Selective Transfection of a Transferrin Receptor-Expressing Cell Line with DNA-Lipid Nanoparticles

Irodiel Vinales; Juan Carlos Silva-Espinoza; Bryan A Medina; Juan E M Urbay; Miguel A Beltran; Dante E Salinas; Marco A Ramirez-Ramos; Rosa A Maldonado; Wilson Poon; Manuel L Penichet; Igor C Almeida; Katja Michael

doi:10.1021/acsomega.4c03541

Selective Transfection of a Transferrin Receptor-Expressing Cell Line with DNA-Lipid Nanoparticles

ACS Omega. 2024 Sep 15;9(38):39533-39545. doi: 10.1021/acsomega.4c03541. eCollection 2024 Sep 24.

Authors

Irodiel Vinales^{1

2}, Juan Carlos Silva-Espinoza^{3

2}, Bryan A Medina^{1

2}, Juan E M Urbay^{1

2}, Miguel A Beltran^{2

3}, Dante E Salinas^{2

3}, Marco A Ramirez-Ramos¹, Rosa A Maldonado^{2

3}, Wilson Poon⁴, Manuel L Penichet^{5

6

7

8

9}, Igor C Almeida^{2

3}, Katja Michael^{1

2}

Affiliations

¹ Department of Chemistry and Biochemistry, University of Texas at El Paso, El Paso, Texas 79968, United States.
² Border Biomedical Research Center, University of Texas at El Paso, El Paso, Texas 79968, United States.
³ Department of Biological Sciences, University of Texas at El Paso, El Paso, Texas 79968, United States.
⁴ Department of Metallurgical, Materials and Biomedical Engineering, University of Texas at El Paso, El Paso, Texas 79968, United States.
⁵ Division of Surgical Oncology, Department of Surgery, David Geffen School of Medicine, University of California, Los Angeles (UCLA), Los Angeles, California 90095, United States.
⁶ Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles (UCLA), Los Angeles, California 90095, United States.
⁷ California Nanosystems Institute, University of California, Los Angeles, Los Angeles, California 90095, United States.
⁸ The Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, United States.
⁹ Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, California 90095, United States.

Abstract

Despite considerable progress in using lipid nanoparticle (LNP) vehicles for gene delivery, achieving selective transfection of specific cell types remains a significant challenge, hindering the advancement of new gene or gene-editing therapies. Although LNPs have been equipped with ligands aimed at targeting specific cellular receptors, achieving complete selectivity continues to be elusive. The exact reasons for this limited selectivity are not fully understood, as cell targeting involves a complex interplay of various cellular factors. Assessing how much ligand/receptor binding contributes to selectivity is challenging due to these additional influencing factors. Nonetheless, such data are important for developing new nanocarriers and setting realistic expectations for selectivity. Here, we have quantified the selective, targeted transfection using two uniquely engineered cell lines that eliminate unpredictable and interfering cellular influences. We have compared the targeted transfection of Chinese ovary hamster (CHO) cells engineered to express the human transferrin receptor 1 (hTfR1), CHO-TRVb-hTfR1, with CHO cells that completely lack any transferrin receptor, CHO-TRVb-neo cells (negative control). Thus, the two cell lines differ only in the presence/absence of hTfR1. The transfection was performed with pDNA-encapsulating LNPs equipped with the DT7 peptide ligand that specifically binds to hTfR1 and enables targeted transfection. The LNP's pDNA encoded for the monomeric GreenLantern (mGL) reporter protein, whose fluorescence was used to quantify transfection. We report a novel LNP composition designed to achieve an optimal particle size and ζ-potential, efficient pDNA encapsulation, hTfR1-targeting capability, and sufficient polyethylene glycol sheltering to minimize random cell targeting. The transfection efficiency was quantified in both cell lines separately through flow cytometry based on the expression of the fluorescent gene product. Our results demonstrated an LNP dose-dependent mGL expression, with a 5-fold preference for the CHO-TRVb-hTfR1 when compared to CHO-TRVb-neo. In another experiment, when both cell lines were mixed at a 1:1 ratio, the DT7-decorated LNP achieved a 3-fold higher transfection of the CHO-TRVb-hTfR1 over the CHO-TRVb-neo cells. Based on the low-level transfection of the CHO-TRVb-neo cells in both experiments, our results suggest that 17-25% of the transfection occurred in a nonspecific manner. The observed transfection selectivity for the CHO-TRVb-hTfR1 cells was based entirely on the hTfR1/DT7 interaction. This work showed that the platform of two engineered cell lines which differ only in the hTfR1 can greatly facilitate the development of LNPs with hTfR1-targeting ligands.