<p>Self-Assembled Nanoparticles Prepared from Low-Molecular-Weight PEI and Low-Generation PAMAM for EGFRvIII-Chimeric Antigen Receptor Gene Loading and T-Cell Transient Modification</p>
Qianru Yu, Maxin Zhang, Yuetan Chen, Xiaolong Chen, Sanyuan Shi, Kang Sun, Ye Ran, Yixi Zheng, Chen Yang, Yuhong Xu, Jinliang Peng
Abstract
Background: 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. Methods: A series of plasmid DNA-loaded self-assembled nanoparticles ( [email protected] x/y ) 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 [email protected] G1/800 were systematically investigated. Thereafter, the epidermal growth factor receptor variant III (EGFRvIII) CAR-expression plasmid vector (pEGFRvIII-CAR) was constructed and encapsulated into SNP G1/800 . The resulting [email protected] G1/800 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. Results: The [email protected] G1/800 showed the highest efficacy in Jurkat cell gene transfection and exhibited low cytotoxicity. [email protected] G1/800 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. Conclusion: These results indicated that [email protected] G1/800 can effectively achieve T-cell transient CAR modification, thereby demonstrating considerable potential in CAR-T cancer therapy. Keywords: self-assembled nanoparticle, PAMAM dendrimer, polyethylenimine, epidermal growth factor receptor variant III, chimeric antigen receptor, T lymphocyte