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Cardiovascular disease (CVD) is a group of health conditions affecting the heart and vascular system with very high prevalence and mortality rates. The presence of CVD is characterised by high levels of inflammation which have previously been associated with increased plasma concentrations of N-acetyl neuraminic acid (Neu5Ac). While Neu5Ac has been studied in the context of CVD, Neu5,9Ac2 has not, despite being the second most abundant sialic acid in human plasma. A small-scale pilot study of thirty plasma samples from patients with diagnosed CVD, and thirty age and sex-matched healthy controls, was designed to gain insight into sialic acids as biomarkers for CVD and potential future areas of study. Each sample was assayed for Neu5Ac and Neu5,9Ac2 concentrations. Mean Neu5Ac and Neu5,9Ac2 concentrations were significantly elevated in patients with CVD compared to healthy controls (Neu5Ac: P < 0.001; Neu5,9Ac2: P < 0.04). Receiver operator curve (ROC) analysis indicated that both Neu5Ac and Neu5,9Ac2 have reasonable predictive power for the presence of CVD (Neu5Ac AUC: 0.86; Neu5,9Ac2 AUC: 0.71). However, while Neu5Ac had both good sensitivity (0.82) and specificity (0.81), Neu5,9Ac2 had equivalent specificity (0.81) but very poor sensitivity (0.44). A combination marker of Neu5Ac + Neu5,9Ac2 showed improvement over Neu5Ac alone in terms of predictive power (AUC: 0.93), sensitivity (0.87), and specificity (0.90). Comparison to a known inflammatory marker, high sensitivity c-reactive protein (hs-CRP: P-value: NS, ROC:0.50) was carried out, showing that both Neu5Ac and Neu5,9Ac2 outperformed this marker. Further to this, hs-CRP values were combined with the three different sialic acid markers to determine any effect on the AUC values. A slight improvement in AUC was noted for each of the combinations, with Neu5Ac + Neu5,9Ac2 + hs-CRP giving the best AUC of 0.97 overall. Thus, Neu5Ac would appear to offer good potential as a predictive marker for the presence of CVD, which the addition of Neu5,9Ac2 predictive power improves, with further improvement seen by the addition of hs-CRP.
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Doenças Cardiovasculares , Ácido N-Acetilneuramínico , Humanos , Proteína C-Reativa/análise , Doenças Cardiovasculares/diagnóstico , Projetos Piloto , Ácidos Siálicos/metabolismo , BiomarcadoresRESUMO
BACKGROUND: The widespread use of nano-biomaterials (NBMs) has increased the chance of human exposure. Although ingestion is one of the major routes of exposure to NBMs, it is not thoroughly studied to date. NBMs are expected to be dramatically modified following the transit into the oral-gastric-intestinal (OGI) tract. How these transformations affect their interaction with intestinal cells is still poorly understood. NBMs of different chemical nature-lipid-surfactant nanoparticles (LSNPs), carbon nanoparticles (CNPs), surface modified Fe3O4 nanoparticles (FNPs) and hydroxyapatite nanoparticles (HNPs)-were treated in a simulated human digestive system (SHDS) and then characterised. The biological effects of SHDS-treated and untreated NBMs were evaluated on primary (HCoEpiC) and immortalised (Caco-2, HCT116) epithelial intestinal cells and on an intestinal barrier model. RESULTS: The application of the in vitro SDHS modified the biocompatibility of NBMs on gastrointestinal cells. The differences between SHDS-treated and untreated NBMs could be attributed to the irreversible modification of the NBMs in the SHDS. Aggregation was detected for all NBMs regardless of their chemical nature, while pH- or enzyme-mediated partial degradation was detected for hydroxyapatite or polymer-coated iron oxide nanoparticles and lipid nanoparticles, respectively. The formation of a bio-corona, which contains proteases, was also demonstrated on all the analysed NBMs. In viability assays, undifferentiated primary cells were more sensitive than immortalised cells to digested NBMs, but neither pristine nor treated NBMs affected the intestinal barrier viability and permeability. SHDS-treated NBMs up-regulated the tight junction genes (claudin 3 and 5, occludin, zonula occludens 1) in intestinal barrier, with different patterns between each NBM, and increase the expression of both pro- and anti-inflammatory cytokines (IL-1ß, TNF-α, IL-22, IL-10). Notably, none of these NBMs showed any significant genotoxic effect. CONCLUSIONS: Overall, the results add a piece of evidence on the importance of applying validated in vitro SHDS models for the assessment of NBM intestinal toxicity/biocompatibility. We propose the association of chemical and microscopic characterization, SHDS and in vitro tests on both immortalised and primary cells as a robust screening pipeline useful to monitor the changes in the physico-chemical properties of ingested NBMs and their effects on intestinal cells.
