80) based NP incorporating the cationic components poly-l-arginine or cationized pullulan, thus allowing the association of siRNA were designed. These NP presented a small size (205 nm) and a negative surface charge (-38 mV). On the other hand, lipid nanocapsules (LNC) associating polymers with lipids and allowing encapsulation of siRNA complexed with lipoplexes were also developed. Their size was of 82 nm with a positive surface charge of +7 mV. Both NP could be frozen with appropriate cryoprotectors. Cytotoxicity and transfection efficiency at different siRNA doses were monitored by evaluating REST expression. An inhibition of around 60% of REST expression was observed with both NP when associating 250 ng/mL of siRNA-REST, as recommended for commercial reagents. Span NP were less toxic for human MSCs than LNCs, but although both NP showed a similar inhibition of REST over time and the induction of neuronal commitment, LNC-siREST induced a higher expression of neuronal markers. Therefore, two different tailored siRNA NP offering great potential for human stem cell

  2. The antifibrotic effects of TGF-{beta}1 siRNA on hepatic fibrosis in rats

    SciTech Connect

    Lang, Qing; Liu, Qi; Xu, Ning; Qian, Ke-Li; Qi, Jing-Hu; Sun, Yin-Chun; Xiao, Lang; Shi, Xiao-Feng

    2011-06-10

    Highlights: {yields} We constructed CCL4 induced liver fibrosis model successfully. {yields} We proofed that the TGF-{beta}1 siRNA had a definite therapy effect to CCL4 induced liver fibrosis. {yields} The therapy effect of TGF-{beta}1 siRNA had dose-dependent. -- Abstract: Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-{beta}1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-{beta}1 in hepatic fibrosis and its mechanism in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague-Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-{beta}1 siRNA 0.125 mg/kg treatment group, TGF-{beta}1 siRNA 0.25 mg/kg treatment group and TGF-{beta}1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin-Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-{beta}1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-{beta}1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-{beta}1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-{beta}1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-{beta}1 siRNA negative control group and the TGF-{beta}1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the m

  3. Target Gene Abundance Contributes to the Efficiency of siRNA-Mediated Gene Silencing

    PubMed Central

    Hong, Sun Woo; Jiang, Yuanyuan; Kim, Soyoun; Li, Chiang J.

    2014-01-01

    The gene-silencing activity of a small interfering RNA (siRNA) is determined by various factors. Considering that RNA interference (RNAi) is an unparalleled technology in both basic research and therapeutic applications, thorough understanding of the factors determining RNAi activity is critical. This report presents observations that siRNAs targeting KRT7 show cell-line-dependent activity, which correlates with the expression level of KRT7 mRNA. By modulating the target mRNA level, it was confirmed that highly expressed genes are more susceptible to siRNA-mediated gene silencing. Finally, several genes that show different expression levels in a cell-line dependent manner were tested, which verified the expression-level-dependent siRNA activities. These results strongly suggest that the abundance of target mRNA is a critical factor that determines the efficiency of the siRNA-mediated gene silencing in a given cellular context. This report should provide practical guidelines for designing RNAi experiments and for selecting targetable genes in RNAi therapeutics studies. PMID:24527979

  4. Development of a novel endosomolytic diblock copolymer for siRNA delivery

    PubMed Central

    Convertine, Anthony J.; Benoit, Danielle S.W.; Duvall, Craig L.; Hoffman, Allan S.; Stayton, Patrick S.

    2011-01-01

    The gene knockdown activity of small interfering RNA (siRNA) has led to their use as target validation tools and as potential therapeutics for a variety of diseases. The delivery of these double-stranded RNA macromolecules has proven to be challenging, however, and in many cases, is a barrier to their deployment. Here we report the development of a new diblock copolymer family that was designed to enhance the systemic and intracellular delivery of siRNA. These diblock copolymers were synthesized using the controlled reversible addition fragmentation chain transfer polymerization (RAFT) method and are composed of a positively-charged block of dimethylaminoethyl methacrylate (DMAEMA) to mediate siRNA condensation, and a second endosomal-releasing block composed of DMAEMA and propylacrylic acid (PAA) in roughly equimolar ratios, together with butyl methacylate (BMA). A related series of diblock compositions were characterized, with the cationic block kept constant, and with the ratio of DMAEMA and PAA to BMA varied. These carriers became sharply hemolytic at endosomal pH regimes, with increasing hemolytic activity seen as the percentage of BMA in the second block was systematically increased. The diblock copolymers condensed siRNA into 80–250 nm particles with slightly positive Zeta potentials. SiRNA-mediated knockdown of a model protein, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH), in HeLa cells generally followed the hemolytic activity trends, with the most hydrophobic second block (highest BMA content) exhibiting the best knockdown. This pH-responsive carrier designed to mediate endosomal release shows significant promise for the intracellular delivery of siRNA. PMID:18973780

  5. Development of a novel endosomolytic diblock copolymer for siRNA delivery.

    PubMed

    Convertine, Anthony J; Benoit, Danielle S W; Duvall, Craig L; Hoffman, Allan S; Stayton, Patrick S

    2009-02-10

    The gene knockdown activity of small interfering RNA (siRNA) has led to their use as target validation tools and as potential therapeutics for a variety of diseases. The delivery of these double-stranded RNA macromolecules has proven to be challenging, however, and in many cases, is a barrier to their deployment. Here we report the development of a new diblock copolymer family that was designed to enhance the systemic and intracellular delivery of siRNA. These diblock copolymers were synthesized using the controlled reversible addition fragmentation chain transfer polymerization (RAFT) method and are composed of a positively-charged block of dimethylaminoethyl methacrylate (DMAEMA) to mediate siRNA condensation, and a second endosomal-releasing block composed of DMAEMA and propylacrylic acid (PAA) in roughly equimolar ratios, together with butyl methacylate (BMA). A related series of diblock compositions were characterized, with the cationic block kept constant, and with the ratio of DMAEMA and PAA to BMA varied. These carriers became sharply hemolytic at endosomal pH regimes, with increasing hemolytic activity seen as the percentage of BMA in the second block was systematically increased. The diblock copolymers condensed siRNA into 80-250 nm particles with slightly positive Zeta potentials. SiRNA-mediated knockdown of a model protein, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH), in HeLa cells generally followed the hemolytic activity trends, with the most hydrophobic second block (highest BMA content) exhibiting the best knockdown. This pH-responsive carrier designed to mediate endosomal release shows significant promise for the intracellular delivery of siRNA. PMID:18973780

  6. Tumor priming enhances siRNA delivery and transfection in intraperitoneal tumors.

    PubMed

    Wang, Jie; Lu, Ze; Yeung, Bertrand Z; Wientjes, M Guillaume; Cole, David J; Au, Jessie L-S

    2014-03-28

    Cancers originating from the digestive system account for 290,000 or ~20% of all new cancer cases annually in the US. We previously developed paclitaxel-loaded tumor-penetrating microparticles (TPM) for intraperitoneal (IP) treatment of peritoneal tumors (Lu et al., 2008; Tsai et al., 2007; Tsai et al., 2013). TPM is undergoing NIH-supported IND-enabling studies for clinical evaluation. The present study evaluated the hypothesis that TPM, via inducing apoptosis and expanding the interstitial space, promotes the delivery and transfection of lipid vectors containing siRNA. The in vivo model was the metastatic human Hs766T pancreatic tumor that, upon IP injection, produced widely distributed solid tumors and ascites in the peritoneal cavity in 100% of animals. The target gene was survivin, an anti-apoptotic protein induced by chemotherapy and associated with metastases and poor prognosis of patients with gastric and colorectal cancers. The siRNA carrier was pegylated liposomes comprising cationic and neutral lipids plus a fusogenic lipid (PCat). PCat-loaded with survivin siRNA (PCat-siSurvivin) was active in cultured cells (decreased survivin mRNA and protein levels, reduced cell clonogenicity, enhanced paclitaxel activity), but lost its activity in vivo; this difference is consistent with the well-known problem of inadequate delivery and transfection of siRNA in vivo. In comparison, single agent TPM prolonged animal survival and, as expected, induced survivin expression in tumors. Addition of PCat-siSurvivin reversed the TPM-induced survivin expression and enhanced the antitumor activity of TPM. The finding that in vivo survivin knockdown by PCat-siSurvivin was successful only when it was given in combination with TPM provides the proof-of-concept that tumor priming promotes the delivery and transfection of liposomal siRNA. The data further suggest the TPM/PCat-siSurvivin combination as a potentially useful chemo-gene therapy for peritoneal cancer. PMID:24462901

  7. A designed recombinant fusion protein for targeted delivery of siRNA to the mouse brain.

    PubMed

    Haroon, Mohamed Mohamed; Dar, Ghulam Hassan; Jeyalakshmi, Durga; Venkatraman, Uthra; Saba, Kamal; Rangaraj, Nandini; Patel, Anant Bahadur; Gopal, Vijaya

    2016-04-28

    RNA interference represents a novel therapeutic approach to modulate several neurodegenerative disease-related genes. However, exogenous delivery of siRNA restricts their transport into different tissues and specifically into the brain mainly due to its large size and the presence of the blood-brain barrier (BBB). To overcome these challenges, we developed here a strategy wherein a peptide known to target specific gangliosides was fused to a double-stranded RNA binding protein to deliver siRNA to the brain parenchyma. The designed fusion protein designated as TARBP-BTP consists of a double-stranded RNA-binding domain (dsRBD) of human Trans Activation response element (TAR) RNA Binding Protein (TARBP2) fused to a brain targeting peptide that binds to monosialoganglioside GM1. Conformation-specific binding of TARBP2 domain to siRNA led to the formation of homogenous serum-stable complex with targeting potential. Further, uptake of the complex in Neuro-2a, IMR32 and HepG2 cells analyzed by confocal microscopy and fluorescence activated cell sorting, revealed selective requirement of GM1 for entry. Remarkably, systemic delivery of the fluorescently labeled complex (TARBP-BTP:siRNA) in ΑβPP-PS1 mouse model of Alzheimer's disease (AD) led to distinctive localization in the cerebral hemisphere. Further, the delivery of siRNA mediated by TARBP-BTP led to significant knockdown of BACE1 in the brain, in both ΑβPP-PS1 mice and wild type C57BL/6. The study establishes the growing importance of fusion proteins in delivering therapeutic siRNA to brain tissues. PMID:26948382

  8. Conjugated polymer nanoparticles for effective siRNA delivery to tobacco BY-2 protoplasts

    PubMed Central

    2010-01-01

    Background Post transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Plant biologists have employed electroporation to deliver artificial siRNAs to plant protoplasts to study gene expression mechanisms at the single cell level. One drawback of electroporation is the extensive loss of viable protoplasts that occurs as a result of the transfection technology. Results We employed fluorescent conjugated polymer nanoparticles (CPNs) to deliver siRNAs and knockdown a target gene in plant protoplasts. CPNs are non toxic to protoplasts, having little impact on viability over a 72 h period. Microscopy and flow cytometry reveal that CPNs can penetrate protoplasts within 2 h of delivery. Cellular uptake of CPNs/siRNA complexes were easily monitored using epifluorescence microscopy. We also demonstrate that CPNs can deliver siRNAs targeting specific genes in the cellulose biosynthesis pathway (NtCesA-1a and NtCesA-1b). Conclusions While prior work showed that NtCesA-1 is a factor involved in cell wall synthesis in whole plants, we demonstrate that the same gene plays an essential role in cell wall regeneration in isolated protoplasts. Cell wall biosynthesis is central to cell elongation, plant growth and development. The experiments presented here shows that NtCesA is also a factor in cell viability. We show that CPNs are valuable vehicles for delivering siRNAs to plant protoplasts to study vital cellular pathways at the single cell level. PMID:21192827

  9. A biomimetic nanovector-mediated targeted cholesterol-conjugated siRNA delivery for tumor gene therapy.

    PubMed

    Ding, Yang; Wang, Wei; Feng, Meiqing; Wang, Yu; Zhou, Jianping; Ding, Xuefang; Zhou, Xin; Liu, Congyan; Wang, Ruoning; Zhang, Qiang

    2012-12-01

    RNA interference holds tremendous potential as a therapeutic approach of malignant tumors. However, safe and efficient nanovectors are extremely lack for systemic delivery of small interfering RNA (siRNA). The study aimed to develop a biomimetic nanovector, reconstituted high density lipoprotein (rHDL), mediating targeted cholesterol-conjugated siRNA (Chol-siRNA) delivery for Pokemon gene silencing therapy. Chol-siRNA-loaded rHDL nanoparticles (rHDL/Chol-siRNA complexes) were prepared using thin-film dispersion method and their characteristics were investigated in detail. RHDL/Chol-siRNA complexes at the optimal volume ratio (lipid: Chol-siRNA) exhibited high Chol-siRNA-loading efficiency (~99%), desirable nanoparticle size and excellent stability in serum. In addition, by analyzing Chol-siRNA release profile, rHDL/Chol-siRNA complexes displayed sustained-release characteristic and storage stability. Observations from FACS and confocal microscopic analyses revealed that rHDL-mediated carboxyfluorescein tagged Chol-siRNA (FAM-Chol-siRNA) transfection resulted in highly efficient uptake and specific cytoplasmic delivery of FAM-Chol-siRNA into human hepatocellular carcinoma cell line HepG2 via HDL-receptor mediated mechanism. In vitro cytotoxicity, apoptosis and Western-blot analyses revealed significant cellular growth inhibition and decrease of Pokemon and Bcl-2 protein expression in HepG2 cells treated with Chol-siRNA-Pokemon-loaded rHDL nanoparticles (rHDL/Chol-siRNA-Pokemon complexes), respectively. In in vivo studies, the near-infrared (NIR) dye Cy5 labeled Chol-siRNA-loaded rHDL nanoparticles (rHDL/Cy5-Chol-siRNA complexes) obviously accumulated in tumor of nude mice after i.v. administration as compared with Cy5-Chol-siRNA-loaded lipoplexes (Lipos/Cy5-Chol-siRNA complexes). Morover, rHDL/Chol-siRNA-Pokemon complexes demonstrated great tumor growth inhibition and significant decrease of Pokemon and Bcl-2 protein expression in vivo. These results suggested that

  10. Evolutions from extremality

    NASA Astrophysics Data System (ADS)

    Booth, Ivan

    2016-04-01

    We examine the evolution of extremal spherically symmetric black holes, developing both general theory as well as the specific cases of (charged) null dust and massless scalar field spacetimes. As matter accretes onto extremal marginally trapped tubes, they generically evolve to become nonextremal, with the initial extremal horizon bifurcating into inner and outer nonextremal horizons. At the start of this process arbitrarily slow matter accretion can cause a geometrically invariant measure of horizon growth to jump from zero to infinity. We also consider dynamical horizons that are extremal throughout their evolution and see that such spacetimes contain two extremal black hole horizons: an inner isolated one and an outer dynamical one. We compare these extremal dynamical horizons with the dynamical extreme event horizon spacetimes of Murata, Reall and Tanahashi.

  11. Capella: Structure and Abundances

    NASA Technical Reports Server (NTRS)

    Brickhouse, Nancy S.

    1999-01-01

    This grant covers the analysis of ASCA spectra of the cool star binary system Capella. This project has also required the analysis of simultaneous EUVE data. The ASCA spectrum of Capella could not be fit with standard models; by imposing models based on strong lines observed with EUVE, a problem wavelength region was identified. Correcting the problem required calculations of atomic collision strengths of higher principal quantum number than had ever been calculated, resulting in a paper in process by Liedahl and Brickhouse. With these new models applied to the ASCA spectrum, better fits were obtained. While solar abundance ratios are generally consistent with the ASCA data, the ratio of Ne/Fe is three to four times lower than solar photospheric values. Whether there is a general First Ionization Potential (FIP) effect or a specific neon anomaly cannot be determined from these data. Detailed discussion has been provided to NASA in the most recent annual report (1997). Two poster presentations have been made regarding modeling requirements. A substantial paper is in the final revision form, following review by six co-authors. The results of this work have wide implications, since the newly calculated emission lines almost certainly contribute to other problems in fitting not only other stellar spectra, but also composite supernova remnants, galaxies, and cooling flow clusters of galaxies. Furthermore, Liedahl and Brickhouse have identified other species for which lines of a similar nature (high principal quantum number) will contribute significant flux. For moderate resolution X-ray spectra, lines left out of the models in relatively isolated bands, will be attributed to continuum flux by spectral fitting engines, causing errors in line-to-continuum ratios. Thus addressing the general theoretical problem is of crucial importance.

  12. Abundances in dwarf irregular galaxies

    NASA Technical Reports Server (NTRS)

    Dufour, Reginald J.

    1986-01-01

    The results of abundance studies of dwarf irregular galaxies and similar objects are reviewed with special attention to variations in the CNO element group. Observations of the forbidden N II and semiforbidden C III lines in the most metal-poor galaxy known, IZw 18, are presented for the first time and CNO abundances are derived via a photoionization model and discussed in the context of the abundances found in other metal-poor H II regions and galaxies.

  13. Delivery of kinesin spindle protein targeting siRNA in solid lipid nanoparticles to cellular models of tumor vasculature

    SciTech Connect

    Ying, Bo; Campbell, Robert B.

    2014-04-04

    Highlights: • siRNA-lipid nanoparticles are solid particles not lipid bilayers with aqueous core. • High, but not low, PEG content can prevent nanoparticle encapsulation of siRNA. • PEG reduces cellular toxicity of cationic nanoparticles in vitro. • PEG reduces zeta potential while improving gene silencing of siRNA nanoparticles. • Kinesin spindle protein can be an effective target for tumor vascular targeting. - Abstract: The ideal siRNA delivery system should selectively deliver the construct to the target cell, avoid enzymatic degradation, and evade uptake by phagocytes. In the present study, we evaluated the importance of polyethylene glycol (PEG) on lipid-based carrier systems for encapsulating, and delivering, siRNA to tumor vessels using cellular models. Lipid nanoparticles containing different percentage of PEG were evaluated based on their physical chemical properties, density compared to water, siRNA encapsulation, toxicity, targeting efficiency and gene silencing in vitro. siRNA can be efficiently loaded into lipid nanoparticles (LNPs) when DOTAP is included in the formulation mixture. However, the total amount encapsulated decreased with increase in PEG content. In the presence of siRNA, the final formulations contained a mixed population of particles based on density. The major population which contains the majority of siRNA exhibited a density of 4% glucose, and the minor fraction associated with a decreased amount of siRNA had a density less than PBS. The inclusion of 10 mol% PEG resulted in a greater amount of siRNA associated with the minor fraction. Finally, when kinesin spindle protein (KSP) siRNA was encapsulated in lipid nanoparticles containing a modest amount of PEG, the proliferation of endothelial cells was inhibited due to the efficient knock down of KSP mRNA. The presence of siRNA resulted in the formation of solid lipid nanoparticles when prepared using the thin film and hydration method. LNPs with a relatively modest amount of

  14. Treatment with connexin 46 siRNA suppresses the growth of human Y79 retinoblastoma cell xenografts in vivo.

    PubMed

    Burr, Diana B; Molina, Samuel A; Banerjee, Debarshi; Low, Derek M; Takemoto, Dolores J

    2011-04-01

    Tumors with a hypoxic component, including human Y79 retinoblastoma cells, express a specific gap junction protein, Connexin 46 (Cx46), which is usually only found in naturally hypoxic tissues such as the differentiated lens. The aim of this study was to investigate if Cx46 downregulation would suppress Y79 tumor formation in vivo. Five-week old nude mice were subcutaneously implanted with human Y79 retinoblastoma cells and treated with intratumor siRNA injections of 30 μg Cx46 siRNA (n = 6), 30 μg non-silencing siRNA (n = 6), or no siRNA treatment (n = 6) every 2 days for a maximum of 10 treatments. Tumor volume (TV) was calculated from the recorded caliper measurements of length and width. Excised tumors were measured and weighed. Western blot analyses were performed to evaluate Cx46 and Cx43 expression in tumors which received Cx46 siRNA, non-silencing siRNA, or no siRNA treatment. Tumor histopathology was used to assess tumor features. Cx46 siRNA treated Y79 tumors had a reduced TV (287 mm(3) ± 77 mm(3)) when compared to the tumors of mice receiving the negative control siRNA (894 mm(3) ± 218 mm(3); P ≤ 0.03) or no siRNA (1068 mm(3) ± 192 mm(3); P ≤ 0.002). A 6-fold knockdown of Cx46 and a 3-fold rise in Cx43 protein expression was observed from western blots of tumors treated with Cx46 siRNA compared to mice treated with non-silencing siRNA. Knockdown of Cx46 with siRNA had an antitumor effect on human Y79 retinoblastoma tumors in the nude mouse model. The results suggest that anti-Cx46 therapy may be a potential target in the future treatment of retinoblastoma. PMID:21320488

  15. Abundance coefficients, a new method for measuring microorganism relative abundance

    USGS Publications Warehouse

    Forester, R.M.

    1977-01-01

    A new method of measuring the relative abundance of microorganisms by using a set of interrelated coefficients, termed 'abundance coefficients' or 'AC', is proposed. These coefficients provide a means of recording abundance for geometric density categories, and each density measurement represents an approximation of the Poisson parameter ??t. The AC is the natural logarithm of a 'characteristic value,' which is a particular number for each geometric density category. The 'characteristic values' are based upon a probabilistic error statement derived from the Poisson formula, and they present evidence for separation of the geometric category boundaries by e = 2.71828. The proposed AC provide a means for recording species abundance in a manner suitable for arithmetic manipulation, for population structure studies, and for the determination of practical limits for defining the presence or absence of a species. Further, these coefficients provide for both intrasample and intersample abundance comparisons. ?? 1977 Plenum Publishing Corporation.

  16. Trek and ECCO: Abundance measurements of ultraheavy galactic cosmic rays

    NASA Astrophysics Data System (ADS)

    Westphal, Andrew J.

    2000-06-01

    Using the Trek detector, we have measured the abundances of the heaviest elements (with Z>70) in the galactic cosmic rays with sufficient charge resolution to resolve the even-Z elements. We find that the abundance of Pb compared to Pt is ~3 times lower than the value expected from the most widely-held class of models of the origin of galactic cosmic ray nuclei, that is, origination in a partially ionized medium with solar-like composition. The low abundance of Pb is, however, consistent with the interstellar gas and dust model of Meyer, Drury and Ellison, and with a source enriched in r-process material, proposed by Binns et al. A high-resolution, high-statistics measurement of the abundances of the individual actinides would distinguish between these models. This is the goal of ECCO, the Extremely Heavy Cosmic-ray Composition Observer, which we plan to deploy on the International Space Station. .

  17. Self-assembled Nanoscale Coordination Polymers Carrying siRNAs and Cisplatin for Effective Treatment of Resistant Ovarian Cancer

    PubMed Central

    He, Chunbai; Liu, Demin; Lin, Wenbin

    2014-01-01

    Resistance to the chemotherapeutic agent cisplatin is a major limitation for the successful treatment of many cancers. Development of novel strategies to overcome intrinsic and acquired resistance to chemotherapy is of critical importance to effective treatment of ovarian cancer and other types of cancers. We have sought to re-sensitize resistant ovarian cancer cells to chemotherapy by co-delivering chemotherapeutics and pooled siRNAs targeting multi-drug resistance (MDR) genes using self-assembled nanoscale coordination polymers (NCPs). In this work, NCP-1 particles with trigger release properties were first constructed by linking cisplatin prodrug-based bisphosphonate bridging ligands with Zn2+ metal-connecting points and then coated with a cationic lipid layer, followed by the adsorption of pooled siRNAs targeting three MDR genes including survivin, Bcl-2, and P-glycoprotein via electrostatic interactions. The resulting NCP-1/siRNA particles promoted cellular uptake of cisplatin and siRNA and enabled efficient endosomal escape in cisplatin-resistant ovarian cancer cells. By down-regulating the expression of MDR genes, NCP-1/siRNAs enhanced the chemotherapeutic efficacy as indicated by cell viability assay, DNA ladder, and flow cytometry. Local administration of NCP-1/siRNAs effectively reduced tumor sizes of cisplatin-resistant SKOV-3 subcutaneous xenografts. This work shows that the NCP-1/siRNA platform holds great promise in enhancing chemotherapeutic efficacy for the effective treatment of drug-resistant cancers. PMID:25315138

  18. Anticancer siRNA cocktails as a novel tool to treat cancer cells. Part (B). Efficiency of pharmacological action.

    PubMed

    Dzmitruk, Volha; Szulc, Aleksandra; Shcharbin, Dzmitry; Janaszewska, Anna; Shcharbina, Natallia; Lazniewska, Joanna; Novopashina, Darya; Buyanova, Marina; Ionov, Maksim; Klajnert-Maculewicz, Barbara; Gómez-Ramirez, Rafael; Mignani, Serge; Majoral, Jean-Pierre; Muñoz-Fernández, Maria Angeles; Bryszewska, Maria

    2015-05-15

    This paper examines a perspective to use newly engineered nanomaterials as effective and safe carriers for gene therapy of cancer. Three different groups of cationic dendrimers (PAMAM, phosphorus, and carbosilane) were complexed with anticancer siRNA and the biophysical properties of the dendriplexes created were analyzed. The potential of the dendrimers as nanocarriers for anticancer Bcl-xl, Bcl-2, Mcl-1 siRNAs and additionally a scrambled sequence siRNA has been explored. Dendrimer/siRNA complexes were characterised by various methods including fluorescence, zeta potential, dynamic light scattering, circular dichroism, gel electrophoresis and transmission electron microscopy. In this part of study, the transfection of complexes in HeLa and HL-60 cells was analyzed using both single apoptotic siRNAs and a mixture (cocktail) of them. Cocktails were more effective than single siRNAs, allowing one to decrease siRNAs concentration in treating cells. The dendrimers were compared as siRNA carriers, the most effective being the phosphorus-based ones. However, they were also the most cytotoxic on their own, so that in this regard the application of all dendrimers in anticancer therapy will be discussed. PMID:25796120

  19. Enhanced cellular uptake and gene silencing activity of siRNA molecules mediated by chitosan-derivative nanocomplexes.

    PubMed

    Guzman-Villanueva, Diana; El-Sherbiny, Ibrahim M; Vlassov, Alexander V; Herrera-Ruiz, Dea; Smyth, Hugh D C

    2014-10-01

    The RNA interference (RNAi) constitutes a conservative mechanism in eukaryotic cells that induces silencing of target genes. In mammalians, the RNAi is triggered by siRNA (small interfering RNA) molecules. Due to its potential in silencing specific genes, the siRNA has been considered a potential alternative for the treatment of genetic and acquired diseases. However, the siRNA therapy has been limited by its low stability and rapid degradation in presence of nucleases, low cellular uptake, and immune response activation. In order to overcome these drawbacks, we propose the synthesis and characterization of non-viral delivery systems using chitosan derivatives to obtain siRNA complexes (polyplexes). The non-viral delivery systems synthesized included PEG-g-OCs (oligochitosan) and PEG-g-Cs (chitosan medium molecular weight). Both systems allowed the formation of siRNA polyplexes, increased the stability of siRNA in the presence of nucleases, enhanced cellular internalization, and showed low toxicity in the A549 cell line. Finally, the complexes obtained with the PEG-g-OCs system showed silencing activity in a GFP model in the cell line A549 in comparison with naked siRNA. PMID:25063077

  20. Noninvasive Drug Delivery Using Ultrasound: Targeting Melanoma Using siRNA Against Mutant (V600E) B-Raf

    NASA Astrophysics Data System (ADS)

    Tran, Melissa A.; Gowda, Raghavendra; Park, Eun-Joo; Adair, James; Smith, Nadine; Kester, Mark; Robertson, Gavin P.

    2009-04-01

    Melanoma is the most deadly form of skin cancer. Currently early surgical removal is the best treatment option for melanoma patients with little hope of successful treatment of late stage melanoma. Clearly new treatment options must be explored. Topical administration of drugs provides the advantage of being able to apply large quantities of drug in close proximity to the tumor without the issue of systemic side effects. However, the natural barrier formed by the skin must first be overcome for topical treatment to become a viable option. With this in mind we have sought to use low-frequency ultrasound to transiently permeabilize the stratum corneum and successfully deliver liposomal siRNA to melanoma cells residing at the basement membrane. B-Raf is one of the most frequently activated genes in melanoma, making it an ideal candidate for targeting via siRNA. The novel liposomes used in this study load siRNA, protect if from the outside environment and lead to knockdown of target message. Combining ultrasound with liposomal siRNA we show that siRNA can be delivered into melanoma cells. Additionally, we show that siRNA to mutant B-Raf can effectively inhibit melanoma growth in reconstructs and in mice by 60% and 30% respectively. Therefore, ultrasound with liposomal siRNA is a potentially valuable treatment option for melanoma patients.

  1. A novel conditional gene silencing method using a tumor-specific and heat-inducible siRNA system.

    PubMed

    Feng, Jing; Wang, Xiaoyu; Liao, Yi; Feng, Jianguo; Tang, Liling

    2016-06-01

    RNAi technology is an invaluable tool for investigating gene function. However, the non-temporal and non-spatial control is the primary limitation, which leads to siRNA leakiness and off-target effects. In this study, we inserted three kinds of HSE into tumor specific promoter hTERT, which aims to construct a temperature-inducible and tumor-specific RNAi plasmid vector. In our system, the expression of mature siRNA is tightly controlled by the heat shock-inducible and tumor-specific promoters. From the expression level of RNA and protein, we determined the efficiency of the inducible siRNA system by targeting SNCG gene in HepG2 and MCF-7 cells. Results showed that the controllable siRNA system could be induced to initiate siRNA expression by heat-induce. The silencing effect of SNCG is on a relative low level (10 %) at 37 °C, while it is significantly increased to 50 or 60 % after heat inducing at 43 °C. This new conditional siRNA system provides a novel approach to drive the siRNA expression by heat-inducible and tumor-specific promoter. PMID:27033537

  2. Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis.

