Aug 2026· International journal of pharmaceutics· pp.
127357
· 0 citations· 50 references
Medicine
TL;DR
Findings indicate that PEGylation reduces ApoE association primarily through steric exclusion and highlight PEGylation as a useful strategy for modulating nanoparticle biodistribution.
Abstract
Due in part to adsorption of apolipoprotein E (ApoE) and subsequent receptor-mediated uptake by hepatocytes, lipid nanoparticles (LNPs) have a propensity to accumulate within the liver. The mechanistic contribution of PEGylation to this phenomenon has yet to be fully elucidated. Using fluorescence quenching, circular dichroism spectroscopy, molecular dynamics simulations, and in vivo bioluminescence imaging, we investigated how PEGylation influences ApoE interactions with DC-Chol-based nanoparticles. Association studies revealed that non-PEGylated formulations bound ApoE ∼ 1.7-fold more strongly than particles with surface PEG moieties, quantified by Stern-Volmer constants of 0.083 vs. 0.048 μM-1, respectively. Circular dichroism measurements demonstrated a greater structural perturbation to ApoE upon binding to non-PEGylated particles (80% helical loss) versus PEGylated LNPs (24-48% helical loss). Molecular dynamics simulations showed that PEG-2000 creates a dynamic steric barrier that reduces direct protein-lipid contact formation by 2.9-fold and decreases the tendency of ApoE to remain closely associated with the nanoparticle surface. Correspondingly, biodistribution studies in mice showed rapid hepatic accumulation of non-PEGylated nanoparticles within 24 h, whereas PEGylated formulations exhibited delayed liver accumulation that became prominent at 48 h. Together, these findings indicate that PEGylation reduces ApoE association primarily through steric exclusion and highlight PEGylation as a useful strategy for modulating nanoparticle biodistribution.
A common observation in drug delivery studies is that preincubation of nanoparticles (NPs) with high concentrations of human plasma (HP) markedly reduces in vitro transfection efficiency (TE). This effect is commonly attributed to the formation of a protein corona (PC), which is assumed to impair cellular uptake or intracellular trafficking of gene delivery systems such as lipid nanoparticles (LNPs). However, the evidence presented here suggests an alternative mechanism underlying this phenomenon. To reproduce conditions in which LNPs encounter an excess of circulating proteins, NPs were incubated in HP prior to exposure to cells in standard calcium-containing culture medium. Under these conditions, plasma undergoes gelation, leading to the formation of a clot-like network. A combination of complementary approaches, including dynamic light scattering (DLS), fluorescence-activated cell sorting (FACS), confocal fluorescence microscopy, raster image correlation spectroscopy (RICS), and functional assays evaluating TE and cell viability, demonstrates that this gel-like matrix restricts LNP diffusion in the extracellular environment. In contrast, coronated LNPs that reach the intracellular space display comparable trafficking behavior, indicating that the PC does not compromise intracellular processing. These findings highlight the importance of extracellular factors when evaluating the impact of the PC and extrapolating in vitro results to in vivo settings.
Serena Renzi, L. Digiacomo, Francesca Giulimondi et al.· Advanced Healthcare Material...· 0 citations
Extrahepatic delivery of lipid nanoparticles (LNPs) to non-phagocytic cells is a major challenge, with the leading strategy involving surface functionalization with target-specific monoclonal antibody (mAb) ligands. We investigate the stability of mAb-conjugated LNPs using two anchoring systems: the commonly used DSPE-PEG2kDa-maleimide and a block copolymer, PCL5kDa-b-PEG2kDa -maleimide, with the hypothesis that conjugation to a 150,000 Da antibody could overwhelm the relatively small ∼600 Da aliphatic anchor on the PEG-lipid in vivo. Shedding of the mAB would compromise targeting. Conjugation integrity following IV injection was assessed by tagging LNPs and mAbs with metal ion tracers that could be quantified by ICP-MS. Results show that DSPE-PEG-mAb rapidly (within 1h) dissociates from LNPs in blood, leading to accelerated LNP clearance. In contrast, mAbs conjugated using PCL-b-PEG remained stably associated with the LNP over the 24h circulation and clearance of the construct. Results are connected to a thermodynamic model that reproduces experimental findings for PEG-anchor(-mAb) shedding in vitro and in vivo. This study identifies anchoring strength as a critical, unconsidered parameter for in vivo performance when conjugating mAbs to LNPs for extrahepatic delivery. Graphical abstract
Brian K. Wilson, Lucas D. Johnson, Jason Liu et al.· bioRxiv· 0 citations
Surface chemistry affects the in vivo behavior of lipid-based nanocarriers, through lipid-PEGylation (polyethylene glycol, PEG), which forms a steric barrier at the nanoparticle interface and extends systemic circulation. This is relevant for clinically approved nanocarriers products, such as liposomes and lipid nanoparticles (LNPs), as many are PEGylated. However, drawbacks have emerged due to the widespread PEG use, including anti-PEG antibodies accelerated blood clearance (ABC) upon repeat dosing, and hypersensitivity reactions (HSRs) such as complement activation-related pseudoallergy (CARPA). Moreover, the steric barrier providing stealth properties hinders cargo delivery by reducing cell interactions and limiting endosomal escape. Numerous studies link lipid-PEG chemistry to these outcomes, although results vary across formulations and remain debated. Here, we review stealth strategies for lipid nanocarriers, focusing on liposomes and LNPs and compare PEG engineering with selected amide-based polymer alternatives. We summarize key PEGylation design levers: lipid-PEG anchor modification, end-group chemistry, chain length and branched architectures, and cleavable linkers aimed at preserving stealth while mitigating immune responses and intracellular delivery barriers. We then examine three amide-containing polymer families (polysarcosine, poly(2-oxazoline)s, and poly(N-vinylamide)s) selected for their PEG-like surface behavior. We relate biological outcomes and delivery efficiency to practical manufacturing factors, synthetic feasibility and the tunability of lipid-polymer conjugates.