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Materiais Biocompatíveis , Mucosa Intestinal , Materiais Biocompatíveis/farmacologia , Células CACO-2 , Digestão , Humanos , Hidroxiapatitas/farmacologia , Lipossomos , Nanopartículas , Permeabilidade , Junções ÍntimasRESUMO
Manufactured nanomaterials (MNMs) selected from a library of over 120 different MNMs with varied compositions, sizes, and surface coatings were tested by four different laboratories for toxicity by high-throughput/-content (HT/C) techniques. The selected particles comprise 14 MNMs composed of CeO2, Ag, TiO2, ZnO and SiO2 with different coatings and surface characteristics at varying concentrations. The MNMs were tested in different mammalian cell lines at concentrations between 0.5 and 250 µg/mL to link physical-chemical properties to multiple adverse effects. The cell lines are derived from relevant organs such as liver, lung, colon and the immune system. Endpoints such as viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential, lysosomal acidification and steatosis have been studied. Soluble MNMs, Ag and ZnO, were toxic in all cell types. TiO2 and SiO2 MNMs also triggered toxicity in some, but not all, cell types and the cell type-specific effects were influenced by the specific coating and surface modification. CeO2 MNMs were nearly ineffective in our test systems. Differentiated liver cells appear to be most sensitive to MNMs, Whereas most of the investigated MNMs showed no acute toxicity, it became clear that some show adverse effects dependent on the assay and cell line. Hence, it is advised that future nanosafety studies utilise a multi-parametric approach such as HT/C screening to avoid missing signs of toxicity. Furthermore, some of the cell type-specific effects should be followed up in more detail and might also provide an incentive to address potential adverse effects in vivo in the relevant organ.
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Ensaios de Triagem em Larga Escala , Microscopia , Nanoestruturas/toxicidade , Testes de Toxicidade/métodos , Células A549 , Animais , Relação Dose-Resposta a Droga , Células HCT116 , Células Hep G2 , Humanos , Nanopartículas Metálicas/toxicidade , Camundongos , Células RAW 264.7RESUMO
Most inhaled nanomedicines in development are for the treatment of lung disease, yet little is known about their interaction with the respiratory tract lining fluids (RTLFs). Here we combined the use of nano-silica, as a protein concentrator, with label-free snapshot proteomics (LC-MS/MS; key findings confirmed by ELISA) to generate a quantitative profile of the RTLF proteome and provided insight into the evolved corona; information that may be used in future to improve drug targeting to the lungs by inhaled medicines. The asthmatic coronal proteome displayed a reduced contribution of surfactant proteins (SP-A and B) and a higher contribution of α1-antitrypsin. Pathway analysis suggested that asthmatic RTLFs may also be deficient in proteins related to metal handling (e.g. lactoferrin). This study demonstrates how the composition of the corona acquired by inhaled nanoparticles is modified in asthma and suggests depressed mucosal immunity even in mild airway disease.
