Plasma pre-incubation: Concentration-dependent regulation of the biomolecular corona on PEGylated liposome and cellular uptake.

Understanding biomolecular coronas that spontaneously occur around liposomes in biological fluids is critical as both the lipid and protein coronas influence liposome behavior in biological systems. Herein, PEGylated liposomes were pre-incubated at varying plasma concentrations followed by in vitro dynamic simulation incubation. It was found that increasing plasma concentration from 10 % to 300 % resulted in reduced liposomes uptake by immune cells. Most plasma-derived lipids were found to be retained in the coronas but with altered abundances. Lipids such as CE 18:1 and PC 16:0/18:2 exhibited plasma pre-incubation concentration-dependent changes. Changes in the plasma concentration resulted in the emergence of unique proteins in the final protein corona. Using cross-linking mass spectrometry with two crosslinkers and time-limited proteolysis-mass spectrometry, the associations between plasma pre-incubation concentrations and the topological network of the protein corona, the molecular orientation of proteins including alpha-actinin-4, apolipoprotein A1, etc., on the liposome surface, as well as domain proximity, were identified based on peptide-level structural resolution. Among the detected protein complexes with inter-protein crosslinks, over 85 % were not documented in the BioGRID and STRING databases, and more than 90 % of these complexes failed to align with the predictive models generated by AlphaFold Multimer. These results underscore the characteristic weak interactions between proteins within the protein corona on the surface of PEGylated liposomes. The biomolecular corona on the surface of PEGylated liposomes retains a historical imprint of the microenvironment it has experienced, ultimately shaping the authentic biological identity of the liposomes.