Abstract Body (Do not enter title and authors here): Introduction: Very Low-Density Lipoproteins (VLDLs) serve as crucial transporters of triglycerides (TG) and cholesterol (C) from the liver to peripheral tissues, thereby contributing significantly to metabolic disorders such as atherosclerosis. Assessing the dynamic of VLDLs function involves evaluating its clearance from the bloodstream and distribution across various organs. Although VLDLs clearance has been extensively studied using dual radioactive isotopes ³H and ¹⁴C to label TG and C, this method requires large amounts of expensive tracers, compliance, and technical complexities. Our objective is to design and test a fluorescence-based approach to simultaneously assess and visualize VLDLs kinetics in a mouse model. Through this approach, we aim to unravel the intricate interplay between thermogenesis and VLDLs metabolism and provide insights into their collective influence on the progression of atherosclerosis in vivo.

Method: Fluorescence-labeled VLDLs-like TG-rich nanoparticles (F-VLDLs) were prepared at various intensity by incorporating lissamine rhodamine-TG (Rhoda-TG, 1-5% of total TG) and TopFluor®-C (TopF-C, 20-80% of total C) to a mixture containing glycerol trioleate (7mg), egg yolk phosphatidylcholine (2.3mg), lysophosphatidylcholine (0.23mg), cholesteryl oleate (0.3mg), cholesterol (0.2mg) and kolliphor HS15 (2mg). B6 mice were fasted for 4 hours and injected via the tail vein with various intensities of F-VLDLs (2 mg TG/mouse). Blood samples were collected at 5-, 10- and 15-min post-injection to determine the plasma clearance of Rhoda-TG and TopF-C, after which mice were euthanized, and tissues harvested and imaged using an IVIS® spectrum in vivo imaging system.

Results: The mean particle size of F-VLDLs was 80nm, with polydispersity index values of about 0.2. The Zeta potential of freshly made F-VLDLs was about −20 mV. In B6 mice, both Rhoda-TG and TopF-C signal intensities peaked at 5 minutes post-injection and subsequently declined in an intensity-dependent manner. In the collected tissues, Rhoda-TG and TopF-C intensity was as follows: liver>brown fat>subcutaneous white fat >visceral white fat.

Conclusion: We have designed novel fluorescence-labeled VLDVs-like particles as a viable approach for quantifying and visualizing dynamic VLDLs-TG and C flux in vivo. This approach will facilitate the assessment of how various metabolic interventions impact VLDLs metabolism and atherosclerosis.