Formation and Temporal Evolution of Serum-Derived Lipid Coronas on Nanomedicines Revealed by High-Resolution Lipidomics Mass Spectrometry

다국어 초록 (Multilingual Abstract)

Upon immediate exposure to biological fluids such as serum or plasma, nanoparticles (NPs) become coated by spontaneously adsorbing biomolecules— proteins, lipids, carbohydrates, and small-molecule metabolites—forming a multilayered “biomolecular corona.” This corona furnishes the particle with an effective biological identity (the surface actually presented to living systems) and, in turn, governs circulation behavior, cellular interactions, and drug-delivery performance, ultimately determining the particle’s in vivo fate. Operationally, the corona is classified into soft and hard layers according to binding strength and exchange kinetics; these layers coexist in a dynamic quasi-equilibrium, undergoing continual exchange and reorganization. Advances in high-resolution mass- spectrometry proteomics have substantially deepened understanding of the protein corona, especially the abundance of apolipoproteins and their receptor-mediated roles. Yet the corona is not restricted to proteins: lipids and a variety of small metabolites also shape interfacial identity through their intrinsic functions and interactions. Notably, the “metabolite corona” remains comparatively underexplored; standardized analytical frameworks and robust quantitative or mechanistic evidence are still limited. Consequently, a fully integrated, “whole-corona” perspective has yet to be firmly established. To address this gap, we pursued a unified, stepwise program that progressively broadened scope: (1) interrogating curvature-dependent lipoprotein–NP interactions to elucidate the initiating events of corona formation; (2) defining the surface selectivity of metabolite and lipid coronas on liposomal drug carriers; and (3) resolving the time-dependent remodeling of the hard-corona lipidome. Collectively, these studies extend protein-centric interpretations to lipidomic and metabolomic dimensions and advance a dynamic, whole-corona understanding. First, quantitative measurements of interactions between PEG-coated gold NPs (PEG-AuNPs; 20–150 nm in diameter) and high-density lipoprotein (HDL) revealed a clear size dependence of HDL adsorption governed by the available physical contact area. Competition assays with human serum albumin (HSA) further indicated that the initial adsorbate is the intact HDL complex—not its constituent apolipoprotein A-I (ApoA-I)—thereby demonstrating that nascent corona formation on spherical PEG- AuNPs is initiated by complex-level contact events at the interface. Next, serum-derived lipid and metabolite coronas were profiled using doxorubicin-encapsulated liposomes whose surface charge and PEGylation density were systematically varied. After applying identification-confidence and quantitative quality filters, 220 lipids and 88 polar metabolites were retained for final analysis. The isolated coronas were dominated by low-molecular-weight species rather than proteins; across conditions, most metabolites were consistently incorporated into the corona, although their relative abundances varied. Class-resolved lipid patterns depended on particle surface properties, and, overall, more negatively charged and more densely PEGylated surfaces preferentially accumulated both lipids and polar metabolites. Finally, we tracked the lipid composition of the hard corona on DNA–lipoplexes and IgG-conjugated AuNPs over 48 h of serum incubation by untargeted lipidomics. Hydrodynamic-diameter measurements indicated that the hard corona formed rapidly and remained comparatively stable, whereas its lipid composition underwent pronounced time-dependent remodeling. These changes varied by particle type and were more pronounced at the lipid-class level than at the level of individual lipid species, consistent with sustained exchange between soft and hard layers throughout incubation. On AuNPs in particular, chemical transformations—most notably lipid peroxidation of highly polyunsaturated phosphatidylethanolamines (PEs)— introduced an additional layer of complexity to the observed evolution. Taken together, (i) lipoprotein complexes initiate corona formation through curvature-dependent contact mechanics; (ii) on liposomal drug carriers, lipid and metabolite coronas constitute a substantial component and exhibit surface selectivity; and (iii) the hard corona remains stable at the level of hydrodynamic size, yet its internal lipidome undergoes time-dependent reorganization. These integrated findings identify controllable design levers—curvature, surface charge, PEGylation, and incubation time—and, by moving beyond protein-centric interpretations, establish a foundation for a comprehensive understanding of biomolecular corona formation and remodeling that supports the rational design and development of nanomedicines.

