纳米载体介导的PiggyBac转座子制备CAR-NK细胞

目的：探索新型阳离子聚合物纳米载体mPEG-P(Asp-AED-g-HFB) (PAEF)和PiggyBac转座子介导嵌合抗原受体 （chimeric antigen receptor，CAR）修饰自然杀伤（natural killer，NK）细胞的基因转染方法，为肿瘤细胞免疫治疗制剂研发提供新策 略。方法：制备PAEF/DNA（转座酶+转座子）复合物，通过Nano-ZSE动态光散射系统（Malvern Instruments）测量PAEF/DNA复 合物的粒径分布和表面电位；DNA凝胶电泳验证PAEF的DNA包封率、释放性和稳定性，结合粒径电位选择进入细胞合适的N/P 值；CCK-8细胞毒性实验分析不同N/P值条件下PAEF/DNA复合物的细胞毒性；荧光显微镜及流式细胞术检测细胞转染效率， 评 估PAEF基因转染载体的可行性。结果：PAEF可包裹DNA形成粒径100~150 nm的纳米复合物，后者易于介导DNA进入细胞； 当N/P值为20时，PAEF即可实现DNA的完全包裹；在还原剂二硫苏糖醇（dithiothreotol，DTT）存在下，PAEF对DNA有良好的释 放能力；N/P值为80时，PAEF/DNA复合物转染组的NK-92细胞存活率显著高于脂质体转染组[（72.50±3.9） % vs（64.03±1.8） %， P<0.05]；荧光显微镜下观察发现，PAEF/DNA组荧光多、荧光强度大；流式细胞术显示最高转染效率为83.4%。结论：纳米载体 PAEF通过静电吸附作用能够很好地包裹DNA，且生物相容性好，基因转导效率较高，成功制备的CAR-NK细胞为过继免疫治疗 提供良好的实验基础。

Nanocarrier-mediated PiggyBac transposon system for preparation of CAR-NK cells

Objective: To explore the gene transduction method of chimeric antigen receptor (CAR) mediated by novel cationic polymer nanocarrier mPEG-P (Asp-AED-g-HFB) (PAEF) and PigyBac transposon system to modify natural killer (NK) cells, providing a new strategy for immunotherapy of cancer cells. Methods: PAEF/DNA (transposase+transposon) complex were prepared. The particle size distribution and surface potential of PAEF/DNA complexes were measured with Nano-ZSE Dynamic Light Scattering System (Malvern Instruments). The DNA encapsulation rate, release and stability of PAEF were evaluated by DNA gel electrophoresis, and then by combiningwithparticlesizeandsurfacepotentialtodeterminethepreferentialN/PratiotoenterNKcells.Thecell cytotoxicity of PAEF/DNA complexes under different N/P ratios was analyzed by CCK-8 cytotoxicity test. Transduction efficiency of NK cells was evaluated by Fluorescence microscopy and Flow cytometry, and the feasibility of PAEF gene transfection vectors was assessed. Results: PAEF could encapsulate DNA to form nano-complexes with the diameter of 100-150 nm, which was suitable to mediate DNA entering into cells. PAEF could completely encapsulate DNA with N/P ratio of 20. In the presence of reducing agent dithiothreitol (DTT), PAEF had a good ability to release DNA. NK-92 cells transfected with PAEF/DNA complex, which was formed at the N/P ratio of 80, attained a significantly higher cell viability than cells of lipofectamine transfection group [(72.50±3.9)% vs (64.03±1.8)%, P<0.05]; Fluorescence microscopic observation showed more fluorescence and higher fluorescence intensity in cells of PAEF/DNA group; Flow cytometry showed the highest transfection efficiency of 83.4%. Conclusions: Nanocarrier PAEF can encapsulate DNA well by electrostatic adsorption, and has good biocompatibility and high efficiency for gene transduction. It provides a good experimental basis for adoptive immunotherapy.