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Asma/metabolismo , Pulmão/metabolismo , Nanopartículas/metabolismo , Coroa de Proteína/metabolismo , Dióxido de Silício/metabolismo , Administração por Inalação , Humanos , Coroa de Proteína/análise , Proteoma/análise , Proteoma/metabolismo , ProteômicaRESUMO
The bivalve Mytilus galloprovincialis has proven as a suitable model invertebrate for evaluating the potential impact of nanoparticles (NPs) in the marine environment. In particular, in mussels, the immune system represents a sensitive target for different types of NPs. In environmental conditions, both NP intrinsic properties and those of the receiving medium will affect particle behavior and consequent bioavailability/uptake/toxicity. However, the evaluation of the biological effects of NPs requires additional understanding of how, once within the organism, NPs interact at the molecular level with cells in a physiological environment. In mammalian systems, different NPs associate with serum soluble components, organized into a "protein corona", which affects particle interactions with target cells. However, no information is available so far on the interactions of NPs with biological fluids of aquatic organisms. In this work, the influence of hemolymph serum (HS) on the in vitro effects of amino modified polystyrene NPs (PS-NH2) on Mytilus hemocytes was investigated. Hemocytes were incubated with PS-NH2 suspensions in HS (1, 5 and 50µg/mL) and the results were compared with those obtained in ASW medium. Cell functional parameters (lysosomal membrane stability, oxyradical production, phagocytosis) were evaluated, and morphological changes were investigated by TEM. The activation state of the signalling components involved in Mytilus immune response (p38 MAPK and PKC) was determined. The results show that in the presence of HS, PS-NH2 increased cellular damage and ROS production with respect to ASW medium. The effects were apparently mediated by disregulation of p38 MAPK signalling. The formation of a PS-NH2-protein corona in HS was investigated by centrifugation, and 1D- gel electrophoresis and nano-HPLC-ESI-MS/MS. The results identified the Putative C1q domain containing protein (MgC1q6) as the only component of the PS-NH2 hard protein corona in Mytilus hemolymph. These data represent the first evidence for the formation of a NP bio-corona in aquatic organisms and underline the importance of the recognizable biological identity of NPs in physiological exposure medium when testing their potential impact environmental model organisms. Although the results obtained in vitro do not entirely reflect a realistic exposure scenario and the more complex formation of a bio-corona that is likely to occur in vivo, these data will contribute to a better understanding of the effects of NPs in marine invertebrates.
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Hemócitos/efeitos dos fármacos , Mytilus/efeitos dos fármacos , Nanopartículas/toxicidade , Poliestirenos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Cátions/toxicidade , Hemócitos/metabolismo , Hemolinfa/efeitos dos fármacos , Hemolinfa/metabolismo , Mytilus/metabolismo , Proteínas/metabolismoRESUMO
Nanoparticles (NP), when exposed to biological fluids, are coated by specific proteins that form the so-called protein corona. While some adsorbing proteins exchange with the surroundings on a short time scale, described as a "dynamic" corona, others with higher affinity and long-lived interaction with the NP surface form a "hard" corona (HC), which is believed to mediate NP interaction with cellular machineries. In-depth NP protein corona characterization is therefore a necessary step in understanding the relationship between surface layer structure and biological outcomes. In the present work, we evaluate the protein composition and stability over time and we systematically challenge the formed complexes with surfactants. Each challenge is characterized through different physicochemical measurements (dynamic light scattering, ζ-potential, and differential centrifugal sedimentation) alongside proteomic evaluation in titration type experiments (surfactant titration). 100 nm silicon oxide (Si) and 100 nm carboxylated polystyrene (PS-COOH) NPs cloaked by human plasma HC were titrated with 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, zwitterionic), Triton X-100 (nonionic), sodium dodecyl sulfate (SDS, anionic), and dodecyltrimethylammonium bromide (DTAB, cationic) surfactants. Composition and density of HC together with size and ζ-potential of NP-HC complexes were tracked at each step after surfactant titration. Results on Si NP-HC complexes showed that SDS removes most of the HC, while DTAB induces NP agglomeration. Analogous results were obtained for PS NP-HC complexes. Interestingly, CHAPS and Triton X-100, thanks to similar surface binding preferences, enable selective extraction of apolipoprotein AI (ApoAI) from Si NP hard coronas, leaving unaltered the dispersion physicochemical properties. These findings indicate that surfactant titration can enable the study of NP-HC stability through surfactant variation and also selective separation of certain proteins from the HC. This approach thus has an immediate analytical value as well as potential applications in HC engineering.