목차 (Table of Contents)

• CHAPTER 1. General Introduction 1
• 1.1 What is the Biomolecular Corona 1
• 1.2 Factors Influencing Biomolecular Corona Formation 3
• 1.3 Dynamics of the Biomolecular Corona 5
• 1.4 Corona Composition: Protein-Centric Perspectives 6
• 1.5 Limitations of Protein-Centric Perspectives 7
• 1.6 Corona Composition: Beyond Protein-Centric Perspectives (Research Roadmap) 9
• CHAPTER 2. Adsorption of High-Density Lipoprotein on Nanoparticles in Serum Corona Formation 12
• 2.1 Introduction 12
• 2.2 Experimental Methods 15
• 2.2.1 Reagents 15
• 2.2.2 Surface PEG functionalization of citrate-capped AuNPs 16
• 2.2.3 Formation of an HDL corona on PEG-AuNPs during incubation 17
• 2.2.4 Physicochemical characterization of citrate-capped AuNPs, PEG-modified AuNPs, and corona-coated PEG-AuNPs 17
• 2.2.5 Proteomic profiling of protein coronas associated with PEG-AuNPs 18
• 2.2.6 SDS–PAGE-based quantification of corona proteins 19
• 2.3 Results & Discussion 20
• 2.3.1 Generation of HDL corona–coated PEG-AuNPs 20
• 2.3.2 Proteomic profiling of serum-derived protein coronas on PEG-AuNPs 25
• 2.3.3 Effect of nanoparticle size on corona assembly 25
• 2.3.4 Size effect on competitive binding of HDL/APOA1 and HSA 29
• 2.3.5 Effect of size-dependent binding on HDL adsorption to PEG-AuNPs/ APOA1 and HSA 31
• 2.4 Conclusion 33
• CHAPTER 3. Surface-Dependent Serum-Corona Metabolomes on Liposomal Drugs 35
• 3.1 Introduction 35
• 3.2 Experimental Methods 40
• 3.2.1 Reagents 40
• 3.2.2 Liposome synthesis 41
• 3.2.3 DOX loading and serum incubation for corona formation 42
• 3.2.4 Isolation of corona-bearing DOX-liposomes from serum 42
• 3.2.5 Physicochemical characterization of bare DOX-liposomes and corona-coated DOX-liposomes 44
• 3.2.6 Sample extraction procedure 44
• 3.2.7 Untargeted metabolomics by LC-MS/MS 45
• 3.2.8 Bioassay-based measurement of total proteins, TGs, and cholesterols 46
• 3.3 Results & Discussion 48
• 3.3.1 Properties of serum coronas on PEGylated liposomal drugs 48
• 3.3.2 Bioassay quantification of corona protein, TG, and cholesterol contents 52
• 3.3.3 Untargeted metabolomics profiling by mass spectrometry 56
• 3.4 Conclusion 64
• CHAPTER 4. Class-Dependent Temporal Evolution of the Lipidome in Biomolecular Coronas on Nano Drugs 66
• 4.1 Introduction 66
• 4.2 Experimental Methods 70
• 4.2.1 Reagents 70
• 4.2.2. Formation of Cy5-tagged plasmid DNA lipoplexes 71
• 4.2.3 Synthesis of PEG-AuNPs carrying Cy5-labeled IgG 72
• 4.2.4 Incubation of NPs with serum to build biomolecular coronas 73
• 4.2.5 NTA analysis of bare and corona-coated NPs 73
• 4.2.6 Extraction of lipid metabolites from serum-derived coronas on NPs 74
• 4.2.7 Untargeted LC–MS/MS lipidomics 74
• 4.2.8 Bioassay-based quantification of total proteins, TGs, cholesterol, and UFAs 75
• 4.3 Results & Discussion 76
• 4.3.1 Time-course of in situ and hard corona growth on NPs by NTA 76
• 4.3.2 Time-dependent changes in hard-corona biomolecular classes measured by bioassays 78
• 4.3.3 Untargeted MS-based lipidomics of serum medium 82
• 4.3.4 Time-dependent shifts in corona lipid classes: PCA and MS level 1 data 84
• 4.3.5 Temporal changes in the corona lipidome: MS level 2 data 91
• 4.3.6 Time-course of individual lipid species in the corona lipidome: heat-map analysis 92
• 4.4 Conclusion 96
• CHAPTER 5. Overall Conclusion 98
• References 100
• 국문초록 119