Brain-targeted drug delivery poses a great challenge due to the blood-brain barrier (BBB). In a previous study, we have developed a peptide-modified stealth liposome (SP-sLip) to enhance BBB penetration via the adsorption of apolipoproteins in plasma. SP is an 11-amino acid peptide derived from 25 to 35 of the Amyloid β peptide (Aβ1-42), which is the nature ligand of apolipoproteins. Although SP-sLip exhibited efficient brain targeting performance, self-aggregation and instability of storage limited its further application. In this study, we developed a D-peptide ligand according to the reverse sequence of SP with D-amino acids, known as DSP, to solve the problems. Notably, DSP exhibited a reduced tendency for self-aggregation and exceptional stability compared to the SP peptide. Furthermore, compared to SP-sLip, DSP-modified sLip (DSP-sLip) demonstrated enhanced stability (>2 weeks), prolonged blood circulation (AUC increased 44.4%), reduced liver and spleen accumulation (reduced by 2.23 times and 1.86 times) with comparable brain-targeting efficiency. Similar to SP-sLip, DSP-sLip selectively adsorbed apolipoprotein A1, E, and J in the blood to form functionalized protein corona, thus crossing BBB via apolipoprotein receptor-mediated transcytosis. These findings underscored the importance of ligand stability in the in vitro and in vivo performance of brain-targeted liposomes, therefore paving the way for the design and optimization of efficient and stable nanocarriers.

This review describes the formation of a protein corona (or its absence) on different classes of nanoparticles, its basic principles, and its consequences for nanomedicine. For this purpose, it describes general concepts to control (guide/minimize) the interaction between artificial nanoparticles and plasma proteins to reduce protein corona formation. Thereafter, methods for the qualitative or quantitative determination of protein corona formation are presented, as well as the properties of nanoparticle surfaces, which are relevant for protein corona prevention (or formation). Thereby especially the role of grafting density of hydrophilic polymers on the surface of the nanoparticle is discussed to prevent the formation of a protein corona. In this context also the potential of detergents (surfactants) for a temporary modification as well as grafting-to and grafting-from approaches for a permanent modification of the surface are discussed. The review concludes by highlighting several promising avenues. This includes (i) the use of nanoparticles without protein corona for active targeting, (ii) the use of synthetic nanoparticles without protein corona formation to address the immune system, (iii) the recollection of nanoparticles with a defined protein corona after in vivo application to sample the blood proteome and (iv) further concepts to reduce protein corona formation.

Upconversion nanoparticles (UCNPs) are materials that provide unique advantages for biomedical applications. There are constantly emerging customized UCNPs with varying compositions, coatings, and upconversion mechanisms. Cellular uptake is a key parameter for the biological application of UCNPs. Uptake experiments have yielded highly varying results, and correlating trends between cellular uptake with different types of UCNP coatings remains challenging. In this report, the impact of surface polymer coatings on the formation of protein coronas and subsequent cellular uptake of UCNPs by macrophages and cancer cells was investigated. Luminescence confocal microscopy and elemental analysis techniques were used to evaluate the different coatings for internalization within cells. Pathway inhibitors were used to unravel the specific internalization mechanisms of polymer-coated UCNPs. Coatings were chosen as the most promising for colloidal stability, conjugation chemistry, and biomedical applications. PIMA-PEG (poly(isobutylene-alt-maleic) anhydride with polyethylene glycol)-coated UCNPs were found to have low cytotoxicity, low uptake by macrophages (when compared with PEI, poly(ethylenimine)), and sufficient uptake by tumor cells for surface-loaded drug delivery applications. Inductively coupled plasma-optical emission spectroscopy (ICP-OES) studies revealed that PIMA-coated NPs were preferentially internalized by the clathrin- and caveolar-independent pathways, with a preference for clathrin-mediated uptake at longer time points. PMAO-PEG (poly(maleic anhydride-alt-1-octadecene) with polyethylene glycol)-coated UCNPs were internalized by energy-dependent pathways, while PAA- (poly(acrylic acid)) and PEI-coated NPs were internalized by multifactorial mechanisms of internalization. The results indicate that copolymers of PIMA-PEG coatings on UCNPs were well suited for the next-generation of biomedical applications.

