MRI-based microthrombi detection in stroke with polydopamine iron oxide.

In acute ischemic stroke, even when successful recanalization is obtained, downstream microcirculation may still be obstructed by microvascular thrombosis, which is associated with compromised brain reperfusion and cognitive decline. Identifying these microthrombi through non-invasive methods remains challenging. We developed the PHySIOMIC (Polydopamine Hybridized Self-assembled Iron Oxide Mussel Inspired Clusters), a MRI-based contrast agent that unmasks these microthrombi. In a mouse model of thromboembolic ischemic stroke, our findings demonstrate that the PHySIOMIC generate a distinct hypointense signal on T2*-weighted MRI in the presence of microthrombi, that correlates with the lesion areas observed 24 hours post-stroke. Our microfluidic studies reveal the role of fibrinogen in the protein corona for the thrombosis targeting properties. Finally, we observe the biodegradation and biocompatibility of these particles. This work demonstrates that the PHySIOMIC particles offer an innovative and valuable tool for non-invasive in vivo diagnosis and monitoring of microthrombi, using MRI during ischemic stroke.

Cellular uptake and in vitro antibacterial activity of lipid-based nanoantibiotics are influenced by protein corona.

Bacteria have evolved survival mechanisms that enable them to live within host cells, triggering persistent intracellular infections that present significant clinical challenges due to the inability for conventional antibiotics to permeate cell membranes. In recent years, antibiotic nanocarriers or 'nanoantibiotics' have presented a promising strategy for overcoming intracellular infections by facilitating cellular uptake of antibiotics, thus improving targeting to the bacteria. However, prior to reaching host cells, nanocarriers experience interactions with proteins that form a corona and alter their physiological response. The influence of this protein corona on the cellular uptake, drug release and efficacy of nanoantibiotics for intracellular infections is poorly understood and commonly overlooked in preclinical studies. In this study, protein corona influence on cellular uptake was investigated for two nanoparticles; liposomes and cubosomes in macrophage and epithelial cells that are commonly infected with pathogens. Studies were conducted in presence of fetal bovine serum (FBS) to form a biologically relevant protein corona in an in vitro setting. Protein corona impact on cellular uptake was shown to be nanoparticle-dependent, where reduced internalization was observed for liposomes, the opposite was observed for cubosomes. Subsequently, vancomycin-loaded cubosomes were explored for their drug delivery performance against intracellular small colony variants of Staphylococcus aureus. We demonstrated improved bacterial killing in macrophages, with greater reduction in bacterial viability upon internalization of cubosomes mediated by the protein corona. However, no differences in efficacy were observed in epithelial cells. Thus, this study provides insights and evidence to the role of protein corona in modulating the performance of nanoparticles in a dynamic manner; these findings will facilitate improved understanding and translation of future investigations from in vitro to in vivo.

Nanoplastic Size and Surface Chemistry Dictate Decoration by Human Saliva Proteins.

Environmental pollution with plastic polymers has become a global problem, leaving no continent and habitat unaffected. Plastic waste is broken down into smaller parts by environmental factors, which generate micro- and nanoplastic particles (MNPPs), ultimately ending up in the human food chain. Before entering the human body, MNPPs make their first contact with saliva in the human mouth. However, it is unknown what proteins attach to plastic particles and whether such protein corona formation is affected by the particle's biophysical properties. To this end, we employed polystyrene MNPPs of two different sizes and three different charges and incubated them individually with saliva donated by healthy human volunteers. Particle zeta potential and size analyses were performed using dynamic light scattering complemented by nanoliquid chromatography high-resolution mass spectrometry (nLC/HRMS) to qualitatively and quantitatively reveal the protein soft and hard corona for each particle type. Notably, protein profiles and relative quantities were dictated by plastic particle size and charge, which in turn affected their hydrodynamic size, polydispersity, and zeta potential. Strikingly, we provide evidence of the latter to be dynamic processes depending on exposure times. Smaller particles seemed to be more reactive with the surrounding proteins, and cultures of the particles with five different cell lines (HeLa, HEK293, A549, HepG2, and HaCaT) indicated protein corona effects on cellular metabolic activity and genotoxicity. In summary, our data suggest nanoplastic size and surface chemistry dictate the decoration by human saliva proteins, with important implications for MNPP uptake in humans.

