Lipidomic profiling of cellular lipid post LENN formulation treatment

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<p data-end="926" data-start="457">To elucidate the lipid-mediated mechanisms underlying the uptake and endosomal escape of mRNA cargo delivered via LENN nanoparticles, we conducted a comprehensive lipid profiling analysis of T24 bladder carcinoma cells treated with V24-EGF LENN, V40 LENN, or polyion complexes. Here, we provide an MRM profiling dataset for those conditions.</p>

<p data-end="1910" data-start="928">The dataset consists of 10 conditions, including treated cells and blank controls in replicates of 3, collected at multiple time points. Treatments included V24-EGF LENN, V40 LENN, and polyion complexes, each at 0.5 mg/mL mRNA per well. After 1 h of treatment, cells were harvested at 0, 1, and 4 h post-treatment to capture dynamic changes in lipid composition. All samples were subjected to lipid extraction using a modified Bligh & Dyer method. Extracted lipids were resuspended in injection solvent  and analyzed using MRM (multiple reaction monitoring) profiling on an Agilent 6410 QQQ for 1.5 h. Data were normalized and statistically analyzed using univariate and cluster-based approaches in MetaboAnalyst 6.0. </p>

<p data-end="2900" data-start="1912">The study was conducted in two parts, first was the discovery step, where MRM was conducted for 3 different categories of lipids (M1, M2 and M3). Pooled samples for each treatment and time point were used to identify lipid species exhibiting ion intensities exceeding 20% of the corresponding blank. This step enabled detection of major lipid classes, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin (SM), and carnitines (CAR) etc. In the screening step, these identified MRMs from the discovery step were interrogated across all individual samples to characterize time-dependent changes induced by LENN formulations. Collectively, this dataset provides high-resolution insight into lipid-mediated processes during nanoparticle uptake and endosomal processing. By making both discovery and screening datasets available, this work enables the interrogation of specific lipid species implicated in membrane curvature, fusogenicity, and intracellular signaling during receptor-mediated endocytosis and mRNA delivery.</p>

<p data-end="3299" data-start="2902">This project consists of files that include the: 1) discovery excel file for the first run, 2) the discovery file post run (lipids that exceeded 30% ion intensities), and 3) lipid count and experiment design. Further, from the screening, the 4) absolute intensity files for the analyzed lipids are made available from all the 3 classes (Absolute intensity for M1, M2 and M3). The absolute intensity files were normalized and ran on the MetaboAnalyst to generate figures.</p>