Development of low cytotoxic and high efficient disulfide-based polyethylenimine non-viral vectors for in-vitro gene transfection.

• Other Authors: Deng, Rui
• Format: Others
• Language: English; Chinese
• Published: 2010
• Subjects: Gene therapy; Genetic transformation; Gene Transfer Techniques; Genetic Therapy; Polyethyleneimine
• Online Access: http://library.cuhk.edu.hk/record=b6074905; http://repository.lib.cuhk.edu.hk/en/item/cuhk-344538

Due to recent advances in molecular biology and genomic research, numerous diseases have been given their genetic identities for which gene therapy may be a possible prescription. Gradually, the development of viral and non-viral vectors to translocate genes has become a bottleneck. For non-viral vectors, polyethylenimine (PEI) is considered as a potential vector candidate for gene delivery because of its ability to compact DNA and its intrinsic pH buffering capacity. PEI and its derivates have been widely tested in both in-vitro and in-vivo gene transfection experiments. The progress is limited due to the lack of a better understanding of the intracellular mechanism. So far, their cytotoxicity is relatively high and gene transfection efficiency is low. This study was designed to modify PEI and optimize its cytotoxicity and gene transfection efficiency. === During the complexes formation, both LLS and zeta-potential were used to follow the process. The results showed that most of anionic DNA are complexed by cationic PEI-based polymers when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) reaches ∼3, but the gene transfection reaches the highest efficiency when N:P ∼10. When N:P > 3, there exist two population of PEI chains in the solution mixture: bound to DNA and free in the solution. The bound PEI chains condense and protect DNA. Our current study confirms that it is those free PEI chains that play a vital role in promoting the gene transfection. Our preliminary data shows that the promotion mainly occurs in the intracellular space. The detailed mechanism is still lacking at this moment. Nevertheless, our finding leads to a totally different way in the development of non-viral vectors. === Further, we grafted PEI with polyethylene glycol (PEG), respectively via a reductive disulfide -S-S- and a non-degradable -C-C- bond to form two copolymer vectors. A comparative study shows that the polyplexes formed between the two copolymers and DNA are more stable than that formed between unmodified PEI and DNA under the physiological condition, presumably because the grated PEG chains form a protective hydrophilic shell on the PEI/DNA polyplexes. However, PEGylation reduces the internalization of the copolymer/DNA polyplexes in in-vitro experiments. For the two copolymer vectors, PEG-SS-PEI is 2-8 times more effective than its counterpart (PEG-CC-PEI) in the gene transfection, presumably due to the cleavage of the grafted PEG chains inside the reductive cytosol, which promotes the release and translocation of DNA. Our results demonstrate that using the disulfide as a linker is a promising approach to overcome the PEGylation dilemma in the development of low cytotoxic and high efficient non-viral polymeric vectors. === It has been known that short PEI chains are less toxic, but long chains are more effective in gene transfection. Therefore, we decide to use the disulfide bond (-S-S-) to extend short PEI chains to increase efficiency and also utilize the reductive cytosol environment to cleave such extended PEI chains to reduce their cytotoxicity inside the cell. Laser light scattering (LLS) was used to in-situ monitor the linking reaction between short PEI chains (M w = 2000 g/mol) and dithiobis(succinimidyl propionate) (DSP). The molar mass and crosslinking degree of the extended PEI chains was controlled by either the amounts or the adding rate of DSP. A comparative study of two linked PEI samples (PEI-7K-L and PEI-400K-L, respectively with M w = 6.5 x 103 and 3.8 x 10 5 g/mol) reveals that cytotoxicity and gene transfection efficiency of such extended PEI chains are related to the chain length and structure. Namely, PEI-7K-L with an extended chain structure is less cytotoxic and 2--10 times more effective in the gene transfection than the "golden standard" (PEI25K) and the widely used commercial vector, Lipofectamine 2000RTM. Comparatively, PEI-400K-L with a spherical microgel structure is ineffective in spite of its non-toxicity. Our study clearly demonstrates that a proper control of the chain length and structure is important. === by Deng, Rui. === Adviser: Chi Wu. === Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . === Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. === Includes bibliographical references. === Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. === Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. === Abstract also in Chinese.