Construction And Evaluation Of Cationic Cholesterol Derivatives As Gene Vector

In the past decades,gene therapy has shown a good prospect in genetic diseases,cancer treatment and got a widespread concern.Finding an efficient,low-toxicity gene transfection vector which deliver the gene-specific drug to cells and maintain its function stably is to be faced the bottleneck problem for Gene therapy.Currently,gene vectors are generally divided into non-viral vectors and viral vectors,and cationic liposome is most potential in non-viral vector.We investigated the infuence of spacer moiety and Chain length on the transfection efficiencies.In this article,we reported on the synthesis of four cationiccholesterolderivatives3?-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol(DC-Chol)andit'shomologues3?-[N-(N',N'-dimethylaminopropane)-carbamoyl]cholesterol (DMAPA-Chol),N-(cholesterylhemisuccinoylamino-2-ethyl)-N,N-dimethy lamine(DC-CHEMS)and it's homologues N-(cholesterylhemisuccinoyl-amino-3-propyl)-N,N-dimethylamine(DMAPA-CHEMS) in which a ester spacer or succinic anhydride separates the cationic trimethylamino head group from the cholesteryl ring system.Products were analyzed by TLC,mass spectrometry and ~1H-HMR spectrum and identified as the intent products.liposomes were prepared by depositing cationic cholesterol derivatives and Soy lecithin S100 as a thin film with the method of film dispersion method,and their physicochemical properties and cytotoxicity?intracellular uptake and transfection were evaluated.The sizes of DC-Chol?DMAPA-Chol?DC-CHEMS?DMAPA-CHEMS liposomes'were115.8±9.3nm?118.0±15.3nm?120.0±14.2nm?124.1±12.8nm,and zeta potential were 16.94±1.56mv?17.40±0.76mv?23.63±2.52mv?26.76±3.60mv.The shape of four liposomes were all round and regular.Four liposome/pDNA complexes were completely blocked in the sample holes and owned strong ability to protect pDNA when the+/-charge ratio was more than 4:1.Hemolysis tests showed that four liposomes were almost no cytotoxicity because hemolysis rate was less 2%.MTT assay showed that cell viability of four liposomes were more than 80%and expressed low cytotoxicity when the phospholipid concentration was less than 0.05mg/ml.The intracellular fluorescence intensity of the liposome/F-ODN complexes increased with liposomes in a certain range of charge ratio,which were greater than commercially available reagent Lipofectamine?2000 in the lowest charge ratio.The transfection efficiency of four liposomes was all low,and lower than the commercially available reagents Lipofectamine?2000 significantly.Common cationic cholesterol liposomes which were incubated with R8 could improve capacity of binding DNA;Although cytotoxicity was relative increased,the range was not large,toxicity of DC-CHEMS group almost unchanged,the survival rate was 100%,when the Phospholipid concentration was less than 0.01mg/ml,Cell viability of DC-Chol liposome was more than 80%;Liposomes/R8 complexes could promote endocytosis,the promoting effect of DMAPA-Chol liposome was most obvious,mean fluorescence intensity increased 2.14 times,and DC-Chol,DC-CHEMS,DMAPA-CHEMS liposomes group were 1.50 times,1.21times,1.43 times;However,the ability of transfection did not improved than before.Only a small amount of pDNA expressed and transfection efficiency were lower than Lipofectamine?2000.While R8 could promote endocytosis,but the fate of the target gene in the cell did not change,the transfection efficiency was still low.cationic liposomes containing cationic cholesterol derivatives and DOPE were prepared and their physicochemical properties and cellular level were evaluated.The particle sizes of DC-Chol,DMAPA-Chol,DC-CHEMS,DMAPA-CHEMSliposomes were173.3±6.4nm?179.9±8.1nm?147.6±5.9nm?149.1±5.1nm,and zeta potential were24.46±4.50mv?25.33±5.19mv?27.38±4.22mv?29.21±3.69mv.When the+/-charge ratio were in the rang of 1:8 to 8:1,four complexes had a highest transfection efficiency which were better than Lipofectamine?2000significantly;We also found that transfection efficiency was better without R8 in the same+/-charge ratio.The results of intracellular distribution could analysis this phenomenon preliminary,liposome(DOPE)mediated F-ODN mainly located in the nucleus,and liposome(DOPE)/R8 comlexes mediated F-ODN mainly located around the nucleus,while the liposome(S100)mediated F-ODN was the lowest in the nucleus.Western blot showed that DC-CHEMS and DMAPA-CHEMS group had a better transfection efficiency than DC-Chol and DMAPA-Chol groups because the color of DC-CHEMS and DMAPA-CHEMS groups,protein bands were shallower than DC-Chol and DMAPA-Chol groups,but cationic liposomes which had same spacer and different chain length showed transfection efficiency irregularly,the transfection efficiency of DC-Chol was better than DMAPA-Chol,and DMAPA-CHEMS was better than the DC-CHEMS.A further illustrated that transfection efficiency was largely depend on the types of spacer moiety,and the spacer of succinyl had a better potential as a gene carrier material than ester spacer.