Poster X19 - Development of Chemical Tags for Universal Lipid Nanoparticle Visualization and Tracking in 2D and 3D Bio-imaging

Introduction: To achieve effective disease diagnosis and treatment, precise delivery of therapeutic agents to specific cells or sub-cellular compartments is crucial. Lipid nanoparticles have emerged as promising nano-carriers for drugs and genes. However, understanding their transport and distribution within cells and tissues post-administration remains limited due to the lack of methods for visualizing and tracking them at high resolution or over large tissue volumes. In this project, we developed a fluorescent peptide tag for lipid nanoparticles tailored for compatibility with visualization and tracking using advanced optical fluorescence imaging techniques that involve lipid disruption steps, such as cell permeabilization or tissue clearing. We named these tags SPARKLE (Strategic Peptide Anchored, Retained, and Kept after Lipid Elimination). Lipid nanoparticles labelled with SPARKLE tags can be crosslinked and fixed within cells and tissues. In contrast to conventional lipophilic dye tags that wash out during lipid disruption, the SPARKLE tag will be retained and fluorescently mark the position of lipid nanoparticles.

Materials and

Methods: Initially, we optimized the design and conjugation scheme of the SPARKLE tags to lipid nanoparticles. We synthesized lipid nanoparticles with the conventional 4-lipid formulation (SM-102, 1-2,DSPC, cholesterol, and DMG-PEG(2000) with an additional 5th lipid (DSPE-PEG(2000)-maleimide) using microfluidic mixing. Next, we performed thiol-maleimide conjugation of lipid nanoparticles to the cysteine-terminated peptide-based SPARKLE tags containing various lengths of lysines (i.e.: CK4, CK7, and CK15), and then amine coupling of the lysines to NHS-ester Alexa Fluor dyes. Lipid nanoparticles were characterized for size, zeta potential, and polydispersity by dynamic light scattering to confirm conjugation and colloidal stability. SPARKLE-tagged LNPs were then administered to A549 and U87 cancer cells in vitro and subjected to membrane permeabilization staining steps as part of the cell painting assay.

Results, Conclusions, and Discussions: Our results showed that SPARKLE tags could be successfully conjugated to LNPs containing 0.25% - 0.5% mol DSPE-PEG(2000)-maleimide by adjusting mol % of DMG-PEG(2000) or cholesterol. LNPs with SPARKLE tags containing only 4 lysines were insufficient to be crosslinked into cells, whereas LNPs with SPARKLE tags containing 7 and 15 lysines were retained at close to 100% in cells. As a proof of concept, we were able to track SPARKLE-tagged LNPs to sub-cellular resolution using the cell painting assay in A549 and U87 cancer cells. Conventional DiI- or DiD-dye loaded LNPs were not able to be visualized in cells in comparison. Currently, we are working to validate SPARKLE tags for in vivo applications by intravenously administering SPARKLE-tagged LNPs into BALB/c mice and collecting tissues for subsequent CLARITY tissue clearing and 3D optical imaging using lightsheet microscopy. We anticipate future experiments using SPARKLE tags will provide insights into how lipid nanoparticles interact with biological environments in the body, aiding in understanding their journey to the desired target and the release of cargo, ultimately benefiting patients.

Acknowledgements (Optional): We would like to acknowledge Dr. Irodiel Vinales and Dr. Katja Michael for technical assistance in reagent quality assessment and processing. We acknowledge funding support from the University of Texas System STARs Program, as well as the University Research Institute (URI) award 14648682 and COURI student funding from the University of Texas at El Paso. We also acknowledge equipment use at the Border Biomedical Research Center, which is generously supported in part by NIH-NIMHD grant no. U54MD007592.