    PubMed

    Borsani, Omar; Zhu, Jianhua; Verslues, Paul E; Sunkar, Ramanjulu; Zhu, Jian-Kang

    2005-12-29

    In higher eukaryotes, miRNAs and siRNAs guide translational inhibition, mRNA cleavage, or chromatin regulation. We found that the antisense overlapping gene pair of Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH), a stress-related gene, and SRO5, a gene of unknown function, generates two types of siRNAs. When both transcripts are present, a 24-nt siRNA is formed by a biogenesis pathway dependent on DCL2, RDR6, SGS3, and NRPD1A. Initial cleavage of the P5CDH transcript guided by the 24-nt siRNA establishes a phase for the subsequent generation of 21-nt siRNAs by DCL1 and further cleavage of P5CDH transcripts. The expression of SRO5 is induced by salt, and this induction is required to initiate siRNA formation. Our data suggest that the P5CDH and SRO5 proteins are also functionally related, and that the P5CDH-SRO5 gene pair defines a mode of siRNA function and biogenesis that may be applied to other natural cis-antisense gene pairs in eukaryotic genomes. PMID:16377568

  3. Targeted siRNA Delivery Using a Lipo-Oligoaminoamide Nanocore with an Influenza Peptide and Transferrin Shell.

    PubMed

    Zhang, Wei; Müller, Katharina; Kessel, Eva; Reinhard, Sören; He, Dongsheng; Klein, Philipp M; Höhn, Miriam; Rödl, Wolfgang; Kempter, Susanne; Wagner, Ernst

    2016-06-01

    Developing RNA-interference-based therapeutic approaches with efficient and targeted cytosolic delivery of small interfering RNA (siRNA) is remaining a critical challenge since two decades. Herein, a multifunctional transferrin receptor (TfR)-targeted siRNA delivery system (Tf&INF7) is designed based on siRNA complexes formed with the cationic lipo-oligoamino amide 454, sequentially surface-modified with polyethylene glycol-linked transferrin (Tf) for receptor targeting and the endosomolytic peptide INF7 for efficient cytosolic release of the siRNA. Effective Tf&INF7 polyplex internalization and target gene silencing are demonstrated for the TfR overexpressing tumor cell lines (K562, D145, and N2a). Treatment with antitumoral EG5 siRNA results in a block of tumor cell growth and triggered apoptosis. Tf-modified polyplexes are far more effective than the corresponding albumin- (Alb) or nonmodified 454 polyplexes. Competition experiments with excess of Tf demonstrate TfR target specificity. As alternative to the ligand Tf, an anti-murine TfR antibody is incorporated into the polyplexes for specific targeting and gene silencing in the murine N2a cell line. In vivo distribution studies not only demonstrate an enhanced tumor residence of siRNA in N2a tumor-bearing mice with the Tf&INF7 as compared to the 454 polyplex group but also a reduced siRNA nanoparticle stability limiting the in vivo performance. PMID:27109317

  4. Gut Microbiota and Extreme Longevity.

    PubMed

    Biagi, Elena; Franceschi, Claudio; Rampelli, Simone; Severgnini, Marco; Ostan, Rita; Turroni, Silvia; Consolandi, Clarissa; Quercia, Sara; Scurti, Maria; Monti, Daniela; Capri, Miriam; Brigidi, Patrizia; Candela, Marco

    2016-06-01

    The study of the extreme limits of human lifespan may allow a better understanding of how human beings can escape, delay, or survive the most frequent age-related causes of morbidity, a peculiarity shown by long-living individuals. Longevity is a complex trait in which genetics, environment, and stochasticity concur to determine the chance to reach 100 or more years of age [1]. Because of its impact on human metabolism and immunology, the gut microbiome has been proposed as a possible determinant of healthy aging [2, 3]. Indeed, the preservation of host-microbes homeostasis can counteract inflammaging [4], intestinal permeability [5], and decline in bone and cognitive health [6, 7]. Aiming at deepening our knowledge on the relationship between the gut microbiota and a long-living host, we provide for the first time the phylogenetic microbiota analysis of semi-supercentenarians, i.e., 105-109 years old, in comparison to adults, elderly, and centenarians, thus reconstructing the longest available human microbiota trajectory along aging. We highlighted the presence of a core microbiota of highly occurring, symbiotic bacterial taxa (mostly belonging to the dominant Ruminococcaceae, Lachnospiraceae, and Bacteroidaceae families), with a cumulative abundance decreasing along with age. Aging is characterized by an increasing abundance of subdominant species, as well as a rearrangement in their co-occurrence network. These features are maintained in longevity and extreme longevity, but peculiarities emerged, especially in semi-supercentenarians, describing changes that, even accommodating opportunistic and allochthonous bacteria, might possibly support health maintenance during aging, such as an enrichment and/or higher prevalence of health-associated groups (e.g., Akkermansia, Bifidobacterium, and Christensenellaceae). PMID:27185560

  5. Precise engineering of siRNA delivery vehicles to tumors using polyion complexes and gold nanoparticles.

    PubMed

    Kim, Hyun Jin; Takemoto, Hiroyasu; Yi, Yu; Zheng, Meng; Maeda, Yoshinori; Chaya, Hiroyuki; Hayashi, Kotaro; Mi, Peng; Pittella, Frederico; Christie, R James; Toh, Kazuko; Matsumoto, Yu; Nishiyama, Nobuhiro; Miyata, Kanjiro; Kataoka, Kazunori

    2014-09-23

    For systemic delivery of siRNA to solid tumors, a size-regulated and reversibly stabilized nanoarchitecture was constructed by using a 20 kDa siRNA-loaded unimer polyion complex (uPIC) and 20 nm gold nanoparticle (AuNP). The uPIC was selectively prepared by charge-matched polyionic complexation of a poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) copolymer bearing ∼40 positive charges (and thiol group at the ω-end) with a single siRNA bearing 40 negative charges. The thiol group at the ω-end of PEG-PLL further enabled successful conjugation of the uPICs onto the single AuNP through coordinate bonding, generating a nanoarchitecture (uPIC-AuNP) with a size of 38 nm and a narrow size distribution. In contrast, mixing thiolated PEG-PLLs and AuNPs produced a large aggregate in the absence of siRNA, suggesting the essential role of the preformed uPIC in the formation of nanoarchitecture. The smart uPIC-AuNPs were stable in serum-containing media and more resistant against heparin-induced counter polyanion exchange, compared to uPICs alone. On the other hand, the treatment of uPIC-AuNPs with an intracellular concentration of glutathione substantially compromised their stability and triggered the release of siRNA, demonstrating the reversible stability of these nanoarchitectures relative to thiol exchange and negatively charged AuNP surface. The uPIC-AuNPs efficiently delivered siRNA into cultured cancer cells, facilitating significant sequence-specific gene silencing without cytotoxicity. Systemically administered uPIC-AuNPs showed appreciably longer blood circulation time compared to controls, i.e., bare AuNPs and uPICs, indicating that the conjugation of uPICs onto AuNP was crucial for enhancing blood circulation time. Finally, the uPIC-AuNPs efficiently accumulated in a subcutaneously inoculated luciferase-expressing cervical cancer (HeLa-Luc) model and achieved significant luciferase gene silencing in the tumor tissue. These results demonstrate the strong

  6. A Genome-Wide siRNA Screen to Identify Modulators of Insulin Sensitivity and Gluconeogenesis

    PubMed Central

    Yang, Ruojing; Lacson, Raul G.; Castriota, Gino; Zhang, Xiaohua D.; Liu, Yaping; Zhao, Wenqing; Einstein, Monica; Camargo, Luiz Miguel; Qureshi, Sajjad; Wong, Kenny K.; Zhang, Bei B.; Ferrer, Marc; Berger, Joel P.

    2012-01-01

    Background Hepatic insulin resistance impairs insulin’s ability to suppress hepatic glucose production (HGP) and contributes to the development of type 2 diabetes (T2D). Although the interests to discover novel genes that modulate insulin sensitivity and HGP are high, it remains challenging to have a human cell based system to identify novel genes. Methodology/Principal Findings To identify genes that modulate hepatic insulin signaling and HGP, we generated a human cell line stably expressing beta-lactamase under the control of the human glucose-6-phosphatase (G6PC) promoter (AH-G6PC cells). Both beta-lactamase activity and endogenous G6PC mRNA were increased in AH-G6PC cells by a combination of dexamethasone and pCPT-cAMP, and reduced by insulin. A 4-gene High-Throughput-Genomics assay was developed to concomitantly measure G6PC and pyruvate-dehydrogenase-kinase-4 (PDK4) mRNA levels. Using this assay, we screened an siRNA library containing pooled siRNA targeting 6650 druggable genes and identified 614 hits that lowered G6PC expression without increasing PDK4 mRNA levels. Pathway analysis indicated that siRNA-mediated knockdown (KD) of genes known to positively or negatively affect insulin signaling increased or decreased G6PC mRNA expression, respectively, thus validating our screening platform. A subset of 270 primary screen hits was selected and 149 hits were confirmed by target gene KD by pooled siRNA and 7 single siRNA for each gene to reduce G6PC expression in 4-gene HTG assay. Subsequently, pooled siRNA KD of 113 genes decreased PEPCK and/or PGC1alpha mRNA expression thereby demonstrating their role in regulating key gluconeogenic genes in addition to G6PC. Last, KD of 61 of the above 113 genes potentiated insulin-stimulated Akt phosphorylation, suggesting that they suppress gluconeogenic gene by enhancing insulin signaling. Conclusions/Significance These results support the proposition that the proteins encoded by the genes identified in our cell

  7. Erratum: Interstellar Abundance Standards Revisited

    NASA Astrophysics Data System (ADS)

    Sofia, U. J.; Meyer, D. M.

    2001-09-01

    In the Letter ``Interstellar Abundance Standards Revisited'' by U. J. Sofia and D. M. Meyer (ApJ, 554, L221 [2001]), Table 2 and its footnotes contain several typographical errors. The corrected table is shown below. We note that the solar reference standard now implies a positive abundance of nitrogen in halo dust.

  8. Pulmonary Codelivery of Doxorubicin and siRNA by pH-Sensitive Nanoparticles for Therapy of Metastatic Lung Cancer.

    PubMed

    Xu, Caina; Wang, Ping; Zhang, Jingpeng; Tian, Huayu; Park, Kinam; Chen, Xuesi

    2015-09-01

    A pulmonary codelivery system that can simultaneously deliver doxorubicin (DOX) and Bcl2 siRNA to the lungs provides a promising local treatment strategy for lung cancers. In this study, DOX is conjugated onto polyethylenimine (PEI) by using cis-aconitic anhydride (CA, a pH-sensitive linker) to obtain PEI-CA-DOX conjugates. The PEI-CA-DOX/siRNA complex nanoparticles are formed spontaneously via electrostatic interaction between cationic PEI-CA-DOX and anionic siRNA. The drug release experiment shows that DOX releases faster at acidic pH than at pH 7.4. Moreover, PEI-CA-DOX/Bcl2 siRNA complex nanoparticles show higher cytotoxicity and apoptosis induction in B16F10 cells than those treated with either DOX or Bcl2 siRNA alone. When the codelivery systems are directly sprayed into the lungs of B16F10 melanoma-bearing mice, the PEI-CA-DOX/Bcl2 siRNA complex nanoparticles exhibit enhanced antitumor efficacy compared with the single delivery of DOX or Bcl2 siRNA. Compared with systemic delivery, most drug and siRNA show a long-term retention in the lungs via pulmonary delivery, and a considerable number of the drug and siRNA accumulate in tumor tissues of lungs, but rarely in normal lung tissues. The PEI-CA-DOX/Bcl2 siRNA complex nanoparticles are promising for the treatment of metastatic lung cancer by pulmonary delivery with low side effects on the normal tissues. PMID:26136261

  9. pH-Sensitive carboxymethyl chitosan-modified cationic liposomes for sorafenib and siRNA co-delivery

    PubMed Central

    Yao, Yao; Su, Zhihui; Liang, Yanchao; Zhang, Na

    2015-01-01

    Combination of chemotherapeutic drug and small interfering RNA (siRNA) can affect multiple disease pathways and has been proven effective in suppressing tumor progression. Co-delivery of drug and siRNA within a same nanocarrier is a vital means in this field. The present study aimed at the development of a pH-sensitive liposome to co-deliver drug and siRNA to tumor region. Driven by the electrostatic interaction, the pH-sensitive material, carboxymethyl chitosan (CMCS), was coated onto the surface of the cationic liposome (CL) preloaded with sorafenib (Sf) and siRNA (Si). To evaluate whether the resulting CMCS-modified Sf and siRNA co-delivery cationic liposome (CMCS-SiSf-CL) enhanced antitumor efficiency after systematic administration, in vitro and in vivo experiments were evaluated in HepG2 cells and the H22 cells-bearing Kunming mice model. The experimental results demonstrated that CMCS-SiSf-CL was able to condense siRNA efficiently and protect siRNA from being degraded by serum and RNase. The release rate of Sf from CMCS-modified liposome exhibited pH-sensitive release behavior. Furthermore, in vitro cellular uptake results showed that CMCS-SiSf-CL yielded higher fluorescence intensity at pH 6.5 than at pH 7.4, and that siRNA could be delivered to tumor site by CMCS-SiSf-CL in vivo. The in vivo antitumor efficacy showed that CMCS-Sf-CL inhibits tumor growth effectively when compared with free Sf solution. In current experimental conditions, this liposomal formulation did not show significant toxicity both in vitro and in vivo. Therefore, co-delivering Sf with siRNA by CMCS-SiSf-CL might provide a promising approach for tumor therapy. PMID:26491291

  10. pH-Sensitive carboxymethyl chitosan-modified cationic liposomes for sorafenib and siRNA co-delivery.

    PubMed

    Yao, Yao; Su, Zhihui; Liang, Yanchao; Zhang, Na

    2015-01-01

    Combination of chemotherapeutic drug and small interfering RNA (siRNA) can affect multiple disease pathways and has been proven effective in suppressing tumor progression. Co-delivery of drug and siRNA within a same nanocarrier is a vital means in this field. The present study aimed at the development of a pH-sensitive liposome to co-deliver drug and siRNA to tumor region. Driven by the electrostatic interaction, the pH-sensitive material, carboxymethyl chitosan (CMCS), was coated onto the surface of the cationic liposome (CL) preloaded with sorafenib (Sf) and siRNA (Si). To evaluate whether the resulting CMCS-modified Sf and siRNA co-delivery cationic liposome (CMCS-SiSf-CL) enhanced antitumor efficiency after systematic administration, in vitro and in vivo experiments were evaluated in HepG2 cells and the H22 cells-bearing Kunming mice model. The experimental results demonstrated that CMCS-SiSf-CL was able to condense siRNA efficiently and protect siRNA from being degraded by serum and RNase. The release rate of Sf from CMCS-modified liposome exhibited pH-sensitive release behavior. Furthermore, in vitro cellular uptake results showed that CMCS-SiSf-CL yielded higher fluorescence intensity at pH 6.5 than at pH 7.4, and that siRNA could be delivered to tumor site by CMCS-SiSf-CL in vivo. The in vivo antitumor efficacy showed that CMCS-Sf-CL inhibits tumor growth effectively when compared with free Sf solution. In current experimental conditions, this liposomal formulation did not show significant toxicity both in vitro and in vivo. Therefore, co-delivering Sf with siRNA by CMCS-SiSf-CL might provide a promising approach for tumor therapy. PMID:26491291

  11. 21st Birthday Drinking: Extremely Extreme

    ERIC Educational Resources Information Center

    Rutledge, Patricia C.; Park, Aesoon; Sher, Kenneth J.

    2008-01-01

    Despite public recognition of the hazards of 21st birthday drinking, there is little empirical information concerning its prevalence, severity, and risk factors. Data from a sample of 2,518 college students suggest that 21st birthday drinking poses an extreme danger: (a) 4 of every 5 participants (83%) reported drinking to celebrate, (b) birthday…

  12. How extreme is extreme hourly precipitation?

    NASA Astrophysics Data System (ADS)

    Papalexiou, Simon Michael; Dialynas, Yannis G.; Pappas, Christoforos

    2016-04-01

    The importance of accurate representation of precipitation at fine time scales (e.g., hourly), directly associated with flash flood events, is crucial in hydrological design and prediction. The upper part of a probability distribution, known as the distribution tail, determines the behavior of extreme events. In general, and loosely speaking, tails can be categorized in two families: the subexponential and the hyperexponential family, with the first generating more intense and more frequent extremes compared to the latter. In past studies, the focus has been mainly on daily precipitation, with the Gamma distribution being the most popular model. Here, we investigate the behaviour of tails of hourly precipitation by comparing the upper part of empirical distributions of thousands of records with three general types of tails corresponding to the Pareto, Lognormal, and Weibull distributions. Specifically, we use thousands of hourly rainfall records from all over the USA. The analysis indicates that heavier-tailed distributions describe better the observed hourly rainfall extremes in comparison to lighter tails. Traditional representations of the marginal distribution of hourly rainfall may significantly deviate from observed behaviours of extremes, with direct implications on hydroclimatic variables modelling and engineering design.

  13. NIR light controlled photorelease of siRNA and its targeted intracellular delivery based on upconversion nanoparticles

    NASA Astrophysics Data System (ADS)

    Yang, Yanmei; Liu, Fang; Liu, Xiaogang; Xing, Bengang

    2012-12-01

    The most notable role of small interfering RNA (siRNA) is in RNA interference (RNAi) and post-transcriptional gene silencing, which leads to a surge of interest in RNAi for both biomedical research and therapeutic applications. However, ``naked'' siRNA cannot cross cellular membranes freely because of highly negative charges which limits its utility for gene therapy. In this work, a system of near-infrared (NIR) light-induced siRNA release from silica coated upconversion nanoparticles (Si-UCNPs) is presented. These Si-UCNPs were functionalized with cationic photocaged linkers through covalent bonding, which could effectively adsorb anionic siRNA through electrostatic attractions and were easily internalized by living cells. Upon NIR light irradiation, the photocaged linker on the Si-UCNPs surface could be cleaved by the upconverted UV light and thus initiated the intracellular release of the siRNA. The in vitro agarose gel electrophoresis and intracellular imaging results indicated that the Si-UCNPs-based gene carrier system allowed effective siRNA delivery and the applications of NIR light instead of direct high energy UV irradiation may greatly guarantee less cell damage.The most notable role of small interfering RNA (siRNA) is in RNA interference (RNAi) and post-transcriptional gene silencing, which leads to a surge of interest in RNAi for both biomedical research and therapeutic applications. However, ``naked'' siRNA cannot cross cellular membranes freely because of highly negative charges which limits its utility for gene therapy. In this work, a system of near-infrared (NIR) light-induced siRNA release from silica coated upconversion nanoparticles (Si-UCNPs) is presented. These Si-UCNPs were functionalized with cationic photocaged linkers through covalent bonding, which could effectively adsorb anionic siRNA through electrostatic attractions and were easily internalized by living cells. Upon NIR light irradiation, the photocaged linker on the Si-UCNPs surface

  14. Permanent acceptance of mouse cardiac allografts with CD40 siRNA to induce regulatory myeloid cells by use of a novel polysaccharide siRNA delivery system.

    PubMed

    Zhang, Q; Ichimaru, N; Higuchi, S; Cai, S; Hou, J; Fujino, M; Nonomura, N; Kobayashi, M; Ando, H; Uno, A; Sakurai, K; Mochizuki, S; Adachi, Y; Ohno, N; Zou, H; Xu, J; Li, X-K; Takahara, S

    2015-03-01

    The CD40/CD154 co-stimulatory pathway is crucial in alloimmune response. We developed a novel small interfering RNA (siRNA) delivery system with a poly-dA extension at the 5'-end of the siRNA sense strand that was stably incorporated into 1,3-β-glucan (schizophyllan, SPG). This was captured and incorporated into dendritic cells (DCs) through its receptor, Dectin-1, specifically silencing CD40 genes (siCD40) to exert immunoregulatory activity. siCD40/SPG-treated CBA mice permanently accepted B10 fully mismatched cardiac allografts. Consistent with graft survival, the infiltration of CD4(+), CD8(+) T cells into the graft was lower, and that the numbers of CD40(low)CD11c(+) DCs cells and CD4(+)Foxp3(+)cells were increased in both the graft and in the recipient spleen. In addition, naive CBA recipients given an adoptive transfer of splenocytes from the primary recipients with siCD40/SPG accepted a heart graft from donor-type B10, but not third-party Balb/c mice. In conclusion, the treatment with siCD40/SPG targeting DCs could generate antigen-specific Tregs, resulting in the permanent acceptance of mouse cardiac allografts. These findings have important implications for clarifying the mechanism underlying the induction of tolerance in DCs, and also highlight the potential of immunomodulation and the feasibility of siRNA-based clinical therapy in the transplantation field. PMID:25567536

  15. VLT spectroscopy of low-metallicity emission-line galaxies: abundance patterns and abundance discrepancies

    NASA Astrophysics Data System (ADS)

    Guseva, N. G.; Izotov, Y. I.; Stasińska, G.; Fricke, K. J.; Henkel, C.; Papaderos, P.

    2011-05-01

    Context. We present deep spectroscopy of a large sample of low-metallicity emission-line galaxies. Aims: The main goal of this study is to derive element abundances in these low-metallicity galaxies. Methods: We analyze 121 VLT spectra of H ii regions in 46 low-metallicity emission-line galaxies. Of these spectra 83 are archival VLT/FORS1+UVES spectra of H ii regions in 31 low-metallicity emission-line galaxies that are studied for the first time with standard direct methods to determine the electron temperatures, the electron number densities, and the chemical abundances. Results: The oxygen abundance of the sample lies in the range 12 + log O/H = 7.2-8.4. We confirm previous findings that Ne/O increases with increasing oxygen abundance, likely because of a higher depletion of oxygen in higher-metallicity galaxies. The Fe/O ratio decreases from roughly solar at the lowest metallicities to about one tenth of solar, indicating that the degree of depletion of iron into dust grains depends on metallicity. The N/O ratio in extremely low-metallicity galaxies with 12 + log O/H < 7.5 shows a slight increase with decreasing oxygen abundance, which could be the signature of enhanced production of primary nitrogen by rapidly rotating stars at low metallicity. We present the first empirical relation between the electron temperature derived from [S iii]λ6312/λ9069 or [N ii]λ5755/λ6583 and the one derived from [O iii]λ4363/λ(4959+5007) in low-metallicity galaxies. We also present an empirical relation between te derived from [O ii]λ3727/(λ7320 + λ7330) or [S ii]λ4068/(λ6717 + λ6730) and [O iii]λ4363/λ(4959+5007). The electron number densities Ne(Cl iii) and Ne(Ar iv) were derived in a number of objects and are found to be higher than Ne(O ii) and Ne(S ii). This has potential implications for the derivation of the pregalactic helium abundance. In a number of objects, the abundances of C++ and O++ could be derived from recombination lines. Our study confirms the

  16. siRNA targeting PLK-1 induces apoptosis of synoviocytes in rheumatoid arthritis

    SciTech Connect

    Wada, Makoto; Kawahito, Yutaka . E-mail: kawahity@koto.kpu-m.ac.jp; Kimura, Shinya; Kohno, Masataka; Ishino, Hidetaka; Kimura, Mizuho; Omoto, Atsushi; Yamamoto, Aihiro; Hamaguchi, Masahide; Tsubouchi, Yasunori; Tokunaga, Daisaku; Hojo, Tatsuya; Ashihara, Eishi; Maekawa, Taira; Yoshikawa, Toshikazu

    2007-06-01

    Polo-like kinase-1 (PLK-1) is a member of the PLK family and participates in the control of cell mitosis. Here, we show that immunoreactive PLK-1 is strongly expressed in synoviocytes and some infiltrative mononuclear cells in synovial tissues from patients with rheumatoid arthritis (RA), while patients with osteoarthritis and injury show little or no expression of PLK-1 in synovial tissues. Western blot analysis shows that PLK is expressed and its expression is enhanced by IL-1{beta} in RA synoviocytes. IL-1{beta} also enhanced the cell growth of RA synoviocytes. Moreover, siRNA targeted against PLK-1 significantly decreases the expression of PLK-1 of RA synoviocytes stimulated by IL-1{beta} and suppresses the proliferation of these synoviocytes through apoptosis. These findings suggest that PLK-1 plays a critical role in the proliferation of RA synoviocytes leading to bone destruction, and siRNA against PLK-1 is potentially useful for the treatment of RA.

  17. Codelivery of anticancer drugs and siRNA by mesoporous silica nanoparticles.

    PubMed

    Hanafi-Bojd, Mohammad Yahya; Ansari, Legha; Malaekeh-Nikouei, Bizhan

    2016-09-01

    The most common method for cancer treatment is chemotherapy. Multidrug resistance (MDR) is one of the major obstacles in chemotherapeutic treatment of many human cancers. One strategy to overcome this challenge is the delivery of anticancer drugs and siRNA simultaneously using nanoparticles. Mesoporous silica nanoparticles are one of the most popular nanoparticles for cargo delivery because of their intrinsic porosity. This paper highlights recent advances in codelivery of chemotherapeutic and siRNA with mesoporous silica nanoparticles for cancer therapy. In addition, synthesis and functionalization approaches of these nanoparticles are summarized. This review presents insight into the utilization of nanoparticles and combination therapy to achieve more promising results in chemotherapy. PMID:27582236

  18. Silencing of Inducible Immunoproteasome Subunit Expression by Chemically Modified siRNA and shRNA.

    PubMed

    Gvozdeva, Olga V; Prassolov, Vladimir S; Zenkova, Marina A; Vlassov, Valentin V; Chernolovskaya, Elena L

    2016-08-01

    Overexpression of inducible subunits of immunoproteasome is related to pathogenesis of some chronic diseases. Specific inhibition of the immunosubunits may be used for the treatment of these diseases and RNA interference is one of the potent methods used in this area. We designed 2'-O-methyl modified siRNAs with selectively protected nuclease-sensitive sites, which efficiently silence LMP2, LMP7, and MECL-1 genes expression. To provide stable long-lasting inhibition of target genes, short-hairpin RNAs (shRNA) expressed by lentiviral vectors were constructed. Our results demonstrated that chemically modified siRNAs inhibited the expression of target genes with similar efficiency or with efficiency exceeding that of corresponding shRNAs and provide silencing effect for 5 days. PMID:27351110

  19. Non-viral nanocarriers for siRNA delivery in breast cancer

    PubMed Central

    Zhang, Jing; Li, Xiang; Huang, Leaf

    2014-01-01

    Breast cancer is the most frequently diagnosed malignancy in American women. While significant progress has been made in development of modern diagnostic tools and surgical treatments, only marginal improvements have been achieved with relapsed metastatic breast cancer. Small interfering RNAs (siRNAs) mediate gene silencing of a target protein by disrupting messenger RNAs in an efficient and sequence-specific manner. One application of this technology is the knockdown of genes responsible for tumorigenesis, including those driving oncogenesis, survival, proliferation and death of cells, angiogenesis, invasion and metastasis, and resistance to treatment. Non-viral nanocarriers have attracted attention based on their potential for targeted delivery of siRNA and efficient gene silencing without toxicity. Here, we review promising, non-viral delivery strategies employing liposomes, nanoparticles and inorganic materials in breast cancer. PMID:24874288

  20. Amino Acid-Functionalized Dendritic Polyglycerol for Safe and Effective siRNA Delivery.

    PubMed

    Zeng, Hanxiang; Schlesener, Cathleen; Cromwell, Olivia; Hellmund, Markus; Haag, Rainer; Guan, Zhibin

    2015-12-14

    The development of safe and effective delivery vectors is a great challenge for the medicinal application of RNA interference (RNAi). In this study, we aimed to develop new synthetic transfection agents based on dendritic polyglycercol (dPG), which has shown great biocompatibility in several biomaterial applications. Histidine and aromatic amino acids were conjugated to the amine-terminated dPGs through amide bonds. We systematically tuned the amino acid combination, functionalization ratio, ligand density, and dPG core size to find optimal vectors. It was found that histidine-tryptophan-functionalized dPGs exhibited improved delivery efficiency and greatly reduced toxicity over simple amine-terminated dPGs. Furthermore, the optimized vectors exhibited strong siRNA binding and high transfection efficiency in serum containing media. The results indicate that the current amino acid-functionalized dPG system is a promising candidate for in vivo siRNA delivery applications. PMID:26569043

  1. Self-assembled lipid nanomedicines for siRNA tumor targeting.

    PubMed

    Tseng, Yu-Cheng; Huang, Leaf

    2009-08-01

    Lipid-based nanoparticle technology has developed from chemical drug carrier into an efficient multifunctional siRNA tumor targeting delivery system. In this review, we start with an overview of the lipid-based nanomedicine history and the two classes of lipidic vectors for DNA or siRNA delivery. Then we discuss the features of lipid-based nanomedicine that lead to effective tumor targeting and the principles behind. We also discuss nanoparticle surface modification, classes of tumor targeting ligands, and other state-of-the-art strategies for enhancing endosome release primarily focused on lipid-based systems. At the end, we show that multifunctional self-assembled lipid-based nanoparticles could also be versatile delivery vehicles for cancer molecular imaging probes. PMID:20055081

  2. Preparation of Polyion Complex Micelles Using Block Copolymers for SiRNA Delivery.

    PubMed

    Kim, Hyun Jin; Zheng, Meng; Miyata, Kanjiro; Kataoka, Kazunori

    2016-01-01

    Polyion complex (PIC) micelles can be prepared through the spontaneous assembly of cationic block copolymers with oppositely charged short interfering RNAs (SiRNAs). Their core-shell architectures offer a delivery platform for vulnerable SiRNA, improving their biological activities for medicinal applications such as tumor-targeted therapy. Here, we report a protocol for the preparation of SiRNA-loaded PIC micelles using a poly(ethylene glycol)-block-poly(aspartamide) derivative, providing the physicochemical criteria for well-defined micellar formulation. In addition, we describe protocols for a stability assay for SiRNA-loaded PIC micelles in the presence of serum using fluorescence correlation spectroscopy and a luciferase assay for cultured cancer cells stably expressing luciferase, thus providing the biological criteria for further medicinal applications. PMID:26472445

  3. Inhibition of Hepatitis B virus cccDNA replication by siRNA

    SciTech Connect

    Li Guiqiu; Gu Hongxi . E-mail: hxgu2432@163.com; Li Di; Xu Weizhen

    2007-04-06

    The development of an effective therapy for Hepatitis B virus (HBV) infection is still a challenge. Progress in RNA interference (RNAi) has shed slight on developing a new anti-HBV strategy. Here, we present a series of experiments showing a significant reduction in HBV transcripts and replication intermediates in HepG2.2.15 cells by vector-based siRNA targeted nuclear localization signal (NLS) region. More importantly, we showed that siRNA1 markedly inhibited HBV covalently closed circular DNA (cccDNA) replication. Our results indicated that HBV NLS may serve as a novel RNAi target to combat HBV infection, which can enhance anti-HBV efficacy and overcome the drawbacks of current therapies.

  4. Multifunctional polymeric micelles for delivery of drugs and siRNA

    PubMed Central

    Jhaveri, Aditi M.; Torchilin, Vladimir P.

    2014-01-01

    Polymeric micelles, self-assembling nano-constructs of amphiphilic copolymers with a core-shell structure have been used as versatile carriers for delivery of drugs as well as nucleic acids. They have gained immense popularity owing to a host of favorable properties including their capacity to effectively solubilize a variety of poorly soluble pharmaceutical agents, biocompatibility, longevity, high stability in vitro and in vivo and the ability to accumulate in pathological areas with compromised vasculature. Moreover, additional functions can be imparted to these micelles by engineering their surface with various ligands and cell-penetrating moieties to allow for specific targeting and intracellular accumulation, respectively, to load them with contrast agents to confer imaging capabilities, and incorporating stimuli-sensitive groups that allow drug release in response to small changes in the environment. Recently, there has been an increasing trend toward designing polymeric micelles which integrate a number of the above functions into a single carrier to give rise to “smart,” multifunctional polymeric micelles. Such multifunctional micelles can be envisaged as key to improving the efficacy of current treatments which have seen a steady increase not only in hydrophobic small molecules, but also in biologics including therapeutic genes, antibodies and small interfering RNA (siRNA). The purpose of this review is to highlight recent advances in the development of multifunctional polymeric micelles specifically for delivery of drugs and siRNA. In spite of the tremendous potential of siRNA, its translation into clinics has been a significant challenge because of physiological barriers to its effective delivery and the lack of safe, effective and clinically suitable vehicles. To that end, we also discuss the potential and suitability of multifunctional polymeric micelles, including lipid-based micelles, as promising vehicles for both siRNA and drugs. PMID:24795633

  5. Tunable Delivery of siRNA from a Biodegradable Scaffold to Promote Angiogenesis In Vivo

    PubMed Central

    Nelson, Christopher E.; Kim, Arnold J.; Adolph, Elizabeth J.; Gupta, Mukesh K.; Yu, Fang; Hocking, Kyle M.; Davidson, Jeffrey M.; Guelcher, Scott A.; Duvall, Craig L.

    2014-01-01

    A system has been engineered for temporally controlled delivery of siRNA from biodegradable tissue regenerative scaffolds. Therapeutic application of this approach to silence prolyl hydroxylase domain 2 promoted expression of pro-angiogenic genes controlled by HIF1α and enhanced scaffold vascularization in vivo. This technology provides a new standard for efficient and controllable gene silencing to modulate host response within regenerative biomaterials. PMID:24338842

  6. Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery.

    PubMed

    Li, Jun; Chen, Yun-Ching; Tseng, Yu-Cheng; Mozumdar, Subho; Huang, Leaf

    2010-03-19

    A lipid coated calcium phosphate (LCP) nanoparticle (NP) formulation was developed for efficient delivery of small interfering RNA (siRNA) to a xenograft tumor model by intravenous administration. Based on the previous formulation, liposome-polycation-DNA (LPD), which was a DNA-protamine complex wrapped by cationic liposome followed by post-insertion of PEG, LCP was similar to LPD NP except that the core was replaced by a biodegradable nano-sized calcium phosphate precipitate prepared by using water-in-oil micro-emulsions in which siRNA was entrapped. We hypothesized that after entering the cells, LCP would de-assemble at low pH in the endosome, which would cause endosome swelling and bursting to release the entrapped siRNA. Such a mechanism was demonstrated by the increase of intracellular Ca(2+) concentration as shown by using a calcium specific dye Fura-2. The LCP NP was further modified by post-insertion of polyethylene glycol (PEG) with or without anisamide, a sigma-1 receptor ligand for systemic administration. Luciferase siRNA was used to evaluate the gene silencing effect in H-460 cells which were stably transduced with a luciferase gene. The anisamide modified LCP NP silenced about 70% and 50% of luciferase activity for the tumor cells in culture and those grown in a xenograft model, respectively. The untargeted NP showed a very low silencing effect. The new formulation improved the in vitro silencing effect 3-4 folds compared to the previous LPD formulation, but had a negligible immunotoxicity. PMID:19919845

  7. Acid-Degradable Cationic Dextran Particles for the Delivery of siRNA Therapeutics

    PubMed Central

    Cohen, Jessica L.; Schubert, Stephanie; Wich, Peter R.; Cui, Lina; Cohen, Joel A.; Mynar, Justin L.; Fréchet, Jean M. J.

