Selective Transfection of a Transferrin Receptor-Expressing\nCell Line with DNA–Lipid Nanoparticles

Despite considerable\nprogress in using lipid nanoparticle (LNP)\nvehicles for gene delivery, achieving selective transfection of specific\ncell types remains a significant challenge, hindering the advancement\nof new gene or gene-editing therapies. Although LNPs have been equipped\nwith ligands aimed at targeting specific cellular receptors, achieving\ncomplete selectivity continues to be elusive. The exact reasons for\nthis limited selectivity are not fully understood, as cell targeting\ninvolves a complex interplay of various cellular factors. Assessing\nhow much ligand/receptor binding contributes to selectivity is challenging\ndue to these additional influencing factors. Nonetheless, such data\nare important for developing new nanocarriers and setting realistic\nexpectations for selectivity. Here, we have quantified the selective,\ntargeted transfection using two uniquely engineered cell lines that\neliminate unpredictable and interfering cellular influences. We have\ncompared the targeted transfection of Chinese ovary hamster (CHO)\ncells engineered to express the human transferrin receptor 1 (hTfR1),\nCHO-TRVb-hTfR1, with CHO cells that completely lack any transferrin\nreceptor, CHO-TRVb-neo cells (negative control). Thus, the two cell\nlines differ only in the presence/absence of hTfR1. The transfection\nwas performed with pDNA-encapsulating LNPs equipped with the DT7 peptide\nligand that specifically binds to hTfR1 and enables targeted transfection.\nThe LNP’s pDNA encoded for the monomeric GreenLantern (mGL)\nreporter protein, whose fluorescence was used to quantify transfection.\nWe report a novel LNP composition designed to achieve an optimal particle\nsize and ζ-potential, efficient pDNA encapsulation, hTfR1-targeting\ncapability, and sufficient polyethylene glycol sheltering to minimize\nrandom cell targeting. The transfection efficiency was quantified\nin both cell lines separately through flow cytometry based on the\nexpression of the fluorescent gene product. Our results demonstrated\nan LNP dose-dependent mGL expression, with a 5-fold preference for\nthe CHO-TRVb-hTfR1 when compared to CHO-TRVb-neo. In another experiment,\nwhen both cell lines were mixed at a 1:1 ratio, the DT7-decorated\nLNP achieved a 3-fold higher transfection of the CHO-TRVb-hTfR1 over\nthe CHO-TRVb-neo cells. Based on the low-level transfection of the\nCHO-TRVb-neo cells in both experiments, our results suggest that 17–25%\nof the transfection occurred in a nonspecific manner. The observed\ntransfection selectivity for the CHO-TRVb-hTfR1 cells was based entirely\non the hTfR1/DT7 interaction. This work showed that the platform of\ntwo engineered cell lines which differ only in the hTfR1 can greatly\nfacilitate the development of LNPs with hTfR1-targeting ligands.