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   https://doi.org/10.1021/acs.analchem.8b03911  

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2. Fluorescence cross-correlation spectroscopy of lipid-peptide interactions on supported lipid bilayers

   https://doi.org/bs.abl.2019.01.005  

   Xiaosi Li, Xiaojun Shi (March 2019, Advances in biomembranes and lipid self-assembly)

   Electrostatic interactions drive molecular assembly and organization in the plasma membrane. Specific protein-lipid interactions, however, are difficult to resolve. Here we report on a unique approach to investigate these interactions with time-resolved fluorescence spectroscopy. The experiments were performed on a model membrane system consisting of a supported lipid bilayer with an asymmetric distribution of PIP2 in the upper leaflet of the bilayer. The bilayer also contained nickel-chelating lipids that bind to a histidine-tagged peptide of interest. Both the peptide and the lipid were labeled with orthogonal fluorescent probes, so that diffusion and binding could be measured with two-color, pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Our PIE-FCCS data showed significant lipid-peptide cross-correlation between PIP2 lipids and membrane-bound cationic peptides. Cross-correlation is a direct indication of lipid-peptide binding and complexation. Together with mobility data, we quantified the degree of binding, which offers new insight into this class of lipid-peptide interactions. Overall, this is the first report of lipid-peptide cross-correlation by FCCS, and provides a new route to quantifying the interactions between proteins and lipid membranes, a key interface in cell signaling. 

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3. Spatial arrangements of spherical nanoparticles on lipid vesicles

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5. Influence of ionizable lipid tail length on lipid nanoparticle delivery of mRNA of varying length

   https://doi.org/10.1002/jbm.a.37705  

   Mrksich, Kaitlin; Padilla, Marshall S; Joseph, Ryann A; Han, Emily L; Kim, Dongyoon; Palanki, Rohan; Xu, Junchao; Mitchell, Michael J (September 2024, Journal of Biomedical Materials Research Part A)

   Abstract RNA‐based therapeutics have gained traction for the prevention and treatment of a variety of diseases. However, their fragility and immunogenicity necessitate a drug carrier. Lipid nanoparticles (LNPs) have emerged as the predominant delivery vehicle for RNA therapeutics. An important component of LNPs is the ionizable lipid (IL), which is protonated in the acidic environment of the endosome, prompting cargo release into the cytosol. Currently, there is growing evidence that the structure of IL lipid tails significantly impacts the efficacy of LNP‐mediated mRNA translation. Here, we optimized IL tail length for LNP‐mediated delivery of three different mRNA cargos. Using C12‐200, a gold standard IL, as a model, we designed a library of ILs with varying tail lengths and evaluated their potency in vivo. We demonstrated that small changes in lipophilicity can drastically increase or decrease mRNA translation. We identified that LNPs formulated with firefly luciferase mRNA (1929 base pairs) and C10‐200, an IL with shorter tail lengths than C12‐200, enhance liver transfection by over 10‐fold. Furthermore, different IL tail lengths were found to be ideal for transfection of LNPs encapsulating mRNA cargos of varying sizes. LNPs formulated with erythropoietin (EPO), responsible for stimulating red blood cell production, mRNA (858 base pairs), and the C13‐200 IL led to EPO translation at levels similar to the C12‐200 LNP. The LNPs formulated with Cas9 mRNA (4521 base pairs) and the C9‐200 IL induced over three times the quantity of indels compared with the C12‐200 LNP. Our findings suggest that shorter IL tails may lead to higher transfection of LNPs encapsulating larger mRNAs, and that longer IL tails may be more efficacious for delivering smaller mRNA cargos. We envision that the results of this project can be utilized as future design criteria for the next generation of LNP delivery systems for RNA therapeutics. 

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