    2011-01-01

    We report a new acid-sensitive, biocompatible and biodegradable microparticulate delivery system, spermine modified acetalated-dextran (Spermine-Ac-DEX), which can be used to efficiently encapsulate siRNA. These particles demonstrated efficient gene knockdown in HeLa-luc cells with minimal toxicity. This knockdown was comparable to that obtained using Lipofectamine, a commercially available transfection reagent generally limited to in vitro use due to its high toxicity. PMID:21539393

  8. Multifunctional polymeric micelles for delivery of drugs and siRNA.

    PubMed

    Jhaveri, Aditi M; Torchilin, Vladimir P

    2014-01-01

    Polymeric micelles, self-assembling nano-constructs of amphiphilic copolymers with a core-shell structure have been used as versatile carriers for delivery of drugs as well as nucleic acids. They have gained immense popularity owing to a host of favorable properties including their capacity to effectively solubilize a variety of poorly soluble pharmaceutical agents, biocompatibility, longevity, high stability in vitro and in vivo and the ability to accumulate in pathological areas with compromised vasculature. Moreover, additional functions can be imparted to these micelles by engineering their surface with various ligands and cell-penetrating moieties to allow for specific targeting and intracellular accumulation, respectively, to load them with contrast agents to confer imaging capabilities, and incorporating stimuli-sensitive groups that allow drug release in response to small changes in the environment. Recently, there has been an increasing trend toward designing polymeric micelles which integrate a number of the above functions into a single carrier to give rise to "smart," multifunctional polymeric micelles. Such multifunctional micelles can be envisaged as key to improving the efficacy of current treatments which have seen a steady increase not only in hydrophobic small molecules, but also in biologics including therapeutic genes, antibodies and small interfering RNA (siRNA). The purpose of this review is to highlight recent advances in the development of multifunctional polymeric micelles specifically for delivery of drugs and siRNA. In spite of the tremendous potential of siRNA, its translation into clinics has been a significant challenge because of physiological barriers to its effective delivery and the lack of safe, effective and clinically suitable vehicles. To that end, we also discuss the potential and suitability of multifunctional polymeric micelles, including lipid-based micelles, as promising vehicles for both siRNA and drugs. PMID:24795633

  9. Low generation polyamine dendrimers bearing flexible tetraethylene glycol as nanocarriers for plasmids and siRNA

    NASA Astrophysics Data System (ADS)

    Sharma, Rishi; Zhang, Issan; Shiao, Tze Chieh; Pavan, Giovanni M.; Maysinger, Dusica; Roy, René

    2016-02-01

    Low G1 generation polyamine dendrimers built around programmable, flexible, and short tetraethyleneglycol branches were readily prepared in a divergent manner using a combination of orthogonal AB3 or AB5 units and highly efficient chemical transformations based on Cu(i) catalyzed alkyne-azide cycloaddition (CUAAC) and thiol-ene click reactions. The constructs showed that the G1 polyamines with only twelve and eighteen amine surface groups can successfully deliver siRNA in human cells, with transfection efficiency comparable to that of Lipofectamine 2000®. Measurements of cell viability following transfection of plasmid DNA and siRNA showed that the dendritic polyamines are less cytotoxic than Lipofectamine 2000® and are thus preferable for biological applications.Low G1 generation polyamine dendrimers built around programmable, flexible, and short tetraethyleneglycol branches were readily prepared in a divergent manner using a combination of orthogonal AB3 or AB5 units and highly efficient chemical transformations based on Cu(i) catalyzed alkyne-azide cycloaddition (CUAAC) and thiol-ene click reactions. The constructs showed that the G1 polyamines with only twelve and eighteen amine surface groups can successfully deliver siRNA in human cells, with transfection efficiency comparable to that of Lipofectamine 2000®. Measurements of cell viability following transfection of plasmid DNA and siRNA showed that the dendritic polyamines are less cytotoxic than Lipofectamine 2000® and are thus preferable for biological applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06757j

  10. Liquid crystalline phase nanodispersions enable skin delivery of siRNA.

    PubMed

    Vicentini, Fabiana Testa Moura de Carvalho; Depieri, Lívia Vieira; Polizello, Ana Cristina Morseli; Del Ciampo, José Orestes; Spadaro, Augusto César Cropanese; Fantini, Márcia C A; Vitória Lopes Badra Bentley, Maria

    2013-01-01

    The ability of small interfering RNAs (siRNAs) to potently but reversibly silence genes in vivo has made them particularly well suited as a new class of drugs that interfere with disease-causing or disease-promoting genes. However, the largest remaining hurdle for the widespread use of this technology in skin is the lack of an effective delivery system. The aim of the present study was to evaluate nanodispersed systems in liquid crystalline phases that deliver siRNA into the skin. The proposed systems present important properties for the delivery of macromolecules in a biological medium, as they are formed by substances that have absorption-enhancing and fusogenic effects; additionally, they facilitate entrapment by cellular membranes due to their nano-scale structure. The cationic polymer polyethylenimine (PEI) or the cationic lipid oleylamine (OAM) were added to monoolein (MO)-based systems in different concentrations, and after dispersion in aqueous medium, liquid crystalline phase nanodispersions were obtained and characterized by their physicochemical properties. Then, in vitro penetration studies using diffusion cell and pig ear skin were carried out to evaluate the effect of the nanodispersions on the skin penetration of siRNA; based on these results, the nanodispersions containing MO/OA/PEI/aqueous phase (8:2:5:85, w/w/w/w) and MO/OA/OAM/aqueous phase (8:2:2:88, w/w/w/w) were selected. These systems were investigated in vivo for skin penetration, skin irritation, and the ability to knockdown glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein levels in animal models. The results showed that the studied nanodispersions may represent a promising new non-viral vehicle and can be considered highly advantageous in the treatment of skin disorders; they were effective in optimizing the skin penetration of siRNA and reducing the levels of the model protein GAPDH without causing skin irritation. PMID:23010565

  11. Detection of small interfering RNA (siRNA) by mass spectrometry procedures in doping controls.

    PubMed

    Thomas, Andreas; Walpurgis, Katja; Delahaut, Philippe; Kohler, Maxie; Schänzer, Wilhelm; Thevis, Mario

    2013-01-01

    Uncovering manipulation of athletic performance via small interfering (si)RNA is an emerging field in sports drug testing. Due to the potential to principally knock down every target gene in the organism by means of the RNA interference pathway, this facet of gene doping has become a realistic scenario. In the present study, two distinct model siRNAs comprising 21 nucleotides were designed as double strands which were perfect counterparts to a sequence of the respective messenger RNA coding the muscle regulator myostatin of Rattus norvegicus. Several modified nucleotides were introduced in both the sense and the antisense strand comprising phosphothioates, 2'-O-methylation, 2'-fluoro-nucleotides, locked nucleic acids and a cholesterol tag at the 3'-end. The model siRNAs were applied to rats at 1 mg/kg (i.v.) and blood as well as urine samples were collected. After isolation of the RNA by means of a RNA purification kit, the target analytes were detected by liquid chromatography - high resolution/high accuracy mass spectrometry (LC-HRMS). Analytes were detected as modified nucleotides after alkaline hydrolysis, as intact oligonucleotide strands (top-down) and by means of denaturing SDS-PAGE analysis. The gel-separated siRNA was further subjected to in-gel hydrolysis with different RNases and subsequent identification of the fragments by untargeted LC-HRMS analysis (bottom-up, 'experimental RNomics'). Combining the results of all approaches, the identification of several 3'-truncated urinary metabolites was accomplished and target analytes were detected up to 24 h after a single administration. Simultaneously collected blood samples yielded no promising results. The methods were validated and found fit-for-purpose for doping controls. PMID:23913913

  12. Extremity x-ray

    MedlinePlus

    An extremity x-ray is an image of the hands, wrist, feet, ankle, leg, thigh, forearm humerus or upper arm, hip, shoulder ... term "extremity" often refers to a human limb. X-rays are a form of radiation that passes through ...

  13. Bivariate extreme value distributions

    NASA Technical Reports Server (NTRS)

    Elshamy, M.

    1992-01-01

    In certain engineering applications, such as those occurring in the analyses of ascent structural loads for the Space Transportation System (STS), some of the load variables have a lower bound of zero. Thus, the need for practical models of bivariate extreme value probability distribution functions with lower limits was identified. We discuss the Gumbel models and present practical forms of bivariate extreme probability distributions of Weibull and Frechet types with two parameters. Bivariate extreme value probability distribution functions can be expressed in terms of the marginal extremel distributions and a 'dependence' function subject to certain analytical conditions. Properties of such bivariate extreme distributions, sums and differences of paired extremals, as well as the corresponding forms of conditional distributions, are discussed. Practical estimation techniques are also given.

  14. Screening of efficient siRNA carriers in a library of surface-engineered dendrimers.

    PubMed

    Liu, Hongmei; Chang, Hong; Lv, Jia; Jiang, Cong; Li, Zhenxi; Wang, Fei; Wang, Hui; Wang, Mingming; Liu, Chongyi; Wang, Xinyu; Shao, Naimin; He, Bingwei; Shen, Wanwan; Zhang, Qiang; Cheng, Yiyun

    2016-01-01

    Polymers are widely used as non-viral carriers for siRNA delivery, but concern has also arisen in their limited efficacy and inherent toxicity. Whilst many of previous efforts have been documented towards improving the performance of polymers via chemical modifications, the structure-activity relationships (SAR) of these ligand-modified polymers are not well understood. To address this issue, we systemically prepared a library of surface-engineered dendrimers (>300) as the screening pool to discover efficient siRNA carriers. The modified ligands include alkyls and fluoroalkyls, amino acids, benzene derivatives and heterocyclic compounds. Gene silencing results showed that the lead material shows excellent efficacy even in hard-to-transfect cells such as mesenchymal stem cells. The SAR studies revealed that ligands containing appropriate hydrophobicity, or ligands with both hydrophobic and functional atoms/groups are essential for polymers to achive efficient knockdown efficacy. A second-generation library designed based on the above principles further confirms the proposed design criteria. The results enable the future rational design of potent siRNA carriers. PMID:27121799

  15. Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery

    NASA Astrophysics Data System (ADS)

    Lee, Hyukjin; Lytton-Jean, Abigail K. R.; Chen, Yi; Love, Kevin T.; Park, Angela I.; Karagiannis, Emmanouil D.; Sehgal, Alfica; Querbes, William; Zurenko, Christopher S.; Jayaraman, Muthusamy; Peng, Chang G.; Charisse, Klaus; Borodovsky, Anna; Manoharan, Muthiah; Donahoe, Jessica S.; Truelove, Jessica; Nahrendorf, Matthias; Langer, Robert; Anderson, Daniel G.

    2012-06-01

    Nanoparticles are used for delivering therapeutics into cells. However, size, shape, surface chemistry and the presentation of targeting ligands on the surface of nanoparticles can affect circulation half-life and biodistribution, cell-specific internalization, excretion, toxicity and efficacy. A variety of materials have been explored for delivering small interfering RNAs (siRNAs)--a therapeutic agent that suppresses the expression of targeted genes. However, conventional delivery nanoparticles such as liposomes and polymeric systems are heterogeneous in size, composition and surface chemistry, and this can lead to suboptimal performance, a lack of tissue specificity and potential toxicity. Here, we show that self-assembled DNA tetrahedral nanoparticles with a well-defined size can deliver siRNAs into cells and silence target genes in tumours. Monodisperse nanoparticles are prepared through the self-assembly of complementary DNA strands. Because the DNA strands are easily programmable, the size of the nanoparticles and the spatial orientation and density of cancer-targeting ligands (such as peptides and folate) on the nanoparticle surface can be controlled precisely. We show that at least three folate molecules per nanoparticle are required for optimal delivery of the siRNAs into cells and, gene silencing occurs only when the ligands are in the appropriate spatial orientation. In vivo, these nanoparticles showed a longer blood circulation time (t1/2 ~ 24.2 min) than the parent siRNA (t1/2 ~ 6 min).

  16. Efficient Inhibition of wear debris-induced inflammation by locally delivered siRNA

    SciTech Connect

    Peng Xiaochun; Tao Kun; Cheng Tao; Zhu Junfeng; Zhang Xianlong

    2008-12-12

    Aseptic loosening is the most common long-term complication of total joint replacement, which is associated with the generation of wear debris. The purpose of this study was to investigate the inhibitory effect of small interfering RNA (siRNA) targeting tumor necrosis factor-{alpha} (TNF-{alpha}) on wear debris-induced inflammation. A local delivery of lentivirus-mediated TNF-{alpha} siRNA into the modified murine air pouch, which was stimulated by polymethylmethacrylate (PMMA) particles, resulted in significant blockage of TNF-{alpha} both in mRNA and protein levels for up to 4 weeks. In addition, significant down-regulation of interleukin-1 (IL-1) and interleukin-6 (IL-6) was observed in TNF-{alpha} siRNA-treated pouches. The safety profile of gene therapy was proven by Bioluminescent assay and quantitative fluorescent flux. Histological analysis revealed less inflammatory responses (thinner pouch membrane and decreased cellular infiltration) in TNF-{alpha} siRNA-treated pouches. These findings suggest that local delivery of TNF-{alpha} siRNA might be an excellent therapeutic candidate to inhibit particle-induced inflammation.

  17. Notch1 targeting siRNA delivery nanoparticles for rheumatoid arthritis therapy.

    PubMed

    Kim, Min Ju; Park, Jong-Sung; Lee, So Jin; Jang, Jiyeon; Park, Jin Su; Back, Seung Hyun; Bahn, Gahee; Park, Jae Hyung; Kang, Young Mo; Kim, Sun Hwa; Kwon, Ick Chan; Jo, Dong-Gyu; Kim, Kwangmeyung

    2015-10-28

    Notch pathway plays a pivotal role in synoviocytes involved in progression of rheumatoid arthritis (RA). Herein, we designed the Notch1 targeting siRNA delivery nanoparticles (siRNA-NPs) in order to confirm the anti-inflammatory effect in collagen-induced arthritis (CIA) model. The siRNA-NPs were successfully produced by encapsulating polymerized siRNA (poly-siRNA) into thiolated glycol chitosan (tGC) nanoparticles in aqueous condition. The in vitro Notch1 inhibition of siRNA-NPs in murine macrophage cell (RAW 264.7) was confirmed using confocal microscopy and real time PCR. Fluorescently labeled siRNA-NPs were successfully transfected in RAW 264.7 and modulated the expression of Notch1 in mRNA level. For in vivo study, siRNA-NPs exhibited the higher targeting efficiency in the arthritic joins of CIA mice, confirmed by the near-infrared fluorescence (NIRF) imaging. Furthermore, inhibition of Notch1 with siRNA-NPs resulted in retarded progression of inflammation, bone erosion, and cartilage damage in CIA mice. Novel Notch1 targeting siRNA delivery system of siRNA-NPs showed effective RA treatment by suppressing Notch1 signaling pathway without undesirable severe toxicity. Thus, Notch1 inhibiting siRNA-NPs demonstrated the great potential in RA therapeutics that was hard to be achieved using conventional drugs. PMID:26282098

  18. Multifunctional, self-assembling anionic peptide-lipid nanocomplexes for targeted siRNA delivery.

    PubMed

    Tagalakis, Aristides D; Lee, Do Hyang D; Bienemann, Alison S; Zhou, Haiyan; Munye, Mustafa M; Saraiva, Luisa; McCarthy, David; Du, Zixiu; Vink, Conrad A; Maeshima, Ruhina; White, Edward A; Gustafsson, Kenth; Hart, Stephen L

    2014-09-01

    Formulations of cationic liposomes and polymers readily self-assemble by electrostatic interactions with siRNA to form cationic nanoparticles which achieve efficient transfection and silencing in vitro. However, the utility of cationic formulations in vivo is limited due to rapid clearance from the circulation, due to their association with serum proteins, as well as systemic and cellular toxicity. These problems may be overcome with anionic formulations but they provide challenges of self-assembly and transfection efficiency. We have developed anionic, siRNA nanocomplexes utilizing anionic PEGylated liposomes and cationic targeting peptides that overcome these problems. Biophysical measurements indicated that at optimal ratios of components, anionic PEGylated nanocomplexes formed spherical particles and that, unlike cationic nanocomplexes, were resistant to aggregation in the presence of serum, and achieved significant gene silencing although their non-PEGylated anionic counterparts were less efficient. We have evaluated the utility of anionic nanoparticles for the treatment of neuronal diseases by administration to rat brains of siRNA to BACE1, a key enzyme involved in the formation of amyloid plaques. Silencing of BACE1 was achieved in vivo following a single injection of anionic nanoparticles by convection enhanced delivery and specificity of RNA interference verified by 5' RACE-PCR and Western blot analysis of protein. PMID:24985735

  19. In vivo silencing of alpha-synuclein using naked siRNA

    PubMed Central

    Lewis, Jada; Melrose, Heather; Bumcrot, David; Hope, Andrew; Zehr, Cynthia; Lincoln, Sarah; Braithwaite, Adam; He, Zhen; Ogholikhan, Sina; Hinkle, Kelly; Kent, Caroline; Toudjarska, Ivanka; Charisse, Klaus; Braich, Ravi; Pandey, Rajendra K; Heckman, Michael; Maraganore, Demetrius M; Crook, Julia; Farrer, Matthew J

    2008-01-01

    Background Overexpression of α-synuclein (SNCA) in families with multiplication mutations causes parkinsonism and subsequent dementia, characterized by diffuse Lewy Body disease post-mortem. Genetic variability in SNCA contributes to risk of idiopathic Parkinson's disease (PD), possibly as a result of overexpression. SNCA downregulation is therefore a valid therapeutic target for PD. Results We have identified human and murine-specific siRNA molecules which reduce SNCA in vitro. As a proof of concept, we demonstrate that direct infusion of chemically modified (naked), murine-specific siRNA into the hippocampus significantly reduces SNCA levels. Reduction of SNCA in the hippocampus and cortex persists for a minimum of 1 week post-infusion with recovery nearing control levels by 3 weeks post-infusion. Conclusion We have developed naked gene-specific siRNAs that silence expression of SNCA in vivo. This approach may prove beneficial toward our understanding of the endogenous functional equilibrium of SNCA, its role in disease, and eventually as a therapeutic strategy for α-synucleinopathies resulting from SNCA overexpression. PMID:18976489

  20. Delivery of Therapeutic siRNA to the Lung Endothelium via Novel Lipoplex Formulation DACC

    PubMed Central

    Fehring, V; Schaeper, U; Ahrens, K; Santel, A; Keil, O; Eisermann, M; Giese, K; Kaufmann, Jörg

    2014-01-01

    Posttranscriptional gene silencing by RNA interference can be therapeutically exploited to inhibit pathophysiological gene expression. However, in contrast to the established effectiveness of RNAi in vitro, safe and effective delivery of siRNAs to specific organs and cell types in vivo remains the major hurdle. Here, we report the development and in vivo characterization of a novel siRNA delivery system (DACC lipoplex) suitable for modulating target gene expression specifically in the lung vasculature. Systemic administration of DACC in mice delivered siRNA cargo functionally to the lung pulmonary endothelium. A single dose of DACC lipoplexes administered by bolus injection or by infusion was sufficient to specifically silence genes expressed in pulmonary endothelial cells such as CD31, Tie-2, VE-cadherin, or BMP-R2. When tested in a mouse model for lung cancer, repeated treatment with DACC/siRNACD31 reduced formation of lung metastases and increased life span in a mouse model of experimental lung metastasis. PMID:24390281

  1. siRNA Targeting Hes5 Augments Hair Cell Regeneration in Aminoglycoside-damaged Mouse Utricle

    PubMed Central

    Jung, Jae Yun; Avenarius, Matt R.; Adamsky, Swetlana; Alpert, Evgenia; Feinstein, Elena; Raphael, Yehoash

    2013-01-01

    Notch signaling is active during the development of mosaic epithelial sheets and during their turnover and regeneration. After the loss of hair cells in the mosaic sheet of the vestibular sensory epithelium, new hair cells can be spontaneously generated by transdifferentiation of supporting cells. This regenerative process involves downregulation of the Hes5 gene and is known to be limited and incomplete, especially when the lesion is severe. Here, we test whether further downregulation of Hes5 gene accomplished by the use of siRNA after a severe lesion induced by an aminoglycoside in the mouse utricle can enhance the transdifferentiation of supporting cells and lead to the increased production of new hair cells. We demonstrate that Hes5 levels in the utricle decreased after the application of siRNA and that the number of hair cells in these utricles was significantly larger than following control treatment. The data suggest that siRNA technology may be useful for inducing repair and regeneration in the inner ear and that the Notch signaling pathway is a potentially useful target for specific gene expression inhibition. PMID:23439501

  2. Inter-molecular β-sheet structure facilitates lung-targeting siRNA delivery

    PubMed Central

    Zhou, Jihan; Li, Dong; Wen, Hao; Zheng, Shuquan; Su, Cuicui; Yi, Fan; Wang, Jue; Liang, Zicai; Tang, Tao; Zhou, Demin; Zhang, Li-He; Liang, Dehai; Du, Quan

    2016-01-01

    Size-dependent passive targeting based on the characteristics of tissues is a basic mechanism of drug delivery. While the nanometer-sized particles are efficiently captured by the liver and spleen, the micron-sized particles are most likely entrapped within the lung owing to its unique capillary structure and physiological features. To exploit this property in lung-targeting siRNA delivery, we designed and studied a multi-domain peptide named K-β, which was able to form inter-molecular β-sheet structures. Results showed that K-β peptides and siRNAs formed stable complex particles of 60 nm when mixed together. A critical property of such particles was that, after being intravenously injected into mice, they further associated into loose and micron-sized aggregates, and thus effectively entrapped within the capillaries of the lung, leading to a passive accumulation and gene-silencing. The large size aggregates can dissociate or break down by the shear stress generated by blood flow, alleviating the pulmonary embolism. Besides the lung, siRNA enrichment and targeted gene silencing were also observed in the liver. This drug delivery strategy, together with the low toxicity, biodegradability, and programmability of peptide carriers, show great potentials in vivo applications. PMID:26955887

  3. Multivalent dendritic polyglycerolamine with arginine and histidine end groups for efficient siRNA transfection

    PubMed Central

    Sheikhi Mehrabadi, Fatemeh; Zeng, Hanxiang; Johnson, Mark; Schlesener, Cathleen

    2015-01-01

    Summary The success of siRNA-based therapeutics highly depends on a safe and efficient delivery of siRNA into the cytosol. In this study, we post-modified the primary amines on dendritic polyglycerolamine (dPG-NH2) with different ratios of two relevant amino acids, namely, arginine (Arg) and histidine (His). To investigate the effects from introducing Arg and His to dPG, the resulting polyplexes of amino acid functionalized dPG-NH2s (AAdPGs)/siRNA were evaluated regarding cytotoxicity, transfection efficiency, and cellular uptake. Among AAdPGs, an optimal vector with (1:3) Arg to His ratio, showed efficient siRNA transfection with minimal cytotoxicity (cell viability ≥ 90%) in NIH 3T3 cells line. We also demonstrated that the cytotoxicity of dPG-NH2 decreased as a result of amino acid functionalization. While the incorporation of both cationic (Arg) and pH-responsive residues (His) are important for safe and efficient siRNA transfection, this study indicates that AAdPGs containing higher degrees of His display lower cytotoxicity and more efficient endosomal escape. PMID:26124878

  4. Utility of MicroRNAs and siRNAs in Cervical Carcinogenesis

    PubMed Central

    Díaz-González, Sacnite del Mar; Benítez-Boijseauneau, Odelia; Gómez-Cerón, Claudia; Bermúdez-Morales, Victor Hugo; Rodríguez-Dorantes, Mauricio; Pérez-Plasencia, Carlos; Peralta-Zaragoza, Oscar

    2015-01-01

    MicroRNAs and siRNAs belong to a family of small noncoding RNAs which bind through partial sequence complementarity to 3′-UTR regions of mRNA from target genes, resulting in the regulation of gene expression. MicroRNAs have become an attractive target for genetic and pharmacological modulation due to the critical function of their target proteins in several signaling pathways, and their expression profiles have been found to be altered in various cancers. A promising technology platform for selective silencing of cell and/or viral gene expression using siRNAs is currently in development. Cervical cancer is the most common cancer in women in the developing world and sexually transmitted infection with HPV is the cause of this malignancy. Therefore, a cascade of abnormal events is induced during cervical carcinogenesis, including the induction of genomic instability, reprogramming of cellular metabolic pathways, deregulation of cell proliferation, inhibition of apoptotic mechanisms, disruption of cell cycle control mechanisms, and alteration of gene expression. Thus, in the present review article, we highlight new research on microRNA expression profiles which may be utilized as biomarkers for cervical cancer. Furthermore, we discuss selective silencing of HPV E6 and E7 with siRNAs which represents a potential gene therapy strategy against cervical cancer. PMID:25874209

  5. Utility of microRNAs and siRNAs in cervical carcinogenesis.

    PubMed

    Díaz-González, Sacnite del Mar; Deas, Jessica; Benítez-Boijseauneau, Odelia; Gómez-Cerón, Claudia; Bermúdez-Morales, Victor Hugo; Rodríguez-Dorantes, Mauricio; Pérez-Plasencia, Carlos; Peralta-Zaragoza, Oscar

    2015-01-01

    MicroRNAs and siRNAs belong to a family of small noncoding RNAs which bind through partial sequence complementarity to 3'-UTR regions of mRNA from target genes, resulting in the regulation of gene expression. MicroRNAs have become an attractive target for genetic and pharmacological modulation due to the critical function of their target proteins in several signaling pathways, and their expression profiles have been found to be altered in various cancers. A promising technology platform for selective silencing of cell and/or viral gene expression using siRNAs is currently in development. Cervical cancer is the most common cancer in women in the developing world and sexually transmitted infection with HPV is the cause of this malignancy. Therefore, a cascade of abnormal events is induced during cervical carcinogenesis, including the induction of genomic instability, reprogramming of cellular metabolic pathways, deregulation of cell proliferation, inhibition of apoptotic mechanisms, disruption of cell cycle control mechanisms, and alteration of gene expression. Thus, in the present review article, we highlight new research on microRNA expression profiles which may be utilized as biomarkers for cervical cancer. Furthermore, we discuss selective silencing of HPV E6 and E7 with siRNAs which represents a potential gene therapy strategy against cervical cancer. PMID:25874209

  6. Molecular Properties, Functional Mechanisms, and Applications of Sliced siRNA

    PubMed Central

    Sun, Guihua; Yeh, Spencer Yele; Yuan, Christine Wan-Yin; Chiu, Margaret Jean-Yih; Yung, Bryan Shing Hei; Yen, Yun

    2015-01-01

    Using pre-miR-451 as a model molecule, we have characterized the general molecular properties of small hairpin RNAs that are processed into potent small interfering RNAs (siRNA) by Argonaute2 (Ago2). The Ago2-sliced siRNAs (sli-siRNAs) have the same silencing potency as the classical Dicer diced siRNAs (di-siRNAs) but have dramatically reduced unwanted sense strand activities. We have built vectors with the constitutive or inducible U6 promoter that can express sli-siRNAs in mammalian cells, in which the sli-siRNAs can be correctly processed to repress target genes. As a proof of principle for potential applications of sli-siRNAs in vivo, we show that the expression of one Ago2 shRNA-1148 in HCT-116 colon cancer cells knocked down RRM2 expression and reduced the proliferation and invasiveness of the cells. The defined sli-siRNA model molecules and the expression systems established in this study will facilitate the design and application of sli-siRNAs as novel potent RNAi triggers with reduced off-target effects. PMID:25602583

  7. Inter-molecular β-sheet structure facilitates lung-targeting siRNA delivery

    NASA Astrophysics Data System (ADS)

    Zhou, Jihan; Li, Dong; Wen, Hao; Zheng, Shuquan; Su, Cuicui; Yi, Fan; Wang, Jue; Liang, Zicai; Tang, Tao; Zhou, Demin; Zhang, Li-He; Liang, Dehai; Du, Quan

    2016-03-01

    Size-dependent passive targeting based on the characteristics of tissues is a basic mechanism of drug delivery. While the nanometer-sized particles are efficiently captured by the liver and spleen, the micron-sized particles are most likely entrapped within the lung owing to its unique capillary structure and physiological features. To exploit this property in lung-targeting siRNA delivery, we designed and studied a multi-domain peptide named K-β, which was able to form inter-molecular β-sheet structures. Results showed that K-β peptides and siRNAs formed stable complex particles of 60 nm when mixed together. A critical property of such particles was that, after being intravenously injected into mice, they further associated into loose and micron-sized aggregates, and thus effectively entrapped within the capillaries of the lung, leading to a passive accumulation and gene-silencing. The large size aggregates can dissociate or break down by the shear stress generated by blood flow, alleviating the pulmonary embolism. Besides the lung, siRNA enrichment and targeted gene silencing were also observed in the liver. This drug delivery strategy, together with the low toxicity, biodegradability, and programmability of peptide carriers, show great potentials in vivo applications.

  8. PEGylated cyclodextrins as novel siRNA nanosystems: correlations between polyethylene glycol length and nanoparticle stability.

    PubMed

    Godinho, Bruno M D C; Ogier, Julien R; Quinlan, Aoife; Darcy, Raphael; Griffin, Brendan T; Cryan, John F; O'Driscoll, Caitriona M

    2014-10-01

    Silencing disease-related genes in the central nervous system (CNS) using short interfering RNA (siRNA) holds great promise for treating neurological disorders. Yet, delivery of RNAi therapeutics to the brain poses major challenges to non-viral systems, especially when considering systemic administration. Cationic nanoparticles have been widely investigated for siRNA delivery, but the tendency of these to aggregate in physiological environments limits their intravenous application. Thus, strategies to increase the stability of nanoparticles have been developed. Here, we investigated the ability of modified cationic amphiphilic or PEGylated amphiphilic cyclodextrins (CD) to formulate stable CD.siRNA nanoparticles. To this end, we describe a simple method for post-modification of pre-formed cationic CD.siRNA nanoparticles at their surface using PEGylated CDs of different PEG lengths. PEGylated CD.siRNA nanoparticles presented reduced surface charges and increased stability in physiological salt conditions. Stability of PEGylated CD.siRNA nanoparticles in vitro increased with both PEG length and PEG density at the surface. Furthermore, in a comparative pharmacokinetic study, increased systemic exposure and reduced clearance were achieved with CD-formulations when compared to naked siRNAs. However, no significant differences were observed among non-PEGylated and PEGylated CD.siRNAs suggesting that longer PEG lengths might be required for improving stability in vivo. PMID:24992319

  9. A combinatorial approach to determine functional group effects on lipidoid-mediated siRNA delivery

    PubMed Central

    Mahon, Kerry P.; Love, Kevin T.; Whitehead, Kathryn A.; Qin, June; Akinc, Akin; Leshchiner, Elizaveta; Leshchiner, Ignaty; Langer, Robert

    2010-01-01

    The application of RNA interference (RNAi), either in the clinic or laboratory, requires safe and effective delivery methods. Here we develop a combinatorial approach to synthesize a library of delivery vectors based on two lipid-like substrates with known siRNA delivery capabilities. Members of this library have a mixture of lipid-like tails and feature appendages containing hydroxyl, carbamate, ether or amine functional groups as well as variations in alkyl chain length and branching. Using a luciferase reporter system in HeLa cells, we study the relationship between lipid chemical modification and delivery performance in vitro. The impact of the functional group was shown to vary depending on the overall amine content and tail number of the delivery vector. Additionally, in vivo performance was evaluated using a Factor VII knockdown assay. Two library members, each containing ether groups, were found to knock down the target protein at levels comparable to the parent delivery vector. These results demonstrate that small chemical changes to the delivery vector impact knockdown efficiency and cell viability both in vitro and in vivo. The work described here identifies new materials for siRNA delivery, as well as provides new insight into the parameters for optimized chemical makeup of lipid-like siRNA delivery materials. PMID:20715849

  10. Inter-molecular β-sheet structure facilitates lung-targeting siRNA delivery.

    PubMed

    Zhou, Jihan; Li, Dong; Wen, Hao; Zheng, Shuquan; Su, Cuicui; Yi, Fan; Wang, Jue; Liang, Zicai; Tang, Tao; Zhou, Demin; Zhang, Li-He; Liang, Dehai; Du, Quan

    2016-01-01

    Size-dependent passive targeting based on the characteristics of tissues is a basic mechanism of drug delivery. While the nanometer-sized particles are efficiently captured by the liver and spleen, the micron-sized particles are most likely entrapped within the lung owing to its unique capillary structure and physiological features. To exploit this property in lung-targeting siRNA delivery, we designed and studied a multi-domain peptide named K-β, which was able to form inter-molecular β-sheet structures. Results showed that K-β peptides and siRNAs formed stable complex particles of 60 nm when mixed together. A critical property of such particles was that, after being intravenously injected into mice, they further associated into loose and micron-sized aggregates, and thus effectively entrapped within the capillaries of the lung, leading to a passive accumulation and gene-silencing. The large size aggregates can dissociate or break down by the shear stress generated by blood flow, alleviating the pulmonary embolism. Besides the lung, siRNA enrichment and targeted gene silencing were also observed in the liver. This drug delivery strategy, together with the low toxicity, biodegradability, and programmability of peptide carriers, show great potentials in vivo applications. PMID:26955887

  11. Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates

    PubMed Central

    Dong, Yizhou; Love, Kevin T.; Dorkin, J. Robert; Sirirungruang, Sasilada; Zhang, Yunlong; Chen, Delai; Bogorad, Roman L.; Yin, Hao; Chen, Yi; Vegas, Arturo J.; Alabi, Christopher A.; Sahay, Gaurav; Olejnik, Karsten T.; Wang, Weiheng; Schroeder, Avi; Lytton-Jean, Abigail K. R.; Siegwart, Daniel J.; Akinc, Akin; Barnes, Carmen; Barros, Scott A.; Carioto, Mary; Fitzgerald, Kevin; Hettinger, Julia; Kumar, Varun; Novobrantseva, Tatiana I.; Qin, June; Querbes, William; Koteliansky, Victor; Langer, Robert; Anderson, Daniel G.