Liposomes are versatile drug delivery systems in clinical use for cancer and many other diseases. Unfortunately, PEGylated liposomal doxorubicin (sLip/DOX) exhibits serious dose-limiting cutaneous toxicities, which are closely related to the extravascular accumulation of sLip/DOX in the dermis. No clinical interventions have been proposed for cutaneous toxicities due to the elusive transport pathways. Herein, we showed that the reciprocal interaction between liposomes and neutrophils played pivotal roles in liposome extravasation into the dermis. Neutrophils captured liposomes via the complement receptor 3 (CD11b/CD18) recognizing the fragment of complement component C3 (iC3b) deposited on the liposomal surface. Uptake of liposomes also activated neutrophils to induce CD11b upregulation and enhanced the ability of neutrophils to migrate outside the capillaries. Furthermore, inhibition of complement activation either by CRIg-L-FH (a C3b/iC3b targeted complement inhibitor) or blocking the phosphate negative charge in mPEG-DSPE could significantly reduce liposome uptake by neutrophils and alleviate the cutaneous accumulation of liposomes. These results validated the liposome extravasation pathway mediated by neutrophils and provided potential solutions to the devastating cutaneous toxicities occurring during sLip/DOX treatment.

Liposomes represent one of the most extensively studied nano-carriers due to their potential in targeted drug delivery. However, the complex in vivo fate, particularly under pathological conditions, presents challenges for clinical translation of liposomal therapeutics. Liver serves as the most important organ for liposome accumulation and metabolism. Unfortunately, the fate of liposomes under pathological liver conditions has been significantly overlooked. This study aimed to investigate the in vivo pharmacokinetic profile and biodistribution profile of liposomes under drug-induced liver injury (DILI) conditions. Two classic DILI animal models, i.e. acetaminophen-induced acute liver injury (AILI) and triptolide-induced subacute liver injury (TILI), were established to observe the effect of pathological liver conditions on the in vivo performance of liposomes. The study revealed significant changes in the in vivo fate of liposomes following DILI, including prolonged blood circulation and enhanced hepatic accumulation of liposomes. Changes in the composition of plasma proteins and mononuclear phagocyte system (MPS)-related cell subpopulations collectively led to the altered in vivo fate of liposomes under liver injury conditions. Despite liver injury, macrophages remained the primary cells responsible for liposomes uptake in liver, with the recruited monocyte-derived macrophages exhibiting enhanced ability to phagocytose liposomes under pathological conditions. These findings indicated that high capture of liposomes by the recruited hepatic macrophages not only offered potential solutions for targeted delivery, but also warned the clinical application of patients under pathological liver conditions.

Antithrombin (AT) is a glycoprotein produced by the liver and a principal antagonist of active clotting proteases. A deficit in AT function leads to AT qualitative deficiency, challenging to diagnose. Here we report that active AT may travel physiosorbed on the surface of plasma extracellular vesicles (EVs), contributing to form the \"EV-protein corona.\" The corona is enriched in specific AT glycoforms, thus suggesting glycosylation to play a key role in AT partitioning between EVs and plasma. Differences in AT glycoform composition of the corona of EVs separated from plasma of healthy and AT qualitative deficiency-affected subjects were also noticed. This suggests deconstructing the plasma into its nanostructured components, as EVs, could suggest novel directions to unravel pathophysiological mechanisms.

