핵산 전달을 위한 세포 내부 자극에 감응하는 나노 전달체의 개발

Abstract

Successful gene therapy must cater the safety concern and in this aspect the non-viral polymeric vector are highly preferred over viral vectors owing to the associated toxicity and immunogenicity with viral vectors. Since its emergence polymeric vector-mediated gene therapy has come through a significant evolution, but yet need further refinements and advancements in each of the key steps such as condensation of nucleic acids, stability in physiological conditions, transportation to specific target cells, cellular uptake and release of therapeutic pay load. Polymeric vectors due to their facile production, high loading capacity, low safety concerns and high tunability to integrate multiple functional traits to address various impediments, have captured huge attention in recent research forays. In Part I, We presented that the rational design of polymeric vectors must render co-existence of two inter-conflicting attributes namely condensation and release of nucleic acids. Cationic polymers possess the abilities to form compact polymer complex (polyplex) with nucleic acids, trigger efficient cell internalization and endosomal escape: however such strong complexation impedes the release of nucleic acids and retards the dissociation of polyplexes at the cytoplasm entailing a major hurdle to initiate transcription and enhance gene expression. The main objective of Chapter I is to build a comparative account of two PLGA nanoparticle (NP)-based gene delivery systems namely, plasmid DNA (pDNA) encapsulated PLGA NPs (PLGA-E) and surface adsorbed pDNA on PLGA-BPEI NPs (PLGA-BPEI), with respect to the extent of internalization and intracellular release of pDNA. Several formulations have also been evaluated systematically for determination of the optimal transfection efficiency. The zeta-potential, particle size measurements and DNase I protection assay established the importance of the BPEI chain length in regulating the effective loading and condensation of pDNA with PLGA-BPEI NPs and pDNA protection ability of PLGA-BPEI NPs. The colloidal stability of these formulations was also investigated as a function of serum concentration. The in vitro release of pDNA from both kind of formulations was studied at pH 7.4 and pH 5.0 by comparing the kinetics of pH-triggered release of pDNA from PLGA-BPEI-25K(5) and PLGA-E. In vitro time-dependent gene transfection efficiencies were studied in presence as well as in absence of serum for NIH3T3 and HEK293 cells. The cell viability and intracellular localization were also investigated using MTT assay and confocal microscopy study, respectively.In Chapter II, the work demonstrated development of multifunctional gene carrier which has incorporated reducible moiety, tumor targeting ligands as well as PEG to achieve efficient release of pDNA, enhanced tumor-specificity and long circulation, respectively. In our successful one-pot synthesis of multifunctional polymer, low molecular weight branched polyethylenimine was thiolated with propylene sulfide, and mixed with α-Maleimide-ω-N-hydroxysuccinimide ester polyethylene glycol (MAL–PEG–NHS, MW: 5000), cyclic NGR peptide. The structural elucidation of the polymer was done by NMR and GPC study. Complex formation as well as reducible property of the polymer was confirmed by gel retardation assay. In order to achieve efficient tumor targeting, we have used cNGR peptide which is known to bind to CD13 overexpressed in neovasculature endothelial cells. Tumor target-specificity of polymer was established by carrying out competitive inhibition assay with free cNGR peptide. Cellular uptake of polymers was evaluated by confocal laser scanning microscope (CLSM). Finally, addition of free cNGR and buthionine sulfoximine (BSO) reduced transfection efficiency synergistically, which implied that multifunctional polymer-mediated gene transfection took place tumor-specifically and via GSH-dependent pathway.In Chapter III, the work demonstrated the successful delivery of gene to mouse brain overcoming the blood-brain barrier (BBB) through expedient vector construct having RVG peptide as targeting ligand for neuronal cells. The newly developed delivery vector was designed to impart bioreducibility for greater intracellular pDNA release, higher serum stability and efficient complexing ability by incorporating disulfide linkage, PEG and low molecular weight polyethylenimine, respectively. The physiochemical properties of the polyplex, its cytotoxicity and the in vitro transfection efficiency on Neuro2a cell were studied prior to the successful in vivo study. Observed luciferase expression substantiated the permeation of the pDNA loaded polymeric vector through the BBB. The RVG-mediated target specific cellular uptake of polymeric vector was established conclusively by competitive assay.In Part II, we present the rational design, construction and operation of artificial nucleic acids based molecular nanodevice which works and heals inside a living cell. The working principle of our nanodevice is i-motif based switches which transduce the chemical change of the pH value into conformational changes, whereby they can form reversible Au NPs clusters being exploited as colorimetric assay and photothermal therapy. The efficient pH dependent dynamic behavior and physicochemical properties were extensively evaluated and further validated in living cells. Actually, the realization of constructing the medicinal DNA nanomachine will be potentiated through the installing of qualified features as a drug carrier as well as a nanomachine. By combining the precise programmability of nucleic acids and the diverse optical properties and functionality of Au NPs, these hybrid nanostructures will provide a robust platform for the engineering of highly sophisticated multi-functional nanodevices.