    2014-01-01

    siRNA therapeutics have promise for the treatment of a wide range of genetic disorders. Motivated by lipoproteins, we report lipopeptide nanoparticles as potent and selective siRNA carriers with a wide therapeutic index. Lead material cKK-E12 showed potent silencing effects in mice (ED50 ∼ 0.002 mg/kg), rats (ED50 < 0.01 mg/kg), and nonhuman primates (over 95% silencing at 0.3 mg/kg). Apolipoprotein E plays a significant role in the potency of cKK-E12 both in vitro and in vivo. cKK-E12 was highly selective toward liver parenchymal cell in vivo, with orders of magnitude lower doses needed to silence in hepatocytes compared with endothelial cells and immune cells in different organs. Toxicity studies showed that cKK-E12 was well tolerated in rats at a dose of 1 mg/kg (over 100-fold higher than the ED50). To our knowledge, this is the most efficacious and selective nonviral siRNA delivery system for gene silencing in hepatocytes reported to date. PMID:24516150

  12. Acid Sensitive Polymeric Micelles Combining Folate and Bioreducible Conjugate for Specific Intracellular siRNA Delivery.

    PubMed

    Yang, Yanfang; Xia, Xuejun; Dong, Wujun; Wang, Hongliang; Li, Lin; Ma, Panpan; Sheng, Wei; Xu, Xueqing; Liu, Yuling

    2016-05-01

    An efficiently siRNA transporting nanocarrier still remains to be developed. In this study, utilizing the dual stimulus of acid tumor extracellular environment and redox effect of glutathione in the cytosol, a new siRNA transporting system combining triple effects of folate targeting, acid sensitive polymer micelles, and bio-reducible disulfide bond linked siRNA-cell penetrating peptides (CPPs) conjugate is developed to suppress c-myc gene expression of breast cancer (MCF-7 cells) both in vitro and in vivo. Subsequent research demonstrates that the vesicle has particle size of about 100 nm and siRNA entrapment efficiency of approximately 80%. In vitro studies verified over 90% of encapsulated siRNA-CPPs can be released and the vesicle shows higher cellular uptake in response to the tumorous zone. Determination of gene expression at both mRNA and protein levels indicates the constructed vesicle exhibited enhanced cancer cell apoptosis and improved therapeutic efficacy in vitro and in vivo. PMID:26822264

  13. PolyMetformin combines carrier and anticancer activities for in vivo siRNA delivery

    PubMed Central

    Zhao, Yi; Wang, Wei; Guo, Shutao; Wang, Yuhua; Miao, Lei; Xiong, Yang; Huang, Leaf

    2016-01-01

    Metformin, a widely implemented anti-diabetic drug, exhibits potent anticancer efficacies. Herein a polymeric construction of Metformin, PolyMetformin (PolyMet) is successfully synthesized through conjugation of linear polyethylenimine (PEI) with dicyandiamide. The delocalization of cationic charges in the biguanide groups of PolyMet reduces the toxicity of PEI both in vitro and in vivo. Furthermore, the polycationic properties of PolyMet permits capture of siRNA into a core-membrane structured lipid-polycation-hyaluronic acid (LPH) nanoparticle for systemic gene delivery. Advances herein permit LPH-PolyMet nanoparticles to facilitate VEGF siRNA delivery for VEGF knockdown in a human lung cancer xenograft, leading to enhanced tumour suppressive efficacy. Even in the absence of RNAi, LPH-PolyMet nanoparticles act similarly to Metformin and induce antitumour efficacy through activation of the AMPK and inhibition of the mTOR. In essence, PolyMet successfully combines the intrinsic anticancer efficacy of Metformin with the capacity to carry siRNA to enhance the therapeutic activity of an anticancer gene therapy. PMID:27264609

  14. Screening of efficient siRNA carriers in a library of surface-engineered dendrimers

    PubMed Central

    Liu, Hongmei; Chang, Hong; Lv, Jia; Jiang, Cong; Li, Zhenxi; Wang, Fei; Wang, Hui; Wang, Mingming; Liu, Chongyi; Wang, Xinyu; Shao, Naimin; He, Bingwei; Shen, Wanwan; Zhang, Qiang; Cheng, Yiyun

    2016-01-01

    Polymers are widely used as non-viral carriers for siRNA delivery, but concern has also arisen in their limited efficacy and inherent toxicity. Whilst many of previous efforts have been documented towards improving the performance of polymers via chemical modifications, the structure-activity relationships (SAR) of these ligand-modified polymers are not well understood. To address this issue, we systemically prepared a library of surface-engineered dendrimers (>300) as the screening pool to discover efficient siRNA carriers. The modified ligands include alkyls and fluoroalkyls, amino acids, benzene derivatives and heterocyclic compounds. Gene silencing results showed that the lead material shows excellent efficacy even in hard-to-transfect cells such as mesenchymal stem cells. The SAR studies revealed that ligands containing appropriate hydrophobicity, or ligands with both hydrophobic and functional atoms/groups are essential for polymers to achive efficient knockdown efficacy. A second-generation library designed based on the above principles further confirms the proposed design criteria. The results enable the future rational design of potent siRNA carriers. PMID:27121799

  15. Gold nanorod delivery of LSD1 siRNA induces human mesenchymal stem cell differentiation.

    PubMed

    Zhao, Xiongfei; Huang, Qianying; Jin, Yiqiang

    2015-09-01

    Over the past decade, theranostic nanoparticles with microsize and multifunctional ability have emerged as a new platform in biomedical field, such as cancer therapy, optical imaging and gene therapy. Gene therapy has been recently shown as a promising tool for tissue engineering as safe and effective nanotechnology-based delivery methods are developed. Controlling adhesion and differentiation of stem cells is critical for tissue regeneration. In this study, we have developed poly-sodium 4-styrenesulfonate (PSS) and poly-allylamine hydrochloride (PAH) coated AuNR-based nanocarriers, which are capable of delivering small interfering RNA (siRNA) against LSD1 to induce the differentiation of human mesenchymal stem cells. To further study the mechanism, we tested the stemness and differentiation genes and found that they have been changed with LSD1 down-regulation. In addition, with the hepatocyte growth factor (HGF), LSD1 siRNA delivery by AuNRs could promote the differentiation of the human mesenchymal stem cells (human MSCs) into a hepatocyte lineage in vitro. Our results suggest for the first time use of AuNRs as nanocarriers of delivery LSD1 siRNA to induce the differentiation of human MSCs into a hepatocyte lineage, and envision the potential application of nanotechnology in tissue remodeling (such as liver and bone) in vivo, eventually translating to clinical applications. PMID:26046277

  16. Li-7 abundances in halo stars: Testing stellar evolution models and the primordial Li-7 abundance

    NASA Technical Reports Server (NTRS)

    Chaboyer, Brian; Demarque, P.

    1994-01-01

    A large number of stellar evolution models with (Fe/H) = -2.3 and -3.3 have been calculated in order to determine the primordial Li-7 abundance and to test current stellar evolution models by a comparison to the extensive database of accurate Li abundances in extremely metal-poor halo stars observed by Thorburn (1994). Standard models with gray atmospheres do a very good job of fitting the observed Li abundances in stars hotter than approximately 5600 K. They predict a primordial. Li-7 abundance of log N(Li) = 2.24 +/- 0.03. Models which include microscopic diffusion predict a downward curvature in the Li-7 destruction isochrones at hot temperatures which is not present in the observations. Thus, the observations clearly rule out models which include uninhibited microscopic diffusion of Li-7 from the surface of the star. Rotational mixing inhibits the microscopic diffusion and the (Fe/H) = -2.28 stellar models which include both diffusion and rotational mixing provide an excellent match to the mean trend in T(sub eff) which is present in the observations. Both the plateau stars and the heavily depleted cool stars are well fit by these models. The rotational mixing leads to considerable Li-7 depletion in these models and the primordial Li-7 abundance inferred from these models is log N(Li) = 3.08 +/- 0.1. However, the (Fe/H) = -3.28 isochrones reveal problems with the combined models. These isochrones predict a trend of decreasing log N(Li) with increasing T(sub eff) which is not present in the observations. Possible causes for this discrepancy are discussed.

  17. Li-7 abundances in halo stars: Testing stellar evolution models and the primordial Li-7 abundance

    NASA Astrophysics Data System (ADS)

    Chaboyer, Brian; Demarque, P.

    1994-10-01

    A large number of stellar evolution models with (Fe/H) = -2.3 and -3.3 have been calculated in order to determine the primordial Li-7 abundance and to test current stellar evolution models by a comparison to the extensive database of accurate Li abundances in extremely metal-poor halo stars observed by Thorburn (1994). Standard models with gray atmospheres do a very good job of fitting the observed Li abundances in stars hotter than approximately 5600 K. They predict a primordial. Li-7 abundance of log N(Li) = 2.24 +/- 0.03. Models which include microscopic diffusion predict a downward curvature in the Li-7 destruction isochrones at hot temperatures which is not present in the observations. Thus, the observations clearly rule out models which include uninhibited microscopic diffusion of Li-7 from the surface of the star. Rotational mixing inhibits the microscopic diffusion and the (Fe/H) = -2.28 stellar models which include both diffusion and rotational mixing provide an excellent match to the mean trend in Teff which is present in the observations. Both the plateau stars and the heavily depleted cool stars are well fit by these models. The rotational mixing leads to considerable Li-7 depletion in these models and the primordial Li-7 abundance inferred from these models is log N(Li) = 3.08 +/- 0.1. However, the (Fe/H) = -3.28 isochrones reveal problems with the combined models. These isochrones predict a trend of decreasing log N(Li) with increasing Teff which is not present in the observations. Possible causes for this discrepancy are discussed.

  18. Effects of chitosan nanoparticle-mediated BRAF siRNA interference on invasion and metastasis of gastric cancer cells.

    PubMed

    Huo, Jian

    2016-08-01

    To observe the changes in invasion capacity of gastric cancer BGC823 cells after being treated with chitosan-encapsulated BRAF siRNA nanoparticles, and to evaluate the effects of the nanoparticle-mediated BRAF siRNA interference on cell invasion and metastasis, BRAF siRNA was encapsulated with chitosan into nanoparticles sized 350 nm to treat gastric cancer cells. Silencing of BRAF was detected by Western blot and PCR, and cell invasion was observed by the Transwell assay. The nanoparticles significantly downregulated BRAF expression in BGC823 cells (P < 0.01) and inhibited their invasion (P < 0.001). Chitosan nanoparticle-mediated BRAF siRNA interference evidently reduced the invasion capacity of gastric cancers. PMID:25794798

  19. Recent In Vivo Evidences of Particle-Based Delivery of Small-Interfering RNA (siRNA) into Solid Tumors

    PubMed Central

    2014-01-01

    Small-interfering RNA (siRNA) is both a powerful tool in research and a promising therapeutic platform to modulate expression of disease-related genes. Malignant tumors are attractive disease targets for nucleic acid-based therapies. siRNA directed against oncogenes, and genes driving metastases or angiogenesis have been evaluated in animal models and in some cases, in humans. The outcomes of these studies indicate that drug delivery is a significant limiting factor. This review provides perspectives on in vivo validated nanoparticle-based siRNA delivery systems. Results of recent advances in liposomes and polymeric and inorganic formulations illustrate the need for mutually optimized attributes for performance in systemic circulation, tumor interstitial space, plasma membrane, and endosomes. Physiochemical properties conducive to efficient siRNA delivery are summarized and directions for future research are discussed. PMID:25221632

  20. Atelocollagen-mediated systemic administration of myostatin-targeting siRNA improves muscular atrophy in caveolin-3-deficient mice.

    PubMed

    Kawakami, Emi; Kinouchi, Nao; Adachi, Taro; Ohsawa, Yutaka; Ishimaru, Naozumi; Ohuchi, Hideyo; Sunada, Yoshihide; Hayashi, Yoshio; Tanaka, Eiji; Noji, Sumihare

    2011-01-01

    Small interfering RNA (siRNA)-mediated silencing of gene expression is rapidly becoming a powerful tool for molecular therapy. However, the rapid degradation of siRNAs and their limited duration of activity require efficient delivery methods. Atelocollagen (ATCOL)-mediated administration of siRNAs is a promising approach to disease treatment, including muscular atrophy. Herein, we report that ATCOL-mediated systemic administration of a myostatin-targeting siRNA into a caveolin-3-deficient mouse model of limb-girdle muscular dystrophy 1C (LGMD1C) induced a marked increase in muscle mass and a significant recovery of contractile force. These results provide evidence that ATCOL-mediated systemic administration of siRNAs may be a powerful therapeutic tool for disease treatment, including muscular atrophy. PMID:21261610

  1. Unbinding forces and energies between a siRNA molecule and a dendrimer measured by force spectroscopy.

    PubMed

    Dumitru, Andra C; Herruzo, Elena T; Rausell, Estrella; Ceña, Valentin; Garcia, Ricardo

    2015-12-21

    We have measured the intermolecular forces between small interference RNA (siRNA) and polyamidoamine dendrimers at the single molecular level. A single molecule force spectroscopy approach has been developed to measure the unbinding forces and energies between a siRNA molecule and polyamidoamine dendrimers deposited on a mica surface in a buffer solution. We report three types of unbinding events which are characterized by forces and free unbinding energies, respectively, of 28 pN, 0.709 eV; 38 pN, 0.722 eV; and 50 pN, 0.724 eV. These events reflect different possible electrostatic interactions between the positive charges of one or two dendrimers and the negatively charged phosphate groups of a single siRNA. We have evidence of a high binding affinity of siRNA towards polyamidoamine dendrimers that leads to a 45% probability of measuring specific unbinding events. PMID:26580848

  2. Unbinding forces and energies between a siRNA molecule and a dendrimer measured by force spectroscopy

    NASA Astrophysics Data System (ADS)

    Dumitru, Andra C.; Herruzo, Elena T.; Rausell, Estrella; Ceña, Valentin; Garcia, Ricardo

    2015-11-01

    We have measured the intermolecular forces between small interference RNA (siRNA) and polyamidoamine dendrimers at the single molecular level. A single molecule force spectroscopy approach has been developed to measure the unbinding forces and energies between a siRNA molecule and polyamidoamine dendrimers deposited on a mica surface in a buffer solution. We report three types of unbinding events which are characterized by forces and free unbinding energies, respectively, of 28 pN, 0.709 eV; 38 pN, 0.722 eV; and 50 pN, 0.724 eV. These events reflect different possible electrostatic interactions between the positive charges of one or two dendrimers and the negatively charged phosphate groups of a single siRNA. We have evidence of a high binding affinity of siRNA towards polyamidoamine dendrimers that leads to a 45% probability of measuring specific unbinding events.

  3. SiRNA In Vivo-Targeted Delivery to Murine Dendritic Cells by Oral Administration of Recombinant Yeast.

    PubMed

    Xu, Kun; Liu, Zhongtian; Zhang, Long; Zhang, Tingting; Zhang, Zhiying

    2016-01-01

    SiRNA therapeutics promise a future where any target in the transcriptome could be potentially addressed. However, the delivery of SiRNAs and targeting of particular cell types or organs are major challenges. A novel, efficient, and safe delivery system for promising the introduction of SiRNAs into particular cell types within living organisms is of great significance. Our previous studies have proved that recombinant protein (MSTN) and exogenous gene (EGFP) as vaccines, and furthermore functional CD40 shRNA expression can be delivered into dendritic cells (DCs) in mouse by oral administration of recombinant yeast (Saccharomyces cerevisiae). Here, we describe the details of the promising and innovative approach based on oral administration of recombinant yeast that allows in vivo-targeted delivery of functional SiRNA to murine intestinal DCs. PMID:26472450

  4. siVirus: web-based antiviral siRNA design software for highly divergent viral sequences

    PubMed Central

    Naito, Yuki; Ui-Tei, Kumiko; Nishikawa, Toru; Takebe, Yutaka; Saigo, Kaoru

    2006-01-01

    siVirus () is a web-based online software system that provides efficient short interfering RNA (siRNA) design for antiviral RNA interference (RNAi). siVirus searches for functional, off-target minimized siRNAs targeting highly conserved regions of divergent viral sequences. These siRNAs are expected to resist viral mutational escape, since their highly conserved targets likely contain structurally/functionally constrained elements. siVirus will be a useful tool for designing optimal siRNAs targeting highly divergent pathogens, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus and SARS coronavirus, all of which pose enormous threats to global human health. PMID:16845046

  5. siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference

    PubMed Central

    Naito, Yuki; Yamada, Tomoyuki; Ui-Tei, Kumiko; Morishita, Shinichi; Saigo, Kaoru

    2004-01-01

    siDirect (http://design.RNAi.jp/) is a web-based online software system for computing highly effective small interfering RNA (siRNA) sequences with maximum target-specificity for mammalian RNA interference (RNAi). Highly effective siRNA sequences are selected using novel guidelines that were established through an extensive study of the relationship between siRNA sequences and RNAi activity. Our efficient software avoids off-target gene silencing to enumerate potential cross-hybridization candidates that the widely used BLAST search may overlook. The website accepts an arbitrary sequence as input and quickly returns siRNA candidates, providing a wide scope of applications in mammalian RNAi, including systematic functional genomics and therapeutic gene silencing. PMID:15215364

  6. Non-Covalently Functionalized of Single-Walled Carbon Nanotubes by DSPE-PEG-PEI for SiRNA Delivery.

    PubMed

    Siu, King Sun; Zhang, Yujuan; Zheng, Xiufen; Koropatnick, James; Min, Wei-Ping

    2016-01-01

    The expression of a gene can be specifically downregulated by small interfering RNA (SiRNA). Modified carbon nanotubes (CNT) can be used to protect SiRNA and facilitate its entry into cells. Regardless of that, simple and efficient functionalization of CNT is lacking. Effective SiRNA delivery can be carried out using non-covalently functionalized CNT, where non-covalent (versus covalent) functionalization is simpler and more expeditious. Non-covalently functionalized single walled carbon nanotubes (SWCNT) that include a lipopolymer are described here. Polyethylenimine (PEI) conjugated to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) was generated and the products used to disperse CNT to form DSPE-PEG-PEI/CNT (DGI/C), an agent capable of facilitating SiRNA delivery to cells in vitro and organs and cells in vivo. PMID:26472449

  7. Small RNA transcriptomes of mangroves evolve adaptively in extreme environments

    PubMed Central

    Wen, Ming; Lin, Xingqin; Xie, Munan; Wang, Yushuai; Shen, Xu; Liufu, Zhongqi; Wu, Chung-I; Shi, Suhua; Tang, Tian

    2016-01-01

    MicroRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) are key players in plant stress responses. Here, we present the sRNA transcriptomes of mangroves Bruguiera gymnorrhiza and Kandelia candel. Comparative computational analyses and target predictions revealed that mangroves exhibit distinct sRNA regulatory networks that differ from those of glycophytes. A total of 32 known and three novel miRNA families were identified. Conserved and mangrove-specific miRNA targets were predicted; the latter were widely involved in stress responses. The known miRNAs showed differential expression between the mangroves and glycophytes, reminiscent of the adaptive stress-responsive changes in Arabidopsis. B. gymnorrhiza possessed highly abundant but less conserved TAS3 trans-acting siRNAs (tasiRNAs) in addition to tasiR-ARFs, with expanded potential targets. Our results indicate that the evolutionary alteration of sRNA expression levels and the rewiring of sRNA-regulatory networks are important mechanisms underlying stress adaptation. We also identified sRNAs that are involved in salt and/or drought tolerance and nutrient homeostasis as possible contributors to mangrove success in stressful environments. PMID:27278626

  8. Small RNA transcriptomes of mangroves evolve adaptively in extreme environments.

    PubMed

    Wen, Ming; Lin, Xingqin; Xie, Munan; Wang, Yushuai; Shen, Xu; Liufu, Zhongqi; Wu, Chung-I; Shi, Suhua; Tang, Tian

    2016-01-01

    MicroRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) are key players in plant stress responses. Here, we present the sRNA transcriptomes of mangroves Bruguiera gymnorrhiza and Kandelia candel. Comparative computational analyses and target predictions revealed that mangroves exhibit distinct sRNA regulatory networks that differ from those of glycophytes. A total of 32 known and three novel miRNA families were identified. Conserved and mangrove-specific miRNA targets were predicted; the latter were widely involved in stress responses. The known miRNAs showed differential expression between the mangroves and glycophytes, reminiscent of the adaptive stress-responsive changes in Arabidopsis. B. gymnorrhiza possessed highly abundant but less conserved TAS3 trans-acting siRNAs (tasiRNAs) in addition to tasiR-ARFs, with expanded potential targets. Our results indicate that the evolutionary alteration of sRNA expression levels and the rewiring of sRNA-regulatory networks are important mechanisms underlying stress adaptation. We also identified sRNAs that are involved in salt and/or drought tolerance and nutrient homeostasis as possible contributors to mangrove success in stressful environments. PMID:27278626

  9. Circular RNAs are abundant, conserved, and associated with ALU repeats

    PubMed Central

    Jeck, William R.; Sorrentino, Jessica A.; Wang, Kai; Slevin, Michael K.; Burd, Christin E.; Liu, Jinze; Marzluff, William F.; Sharpless, Norman E.

    2013-01-01

    Circular RNAs composed of exonic sequence have been described in a small number of genes. Thought to result from splicing errors, circular RNA species possess no known function. To delineate the universe of endogenous circular RNAs, we performed high-throughput sequencing (RNA-seq) of libraries prepared from ribosome-depleted RNA with or without digestion with the RNA exonuclease, RNase R. We identified >25,000 distinct RNA species in human fibroblasts that contained non-colinear exons (a “backsplice”) and were reproducibly enriched by exonuclease degradation of linear RNA. These RNAs were validated as circular RNA (ecircRNA), rather than linear RNA, and were more stable than associated linear mRNAs in vivo. In some cases, the abundance of circular molecules exceeded that of associated linear mRNA by >10-fold. By conservative estimate, we identified ecircRNAs from 14.4% of actively transcribed genes in human fibroblasts. Application of this method to murine testis RNA identified 69 ecircRNAs in precisely orthologous locations to human circular RNAs. Of note, paralogous kinases HIPK2 and HIPK3 produce abundant ecircRNA from their second exon in both humans and mice. Though HIPK3 circular RNAs contain an AUG translation start, it and other ecircRNAs were not bound to ribosomes. Circular RNAs could be degraded by siRNAs and, therefore, may act as competing endogenous RNAs. Bioinformatic analysis revealed shared features of circularized exons, including long bordering introns that contained complementary ALU repeats. These data show that ecircRNAs are abundant, stable, conserved and nonrandom products of RNA splicing that could be involved in control of gene expression. PMID:23249747

  10. ADAM17-siRNA inhibits MCF-7 breast cancer through EGFR-PI3K-AKT activation.

    PubMed

    Meng, Xiangchao; Hu, Baoshan; Hossain, Mohammad Monir; Chen, Guofu; Sun, Ying; Zhang, Xuepeng

    2016-08-01

    A disintegrin and metalloproteinase-17 (ADAM17) can cut and release a wide variety of epidermal growth factor receptor (EGFR) ligands to promote survival, invasion and proliferation of cancer cell, and therefore, is considered to be a potential therapeutic target for cancer. The main goal of the present study was to observe the effects of ADAM17 small interfering RNA (ADAM17-siRNA) on human MCF-7 breast cancer and investigate its activation pathway. In vitro, MCF-7 cells were divided into ADAM17-siRNA groups, nonsense siRNA groups, AG1478 (selective EGFR blocker) groups, LY294002 [phosphatidylinositol 3-kinase (PI3K) phosphorylation inhibitor] groups, PD0325901 [mitogen extracellular kinase (MEK) inhibitor] groups and control groups. In vivo, MCF-7 cells were implanted subcutaneously into nude mice and then these mice were randomly divided into ADAM17-siRNA groups, vector groups and control groups. Our data showed that compared with the control groups, ADAM17-siRNA, AG1478 and LY294002 could inhibit the migration and proliferation of MCF-7 cells, but PD0325901 and nonsense siRNA did not show this effect. Except that specific ADAM17-siRNA could inhibit the expression of ADAM17 mRNA, others did not change it. Western blot analysis further confirmed that EGFR-PI3K-AKT signaling pathway is involved in ADAM17-siRNA inhibiting migration and proliferation of MCF-7 cells. Similarly to the former, the growth of MCF-7 breast cancer in nude mice was significantly inhibited by ADAM17-siRNA. Compared with the control group and the vector group, the tumor volume was smaller in the ADAM17-siRNA group, the tissues developed large areas of necrosis, immunohistochemistry showed low expressions of ADAM17 and Ki-67 and western blot analysis proved that the expression of ADAM17 protein in the tissue was also reduced. The present study suggests that ADAM17-siRNA inhibits MCF-7 breast cancer and is activated through the EGFR-PI3K-AKT signaling pathway. PMID:27221510

  11. The boron abundance of Procyon

    NASA Technical Reports Server (NTRS)

    Lemke, Michael; Lambert, David L.; Edvardsson, Bengt

    1993-01-01

    The B I 2496.8 A resonance line and HST/GHRS echelle spectra are used with model atmospheres and synthetic spectra to derive the B abundance of the F dwarfs Procyon (Alpha Canis Minoris), Theta Ursae Majoris, and Iota Pegasi. The B abundance of Theta UMa and Iota Peg is similar to that derived by Boesgaard and Heacox (1978) from the B II resonance line in spectra of A- and B-type stars. These two dwarfs show normal abundances of Li, Be, and B. Procyon, which is highly depleted in Li and Be, is depleted in B by a factor of at least 3. Comparison of the spectra of Procyon and the halo dwarf HD 140283 shows that the B abundance assigned by Duncan et al. (1992) to three halo dwarfs is not greatly overestimated as a result of contamination of the B I line by an unidentified line.

  12. Hyaluronic acid based self-assembling nanosystems for CD44 target mediated siRNA delivery to solid tumors

    PubMed Central

    Ganesh, Shanthi; Iyer, Arun K.; Morrissey, David V.; Amiji, Mansoor M.

    2013-01-01

    Anticancer therapeutics employing RNA interference mechanism holds promising potentials for sequence-specific silencing of target genes. However targeted delivery of siRNAs to tumor tissues and cells and more importantly, their intracellular release at sites of interest still remains a major challenge that needs to be addressed before this technique could become a clinically viable option. In the current study, we have engineered and screened a series of CD44 targeting hyaluronic acid (HA) based self-assembling nanosystems for targeted siRNA delivery. The HA polymer was functionalized with lipids of varying carbon chain lengths/nitrogen content, as well as polyamines for assessing siRNA encapsulation. From the screens, several HA-derivatives were identified that could stably encapsulate/complex siRNAs and form self-assembled nanosystems, as determined by gel retardation assays and dynamic light scattering. Many HA derivatives could transfect siRNAs into cancer cells overexpressing CD44 receptors. Interestingly, blocking the CD44 receptors on the cells using free excess soluble HA prior to incubation of cy3-labeled-siRNA loaded HA nano-assemblies resulted in >90% inhibition of the receptor mediated uptake, confirming target specificity. In addition, SSB/PLK1 siRNA encapsulated in HA-PEI/PEG nanosystems demonstrated dose dependent and target specific gene knockdown in both sensitive and resistant A549 lung cancer cells overexpressing CD44 receptors. More importantly, these siRNA encapsulated nanosystems demonstrated tumor selective uptake and target specific gene knock down in vivo in solid tumors as well as in metastatic tumors. The HA based nanosystems thus portend to be promising siRNA delivery vectors for systemic targeting of CD44 overexpressing cancers including tumor initiating (stem-) cells and metastatic lesions. PMID:23410679

  13. Combination siRNA therapy against feline coronavirus can delay the emergence of antiviral resistance in vitro.

    PubMed

    McDonagh, Phillip; Sheehy, Paul A; Norris, Jacqueline M

    2015-03-23

    Virulent biotypes of feline coronavirus (FCoV), commonly referred to as feline infectious peritonitis virus (FIPV), can result in the development of feline infectious peritonitis (FIP), a typically fatal immune mediated disease for which there is currently no effective antiviral treatment. We previously reported the successful in vitro inhibition of FIPV replication by synthetic siRNA mediated RNA interference (RNAi) in an immortalised cell line (McDonagh et al., 2011). A major challenge facing the development of any antiviral strategy is that of resistance, a problem which is particularly acute for RNAi based therapeutics due to the exquisite sequence specificity of the targeting mechanism. The development of resistance during treatment can be minimised using combination therapy to raise the genetic barrier or using highly potent compounds which result in a more rapid and pronounced reduction in the viral replication rate, thereby reducing the formation of mutant, and potentially resistant viruses. This study investigated the efficacy of combination siRNA therapy and its ability to delay or prevent viral escape. Virus serially passaged through cells treated with a single or dual siRNAs rapidly acquired resistance, with mutations identified in the siRNA target sites. Combination therapy with three siRNA prevented viral escape over the course of five passages. To identify more potent silencing molecules we also compared the efficacy, in terms of potency and duration of action, of canonical versus Dicer-substrate siRNAs for two previously identified effective viral motifs. Dicer-substrate siRNAs showed equivalent or better potency than canonical siRNAs for the target sites investigated, and may be a more appropriate molecule for in vivo use. Combined, these data inform the potential therapeutic application of antiviral RNAi against FIPV. PMID:25596968

  14. Anti-tumor effects in mice induced by survivin-targeted siRNA delivered through polysaccharide nanoparticles.

    PubMed

    Yang, Feifei; Huang, Wei; Li, Yunfei; Liu, Shan; Jin, Mingji; Wang, Yuli; Jia, Lihua; Gao, Zhonggao

    2013-07-01

    Recently, survivin has been attracting great attention because it plays an important role in inhibiting the apoptosis process of tumor cells. Down-regulating the expression of survivin gene by small interfering RNA (siRNA) offers a promising method for anti-tumor therapy. However, lack of appropriate siRNA delivery vector has significantly hindered the successful application of survivin-targeted siRNA in anti-tumor therapy. The purpose of this study was to use polysaccharide vector TAT-g-CS we synthesized to deliver functional siRNA and evaluate its in vivo anti-tumor activity. TAT-g-CS vector was firstly synthesized and well structurally characterized. MTT assay showed that TAT-g-CS vector exhibited good biocompatibility. TAT-g-CS complexed with siRNA offering nanoparticles with an average particle size of 212.2 nm and a polydispersity index of 0.121, and the zeta potential of the nanoparticles was +18.58 mV. Results from reporter gene assay suggested that luciferase-targeted siRNA when delivered by TAT-g-CS could down-regulate the expression of luciferase gene with 75.3% reduction. Most importantly, we use siRNA(Sur) targeting survivin gene to assess the in vitro and in vivo delivery capacity of TAT-g-CS and its anti-tumor effects. Our results demonstrated that TAT-g-CS/siRNA(Sur) nanoparticles not only strongly inhibited the in vitro proliferation of 4T1-Luc tumor cells via inducing cell apoptosis, but also effectively inhibited the in vivo growth and metastasis of malignant breast tumor, which suggested that TAT-g-CS/siRNA nanoparticle was a highly efficient non-viral system for siRNA delivery, especially for anti-tumor therapy based on siRNA therapeutics. PMID:23632321

  15. A molecular dynamics simulation study on the effect of lipid substitution on polyethylenimine mediated siRNA complexation.