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Nanopartículas/química , Proteínas/química , Tensoativos/química , Eletroforese em Gel Bidimensional , HumanosRESUMO
Nanoparticles in contact with cells and living organisms generate quite novel interactions at the interface between the nanoparticle surface and the surrounding biological environment. However, a detailed time resolved molecular level description of the evolving interactions as nanoparticles are internalized and trafficked within the cellular environment is still missing and will certainly be required for the emerging arena of nanoparticle-cell interactions to mature. In this paper promising methodologies to map out the time resolved nanoparticle-cell interactome for nanoparticle uptake are discussed. Thus silica coated magnetite nanoparticles are presented to cells and their magnetic properties used to isolate, in a time resolved manner, the organelles containing the nanoparticles. Characterization of the recovered fractions shows that different cell compartments are isolated at different times, in agreement with imaging results on nanoparticle intracellular location. Subsequently the internalized nanoparticles can be further isolated from the recovered organelles, allowing the study of the most tightly nanoparticle-bound biomolecules, analogous to the 'hard corona' that so far has mostly been characterized in extracellular environments. Preliminary data on the recovered nanoparticles suggest that significant portion of the original corona (derived from the serum in which particles are presented to the cells) is preserved as nanoparticles are trafficked through the cells.
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Magnetismo , Nanopartículas , Organelas/metabolismo , Linhagem Celular Tumoral , Humanos , Microscopia Eletrônica de TransmissãoRESUMO
Engineered nanoparticles (NPs) pose a broad spectrum of interesting properties that make them useful for many applications. However, continuous exposure to NPs requires the need to deeply understand the outcomes when these NPs interact with different biological environments. After exposure within (to) these environments, the pristine surfaces of NPs strongly interact with the molecules from the surrounding medium, including metabolites, lipids, glycan, and proteins, forming the so-called protein corona (PC). It is well established that the NP-PC strongly influences the biological fate of various NPs types, including cellular uptake, toxicity, and biodistribution. Thus, for a proper assessment of potential hazards associated with engineered NPs, it is mandatory to study and evaluate the PC that forms around NPs. Herein, we describe protocols in detail for the isolation and characterization of NP-PC complexes and cover the following aspects: 1) isolation protocols for different nanomaterials in a range of exposing media, including magnetic isolation methods for superparamagnetic NPs, 2) NP physico-chemical characterization using advanced and standard techniques available in regular laboratories, and 3) NP- PC characterization of the protein and glycan components.
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The adsorption of proteins onto the surface of nanoparticle (NP) leads to the formation of the so-called "protein corona" as consisting both loosely and tightly bound proteins. It is well established that the biological identity of NPs that may be acquired after exposure to a biological matrix is mostly provided by the components of the hard corona as the pristine surface is generally less accessible for binding. For that reason, the isolation and the characterisation of the NP-corona complexes and identification of the associated biomolecules can help in understanding its biological behaviour. Established methods for the isolation of the NP-HC complexes are time-demanding and can lead to different results based on the isolation method applied. Herein, we have developed a fast and simple method using ferromagnetic beads isolated from commercial MACS column and used for the isolation of superparamagnetic NP following exposure to different types of biological milieu. We first demonstrated the ability to easily isolate superparamagnetic iron oxide NPs (IONPs) from different concentrations of human blood plasma, and also tested the method on the corona isolation using more complex biological matrices, such as culture medium containing pulmonary mucus where the ordinary corona methods cannot be applied. Our developed method showed less than 20% difference in plasma corona composition when compared with centrifugation. It also showed effective isolation of NP-HC complexes from mucus-containing culture media upon comparing with centrifugation and MACS columns, which failed to wash out the unbound proteins. Our study was supported with a full characterisation profile including dynamic light scattering, nanoparticle tracking analysis, analytical disk centrifuge, and zeta potentials. The biomolecules/ proteins composing the HC were separated by vertical gel electrophoresis and subsequently analysed by liquid chromatography-tandem mass spectrometry. In addition to our achievements in comparing different isolation methods to separate IONPs with corona from human plasma, this is the first study that provides a complete characterisation profile of particle protein corona after exposure in vitro to pulmonary mucus-containing culture media.