In the last years, extracellular vesicles (EVs), secreted by various cells and body fluids have shown extreme potential in biomedical applications. Increasing number of studies suggest that a protein corona could adhere to the surface of EVs which can have a fundamental effect on their function, targeting and therapeutical efficacy. However, removing and identifying these corona members is currently a challenging task to achieve. In this study we have employed red blood cell-derived extracellular vesicles (REVs) as a model system and three membrane active antimicrobial peptides (AMPs), LL-37, FK-16 and CM15, to test whether they can be used to remove protein corona members from the surface of vesicles. These AMPs were reported to preferentially exert their membrane-related activity via one of the common helical surface-covering models and do not significantly affect the interior of lipid bilayer bodies. The interaction between the peptides and the REVs was followed by biophysical techniques, such as flow-linear dichroism spectroscopy which provided the effective applicable peptide concentration for protein removal. REV samples were then subjected to subsequent size exclusion chromatography and to proteomics analysis. Based on the comparison of control REVs with the peptide treated samples, seventeen proteins were identified as external protein corona members. From the three investigated AMPs, FK-16 can be considered as the best candidate to further optimize EV-related applicability of AMPs. Our results on the REV model system envisage that membrane active peptides may become a useful set of tools in engineering and modifying surfaces of EVs and other lipid-based natural particles.

Background: Nanoparticles conjugated with fluorescent probes have versatile applications, serving not only for targeted fluorescent imaging but also for evaluating the in vivo profiles of designed nanoparticles. However, the relationship between fluorophore density and nanoparticle behavior remains unexplored. Methods: The IR783-modified liposomes (IR783-sLip) were prepared through a modified ethanol injection and extrusion method. The cellular uptake efficiency of IR783-sLip was characterized by flow cytometry and fluorescence microscope imaging. The effects of IR783 density on liposomal in vivo behavior were investigated by pharmacokinetic studies, biodistribution studies, and in vivo imaging. The constitution of protein corona was analyzed by the Western blot assay. Results: Dense IR783 modification improved cellular uptake of liposomes in vitro but hindered their blood retention and tumor imaging performance in vivo. We found a correlation between IR783 density and protein corona absorption, particularly IgM, which significantly impacted the liposome performance. Meanwhile, we observed that increasing IR783 density did not consistently improve the effectiveness of tumor imaging. Conclusions: Increasing the density of modified IR783 on liposomes is not always beneficial for tumor near-infrared (NIR) imaging yield. It is not advisable to prematurely evaluate novel nanomaterials through fluorescence dye conjugation without carefully optimizing the density of the modifications.

Abundant biochar colloids (BCs) produced from a wide range of feedstocks, resulting from forest fires, agricultural production, and environmental restoration, exhibit varying aggregation behaviors influenced by feedstock type and natural organic matter. However, the impact of natural organic matter on the colloidal stability of BCs derived from different feedstocks remains poorly understood. In this study, six selected biochars were derived from various feedstocks as follows: sewage sludge (SS), rice husk (RH), oil seed rape straw pellets (OSR), wheat straw pellets (WS), miscanthus straw pellets (MS) and softwood pellets (SW). The colloidal stability of BCs, with the exogenous addition of organic matter, was further determined. The order of critical coagulation concentrations (CCCs) of BCs with the presence of humic acid (HA) was as follows: RH (989.48 mM) < MS (1084.69 mM) < SS (1149.76 mM) < WS (1338.99 mM) < OSR (2402.98 mM) < SW (3151.32 mM). This order was significantly positively correlated with the specific surface area and negatively correlated with the ash content of the bulk biochar. Compared to HA, bovine serum albumin (BSA) more effectively inhibited the aggregation behavior of BCs due to steric hindrance. The initial aggregation rate constant (k) of BCs at 3000 mM NaCl was as follows: MS (0.238 nm/s) > OSR (0.142 nm/s) > WS (0.128 nm/s) > SS (0.126 nm/s) > RH (0.118 nm/s) > SW (0.112 nm/s). The stabilizing effects of BSA on biochar colloids were independent of the physicochemical properties of bulk biochar. In the presence of BSA, a thin layer of protein corona significantly enhanced the stability of biochar colloids, particularly the BCs derived from MS. Our results underscore the importance of considering feedstock resources and natural organic matter type when assessing the aggregation and potential risks of BCs in aquatic systems.

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.