siRNA Synthesis, synthetic oligos, RNA interference
However, translation of siRNA into the clinic is dependent on the availability of an effective delivery system.
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Intracellular uptake of siRNA/polymer complexes was measured using flow cytometry (Becton Dickinson LSR benchtop analyzer). Hela cells were seeded at 12,000 cells/cm2 (24-well plates) in folate-free RPMI media and allowed to adhere overnight. FAM labeled siRNA (Ambion) was complexed with control diblock or folate diblock at a charge ratio of 4:1 for 30 min at room temperature and then added to the plated Hela cells at a final siRNA concentration of 25 nM (500 µl volume). To a subset of the folate diblock/siRNA treatments, 10 µg/ml of free folate was added to the media as a control. After incubation with the complexes for 1 h, the cells were trypsinized and resuspended in PBS with 0.5% BSA and 0.01% trypan blue. Trypan blue was utilized as previously described for quenching of extracellular fluorescence and discrimination of complexes that have been endocytosed by cells. Ten thousand cells were analyzed per sample and fluorescence gating was determined using samples receiving no treatment and treated with polymer alone.
To assess the siRNA binding efficiency of MnHAP, agel retardation assay was carried out at six weight ratios of MnHAPto siRNA while maintaining the amount of siRNA. illustrates that with the increaseof MnHAP/ siRNA weight ratio, the migration of siRNA bands wassignificantly retarded. When the ratio of the MnHAP/siRNA was ≥2:1,no free siRNA could be detected, which indicated that the siRNA wascompletely confined to the sample well. Therefore, the 2:1 ratiocould be the minimal ratio for siRNA being totally bound by MnHAPand this ratio was used in the subsequent experiments.
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Typically, cationic carriers have been employed for siRNA delivery.,,– Through electrostatically complexation with negatively-charged phosphate groups of nucleic acids, cationic carriers imbibe siRNA with protection against enzymatic degradation and a means to enter cells through endocytosis. However, this approach renders a delivery system that lacks cellular specificity. Folic acid has shown great promise as a targeting ligand for a number of in vivo applications.– While folic acid is internalized by the low affinity reduced folate carrier expressed by nearly all cells (Km ~ 10−6 M), it has greater affinity (Kd ~ 10−10 M), towards the folate receptor, which is overexpressed in a number of tumors and cancer cell lines–. Folic acid is internalized via the folate receptor in two steps. First, the folate binds to the receptor and then it is transferred into the cell by receptor-mediated endocytosis, allowing large drug carrier systems, including micelles, proteins, liposomes, and nanoparticles to enter the cell via endo-lysosomal trafficking. However, irrespective of specificity, once trafficked to the endo-lysosomal pathway, cargo typically becomes degraded in lysosomes. Thus an effective, efficient in vivo siRNA delivery system must be multifunctional and provide protection against enzymatic degradation, be targeted to and internalized by the desired cell type, and result in intracellular trafficking to the intracellular environment where the target is located.
We have previously reported the development of cationic and pH-responsive, endosomolytic diblock copolymers as potent siRNA delivery systems in vitro.,,, These carriers were formed using reversible addition-fragmentation chain transfer polymerization (RAFT), a type of living radical polymerization (LRP). A distinct advantage of polymeric carrier systems, and particularly polymers synthesized via LRPs, is the ease with which modularity can be introduced to incorporate many functionalities.,– LRPs provide polymers with narrow molecular weight distributions and a wide range of functional polymers with defined architectures.,,– RAFT, similar to other LRPs, uses a chain transfer agent (CTA), which typically consists of a dithioester or trithiocarbonate, and reactive Rand Z-groups, where the resultant polymer contains the same R and Z functionalities at its chain-ends. Reactive R- and Z-group modified CTAs have been employed to prepare alpha and omega-functional polymers, respectively, that are directly reactive toward biomolecules., More recently, this approach has been used to exploit pyridyl-disulfide groups to enable reversible conjugation of siRNA and proapoptotic peptides18 to polymers for enhanced delivery characteristics. In addition, chain extension strategies have been employed to specifically and reproducibly functionalize the omega-end of RAFT polymers with a multitude of functional groups, including folic acid. However, these synthetic schemes requires subsequent conjugation steps to introduce the targeting or drug functionality whereas the current approach achieves functionalization concurrently with RAFT polymer synthesis.
