Construction Of RGD - PEG - SS - PEI / DNA Gene Delivery System Based On Redox Response

The ideal non-viral vector gene delivery system is efficient in reducing phagocytosis of reticuloendothelial system and degradation of DNase in plasma, penetrating cell membrane, increasing the ability of endosomal escape and taking DNA from cytoplasm into the nucleus successfully. Polyethylenimine (PEI) is one of the most successful and extensively used synthetic carriers for gene delivery. Its main advantage is high transfection efficiency due to its endosomal buffering capacity. However, with increasing of PEI molecular weight, the potential cytotoxicity increases significantly against various cell lines, especially PEI of average molecular weight25,000.Attachment of hydrophilic polymers such as polyethylene glycol (PEG) has been found to be very useful for improving pharmacokinetics of recombinant protein, liposomes and some nanocarriers. PEGylation of PEI/DNA complex reduced the surface charge and interaction with blood components, therefore extended circulation in blood and reduced cytotoxicity. However, it could also interfere with the mechanism that PEI acts with endosomes as proton sponges and trigger endosomal release of the complexes into the cytoplasm, which leads to lower transfection efficiency. Dissociating PEG from PEI inside endosomes might solve this problem.Some scholars have designed releasable PEG modifications of nucleic acid complexes to overcome this dilemma. PEG-shielded pH sensitive PEI was developed where the low pH of endosome can trigger the removal of PEG. These complexes with reversible shielding showed higher transfection efficiency than that of stably shielded particles. Disulfide linkage is one of most promising biodegradable bonds applied in designing gene delivery vector. The intracellular GSH concentration (10mM) is known to be substantially higher than the level in the cellular exterior (2μM). Disulfide linkage can keep stable in extracellular environment, while once in the cytoplasm or endosomes, can be degraded in the presence of intracellular reductive glutathione.αvβ3integrin receptors are highly expressed in some activated endothelial cells and tumor cells, such as U87MG glioblastoma cells. Cyclic RGD sequence has been identified to facilitate interaction between drug delivery systems including micelles and some integrins as an essential binding motif. Based on above, the objective of this study is to establish mPEG-SS-PEI and RGD-PEG-SS-PEI containing disulfide linkage. It is supposed to break the disulfide linkage intracellular to enhance the gene transfection efficiency and treat glioblastoma multiforme via RGD which could target U87glioblastoma cells and endothelial cells.The present work includes four chapters.In chapter one, mPEG-PEI, mPEG-SS-PEI, RGD-PEG-PEI and RGD-PEG-SS-PEI were prepared and characterized by1H-NMR. Data showed that, each PEI molecules are conjugated with4.89,5.04,5.12and5.14PEG molecules respectively. mPEG-SS-PEI and RGD-PEG-SS-PEI were reduced by glutathione (GSH) and then disulfide linkage were certified by thin layer chromatography (TLC).In chapter two, PEI/DNA, mPEG-PEI/DNA, mPEG-SS-PEI/DNA, RGD-PEG-PEI/DNA and RGD-PEG-SS-PEI/DNA complexes were prepared and characterized. The results show that PEI/DNA, mPEG-PEI/DNA, mPEG-SS-PEI/DNA, RGD-PEG-PEI/DNA and RGD-PEG-SS-PEI/DNA complexes were stable in PBS or DMEM containing10%FBS at N/P12. The size of complexes decreased and zeta potential increased from N/P4to16. The size and zeta potential of complexes composed of PEG modified vectors are decreased compared with that of PEI at the same N/P. Transmission electron micrograph showed that the shape of mPEG-SS-PEI/pEGFP and RGD-PEG-SS-PEI/pEGFP complexes are regular and round and size is about200nm.In chapter three, transfection efficiency of PEI/DNA, mPEG-PEI/DNA, mPEG-SS-PEI/DNA, RGD-PEG-PEI/DNA and RGD-PEG-SS-PEI/DNA complexes were investigated. The result of GFP expression indicated that mPEG-SS-PEI was better than mPEG-PEI and RGD-PEG-SS-PEI was better than RGD-PEG-PEI. The result of PGL3expression is consistent with that of GFP. In the cytotoxicity experiment, mPEG-SS-PEI and RGD-PEG-SS-PEI showed the same cytotoxicity with that of mPEG-PEI and RGD-PEG-PEI, which indicated that disulfide linkage would not increase the cytotoxicity. Cellular uptake experiment verified that disulfide linkage could enhance the transfection efficiency via breaking disulfide bonds and dissociating PEG from PEI.In chapter four, model mice of bearing glioblastoma multiforme were established and brain sections stained with H&E demonstrated that models were successfully established. In vivo transfection study, RGD-PEG-SS-PEI/pDsRED enhanced the transfection efficiency compared with RGD-PEG-PEI/pDsRED. Anti-glioblastoma study showed that RGD-PEG-SS-PEI/pTRAIL complexes could elongate the lifetime of U87grafted mice significantly compared with RGD-PEG-PEI/pTRAIL.Briefly, redox sensitive RGD-PEG-SS-PEI/DNA gene delivery system was established. Both the in vitro and in vivo data showed that it could enhance the gene transfection effiency. This work plays a great role in biological response gene delivery system.