An antifouling membrane-fusogenic liposome for effective intracellular delivery in vivo.

The membrane-fusion-based internalization without lysosomal entrapment is advantageous for intracellular delivery over endocytosis. However, protein corona formed on the membrane-fusogenic liposome surface converts its membrane-fusion performance to lysosome-dependent endocytosis, causing poorer delivery efficiency in biological conditions. Herein, we develop an antifouling membrane-fusogenic liposome for effective intracellular delivery in vivo. Leveraging specific lipid composition at an optimized ratio, such antifouling membrane-fusogenic liposome facilitates fusion capacity even in protein-rich conditions, attributed to the copious zwitterionic phosphorylcholine groups for protein-adsorption resistance. Consequently, the antifouling membrane-fusogenic liposome demonstrates robust membrane-fusion-mediated delivery in the medium with up to 38% fetal bovine serum, outclassing two traditional membrane-fusogenic liposomes effective at 4% and 6% concentrations. When injected into mice, antifouling membrane-fusogenic liposomes can keep their membrane-fusion-transportation behaviors, thereby achieving efficient luciferase transfection and enhancing gene-editing-mediated viral inhibition. This study provides a promising tool for effective intracellular delivery under complex physiological environments, enlightening future nanomedicine design.

[Progress in the mechanism and influencing factors of the formation of protein corona on nanoparticle surfaces].

Nanoparticles, as a novel material, have a wide range of applications in the food and biomedical fields. Nanoparticles spontaneously adsorb proteins in the biological environment, and tens or even hundreds of proteins can form protein corona on the surface of nanoparticles. The formation of protein corona on the surface of nanoparticles is one of the key factors affecting the stability, biocompatibility, targeting, and drug release properties of nanoparticles. The formation mechanism of protein corona is affected by a variety of factors, including the surface chemical properties, sizes, and shapes of nanoparticles and the types, concentrations, and pH of proteins. Studies have shown that the protein structure is associated with protein distribution on the nanoparticle surface, while the protein conformation affects the binding mode and stability of the protein on the nanoparticle surface. Since the mechanism of the formation of protein corona on the surface of nanoparticles is complex, the roles of multiple factors need to be considered comprehensively. Understanding the mechanisms and influencing factors of the formation of protein corona will help us to understand the process of protein corona formation and control the formation of protein corona for specific needs. In this paper, we summarize the recent studies on the mechanisms and influencing factors of the formation of protein corona on the surface of nanoparticles, with a view to providing a theoretical basis for in-depth research on protein corona.

Inflammatory bowel disease alters in vivo distribution of orally administrated nanoparticles: Revealing via SERS tag labeling technique.

Nanoparticles (NPs) could be uptake orally and exposed to digestive tract through various sources such as particulate pollutant, nanomedicine and food additive. Inflammatory bowel disease (IBD), as a global disease, induced disruption of the intestinal mucosal barrier and thus altered in vivo distribution of NPs as a possible consequence. However, related information was relatively scarce. Herein, in vivo distribution of typical silica (SiO2) and titania (TiO2) NPs was investigated in healthy and IBD models at cell and animal levels via a surface-enhanced Raman scattering (SERS) tag labeling technique. The labeled NPs were composed of gold SERS tag core and SiO2 (or TiO2) shell, demonstrating sensitive and characteristic SERS signals ideal to trace the NPs in vivo. Cell SERS mapping revealed that protein corona from IBD intestinal fluid decreased uptake of NPs by lipopolysaccharide-induced RAW264.7 cells compared with normal intestinal fluid protein corona. SERS signal detection combined with inductively coupled plasma mass spectrometry (ICP-MS) analysis of mouse tissues (heart, liver, spleen, lung and kidney) indicated that both NPs tended to accumulate in lung specifically after oral administration for IBD mouse (6 out of 20 mice for SiO2 and 4 out of 16 mice for TiO2 were detected in lung). Comparatively, no NP signals were detected in all tissues from healthy mice. These findings suggested that there might be a greater risk associated with the oral uptake of NPs in IBD patients due to altered in vivo distribution of NPs.