    PubMed

    Sun, Chongbo; Tang, Tian; Uludag, Hasan

    2013-04-01

    Polycations have been explored as non-viral carriers for effective delivery of small interfering RNA (siRNA). Modifying polycations such as polyethylenimine (PEI) with lipid substitution was found to improve the siRNA delivery efficiency of polycationic carriers. However, the role of such lipid modification is not clear and remains to be probed at the atomistic level. In this work, we elucidate the role of lipid modification through a series of all-atom molecular dynamics simulations on siRNA complexation mediated by a native PEI and four analogous obtained by different lipid modifications. The lipid modification does not affect PEI's capability of neutralizing the siRNA charge, neither does it affect the polyion bridging which plays an important role in siRNA complexation. Significant linkages among the lipid modified PEIs via association of lipid side-groups are observed and this results in more stable and compact PEI/siRNA polyplexes. The lipid associations between short lipids form and break frequently while the lipid associations between long lipids are more stable. For PEIs modified with short lipids, increasing the lipid substitution level results in more compact and stable siRNA structure. For PEIs modified with long lipids, increasing the lipid substitution does not change the amount of PEI linkage via lipid association, and has a reverse effect on compacting siRNA structure due to increased steric hindrance brought by the lipid association on individual PEIs. The simulation results generally correlate well with experimental data and suggest a framework of designing and systematic evaluation of polycation-based siRNA carriers using molecular dynamics simulations. PMID:23352043

  16. Elemental Abundances of Mercury-Manganese Stars

    NASA Astrophysics Data System (ADS)

    Adelman, Saul J.

    We propose to obtain a carefully planned set of multiple high dispersion exposures of three MercuryManganese stars in both the SWP and LWP cameras. These observations will be coadded to increase the S/N ratio so that accurate elemental abundances can be derived for these examples of HgMn stars, each of which represents some extreme class aspect. This will increase of sample of HgMn stars from four to seven. Of particular interest are the abundances of N and Co in which some HgMn stars have shown remarkable underabundances. Comparison of the UV and optical spectra features due to light elements such as N (and C and 0) provide an observational framework to test NLTE models such as those of Takada (1993). This work has already shed some light on some of earlier findings for normal stars. The ability to accurately determine the surface chemical composition of the late B stars through such studies will lead to better tests for theories purporting to explain the origin of the chemical peculiarities seen in this temperature domain.

  17. Intracellular delivery of peptides and siRNAs using microbubble enhanced focused ultrasound

    NASA Astrophysics Data System (ADS)

    Kinoshita, Manabu; Hynynen, Kullervo

    2006-05-01

    Bioactive substances such as peptides and nucleic acid based agents have attracted great attention for the next generation drug for various diseases. However, the greatest challenge for using these bioactive substances is the development of their delivery system, especially the method for delivering these substances through the cell membrane. With the advancement of ultrasound and ultrasound contrast agent technology, it has become possible to transiently change the permeability of the cell membrane. Moreover, using a focused ultrasound transducer, it is possible to narrow and focus the ultrasound energy within a small target, avoiding damage to the surrounding tissue. In this research we have searched the possibility of delivering the Bak BH3 peptide, the death domain of the Bc1-2 family of proteins, or the short interfering RNA (siRNA) targeting the enhanced green fluorescent protein (EGFP) using microbubble-enhanced focused ultrasound in an in vitro setting. Using a 1.696 MHz focused ultrasound and a microbubble ultrasound contrast agent OPTISON®, we first tested the stability of BH3 peptide under microbubble-enhanced focused ultrasound exposure and proved that the peptide is stable under these circumstances. Next, we have tested the cell-killing effect of the intracellularly delivered Bak BH3 peptide in HeLa and BJAB cell line and observed a statistically enhanced cell death in BJAB cells but not in HeLa cells, leading to the conclusion that intracellularly delivered BH3 peptide by microbubble-enhanced ultrasound can exert its cell killing effect in some cells. We also investigated if we can silence the EGFP expression in the cell by delivering siRNA targeting the EGFP in both transient and stable EGFP expression cell line. Using a 1.653 MHz focused ultrasound and OPTISON®, in both cases, intracellularly delivered siRNA by microbubble-enhanced ultrasound was able to knock down the EGFP expression, which demonstrates the feasibility of using this novel method

  18. Effects of a human plasma membrane-associated sialidase siRNA on prostate cancer invasion

    SciTech Connect

    Li, Xiaojie; Zhang, Ling; Shao, Yueting; Liang, Zuowen; Shao, Chen; Wang, Bo; Guo, Baofeng; Li, Na; Zhao, Xuejian; Li, Yang; Xu, Deqi

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Neu3 is as one of the sialidases and regulates cell surface functions. Black-Right-Pointing-Pointer A Neu3-specific siRNA inhibited prostrate cancer cell invasion and migration. Black-Right-Pointing-Pointer The Neu3-specific siRNA inhibited prostate cancer metastasis in mice. Black-Right-Pointing-Pointer Targeting Neu3 may have utility for gene-based therapy of human cancer metastasis. -- Abstract: Human plasma membrane-associated sialidase (Neu3) is one of several sialidases that hydrolyze sialic acids in the terminal position of the carbohydrate groups of glycolipids and glycoproteins. Neu3 is mainly localized in plasma membranes and plays crucial roles in the regulation of cell surface functions. In this study, we investigated the effects and molecular mechanisms of Neu3 on cell invasion and migration in vivo and in vitro. Initially, we found that the levels of Neu3 expression were higher in prostate cancer tissues and cell lines than in normal prostate tissues based on RT-PCR and Western blotting analyses. We then applied a Neu3 siRNA approach to block Neu3 signaling using PC-3M cells as model cells. Transwell invasion assays and wound assays showed significantly decreased invasion and migration potential in the Neu3 siRNA-transfected cells. RT-PCR and Western blotting analyses revealed that Neu3 knockdown decreased the expressions of the matrix metalloproteinases MMP-2 and MMP-9. In vivo, mice injected with PC-3M cell tumors were evaluated by SPECT/CT to determine the presence of bone metastases. Mice treated with attenuated Salmonella carrying the Neu3 siRNA developed fewer bone metastases than mice treated with attenuated Salmonella carrying a control Scramble siRNA, attenuated Salmonella alone or PBS. The results for bone metastasis detection by pathology were consistent with the data obtained by SPECT/CT. Tumor blocks were evaluated by histochemical, RT-PCR and Western blotting analyses. The results revealed

  19. Targeted delivery of CXCR4-siRNA by scFv for HER2(+) breast cancer therapy.

    PubMed

    Jiang, Kuo; Li, Jia; Yin, Jipeng; Ma, Qiong; Yan, Bo; Zhang, Xiang; Wang, Lei; Wang, Lifeng; Liu, Tao; Zhang, Yinglong; Fan, Qingyu; Yang, Angang; Qiu, Xiuchun; Ma, Baoan

    2015-08-01

    Therapeutics based on short interfering RNAs (siRNAs) have great potential to treat human diseases. However, the clinical application of siRNAs has been limited by their poor intracellular uptake, low serum stability, and inability to target specific cells. In this study, we addressed this lack of specificity by synthesizing a molecularly targeted CXCR4-siRNA (CXCR4si) for the treatment of HER2(+) breast cancers using a HER2-scFv-arginine nonamer peptide fusion protein (e23sFv-9R) as an siRNA carrier. The e23sFv-9R binding siRNA is able to specifically deliver the siRNA to HER2(+) breast cancer cells and concentrate and persist in orthotopic HER2(+) breast cancer xenografts for at least 36 h. CXCR4si delivered by e23sFv-9R inhibited CXCR4 gene expression, reduced proliferation and metastasis and induced apoptosis in the HER2(+) breast cancer BT-474 cell line in vitro. Moreover, the systemic delivery of CXCR4si by e23sFv-9R is able to suppress tumor growth, reduce metastasis and prolong survival in mice bearing HER2(+) xenografts. This approach causes no systemic toxicity and does not activate the innate immune response, suggesting that a fusion protein carrying CXCR4si shows promise in the treatment of HER2-overexpressing breast cancer. PMID:25956853

  20. Evaluation of dendrimer type bio-reducible polymer as a siRNA delivery carrier for cancer therapy

    PubMed Central

    Nam, Joung-Pyo; Nam, Kihoon; Jung, Simhyun; Nah, Jae-Woon; Kim, Sung Wan

    2015-01-01

    Small interfering ribonucleic acid (siRNA), 20 – 25 base pairs in length, can interfere with the expression of specific genes. Recently, many groups reported the therapeutic intervention of siRNA in various cancer cells. In this study, dendrimer type bio-reducible polymer (PAM-ABP) which was synthesized using arginine grafted bio-reducible poly(cystaminebisacrylamide-diaminohexane) (ABP) and polyamidoamine (PAMAM) was used to deliver anti-VEGF siRNA into cancer cell lines including human hepatocarcinoma (Huh-7), human lung adenocarcinoma (A549), and human fibrosarcoma (HT1080) cells and access their potential as a siRNA delivery carrier for cancer therapy. PAM-ABP and siRNA formed polyplexes with average diameter of 116 nm and charge of around +24.6 mV. The siRNA in the PAM-ABP/siRNA polyplex released by 5 mM DTT and heparin. VEGF gene silencing efficiency of PAM-ABP/siRNA polyplexes was shown to be more effective than PEI/siRNA polyplexes in three cell lines with the following order HT1080>A549>Huh-7. PMID:25937533

  1. NIR light controlled photorelease of siRNA and its targeted intracellular delivery based on upconversion nanoparticles.

    PubMed

    Yang, Yanmei; Liu, Fang; Liu, Xiaogang; Xing, Bengang

    2013-01-01

    The most notable role of small interfering RNA (siRNA) is in RNA interference (RNAi) and post-transcriptional gene silencing, which leads to a surge of interest in RNAi for both biomedical research and therapeutic applications. However, "naked" siRNA cannot cross cellular membranes freely because of highly negative charges which limits its utility for gene therapy. In this work, a system of near-infrared (NIR) light-induced siRNA release from silica coated upconversion nanoparticles (Si-UCNPs) is presented. These Si-UCNPs were functionalized with cationic photocaged linkers through covalent bonding, which could effectively adsorb anionic siRNA through electrostatic attractions and were easily internalized by living cells. Upon NIR light irradiation, the photocaged linker on the Si-UCNPs surface could be cleaved by the upconverted UV light and thus initiated the intracellular release of the siRNA. The in vitro agarose gel electrophoresis and intracellular imaging results indicated that the Si-UCNPs-based gene carrier system allowed effective siRNA delivery and the applications of NIR light instead of direct high energy UV irradiation may greatly guarantee less cell damage. PMID:23154830

  2. Iron-Oxide-Based Nanovector for Tumor Targeted siRNA Delivery in an Orthotopic Hepatocellular Carcinoma Xenograft Mouse Model.

    PubMed

    Wang, Kui; Kievit, Forrest M; Sham, Jonathan G; Jeon, Mike; Stephen, Zachary R; Bakthavatsalam, Arvind; Park, James O; Zhang, Miqin

    2016-01-27

    Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. Small interfering RNA (siRNA) holds promise as a new class of therapeutics for HCC, as it can achieve sequence-specific gene knockdown with low cytotoxicity. However, the main challenge in the clinical application of siRNA lies in the lack of effective delivery approaches that need to be highly specific and thus incur low or no systemic toxicity. Here, a nonviral nanoparticle-based gene carrier is presented that can specifically deliver siRNA to HCC. The nanovector (NP-siRNA-GPC3 Ab) is made of an iron oxide core coated with chitosan-polyethylene glycol (PEG) grafted polyethyleneimine copolymer, which is further functionalized with siRNA and conjugated with a monoclonal antibody (Ab) against human glypican-3 (GPC3) receptor highly expressed in HCC. A rat RH7777 HCC cell line that coexpresses human GPC3 and firefly luciferase (Luc) is established to evaluate the nanovector. The nanoparticle-mediated delivery of siRNA against Luc effectively suppresses Luc expression in vitro without notable cytotoxicity. Significantly, NP-siLuc-GPC3 Ab administered intravenously in an orthotopic model of HCC is able to specifically bound to tumor and induce remarkable inhibition of Luc expression. The findings demonstrate the potential of using this nanovector for targeted delivery of therapeutic siRNA to HCC. PMID:26641029

  3. Nanoparticle siRNA against BMI-1 with a Polyethylenimine-Laminarin Conjugate for Gene Therapy in Human Breast Cancer.

    PubMed

    Ren, Xueling; Liu, Lei; Zhou, Yuxue; Zhu, Yan; Zhang, Hong; Zhang, Zhenzhong; Li, Huixiang

    2016-01-20

    The B-cell-specific Moloney leukemia virus inset site 1 gene (BMI-1) has attracted considerable attention in recent years because of its key role in breast cancer development and metastasis. The downregulation of BMI-1 expression via small interfering RNA (siRNA) effectively inhibits tumor growth. However, the successful application of this therapy is limited by the unavailability of an appropriate vector for siRNA transfer. Therefore, this study aimed to construct a novel laminarin-based nonviral gene transfer vector to carry a constructed BMI-1-targeting siRNA and to investigate the in vitro and in vivo antitumor effects of this siRNA on breast cancer cells. To enhance the siRNA-carrying capacity, we introduced polyethylenimine (PEI) to laminarin's surface via N,N'-carbonyldiimidazole, which produced the cationic PEI-modified laminarin conjugate nLP. Subsequent in vitro experiments indicated that nLP not only formed a nanoparticle with a diameter of 200 nm through electrostatic interactions with siRNA but also showed high efficiency (95.0%) in the delivery siRNA to MCF-7 cells. The nanoparticle targeting BMI-1 (nLP/siBMI-2) reduced BMI-1 expression in breast MCF-7 cells by 90.9% reduction. An in vivo tumor suppression experiment demonstrated that the nLP/siBMI-2 nanoparticle had relatively low toxicity and good gene-therapeutic efficacy, with a tumor inhibition rate of 46.6%. PMID:26629893

  4. The Inheritance Pattern of 24 nt siRNA Clusters in Arabidopsis Hybrids Is Influenced by Proximity to Transposable Elements

    PubMed Central

    Li, Ying; Varala, Kranthi; Moose, Stephen P.; Hudson, Matthew E.

    2012-01-01

    Hybrids often display increased size and growth, and thus are widely cultivated in agriculture and horticulture. Recent discoveries demonstrating the important regulatory roles of small RNAs have greatly improved our understanding of many basic biological questions, and could illuminate the molecular basis for the enhanced growth and size of hybrid plants. We profiled small RNAs by deep sequencing to characterize the inheritance patterns of small RNA levels in reciprocal hybrids of two Arabidopsis thaliana accessions, Columbia and Landsberg erecta. We find 24-nt siRNAs predominate among those small RNAs that are differentially expressed between the parents. Following hybridization, the transposable element (TE)-derived siRNAs are often inherited in an additive manner, whereas siRNAs associated with protein-coding genes are often down-regulated in hybrids to the levels observed for the parent with lower relative siRNA levels. Among the protein-coding genes that exhibit this pattern, genes that function in pathogen defense, abiotic stress tolerance, and secondary metabolism are significantly enriched. Small RNA clusters from protein-coding genes where a TE is present within one kilobase show a different predominant inheritance pattern (additive) from those that do not (low-parent dominance). Thus, down-regulation in the form of low-parent dominance is likely the default pattern of inheritance for genic siRNA, and a different inheritance mechanism for TE siRNA is suggested. PMID:23118865

  5. Spray-dried powders enhance vaginal siRNA delivery by potentially modulating the mucus molecular sieve structure

    PubMed Central

    Wu, Na; Zhang, Xinxin; Li, Feifei; Zhang, Tao; Gan, Yong; Li, Juan

    2015-01-01

    Vaginal small interfering RNA (siRNA) delivery provides a promising strategy for the prevention and treatment of vaginal diseases. However, the densely cross-linked mucus layer on the vaginal wall severely restricts nanoparticle-mediated siRNA delivery to the vaginal epithelium. In order to overcome this barrier and enhance vaginal mucus penetration, we prepared spray-dried powders containing siRNA-loaded nanoparticles. Powders with Pluronic F127 (F127), hydroxypropyl methyl cellulose (HPMC), and mannitol as carriers were obtained using an ultrasound-assisted spray-drying technique. Highly dispersed dry powders with diameters of 5–15 μm were produced. These powders showed effective siRNA protection and sustained release. The mucus-penetrating properties of the powders differed depending on their compositions. They exhibited different potential of opening mesh size of molecular sieve in simulated vaginal mucus system. A powder formulation with 0.6% F127 and 0.1% HPMC produced the maximum increase in the pore size of the model gel used to simulate vaginal mucus by rapidly extracting water from the gel and interacting with the gel; the resulting modulation of the molecular sieve effect achieved a 17.8-fold improvement of siRNA delivery in vaginal tract and effective siRNA delivery to the epithelium. This study suggests that powder formulations with optimized compositions have the potential to alter the steric barrier posed by mucus and hold promise for effective vaginal siRNA delivery. PMID:26347257

  6. Phenylboronic acid-functionalized polyamidoamine-mediated Bcl-2 siRNA delivery for inhibiting the cell proliferation.

    PubMed

    Wu, Di; Yang, Jiebing; Xing, Zhen; Han, Haobo; Wang, Tingting; Zhang, Aijun; Yang, Yan; Li, Quanshun

    2016-10-01

    In this study, the conjugation of phenylboronic acid (PBA) to amine-terminated polyamidoamine (PAMAM) was successfully conducted to prepare a tumor-targeted gene carrier PBA-functionalized PAMAM (PPP) for Bcl-2 siRNA delivery, using a heterobifunctional crosslinker NHS-PEG5k-Mal. The carrier possessed favorable capacity for siRNA condensation and could protect siRNA from the degradation against RNase and serum. The introduction of PBA could facilitate the cellular uptake and further transfection of Bcl-2 siRNA demonstrated by confocal laser scanning microscopy and flow cytometry. Meanwhile, PPP-mediated transfection of Bcl-2 siRNA could significantly inhibit the expression of Bcl-2 gene at both mRNA and protein levels. Furthermore, owing to the knock-down of Bcl-2, PPP/siRNA could significantly inhibit the cell proliferation by inducing the cell apoptosis, and also enhance the antitumor efficiency of doxorubicin by suppressing the resistance of tumor cells to chemotherapeutics. In conclusion, the PPP-mediated Bcl-2 siRNA delivery could potentially be an effective platform for solving the drug resistance and further achieving the combined chemotherapy and gene therapy in tumor treatment. PMID:27371891

  7. Silver nanoparticles-quercetin conjugation to siRNA against drug-resistant Bacillus subtilis for effective gene silencing: in vitro and in vivo.

    PubMed

    Sun, Dongdong; Zhang, Weiwei; Li, Nuan; Zhao, Zhiwei; Mou, Zhipeng; Yang, Endong; Wang, Weiyun

    2016-06-01

    Quercetin (Qe) exhibited extremely low water solubility, and thus, it was modified using silver nanoparticles (AgNPs). We fabricated AgNPs combined with Qe (AgNPs-Qe). The modification suggested that the synergistic properties of Qe enhanced the antibacterial activity of AgNPs. However, AgNPs-Qe exerted no effect on many kinds of drug-resistant bacteria, including Pseudomonas aeruginosa and Bacillus subtilis. RNA interference has considerable therapeutic potential because of its high specificity and potential capability to evade drug resistance. Therefore, we stabilized AgNPs-Qe with a layer of molecules (siRNA). The newly fabricated nanoparticles exerted improved effect on many kinds of bacteria, including the most prominent drug-resistant species B. subtilis. Agarose gel electrophoresis showed that the highest critical nitrogen-to-phosphorus (N/P) ratio occurred at a vector/siRNA with a w/w ratio of 7:1. Characterization experiment indicated that the diameter of siRNA/AgNPs-Qe was approximately 40 nm (40 ± 10 nm). Moreover, AgNPs-Qe were stabilized with a layer of siRNA that was approximately 10nm thick. Results of the in vitro study suggested that siRNA/AgNPs-Qe could destroy the cell wall and inhibit bacterial propagation. Meanwhile, the in vivo experiment on the animal bacteremia model, as well as the optical imaging of nude mice and their isolated organs, demonstrated that bacteria accumulated in the blood, heart, liver, spleen, lungs, and kidneys after the intravenous injection of B. subtilis. The bacteria in the blood and organs, as well as the inflamed cells in the tissues, gradually decreased after the mice received intravenous tail injection of siRNA/AgNPs-Qe for treatment. Both the in vitro and the in vivo studies exhibit that siRNA/AgNPs-Qe can be a potential nanoscale drug delivery system for B. subtilis targeting bacterimia. PMID:27040247

  8. Survival of extreme opinions

    NASA Astrophysics Data System (ADS)

    Hsu, Jiann-wien; Huang, Ding-wei

    2009-12-01

    We study the survival of extreme opinions in various processes of consensus formation. All the opinions are treated equally and subjected to the same rules of changing. We investigate three typical models to reach a consensus in each case: (A) personal influence, (B) influence from surroundings, and (C) influence to surroundings. Starting with uniformly distributed random opinions, our calculated results show that the extreme opinions can survive in both models (A) and (B), but not in model (C). We obtain a conclusion that both personal influence and passive adaptation to the environment are not sufficient enough to eradicate all the extreme opinions. Only the active persuasion to change the surroundings eliminates the extreme opinions completely.

  9. Improving extreme value statistics.

    PubMed

    Shekhawat, Ashivni

    2014-11-01

    The rate of convergence in extreme value statistics is nonuniversal and can be arbitrarily slow. Further, the relative error can be unbounded in the tail of the approximation, leading to difficulty in extrapolating the extreme value fit beyond the available data. We introduce the T method, and show that by using simple nonlinear transformations the extreme value approximation can be rendered rapidly convergent in the bulk, and asymptotic in the tail, thus fixing both issues. The transformations are often parametrized by just one parameter, which can be estimated numerically. The classical extreme value method is shown to be a special case of the proposed method. We demonstrate that vastly improved results can be obtained with almost no extra cost. PMID:25493780

  10. Extreme environments and exobiology

    NASA Technical Reports Server (NTRS)

    Friedmann, E. I.

    1993-01-01

    Ecological research on extreme environments can be applied to exobiological problems such as the question of life on Mars. If life forms (fossil or extant) are found on Mars, their study will help to solve fundamental questions about the nature of life on Earth. Extreme environments that are beyond the range of adaptability of their inhabitants are defined as "absolute extreme". Such environments can serve as terrestrial models for the last stages of life in the history of Mars, when the surface cooled down and atmosphere and water disappeared. The cryptoendolithic microbial community in porous rocks of the Ross Desert in Antarctica and the microbial mats at the bottom of frozen Antarctic lakes are such examples. The microbial communities of Siberian permafrost show that, in frozen but stable communities, long-term survival is possible. In the context of terraforming Mars, selected microorganisms isolated from absolute extreme environments are considered for use in creation of a biological carbon cycle.

  11. Hardware removal - extremity

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/007644.htm Hardware removal - extremity To use the sharing features on this page, please enable JavaScript. Surgeons use hardware such as pins, plates, or screws to help ...

  12. In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes

    PubMed Central

    ElHefnawi, Mahmoud; Kim, TaeKyu; Kamar, Mona A.; Min, Saehong; Hassan, Nafisa M.; El-Ahwany, Eman; Kim, Heeyoung; Zada, Suher; Amer, Marwa; Windisch, Marc P.

    2016-01-01

    RNA interference (RNAi) is a post-transcriptional gene silencing mechanism that mediates the sequence-specific degradation of targeted RNA and thus provides a tremendous opportunity for development of oligonucleotide-based drugs. Here, we report on the design and validation of small interfering RNAs (siRNAs) targeting highly conserved regions of the hepatitis C virus (HCV) genome. To aim for therapeutic applications by optimizing the RNAi efficacy and reducing potential side effects, we considered different factors such as target RNA variations, thermodynamics and accessibility of the siRNA and target RNA, and off-target effects. This aim was achieved using an in silico design and selection protocol complemented by an automated MysiRNA-Designer pipeline. The protocol included the design and filtration of siRNAs targeting highly conserved and accessible regions within the HCV internal ribosome entry site, and adjacent core sequences of the viral genome with high-ranking efficacy scores. Off-target analysis excluded siRNAs with potential binding to human mRNAs. Under this strict selection process, two siRNAs (HCV353 and HCV258) were selected based on their predicted high specificity and potency. These siRNAs were tested for antiviral efficacy in HCV genotype 1 and 2 replicon cell lines. Both in silico-designed siRNAs efficiently inhibited HCV RNA replication, even at low concentrations and for short exposure times (24h); they also exceeded the antiviral potencies of reference siRNAs targeting HCV. Furthermore, HCV353 and HCV258 siRNAs also inhibited replication of patient-derived HCV genotype 4 isolates in infected Huh-7 cells. Prolonged treatment of HCV replicon cells with HCV353 did not result in the appearance of escape mutant viruses. Taken together, these results reveal the accuracy and strength of our integrated siRNA design and selection protocols. These protocols could be used to design highly potent and specific RNAi-based therapeutic oligonucleotide

  13. A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects

    PubMed Central

    Bramsen, Jesper B.; Pakula, Malgorzata M.; Hansen, Thomas B.; Bus, Claus; Langkjær, Niels; Odadzic, Dalibor; Smicius, Romualdas; Wengel, Suzy L.; Chattopadhyaya, Jyoti; Engels, Joachim W.; Herdewijn, Piet; Wengel, Jesper; Kjems, Jørgen

    2010-01-01

    Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2–8 of either siRNA strand counting from the 5′-end) and complementary sequences in the 3′UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA–target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low. PMID:20453030

  14. Co-delivery of doxorubicin and siRNA using octreotide-conjugated gold nanorods for targeted neuroendocrine cancer therapy

    NASA Astrophysics Data System (ADS)

    Xiao, Yuling; Jaskula-Sztul, Renata; Javadi, Alireza; Xu, Wenjin; Eide, Jacob; Dammalapati, Ajitha; Kunnimalaiyaan, Muthusamy; Chen, Herbert; Gong, Shaoqin

    2012-10-01

    A multifunctional gold (Au) nanorod (NR)-based nanocarrier capable of co-delivering small interfering RNA (siRNA) against achaete-scute complex-like 1 (ASCL1) and an anticancer drug (doxorubicin (DOX)) specifically to neuroendocrine (NE) cancer cells was developed and characterized for combined chemotherapy and siRNA-mediated gene silencing. The Au NR was conjugated with (1) DOX, an anticancer drug, via a pH-labile hydrazone linkage to enable pH-controlled drug release, (2) polyarginine, a cationic polymer for complexing siRNA, and (3) octreotide (OCT), a tumor-targeting ligand, to specifically target NE cancer cells with overexpressed somatostatin receptors. The Au NR-based nanocarriers exhibited a uniform size distribution as well as pH-sensitive drug release. The OCT-conjugated Au NR-based nanocarriers (Au-DOX-OCT, targeted) exhibited a much higher cellular uptake in a human carcinoid cell line (BON cells) than non-targeted Au NR-based nanocarriers (Au-DOX) as measured by both flow cytometry and confocal laser scanning microscopy (CLSM). Moreover, Au-DOX-OCT-ASCL1 siRNA (Au-DOX-OCT complexed with ASCL1 siRNA) resulted in significantly higher gene silencing in NE cancer cells than Au-DOX-ASCL1 siRNA (non-targeted Au-DOX complexed with ASCL1 siRNA) as measured by an immunoblot analysis. Additionally, Au-DOX-OCT-ASCL1 siRNA was the most efficient nanocarrier at altering the NE phenotype of NE cancer cells and showed the strongest anti-proliferative effect. Thus, combined chemotherapy and RNA silencing using NE tumor-targeting Au NR-based nanocarriers could potentially enhance the therapeutic outcomes in treating NE cancers.A multifunctional gold (Au) nanorod (NR)-based nanocarrier capable of co-delivering small interfering RNA (siRNA) against achaete-scute complex-like 1 (ASCL1) and an anticancer drug (doxorubicin (DOX)) specifically to neuroendocrine (NE) cancer cells was developed and characterized for combined chemotherapy and siRNA-mediated gene silencing. The

  15. Solar and stellar photospheric abundances

    NASA Astrophysics Data System (ADS)

    Allende Prieto, Carlos

    2016-07-01

    The determination of photospheric abundances in late-type stars from spectroscopic observations is a well-established field, built on solid theoretical foundations. Improving those foundations to refine the accuracy of the inferred abundances has proven challenging, but progress has been made. In parallel, developments on instrumentation, chiefly regarding multi-object spectroscopy, have been spectacular, and a number of projects are collecting large numbers of observations for stars across the Milky Way and nearby galaxies, promising important advances in our understanding of galaxy formation and evolution. After providing a brief description of the basic physics and input data involved in the analysis of stellar spectra, a review is made of the analysis steps, and the available tools to cope with large observational efforts. The paper closes with a quick overview of relevant ongoing and planned spectroscopic surveys, and highlights of recent research on photospheric abundances.

  16. Robust Abundance Estimation in Animal Abundance Surveys with Imperfect Detection

    EPA Science Inventory

    Surveys of animal abundance are central to the conservation and management of living natural resources. However, detection uncertainty complicates the sampling process of many species. One sampling method employed to deal with this problem is depletion (or removal) surveys in whi...

  17. Extreme Programming: Maestro Style

    NASA Technical Reports Server (NTRS)

    Norris, Jeffrey; Fox, Jason; Rabe, Kenneth; Shu, I-Hsiang; Powell, Mark

    2009-01-01

    "Extreme Programming: Maestro Style" is the name of a computer programming methodology that has evolved as a custom version of a methodology, called extreme programming that has been practiced in the software industry since the late 1990s. The name of this version reflects its origin in the work of the Maestro team at NASA's Jet Propulsion Laboratory that develops software for Mars exploration missions. Extreme programming is oriented toward agile development of software resting on values of simplicity, communication, testing, and aggressiveness. Extreme programming involves use of methods of rapidly building and disseminating institutional knowledge among members of a computer-programming team to give all the members a shared view that matches the view of the customers for whom the software system is to be developed. Extreme programming includes frequent planning by programmers in collaboration with customers, continually examining and rewriting code in striving for the simplest workable software designs, a system metaphor (basically, an abstraction of the system that provides easy-to-remember software-naming conventions and insight into the architecture of the system), programmers working in pairs, adherence to a set of coding standards, collaboration of customers and programmers, frequent verbal communication, frequent releases of software in small increments of development, repeated testing of the developmental software by both programmers and customers, and continuous interaction between the team and the customers. The environment in which the Maestro team works requires the team to quickly adapt to changing needs of its customers. In addition, the team cannot afford to accept unnecessary development risk. Extreme programming enables the Maestro team to remain agile and provide high-quality software and service to its customers. However, several factors in the Maestro environment have made it necessary to modify some of the conventional extreme

  18. Electronics for Extreme Environments

    NASA Astrophysics Data System (ADS)

    Patel, J. U.; Cressler, J.; Li, Y.; Niu, G.

