Nanoanalytical analysis of bisphosphonate-driven alterations of microcalcifications using a 3D hydrogel system and in vivo mouse model.

Vascular calcification predicts atherosclerotic plaque rupture and cardiovascular events. Retrospective studies of women taking bisphosphonates (BiPs), a proposed therapy for vascular calcification, showed that BiPs paradoxically increased morbidity in patients with prior acute cardiovascular events but decreased mortality in event-free patients. Calcifying extracellular vesicles (EVs), released by cells within atherosclerotic plaques, aggregate and nucleate calcification. We hypothesized that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the risk of plaque rupture. Three-dimensional (3D) collagen hydrogels incubated with calcifying EVs were used to mimic fibrous cap calcification in vitro, while an ApoE-/- mouse was used as a model of atherosclerosis in vivo. EV aggregation and formation of stress-inducing microcalcifications was imaged via scanning electron microscopy (SEM) and atomic force microscopy (AFM). In both models, BiP (ibandronate) treatment resulted in time-dependent changes in microcalcification size and mineral morphology, dependent on whether BiP treatment was initiated before or after the expected onset of microcalcification formation. Following BiP treatment at any time, microcalcifications formed in vitro were predicted to have an associated threefold decrease in fibrous cap tensile stress compared to untreated controls, estimated using finite element analysis (FEA). These findings support our hypothesis that BiPs alter EV-driven calcification. The study also confirmed that our 3D hydrogel is a viable platform to study EV-mediated mineral nucleation and evaluate potential therapies for cardiovascular calcification.

Multiparametric Orthogonal Characterization of Extracellular Vesicles by Liquid Chromatography Combined with In-Line Light Scattering and Fluorescence Detection.

Extracellular vesicles (EVs) are membrane-enclosed biological nanoparticles with potential as diagnostic markers and carriers for therapeutics. Characterization of EVs poses severe challenges due to their complex structure and composition, requiring the combination of orthogonal analytical techniques. Here, we demonstrate how liquid chromatography combined with multi-angle light scattering (MALS) and fluorescence detection in one single apparatus can provide multiparametric characterization of EV samples, including concentration of particles, average diameter of the particles, protein amount to particle number ratio, presence of EV surface markers and lipids, EV shape, and sample purity. The method requires a small amount of sample of approximately 107 EVs, limited handling of the sample and data analysis time in the order of minutes; it is fully automatable and can be applied to both crude and purified samples.

AFM-Based High-Throughput Nanomechanical Screening of Single Extracellular Vesicles.

The mechanical properties of extracellular vesicles (EVs) are known to influence their biological function, in terms of, e.g., cellular adhesion, endo/exocytosis, cellular uptake, and mechanosensing. EVs have a characteristic nanomechanical response which can be probed via force spectroscopy (FS) and exploited to single them out from nonvesicular contaminants or to discriminate between subtypes. However, measuring the nanomechanical characteristics of individual EVs via FS is a labor-intensive and time-consuming task, usually limiting this approach to specialists. Herein, we describe a simple atomic force microscopy based experimental procedure for the simultaneous nanomechanical and morphological analysis of several hundred individual nanosized EVs within the hour time scale, using basic AFM equipment and skills and only needing freely available software for data analysis. This procedure yields a "nanomechanical snapshot" of an EV sample which can be used to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample and between populations of vesicles with similar sizes but different mechanical characteristics. We demonstrate the applicability of the proposed approach to EVs obtained from three very different sources (human colorectal carcinoma cell culture, raw bovine milk, and Ascaris suum nematode excretions), recovering size and stiffness distributions of individual vesicles in a sample. EV stiffness values measured with our high-throughput method are in very good quantitative accord with values obtained by FS techniques which measure EVs one at a time. We show how our procedure can detect EV samples contamination by nonvesicular aggregates and how it can quickly attest the presence of EVs even in samples for which no established assays and/or commercial kits are available (e.g., Ascaris EVs), thus making it a valuable tool for the rapid assessment of EV samples during the development of isolation/enrichment protocols by EV researchers. As a side observation, we show that all measured EVs have a strikingly similar stiffness, further reinforcing the hypothesis that their mechanical characteristics could have a functional role.

Uptake Profiles of Human Serum Exosomes by Murine and Human Tumor Cells through Combined Use of Colloidal Nanoplasmonics and Flow Cytofluorimetric Analysis.

Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capital importance for both EV basic biology and clinical translation, but still presents analytical hurdles, such as undetermined purity grade and/or concentration of the EV samples and lack of standard protocols. We report an accessible, robust, and versatile method for resolving dose-dependent uptake profiles of exosomes-the nanosized (30-150 nm) subtypes of EVs of intracellular origin which are more intensively investigated for diagnostic and therapeutic applications-by cultured cells. The method is based on incubating recipient cells with consistently increasing doses of exosomes which are graded for purity and titrated by a COlorimetric NANoplasmonic (CONAN) assay followed by cell flow cytofluorimetric analysis. The proposed method allowed evaluation and comparison of the uptake of human serum exosomes by cancer cell lines of murine (TRAMP-C2) and human (LNCaP, DU145, MDA-MB-231, and A375) origin, setting a firmer footing for better characterization and understanding of exosome biology in different in vitro and (potentially) in vivo models of cancer growth.

