Core-shell type lipid/rPAA-Chol polymer hybrid nanoparticles for in vivo siRNA delivery.

Our previous study had reported that cholesterol-grafted poly(amidoamine) (rPAA-Chol polymer) was able to self-assemble into cationic nanoparticles and act as a potential carrier for siRNA transfection. In this study, the core-shell type lipid/rPAA-Chol hybrid nanoparticles (PEG-LP/siRNA NPs and T7-LP/siRNA NPs) were developed for improving in vivo siRNA delivery by modifying the surface of rPAA-Chol/siRNA nanoplex core with a lipid shell, followed by post-insertion of polyethylene glycol phospholipid (DSPE-PEG) and/or peptide (HAIYPRH, named as T7) modified DSPE-PEG-T7. The integrative hybrid nanostructures of LP/siRNA NPs were evidenced by dynamic light scattering (DLS), confocal laser scanning microscope (CLSM), cryo-transmission electron microscope (Cryo-TEM) and surface plasmon resonance (SPR) assay. It was demonstrated that the T7 peptide modified LP/siRNA NPs (T7-LP/siRNA NPs) exhibited uniform and spherical structures with particle size of 99.39 ± 0.65 nm and surface potential of 42.53 ± 1.03 mV, and showed high cellular uptake efficiency and rapid endosomal/lysosomal escape ability in MCF-7 cells. Importantly, in vitro gene silencing experiment demonstrated that both of pegylated and targeted LP/siEGFR NPs exhibited significantly stronger downregulation of EGFR protein expression level in MCF-7 cells, compared to that of the physical mixture of siRNA lipoplexes and rPAA-Chol/siRNA nanoplexes. In vivo tumor therapy on nude mice bearing MCF-7 tumors further confirmed that the targeted T7-LP/siEGFR NPs exhibited the greatest inhibition on tumor growth via transferrin receptor-mediated targeting delivery, without any activation of immune responses and significant body weight loss following systemic administration. These findings indicated that the core-shell type T7-LP/siRNA nanoparticles would be promising siRNA delivery systems for in vivo tumor-targeted therapy.

Self-assembly cationic nanoparticles based on cholesterol-grafted bioreducible poly(amidoamine) for siRNA delivery.

In this study, a series of bioreducible poly(amidoamine)s grafting different percentages of cholesterol (rPAA-Ch14: 14%, rPAA-Ch29: 29%, rPAA-Ch57: 57% and rPAA-Ch87: 87%) was synthesized and used for siRNA delivery. These amphiphilic polymers were able to self-assemble into cationic nanoparticles in aqueous solution at low concentrations. The nanoparticle formation was evidenced via cryo-transmission electron microscope (Cryo-TEM) and dynamic light scattering analysis. The average hydrodynamic size of rPAA-Ch blank nanoparticles was about 80-160 nm with zeta potential of 50-60 mV. Also, the effects of different percentages of cholesterol grafted onto rPAA on physicochemical characteristics, in vitro cytotoxicity, cellular uptake, VEGF gene silencing efficacy and translocation mechanism of rPAA-Ch/siRNA complexes were investigated. The results showed that rPAA-Ch57 polymer was not only able to form stable nanocomplexes and possess high cell uptake, but also to exhibit the best in vitro VEGF gene silencing efficacy and the best in vivo tumor growth inhibition effect when it was formulated with VEGF-siRNA. Moreover, the observations of confocal laser scanning microscope (CLSM) and the study of cholesterol competitive inhibition demonstrated that endosomal/lysosomal escape and cytoplasmic dissociation of rPAA-Ch57/siRNA complexes were dependent on the "proton sponge effect" and disulfide cleavage, following internalization with cholesterol-related endocytosis pathway and subsequent transportion into endosomes/lysosomes. These findings indicated that the rPAA-Ch57 polymer should be a promising and potent carrier for siRNA delivery.

A comparative study of three ternary complexes prepared in different mixing orders of siRNA/redox-responsive hyperbranched poly (amido amine)/hyaluronic acid.

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.

Self-assembly nanomicelles based on cationic mPEG-PLA-b-Polyarginine(R15) triblock copolymer for siRNA delivery.

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 (mPEG(2000)-PLA(3000)-b-R(15)) 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 (1)H 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 R(15) 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 mPEG(2000)-PLA(3000)-b-R(15) copolymer would be a safe and efficient nanocarrier for in vivo delivery of therapeutic siRNA.