阳离子固体脂质纳米粒/pDNA二元复合物的初步研究

Preliminary Studies on Cationic Solid Lipid Nanoparticles/pDNA Binary Complex

目的制备阳离子固体脂质纳米粒/pDNA二元复合物(SLNs-pDNA),并研究该复合物的药剂学和生物学特征。方法复乳法制备空白阳离子固体脂质纳米粒,与报告基因复合,加入不同浓度Ca^(2+)调节体系的荷正电量,分别对纳米粒的相关理化性质,细胞毒性,体外释药特性及其保护pDNA抵抗核酸酶降解的能力进行考察,通过体外基因转染实验对复合物在COS-7细胞中的转染效率进行了初步评价。结果本实验中制备的纳米粒呈球形或类球形,平均粒径(51.9±0.58)nm,Zeta电位(47.4±1.1)mV,加入Ca^(2+)显著提高了纳米粒复合pDNA的能力,制得的复合物平均粒径为(91.6±5.3)nm,Zeta电位为(31.5±1.4)mV。细胞毒性实验表明,纳米粒对COS-7细胞的毒性较小,凝胶阻滞分析表明,阳离子固体脂质纳米粒能够充分结合pDNA,形成稳定的复合物。pDNA保护性实验表明,该复合物对pDNA有很好的保护作用。体外释放实验结果表明,SLNs-pDNA具有缓释能力。体外基因转染实验表明,该复合物能够转染COS-7细胞,复合物所荷基因能够在该细胞中表达。结论阳离子固体脂质纳米粒/DNA复合物是一种制备工艺简单,复合物粒径较小,细胞毒性小,对pDNA保护作用强,所载DNA能够从复合物中缓慢释放,体外转染效率较高的具有一定开发前景的非病毒纳米基因载体。

OBJECTIVE To prepare the binary complex of cationic solid lipid nanoparticles/pDNA （SLNs-pDNA） and investigate the pharmaceutical and biological characteristics of the complex. METHODS Cationic solid lipid nanopartieles （ SLNs ） was prepared by double emulsion method, and the reported gene was condensed using the cationic SLNs via electrostatic force leading to the formation of the SLNs-pDNA complex. The electric quantity of the system was modified by addition of various concentrations of Ca2＋. The physicochemical properties, in vitro cytotoxicity, release behaviour as well as protection of the SLNs-pDNA complex to pDNA from nuclease degradation were evaluated respectively. Transfection activity of SLN-pDNA complex was determined by gene transfection experiment in vitro in COS-7 cells. RESULTS The morphology of SLNs and the SLNs-pDNA complex were approximately spherical, the average particle sizes of SLNs and the complex were （ 51.9 ± 0. 58 ） and （ 91.6 ± 5. 3 ） nm, respectively. The Zeta potentials were （47.4 ± 1.1 ） and （31.5 ± 1.4）mV, respectively. The cell viability assay showed nanoparticles exhibited a low cytotoxicity to COS-7 cells. The nuclease degradation test results confirmed that the pDNA was protected considerably. SLNs-pDNA maintained sustained-release of pDNA for several days in vitro . CONCLUSION SLNs-pDNA complex could be prepared easily with small particle sizes, low cytotoxicity and high protection to pDNA. The gene transfection experiment in vitro suggested that SLN-pDNA complex could transfer the loaded gene into COS-7 cells, and the gene could express well inside the cells. The complex could be a promising non-virus nano-device, which has the potential to make in vivo cancer gene therapy achievable.