Exosomes Secreted by HeLa Cells Shuttle on Their Surface the Plasma Membrane-Associated Sialidase NEU3.

Sialidases are glycohydrolases that remove terminal sialic acid residues from oligosaccharides, glycolipids, and glycoproteins. The plasma membrane-associated sialidase NEU3 is involved in the fine-tuning of sialic acid-containing glycans directly on the cell surface and plays relevant roles in important biological phenomena such as cell differentiation, molecular recognition, and cancer transformation. Extracellular vesicles are membranous structures with a diameter of 0.03-1 µm released by cells and can be detected in blood, urine, and culture media. Among extracellular vesicles, exosomes play roles in intercellular communication and maintenance of several physiological and pathological conditions, including cancer, and could represent a useful diagnostic tool for personalized nanomedicine approaches. Using inducible expression of the murine form of NEU3 in HeLa cells, a study of the association of the enzyme with exosomes released in the culture media has been performed. Briefly, NEU3 is associated with highly purified exosomes and localizes on the external leaflet of these nanovesicles, as demonstrated by enzyme activity measurements, Western blot analysis, and dot blot analysis using specific protein markers. On the basis of these results, it is plausible that NEU3 activity on exosome glycans enhances the dynamic biological behavior of these small extracellular vesicles by modifying the negative charge and steric hindrance of their glycocalyx. The presence of NEU3 on the exosomal surface could represent a useful marker for the detection of these nanovesicles and a tool for improving our understanding of the biology of these important extracellular carriers in physiological and pathological conditions.

Cultured human amniocytes express hTERT, which is distributed between nucleus and cytoplasm and is secreted in extracellular vesicles.

BACKGROUND: An increasing number of studies on stem cells suggests that the therapeutic effect they exert is primarily mediated by a paracrine regulation through extracellular vesicles (EVs) giving solid grounds for stem cell EVs to be exploited as agents for treating diseases or for restoring damaged tissues and organs. Due to their capacity to differentiate in all embryonic germ layers, amniotic fluid stem cells (AFCs), represent a highly promising cell type for tissue regeneration, which however is still poorly studied and in turn underutilized. In view of this, we conducted a first investigation on the expression of human hTERT gene - known to be among the key triggers of organ regeneration - in AFCs and in the EVs they secrete. METHODS: Isolated AFCs were evaluated by RT-qPCR for hTERT expression. The clones expressing the highest levels of transcript, were analyzed by Immunofluorescence imaging and Nuclear/cytoplasmic fractionation in order to evaluate hTERT subcellular localization. We then separated EVs from FBS depleted culture medium by serial (ultra) centrifugations steps and characterized them using Western blotting, Atomic force Microscopy and Nanoplasmonic assay. RESULTS: We first demonstrated that primary cultures of AFCs express the gene hTERT at different levels. Then we evidenced that in AFCs with the higher transcript levels, the hTERT protein is present in the nuclear and cytoplasmic compartment. Finally, we found that cytosolic hTERT is embodied in the EVs that AFCs secrete in the extracellular milieu. CONCLUSIONS: Our study demonstrates for the first time the expression of the full protein hTERT by AFCs and its release outside the cell mediated by EVs, indicating a new extra telomeric role for this protein. This finding represents an initial but crucial evidence for considering AFCs derived EVs as new potential sources for tissue regeneration.

Colorimetric nanoplasmonic assay to determine purity and titrate extracellular vesicles.

Extracellular Vesicles (EVs) - cell secreted vesicles that carry rich molecular information of the parental cell and constitute an important mode of intercellular communication - are becoming a primary topic in translational medicine. EVs (that comprise exosomes and microvesicles/microparticles) have a size ranging from 40 nm to 1 µm and share several physicochemical proprieties, including size, density, surface charge, and light interaction, with other nano-objects present in body fluids, such as single and aggregated proteins. This makes separation, titration, and characterization of EVs challenging and time-consuming. Here we present a cost-effective and fast colorimetric assay for probing by eye protein contaminants and determine the concentration of EV preparations, which exploits the synergy between colloidal gold nanoplasmonics, nanoparticle-protein corona, and nanoparticle-membrane interaction. The assay hits a limit of detection of protein contaminants of 5 ng/µL and has a dynamic range of EV concentration ranging from 35 fM to 35 pM, which matches the typical range of EV concentration in body fluids. This work provides the first example of the exploitation of the nanoparticle-protein corona in analytical chemistry.

Surfactant titration of nanoparticle-protein corona.

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.

Cavitand-grafted silicon microcantilevers as a universal probe for illicit and designer drugs in water.

The direct, clean, and unbiased transduction of molecular recognition into a readable and reproducible response is the biggest challenge associated to the use of synthetic receptors in sensing. All possible solutions demand the mastering of molecular recognition at the solid-liquid interface as prerequisite. The socially relevant issue of screening amine-based illicit and designer drugs is addressed by nanomechanical recognition at the silicon-water interface. The methylamino moieties of different drugs are all first recognized by a single cavitand receptor through a synergistic set of weak interactions. The peculiar recognition ability of the cavitand is then transferred with high fidelity and robustness on silicon microcantilevers and harnessed to realize a nanomechanical device for label-free detection of these drugs in water.

