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Abstract: Due to the absence of safe and effective carriers for in vivo delivery, the applications of small interference RNA (siRNA) in clinic for therapeutic purposes have been limited. In this study, a biodegradable amphiphilic tri-block copolymer (mPEG2000-PLA3000-b-R15) composed of monomethoxy poly(ethylene glycol), poly(d,l-lactide) and polyarginine was synthesized and further self-assembled to cationic polymeric nanomicelles for in vivo siRNA delivery, with an average diameter of 54.30 ± 3.48 nm and a zeta potential of approximately 34.8 ± 1.77 mV. The chemical structures of the copolymers were well characterized by 1H NMR spectroscopy and FT-IR spectra. In vitro cytotoxicity and hemolysis assays demonstrated that the polymeric nanomicelles showed greater cell viability and haemocompatibility than those of polyethyleneimine (PEI) or R15 peptide. In vitro experiments demonstrated that EGFR targeted siRNA formulated in micelleplexes exhibited approximately 65% inhibition of EGFR expression on MCF-7 cells in a sequence-specific manner, which was comparable to Lipofectamine™ 2000. The results of intravenous administration showed Micelleplex/EGFR-siRNA significantly inhibited tumor growth in nude mice xenografted MCF-7 tumors, with a remarkable inhibition of EGFR expression. Furthermore, no positive activation of the innate immune responses and no significant body weight loss was observed during treatment suggested that this polymeric micelle delivery system is non-toxic. In conclusion, the present nanomicelles based on cationic mPEG2000-PLA3000-b-R15 copolymer would be a safe and efficient nanocarrier for in vivo delivery of therapeutic siRNA. [Copyright &y& Elsevier]

In this study, a novel redox-responsive hyperbranched poly(amido amine) (named PCD) was synthesized and used as a cationic polymer to form a ternary complex with small interfering RNA (siRNA) and hyaluronic acid (HA) for siRNA delivery. Here, it is hypothesized that different mixing orders result in different assembly structures, which may affect the siRNA delivery efficiency. To investigate the effects of mixing orders on siRNA delivery efficiency in two human breast cancer cell lines, three ternary complexes with different mixing orders of siRNA/PCD/HA were prepared and characterized: mixing order I (initially prepared siRNA/PCD binary complex further coated by negatively charged HA), mixing order II ( initially prepared HA/PCD binary complex further incubated with siRNA), and mixing order III ( initially prepared siRNA/HA mixture further electrostatically compacted by positively charged PCD). With an optimized siRNA/PCD/HA charge ratio of 1/20/16, the particle sizes and zeta potentials of these ternary complexes were 124.8 nm and 27.3 mV (mixing order I), 147.5 nm and 29.9 mV (mixing order II), and 128.8 nm and 19.4 mV (mixing order III). Also, the effects on stability, cellular uptake, and gene silencing efficiency of siRNA formulated in ternary complexes with different mixing orders were investigated. The results showed that mixing orders I and III displayed better siRNA transfection and protection than mixing order II in human breast cancer MCF-7 and MDA-MB-231 cells. More interesting, at the siRNA/PCD/HA charge ratio of 1/20/16, the gene silencing effects on vascular endothelial growth factor expression in MDA-MB- 231 cells were as follows: mixing order III > mixing order I > mixing order II. Based on these results, a likely explanation for the difference in functionality dependent on mixing orders is the formation of different assembly structures. These results may help future optimization of siRNA ternary complexes for achieving better delivery efficiencies, especially for target-specific siRNA delivery to cells with HA receptor overexpression.