    2001-01-01

    Most of the NASA missions involve extreme environments comprising radiation and low or high temperatures. Current practice of providing friendly ambient operating environment to electronics costs considerable power and mass (for shielding). Immediate missions such as the Europa orbiter and lander and Mars landers require the electronics to perform reliably in extreme conditions during the most critical part of the mission. Some other missions planned in the future also involve substantial surface activity in terms of measurements, sample collection, penetration through ice and crust and the analysis of samples. Thus it is extremely critical to develop electronics that could reliably operate under extreme space environments. Silicon On Insulator (SOI) technology is an extremely attractive candidate for NASA's future low power and high speed electronic systems because it offers increased transconductance, decreased sub-threshold slope, reduced short channel effects, elimination of kink effect, enhanced low field mobility, and immunity from radiation induced latch-up. A common belief that semiconductor devices function better at low temperatures is generally true for bulk devices but it does not hold true for deep sub-micron SOI CMOS devices with microscopic device features of 0.25 micrometers and smaller. Various temperature sensitive device parameters and device characteristics have recently been reported in the literature. Behavior of state of the art technology devices under such conditions needs to be evaluated in order to determine possible modifications in the device design for better performance and survivability under extreme environments. Here, we present a unique approach of developing electronics for extreme environments to benefit future NASA missions as described above. This will also benefit other long transit/life time missions such as the solar sail and planetary outposts in which electronics is out open in the unshielded space at the ambient space

  19. PEGylated siRNA lipoplexes for silencing of BLIMP-1 in Primary Effusion Lymphoma: In vitro evidences of antitumoral activity.

    PubMed

    Belletti, Daniela; Tosi, Giovanni; Forni, Flavio; Lagreca, Ivana; Barozzi, Patrizia; Pederzoli, Francesca; Vandelli, Maria Angela; Riva, Giovanni; Luppi, Mario; Ruozi, Barbara

    2016-02-01

    Silencing of the B lymphocyte-induced maturation protein 1 (Blimp-1), a pivotal transcriptional regulator during terminal differentiation of B cells into plasma cells with siRNAs is under investigation as novel therapeutic approach in Primary Effusion Lymphoma (PEL), a HHV-8 related and aggressive B cell Lymphoma currently lacking of an efficacious therapeutic approach. The clinical application of small interfering RNA (siRNA) in cancer therapy is limited by the lack of an efficient systemic siRNA delivery system. In this study we aim to develop pegylated siRNA lipoplexes formed using the cationic lipid DOTAP and DSPE-PEG2000, capable to effectively stabilize anti-Blimp-1 siRNA and suitable for systemic administration. Two types of pegylated lipoplexes using a classic (C-PEG Lipoplexes) or a post-pegylation method (P-PEG-Lipoplexes) were formulated and compared in their physicochemical properties (size, zeta potential, morphology and structure) and efficiency on PEL cell lines. A stable siRNAs protection was obtained with post pegylation approach (2% molar of DSPE-PEG2000 with respect to lipid) resulting in structures with diameters of 300 nm and a complexation efficiency higher that 80% (0.08 nmol/10 nmol of lipid). In vitro studies on PEL cell lines suggested that empty liposomes were characterized by a low cell toxicity also after PEG modification (cell viability and cell density over 85% after treatment with 10 μM of lipid). We demonstrated that P-PEG-Lipoplexes were able to significantly reduce the levels of BLIMP-1 protein leading to reduction of viability (less that 15% after transfection with 100 nM of complexed siRNAs) and activation of apoptosis. In vitro efficiency encourages us to further test the in vivo potential of P-PEG-Lipoplexes in PEL therapy. PMID:26625717

  20. Therapeutic aerosol bioengineering of siRNA for the treatment of inflammatory lung disease by TNFα gene silencing in macrophages.

    PubMed

    Kelly, Ciara; Yadav, Awadh B; Lawlor, Ciaran; Nolan, Katie; O'Dwyer, Joanne; Greene, Catherine M; McElvaney, Noel G; Sivadas, Neeraj; Ramsey, Joanne M; Cryan, Sally-Ann

    2014-11-01

    The development of small interfering RNA (siRNA) to silence specific genes offers a new means of understanding and treating a range of respiratory diseases, including inflammatory lung disease. The alveolar macrophage (AM) is a key component of the inflammatory process in the lungs, associated with high levels of gene expression in inflammatory lung disease and therefore an attractive target for therapeutic siRNA. Delivery of siRNA to macrophages presents a significant delivery challenge, as fully differentiated alveolar macrophages are difficult to access and transfect. In this study we engineered particles suitable for inhalation that would efficiently transfect macrophages postinhalation. The process for encapsulation of siRNA in poly(lactic-co-glycolic acid) microparticles (MPs) was optimized using a double emulsion technique, and the resulting particles were characterized for size, shape, aerosol characteristics, encapsulation efficiency, and integrity of encapsulated siRNA. The cell uptake of the siRNA-loaded microparticles was determined by flow cytometry, confocal laser scanning microscopy (CLSM), and high-content analysis (HCA) with MPs capable of transfecting up to 55% of cells. Anti-TNFα siRNA-MPs were then prepared to study the functional activity of encapsulated siRNA in LPS-stimulated macrophages as a model of inflammation. The anti-TNFα siRNA-MPs were able to decrease TNFα expression by 45% over 48 h in the differentiated human monocytic cell line THP-1 compared to negligible knockdown using commercial transfection reagents and offered significant, sustained siRNA knockdown of TNFα in primary monocytes for up to 72 h. PMID:25243784

  1. PEGylated carboxymethyl chitosan/calcium phosphate hybrid anionic nanoparticles mediated hTERT siRNA delivery for anticancer therapy.

    PubMed

    Xie, Ying; Qiao, Hongzhi; Su, Zhigui; Chen, Minglei; Ping, Qineng; Sun, Minjie

    2014-09-01

    Lack of safe and effective delivery vehicle is the main obstacle for siRNA mediated cancer therapy. In this study, we synthesized a pH-sensitive polymer of PEG grafted carboxymethyl chitosan (PEG-CMCS) and developed anionic-charged hybrid nanoparticles of PEG-CMCS and calcium phosphate (CaP) for siRNA delivery through a single-step self-assembly method in aqueous condition. The formed nanoparticles with charge of around -8.25 mv and average diameter of 102.1 nm exhibited efficient siRNA encapsulation and enhanced colloidal and serum stability. The test in vitro indicated that the nanoparticles entered into HepG2 cells by endocytosis, and achieved endosomal escape of siRNA effectively due to the pH-responsive disassembly of nanoparticles and dissolution of CaP in the endosome. Reporter gene silencing assay showed that luciferase siRNA delivered by the anionic nanoparticles could achieve gene silencing efficacy comparable to that of conventional Lipofectamine 2000. Additionally, dramatic hTERT knockdown mediated by the anionic nanoparticles transfection induced significant apoptosis of HepG2 cells in vitro. After intravenous injection in tumor-bearing BALB/c nude mice, the nanoparticles specifically accumulated into tumor regions by EPR effect, leading to efficient and specific gene silencing sequentially. Most importantly, the nanoparticles carrying hTERT siRNA inhibited tumor growth significantly via silencing hTERT expression and inducing cells apoptosis in HepG2 tumor xenograft. Moreover, comprehensive safety studies of the nanoparticles confirmed their superior safety both in vitro and in vivo. We concluded that the PEG-CMCS/CaP hybrid anionic nanoparticles possessed potential as a safe and effective siRNA delivery system for anticancer therapy. PMID:24939077

  2. An albumin-mediated cholesterol design-based strategy for tuning siRNA pharmacokinetics and gene silencing.

    PubMed

    Bienk, Konrad; Hvam, Michael Lykke; Pakula, Malgorzata Maria; Dagnæs-Hansen, Frederik; Wengel, Jesper; Malle, Birgitte Mølholm; Kragh-Hansen, Ulrich; Cameron, Jason; Bukrinski, Jens Thostrup; Howard, Kenneth A

    2016-06-28

    Major challenges for the clinical translation of small interfering RNA (siRNA) include overcoming the poor plasma half-life, site-specific delivery and modulation of gene silencing. In this work, we exploit the intrinsic transport properties of human serum albumin to tune the blood circulatory half-life, hepatic accumulation and gene silencing; based on the number of siRNA cholesteryl modifications. We demonstrate by a gel shift assay a strong and specific affinity of recombinant human serum albumin (rHSA) towards cholesteryl-modified siRNA (Kd>1×10(-7)M) dependent on number of modifications. The rHSA/siRNA complex exhibited reduced nuclease degradation and reduced induction of TNF-α production by human peripheral blood mononuclear cells. The increased solubility of heavily cholesteryl modified siRNA in the presence of rHSA facilitated duplex annealing and consequent interaction that allowed in vivo studies using multiple cholesteryl modifications. A structural-activity-based screen of in vitro EGFP-silencing was used to select optimal siRNA designs containing cholesteryl modifications within the sense strand that were used for in vivo studies. We demonstrate plasma half-life extension in NMRI mice from t1/2 12min (naked) to t1/2 45min (single cholesteryl) and t1/2 71min (double cholesteryl) using fluorescent live bioimaging. The biodistribution showed increased accumulation in the liver for the double cholesteryl modified siRNA that correlated with an increase in hepatic Factor VII gene silencing of 28% (rHSA/siRNA) compared to 4% (naked siRNA) 6days post-injection. This work presents a novel albumin-mediated cholesteryl design-based strategy for tuning pharmacokinetics and systemic gene silencing. PMID:27084489

  3. The Promising Nanocarrier for Doxorubicin and siRNA Co-delivery by PDMAEMA-based Amphiphilic Nanomicelles.

    PubMed

    Cheng, Qiang; Du, Lili; Meng, Lingwei; Han, Shangcong; Wei, Tuo; Wang, Xiaoxia; Wu, Yidi; Song, Xinyun; Zhou, Junhui; Zheng, Shuquan; Huang, Yuanyu; Liang, Xing-jie; Cao, Huiqing; Dong, Anjie; Liang, Zicai

    2016-02-01

    Synergistic effects of anticancer drug and siRNA have displayed superior advantages for cancer therapy. Herein, we deeply analyzed the feasibility that whether doxorubicin (DOX) and siRNA could be co-delivered by mPEG-PCL-graft-PDMAEMA (PECD) micelles, which mediated excellent DNA/siRNA delivery in vitro and in vivo reported in our previous work. DOX-loaded NPs (PECD-D) were developed by nanoprecipitation technology and exhibited high drug loading content (DLC, 9.5%). In vitro cytotoxicity study in MDA-MB-231 cells, PECD-D treated groups had lower IC50 compared to free DOX groups (F-DOX) at different transfection time (24, 48, and 72h), which maybe attribute to its high cellular uptake and endosomal escape properties. The speculation was confirmed with the results of drug release profile in acidic media, flow cytometry analysis and confocal images. Futhermore, Cy5 labeled siRNA was introduced in PECD-D micelles (PECD-D/siRNA) to track the behavior of dual-loaded nanodrug in vitro and in vivo. Flow cytometry analysis presented that DOX and siRNA were successfully co-delivered into cells, the positive cells ratio were 94.6 and 99.5%, respectively. Confocal images showed that not only DOX and siRNA existed in cytoplasm, but DOX traversed endosome/lysosome and entered into cell nucleus. For in vivo tumor-targeting evaluation in BALB/c nude mice, both DOX and Cy5-siRNA could be detected in tumor sites after intravenous injection with PECD-D/siRNA formulation. Therefore, we believed that PECD micelles have a potential ability as DOX and siRNA co-delivery carrier for cancer therapy. PMID:26835788

  4. Coronal abundances and their variation

    NASA Technical Reports Server (NTRS)

    Saba, Julia L. R.

    1995-01-01

    This contract supports the investigation of elemental abundances in the solar corona, principally through analysis of high-resolution soft X-ray spectra from the Flat Crystal Spectrometer on the Solar Maximum Mission. The goals of the study are a characterization of the mean values of relative abundances of elements accessible in the FCS data, and information on the extent and circumstances of their variability. This report is a summation of the data analysis and reporting activities which occurred during the period of 15 April 1994 to 15 April 1995.

  5. The solar abundance of beryllium

    NASA Technical Reports Server (NTRS)

    Ross, J. E.; Aller, L. H.

    1974-01-01

    The solar abundance of beryllium is deduced from high-resolution Kitt Peak observations of the 3130.43- and 3131.08-A lines of Be II interpreted by the method of spectrum synthesis. The results are in good agreement with those previously obtained by Grevesse (1968) and by Hauge and Engvold (1968) and indicate that in the photospheric layers, beryllium is depleted below the chondritic value by a factor of about two. It is found that the beryllium abundance is equal to logN(Be)/N(H) + 12 = 1.08 plus or minus 0.05.

  6. Chemical Abundances of Symbiotic Giants

    NASA Astrophysics Data System (ADS)

    Gałan, C.; Mikołajewska, J.; Hinkle, K. H.; Joyce, R. R.

    2015-12-01

    High resolution (R ˜ 50000), near-IR spectra were used to measure photospheric abundances of CNO and elements around the iron peak for 24 symbiotic giants. Spectrum synthesis was employed using local thermal equilibrium and hydrostatic model atmospheres. The metallicities are distributed in a wide range with maximum around [Fe/H] ˜-0.4 - - 0.3 dex. Enrichment in 14N indicates that all the sample giants have experienced the first dredge-up. The relative abundance of [Ti/Fe] is generally large in red symbiotic systems.

  7. Coronal Abundances and Their Variation

    NASA Technical Reports Server (NTRS)

    Saba, Julia L. R.

    1996-01-01

    This contract supported the investigation of elemental abundances in the solar corona, principally through analysis of high-resolution soft X-ray spectra from the Flat Crystal Spectrometer on NASA's Solar Maximum Mission. The goals of the study were a characterization of the mean values of relative abundances of elements accessible in the FCS data, and information on the extent and circumstances of their variability. This is the Final Report, summarizing the data analysis and reporting activities which occurred during the period of performance, June 1993 - December 1996.

  8. SOLAR MODELS WITH REVISED ABUNDANCE

    SciTech Connect

    Bi, S. L.; Li, T. D.; Yang, W. M.; Li, L. H.

    2011-04-20

    We present new solar models in which we use the latest low abundances and further include the effects of rotation, magnetic fields, and extra-mixing processes. We assume that the extra-element mixing can be treated as a diffusion process, with the diffusion coefficient depending mainly on the solar internal configuration of rotation and magnetic fields. We find that such models can well reproduce the observed solar rotation profile in the radiative region. Furthermore, the proposed models can match the seismic constraints better than the standard solar models, also when these include the latest abundances, but neglect the effects of rotation and magnetic fields.

  9. Plants Encode a General siRNA Suppressor That Is Induced and Suppressed by Viruses

    PubMed Central

    Charbonnel, Cyril; Elvira-Matelot, Emilie; Bochnakian, Aurore; Comella, Pascale; Mallory, Allison C.; Lepère, Gersende; Sáez-Vásquez, Julio; Vaucheret, Hervé

    2015-01-01

    Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant–virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses. PMID:26696443

  10. Plants Encode a General siRNA Suppressor That Is Induced and Suppressed by Viruses.

    PubMed

    Shamandi, Nahid; Zytnicki, Matthias; Charbonnel, Cyril; Elvira-Matelot, Emilie; Bochnakian, Aurore; Comella, Pascale; Mallory, Allison C; Lepère, Gersende; Sáez-Vásquez, Julio; Vaucheret, Hervé

    2015-12-01

    Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant-virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses. PMID:26696443

  11. Reduction of bilirubin by targeting human heme oxygenase-1 through siRNA.

    PubMed

    Xia, Zhen-Wei; Li, Chun-E; Jin, You-Xin; Shi, Yi; Xu, Li-Qing; Zhong, Wen-Wei; Li, Yun-Zhu; Yu, Shan-Chang; Zhang, Zi-Li

    2007-04-01

    Neonatal hyperbilirubinemia is a common clinical condition caused mainly by the increased production and decreased excretion of bilirubin. Current treatment is aimed at reducing the serum levels of bilirubin. Heme oxygenase-1 (HO-1) is a rate-limiting enzyme that generates bilirubin. In this study we intended to suppress HO-1 using the RNA interference technique. Small interfering RNA (siRNA)-A, -B, and -C were designed based on human HO-1 (hHO-1) mRNA sequences. siRNA was transfected into a human hepatic cell line (HL-7702). hHO-1 transcription and protein levels were then determined. In addition, the inhibitory effect of siRNA on hHO-1 was assessed in cells treated with hemin or transfected with an hHO-1 plasmid. siRNA-C showed the most potent suppressive effect on hHO-1. This inhibition is dose and time dependent. Compared with control, both hemin and hHO-1 plasmids up-regulated hHO-1 expression in HL-7702 cells. However, the up-regulation was significantly attenuated by siRNA-C. Furthermore, the decrease in hHO-1 activity was coincident with the suppression of its transcription. Finally, siRNA-C was shown to reduce hHO-1 enzymatic activity and bilirubin levels. Thus, this study provides a novel therapeutic rationale by blocking bilirubin formation via siRNA for preventing and treating neonatal hyperbilirubinemia and bilirubin encephalopathy at an early clinical stage. PMID:17392485

  12. Polymersome Carriers: from Self-Assembly to siRNA and Protein Therapeutics

    PubMed Central

    Christian, David A.; Cai, Shenshen; Bowen, Diana M.; Kim, Younghoon; Pajerowski, J. David; Discher, Dennis E.

    2009-01-01

    Polymersomes are polymer-based vesicular shells that form upon hydration of amphiphilic block copolymers. These high molecular weight amphiphiles impart physicochemical properties that allow polymersomes to stably encapsulate or integrate a broad range of active molecules. This robustness together with recently described mechanisms for controlled breakdown of degradable polymersomes as well as escape from endolysosomes suggests that polymersomes might be usefully viewed as having structure/property/function relationships somewhere between lipid vesicles and viral capsids. Here we summarize the assembly and development of controlled release polymersomes to encapsulate therapeutics ranging from small molecule anti-cancer drugs to siRNA and therapeutic proteins. PMID:18977437

  13. Cationic Mucic Acid Polymer-Based siRNA Delivery Systems.

    PubMed

    Pan, Dorothy W; Davis, Mark E

    2015-08-19

    Nanoparticle (NP) delivery systems for small interfering RNA (siRNA) that have good systemic circulation and high nucleic acid content are highly desired for translation into clinical use. Here, a family of cationic mucic acid-containing polymers is synthesized and shown to assemble with siRNA to form NPs. A cationic mucic acid polymer (cMAP) containing alternating mucic acid and charged monomers is synthesized. When combined with siRNA, cMAP forms NPs that require steric stabilization by poly(ethylene glycol) (PEG) that is attached to the NP surface via a 5-nitrophenylboronic acid linkage (5-nitrophenylboronic acid-PEGm (5-nPBA-PEGm)) to diols on mucic acid in the cMAP in order to inhibit aggregation in biological fluids. As an alternative, cMAP is covalently conjugated with PEG via two methods. First, a copolymer is prepared with alternating cMAP-PEG units that can form loops of PEG on the surface of the formulated siRNA-containing NPs. Second, an mPEG-cMAP-PEGm triblock polymer is synthesized that could lead to a PEG brush configuration on the surface of the formulated siRNA-containing NPs. The copolymer and triblock polymer are able to form stable siRNA-containing NPs without and with the addition of 5-nPBA-PEGm. Five formulations, (i) cMAP with 5-nPBA-PEGm, (ii) cMAP-PEG copolymer both (a) with and (b) without 5-nPBA-PEGm, and (iii) mPEG-cMAP-PEGm triblock polymer both (a) with and (b) without 5-nPBA-PEGm, are used to produce NPs in the 30-40 nm size range, and their circulation times are evaluated in mice using tail vein injections. The mPEG-cMAP-PEGm triblock polymer provides the siRNA-containing NP with the longest circulation time (5-10% of the formulation remains in circulation at 60 min postdosing), even when a portion of the excess cationic components used in the formulation is filtered away prior to injection. A NP formulation using the mPEG-cMAP-PEGm triblock polymer that is free of excess components could contain as much as ca. 30 wt % siRNA. PMID

  14. THE SOLAR FLARE IRON ABUNDANCE

    SciTech Connect

    Phillips, K. J. H.; Dennis, B. R. E-mail: Brian.R.Dennis@nasa.gov

    2012-03-20

    The abundance of iron is measured from emission line complexes at 6.65 keV (Fe line) and 8 keV (Fe/Ni line) in RHESSI X-ray spectra during solar flares. Spectra during long-duration flares with steady declines were selected, with an isothermal assumption and improved data analysis methods over previous work. Two spectral fitting models give comparable results, viz., an iron abundance that is lower than previous coronal values but higher than photospheric values. In the preferred method, the estimated Fe abundance is A(Fe) = 7.91 {+-} 0.10 (on a logarithmic scale, with A(H) = 12) or 2.6 {+-} 0.6 times the photospheric Fe abundance. Our estimate is based on a detailed analysis of 1898 spectra taken during 20 flares. No variation from flare to flare is indicated. This argues for a fractionation mechanism similar to quiet-Sun plasma. The new value of A(Fe) has important implications for radiation loss curves, which are estimated.

  15. Adventure and Extreme Sports.

    PubMed

    Gomez, Andrew Thomas; Rao, Ashwin

    2016-03-01

    Adventure and extreme sports often involve unpredictable and inhospitable environments, high velocities, and stunts. These activities vary widely and include sports like BASE jumping, snowboarding, kayaking, and surfing. Increasing interest and participation in adventure and extreme sports warrants understanding by clinicians to facilitate prevention, identification, and treatment of injuries unique to each sport. This article covers alpine skiing and snowboarding, skateboarding, surfing, bungee jumping, BASE jumping, and whitewater sports with emphasis on epidemiology, demographics, general injury mechanisms, specific injuries, chronic injuries, fatality data, and prevention. Overall, most injuries are related to overuse, trauma, and environmental or microbial exposure. PMID:26900120

  16. Extremal entanglement witnesses

    NASA Astrophysics Data System (ADS)

    Hansen, Leif Ove; Hauge, Andreas; Myrheim, Jan; Sollid, Per Øyvind

    2015-02-01

    We present a study of extremal entanglement witnesses on a bipartite composite quantum system. We define the cone of witnesses as the dual of the set of separable density matrices, thus TrΩρ≥0 when Ω is a witness and ρ is a pure product state, ρ=ψψ† with ψ=ϕ⊗χ. The set of witnesses of unit trace is a compact convex set, uniquely defined by its extremal points. The expectation value f(ϕ,χ)=TrΩρ as a function of vectors ϕ and χ is a positive semidefinite biquadratic form. Every zero of f(ϕ,χ) imposes strong real-linear constraints on f and Ω. The real and symmetric Hessian matrix at the zero must be positive semidefinite. Its eigenvectors with zero eigenvalue, if such exist, we call Hessian zeros. A zero of f(ϕ,χ) is quadratic if it has no Hessian zeros, otherwise it is quartic. We call a witness quadratic if it has only quadratic zeros, and quartic if it has at least one quartic zero. A main result we prove is that a witness is extremal if and only if no other witness has the same, or a larger, set of zeros and Hessian zeros. A quadratic extremal witness has a minimum number of isolated zeros depending on dimensions. If a witness is not extremal, then the constraints defined by its zeros and Hessian zeros determine all directions in which we may search for witnesses having more zeros or Hessian zeros. A finite number of iterated searches in random directions, by numerical methods, leads to an extremal witness which is nearly always quadratic and has the minimum number of zeros. We discuss briefly some topics related to extremal witnesses, in particular the relation between the facial structures of the dual sets of witnesses and separable states. We discuss the relation between extremality and optimality of witnesses, and a conjecture of separability of the so-called structural physical approximation (SPA) of an optimal witness. Finally, we discuss how to treat the entanglement witnesses on a complex Hilbert space as a subset of the

  17. Extreme black hole holography

    NASA Astrophysics Data System (ADS)

    Hartman, Thomas Edward

    The connection between black holes in four dimensions and conformal field theories (CFTs) in two dimensions is explored, focusing on zero temperature (extreme) black holes and their low-temperature cousins. It is shown that extreme black holes in a theory of quantum gravity are holographically dual to field theories living in two dimensions without gravity, and that the field theory reproduces a variety of black hole phenomena in detail. The extreme black hole/CFT correspondence is derived from a symmetry analysis near the horizon of a Kerr black hole with mass M and maximal angular momentum J=M 2. The asymptotic symmetry generators form one copy of the Virasoro algebra with central charge c=12J, which implies that the near-horizon quantum states are identical to those of a two-dimensional CFT. We discuss extensions of this result to near-extreme black holes and cosmological horizons. Astrophysical black holes are never exactly extremal, but the black hole GRS1915+105 observed through X-ray and radio telescopy is likely within 1% of the extremal spin, suggesting that this extraordinary and well studied object is approximately dual to a two-dimensional CFT with c˜1079. As evidence for the correspondence, microstate counting in the CFT is used to derive the Bekenstein-Hawking area law for the Kerr entropy, S=Horizon area/4. Furthermore, the correlators in the dual CFT are shown to reproduce the scattering amplitudes of a charged scalar or spin-½ field by a near-extreme Kerr-Newman black hole, and a neutral spin-1 or spin-2 field by a near-extreme Kerr black hole. Scattering amplitudes probe the vacuum of fields living on the black hole background. For scalars, bound superradiant modes lead to an instability, while for fermions, it is shown that the bound superradiant modes condense and form a Fermi sea which extends well outside the ergosphere. Assuming no further instabilities, the low energy effective theory near the black hole is described by ripples in the

  18. One-step encapsulation of siRNA between lipid-layers of multi-layer polycation liposomes by lipoplex freeze-thawing.

    PubMed

    Koide, Hiroyuki; Okamoto, Ayaka; Tsuchida, Hiroki; Ando, Hidenori; Ariizumi, Saki; Kiyokawa, Chiaki; Hashimoto, Masahiro; Asai, Tomohiro; Dewa, Takehisa; Oku, Naoto

    2016-04-28

    Small interfering RNA (siRNA) has the potential to be a candidate as a cure for intractable diseases. However, an appropriate vector is required for siRNA delivery because of the low transfection efficiency of siRNA without a vector and its easy degradation in vivo. Here, we report a simple, only one step, and efficient method for siRNA encapsulation into a lipidic nanocarrier by freeze-thawing: siRNA was entrapped between the lipid layers of multi-layer liposomes by freeze-thawing of lipoplexes composed of polycation liposomes (PCLs) and siRNA. siRNA-holding capacity to the PCL was increased by repeating freeze-thaw of the lipoplex up to 5cycles. Although siRNA in the conventional lipoplex was degraded after incubation in 90% fetal bovine serum for 72h, siRNA in the frozen and thawed lipoplex was not degraded. Interestingly, we found that the lipoplex formed a "packed multi-layer" structure after the freeze-thawing of "single-layer" PCL and siRNA complex, suggesting that siRNA exists between the lipid layers working as a binder. The frozen and thawed lipoplex showed significantly higher knockdown efficacy compared with the conventional lipoplex. In addition, PEGylated freeze-thawed lipoplexes delivered a higher amount of siRNA to a tumor in vivo compared with the PEGylated conventional ones. These results provide an attractive strategy for "one-step" encapsulation of siRNA into liposomes by freeze-thawing. PMID:26826309

  19. A role for peptides in overcoming endosomal entrapment in siRNA delivery - A focus on melittin.

    PubMed

    Hou, Kirk K; Pan, Hua; Schlesinger, Paul H; Wickline, Samuel A

    2015-11-01

    siRNA has the possibility to revolutionize medicine by enabling highly specific and efficient silencing of proteins involved in disease pathogenesis. Despite nearly 20 years of research dedicated to translating siRNA from a research tool into a clinically relevant therapeutic, minimal success has been had to date. Access to RNA interference machinery located in the cytoplasm is often overlooked, but must be considered when designing the next generation of siRNA delivery strategies. Peptide transduction domains (PTDs) have demonstrated moderate siRNA transfection, which is primarily limited by endosomal entrapment. Strategies aimed at overcoming endosomal entrapment associated with peptide vectors are reviewed here, including osmotic methods, lipid conjugation, and fusogenic peptides. As an alternative to traditional PTD, the hemolytic peptide melittin exhibits the native capacity for endosomal disruption but causes cytotoxicity. However, appropriate packaging and protection of melittin with activation and release in the endosomal compartment has allowed melittin-based strategies to demonstrate both in vitro and in vivo safety and efficacy. These data suggest that melittin's membrane disruptive properties can enable safe and effective endosomolysis, building a case for melittin as a key component in a new generation of siRNA therapeutics. PMID:26025036

  20. Targeted exosome-mediated delivery of opioid receptor Mu siRNA for the treatment of morphine relapse

    PubMed Central

    Liu, Yuchen; Li, Dameng; Liu, Zhengya; Zhou, Yu; Chu, Danping; Li, Xihan; Jiang, Xiaohong; Hou, Dongxia; Chen, Xi; Chen, Yuda; Yang, Zhanzhao; Jin, Ling; Jiang, Waner; Tian, Chenfei; Zhou, Geyu; Zen, Ke; Zhang, Junfeng; Zhang, Yujing; Li, Jing; Zhang, Chen-Yu

    2015-01-01

    Cell-derived exosomes have been demonstrated to be efficient carriers of small RNAs to neighbouring or distant cells, highlighting the preponderance of exosomes as carriers for gene therapy over other artificial delivery tools. In the present study, we employed modified exosomes expressing the neuron-specific rabies viral glycoprotein (RVG) peptide on the membrane surface to deliver opioid receptor mu (MOR) siRNA into the brain to treat morphine addiction. We found that MOR siRNA could be efficiently packaged into RVG exosomes and was associated with argonaute 2 (AGO2) in exosomes. These exosomes efficiently and specifically delivered MOR siRNA into Neuro2A cells and the mouse brain. Functionally, siRNA-loaded RVG exosomes significantly reduced MOR mRNA and protein levels. Surprisingly, MOR siRNA delivered by the RVG exosomes strongly inhibited morphine relapse via the down-regulation of MOR expression levels. In conclusion, our results demonstrate that targeted RVG exosomes can efficiently transfer siRNA to the central nervous system and mediate the treatment of morphine relapse by down-regulating MOR expression levels. Our study provides a brand new strategy to treat drug relapse and diseases of the central nervous system. PMID:26633001

  1. Delivery of siRNAs to Dendritic Cells Using DEC205-Targeted Lipid Nanoparticles to Inhibit Immune Responses.

    PubMed

    Katakowski, Joseph A; Mukherjee, Gayatri; Wilner, Samantha E; Maier, Keith E; Harrison, Michael Travis; DiLorenzo, Teresa P; Levy, Matthew; Palliser, Deborah

    2016-02-01

    Due to their ability to knock down the expression of any gene, siRNAs have been heralded as ideal candidates for treating a wide variety of diseases, including those involving "undruggable" targets. However, the therapeutic potential of siRNAs remains severely limited by a lack of effective delivery vehicles. Recently, lipid nanoparticles (LNPs) containing ionizable cationic lipids have been developed for hepatic siRNA delivery. However, their suitability for delivery to other cell types has not been determined. We have modified LNPs for preferential targeting to dendritic cells (DCs), central regulators of immune responses. To achieve directed delivery, we coated LNPs with a single-chain antibody (scFv; DEC-LNPs), specific to murine DEC205, which is highly expressed on distinct DC subsets. Here we show that injection of siRNAs encapsulated in DEC-LNPs are preferentially delivered to DEC205(+) DCs. Gene knockdown following uptake of DEC-LNPs containing siRNAs specific for the costimulatory molecules CD40, CD80, and CD86 dramatically decreases gene expression levels. We demonstrate the functionality of this knockdown with a mixed lymphocyte response (MLR). Overall, we report that injection of LNPs modified to restrict their uptake to a distinct cell population can confer profound gene knockdown, sufficient to inhibit powerful immune responses like the MLR. PMID:26412590

  2. Hydrolytic charge-reversal of PEGylated polyplexes enhances intracellular un-packaging and activity of siRNA.

    PubMed

    Werfel, Thomas A; Swain, Corban; Nelson, Christopher E; Kilchrist, Kameron V; Evans, Brian C; Miteva, Martina; Duvall, Craig L

    2016-04-01

    Hydrolytically degrading nano-polyplexes (HDG-NPs) that reverse charge through conversion of tertiary amines to carboxylic acids were investigated to improve intracellular un-packaging of siRNA and target gene silencing compared to a non-degradable analog (non-HDG-NPs). Both NP types comprised reversible addition-fragmentation chain-transfer (RAFT) synthesized diblock copolymers of a poly(ethylene glycol) (PEG) corona-forming block and a cationic block for nucleic acid packaging that incorporated butyl methacrylate (BMA) and either dimethylaminoethyl methacrylate (DMAEMA, non-HDG-NPs) or dimethylaminoethyl acrylate (DMAEA, HDG-NPs). HDG-NPs decreased significantly in size and released significantly more siRNA (∼40%) than non-HDG-NPs after 24 h in aqueous solution. While both HDG-NPs and non-HDG-NPs had comparable uptake and cytotoxicity up to 150 nM siRNA doses, HDG-NPs achieved significantly higher target gene silencing of the model gene luciferase in vitro. High resolution FRET confocal microscopy was used to monitor the intracellular un-packaging of siRNA. Non-HDG-NPs had significantly higher FRET efficiency than HDG-NPs, indicating that siRNA delivered from HDG-NPs was more fully un-packaged and therefore had improved intracellular bioavailability. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 917-927, 2016. PMID:26691570

  3. [Inhibition of proliferation of H5N1 subtype AIV in CEF by chemosynthetic siRNA].