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Nanopartículas , Coroa de Proteína , Humanos , Coroa de Proteína/química , Proteínas/química , Nanopartículas Magnéticas de Óxido de Ferro , Nanopartículas/química , Meios de CulturaRESUMO
The 'QuantitatEVs: multiscale analyses, from bulk to single vesicle' workshop aimed to discuss quantitative strategies and harmonized wet and computational approaches toward the comprehensive analysis of extracellular vesicles (EVs) from bulk to single vesicle analyses with a special focus on emerging technologies. The workshop covered the key issues in the quantitative analysis of different EV-associated molecular components and EV biophysical features, which are considered the core of EV-associated biomarker discovery and validation for their clinical translation. The in-person-only workshop was held in Trento, Italy, from January 31st to February 2nd, 2023, and continued in Milan on February 3rd with "Next Generation EVs", a satellite event dedicated to early career researchers (ECR). This report summarizes the main topics and outcomes of the workshop.
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Put your coat on: It is well recognized that the surfaces of nanomaterials in biological media are covered by various biomolecules (e.g., proteins). A) The protein corona creates a shell over different nanomaterials, regardless of their physicochemical properties (e.g., composition and shape), resulting in reduced levels of amyloid beta fibril formation. B) Pristine nanomaterials might have acceleratory effects on the fibrillation of amyloid beta.
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Peptídeos beta-Amiloides/metabolismo , Nanoestruturas/química , Peptídeos beta-Amiloides/química , Fulerenos/química , Nanoestruturas/ultraestrutura , Nanotubos de Carbono/química , Poliestirenos/químicaRESUMO
PAMAM (polyamidoamine) dendrimers are promising in biomedical applications that can interact with both the bilayer and . Here we employed giant unilamellar vesicles (GUVs) of two different charge densities to study the effect of albumin, one of the major in blood plasma, on the interactions between PAMAM dendrimers and membranes. The results show that albumin exacerbates the effect of dendrimers on the destabilization of the vesicles in terms of leakage, aggregation and collapse in particular for negatively charged vesicles while neutrally charged membranes are not affected. We conclude that the higher affinity of both albumin and PAMAM G6 towards negatively charged membranes explains their synergistic behavior in this case. In the case of neutral vesicles, the affinity between PAMAM G6 and albumin is stronger than that between PAMAM G6 (or albumin) and neutral vesicles, and thus no synergism is observed for the mixture during the interaction with neutral membranes.
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Nanoparticles have unique capacities of interacting with the cellular machinery and entering cells. To be able to exploit this potential, it is essential to understand what controls the interactions at the interface between nanoparticles and cells: it is now established that nanoparticles in biological media are covered by proteins and other biomolecules forming a "corona" on the nanoparticle surface, which confers a new identity to the nanoparticles. By labelling the proteins of the serum, using positively-charged polystyrene, we now show that this adsorbed layer is strong enough to be retained on the nanoparticles as they enter cells and is trafficked to the lysosomes on the nanoparticles. There, the corona is degraded and this is followed by lysosomal damage, leading to cytosolic release of lysosomal content, and ultimately apoptosis. Thus the corona protects the cells from the damage induced by the bare nanoparticle surface until enzymatically cleared in the lysosomes. FROM THE CLINICAL EDITOR: This study investigates the effects of protein corona that normally forms on the surface of nanoparticles during in vivo use, describing the steps of intracellular processing of such particles, to enhance our understanding of how these particles interact with the cellular machinery.
