Abstract
The main object of cancer gene therapy is to eradicate tumor cells, while minimizing the accumulation to the surrounding normal tissues. However, cationic lipid and polymeric carrier systems have a common dilemma in that these systems which was specific gene expression on cancer tissue. Photochemical internalization (PCI) is a unique procedure for site-specific delivery of several types of membrane-impermeable molecules to the cytosol of target cells. The technology is based on photochemical-induced release of endocytosed macromolecules from endosomes and lysosomes into the cytosol. This study is divided into three major topics, including (1) preparation of a high tumor targeting photosensitive (20FTPP) drug carrier (20FTPP-micelles) system, (2) the feasibility of sterically polymer-based liposomal complexes (SPLexes) for siRNA transfer was proposed, and (3) PCI enhanced the siRNA delivery was cooperation of twe carriers, 20FTPP-micelles and SPLexes. (1) Dual stealth nanodevice for PDT drug delivery base on hexagonal nanoprism and pegylation for immune shelter and cancer imaging. Target geometry for mitigating phagocytosis has garnered considerable attention recently in the drug delivery field. This study examined nanoparticles (NPs) with same volume but different shapes, namely, spherical NPs (SNPs) and hexagonal nanoprisms (HNPs), and analyzed their behaviors in vitro and in vivo. These NPs were constructed with a multifunctional block copolymer component, mPEG-b-P(HEMA-co-histidine-PLA). Geometry of SNPs and HNPs was controlled by adjusting copolymer properties and particle size was controlled by adjusting formulation parameters. Nanoparticle morphology had no effect in mitigating phagocytosis when NP size was 70 nm; however, morphology had a significant effect when NP size was 120 nm. The in vitro and in vivo studies reveal that dual stealth characteristics, pegylation and hexagonal prism structure, of nanocarriers can be adopted in clinical application for safe and efficient delivery of cancer therapy. (2) Establishment of liposomal system stabilized via polyelectrolyte copolymer for the siRNA delivery This part demonstrated the feasibility of sterically polymer-based liposomal complexes (SPLexes) for siRNA transfer. The stabilized SPLexes causing the corss-linkeding polyion polymers, cholesterol-P(HEMA-lysine) (PI) and methoxyl/functionalized PEG-P(HEMA-Histidine-co-MAAc) (PII), were formed, and a dual-shelled structure was obtained by TEM image. The anionic SPLexes consisting DOPE and PI could stabilize liposome mechanical strength and encapsulate with VEGF-siRNA. After associating with PII via the electronic interaction, this structure had the versatility to provide stability in vitro (4 % FBS) and mitigate the phagocytosis. Additionally, PII is disassociated from the SPLexes surface, as induced by pH changes, causing anionic SPLexes to fuse endo/lysosomes directly into the cytoplasm. Encapsulated with VEGF-siRNA, the folated SPLexes inhibited the VEGF protein expression to 74.5 ± 4.5 % and induced the apoptosis efficiently by western blot and flow cytometry analysis. In vivo study, the trace of isotope labeling nanoparticles showed that the accumulation of folate SPLexes in tumor site was higher obviously than nonfolated SPLexes did. Results of this study significantly contribute to efforts to develop lipoplexes based siRNA delivery systems. (3) siRNA Photochemical internalization delivery based the SPLexes system for cancer therapy Many potentially therapeutic macromolecules are taken into the cells by endocytosis, and have to be released from endocytic vesicles in order to express a therapeutic function. Photochemical internalization (PCI) is a novel technology based on photo-chemical reactions inducing rupture of endocytic vesicles. The aim of this study was to clarify which mechanisms and characters of photosensitizers applied the PCI effect for improving gene transfection. The results that the 20FTPP-micelles were low photocytotoxic and a potential candidate applying the PCI effect contrast to the high photochemical sensitive of hydrophobic/amphiphilic photosensitisers. According to the variation and production of reactive oxygen species in cytoplasm, the 20FTPP-micelles provided effect for leakage from the endocytic compartments and at the level of 80% cell viability. The results of CLSM images show that the siRNA incorporating into lipoplexes would fuse with the endo/lysosome existing the 20FTPP, and siRNA release from the chasmal endo/lysosome. Finally, the cooperation with PCI effect enhanced the siRNA-VEGF transfection and induced the VEGF expression from the western blotting analysis.