    PubMed

    Li, Ru-Shu; Yu, Dan; Luo, Bao-Zheng; Bo, Qing-Ru; Xu, Hai-Nie; Sha, Cai-Hua; Liao, Xiu-Yun

    2013-06-01

    In order to study the proliferation inhibition effect of H5N1 subtype avian influenza virus (AIV) with small interfere RNA (siRNA), a total of 4 siRNAs were designed in accordance with the NP and PA genes of H5N1 subtype AIV, the siRNAs were then transfected to chicken embryo fibroblast(CEF), CEF was infected with H5N1 subtype AIV after 6 hrs. Virus titer of cell supernatant was tested at 16-56hrs post infection, and pathological changes of the cells was observed; mRNA levels of NP, PA, HA and p13-actin gene were tested at 36hrs post infection. The results showed that these 4 siRNAs could inhibit the prolif-eration of H5N1 subtype AIV in CEF in varying degrees, and one siRNA targeting PA was best per-formed. The experimental results also showed that the inhibition effect was decreased with the time prolonged. This research provides a basis for further studying RNAi on AIV prevention and control. PMID:23895002

  4. Targeted exosome-mediated delivery of opioid receptor Mu siRNA for the treatment of morphine relapse.

    PubMed

    Liu, Yuchen; Li, Dameng; Liu, Zhengya; Zhou, Yu; Chu, Danping; Li, Xihan; Jiang, Xiaohong; Hou, Dongxia; Chen, Xi; Chen, Yuda; Yang, Zhanzhao; Jin, Ling; Jiang, Waner; Tian, Chenfei; Zhou, Geyu; Zen, Ke; Zhang, Junfeng; Zhang, Yujing; Li, Jing; Zhang, Chen-Yu

    2015-01-01

    Cell-derived exosomes have been demonstrated to be efficient carriers of small RNAs to neighbouring or distant cells, highlighting the preponderance of exosomes as carriers for gene therapy over other artificial delivery tools. In the present study, we employed modified exosomes expressing the neuron-specific rabies viral glycoprotein (RVG) peptide on the membrane surface to deliver opioid receptor mu (MOR) siRNA into the brain to treat morphine addiction. We found that MOR siRNA could be efficiently packaged into RVG exosomes and was associated with argonaute 2 (AGO2) in exosomes. These exosomes efficiently and specifically delivered MOR siRNA into Neuro2A cells and the mouse brain. Functionally, siRNA-loaded RVG exosomes significantly reduced MOR mRNA and protein levels. Surprisingly, MOR siRNA delivered by the RVG exosomes strongly inhibited morphine relapse via the down-regulation of MOR expression levels. In conclusion, our results demonstrate that targeted RVG exosomes can efficiently transfer siRNA to the central nervous system and mediate the treatment of morphine relapse by down-regulating MOR expression levels. Our study provides a brand new strategy to treat drug relapse and diseases of the central nervous system. PMID:26633001

  5. Amphiphilic gold nanoparticles displaying flexible bifurcated ligands as a carrier for siRNA delivery into the cell cytosol.

    PubMed

    Niikura, Kenichi; Kobayashi, Kenya; Takeuchi, Chie; Fujitani, Naoki; Takahara, Shuko; Ninomiya, Takafumi; Hagiwara, Kyoji; Mitomo, Hideyuki; Ito, Yoshihiro; Osada, Yoshihito; Ijiro, Kuniharu

    2014-12-24

    The nanoparticle-based delivery of siRNA with a noncationic outermost surface at a low particle concentration is greatly desired. We newly synthesized a bifurcated ligand (BL) possessing hydrophobic and hydrophilic arms as a surface ligand for gold nanoparticles (AuNPs) to allow siRNA delivery. The concept underlying the design of this ligand is that amphiphilic property should allow AuNPs to permeate the cell cytosol thorough the endosomal membrane. BLs and quaternary cationic ligands were codisplayed on 40 nm AuNPs, which were subsequently coated with siRNA via electrostatic interaction. The number of siRNAs immobilized on a single nanoparticle was 26, and the conjugate showed a negative zeta potential due to siRNAs on the outermost surface of the AuNPs. Apparent gene silencing of luciferase expression in HeLa cells was achieved at an AuNP concentration as low as 60 pM. Almost no gene silencing was observed for AuNPs not displaying BLs. To reveal the effect of the BL, we compared the number of AuNPs internalized into HeLa cells and the localization in the cytosol between AuNPs displaying and those not displaying BLs. These analyses indicated that the role of BLs is not only the simple promotion of cellular uptake but also involves the enhancement of AuNPs permeation into the cytosol from the endosomes, leading to effective gene silencing. PMID:25466488

  6. Protection of mice against lethal rabies virus challenge using short interfering RNAs (siRNAs) delivered through lentiviral vector.

    PubMed

    Singh, Niraj K; Meshram, Chetan D; Sonwane, Arvind A; Dahiya, Shyam S; Pawar, Sachin S; Chaturvedi, V K; Saini, Mohini; Singh, R P; Gupta, Praveen K

    2014-02-01

    The antiviral potential of small interfering RNAs (siRNAs) targeting rabies virus (RV) polymerase (L) and nucleoprotein (N) genes delivered through lentiviral vector was investigated. For in vitro evaluation, siRNAs expressing BHK-21 cell lines (BHK-L and BHK-N) were developed using transduction with Lenti-L and Lenti-N lentiviruses encoding siRNAs against RV-L and N genes, respectively. When these cell lines were challenged in vitro with RV Pasteur virus-11 (PV-11) strain, there was reduction in number of RV-specific foci and target gene transcripts indicating inhibitory effect on RV multiplication. For in vivo evaluation, mice were treated intracerebrally with lentiviruses and challenged with 20 LD50 of RV challenge virus standard-11 (CVS-11) strain by intramuscular route in masseter muscle. Five out of eight mice treated with Lenti-N survived indicating 62.5 % protection. The control and Lenti-L-treated mice died within 7-10 days indicating lethal nature of challenge virus and no protection. These results demonstrated that siRNA targeting RV-N could not only inhibit RV multiplication, but also conferred protection in mice against lethal RV challenge. These findings have implication on therapeutic use of siRNA targeting RV-N against RV infection. PMID:23877894

  7. Cationic Polymer Modified Mesoporous Silica Nanoparticles for Targeted SiRNA Delivery to HER2+ Breast Cancer

    PubMed Central

    Ngamcherdtrakul, Worapol; Morry, Jingga; Gu, Shenda; Castro, David J.; Goodyear, Shaun M.; Sangvanich, Thanapon; Reda, Moataz M.; Lee, Richard; Mihelic, Samuel A.; Beckman, Brandon L.; Hu, Zhi; Gray, Joe W.; Yantasee, Wassana

    2015-01-01

    In vivo delivery of siRNAs designed to inhibit genes important in cancer and other diseases continues to be an important biomedical goal. We now describe a new nanoparticle construct that has been engineered for efficient delivery of siRNA to tumors. The construct is comprised of a 47-nm mesoporous silica nanoparticle (MSNP) core coated with a cross-linked PEI-PEG copolymer, carrying siRNA against the HER2 oncogene, and coupled to the anti-HER2 monoclonal antibody (trastuzumab). The construct has been engineered to increase siRNA blood half-life, enhance tumor-specific cellular uptake, and maximize siRNA knockdown efficacy. The optimized anti-HER2-nanoparticles produced apoptotic death in HER2 positive (HER2+) breast cancer cells grown in vitro, but not in HER2 negative (HER2−) cells. One dose of the siHER2-nanoparticles reduced HER2 protein levels by 60% in trastuzumab-resistant HCC1954 xenografts. Multiple doses administered intravenously over 3 weeks significantly inhibited tumor growth (p < 0.004). The siHER2-nanoparticles have an excellent safety profile in terms of blood compatibility and low cytokine induction, when exposed to human peripheral blood mononuclear cells. The construct can be produced with high batch-to-batch reproducibility and the production methods are suitable for large-scale production. These results suggest that this siHER2-nanoparticle is ready for clinical evaluation. PMID:26097445

  8. An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin-5 Inhibitor

    PubMed Central

    Tsui, Melody; Xie, Tiao; Orth, James D.; Carpenter, Anne E.; Rudnicki, Stewart; Kim, Suejong; Shamu, Caroline E.; Mitchison, Timothy J.

    2009-01-01

    Kinesin-5 (also known as Eg5, KSP and Kif11) is required for assembly of a bipolar mitotic spindle. Small molecule inhibitors of Kinesin-5, developed as potential anti-cancer drugs, arrest cell in mitosis and promote apoptosis of cancer cells. We performed a genome-wide siRNA screen for enhancers and suppressors of a Kinesin-5 inhibitor in human cells to elucidate cellular responses, and thus identify factors that might predict drug sensitivity in cancers. Because the drug's actions play out over several days, we developed an intermittent imaging screen. Live HeLa cells expressing GFP-tagged histone H2B were imaged at 0, 24 and 48 hours after drug addition, and images were analyzed using open-source software that incorporates machine learning. This screen effectively identified siRNAs that caused increased mitotic arrest at low drug concentrations (enhancers), and vice versa (suppressors), and we report siRNAs that caused both effects. We then classified the effect of siRNAs for 15 genes where 3 or 4 out of 4 siRNA oligos tested were suppressors as assessed by time lapse imaging, and by testing for suppression of mitotic arrest in taxol and nocodazole. This identified 4 phenotypic classes of drug suppressors, which included known and novel genes. Our methodology should be applicable to other screens, and the suppressor and enhancer genes we identified may open new lines of research into mitosis and checkpoint biology. PMID:19802393

  9. Primary and secondary siRNA synthesis triggered by RNAs from food bacteria in the ciliate Paramecium tetraurelia.

    PubMed

    Carradec, Quentin; Götz, Ulrike; Arnaiz, Olivier; Pouch, Juliette; Simon, Martin; Meyer, Eric; Marker, Simone

    2015-02-18

    In various organisms, an efficient RNAi response can be triggered by feeding cells with bacteria producing double-stranded RNA (dsRNA) against an endogenous gene. However, the detailed mechanisms and natural functions of this pathway are not well understood in most cases. Here, we studied siRNA biogenesis from exogenous RNA and its genetic overlap with endogenous RNAi in the ciliate Paramecium tetraurelia by high-throughput sequencing. Using wild-type and mutant strains deficient for dsRNA feeding we found that high levels of primary siRNAs of both strands are processed from the ingested dsRNA trigger by the Dicer Dcr1, the RNA-dependent RNA polymerases Rdr1 and Rdr2 and other factors. We further show that this induces the synthesis of secondary siRNAs spreading along the entire endogenous mRNA, demonstrating the occurrence of both 3'-to-5' and 5'-to-3' transitivity for the first time in the SAR clade of eukaryotes (Stramenopiles, Alveolates, Rhizaria). Secondary siRNAs depend on Rdr2 and show a strong antisense bias; they are produced at much lower levels than primary siRNAs and hardly contribute to RNAi efficiency. We further provide evidence that the Paramecium RNAi machinery also processes single-stranded RNAs from its bacterial food, broadening the possible natural functions of exogenously induced RNAi in this organism. PMID:25593325

  10. siRNA delivery targeting to the lung via agglutination-induced accumulation and clearance of cationic tetraamino fullerene

    NASA Astrophysics Data System (ADS)

    Minami, Kosuke; Okamoto, Koji; Doi, Kent; Harano, Koji; Noiri, Eisei; Nakamura, Eiichi

    2014-05-01

    The efficient treatment of lung diseases requires lung-selective delivery of agents to the lung. However, lung-selective delivery is difficult because the accumulation of micrometer-sized carriers in the lung often induces inflammation and embolization-related toxicity. Here we demonstrate a lung-selective delivery system of small interfering RNA (siRNA) by controlling the size of carrier vehicle in blood vessels. The carrier is made of tetra(piperazino)fullerene epoxide (TPFE), a water-soluble cationic tetraamino fullerene. TPFE and siRNA form sub-micrometer-sized complexes in buffered solution and these complexes agglutinate further with plasma proteins in the bloodstream to form micrometer-sized particles. The agglutinate rapidly clogs the lung capillaries, releases the siRNA into lung cells to silence expression of target genes, and is then cleared rapidly from the lung after siRNA delivery. We applied our delivery system to an animal model of sepsis, indicating the potential of TPFE-based siRNA delivery for clinical applications.

  11. Selective silencing of gene target expression by siRNA expression plasmids in human cervical cancer cells.

    PubMed

    Peralta-Zaragoza, Oscar; De-la-O-Gómez, Faustino; Deas, Jessica; Fernández-Tilapa, Gloria; Fierros-Zárate, Geny Del Socorro; Gómez-Cerón, Claudia; Burguete-García, Ana; Torres-Poveda, Kirvis; Bermúdez-Morales, Victor Hugo; Rodríguez-Dorantes, Mauricio; Pérez-Plasencia, Carlos; Madrid-Marina, Vicente

    2015-01-01

    RNA interference is a natural mechanism to silence post-transcriptional gene expression in eukaryotic cells in which microRNAs act to cleave or halt the translation of target mRNAs at specific target sequences. Mature microRNAs, 19-25 nucleotides in length, mediate their effect at the mRNA level by inhibiting translation, or inducing cleavage of the mRNA target. This process is directed by the degree of complementary nucleotides between the microRNAs and the target mRNA; perfect complementary base pairing induces cleavage of mRNA, whereas several mismatches lead to translational arrest. Biological effects of microRNAs can be manipulated through the use of small interference RNAs (siRNAs) generated by chemical synthesis, or by cloning in molecular vectors. The cloning of a DNA insert in a molecular vector that will be transcribed into the corresponding siRNAs is an approach that has been developed using siRNA expression plasmids. These vectors contain DNA inserts designed with software to generate highly efficient siRNAs which will assemble into RNA-induced silencing complexes (RISC), and silence the target mRNA. In addition, the DNA inserts may be contained in cloning cassettes, and introduced in other molecular vectors. In this chapter we describe an attractive technology platform to silence cellular gene expression using specific siRNA expression plasmids, and evaluate its biological effect on target gene expression in human cervical cancer cells. PMID:25348304

  12. Delivery of siRNAs to Dendritic Cells Using DEC205-Targeted Lipid Nanoparticles to Inhibit Immune Responses

    PubMed Central

    Katakowski, Joseph A; Mukherjee, Gayatri; Wilner, Samantha E; Maier, Keith E; Harrison, Michael Travis; DiLorenzo, Teresa P; Levy, Matthew; Palliser, Deborah

    2016-01-01

    Due to their ability to knock down the expression of any gene, siRNAs have been heralded as ideal candidates for treating a wide variety of diseases, including those involving “undruggable” targets. However, the therapeutic potential of siRNAs remains severely limited by a lack of effective delivery vehicles. Recently, lipid nanoparticles (LNPs) containing ionizable cationic lipids have been developed for hepatic siRNA delivery. However, their suitability for delivery to other cell types has not been determined. We have modified LNPs for preferential targeting to dendritic cells (DCs), central regulators of immune responses. To achieve directed delivery, we coated LNPs with a single-chain antibody (scFv; DEC-LNPs), specific to murine DEC205, which is highly expressed on distinct DC subsets. Here we show that injection of siRNAs encapsulated in DEC-LNPs are preferentially delivered to DEC205+ DCs. Gene knockdown following uptake of DEC-LNPs containing siRNAs specific for the costimulatory molecules CD40, CD80, and CD86 dramatically decreases gene expression levels. We demonstrate the functionality of this knockdown with a mixed lymphocyte response (MLR). Overall, we report that injection of LNPs modified to restrict their uptake to a distinct cell population can confer profound gene knockdown, sufficient to inhibit powerful immune responses like the MLR. PMID:26412590

  13. Effects of stathmin 1 silencing by siRNA on sensitivity of esophageal cancer cells Eca-109 to paclitaxel.

    PubMed

    Zhu, H W; Jiang, D; Xie, Z Y; Zhou, M H; Sun, D Y; Zhao, Y G

    2015-01-01

    We investigated the effects of stathmin 1 (STMN1) silencing by small interfering (siRNA) on the sensitivity of esophageal cancer cells Eca-109 to paclitaxel. STMN1 siRNA was transiently transfected into Eca-109 cells. The effects of transfection were detected by quantitative polymerase chain reaction and western blotting. The effects of STMN1 silencing by siRNA on the sensitivity of esophageal cancer cells Eca-109 to paclitaxel was tested by MTT and colony formation assays. Hoechst 33258 nuclear staining was used to investigate the differences in Eca-109 cell apoptosis induced by paclitaxel. STMN1 siRNA was successfully transfected and the expression of STMN1 was inhibited. The sensitivity of STMN1 siRNA-transfected Eca-109 cells to paclitaxel was significantly increased (P < 0.01). The apoptosis of Eca-109 cells significantly increased following treatment with paclitaxel (P < 0.01). STMN1 silencing by siRNA may enhance the sensitivity of esophageal cancer cells Eca-109 to paclitaxel and induce apoptosis. PMID:26782519

  14. Pokemon siRNA Delivery Mediated by RGD-Modified HBV Core Protein Suppressed the Growth of Hepatocellular Carcinoma.

    PubMed

    Kong, Jing; Liu, Xiaoping; Jia, Jianbo; Wu, Jinsheng; Wu, Ning; Chen, Jun; Fang, Fang

    2015-10-01

    Hepatocellular carcinoma (HCC) is a deadly human malignant tumor that is among the most common cancers in the world, especially in Asia. Hepatitis B virus (HBV) infection has been well established as a high risk factor for hepatic malignance. Studies have shown that Pokemon is a master oncogene for HCC growth, suggesting it as an ideal therapeutic target. However, efficient delivery system is still lacking for Pokemon targeting treatment. In this study, we used core proteins of HBV, which is modified with RGD peptides, to construct a biomimetic vector for the delivery of Pokemon siRNAs (namely, RGD-HBc-Pokemon siRNA). Quantitative PCR and Western blot assays revealed that RGD-HBc-Pokemon siRNA possessed the highest efficiency of Pokemon suppression in HCC cells. In vitro experiments further indicated that RGD-HBc-Pokemon-siRNA exerted a higher tumor suppressor activity on HCC cell lines, evidenced by reduced proliferation and attenuated invasiveness, than Pokemon-siRNA or RGD-HBc alone. Finally, animal studies demonstrated that RGD-HBc-Pokemon siRNA suppressed the growth of HCC xenografts in mice by a greater extent than Pokemon-siRNA or RGD-HBc alone. Based on the above results, Pokemon siRNA delivery mediated by RGD-modified HBV core protein was shown to be an effective strategy of HCC gene therapy. PMID:26356810

  15. Insight into the relationship between the cell culture model, cell trafficking and siRNA silencing efficiency.

    PubMed

    Capel, Victoria; Vllasaliu, Driton; Watts, Peter; Stolnik, Snow

    2016-08-19

    Despite research efforts, cell uptake processes determining siRNA silencing efficiency remain unclear. Here, we examine the relationship between in vitro cell culture models, cellular trafficking and siRNA silencing efficiency to provide a mechanistic insight on siRNA delivery system design. Model siRNA-polyplexes, based on chitosan as a 'classical' condensing agent, were applied to a panel of lung epithelial cell lines, H1299, A549 and Calu-3 and cell internalization levels, trafficking pathways and gene silencing assessed on exposure to pharmacological inhibitors. The data reveal striking differences in the internalization behaviour and gene silencing efficiency in the tested cell lines, despite their common lung epithelial origins. The model system's silencing was lower where clathrin internalization pathway predominated in Calu-3, relative to silencing in H1299 cells where a non-clathrin internalization appears dominant. Increased silencing on endosomal disruption was apparent in Calu-3 cells, but absent when cellular internalization was not predominantly clathrin-mediated in A549 cells. This highlights that identifying cell trafficking pathways before incorporation of functional components to siRNA delivery systems (e.g. endosomolytic compounds) is crucial. The study hence stresses the importance of selection of appropriate cell culture model, relevant to in vivo target, to assess the gene silencing efficiency and decide which functionalities the 'stratified siRNA silencing vector' requires. PMID:27349867

  16. Liposome-based co-delivery of siRNA and docetaxel for the synergistic treatment of lung cancer.

    PubMed

    Qu, Mei-Hua; Zeng, Rui-Fang; Fang, Shi; Dai, Qiang-Sheng; Li, He-Ping; Long, Jian-Ting

    2014-10-20

    Combination of more than one therapeutic strategy is the standard treatment in clinics. Co-delivery of chemotherapeutic drug and small interfering RNA (siRNA) within a nanoparticulate system will suppress the tumor growth. In the present study, docetaxel (DTX) and BCL-2 siRNA was incorporated in a PEGylated liposome to systemically deliver in a lung cancer model (A549). The resulting nanoparticle (lipo-DTX/siRNA) was stable and exhibited a sustained release profile. The co-delivery of therapeutic moieties inhibited the cell proliferation (A549 and H226) in a time-dependent manner. Moreover, the co-delivery system of DTX and siRNA exhibited a remarkable apoptosis of cancer cells with elevated levels of caspase 3/7 activity (apoptosis markers). Cell cycle analysis further showed remarkable increase in sub-G0/G1 phase, indicating increasing hypodiploids or apoptotic cells. Pharmacokinetic study showed a long circulating profile for DTX from lipo-DTX/siRNA system facilitating the passive tumor targeting. In vivo antitumor study on A549 cell bearing xenograft tumor model exhibited a remarkable tumor regression profile for lipo-DTX/siRNA with 100% survival rate. The favorable tumor inhibition response was attributed to the synergistic effect of DTX potency and MDR reversing ability of BCL-2 siRNA in the tumor mass. Overall, experimental results suggest that co-delivery of DTX and siRNA could be promising approach in the treatment of lung cancers. PMID:25138252

  17. Design of a platform technology for systemic delivery of siRNA to tumours using rolling circle transcription

    PubMed Central

    Jang, Mihue; Kim, Jong Hwan; Nam, Hae Yun; Kwon, Ick Chan; Ahn, Hyung Jun

    2015-01-01

    For therapeutic applications of siRNA, there are technical challenges with respect to targeted and systemic delivery. We here report a new siRNA carrier, RNAtr NPs, in a way that multiple tandem copies of RNA hairpins as a result of rolling circle transcription (RCT) can be readily adapted in tumour-targeted and systemic siRNA delivery. RNAtr NPs provide a means of condensing large amounts of multimeric RNA transcripts into the compact nanoparticles, especially without the aid of polycationic agents, and thus reduce the risk of immunogenicity and cytotoxicity by avoiding the use of synthetic polycationic reagents. This strategy allows the design of a platform technology for systemic delivery of siRNA to tumour sites, because RCT reaction, which enzymatically generates RNA polymers in multiple copy numbers at low cost, can lead to directly accessible routes to targeted and systemic delivery. Therefore, RNAtr NPs suggest great potentials as the siRNA therapeutics for cancer treatment. PMID:26246279

  18. Actinide abundances in ordinary chondrites

    USGS Publications Warehouse

    Hagee, B.; Bernatowicz, T.J.; Podosek, F.A.; Johnson, M.L.; Burnett, D.S.; Tatsumoto, M.

    1990-01-01

    Measurements of 244Pu fission Xe, U, Th, and light REE (LREE) abundances, along with modal petrographic determinations of phosphate abundances, were carried out on equilibrated ordinary chondrites in order to define better the solar system Pu abundance and to determine the degree of variation of actinide and LREE abundances. Our data permit comparison of the directly measured Pu/ U ratio with that determined indirectly as (Pu/Nd) ?? (Nd/U) assuming that Pu behaves chemically as a LREE. Except for Guaren??a, and perhaps H chondrites in general, Pu concentrations are similar to that determined previously for St. Se??verin, although less precise because of higher trapped Xe contents. Trapped 130Xe 136Xe ratios appear to vary from meteorite to meteorite, but, relative to AVCC, all are similar in the sense of having less of the interstellar heavy Xe found in carbonaceous chondrite acid residues. The Pu/U and Pu/Nd ratios are consistent with previous data for St. Se??verin, but both tend to be slightly higher than those inferred from previous data on Angra dos Reis. Although significant variations exist, the distribution of our Th/U ratios, along with other precise isotope dilution data for ordinary chondrites, is rather symmetric about the CI chondrite value; however, actinide/(LREE) ratios are systematically lower than the CI value. Variations in actinide or LREE absolute and relative abundances are interpreted as reflecting differences in the proportions and/or compositions of more primitive components (chondrules and CAI materials?) incorporated into different regions of the ordinary chondrite parent bodies. The observed variations of Th/U, Nd/U, or Ce/U suggest that measurements of Pu/U on any single equilibrated ordinary chondrite specimen, such as St. Se??verin, should statistically be within ??20-30% of the average solar system value, although it is also clear that anomalous samples exist. ?? 1990.

  19. Elemental abundances variations in plume and interplume regions

    NASA Astrophysics Data System (ADS)

    Guennou, Chloé; Savin, Daniel; Hahn, Michael

    2016-07-01

    Plumes are relatively bright, narrow structures in coronal holes that extend along open magnetic field lines far out into the corona. Extensive coronal measurements show abundances anomalies in the solar corona, in which elements with a low first ionization potential (FIP) < 10 eV are enhanced relative to the high FIP elements. Remote sensing spectroscopic measurements show that interplume regions have a photospheric composition. In contrast, the elemental composition of plume material is still unclear, previous spectroscopic measurements have reached contradictory results as to whether the elemental abundances in plumes are the same as or different from interplume regions. In this work, we measured the FIP bias, i.e. the ratio of coronal to photospheric abundances, in both interplumes and plumes using Hinode/Extreme Ultraviolet Imaging Spectrometer (EIS) data. Using spectral line intensities and Differential Emission Measure analysis, we assess the chemical composition of plumes and interplumes over an ~24 hour period in March, 2007. We find that some plumes do show different elemental abundances relative to interplumes. Moreover, the abundance anomaly in plumes is time dependent. If previous studies observed plumes at different stages in their evolution, this time dependence may explain the lack of consistency among previous results. Our work on plume and interplume elemental composition may also enable in situ measurements to answer the longstanding question of whether plumes contribute to the fast solar wind, which originates from coronal holes.

  20. A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity

    PubMed Central

    Bramsen, Jesper B.; Laursen, Maria B.; Nielsen, Anne F.; Hansen, Thomas B.; Bus, Claus; Langkjær, Niels; Babu, B. Ravindra; Højland, Torben; Abramov, Mikhail; Van Aerschot, Arthur; Odadzic, Dalibor; Smicius, Romualdas; Haas, Jens; Andree, Cordula; Barman, Jharna; Wenska, Malgorzata; Srivastava, Puneet; Zhou, Chuanzheng; Honcharenko, Dmytro; Hess, Simone; Müller, Elke; Bobkov, Georgii V.; Mikhailov, Sergey N.; Fava, Eugenio; Meyer, Thomas F.; Chattopadhyaya, Jyoti; Zerial, Marino; Engels, Joachim W.; Herdewijn, Piet; Wengel, Jesper; Kjems, Jørgen

    2009-01-01

    The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3′-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity. PMID:19282453

  1. siRNA as a tool for investigating organogenesis: The pitfalls and the promises.

    PubMed

    Lee, Wen-Chin; Berry, Rachel; Hohenstein, Peter; Davies, Jamie

    2008-07-01

    Removing the function of a specific gene from a developing organ, by making a 'knockout' mouse, is a powerful method for analyzing the molecular pathways that control organogenesis. The technique is expensive, though, in terms of time and money, and complex strategies for producing conditional knockouts are needed for genes that are essential for early development of the embryo, for which an unconditional knockout would be lethal before the organ of interest begins to form. Small interfering RNAs (siRNAs) offer a method of knocking down the expression of specific genes with no need for genomic manipulation. Almost as soon as they had been discovered, siRNAs began to be used to explore the molecular biology of mammalian cells in conventional, two-dimensional culture. They have now also been applied successfully, by several groups, to knock down specific genes in various organ rudiments developing in organ culture. This article reviews the basic technique of siRNA-mediated gene knockdown and how it is being applied to organ culture. It also reviews some of the current problems and challenges in the field, and the ways in which these problems are likely to be overcome. PMID:19279730

  2. Targeting Melanoma Growth and Metastasis with Systemic Delivery of Liposomal Incorporated PAR-1 siRNA

    PubMed Central

    Villares, Gabriel J.; Zigler, Maya; Wang, Hua; Melnikova, Vladislava O.; Wu, Hong; Friedman, Ran; Leslie, Michael C.; Vivas-Mejia, Pablo E.; Lopez-Berestein, Gabriel; Sood, Anil K.; Bar-Eli, Menashe

    2008-01-01

    The thrombin receptor (PAR-1, Protease-Activated-Receptor-1) is over-expressed in highly metastatic melanoma cell lines and in patients with metastatic lesions. Activation of PAR-1 leads to cell signaling and upregulation of genes involved in adhesion, invasion and angiogenesis. Herein, we stably silence PAR-1 through the use of lentiviral shRNA and found significant decreases in both tumor growth (P<.01) and metastasis (P<.001) of highly metastatic melanoma cell lines in vivo. The use of viruses for therapy is not ideal as it can induce toxic immune responses and possible gene alterations following viral integration. Therefore, we also utilized systemic delivery of PAR-1 siRNA incorporated into neutral liposomes (DOPC) to decrease melanoma growth and metastasis in vivo. Significant decreases in tumor growth, weight and metastatic lung colonies (P<.001 for all) were found in mice treated with PAR-1-siRNA-DOPC. The in vivo effects of PAR-1 on invasion and angiogenesis were analyzed via immunohistochemistry. Concomitant decreases in VEGF, IL-8, and MMP-2 expression levels, as well as decreased blood-vessel density (CD31), were found in tumor samples from PAR-1 siRNA-treated mice, suggesting that PAR-1 is a regulator of melanoma cell growth and metastasis by affecting angiogenic and invasive factors. We propose that siRNA incorporated into DOPC nanoparticles could be delivered systemically and used as a new modality for melanoma treatment. PMID:18974154

  3. Micelle-like Nanoparticles as Carriers for DNA and siRNA

    PubMed Central

    Navarro, Gemma; Pan, Jiayi; Torchilin, Vladimir P.

    2015-01-01

    Gene therapy represents a potential efficient approach of disease prevention and therapy. However, due to their poor in vivo stability, gene molecules need to be associated with delivery systems to overcome extracellular and intracellular barriers and allow access to the site of action. Cationic polymeric nanoparticles are popular carriers for small interfering RNA (siRNA) and DNA-based therapeutics for which efficient and safe delivery are important factors that need to be optimized. Micelle-like nanoparticles (MNP) (half micelles, half polymeric nanoparticles) can overcome some of the disadvantages of such cationic carriers by unifying in one single carrier the best of both delivery systems. In this review, we will discuss how the unique properties of MNP including self-assembly, condensation and protection of nucleic acids, improved cell association and gene transfection, and low toxicity may contribute to the successful application of siRNA- and DNA-based therapeutics into the clinic. Recent developments of MNP involving the addition of stimulus-sensitive functions to respond specifically to pathological or externally applied “triggers” (e.g., temperature, pH or enzymatic catalysis, light, or magnetic fields) will be discussed. Finally, we will overview the use of MNP as two-in-one carriers for the simultaneous delivery of different agents (small molecules, imaging agents) and nucleic acid combinations. PMID:25557580

  4. Host Generated siRNAs Attenuate Expression of Serine Protease Gene in Myzus persicae

    PubMed Central

    Bhatia, Varnika; Bhattacharya, Ramcharan; Uniyal, Prem L.; Singh, Rajendra; Niranjan, Rampal S.

