Polyethyleneimine grafted Chitosan blending Chondroitin sulfate as a non-viral gene vector

• Main Authors: Yu, Siang, 虞翔
• Other Authors: 朱一民
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/16769237289868289495

碩士 === 國立清華大學 === 化學工程學系 === 102 === The purpose of this study is to investigate the gene delivery efficacy of a non-viral gene vector by blending an anionic polymer chondroitin sulfate (CS) with a cationic polymer which is composed of polyethyleneimine (PEI, average molecular weight = 600) and chitosan (CHI). These two polymers are linked together via a PEG linker (Poly(ethylene glycol) diglycidyl ether, EX-810) by chemical bonds written as polyethyleneimine grafted chitosan (PEI600-g-CHI). The use of cationic polymers as gene vector has been widely researched and reported in recent years. However, positively charged polymers could have strong electrostatic force to bind negatively charged DNA. This property of cationic polymers makes DNA unable to be released from polymer/DNA polyplexes, and the high charge density of cationic polymer could damage cell membrane. These all lead to lower transfection efficiency. In this study, different amounts of CS (compared to DNA) were blended with PEI600-g-CHI and plasmid DNA (pDNA) to form ternary PEI600-g-CHI/pDNA/CS polyplexes. To know whether the adding of negatively charged polymer CS would reduce the binding strength between PEI600-g-CHI and pDNA or not, gel electrophoresis was done and no plasmid DNA was released due to the addition of CS. Results showed that the adding of anionic polymer does not affect the binding ability of PEI600-g-CHI/pDNA polyplexes.   Particle size and zeta potential measurements of the polyplexes were also performed. With adding low weight ratios of CS to pDNA, the PEI600-g-CHI/pDNA/CS nanoparticle compacted pDNA tightly leading the size smaller than PEI600-g-CHI/pDNA nanoparticle. The particle size of the polyplexes is suitable for cellular uptake. Zeta potential measurements showed that the surface charge of polyplexes was stabilized while low weight ratio of CS added. The morphology of polyplexes was observed by transmission electron microscope (TEM). At low weight ratios of CS to pDNA, nanoparticles have diameters about 140 nm and their shapes are spherical. Cell viability is also a major concern about a good gene vector. Cytotoxicity assay toward human embryonic kidney cell line (HEK 293T cell line) was assayed by MTT method. These tests showed that there was no significant cytotoxicity for binary PEI600-g-CHI/pDNA and ternary PEI600-g-CHI/pDNA/CS polyplexes. Cell viability at any concentration is above ninety percent. In the experiment of transfection efficiency, a green fluorescent protein-encoding gene (pEGFP-N1) was used to be transfected into HEK 293T cells. If gene can be successfully delivered into cell nucleus, it would produce green fluorescent protein (GFP) through steps of transcription and translation. The transfection efficiency is determined by the amount of green fluorescent protein produced. There were two experimental groups with different time of transfection step; the 6th and 24th hour groups. At the 72th hour after transfection step, samples of GFP expression were observed under fluorescence microscope. Then the intensity of expressed GFP was measured by the fluorescence plate reader. Results showed that ternary PEI600-g-CHI/pDNA/CS polyplexes at 0.2 weight ratio of CS to pDNA expresses the largest fluorescence intensity of GFP. The fluorescence intensity is nearly twice as large as the PEI600-g-CHI polyplexes.