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

Area of Science:

• Biotechnology
• Molecular Biology
• Nanomedicine

Background:

• Lipid nanoparticles (LNPs) are promising for gene delivery but lack cell-type selectivity.
• Targeting ligands on LNPs aim to improve specificity, yet true selectivity remains challenging due to complex cellular interactions.
• Quantifying ligand-receptor contributions to selectivity is vital for developing effective nanocarriers.

Purpose of the Study:

• To quantify the selective gene transfection efficiency of engineered LNPs targeting the human transferrin receptor 1 (hTfR1).
• To establish a robust experimental system for evaluating LNP targeting specificity by minimizing confounding cellular factors.

Main Methods:

• Utilized two engineered Chinese hamster ovary (CHO) cell lines: one expressing hTfR1 (CHO-TRVb-hTfR1) and a control lacking it (CHO-TRVb-neo).
• Developed novel LNPs encapsulating plasmid DNA (pDNA) encoding a fluorescent reporter (mGL) and functionalized with a DT7 peptide ligand targeting hTfR1.
• Quantified transfection efficiency via flow cytometry measuring mGL expression in both cell lines separately and in a mixed population.

Main Results:

• Engineered LNPs demonstrated a dose-dependent, 5-fold higher transfection preference for hTfR1-expressing cells (CHO-TRVb-hTfR1) compared to control cells (CHO-TRVb-neo).
• In a mixed-cell experiment, DT7-decorated LNPs achieved a 3-fold greater transfection of CHO-TRVb-hTfR1 cells over CHO-TRVb-neo cells.
• Non-specific transfection was estimated to be 17-25%, indicating high selectivity driven by the hTfR1/DT7 interaction.

Conclusions:

• The developed LNP formulation exhibits significant, ligand-mediated selectivity for hTfR1-expressing cells.
• The use of engineered cell lines differing only in hTfR1 expression provides a powerful platform for LNP targeting development.
• This approach facilitates the creation of more precise nanocarriers for targeted gene delivery and therapeutic applications.