Impact of Protein Corona Formation and Polystyrene Nanoparticle Functionalisation on the Interaction with Dynamic Biomimetic Membranes Comprising of Integrin.

Plastics, omnipresent in the environment, have become a global concern due to their durability and limited biodegradability, especially in the form of microparticles and nanoparticles. Polystyrene (PS), a key plastic type, is susceptible to fragmentation and surface alterations induced by environmental factors or industrial processes. With widespread human exposure through pollution and diverse industrial applications, understanding the physiological impact of PS, particularly in nanoparticle form (PS-NPs), is crucial. This study focuses on the interaction of PS-NPs with model blood proteins, emphasising the formation of a protein corona, and explores the subsequent contact with platelet membrane mimetics using experimental and theoretical approaches. The investigation involves αIIbß3-expressing cells and biomimetic membranes, enabling real-time and label-free nanoscale precision. By employing quartz-crystal microbalance with dissipation monitoring studies, the concentration-dependent cytotoxic effects of differently functionalised ~210 nm PS-NPs on HEK293 cells overexpressing αIIbß3 are evaluated in detail. The study unveils insights into the molecular details of PS-NP interaction with supported lipid bilayers, demonstrating that a protein corona formed in the presence of exemplary blood proteins offers protection against membrane damage, mitigating PS-NP cytotoxicity.

Perspectives on Protein-Nanoparticle Interactions at the In Vivo Level.

The distinct features of nanoparticles have provided a vast opportunity of developing new diagnosis and therapy strategies for miscellaneous diseases. Although a few nanomedicines are available in the market or in the translation stage, many important issues are still unsolved. When entering the body, nanomaterials will be quickly coated by proteins from their surroundings, forming a corona on their surface, the so-called protein corona. Studies have shown that the protein corona has many important biological implications, particularly at the in vivo level. For example, they can promote the immune system to rapidly clear these outer materials and prevent nanoparticles from playing their designed role in therapy. In this Perspective, the available techniques for characterizing protein-nanoparticle interactions are critically summarized. Effects of nanoparticle properties and environmental factors on protein corona formation, which can further regulate the in vivo fate of nanoparticles, are highlighted and discussed. Moreover, recent progress on the biomedical application of protein corona-engineered nanoparticles is introduced, and future directions for this important yet challenging research area are also briefly discussed.

Controlling nanoparticle-induced endothelial leakiness with the protein corona.

Understanding nanoparticle-cell interaction is essential for advancing research in nanomedicine and nanotoxicology. Apart from the transcytotic pathway mediated by cellular recognition and energetics, nanoparticles (including nanomedicines) may harness the paracellular route for their transport by inducing endothelial leakiness at cadherin junctions. This phenomenon, termed as NanoEL, is correlated with the physicochemical properties of the nanoparticles in close association with cellular signalling, membrane mechanics, as well as cytoskeletal remodelling. However, nanoparticles in biological systems are transformed by the ubiquitous protein corona and yet the potential effect of the protein corona on NanoEL remains unclear. Using confocal fluorescence microscopy, biolayer interferometry, transwell, toxicity, and molecular inhibition assays, complemented by molecular docking, here we reveal the minimal to significant effects of the anionic human serum albumin and fibrinogen, the charge neutral immunoglobulin G as well as the cationic lysozyme on negating gold nanoparticle-induced endothelial leakiness in vitro and in vivo. This study suggests that nanoparticle-cadherin interaction and hence the extent of NanoEL may be partially controlled by pre-exposing the nanoparticles to plasma proteins of specific charge and topology to facilitate their biomedical applications.

Deciphering the monocyte-targeting mechanisms of PEGylated cationic liposomes by investigating the biomolecular corona.