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Lisossomos/metabolismo , Nanopartículas/efeitos adversos , Nanopartículas/metabolismo , Adsorção , Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/metabolismo , Cátions/efeitos adversos , Cátions/química , Cátions/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Nanopartículas/química , Peptídeo Hidrolases/metabolismo , Permeabilidade , Proteólise , Propriedades de SuperfícieRESUMO
Gold nanoparticles (GNPs) are considered promising candidates for healthcare applications, however, their toxicity after long-term exposure to the material remains uncertain. Since the liver is the main filter organ for nanomaterials, this work was aimed at evaluating hepatic accumulation, internalisation and overall safety of well-characterised and endotoxin-free GNPs in healthy mice from 15 minutes to 7 weeks after a single administration. Our data demonstrate that GNPs were rapidly segregated into lysosomes of endothelial cells (LSEC) or Kupffer cells regardless of coating or shape but with different kinetics. Despite the long-lasting accumulation in tissues, the safety of GNPs was confirmed by liver enzymatic levels, as they were rapidly eliminated from the blood circulation and accumulated in the liver without inducing hepatic toxicity. Our results demonstrate that GNPs have a safe and biocompatibile profile despite their long-term accumulation.
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Ouro , Nanopartículas Metálicas , Camundongos , Animais , Ouro/toxicidade , Células Endoteliais , Nanopartículas Metálicas/toxicidade , Fígado , Células de KupfferRESUMO
Superparamagnetic iron oxide nanoparticles (SPIONs) have proved their use in many biomedical applications, such as drug delivery, hyperthermia, and MRI (magnetic resonance imaging) contrast agents. Due to their instability in fluids, several surface coatings have been used to both stabilize and tune the properties of these nanoparticles (NPs) according to their applications. These coatings will strongly modify their surface properties and influence their interaction with the environment proteins in a relevant biological medium with a clear impact on their function. It is well-accepted that a protein corona is immediately formed when nanoparticles come in contact with a biological milieu, and the emergent bionano interface represents the biological identity of the particles. Here, we investigate how a different coating on the same magnetic core can influence the protein corona composition and structure with clear relevance to application of these NPs in medicine. In particular, we have studied the structure and composition of the protein corona-SPION complexes of magnetite nanoparticles stabilized with citric acid, poly(acrylic acid), or double layer oleic acid by a range of approaches, including dynamic light scattering, nanoparticle tracking analysis, differential centrifugal sedimentation, infrared spectroscopy, 1-D SDS gel electrophoresis, and mass spectroscopy.
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Compostos Férricos/química , Nanopartículas de Magnetita/química , Proteínas/química , Resinas Acrílicas/química , Ácido Cítrico/química , Ácido Oleico/química , Propriedades de SuperfícieRESUMO
Biomolecular corona is spontaneously formed on the surface of nanoparticles (NPs) when they are in contact with biological fluids. It plays an important role in the colloidal stability of NPs, which is of importance for most of their medical applications and toxicity assessment. While typical studies use either blood plasma or serum from a pooled biobank, it is unclear whether differences in the media, such as cholesterol level or protein concentration, might affect the NP colloidal stability and corona composition. In this study, the silica corona was prepared at particularly low plasma concentrations (3%, v/v-1.98 mg/mL) to identify the critical roles of the protein mass/NP surface ratio and the level of plasma cholesterol on the corona protein pattern and particle stability. While depending on the plasma dilution factor, the corona protein composition could be controlled by keeping the protein/NP constant. The NP colloidal stability was found to strongly correlate with the level of cholesterol in human plasma, particularly due to the high enrichment of high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in the corona. A cohort study on plasma samples from individuals with known cholesterol levels was performed to highlight that association, which could be relevant for all corona systems enriched with the LDL.
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Poly (ethylene glycol) (PEG) is a widely used polymer in a variety of consumer products and in medicine. PEGylation refers to the conjugation of PEG to drugs or nanoparticles to increase circulation time and reduce unwanted host responses. PEG is viewed as being well-tolerated, but previous studies have identified anti-PEG antibodies and so-called pseudoallergic reactions in certain individuals. The increased use of nanoparticles as contrast agents or in drug delivery, along with the introduction of mRNA vaccines encapsulated in PEGylated lipid nanoparticles has brought this issue to the fore. Thus, while these vaccines have proven to be remarkably effective, rare cases of anaphylaxis have been reported, and this has been tentatively ascribed to the PEGylated carriers, which may trigger complement activation in susceptible individuals. Here, we provide a general overview of the use of PEGylated nanoparticles for pharmaceutical applications, and we discuss the activation of the complement cascade that might be caused by PEGylated nanomedicines for a better understanding of these immunological adverse reactions.
