A major bottleneck in the development of siRNA therapies is their delivery to the desired cell type or tissue, followed by effective passage across the cell membrane with subsequent silencing of the targeted mRNA. To address this problem, we describe the synthesis of core/shell hydrogel nanoparticles (nanogels) with surface-localized peptides that specifically target ovarian carcinoma cell lines possessing high expression levels of the Eph2A receptor. These nanogels are also demonstrated to be highly effective in the noncovalent encapsulation of siRNA and enable cellspecific delivery of the oligonucleotides in serum-containing medium. Cell toxicity and viability assays reveal that the nanogel construct is nontoxic under the conditions studied, as no toxicity or decrease in cell proliferation is observed following delivery. Importantly, a preliminary investigation of gene silencing illustrates that nanogel-mediated delivery of siRNA targeted to the EGF receptor results in knockdown of that receptor. Excellent protection of siRNA during endosomal uptake and endosomal escape of the nanogels is suggested by these results since siRNA activity in the cytosol is required for gene silencing.

BackgroundChemoresistance is a major obstacle in cancer treatment. Targeted therapies that enhance cancer cell sensitivity to chemotherapeutic agents have the potential to increase drug efficacy while reducing toxic effects on untargeted cells. Targeted cancer therapy by RNA interference (RNAi) is a relatively new approach that can be used to reversibly silence genes in vivo by selectively targeting genes such as the epidermal growth factor receptor (EGFR), which has been shown to increase the sensitivity of cancer cells to taxane chemotherapy. However, delivery represents the main hurdle for the broad development of RNAi therapeutics.MethodsWe report here the use of core/shell hydrogel nanoparticles (nanogels) functionalized with peptides that specially target the EphA2 receptor to deliver small interfering RNAs (siRNAs) targeting EGFR. Expression of EGFR was determined by immunoblotting, and the effect of decreased EGFR expression on chemosensitization of ovarian cancer cells after siRNA delivery was investigated.ResultsTreatment of EphA2 positive Hey cells with siRNA-loaded, peptide-targeted nanogels decreased EGFR expression levels and significantly increased the sensitivity of this cell line to docetaxel (P < 0.05). Nanogel treatment of SK-OV-3 cells, which are negative for EphA2 expression, failed to reduce EGFR levels and did not increase docetaxel sensitivity (P > 0.05).ConclusionThis study suggests that targeted delivery of siRNAs by nanogels may be a promising strategy to increase the efficacy of chemotherapy drugs for the treatment of ovarian cancer. In addition, EphA2 is a viable target for therapeutic delivery, and the siRNAs are effectively protected by the nanogel carrier, overcoming the poor stability and uptake that has hindered clinical advancement of therapeutic siRNAs.

Small, monodispersed nanogels (~ 50-nm radius) were synthesized by free-radical precipitation polymerization and characterized using a suite of light scattering and chromatography methods. Nanogels were synthesized with either N-isopropylacrylamide or N-isopropylmethacrylamide as the main monomer, with acrylic acid or 4-acrylamidofluorescein as a co-monomer and N, N′-methylenebis(acrylamide) as a cross-linker. By varying the surfactant and initiator concentrations, particle size was controlled while maintaining excellent monodispersity. An amine-containing shell was added to these core particles to facilitate subsequent bioconjugation. Successful conjugation of folic acid to the particles was demonstrated as an example of how such materials might be employed in a targeted drug delivery system.

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