School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.
NanoDrug Platform, Zhejiang California International NanoSystems Institute, Zhejiang University, Hangzhou 310058, People's Republic of China.
Int J Nanomedicine. 2020 Jan 23;15:483-495. doi: 10.2147/IJN.S229858. eCollection 2020.
The complex preparation procedures and severe toxicities are two major obstacles facing the wide use of chimeric antigen receptor-modified T (CAR-T) cells in clinical cancer immunotherapy. The nanotechnology-based T cell temporary CAR modification may be a potential approach to solve these problems and make the CAR-T cell-based tumor therapy feasible and broadly applicable.
A series of plasmid DNA-loaded self-assembled nanoparticles (pDNA@SNPs) prepared from adamantane-grafted polyamidoamine (Ad-PAMAM) dendrimers of different generations (G1 or G5) and cyclodextrin-grafted branched polyethylenimine (CD-PEI) of different molecular weights (800, 2000, or 25,000 Da) were characterized and evaluated. The detailed physicochemical properties, cellular interaction, and cytotoxicity of selected pDNA@SNP were systematically investigated. Thereafter, the epidermal growth factor receptor variant III (EGFRvIII) CAR-expression plasmid vector (pEGFRvIII-CAR) was constructed and encapsulated into SNP. The resulting pEGFRvIII-CAR@SNP was used for Jurkat cell transient transfection, and the EGFRvIII-CAR expressed in transfected cells was measured by flow cytometry and Western blot. Finally, the response of EGFRvIII CAR-positive Jurkat T cell to target tumor cell was evaluated.
The pDNA@SNP showed the highest efficacy in Jurkat cell gene transfection and exhibited low cytotoxicity. pEGFRvIII-CAR@SNP can efficiently deliver pEGFRvIII-CAR into Jurkat T cells, thereby resulting in transient EGFRvIII-CAR expression in transfected cells. EGFRvIII-CAR that is present on the cell membrane enabled Jurkat T cells to recognize and bind specifically with EGFRvIII-positive tumor cells.
These results indicated that pEGFRvIII-CAR@SNP can effectively achieve T-cell transient CAR modification, thereby demonstrating considerable potential in CAR-T cancer therapy.
嵌合抗原受体修饰的 T 细胞（CAR-T）在临床癌症免疫治疗中广泛应用面临着复杂的制备程序和严重的毒性等两大障碍。基于纳米技术的 T 细胞临时 CAR 修饰可能是解决这些问题的一种潜在方法，可以使基于 CAR-T 细胞的肿瘤治疗变得可行并广泛适用。
从不同代数（G1 或 G5）的金刚烷接枝聚酰胺胺（Ad-PAMAM）树枝状大分子和不同分子量（800、2000 或 25000Da）的环糊精接枝支化聚乙烯亚胺（CD-PEI）制备了一系列负载质粒 DNA 的自组装纳米颗粒（pDNA@SNPs），并对其进行了表征和评价。系统研究了所选 pDNA@SNP 的详细理化性质、细胞相互作用和细胞毒性。此后，构建了表皮生长因子受体变异 III（EGFRvIII）CAR 表达质粒载体（pEGFRvIII-CAR）并将其包封到 SNP 中。所得的 pEGFRvIII-CAR@SNP 用于 Jurkat 细胞瞬时转染，通过流式细胞术和 Western blot 测定转染细胞中表达的 EGFRvIII-CAR。最后，评估了 EGFRvIII CAR 阳性 Jurkat T 细胞对靶肿瘤细胞的反应。
pDNA@SNP 在 Jurkat 细胞基因转染中显示出最高的功效，且表现出低细胞毒性。pEGFRvIII-CAR@SNP 可有效将 pEGFRvIII-CAR 递送至 Jurkat T 细胞，从而导致转染细胞中瞬时表达 EGFRvIII-CAR。存在于细胞膜上的 EGFRvIII-CAR 使 Jurkat T 细胞能够特异性识别和结合 EGFRvIII 阳性肿瘤细胞。
这些结果表明，pEGFRvIII-CAR@SNP 可有效实现 T 细胞临时 CAR 修饰，在 CAR-T 癌症治疗中具有很大的潜力。
