PROJECT SUMMARY We propose the acquisition of a new image-enhanced flow cytometer (Attune CytPix) to support the research of 1R01GM143723-01A1: Endosomal escape of lipid-based nanoparticles comprising Gaussian curvature lipids. RNA-based therapies offer significant potential for treating a variety of diseases that have a major impact on human health. These include chronic infections, genetic disorders, specific cancers, and the current COVID-19 pandemic. Non-viral lipid-based nanoparticles (LNPs) are the primary RNA delivery vehicles approved by the FDA and are also being evaluated in numerous clinical trials. LNPs are composed of standard phospholipids, cholesterol, and ionizable lipids (ILs) that become protonated in acidic conditions. Similar to enveloped viruses, LNPs exploit the endocytic pathway to gain entry into cells. The success of RNA delivery depends on the ability of LNPs to fuse with the endosomal membrane and escape the endosome. However, the mechanisms that govern LNP-endosome fusion remain largely unknown. The central goal of 1R01GM143723-01A1 is to test the hypothesis that the inclusion of a new class of structural lipids with single chains and small headgroups like glycerol monooleate onto state-of-the art LNP formulations allows us to prescribe well-defined internal nanostructures of LNPs directly impacting their ability to fuse with endosomal membranes and releasing RNA cargo into the cytosol. Acquiring the new Attune CytPix equipment is vital to the progression of our project for two main reasons. Firstly, the only flow cytometry instrument in close proximity to our primary research location is currently non-operational due to frequent breakdowns and high user demand, resulting in prolonged periods of downtime. Secondly, the Attune CytPix offers simultaneous high throughput flow cytometry and high resolution brightfield imaging through acoustic focusing, which is not available with our current equipment. This feature is particularly important for quantifying LNP cargo loading and delivery efficiency. The high-speed brightfield camera records individual events as they pass through the flow cell, and the Attune Cytometric Software ensures that analyzed events originate from single cells and particles rather than doublets, clumps, or debris. This capability is critical in cell and gene therapy research, as well as other flow cytometry experiments that aim to understand the morphology of each cell population. Through our research, we aim to uncover novel physical insights into the endosomal escape of LNPs and determine the optimal membrane properties of LNPs to enhance fusion in living systems. This will lead to the development of RNA delivery vehicles that are significantly more effective in delivering their cargo.