Release Thiolated Chitosan The Quaternary Ammonium Nanoparticles And Collagen Scaffold-based Gene Delivery Research

1 thiolated trimethyl chitosan as pDNA carriers with high transfection effiency in vitro/vivo and investigation of its transfection mechanismTo combine the advantage of trimethyl chitosan and thiolated chitosan, we prepared trimethyl chitosan-cysteine conjugate(TMC-Cys) as a non-viral vector for gene delivery. TMC-Cys with different molecular weight (30,100 and 200kDa) and quaternization degrees(15% and 30%) was used to form self-assemble nanoparticles with plasmid encoding enhanced green fluorescene protein(pEGFP). Cytoxicity result showed that all derivatives were non-toxic at concentration≤2 mg/ml. Ethidium bromide exclusion assay and agarose gel electrophoresis demonstrated that our TMC-Cys could effectively condense pDNA, and the resultant TMC-Cys/pEGFP nanocomplex with optimal N/P ratio showed particle size of 120nm-200nm and zeta potential of+15mV-+20mV. TMC-Cys/pEGFP NC exhibited physical stability and protected pEGFP against nuclease digestion. Cell binding and musin adsorption of TMC-Cys/pEGFP NC were enhanced average 2.7 and 1.5 folds, respectively, compared to TMC/pEGFP NC, and cellular uptake of TMC-Cys/pEGFP NC was enhanced average 2.6 and 3 folds compared to TMC/pEGFP NC and Lipofectamine 2000. Cellular uptake of TMC-Cys/pEGFP NC at 4℃was reduced by about 75% compared to that measured at 37℃, and pretreatment with sodium azide induced a inhibition in the complex internalization by approximately 30%, implying the cell uptake of TMC-Cys/pEGFP NC might be thermal and energy dependent. Incubation with chlorpromazine yielded a suppression in the complex internalization by nearly 70%, suggesting that clathrin-mediated endocytosis might be involved. The presence of GSH with relatively high concentration maintains a reducing enviroment intracelluarly and enhanced the release of pEGFP from TMC-Cys/pEGFP NC with about 3.5 folds compared to extracelluarly, leading a 3.7-fold increase in nuclear accumulation of pEGFP compared to TMC/pEGFP NC as evidenced by Qulitative and quantitative analysis of CLSM. Consequently, TMC-Cys/pEGFP NC were able to transfect HEK293 cells with greater efficiency than TMC/pEGFP NC 1.4-3.2 folds. Optimal TMC-Cys(100,30)/pEGFP NC showed 1.5-fold enhancement than Lipofectamine 2000. Also, intramuscular adminstration of TMC-Cys/pEGFP NC to mice yielded a 2.3-fold and 4.1 fold higher expression of green fluorescene protein than TMC/pEGFP NC and Lipofectamine 2000, respectively. 2 3D collagen scaffolds embedding TMC-Cys/pSUPER-smad2 NC used for primary treatment of hypertrophic scarHypertrophic scar is a result of abnormal wound healing that often occurs after dermal injury. Hypertrophic scar is characterized by excessive deposition of extracellular matrix especially typeⅠand typeⅢcollagen in the dermis and subcutaneous tissues. Transforming growth factorβ1 (TGFβ1) plays a essential role in the pathogenesis of fibrosis by inducing and sustaining activation of hypertrophic scar fibroblast. Smad proteins are important intracellular mediators of TGFβ1 signaling. The smad pathway plays a fundamental role in regulation of collagen synthesis, and Smads are necessary to mediate TGFβ-dependent stimulation. Therefore, the pSUPER-EGFP plasmid was applied to inhibition TGFβ1 signaling pathway of a collagen scaffold. A cationic gene delivery vector metioned above, TMC-Cys, was used to form complexes with the pSUPER-EGFP plasmid encoding smad2 siRNA. The complexes then incorporated into the collagen scaffold acted as a three-dimensional carrier. The entrapment efficiencies were above 60% and mediated by the feeding dose. TMC-Cys/pSUPER-smad2 NC released in a sustained manner with a total amount of～70% up to 3days. Alamar blue assay showed that Dermal fibroblasts seeded into the collagen scaffold embedded with TMC-Cys/pSUPER-smad2 NC achieved high proliferation. Cell number doubled within 7 days. RT-PCR results demonstrated a significant depression from the TMC-Cys/pSUPER-smad2 NC containing scaffolds in the mRNA levels of smad2, typeⅠand typeⅢcollagen by approximately 80-87%. ELISA assay also proved the down-regulation of typeⅠand typeⅢcollagen in the translation level.