Cationic liposomes specifically target monocytes in blood, rendering them promising drug-delivery tools for cancer immunotherapy, vaccines, and therapies for monocytic leukaemia. The mechanism behind this monocyte targeting ability is, however, not understood, but may involve plasma proteins adsorbed on the liposomal surfaces. To shed light on this, we investigated the biomolecular corona of three different types of PEGylated cationic liposomes, finding all of them to adsorb hyaluronan-associated proteins and proteoglycans upon incubation in human blood plasma. This prompted us to study the role of the TLR4 co-receptors CD44 and CD14, both involved in signalling and uptake pathways of proteoglycans and glycosaminoglycans. We found that separate inhibition of each of these receptors hampered the monocyte uptake of the liposomes in whole human blood. Based on clues from the biomolecular corona, we have thus identified two receptors involved in the targeting and uptake of cationic liposomes in monocytes, in turn suggesting that certain proteoglycans and glycosaminoglycans may serve as monocyte-targeting opsonins. This mechanistic knowledge may pave the way for rational design of future monocyte-targeting drug-delivery platforms.

Engineering the protein corona: Strategies, effects, and future directions in nanoparticle therapeutics.

Nanoparticles (NPs) serve as versatile delivery systems for anticancer, antibacterial, and antioxidant agents. The manipulation of protein-NP interactions within biological systems is crucial to the application of NPs in drug delivery and cancer nanotherapeutics. The protein corona (PC) that forms on the surface of NPs is the interface between biomacromolecules and NPs and significantly influences their pharmacokinetics and pharmacodynamics. Upon encountering proteins, NPs undergo surface alterations that facilitate their clearance from circulation by the mononuclear phagocytic system (MPS). PC behavior depends largely on the biological microenvironment and the physicochemical properties of the NPs. This review describes various strategies employed to engineer PC compositions on NP surfaces. The effects of NP characteristics such as size, shape, surface modification and protein precoating on PC performance were explored. In addition, this study addresses these challenges and guides the future directions of this evolving field.

Molecular Serum Albumin Unmask Nanobio Properties of Molecular Graphenes in Shungite Carbon Nanoparticles.

Serum albumin is a popular macromolecule for studying the effect of proteins on the colloidal stability of nanoparticle (NP) dispersions, as well as the protein-nanoparticle interaction and protein corona formation. In this work, we analyze the specific conformation-dependent phase, redox, and fatty acid delivery properties of bovine albumin in the presence of shungite carbon (ShC) molecular graphenes stabilized in aqueous dispersions in the form of NPs in order to reveal the features of NP bioactivity. The formation of NP complexes with proteins (protein corona around NP) affects the transport properties of albumin for the delivery of fatty acids. Being acceptors of electrons and ligands, ShC NPs are capable of exhibiting both their own biological activity and significantly affecting conformational and phase transformations in protein systems.

The biomolecular corona of nanomedicines: effects on nanomedicine outcomes and emerging opportunities.

Upon administration, nanomedicines adsorb a corona of endogenous biomolecules on their surface, which can affect nanomedicine interactions with cells, targeting, and efficacy. While strategies to reduce protein binding are available, the high selectivity of the adsorbed corona is enabling novel applications, such as for biomarker discovery and rare protein identification. Additionally, the adsorbed molecules can promote interactions with specific cell receptors, thus conferring the nanomedicine new endogenous targeting capabilities. This has been reported for Onpattro, a lipid nanoparticle targeting the hepatocytes via apolipoproteins in its corona. Recently, selective organ-targeting (SORT) nanoparticles have been proposed, which exploit corona-mediated interactions to deliver nanoparticles outside the liver. Strategies for corona seeding and corona engineering are emerging to increase the selectivity of similar endogenous targeting mechanisms.

Unveiling the challenges of engineered protein corona from the proteins' perspective.

It is well known that protein corona affects the "biological identity" of nanoparticles (NPs), which has been seen as both a challenge and an opportunity. Approaches have moved from avoiding protein adsorption to trying to direct it, taking advantage of the formation of a protein corona to favorably modify the pharmacokinetic parameters of NPs. Although promising, the results obtained with engineered NPs still need to be completely understood. While much effort has been put into understanding how the surface of nanomaterials affects protein absorption, less is known about how proteins can affect corona formation due to their specific physicochemical properties. This review addresses this knowledge gap, examining key protein factors influencing corona formation, highlighting current challenges in studying protein-protein interactions, and discussing future perspectives in the field.

Regulating protein corona on nanovesicles by glycosylated polyhydroxy polymer modification for efficient drug delivery.

