Poster O19 - Unnatural Lipids for Simultaneous mRNA Delivery and Metabolic Cell Labeling

Introduction: Lipids have demonstrated tremendous promise for mRNA delivery, as evidenced by the success of Covid-19 mRNA vaccines. However, existing lipids are mostly used as the delivery vehicle and lack the ability to monitor and further modulate the cell of target. Here, for the first time, we report a class of unnatural lipid (azido-DOTAP) that can efficiently deliver mRNAs into cells and meanwhile metabolically label cells with unique chemical tags (e.g., azido groups). The azido tags expressed on the cell membrane enable the monitoring of transfected cells, and can mediate subsequent conjugation of cargos via efficient click chemistry for further modulation of transfected cells. We further demonstrate that the dual-functional unnatural lipid is applicable to different types of cells including dendritic cells, the prominent type of antigen presenting cells, potentially opening a new avenue to developing enhanced mRNA vaccines.

Materials and

Methods: We first chemically synthesized the azido lipids (azido-DDAP and azido-DOTAP) and confirmed their structure by mass spectrum and NMR. We also tested the release kinetics of the azido-DOTAP in vitro via HPLC.
Next, we tested the labeling efficiency of these two lipids on the 4T1 cell line by incubating with DBCO-Cy5 to detect the cell surface azido group. The Cy5 signal was quantified by flow cytometry and the signal location was confirmed by confocal imaging. The lipids of the cells were extracted and analyzed with HPLC with fluorescent detection.
We further fabricated the azido-DOTAP and eGFP mRNA complex and studied their stability. This complex was used to transfect HEK293 and BMDCs to study the dual transfection and labeling function. Trasfected cells were stained with DBCO-Cy5 before running flow cytometry, and the eGFP and Cy5 signals were analyzed. The activation of BMDCs was also studied with flow cytometry after the transfection with CD86 and CD11c antibody staining.

Results, Conclusions, and Discussions: Our results demonstrated the ability of azido-DOTAP to induce the efficient transfection of mRNAs in various types of cells including 4T1 breast cancer cells, HEK293T kidney cells, and BMDCs, while installing azido groups on the cell membrane. We further demonstrated that additional lipids can be incorporated to improve the overall transfection efficiency while maintaining the metabolic labeling capability. The cell-surface azido groups enable targeted conjugation of molecules or materials via efficient click chemistry to further modulate the function of transfected cells. For the first time, we show that cationic lipids not only can deliver nucleic acids but also function as a labeling reagent for the monitoring and manipulation of target cells.

Acknowledgements (Optional): The authors would like to acknowledge the financial support from NSF DMR 21-43673 CAR (H.W.), NIH R01CA274738 (H.W.), NIH R21CA270872 (H.W.), and start-up package (H.W.) from the Department of Materials Science and Engineering at the University of Illinois at Urbana-Champaign and the Cancer Center at Illinois (CCIL).