The Development And Characterization Of Reduction-sensitive Response Gene Carrier

Traditional therapy are always subjected to various drawbacks such as limited therapeutic efficacy and lots of pain and side effects to the patients.As an emerging treatment,gene therapy,characterized by curing diseases at the fundamental genetic level,has gradually attracted people's attention and been proved to an effective therapy.Herein,in this paper,a reduction-sensitive nano-gene delivery system based on disulfide bond was developed to achieve rapid DNA release within cells as well as high transfection efficiency.The main research contents and results of this thesis are as follows:1.Synthesis and characterization of reduction-sensitive response gene carrier.The reduction-sensitive gene carrier PLGA-ss-P[Asp(DET)](abbreviated as P-ss-P for convenience)was obtained by linking the hydrophilic segment(the polyaspartic acid modified by diethylenetriamine)and hydrophobic PLGA with cystamine dihydrochloride containing a disulfide bond.Then,cystamine dihydrochloride was replaced by hexamethylenediamine to synthesize PLGA-P[Asp(DET)](hereinafter referred to as P-P)without disulfide bond as a control group,and P[Asp(DET)]without a hydrophobic segment was used as a positive control group.The results of FTIR and ~1H NMR demonstrated that P-ss-P was successfully synthesized.The TEM result showed that the nanoparticles were uniformly distributed in the shape of a sphere with a particle size about 20-30 nm,and have good stability for a long time period of 144 h.Additionally,the buffer capacity assay showed that P-ss-P has good proton buffer capacity.2.Study on the ability of P-ss-P gene carrier for delivering a exogenous gene.We used the carrier P-ss-P and the reporter gene pDsRed2-N1 to prepare the carrier/pDNA complex and then we explored its properties.The particle size of the P-ss-P/pDNA complex was between100-350 nm,and finally stabilized at about 120 nm.The maximum zeta potential was 30.93mV.Gel retardation experiments proved that P-ss-P can combine with pDNA well.At the same time,the carrier P-ss-P can protect the pDNA from degradation by substances such as nuclease effectively.MTT results indicated that P[Asp(DET)],P-ss-P and P-P all exhibited low cytotoxicity,and P-ss-P displayed better biocompatibility compared with P[Asp(DET)].The results of transfection experiments in vitro showed that both P[Asp(DET)]and P-ss-P could successfully deliver gene into cells and then express it.The transfection efficiencies of P-ss-P/pDsRed2-N1 complex under different N/P(4,8,16)were 9.0%,22.3%,and 26.3%.CLSM and FCM results indicated that P[Asp(DET)]/Cy5-pDNA,P-P/Cy5-pDNA and P-ss-P/Cy5-pDNA complexes could be taken up by HeLa cells.The cellular uptake of P-P and P-ss-P with hydrophobic structure was much higher than that of free DNA,and was significantly higher than that of P[Asp(DET)].3.Study on the reduction-sensitive response of gene carrier.We evaluated the reductive sensitive response behavior of P-ss-P by measuring the change in particle size,the ability of combining with DNA and the transfection efficiency.The results of DLS showed that the mean size of P-ss-P increased with time going on under reductive conditions,which proved that the disulfide bond was broken.However,the size of P-P didn't change significantly under the same condition.The results of gel retardation experiments indicated that the binding ability of P-ss-P to DNA under reductive conditions was weakened with the time increasing,while P-P/pDNA complex was not affected.In terms of the transfection efficiency,P-ss-P/pDsRed2-N1(26.3%)exhibited a better transfection effect than P-P(4.5%).These results proved that P-ss-P has a good reductive sensitivity.4.Study on the effect of P-ss-P in cancer gene therapy in vitro.The anti-cancer effect of P-ss-P with therapeutic gene p53 was evaluated by MTT cytotoxicity assay.Compared with P-P,P-ss-P could inhibit the growth and proliferation of cancer cells more effectively because it can deliver the tumor suppressor gene p53 into cancer cells better.