携带质粒的PLGA纳米粒-超声微泡复合体的构建及细胞相容性研究

Construction of pIRES-tPA-DsRed Express2-encapsulated PLGA Nanoparticles-ultrasound Microbubble Complexes and Study on Their Cell Compatibility

目的构建携带tPA基因质粒的聚乳酸-羟基乙酸共聚物(PLGA)纳米粒-超声微泡复合体,检测其理化性质、体外缓释方式、细胞相容性。方法应用双次乳化法制备携有tPA基因质粒的PLGA纳米粒;应用薄膜水化超声法制备阳离子超声微泡;两者通过静电吸附作用,形成质粒-纳米粒-超声微泡复合体(plasmid encapsulated nanoparticle-mi-crobubble complex,PENMC)。检测PLGA纳米粒的质粒载量,该复合体PENMC的质粒载量;检测PLGA纳米粒及复合体体外缓释质粒的情况;CCK-8法检测纳米粒及复合体PENMC的细胞毒性。结果检测携带质粒的纳米粒粒径为(217.2±2.2)nm,zeta电位为(-15.24±0.83)mV。微泡平均大小为(3.2±1.5)μm,zeta电位为(13.66±2.05)mV。微泡浓度为(4.3±1.1)×108/mL。PENMC平均直径为(4.6±1.7)μm,zeta电位为(2.23±1.45)mV。浓度为(3.0±1.3)×108/mL。1mg PLGA纳米粒载有质粒(42.3±2.1)μg。1mL PENMC中带有质粒(20.5±2.7)μg。PLGA纳米粒及复合体PENMC前7d累计释放量均为(57±3)%。起始段短时间内快速释放,后呈现稳定、缓慢释放。体外细胞毒实验证实PLGA纳米粒及复合体PENMC均未见明显细胞毒性效应。结论所构建的纳米粒-超声微泡复合体显示出较好的缓释效果,较低的细胞毒性,为后续将其应用于基因治疗奠定了实验基础。

Objective To construct pIRES-tPA-DsRed Express2-encapsulated PLGA nanoparticles-ultrasound microbubble complexes and investigate their physical and chemical properties,the in vitro plasmid release manner,and cell compatibility.Methods pIRES-tPA-DsRed Express2-encapsulated PLGA nanoparticles were prepared by double emulsion method.Cationic ultrasound microbubbles were prepared by thin-film hydration method.Plasmid-encapsuled nanoparticle-microbubble complexes（PENMC）were created by electrostatic adsorption.The plasmid loads in PLGA nanoparticles and PENMC were assayed,respectively.The in vitro plasmid release manner and the cytotoxicity of PLGA nanoparticles and PENMC were examined.Results The size and zeta potential of the plasmid-encapsulated PLGA nanoparticles were（217.2±2.2）nm and（-15.24±0.83）mV,respectively.The average size and zeta potential of the cationic microbubbles were（3.2±1.5）μm and（13.66±2.05）mV,while the concentration was（4.3±1.1）×108/mL.The average size of PENMC was（4.6±1.7）μm,the zeta potential（2.23±1.45）mV and the concentration（3.0±1.3）×108/mL.The plasmid loads were（20.5±2.7）μg/mL and（42.3±2.1）μg/mg in PENMC and PLGA nanoparticles,respectively.The cumulative plasmid release rate was（57±3）%in the first seven days in both PLGA nanoparticles and PENMC groups.The plasmids were initially released rapidly and a short time later,slow and stable release pattern was presented.There was little cytotoxicity in PLGA nanoparticles and PENMC.Conclusion The PENMC with a good release pattern and low cytotoxicity may be used for gene therapy in future.