Abstract
The research synthesized the non-viral gene vectors based on poly(2-ethyl-2-oxazo-line) and it’s copolymer.Part 1: This study synthesized the non-viral and pH-sensitive gene carrier, poly((2-ethyl-2-oxazoline)-co-ethylenimine)-block-poly(2- ethyl-2-oxazoline) (P(EOz/EI)-b-PEOz). The gene carrier contains both cationic poly((2-ethyl-2-oxazoline)-co-ethylenimine) (P(EOz/EI)) segments and charge-neutral poly(2-ethyl-2-oxazoline) (PEOz) segments. In this study, PEOz was used as biocompatibility shell and as the core source. A technique using methanesulfonyl poly((2-ethyl-2-oxazoline)-co-ethylenimine, (P(EOz/EI)-OMs) as a macroinitiator to modify the outer shell PEOz segment provided an amino group at the chain terminus. P(EOz/EI)-b-PEOz were coordinated with plasmid DNA, and the resulting complexes were characterized by gel permeation chromatography and 1H NMR spectra. The P(EOz/EI)-b-PEOz polyplexes showed suitable mean particle size, low cytotoxicity, and acceptable transfection because of shielding of PEI by PEOz outer shell. TEM morphology showed that the stable core-shell structure of ternary polyplexes at pH 7.4 collapsed and released plasmid at pH 5. Observations of cell uptake of the B-PEI/DNA polyplex and P(EOz/EI)-b-PEOz/DNA polyplexes by CLSM revealed that P(EOz/EI)-b-PEOz polyplexes started to accumulate after 6 h incubation and accumulated significantly after 12 h. The results indicated that the hydrophilic, charge-neutral PEOz shell stabilized polyplex formation, and enhanced polyplex cell viability. Polyplex transfection efficiencies were as high as those of commercially available transfection reagents. Our results suggest that this novel gene carrier, based on the diblock copolymer P(EOz/EI)-b-PEOz, has potential for in non-viral gene therapy applications.Part 2: This investigation demonstrates new ternary gene delivery systems, based on the pH-responsive diblock copolymer poly(2-ethyl-oxazoline)-block-poly(methacrylic acid) (PEOz-b-PMAA), and the branched-poly (ethylenimine) (B-PEI). The plasmid DNA is complexed with B-PEI and further with PEOz-b-PMAA to obtain ternary polyplexes (DNA/B-PEI/PEOz-b-PMAA). PMAA was partially dissociated at neutral pH with a negative charge, to attach to the positively charged surface of the B-PEI/DNA polyplex. The ternary polyplexes also desorb and return to the original pre-poly complex to help gene release after cell uptake due to PMMA becomes neutral charge under an acid environment in endosome. The ternary polyplexes show suitable mean particle size, low cytotoxicity, and acceptable transfection at pH 7.4 because of shielding of B-PEI by PEOz-b-PMAA. A transmission electron microscopy morphological examination shows that the stable core-shell structure of ternary polyplexes at pH 7.4 collapsed and released plasmid at pH 5. Observations of the cell uptake of the B-PEI/DNA polyplex and ternary polyplexes by confocal laser-scanning microscope revealed that ternary polyplexes started to accumulate after 3 h of incubation and accumulated significantly after 6 h. In conclusion, the ternary polyplex improves the cytotoxicity of the single B-PEI/DNA polyplex, and presents a pH-responsive behavior to enhance gene escape from the polyplex. The ternary polyplex constitutes a useful approach for gene carrier design.Part 3: The primary objective of this study is to explore the feasibility of inhibiting tumor growth via the delivery of endostatin-angiostatin (hEA) fusion gene by above non-viral gene carrer. We first compared above two gene carriers in particles size, zeta potential, cell toxicity and gene transfection efficiency. The results indicated that the gene carrier P(EOz/EI)-b-PEOz from first part had better quality for gene delievery, so we chose P(EOz/EI)-b-PEOz for further research. The results indicated that gene carrier P(EOz/EI)-b-PEOz with hEA led to a higher degree of tumor growth inhibition, and can be easily accumulated in tumor and metabolized. In conclusion, the P(EOz/EI)-b-PEOz constitutes a useful approach for gene carrier design.