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HYPOTHESIS: Following blood administration, the pristine surface of nanoparticles (NPs) associates with biomolecules from the surrounding environment forming the so-called "biomolecular corona". It is well accepted that the biomolecular corona dramatically affects the NP fate in the biological medium while the pristine surface is no longer available for binding. Recent studies have shown that the glycans associated with the proteins forming the corona have a role in the NP interaction with macrophages, but the glycan identities remain unknown. We aim here to identify the glycan composition of the biomolecular corona and to assess the role of these glycans in the interaction of the proteins from the corona with glycan binding biomolecules, such as lectins. EXPERIMENTS: In this study, we have characterized the biomolecular corona of citrate stabilised gold NPs after exposure of the NPs to blood plasma at two different plasma concentrations, mimicking the in vitro and in vivo conditions. We have extensively characterized the biomolecular corona using HILIC chromatography and shotgun proteomics. Following this, a lectin binding assay was carried out using Dynamic Light Scattering (DLS) and Fluorescence Correlation Spectroscopy (FCS) to assess whether proteins with known affinity towards specific glycans would bind to the corona. FINDINGS: Our findings highlighted that the protein corona composition is dependent on the exposing conditions. However, under both plasma concentrations, the biantennary sialylated glycans (A2G2S2) are enriched. DLS and FCS confirmed that the glycans are accessible for binding as the corona interacts with lectins with known affinity towards terminal sialic acids and the enzymatic removal of the glycans leads to a decrease in lectin affinity. This study shows for the first time that the glycans are present in the corona and that they could potentially be responsible for the modulation of NP biological processes as they can directly engage with glycan binding receptors that are highly expressed in an organism.
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Nanopartículas , Coroa de Proteína , Polissacarídeos , Proteínas , Espectrometria de FluorescênciaRESUMO
Antimicrobial peptides (AMPs) are widely studied as therapeutic agents due to their broad-spectrum efficacy against infections. However, their clinical use is hampered by the low in vivo bioavailability and systemic toxicity. Such limitations might be overcome by using appropriate drug delivery systems. Here, the preparation of a drug delivery system (DDS) by physical conjugation of an arginine-rich peptide and hydrothermal carbon nanoparticles (CNPs) has been explored, and its antimicrobial efficacy against Eschericia coli (E. coli) and Staphylococcus aureus investigated in comparison with the unloaded carrier and the free peptide. The mechanism of interaction between CNPs and the bacteria was investigated by scanning electron microscopy and a combined dielectrophoresis-Raman spectroscopy method for real-time analysis. In view of a possible systemic administration, the effect of proteins on the stability of the DDS was investigated by using albumin as a model protein. The peptide was bounded electrostatically to the CNPs surface, establishing an equilibrium modulated by pH and albumin. The DDS exhibited antimicrobial activity toward the two bacterial strains, albeit lower as compared to the free peptide. The decrease in effectiveness toward E. coli was likely due to the rapid formation of a particle-induced extracellular matrix. The present results are relevant for the future development of hydrothermal CNPs as drug delivery agents of AMPs.
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Biomolecular corona formation has emerged as a recurring and important phenomenon in nanomedicine that has been investigated for potential applications in disease diagnosis. In this study, we have combined the "personalized protein corona" with the N-glycosylation profiling that has recently gained considerable interest in human plasma biomarker discovery as a powerful early warning diagnostic and patient stratification tool. We envisioned that the protein corona formation could be exploited as an enrichment step that is critically important in both proteomic and proteoglycomic workflows. By using silica nanoparticles, plasma fibrinogen was enriched to a level in which its proteomic and glycomic "fingerprints" could be traced with confidence. Despite being a more simplified glycan profile compared to full plasma, the corona glycan profile revealed a fibrinogen-derived glycan peak that was found to potentially distinguish lung cancer patients from controls in a pilot study.