The dynamic protein corona formed on nanocarriers has been revealed to strongly affect their in vivo behaviors. Precisely manipulating the formation of protein corona on nanocarriers may provide an alternative impetus for specific drug delivery. Herein, we explore the role of glycosylated polyhydroxy polymer-modified nanovesicles (CP-LVs) with different amino/hydroxyl ratios in protein corona formation and evolution. CP-LVs with an amino/hydroxyl ratio of approximately 0.4 (CP1-LVs) are found to efficiently suppress immunoglobulin adsorption in blood and livers, resulting in prolonged circulation. Moreover, CP1-LVs adsorb abundant tumor distinctive proteins, such as CD44 and osteopontin in tumor interstitial fluids, mediating selective tumor cell internalization. The proteins corona transformation specific to the environment appears to be affected by the electrostatic interaction between CP-LVs and proteins with diverse isoelectric points. Benefiting from surface modification-mediated protein corona regulation, paclitaxel-loaded CP1-LVs demonstrate superior antitumor efficacy to PEGylated liposomes. Our work offers a perspective on rational surface-design of nanocarriers to modulate the protein corona formation for efficient drug delivery.

The in vitro gastrointestinal digestion-associated protein corona of polystyrene nano- and microplastics increases their uptake by human THP-1-derived macrophages.

BACKGROUND: Micro- and nanoplastics (MNPs) represent one of the most widespread environmental pollutants of the twenty-first century to which all humans are orally exposed. Upon ingestion, MNPs pass harsh biochemical conditions within the gastrointestinal tract, causing a unique protein corona on the MNP surface. Little is known about the digestion-associated protein corona and its impact on the cellular uptake of MNPs. Here, we systematically studied the influence of gastrointestinal digestion on the cellular uptake of neutral and charged polystyrene MNPs using THP-1-derived macrophages. RESULTS: The protein corona composition was quantified using LC‒MS-MS-based proteomics, and the cellular uptake of MNPs was determined using flow cytometry and confocal microscopy. Gastrointestinal digestion resulted in a distinct protein corona on MNPs that was retained in serum-containing cell culture medium. Digestion increased the uptake of uncharged MNPs below 500 nm by 4.0-6.1-fold but did not affect the uptake of larger sized or charged MNPs. Forty proteins showed a good correlation between protein abundance and MNP uptake, including coagulation factors, apolipoproteins and vitronectin. CONCLUSION: This study provides quantitative data on the presence of gastrointestinal proteins on MNPs and relates this to cellular uptake, underpinning the need to include the protein corona in hazard assessment of MNPs.

Impact of Whey Protein Corona Formation around TiO2 Nanoparticles on Their Physiochemical Properties and Gastrointestinal Fate.

Previously, we found that whey proteins form biomolecular coronas around titanium dioxide (TiO2) nanoparticles. Here, the gastrointestinal fate of whey protein-coated TiO2 nanoparticles and their interactions with gut microbiota were investigated. The antioxidant activity of protein-coated nanoparticles was enhanced after simulated digestion. The structure of the whey proteins was changed after they adsorbed to the surfaces of the TiO2 nanoparticles, which reduced their hydrolysis under simulated gastrointestinal conditions. The presence of protein coronas also regulated the impact of the TiO2 nanoparticles on colonic fermentation, including promoting the production of short-chain fatty acids. Bare TiO2 nanoparticles significantly increased the proportion of harmful bacteria and decreased the proportion of beneficial bacteria, but the presence of protein coronas alleviated this effect. In particular, the proportion of beneficial bacteria, such as Bacteroides and Bifidobacterium, was enhanced for the coated nanoparticles. Our results suggest that the formation of a whey protein corona around TiO2 nanoparticles may have beneficial effects on their behavior within the colon. This study provides valuable new insights into the potential impact of protein coronas on the gastrointestinal fate of inorganic nanoparticles.

Protein Coronas Derived from Mucus Act as Both Spear and Shield to Regulate Transferrin Functionalized Nanoparticle Transcellular Transport in Enterocytes.

