Photothermal Mid-Infrared Single Molecule Super-Localization Microscopy - Summary Lipid nanoparticle (LNP)-based formulations are widely used for delivering macromolecular therapeutics, including mRNA. Biologics account for ~55-60% of the total current pharmaceutical product market, with all trends pointing to continued increases in market share. However, transfection efficiency of mRNA is severely hindered by various loss mechanisms, including rapid clearance, suboptimal cellular uptake, and incomplete endosomal escape. These processes reduce the effective delivery of mRNA to only a small fraction (~1-2%) of the administered dose. As a consequence, comparatively subtle patient-to-patient differences in loss can translate to large variability in therapeutic dosing, with corresponding variability in efficacy and side-effects. To improve transfection yields and better understand the intracellular barriers to mRNA delivery, particularly during the key step of endosomal escape, there is a pressing need for new tools capable of providing chemically selective, nanoscale insights in the intracellular fates of LNPs and their cargos within live cells. The goal of this project is to develop fluorescence-detected photothermal infrared (F-PTIR) microscopy to track the intracellular processes governing mRNA release from LNPs in real-time and with ultra-high spatial resolution, well below the optical diffraction limit. In brief, fluorescence from labelled mRNA will serve as a local temperature sensor. Upon IR absorption from the surrounding medium, local transient temperature increases result in corresponding reductions in fluorescence quantum yield. Change in fluorescence intensity as the IR wavelength is tuned enables IR absorption spectroscopy with a spatial resolution set by fluorescence imaging and heat transfer. Embedding fluorescently labeled mRNA within deuterated LNPs will yield IR spectra dominated by the CD stretching modes of isolated LNPs. Endosomal uptake will be tracked both by the physical position of fluorescence within single cells and by the changes in the lipid vibrational spectra (e.g., addition of CH-stretching modes from native lipids in endosome membranes). Tight timing control in combination with heat-transport modeling will be used to quantify distances between the fluorescence reporter and different IR absorbers. The relatively rare subset of internalized LNPs capable of releasing cargo intact into the cytosol will be identified by their corresponding change in local microenvironment (e.g., loss of CD stretches for mRNA solubilized within the cytosol) together with their 3D position and mobility measured by single molecule localization microscopy (SMLM). Once developed and validated, the proposed instrumentation can support informed optimization of therapeutic nanoparticle formulations designed to promote dosing yield and therapeutic efficacy.
