Study I: Studies of the potential use of chitosan (CS) as a non-viral carrier for gene delivery have shown that transfection is relatively low. To address this concern, this study developed a ternary system comprising the core of the CS/DNA complex and the outer coating of an anionic polymer, poly(γ-glutamic acid) (γ-PGA). Molecular dynamic (MD) simulations showed that γ-PGA was compacted by its entanglement with excess CS emanating from the surfaces of test complexes. The γ-PGA coating apparently internalized the test complexes and enhanced their transfection efficiency.] Trypsin treatment induced a concentration-dependent decrease in internalization of the γ-PGA-coated complexes, suggesting which suggested the occurrence of a specific protein-mediated endocytosis. Analysis of endocytosis inhibition showed that uptake of test complexes resulted from the effects of γ-glutamyl transpeptidase (GGT) on cell membranes. The amine group in the N-terminal γ-glutamyl unit of γ-PGA revealed an important role in the interaction with GGT. Since the free N-terminal γ-glutamyl unit of γ-PGA in the test complexes became exposed when entangled with CS, it may be possible to accommodate γ-PGA within the γ-glutamyl binding pocket of the membrane GGT. The above experimental results suggest that the γ-PGA coating on CS/DNA complexes substantially enhances their cellular uptake via a specific GGT-mediated pathway. Improved knowledge of the uptake mechanism is needed to develop an efficient vector for gene transfection.

Study II: Many human diseases carry at least two independent gene clinical disorders. Synthesized disulfide bond-conjugated dual PEGylated siRNAs were capable of specifically targeting and silencing two genes simultaneously. For efficient delivery, a ternary complex was formed from the conjugated siRNAs, the cationic CS, and an anionic polymer, γ-PGA. Experimental results indicate that the incorporated γ-PGA significantly increased the efficiency of intracellular delivery and reduced the disulfide bond-conjugated PEGylated siRNAs delivered to the PEGylated siRNAs in the reductive cytoplasmic environment. Compared to unmodified siRNAs, the PEGylated siRNAs showed significantly higher enzymatic tolerability, more effective silencing in multiple genes, and longer duration of gene silencing capability. Silencing different genes simultaneously can substantially improve the effectiveness of treatment for multiple gene disorders, and prolonged gene silencing can reduce the frequency of administrations
.
Study III: The PDT has been studied intensively as a therapeutic treatment for cancer and other diseases; however, it is often accompanied by prolonged phototoxic reactions in the skin owing to the slow clearance of externally administered synthetic photosensitizers (PSs). This study investigated the genetic use of pKillerRed-dmem, delivered by complexes of CS and γ-PGA, for intracellular expression of a membrane-targeted KillerRed protein that has potential use as a PS for PDT. After transfection with CS/pKillerRed/γ-PGA complexes, a red fluorescence protein of KillerRed was clearly visible at the cellular membranes. Upon exposure to green-light irradiation, the KillerRed-positive cells produced excessive reactive oxygen species (ROS) in a time-dependent manner. Viability assays indicated that ROS have important mediating roles in KillerRed-induced cytotoxicity, apoptosis, and anti-proliferation, which suggests that KillerRed has potential use as an intrinsically generated PS for PDT treatments. Notably, the phototoxic reaction induced by KillerRed in the cells became negligible over time, presumably because of its intracellular degradability. The above experimental results demonstrate that genetically-encoded KillerRed is biodegradable and has potential use for PDT-induced destruction of diseased cells.

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