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The development of biodegradable gene delivery systems , which have the ability to effectively deliver therapeutic DNA to a target tissue , is paramount to the success of nonviral gene delivery . One approach to developing biodegradable polymers is to introduce disulfide bonds along the backbone of the polymers to ensure release of the DNA in the reductive environment of the cytoplasm , whilst simultaneously reducing the molecular weight of the polymers . There is a crucial need to develop biocompatible and biodegradable polymers , which have low cytotoxicities so as to maintain cell viability and hence increase transfection efficiencies . Therefore , to produce a biocompatible gene delivery system , we have designed and synthesized novel reducible copolymers of the type (AB )n , which consist of repeating units of the natural amino acid , L -lysine and cystamine bisacrylamide (CBA ) . These novel reducible linear L -lysine copolymers (LLCs ) were then modified with ethylenediamine so as to introduce primary amines for efficient DNA condensation . The molecular weight (MW ) of the copolymers was found to be ~3 .2 kDa with a polydispersity index of ~1 .2 . Gel retardation assays showed complete condensation of DNA at N /P ratios greater than 20 /1 and exceptional LLC /pDNA polyplex stability during incubation with DNase I . To investigate the mechanism of DNA release from the polymer /pDNA complexes , fluorescence spectroscopy studies were performed with 1 ,4 -dithio -DL -threitol (DTT ) . These data showed a significant reduction in fluorescence intensity following the addition of LLCs to DNA . After the addition of DTT , there was a 95 % increase in fluorescence intensity , which indicated the reduction of the disulfide bonds and the release of the DNA from the complexes . The particle sizes of LLC /pDNA polyplexes were found to be between 100 -231 nm with surface charges of 0 .8 -17 mV respectively . The transfection efficiencies of the polyplexes as determined with a luciferase assay showed that LLC polyplexes produced five times higher transfection efficiencies in HDF cells , three times higher transfection efficiencies in MCF -7 cells , and four times higher transfection efficiencies in MA cells as compared to the optimal PLL control . The LLC /pDNA polyplexes showed significantly lower cytotoxicities as compared to the PLL /pDNA control in HDF , MCF -7 , and MA cells at certain N /P ratios .
Finally , in an exvivo study , LLCs were used as a nonviral gene carrier system to generate genetically modified stem cells to produce sufficient amounts of the angiogenic cytokine , vascular endothelial growth factor (VEGF165 ) . These genetically modified stem cells were used to promote revascularization of an infarcted region of the heart , which can reduce myocardial damage and scar formation . A myocardial infarction model was generated in SCID mice deficient in T and B cells by permanent ligation of the left anterior descending coronary (LAD ) artery . Cardiac hemodynamics , H &E staining and immunohistostaining results from this ex vivo study presented improved cardiac contractility , potential differentiation of hMSCs , new blood vessel formation , and a reduction in infarct size after treatment with the LLC genetically modified stem cells compared to the control animals .
In conclusion , these results suggest that these novel LLCs are efficient , reducible and biocompatible polymers for nonviral gene delivery . Moreover , LLCs , as a nonviral gene carrier vector , hold great potential for the treatment of myocardial infarction in conjunction with stem cell therapy . Finally , adoption of novel nano -therapeutics strategies and techniques combining gene and cell therapies together could open the gate towards endless possibilities in the future of therapeutics and medicine . |
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