The epithelial mucosa is a key biological barrier faced by gastrointestinal, intraoral, intranasal, ocular, and vaginal drug delivery. Ligand-modified nanoparticles demonstrate excellent ability on this process, but their efficacy is diminished by the formation of protein coronas (PCs) when they interact with biological matrices. PCs are broadly implicated in affecting the fate of NPs in vivo and in vitro, yet few studies have investigated PCs formed during interactions of NPs with the epithelial mucosa, especially mucus. In this study, we constructed transferrin modified NPs (Tf-NPs) as a model and explored the mechanisms and effects that epithelial mucosa had on PCs formation and the subsequent impact on the transcellular transport of Tf-NPs. In mucus-secreting cells, Tf-NPs adsorbed more proteins from the mucus layers, which masked, displaced, and dampened the active targeting effects of Tf-NPs, thereby weakening endocytosis and transcellular transport efficiencies. In mucus-free cells, Tf-NPs adsorbed more proteins during intracellular trafficking, which enhanced transcytosis related functions. Inspired by soft coronas and artificial biomimetic membranes, we used mucin as an "active PC" to precoat Tf-NPs (M@Tf-NPs), which limited the negative impacts of "passive PCs" formed during interface with the epithelial mucosa and improved favorable routes of endocytosis. M@Tf-NPs adsorbed more proteins associated with endoplasmic reticulum-Golgi functions, prompting enhanced intracellular transport and exocytosis. In summary, mucus shielded against the absorption of Tf-NPs, but also could be employed as a spear to break through the epithelial mucosa barrier. These findings offer a theoretical foundation and design platform to enhance the efficiency of oral-administered nanomedicines.

Cellular uptake and in vivo distribution of mesenchymal-stem-cell-derived extracellular vesicles are protein corona dependent.

Extracellular vesicles (EVs) derived from mesenchymal stem cells are promising nanotherapeutics in liver diseases due to their regenerative and immunomodulatory properties. Nevertheless, a concern has been raised regarding the rapid clearance of exogenous EVs by phagocytic cells. Here we explore the impact of protein corona on EVs derived from two culturing conditions in which specific proteins acquired from media were simultaneously adsorbed on the EV surface. Additionally, by incubating EVs with serum, simulating protein corona formation upon systemic delivery, further resolved protein corona-EV complex patterns were investigated. Our findings reveal the potential influences of corona composition on EVs under in vitro conditions and their in vivo kinetics. Our data suggest that bound albumin creates an EV signature that can retarget EVs from hepatic macrophages. This results in markedly improved cellular uptake by hepatocytes, liver sinusoidal endothelial cells and hepatic stellate cells. This phenomenon can be applied as a camouflage strategy by precoating EVs with albumin to fabricate the albumin-enriched protein corona-EV complex, enhancing non-phagocytic uptake in the liver. This work addresses a critical challenge facing intravenously administered EVs for liver therapy by tailoring the protein corona-EV complex for liver cell targeting and immune evasion.

Overcoming colloidal nanoparticle aggregation in biological milieu for cancer therapeutic delivery: Perspectives of materials and particle design.

Nanoparticles as cancer therapeutic carrier fail in clinical translation due to complex biological environments in vivo consisting of electrolytes and proteins which render nanoparticle aggregation and unable to reach action site. This review identifies the desirable characteristics of nanoparticles and their constituent materials that prevent aggregation from site of administration (oral, lung, injection) to target site. Oral nanoparticles should ideally be 75-100 nm whereas the size of pulmonary nanoparticles minimally affects their aggregation. Nanoparticles generally should carry excess negative surface charges particularly in fasting state and exert steric hindrance through surface decoration with citrate, anionic surfactants and large polymeric chains (polyethylene glycol and polyvinylpyrrolidone) to prevent aggregation. Anionic as well as cationic nanoparticles are both predisposed to protein corona formation as a function of biological protein isoelectric points. Their nanoparticulate surface composition as such should confer hydrophilicity or steric hindrance to evade protein corona formation or its formation should translate into steric hindrance or surface negative charges to prevent further aggregation. Unexpectedly, smaller and cationic nanoparticles are less prone to aggregation at cancer cell interface favoring endocytosis whereas aggregation is essential to enable nanoparticles retention and subsequent cancer cell uptake in tumor microenvironment. Present studies are largely conducted in vitro with simplified simulated biological media. Future aggregation assessment of nanoparticles in biological fluids that mimic that of patients is imperative to address conflicting materials and designs required as a function of body sites in order to realize the future clinical benefits.