Studies On Novel Polyion Complex Micelles For Gene Delivery

Gene therapy holds great potential for the treatment of human diseases such as infectious diseases, genetic-related disorders and cancer. The development of safe and efficient gene vectors is one of the prerequisites for the success of gene therapy. Despite the high transduction efficiency of viral vectors which are derived from viruses by the use of recombinant DNA techniques, their clinical potential should be fully understood in terms of issues related to production, safety and immune response. The safety concerns regarding the use of viral vectors in humans make non-viral gene delivery system an attractive alternative. The non-viral vectors have recently gained increasing attention due to their stability, safety, ease of preparation, easily manufactured for large-scale production but low transfection efficiency. The main objective in gene therapy is to obtain an efficient non-viral gene vector.Among numerous non-viral gene vectors, polyion complex (PIC) micelles possess a unique core-shell structure which has a PEG shell and a polyionic complex core formed through electrostatic interaction between oppositely charged polymers and gene material. As delivery vehicles for DNA, PIC micelles were demonstrated to have remarkable properties such as excellent colloidal stability in protein aceous media, high solubility in aqueous media, high tolerability toward nuclease degradation, minimal interaction with biological components, including proteins and cells, and prolonged blood circulation compared to the other conventional polyplex and lipoplex systems. Furthermore, the termination functionalized PEG also provided the possibility for targeting modification.In the present study, pEGFP was chosen as the reporter gene. Three different structure PIC micelles were prepared respectively using block copolymer poly (ethylene glycol)-block-poly (L-lysine) (PEG-b-PLL), triblock copolymer poly (lactic acid)-poly (ethylene glycol)-poly (L-lysine)(PLA-PEG-PLL), and novel lipid-polycation copolymer DOPE-graft-poly (L-lysine)-block-poly (ethylene glycol) (DOPE-g-PLL-b-PEG) to assemble with negatively charged DNA. They are:(1) PEG-b-PLL/DNA self-assembled PIC micelles; (2) PLA-PEG-PLL/DNA self-assembled PIC micelles with PEG loop structure; (3) the novel lipid-polycation copolymer DOPE-g-PLL-b-PEG/DNA self-assembled lipid modified polyion complex micelles (LPCM). Furthermore, to achieve the active targeting ability, CNGRCG (cNGR) was coupled on the surface of LPCM as targeting ligand. The main methods and results were as follows:1. Studies on PEG-b-PLL/DNA self-assembled polyion complex micellesPEG-b-PLL and DNA self-assembled into PIC micelles through electrostatic interactions between the negative phosphates along the DNA backbone and positive charges displayed on PLL. The physico-chemical properties, stability in plasma, the protection effect on DNA against nuclease degradation, in vitro cytotoxicity as well as transfection activity of the PIC micelles were evaluated respectively. The obtained PIC micelles were approximately spherical in shape with average particle size of 150.3±5.5 nm and zeta potentials of 10.4±1.2 mV. The PIC micelles could stabilize DNA when incubated with plasma and resist the nuclease degradation in vitro. They showed low cytotoxicity to HepG2 cells and the results of the in vitro gene transfection experiment suggested that the self-assembled PIC could transfer the loaded gene into HepG2 cells, and the gene could express well inside the cells, though the transfection efficiency was not comparable to PEI/DNA. In the further study, the block copolymer PEG-b-PLL was needed to be optimized to improve the transfection efficiency.2. Studies on PLA-PEG-PLL/DNA self-assembled PIC with PEG loopCompared with PEG-b-PLL, the tri-block copolymer PLA-PEG-PLL combined the characters of cationic polymer PLL, PLA and PEG:the self-assembled PIC possessed PEG loop structure to give the long-circulating capacity, PLA segments as the hydrophobic core to increase the stability of the PIC, and PLL segments to condense DNA and deposit with the PLA core. Firstly, the blank PLA-PEG-PLL nanoparticles (NPs) were prepared. The obtained nanoparticles charged positively due to the primaryε-amine groups of PLL, zeta potentials of PLA-PEG-PLL NPs were 28.0±2.4 mV. Then the PLA-PEG-PLL NPs/DNA complexes (PIC with PEG loop) were formed by self-assembly method. The physicochemical properties (morphology, particle size and surface charge) and the biological properties (protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in HeLa and HepG2 cells) of the PLA-PEG-PLL NPs/DNA complexes were evaluated. The obtained PLA-PEG-PLL NPs/DNA complexes were approximately spherical in shape with average particle size of 127.4±6.7 nm and zeta potential of 14.9±4.3 mV. Agarose gel electrophoresis assay confirmed that the PLA-PEG-PLL NPs could condense DNA thoroughly and protect DNA from nuclease and human plasma