Manon Degey, Stefano Pedergnana, B. Evrard et al.· Nanomedicine· 0 citations
Lipid nanoparticles (LNPs) have enabled the clinical translation of various nucleic acid cargos, including messenger RNA. Traditional LNP formulations contain a small proportion of lipids conjugated to poly(ethylene glycol), or PEG, which provides advantages of size control, stability, and extended circulation. However, PEGylation of both LNPs and other nano‐carriers raises challenges to efficient gene delivery, including endogenous production of anti‐PEG antibodies, accelerated clearance of multiple doses of LNPs, and reduced cellular uptake and endosomal escape. Here, we design and characterize a fully PEG‐free LNP platform, stabilized alternatively through the electrostatic adsorption of charged polymeric coatings that leverage principles of layer‐by‐layer self‐assembly. A library of carboxylated polyanions confers stability to non‐PEGylated (nonPEG) LNPs under biological stresses. nonPEG layered LNPs (LLNPs) significantly improve transfection of cancer and immune cells in vitro and generate favorable transfection in vivo via multiple routes of administration. In particular, nonPEG LLNPs reduce hepatic transfection by an order of magnitude, a major challenge for targeted LNP gene delivery, while maintaining similar circulation and transfection in other organs. Ultimately, this platform offers a highly modular, tunable approach to incorporate various bioactive moieties to achieve PEG‐free extrahepatic gene delivery.
Cholesterol is a key determinant of membrane stability and fluidity in both natural and synthetic lipid systems. Although widely used, its content varies greatly among commercial liposomes, and its regulatory effects on biological interactions remain insufficiently understood. Here, we reveal that cholesterol content critically governs the protein corona composition and binding affinity of liposomes, thereby dictating their cellular interactions and in vivo fate. Complement proteins exhibit a biphasic adsorption pattern that modulates monocyte and Kupffer cell uptake and systemic clearance. High cholesterol levels enrich apolipoproteins, particularly ApoE, promoting hepatocyte targeting, liver accumulation, and enhanced uptake by dendritic cells in blood and spleen. In contrast, low-cholesterol liposomes form albumin-dominated coronas that favor uptake by liver sinusoidal and pulmonary endothelial cells, leading to preferential lung accumulation. Notably, cholesterol-rich liposomes significantly suppress hepatocellular carcinoma progression through improved liver targeting and prolonged circulation. These findings identify cholesterol as a pivotal regulator of serum protein interactions and biodistribution, providing valuable insights for the rational design of efficient, organ-specific liposomal drug delivery systems.
Fangqin Fu, Yuting Ge, Xuemei Hu et al.· ACS Nano· 0 citations
Lipid nanoparticles (LNPs) are the leading platform for mRNA delivery, with their in vivo performance governed by lipid composition and colloidal stability. While anionic helper lipids can bias LNP expression toward the spleen, weak RNA-lipid interactions during purification often induce nanoparticle rearrangement and reduced activity. These stability limitations effectively narrow the accessible formulation design and screening space, leaving large regions of anionic compositional space underexplored. Here, we extend our cleavable crosslinking strategy to stabilize anionic LNPs without replacing the primary lipid constituents of the parent LNP formulation. By tuning the lengths of the cholesterol-derived acid-cleavable crosslinker and PEG-diamine, we achieved balanced structural stability. The optimized crosslinked formulation exhibited a significant increase in splenic mRNA expression at 12 h compared to the uncrosslinked LNPs. Notably, 33.2% of CD45+ tdTomato+ cells in the spleen were identified as T cells. Mechanistic analyses suggest that controlled mRNA release and altered intracellular processing contribute to the improved transfection efficiency. Together, these findings define a tunable crosslinking window that expands the accessible design landscape for tissue- and cell-specific mRNA delivery.
Yunhe Su, Joseph Choy, Xiang Liu et al.· ACS Applied Materials and In...· 0 citations
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