    2012-01-01

    Background Sap sucking hemipteran aphids damage diverse crop species. Although delivery of ds-RNA or siRNA through microinjection/feeding has been demonstrated, the efficacy of host-mediated delivery of aphid-specific dsRNA in developing aphid resistance has been far from being elucidated. Methodology/Principal Findings Transgenic Arabidopsis expressing ds-RNA of Myzus persicae serine protease (MySP) was developed that triggered the generation of corresponding siRNAs amenable for delivery to the feeding aphids. M. persicae when fed on the transgenic plants for different time intervals under controlled growth conditions resulted in a significant attenuation of the expression of MySP and a commensurate decline in gut protease activity. Although the survivability of these aphids was not affected, there was a noticeable decline in their fecundity resulting in a significant reduction in parthenogenetic population. Conclusions/Significance The study highlighted the feasibility of developing host based RNAi-mediated resistance against hemipteran pest aphids. PMID:23071558

  5. A comprehensive siRNA screen for kinases that suppress macroautophagy in optimal growth conditions.

    PubMed

    Szyniarowski, Piotr; Corcelle-Termeau, Elisabeth; Farkas, Thomas; Høyer-Hansen, Maria; Nylandsted, Jesper; Kallunki, Tuula; Jäättelä, Marja

    2011-08-01

    Macroautophagy is a catabolic process that maintains cellular homeostasis and protects cells against various external stresses including starvation. Except for the identification of the Akt-mTORC1 pathway as a major negative regulator, little is known about signaling networks that control macroautophagy under optimal growth conditions. Therefore, we screened a human kinome siRNA library for siRNAs that increase the number of autophagosomes in normally growing MCF-7 human breast carcinoma cells, and identified 10 kinases as regulators of constitutive macroautophagy. Further analysis of these kinases with respect to the autophagic flux, kinase signaling and endolysosomal function identified WNK2 as a positive regulator of autophagosome maturation and nine others as macroautophagy inhibitors. The depletion of MK2, PACSIN1, DAPK2, CDKL3 and SCYL1 functioned upstream of Akt-mTORC1 pathway, whereas CSNK1A1, BUB1, PKLR and NEK4 suppressed autophagosome formation downstream or independent of mTORC1. Importantly, all identified kinases except for BUB1 regulated macroautophagy also in immortalized MCF-10A breast epithelial cells. The kinases identified here shed light to the complex regulation of macroautophagy and open new possibilities for its pharmacological manipulation. PMID:21508686

  6. Major degradable polycations as carriers for DNA and siRNA.

    PubMed

    Islam, Mohammad Ariful; Park, Tae-Eun; Singh, Bijay; Maharjan, Sushila; Firdous, Jannatul; Cho, Myung-Haing; Kang, Sang-Kee; Yun, Cheol-Heui; Choi, Yun-Jaie; Cho, Chong-Su

    2014-11-10

    Non-viral gene delivery systems are one of the most potential alternatives to viral vectors because of their less immunogenicity, less toxicity and easy productivity in spite of their low capacity of gene transfection using DNA or silencing using siRNA compared to that of viral vectors. Among non-viral systems, the polycationic derivatives are the most popular gene carriers since they can effectively condense nucleic acids to transfer into the cells, especially the polyethylenimine (PEI) which has been used as a golden standard polymer owing to its high buffering ability for endosomal escape of gene to be expressed. However, PEI has severe problems for its toxicity due to the high positive charge density and non-degradability although the toxicity of PEI depends on its molecular weight (MW) and structure. Therefore, a considerable attention has been paid on synthesis of degradable PEI derivatives using low MW one because low MW PEI is much less toxic than high MW PEI. Other degradable polycationic gene carriers such as polyamidoamines (PAA) and cyclodextrin (CD)-based polycations are also in a significant interest because of their high transfection efficiency with low toxicity. This review in detail explains the recent developments on these three major degradable polycations as promising carriers for deoxyribonucleic acid (DNA) and small interfering RNA (siRNA). PMID:24942341

  7. Dry Powder Formulation of Plasmid DNA and siRNA for Inhalation.

    PubMed

    Chow, Michael Y T; Lam, Jenny K W

    2015-01-01

    Nucleic acid therapeutics has huge potential for the treatment of a wide range of diseases including respiratory diseases. Plasmid DNA (pDNA) and small interfering RNA (siRNA) are the two most widely investigated nucleic acids for therapeutic development. However, efficient and safe delivery of nucleic acids is still a major hurdle in translating nucleic acid therapy into clinical practice. For the treatment of respiratory diseases, administration via inhalation is the most direct and effective way to deliver therapeutic nucleic acids to the lungs. Although liquid aerosol formulation is investigated in most of the studies, it is not desirable in terms of maintaining the stability of nucleic acid especially during long-term storage. This problem could be circumvented by formulating the therapeutic nucleic acids into dry powder for inhalation, and should be considered as the future direction of developing inhalable nucleic acids. In this review, the three major particle engineering methods investigated for the preparation of inhalable pDNA and siRNA formulations, including spray drying (SD), spray freeze drying (SFD) and supercritical fluid (SFC) drying, are discussed and compared. Moreover, common assessment methods and the challenges of evaluating the biological activities of inhalable nucleic acid powders are also reviewed. PMID:26290202

  8. Glutamine-chitosan modified calcium phosphate nanoparticles for efficient siRNA delivery and osteogenic differentiation

    PubMed Central

    Choi, Bogyu; Cui, Zhong-Kai; Kim, Soyon; Fan, Jiabing; Wu, Benjamin M.

    2015-01-01

    RNA interference (RNAi)-based therapy using small interfering RNA (siRNA) exhibits great potential to treat diseases. Although calcium phosphate (CaP)-based systems are attractive options to deliver nucleic acids due to their good biocompatibility and high affinity with nucleic acids, they are limited by uncontrollable particle formation and inconsistent transfection efficiencies. In this study, we developed a stable CaP nanocarrier system with enhanced intracellular uptake by adding highly cationic, glutamine-conjugated oligochitosan (Gln-OChi). CaP nanoparticles coated with Gln-OChi (CaP/Gln-OChi) significantly enhanced gene transfection and knockdown efficiency in both immortalized cell line (HeLa) and primary mesenchymal stem cells (MSCs) with minimal cytotoxicity. The osteogenic bioactivity of siRNA-loaded CaP/Gln-OChi particles was further confirmed in three-dimensional environments by using photocrosslinkable chitosan hydrogels encapsulating MSCs and particles loaded with siRNA targeting noggin, a bone morphogenetic protein antagonist. These findings suggest that our CaP/Gln-OChi nanocarrier provides an efficient and safe gene delivery system for therapeutic applications. PMID:26413302

  9. Guanabenz (Wytensin™) selectively enhances uptake and efficacy of hydrophobically modified siRNAs

    PubMed Central

    Osborn, Maire F.; Alterman, Julia F.; Nikan, Mehran; Cao, Hong; Didiot, Marie C.; Hassler, Matthew R.; Coles, Andrew H.; Khvorova, Anastasia

    2015-01-01

    One of the major obstacles to the pharmaceutical success of oligonucleotide therapeutics (ONTs) is efficient delivery from the point of injection to the intracellular setting where functional gene silencing occurs. In particular, a significant fraction of internalized ONTs are nonproductively sequestered in endo-lysosomal compartments. Here, we describe a two-step, robust assay for high-throughput de novo detection of small bioactive molecules that enhance cellular uptake, endosomal escape, and efficacy of ONTs. Using this assay, we screened the LOPAC (Sigma–Aldrich) Library of Pharmacologically Active Compounds and discovered that Guanabenz acetate (Wytensin™), an FDA-approved drug formerly used as an antihypertensive agent, is capable of markedly increasing the cellular internalization and target mRNA silencing of hydrophobically modified siRNAs (hsiRNAs), yielding a ∼100-fold decrease in hsiRNA IC50 (from 132 nM to 2.4 nM). This is one of the first descriptions of a high-throughput small-molecule screen to identify novel chemistries that specifically enhance siRNA intracellular efficacy, and can be applied toward expansion of the chemical diversity of ONTs. PMID:26400165

  10. Degradable cationic nanohydrogel particles for stimuli-responsive release of siRNA.

    PubMed

    Nuhn, Lutz; Braun, Lydia; Overhoff, Iris; Kelsch, Annette; Schaeffel, David; Koynov, Kaloian; Zentel, Rudolf

    2014-12-01

    Well-defined nanogels have become quite attractive as safe and stable carriers for siRNA delivery. However, to avoid nanoparticle accumulation, they need to provide a stimuli-responsive degradation mechanism that can be activated at the payload's site of action. In this work, the synthetic concept for generating well-defined nanohydrogel particles is extended to incorporate disulfide cross-linkers into a cationic nanonetwork for redox-triggered release of oligonucleotide payload as well as nanoparticle degradation under reductive conditions of the cytoplasm. Therefore, a novel disulfide-modified spermine cross-linker is designed that both allows disassembly of the nanogel as well as removal of cationic charge from residual polymer fragments. The degradation process is monitored by scanning electron microscopy (SEM) and fluorescence correlation spectroscopy (FCS). Moreover, siRNA release is analyzed by agarose gel electrophoresis and a fluorescent RNA detection assay. The results exemplify the versatility of the applied nanogel manufacturing process, which allows alternative stimuli-responsive core cross-linkers to be integrated for triggered oligonucleotide release as well as effective biodegradation for reduced nanotoxicity. PMID:25323454

  11. Cationic and PEGylated Amphiphilic Cyclodextrins: Co-Formulation Opportunities for Neuronal Sirna Delivery.

    PubMed

    O'Mahony, Aoife M; Ogier, Julien; Darcy, Raphael; Cryan, John F; O'Driscoll, Caitriona M

    2013-01-01

    Optimising non-viral vectors for neuronal siRNA delivery presents a significant challenge. Here, we investigate a co-formulation, consisting of two amphiphilic cyclodextrins (CDs), one cationic and the other PEGylated, which were blended together for siRNA delivery to a neuronal cell culture model. Co-formulated CD-siRNA complexes were characterised in terms of size, charge and morphology. Stability in salt and serum was also examined. Uptake was determined by flow cytometry and toxicity was measured by MTT assay. Knockdown of a luciferase reporter gene was used as a measure of gene silencing efficiency. Incorporation of a PEGylated CD in the formulation had significant effects on the physical and biological properties of CD.siRNA complexes. Co-formulated complexes exhibited a lower surface charge and greater stability in a high salt environment. However, the inclusion of the PEGylated CD also dramatically reduced gene silencing efficiency due to its effects on neuronal uptake. The co-formulation strategy for cationic and PEGylated CDs improved the stability of the CD.siRNA delivery systems, although knockdown efficiency was impaired. Future work will focus on the addition of targeting ligands to the co-formulated complexes to restore transfection capabilities. PMID:23805220

  12. siRNA screen identifies QPCT as a druggable target for Huntington’s disease

    PubMed Central

    Jimenez-Sanchez, Maria; Lam, Wun; Tarditi, Alessia; Menzies, Fiona; Dami, Teresa Ed; Xu, Catherine; Gonzalez-Couto, Eduardo; Lazzeroni, Giulia; Heitz, Freddy; Diamanti, Daniela; Massai, Luisa; Satagopam, Venkata P.; Marconi, Guido; Caramelli, Chiara; Nencini, Arianna; Andreini, Matteo; Sardone, Gian Luca; Caradonna, Nicola P.; Porcari, Valentina; Scali, Carla; Schneider, Reinhard; Pollio, Giuseppe; O’Kane, Cahir J.; Caricasole, Andrea; Rubinsztein, David C.

    2015-01-01

    Huntington’s disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified novel modifiers of mutant HTT toxicity by performing a large-scale “druggable genome” siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen, and which also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone alpha B-crystallin and reduced the aggregation of diverse proteins. We generated novel QPCT inhibitors using in silico methods followed by in vitro screens, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a novel HD druggable target affecting mutant huntingtin aggregation, and provide proof-of-principle for a discovery pipeline from druggable genome screen to drug development. PMID:25848931

  13. siRNA screen identifies QPCT as a druggable target for Huntington's disease.

    PubMed

    Jimenez-Sanchez, Maria; Lam, Wun; Hannus, Michael; Sönnichsen, Birte; Imarisio, Sara; Fleming, Angeleen; Tarditi, Alessia; Menzies, Fiona; Ed Dami, Teresa; Xu, Catherine; Gonzalez-Couto, Eduardo; Lazzeroni, Giulia; Heitz, Freddy; Diamanti, Daniela; Massai, Luisa; Satagopam, Venkata P; Marconi, Guido; Caramelli, Chiara; Nencini, Arianna; Andreini, Matteo; Sardone, Gian Luca; Caradonna, Nicola P; Porcari, Valentina; Scali, Carla; Schneider, Reinhard; Pollio, Giuseppe; O'Kane, Cahir J; Caricasole, Andrea; Rubinsztein, David C

    2015-05-01

    Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone αB-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development. PMID:25848931

  14. Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity.

    PubMed

    Whitehead, Kathryn A; Dorkin, J Robert; Vegas, Arturo J; Chang, Philip H; Veiseh, Omid; Matthews, Jonathan; Fenton, Owen S; Zhang, Yunlong; Olejnik, Karsten T; Yesilyurt, Volkan; Chen, Delai; Barros, Scott; Klebanov, Boris; Novobrantseva, Tatiana; Langer, Robert; Anderson, Daniel G

    2014-01-01

    One of the most significant challenges in the development of clinically viable delivery systems for RNA interference therapeutics is to understand how molecular structures influence delivery efficacy. Here, we have synthesized 1,400 degradable lipidoids and evaluate their transfection ability and structure-function activity. We show that lipidoid nanoparticles mediate potent gene knockdown in hepatocytes and immune cell populations on IV administration to mice (siRNA EC50 values as low as 0.01 mg kg(-1)). We identify four necessary and sufficient structural and pKa criteria that robustly predict the ability of nanoparticles to mediate greater than 95% protein silencing in vivo. Because these efficacy criteria can be dictated through chemical design, this discovery could eliminate our dependence on time-consuming and expensive cell culture assays and animal testing. Herein, we identify promising degradable lipidoids and describe new design criteria that reliably predict in vivo siRNA delivery efficacy without any prior biological testing. PMID:24969323

  15. Structure-Based Design of Dendritic Peptide Bolaamphiphiles for siRNA Delivery

    PubMed Central

    2015-01-01

    Development of safe and effective delivery vectors is a critical challenge for the application of RNA interference (RNAi)-based biotechnologies. In this study we show the rational design of a series of novel dendritic peptide bolaamphiphile vectors that demonstrate high efficiency for the delivery of small interfering RNA (siRNA) while exhibiting low cytotoxicity and hemolytic activity. Systematic investigation into structure–property relationships revealed an important correlation between molecular design, self-assembled nanostructure, and biological activity. The unique bolaamphiphile architecture proved a key factor for improved complex stability and transfection efficiency. The optimal vector contains a fluorocarbon core and exhibited enhanced delivery efficiency to a variety of cell lines and improved serum resistance when compared to hydrocarbon analogues and lipofectamine RNAiMAX. In addition to introducing a promising new vector system for siRNA delivery, the structure–property relationships and “fluorocarbon effect” revealed herein offer critical insight for further development of novel materials for nucleic acid delivery and other biomaterial applications. PMID:26436138

  16. Non-Condensing Polymeric Nanoparticles for Targeted Gene and siRNA Delivery

    PubMed Central

    Xu, Jing; Ganesh, Shanthi; Amiji, Mansoor

    2011-01-01

    Gene therapy has shown a tremendous potential to benefit patients in a variety of disease conditions. However, finding a safe and effective systemic delivery system is the major obstacle in this area. Although viral vectors showed promise for high transfection rate, the immunogenicity associated with these systems has hindered further development. As an alternative to viral gene delivery, this review focuses on application of novel safe and effective non-condensing polymeric systems that have shown high transgene expression when administered systemically or by the oral route. Type B gelatin-based engineered nanocarriers were evaluated for passive and active tumor-targeted delivery and transfection using both reporter and therapeutic plasmid DNA. Additionally, we have shown that nanoparticles-in-microsphere oral system (NiMOS) can efficiently deliver reporter and therapeutic gene constructs in the gastrointestinal tract. Additionally, there has been a significant recent interest in the use small interfering RNA (siRNA) as a therapeutic system for gene silencing. Both gelatin nanoparticles and NiMOS have shown activity in systemic and oral delivery of siRNA, respectively. PMID:21621597

  17. Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome.

    PubMed

    Roosing, Susanne; Hofree, Matan; Kim, Sehyun; Scott, Eric; Copeland, Brett; Romani, Marta; Silhavy, Jennifer L; Rosti, Rasim O; Schroth, Jana; Mazza, Tommaso; Miccinilli, Elide; Zaki, Maha S; Swoboda, Kathryn J; Milisa-Drautz, Joanne; Dobyns, William B; Mikati, Mohamed A; İncecik, Faruk; Azam, Matloob; Borgatti, Renato; Romaniello, Romina; Boustany, Rose-Mary; Clericuzio, Carol L; D'Arrigo, Stefano; Strømme, Petter; Boltshauser, Eugen; Stanzial, Franco; Mirabelli-Badenier, Marisol; Moroni, Isabella; Bertini, Enrico; Emma, Francesco; Steinlin, Maja; Hildebrandt, Friedhelm; Johnson, Colin A; Freilinger, Michael; Vaux, Keith K; Gabriel, Stacey B; Aza-Blanc, Pedro; Heynen-Genel, Susanne; Ideker, Trey; Dynlacht, Brian D; Lee, Ji Eun; Valente, Enza Maria; Kim, Joon; Gleeson, Joseph G

    2015-01-01

    Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies. PMID:26026149

  18. Lipid nanoparticle siRNA treatment of Ebola-virus-Makona-infected nonhuman primates.

    PubMed

    Thi, Emily P; Mire, Chad E; Lee, Amy C H; Geisbert, Joan B; Zhou, Joy Z; Agans, Krystle N; Snead, Nicholas M; Deer, Daniel J; Barnard, Trisha R; Fenton, Karla A; MacLachlan, Ian; Geisbert, Thomas W

    2015-05-21

    The current outbreak of Ebola virus in West Africa is unprecedented, causing more cases and fatalities than all previous outbreaks combined, and has yet to be controlled. Several post-exposure interventions have been employed under compassionate use to treat patients repatriated to Europe and the United States. However, the in vivo efficacy of these interventions against the new outbreak strain of Ebola virus is unknown. Here we show that lipid-nanoparticle-encapsulated short interfering RNAs (siRNAs) rapidly adapted to target the Makona outbreak strain of Ebola virus are able to protect 100% of rhesus monkeys against lethal challenge when treatment was initiated at 3 days after exposure while animals were viraemic and clinically ill. Although all infected animals showed evidence of advanced disease including abnormal haematology, blood chemistry and coagulopathy, siRNA-treated animals had milder clinical features and fully recovered, while the untreated control animals succumbed to the disease. These results represent the first, to our knowledge, successful demonstration of therapeutic anti-Ebola virus efficacy against the new outbreak strain in nonhuman primates and highlight the rapid development of lipid-nanoparticle-delivered siRNA as a countermeasure against this highly lethal human disease. PMID:25901685

  19. Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity

    NASA Astrophysics Data System (ADS)

    Whitehead, Kathryn A.; Dorkin, J. Robert; Vegas, Arturo J.; Chang, Philip H.; Veiseh, Omid; Matthews, Jonathan; Fenton, Owen S.; Zhang, Yunlong; Olejnik, Karsten T.; Yesilyurt, Volkan; Chen, Delai; Barros, Scott; Klebanov, Boris; Novobrantseva, Tatiana; Langer, Robert; Anderson, Daniel G.

    2014-06-01

    One of the most significant challenges in the development of clinically viable delivery systems for RNA interference therapeutics is to understand how molecular structures influence delivery efficacy. Here, we have synthesized 1,400 degradable lipidoids and evaluate their transfection ability and structure-function activity. We show that lipidoid nanoparticles mediate potent gene knockdown in hepatocytes and immune cell populations on IV administration to mice (siRNA EC50 values as low as 0.01 mg kg-1). We identify four necessary and sufficient structural and pKa criteria that robustly predict the ability of nanoparticles to mediate greater than 95% protein silencing in vivo. Because these efficacy criteria can be dictated through chemical design, this discovery could eliminate our dependence on time-consuming and expensive cell culture assays and animal testing. Herein, we identify promising degradable lipidoids and describe new design criteria that reliably predict in vivo siRNA delivery efficacy without any prior biological testing.

  20. Chitosan nanoparticles for siRNA delivery: optimization of processing/formulation parameters.

    PubMed

    Esmaeilzadeh Gharehdaghi, Elina; Amani, Amir; Khoshayand, Mohammad Reza; Banan, Mehdi; Esmaeilzadeh Gharehdaghi, Elika; Amini, Mohammad Ali; Faramarzi, Mohammad Ali

    2014-12-01

    Chitosan nanoparticles were prepared using ultrasonication methodology at specific amplitudes and times of sonication. Subsequently, small interfering RNA (siRNA) was added to the solution at predetermined values of nitrogen to phosphorous ratio (N/P), and stirring time. Employing response surfaces generated from a statistical model, the effect of sonication time and amplitude, stirring time, and N/P ratio was studied on the particle size, polydispersity, and loading efficiency of prepared siRNA/chitosan nanoparticles. It was found that to obtain the smallest size, amplitude and time of sonication as well as stirring time should be kept at ∼45%, 165 seconds, and 50 minutes, respectively. Minimum polydispersity values were also obtained at similar values of sonication time/amplitude and stirring time in addition to N/P values of ∼28. Also, the maximum proportion of siRNA loading was observed at approximate values of 300 seconds, 80% and 280 for sonication time, amplitude, and N/P ratio, respectively. The optimum conditions (i.e., to prepare a sample with minimum values of particle size and polydispersity index and maximum values of loading efficiency) were determined as 60.6, 30.0 (seconds), 28.0, and 12.5 (minutes) for amplitude, time of sonication, N/P, and stirring time, respectively. In this scrutiny, the predicted values of optimum formulation were 456 nm size, 89.6% loading efficiency, and 0.4 polydispersity index. PMID:25272198

  1. Chitosan Nanoparticles for siRNA Delivery: Optimization of Processing/Formulation Parameters

    PubMed Central

    Esmaeilzadeh Gharehdaghi, Elina; Amani, Amir; Khoshayand, Mohammad Reza; Banan, Mehdi; Esmaeilzadeh Gharehdaghi, Elika; Amini, Mohammad Ali

    2014-01-01

    Chitosan nanoparticles were prepared using ultrasonication methodology at specific amplitudes and times of sonication. Subsequently, small interfering RNA (siRNA) was added to the solution at predetermined values of nitrogen to phosphorous ratio (N/P), and stirring time. Employing response surfaces generated from a statistical model, the effect of sonication time and amplitude, stirring time, and N/P ratio was studied on the particle size, polydispersity, and loading efficiency of prepared siRNA/chitosan nanoparticles. It was found that to obtain the smallest size, amplitude and time of sonication as well as stirring time should be kept at ∼45%, 165 seconds, and 50 minutes, respectively. Minimum polydispersity values were also obtained at similar values of sonication time/amplitude and stirring time in addition to N/P values of ∼28. Also, the maximum proportion of siRNA loading was observed at approximate values of 300 seconds, 80% and 280 for sonication time, amplitude, and N/P ratio, respectively. The optimum conditions (i.e., to prepare a sample with minimum values of particle size and polydispersity index and maximum values of loading efficiency) were determined as 60.6, 30.0 (seconds), 28.0, and 12.5 (minutes) for amplitude, time of sonication, N/P, and stirring time, respectively. In this scrutiny, the predicted values of optimum formulation were 456 nm size, 89.6% loading efficiency, and 0.4 polydispersity index. PMID:25272198

  2. Protein mediated miRNA detection and siRNA enrichment using p19.

    PubMed

    Jin, Jingmin; Cid, Melissa; Poole, Catherine B; McReynolds, Larry A

    2010-06-01

    p19 RNA binding protein from the Carnation Italian ringspot virus (CIRV) is an RNA-silencing suppressor that binds small interfering RNA (siRNA) with high affinity. We created a bifunctional p19 fusion protein with an N-terminal maltose binding protein (MBP), for protein purification, and a C-terminal chitin binding domain (CBD) to bind p19 to chitin magnetic beads. The fusion protein binds dsRNAs in the size range of 20-23 nucleotides, but does not bind ssRNA or dsDNA. Relative affinities of the p19 fusion protein for different-length RNA and DNA substrates were determined. Binding specificity of the p19 fusion protein for small dsRNA allows detection of miRNA:RNA probe duplexes. Using radioactive RNA probes, we were able to detect low levels of miRNAs in the sub-femtomole range and in the presence of a million-fold excess of total RNA. Detection is linear over three logs. Unlike most nucleic acid detection methods, p19 selects for RNA hybrids of correct length and structure. Rules for designing optimal RNA probes for p19 detection of miRNAs were determined by in vitro binding of 18 different dsRNA oligos to p19. These studies demonstrate the potential of p19 fusion protein to detect miRNAs and isolate endogenous siRNAs. PMID:20569217

  3. Hydrological extremes and security

    NASA Astrophysics Data System (ADS)

    Kundzewicz, Z. W.; Matczak, P.

    2015-04-01

    Economic losses caused by hydrological extremes - floods and droughts - have been on the rise. Hydrological extremes jeopardize human security and impact on societal livelihood and welfare. Security can be generally understood as freedom from threat and the ability of societies to maintain their independent identity and their functional integrity against forces of change. Several dimensions of security are reviewed in the context of hydrological extremes. The traditional interpretation of security, focused on the state military capabilities, has been replaced by a wider understanding, including economic, societal and environmental aspects that get increasing attention. Floods and droughts pose a burden and serious challenges to the state that is responsible for sustaining economic development, and societal and environmental security. The latter can be regarded as the maintenance of ecosystem services, on which a society depends. An important part of it is water security, which can be defined as the availability of an adequate quantity and quality of water for health, livelihoods, ecosystems and production, coupled with an acceptable level of water-related risks to people, environments and economies. Security concerns arise because, over large areas, hydrological extremes - floods and droughts - are becoming more frequent and more severe. In terms of dealing with water-related risks, climate change can increase uncertainties, which makes the state's task to deliver security more difficult and more expensive. However, changes in population size and development, and level of protection, drive exposure to hydrological hazards.

  4. Going to Extremes

    ERIC Educational Resources Information Center

    Coy, Mary

    2008-01-01

    In this article, the author describes a project which gave students a chance to explore the idea of using "extreme" materials in a sculpture. While the process was, at times, challenging and stressful for teacher and student alike, the results proved that, with proper planning, even young students can independently demonstrate multiple solutions…

  5. Climate Extremes and Society

    NASA Astrophysics Data System (ADS)

    Mote, Philip

    2009-10-01

    In October 2005, as the United States still was reeling from Hurricane Katrina in August and as the alphabet was too short to contain all of that year's named Atlantic tropical storms (Hurricane Wilma was forming near Jamaica), a timely workshop in Bermuda focused on climate extremes and society (see Eos, 87(3), 25, 17 January 2006). This edited volume, which corresponds roughly to the presentations given at that workshop, offers a fascinating look at the critically important intersection of acute climate stress and human vulnerabilities. A changing climate affects humans and other living things not through the variable that most robustly demonstrates the role of rising greenhouse gases—globally averaged temperature—but through local changes, especially changes in extremes. The first part of this book, “Defining and modeling the nature of weather and climate extremes,” focuses on natural science. The second part, “Impacts of weather and climate extremes,” focuses on societal impacts and responses, emphasizing an insurance industry perspective because a primary sponsor of the workshop was the Risk Prediction Initiative, whose aim is to “support scientific research on topics of interest to its sponsors” (p. 320).

  6. Optimization using Extremal Dynamics

    NASA Astrophysics Data System (ADS)

    Boettcher, Stefan

    2001-03-01

    We explore a new heuristic for finding high-quality solutions to NP-hard optimization problems which we have recently introduced [see ``Nature's Way of Optimizing," Artificial Intelligence 119, 275-286 (2000) and cond-mat/0010337]. The method, called extremal optimization, is inspired by self-organized criticality, a concept introduced to describe emergent complexity in physical systems. Extremal optimization successively replaces extremely undesirable elements of a single sub-optimal solution with new, random ones. Large fluctuations ensue that efficiently explore many local optima. With only one adjustable parameter, its performance has proved competitive with more elaborate methods, especially near phase transitions which are believed to contain the hardest instances. In particular, extremal optimization is superior to simulated annealing in the partitioning of sparse graphs, it finds the overlap of all ground-states at the phase transition of the 3-coloring problem, and it provides independent confirmation for the ground-state energy of spin glasses, previously obtained with elaborate genetic algorithms.

  7. Disarmed anthrax toxin delivers antisense oligonucleotides and siRNA with high efficiency and low toxicity.

    PubMed

    Dyer, Paul D R; Shepherd, Thomas R; Gollings, Alexander S; Shorter, Susan A; Gorringe-Pattrick, Monique A M; Tang, Chun-Kit; Cattoz, Beatrice N; Baillie, Les; Griffiths, Peter C; Richardson, Simon C W

    2015-12-28

    Inefficient cytosolic delivery and vector toxicity contribute to the limited use of antisense oligonucleotides (ASOs) and siRNA as therapeutics. As anthrax toxin (Atx) accesses the cytosol, the purpose of this study was to evaluate the potential of disarmed Atx to deliver either ASOs or siRNA. We hypothesized that this delivery strategy would facilitate improved transfection efficiency while eliminating the toxicity seen for many vectors due to membrane destabilization. Atx complex formation with ASOs or siRNA was achieved via the in-frame fusion of either Saccharomyces cerevisiae GAL4 or Homo sapien sapien PKR (respectively) to a truncation of Atx lethal factor (LFn), which were used with Atx protective antigen (PA). Western immunoblotting confirmed the production of: LFN-GAL4, LFn-PKR and PA which were detected at ~45.9 kDa, ~37 kDa, and ~83 kDa respectively and small angle neutron scattering confirmed the ability of PA to form an annular structure with a radius of gyration of 7.0 ± 1.0 nm when placed in serum. In order to form a complex with LFn-GAL4, ASOs were engineered to contain a double-stranded region, and a cell free in vitro translation assay demonstrated that no loss of antisense activity above 30 pmol ASO was evident. The in vitro toxicity of both PA:LFn-GAL4:ASO and PA:LFn-PKR:siRNA complexes was low (IC50>100 μg/mL in HeLa and Vero cells) and subcellular fractionation in conjunction with microscopy confirmed the detection of LFn-GAL4 or LFn-PKR in the cytosol. Syntaxin5 (Synt5) was used as a model target gene to determine pharmacological activity. The PA:LFn-GAL4:ASO complexes had transfection efficiency approximately equivalent to Nucleofection® over a variety of ASO concentrations (24h post-transfection) and during a 72 h time course. In HeLa cells, at 200 pmol ASO (with PA:LFN-GAL4), 5.4 ± 2.0% Synt5 expression was evident relative to an untreated control after 24h. Using 200 pmol ASOs, Nucleofection® reduced Synt5 expression to 8.1 ± 2

  8. Data on cell growth inhibition induced by anti-VEGF siRNA delivered by Stealth liposomes incorporating G2 PAMAM-cholesterol versus Metafectene® as a function of exposure time and siRNA concentration.

    PubMed

    Golkar, Nasim; Samani, Soliman Mohammadi; Tamaddon, Ali Mohammad

    2016-09-01

    In this data article, carboxyfluorescein-loaded liposomes were prepared and purified from free carboxyfluorescein using gel filtration chromatography in the first part. In the next part, following preparation of anti-VEGF siRNA loaded liposomes incorporating hydrophobically modified G2 PAMAM dendrimer (G2-Chol40%) (Golkar et al., 2016) [1], the cell growth inhibition induced by the formulations (siRNA/Metafectene complexes and siRNA loaded liposomes incorporating hydrophobic G2) was evaluated at two exposure times through MTT assay in a breast cancer cell (SKBR-3) and compared by two-way ANOVA. PMID:275