degradation. PLA-PEG-PLL NPs/DNA complexes exhibited almost no toxicity and higher gene expression than PEI/DNA in HepG2 cells and HeLa cells. These results revealed that the biodegradable tri-block copolymer PLA-PEG-PLL might be a very attractive candidate as a non-viral vector and might alleviate the drawbacks of the conventional cationic vectors/DNA complexes for gene delivery in vivo.3. Studies on novel lipid-polycation copolymer DOPE-g-PLL-b-PEG and self-assembled lipid modified PIC micellesThe lipophilicity lipid DOPE may attribute to the formation of inverted hexagonal phase destabilizing endosomal membrane and facilitating the release of plasmid DNA from lysosomes, the endosomolytic character of the lipid seems to be the reason for the improved transfection efficiency. To enhance the gene transfection efficiency of PEG-b-PLL, the present work was to synthesize a novel lipid-polycation copolymer DOPE-graft-poly (L-lysine)-block-poly(ethylene glycol) (DOPE-g-PLL-b-PEG) and evaluate the potential of this novel hybrid lipid-polycation polymer as efficient gene delivery carrier. DOPE-g-PLL-b-PEG was synthesized by N-acylation of PEG-b-PLL with NHS esters of the lipid, DOPE-GA. The degree of DOPE modification obtained was 16%,30% and 56%, respectively. How the degree of DOPE grafting with PEG-b-PLL affected in vitro gene transfection and cytotoxicity of the transfection vehicle were then illustrated. DOPE-g-PLL-b-PEG and DNA self-assembled into lipid modified polyion complex micelles (LPCM), through electrostatic interactions between the negative phosphates along the DNA backbone and positive charges displayed on PLL. LPCM could protect DNA from plasma and nuclease degradation in vitro. LPCM showed lower cytotoxicity to HepG2 and HeLa cells than PEI/DNA (p<0.05). The results of the gene transfection experiment in vitro suggested that LPCM exhibited higher gene expression than PIC. Especially, when DOPE grafting was 30%, the corresponding LPCM30 displayed the highest transfection efficiency in HeLa cells (p<0.05). These results indicated that the LPCM could facilitate gene transfer to cultured cells, the developed hybrid lipid-polycation DOPE-g-PLL-b-PEG was shown to be beneficial for the development of non-viral gene transfer carrier and might offer an alternative strategy for the future gene therapy.4. Studies on cNGR modified targeting LPCMIn the previous parts, the novel lipid-polycation polymer, DOPE-g-PLL-b-PEG, was synthesized and used as material to prepare LPCM for gene delivery. This kind of carrier achieved relatively lower toxicity and higher transfection efficiency than PEI/DNA. One major problem with LPCM was its lack of cell targeting ability. Thus surface modification was applied to achieve active targeting in this part. It was reported that small peptides containing NGR sequence could specifically bind the CD 13 receptors which was overexpressed on the neovascular endothelial cells. Especially, the circular NGR peptides have more superiority than the linear ones. In this part, based on the DOPE30-g-PLL-b-PEG and DSPE-PEG-NH2, the reported gene pEGFP was compacted into non-targeted LPCM. Then, a circular 6 peptides CNGRCG (cNGR) was incorporated by post-modified method onto the gene nanovectors to form cNGR-targeted LPCM (cNGR-LPCM). The physicochemical properties of both non-targeted LPCM and cNGR-LPCM including morphology, particle size, zeta potential, protection effects of the LPCM to DNA from nuclease degradation as well as the stability in plasma were evaluated respectively. The in vitro cytotoxicity and transfection activity of cNGR-LPCM were determined in HUVEC and HepG2 cells using non-targeted LPCM as control. Both the obtained non-targeted LPCM and cNGR-LPCM were approximately spherical with average particle size of 134.4±6.1 nm and 139.5±7.6nm, PDI of 0.214 and 0.132, zeta potentials of 6.6±3.4 mV and 7.0±3.8 mV, respectively. The obtained cNGR-LPCM could combine DNA thoroughly and protect DNA from nuclease degradation. The results of in vitro studies on cells indicated that the obtained cNGR-LPCM exhibited lower cytotoxicity to HUVEC and HepG2 cells compared with PEI/DNA (p<0.05) at the transfection dosage. The results of the transfection studies demonstrated that cNGR-LPCM exhibited higher transfection efficiency than non-targeted LPCM in the CD 13 positive cells like HUVEC cells. However, in the CD 13 negative cells like HepG2 cells, no difference of the transfect efficiency was observed between the two kinds of nanoparticles. These results showed that the cNGR-LPCM might be promising non-viral gene vectors specifically delivering the therapeutic gene into the targeted site of tumor neovascular.In conclusion, novel PIC micelles could be prepared easily with small particle sizes, efficiently binding and protection to DNA and low cytotoxicity. In vitro transfection studies have showed that the novel PIC micelles could be a promising non-viral gene vector, which has the potential to make in vivo gene therapy achievable.