A New SiRNA Delivery System:the Preparation Of S-?-Carboline-3-acyl-RGDV And Gene Silencing Efficacy Evaluation Of SURVIVIN

SURVIVIN is a member of the inhibitor of apoptosis protein?IAP?gene family.It is also known as baculoviral IAP repeat-containing protein 5?BIRC5?and apoptosis inhibitor 4?API4?,is a bi-functional protein implicated in the regulation of cell division and the suppression of apoptosis.SURVIVIN expression is regulated in a cell cycle-dependent manner with maximum expression occurring during the G₂/M phase of the cell cycle.It is proposed to have roles in tumor formation and tumor cell resistance to anti-cancer agents,and may act as a marker and prognostic indicator for certain cancers.SURVIVIN-siRNA could silence the expression of SURVIVIN by RNAi.To improve the cancer treatment,A new compound named S-?-carboline-3-acyl-RGDV?CRV?has been synthesized,which has amphiphilic and anti-tumor activity.It may be together with lecithin and cholesterol to form the liposomes.After protamine modifing liposome and calf thymus DNA compressing siRNA,then SURVIVIN-siRNA would be loaded in liposomes,and give a new siRNA delivery system,which named LPD?liposome-polycation-DNA?.The following studies are included:1.S-?-carboline-3-acyl-RGDV was synthesized.The structure was determinated by NMR,MS,UV,IR and HPLC.2.Thin film-dispersion method was used for preparation of CRV liposomes.After protamine modifing liposome and calf thymus DNA compressing siRNA,then SURVIVIN-siRNA would be loaded in liposomes,and gave a new siRNA delivery system,which named LPD?liposome-polycation-DNA?.The confocal microscopy and differential scanning calorimeter?DSC?analysis evidenced that SURVIVIN-siRNA was effectively linked to liposomes.The release assays indicated that the release time of SURVIVIN-siRNA in the CRV liposomes was prolonged by 3folds.TEM and SEM observations evidenced that the appearance of the delivery system were spherical.Particle size is between 100-200 nm.Zeta potential is about0 mV.3.In vitro anti-tumor bioassays were conducted on HeLa cells by MTT method,HeLa cells were cultured with different samples and measured the OD values under490 nm.Found that SURVIVIN-siRNA/100%CRV liposomes had better transfection efficiency.4.Gene silencing efficiency at protein level was performed on HeLa cells.HeLa cells were treated with different samples and the secreted SURVIVIN was detected by ELISA.The results showed that compared to the commercially available transfection reagent,the gene silencing efficacy evaluation of SURVIVIN-siRNA/100%CRV liposomes was considerable.5.Gene silencing efficiency at mRNA level was conducted on HeLa cells.HeLa cells were treated with different samples,then the total RNA was abstracted and reverse transcripited to cDNA.At last,the target SURVIVIN cDNA was amplified by RT-PCR.The results showed that compared to the commercially available transfectionreagent,thegenesilencingefficacyevaluationof SURVIVIN-siRNA/100%CRV liposomes was considerable.6.In vivo anti-tumor evaluation was conducted on S₁₈₀80 Sarcoma mice.Through the way of tail vein injection to evaluate the in vivo anti-tumor activity of SURVIVIN-siRNA/100%CRV liposomes.The results showed that,it had definite anti-tumor activity.7.Acute toxicity was performed on normal ICR mice.Through the way of intraperitoneal injection to evaluate the acute toxicity of CRV.The results showed that,CRV had liver and spleen toxicity.8.Apoptosis experiments were conducted on HeLa cells.HeLa cells were treated with different samples.Using Annexin V-FITC/PI double staining quantified apoptosis changes.The results showed,100%CRV liposomes can effectively carry SURVIVIN-siRNA into the cells and induce apoptosis,while the group of the Naked SURVIVIN-siRNA can not.9.Cell cycle experiments was performed on HeLa cells.HeLa cells were treated with SURVIVIN-siRNA/100%CRV liposomes.Use flow cytometry analysised the various phases of cell cycle and the percentage of DNA.The results showed,100%CRV liposomes can effectively carry SURVIVIN-siRNA into cells,inhibit HeLa cells entering into S phase and accompanied by G₂/M phase arresting.Conclusion:this study successfully prepared a new siRNA delivery system,which had anti-tumor activity,can successfully carry SURVINVIN-siRNA transfected cells.Compared with a commercially available transfection reagent,gene silencing efficiency is considerable.