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A formazan-based assay (MTT assay, Sigma-Aldrich)was employed to determine the cytotoxicity of the MnHAP/siRNAmixture (weight ratio at 2:1). BEL-7402 cells were seeded in96-well plates at a density of 10/well with 100 μlculture medium. Cells were incubated for 24 h to allow completeadherence then the medium was replaced with fresh medium containingsiRNA alone, MnHAP/siRNA complexes and Lipofectamine/siRNAcomplexes respectively; control wells received fresh medium. Foreach well, the amount of siRNA was 0.25 μg and the amount of MnHAPwas 0.25 μg or 0.5 μl Lipofectamine. Four replicates of eachcondition were present and analyzed simultaneously.
To our knowledge there have been no reports of targeting strategies introduced using the RAFT chain transfer agent directly. Herein, we report the straightforward de novo synthesis of a RAFT CTA with a folate-functionalized R-group. The strategy is different from reported LRP strategies to make targeted polymers– and involves synthesis of a novel chain transfer agent (CTA) for RAFT polymerization. To achieve potent, pH-responsive siRNA carriers with precise presentation of folate and narrow polydispersities, RAFT polymerization was utilized in the synthesis of a diblock copolymer consisting of dimethylaminoethyl methacrylate-b-dimethylaminoethyl methacrylate-co-butyl methacrylate-co-propylacrylic acid (DMAEMA-b-DMAEMA-co-BMA-co-PAA), a terpolymer previously described for its potency for in vitro intracellular siRNA delivery., The presence of tertiary and partially protonated amines in the first block allows for complexation and protection of siRNA and the second block imbibes pH-responsive endosomolytic behavior. Moreover, each polymer presents targeting moieties meerly through use of the folate-functionalized CTA. As demonstrated through polymer characterization methods, competitive folate binding assays, and gene knockdown studies, this multifunctional polymer provides potent siRNA delivery to cancer cells that overexpress folate receptors.
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Search results for siRNA at Sigma-Aldrich
Akhtar S and Benter IF: Nonviral deliveryof synthetic siRNAs in vivo. J Clin Invest. 117:3623–3632. 2007. : :
siRNA synthesis, lead selection, and lipid nanoparticle preparation
Advanced siRNA design and quality synthesis are crucial for a successful RNA interference experiment
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RNA interference (RNAi) mediated through double stranded small interfering RNA (siRNA) is a promising therapeutic strategy for a variety of diseases. Effective siRNA delivery results in highly specific gene knockdown, providing a means to reduce expression of virtually any protein target with lower doses and less toxicity than other RNAi mechanisms., However, the successful in vivo delivery of siRNA is a formidable challenge. Although a wide variety of carriers for siRNA have been explored and consist of polymers, peptides, and lipids, a bottleneck for efficient siRNA therapy lies in the inability to specifically target active, non-degraded siRNA to tissues of interest.
Design, synthesis and evaluation of VEGF-siRNA/CRS …
BEL-7402 cells were harvested 48 h after siRNAtransfection and lysed with lysis buffer for 30 min on ice. Totalprotein concentration was determined using a BCA protein assay. Forwestern blotting, protein extracts were electrophoresed on SDS-PAGEgels and transferred to nitrocellulose membranes. After blockingwith 5% non-fat milk powder, the membrane was first incubatedovernight at 4°C with a mouse monoclonal antibody against GAPDH(Abcam). The blots were then washed and incubated with a goatanti-mouse IgG secondary antibody (Abcam). Bands were visualizedusing an ECL detection kit (Millipore) and intensities analyzedusing Image J software. The relative expression of GAPDH wasdetermined by dividing the densitometric value of the GAPDH band bythat for its control (Tubulin).
Learn About siRNA's Uses in Molecular Genetics Research
(2016) 'Anisamide-targeted gold nanoparticles for siRNA delivery in prostate cancer - synthesis, physicochemical characterisation and in vitro evaluation', Journal of Materials Chemistry B, 4(13), pp.
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