TATp和CXCR4修饰的载VP3基因超声微泡制备及体外寻靶实验  

Preparation of VP3 gene-loaded ultrasound microbubbles decorated with TATp and SDF-1 and targeting study in vitro

作　　者：曾峥[1] 邱丽华[2] 高志[3] 李攀[4] 何力[1] 易品诗 

机构地区：[1]重庆医科大学附属口腔医院颌面外科/口腔疾病与生物医学重庆市重点实验室,重庆401147 [2]重庆医科大学附属口腔医院颌面外科/重庆市高校市级口腔生物医学工程重点实验室,重庆401147 [3]重庆医科大学附属第二医院口腔科,重庆400016 [4]重庆医科大学超声影像学研究所,重庆400010

出　　处：《重庆医学》2016年第30期4203-4206,共4页Chongqing medicine

基　　金：重庆市卫计委重点基金资助项目(2011-1-060)

摘　　要：目的制备一种载VP3基因、反式激活转录激活肽(TATp)与基质细胞衍生因子-1(SDF-1)同时修饰的新型载基因高分子靶向超声造影剂,表征其性质。方法采用W/O/W双乳化法制备载基因超声微泡。并通过硫醚键将SDF-1与TATp共价连接到聚乳酸/羟基乙酸共聚物(PLGA)微泡的表面,制备成靶向载基因超声微泡。Malvern测量仪测定其粒径、分布及表面电位,流式细胞仪及共聚焦显微镜检测TATp、SDF-1在高分子微泡表面的连接状况,酶切反应实验鉴定其对DNA的保护性,光镜观察及流式细胞仪初步评估其体外寻靶能力,超声考察体外成像。结果靶向载基因超声微泡呈规则球形。粒径为(536.00±16.55)nm,分布集中,表面电位为(-0.08±0.08)mV。平均载药量为0.62%,平均包封率为36.13%。对DNA保护作用持续60min,未见损坏。TATp、SDF-1同时加载于PLGA微泡表面的连接率为69.84%。体外寻靶实验显示,靶向微泡较多地稳定簇聚在舌癌SCC-15细胞膜上,连接率为91.44%。而非靶向微泡较少结合,连接率为12.96%。体外超声显像呈细小点状、均匀高回声。结论成功制备出TATp-SDF-1-VP3-PEG-PLGA微泡,其能在体外高效靶向结合舌鳞癌SCC-15细胞,且短时间穿过细胞膜。体外成像效果较好。Objective To prepare a innovative VP3gene-loaded ultrasound microbubble decorated with TATp and SDF-1,having the extracellular accumulation and intracellular permeation function,and characterize their property.Methods VP3 geneloaded ultrasound microbubbles were prepared with the method of W/O/W double emulsion.SDF-1and TATp were covalently congjugated to the surface of poly-lactic/acid-glycolic acid（PLGA）microbubble though thioether bonds to prepare gene-loaded targeted ultrasound mirobubbles.Their particle size,distribution and surface potential were determined by Malvern measuring instrument.The conjugation status of TATp and SDF-1were evaluated by flow cytometry and confocal microscopy.Their DNA protection were identified by digestion reaction test.The vitro targeting capacity was preliminarily assessed by light microscopy and flow cytometry,and the vitro ultrasound imaging was investigated under high frequency imaging condition.Results The gene-loaded targeted ultrasound mirobubbles showed regularly sphericity.The diameter was（536.00±16.55）nm,and showed a narrow distribution.The zeta potential was（-0.08±0.08）mV.The average gene loading was 0.62%,with the average rate of 36.13% gene encapsulation efficiency.The DNA protective effect sustained 60 min without damage.Connection rate of TATp and SDF-1coupled with PLGA microbubbles surface was 69.84%.The vitro targeting study showed that more targeted microbubbles stably clustered together in the tongue SCC-15 cell membranes with the connection rate of 91.44%,while non-targeted microbubbles combination rate was12.96%.Moreover,the vitro ultrasound imaging was tiny dot,even high echo.Conclusion TATp-SDF-1-VP3-PEG-PLGA microbubbles were prepared successfully,which can efficiently target to tongue SCC-15 cells,and pass through the cell membranes at a short time in company with outstanding ultrasound imagings in vitro.

关 键 词：聚乳酸/羟基乙酸共聚物 超声微泡 靶向舌鳞癌细胞 分子成像 

分 类 号：R739.86[医药卫生—肿瘤]