Characterization of polyethylene terephthalate (PET) and polyamide (PA) true-to-life nanoplastics and their biological interactions.

Plastic and microplastics, including polyethylene (PE), polypropylene (PP), and polystyrene (PS), are major contributors to environmental pollution. However, there is a growing recognition of the need to investigate a wider range of plastic polymers to fully understand the extent and impacts of plastic pollution. This study focuses on the comprehensive characterization of true-to-life nanoplastics (T2LNPs) derived from polyethylene terephthalate (PET) and polyamide (PA) to enhance our understanding of environmental nanoplastics pollution. T2LNPs were produced through cryogenic mechanical fragmentation of everyday items made from these polymers. A solid methodological framework incorporating various characterization techniques was established. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and thermogravimetric analysis (TGA) were employed to study the chemical composition and confirm the absence of chemical modifications possibly occurring during fragmentation. Atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were used to analyze the morphology of the T2LNPs. Additionally, AFM image analysis compared to dynamic light scattering (DLS) measurements provided insights into the size distribution and the stability of the T2LNP suspensions. The results revealed the heterogeneity of T2LNPs derived from PET and PA, emphasizing the importance of studying different plastic compositions to comprehensively understand nanoplastics pollution. Lastly, the distinctive characteristics and morphology of T2LNPs were translated into the realm of biological interactions, offering initial insights into the influence of these disparities on the formation of the protein corona on the surface of T2LNPs. By proposing T2LNPs as test materials and establishing a comprehensive characterization approach, this study aims to bridge the knowledge gap regarding the behavior and toxicity of nanoplastics. Furthermore, it highlights the need for a reliable and transferable analytical package for nanoplastic characterization to facilitate future studies on the environmental impact of nanoplastics.

Advances in Extracellular Vesicle Research Over the Past Decade: Source and Isolation Method are Connected with Cargo and Function.

The evolution of extracellular vesicle (EV) research has introduced nanotechnology into biomedical cell communication science while recognizing what is formerly considered cell "dust" as constituting an entirely new universe of cell signaling particles. To display the global EV research landscape, a systematic review of 20 364 original research articles selected from all 40 684 EV-related records identified in PubMed 2013-2022 is performed. Machine-learning is used to categorize the high-dimensional data and further dissected significant associations between EV source, isolation method, cargo, and function. Unexpected correlations between these four categories indicate prevalent experimental strategies based on cargo connectivity with function of interest being associated with certain EV sources or isolation strategies. Conceptually relevant association of size-based EV isolation with protein cargo and uptake function will guide strategic conclusions enhancing future EV research and product development. Based on this study, an open-source database is built to facilitate further analysis with conventional or AI tools to identify additional causative associations of interest.

International Society for Extracellular Vesicles Workshop. QuantitatEVs: multiscale analyses, from bulk to single extracellular vesicle.

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.

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Surface functionalization of extracellular vesicle nanoparticles with antibodies: a first study on the protein corona "variable".

To be profitably exploited in medicine, nanosized systems must be endowed with biocompatibility, targeting capability, the ability to evade the immune system, and resistance to clearance. Currently, biogenic nanoparticles, such as extracellular vesicles (EVs), are intensively investigated as the platform that naturally recapitulates these highly needed characteristics. EV native targeting properties and pharmacokinetics can be further augmented by decorating the EV surface with specific target ligands as antibodies. However, to date, studies dealing with the functionalization of the EV surface with proteins have never considered the protein corona "variable", namely the fact that extrinsic proteins may spontaneously adsorb on the EV surface, contributing to determine the surface, and in turn the biological identity of the EV. In this work, we explore and compare the two edge cases of EVs modified with the antibody Cetuximab (CTX) by chemisorption of CTX (through covalent binding via biorthogonal click-chemistry) and by formation of a physisorbed CTX corona. The results indicate that (i) no differences exist between the two formulations in terms of binding affinity imparted by molecular recognition of CTX versus its natural binding partner (epidermal growth factor receptor, EGFR), but (ii) significant differences emerge at the cellular level, where CTX-EVs prepared by click chemistry display superior binding and uptake toward target cells, very likely due to the higher robustness of the CTX anchorage.

Probing the coverage of nanoparticles by biomimetic membranes through nanoplasmonics.

Although promising for biomedicine, the clinical translation of inorganic nanoparticles (NPs) is limited by low biocompatibility and stability in biological fluids. A common strategy to circumvent this drawback consists in disguising the active inorganic core with a lipid bilayer coating, reminiscent of the structure of the cell membrane to redefine the chemical and biological identity of NPs. While recent reports introduced membrane-coating procedures for NPs, a robust and accessible method to quantify the integrity of the bilayer coverage is not yet available. To fill this gap, we prepared SiO2 nanoparticles (SiO2NPs) with different membrane coverage degrees and monitored their interaction with AuNPs by combining microscopic, scattering, and optical techniques. The membrane-coating on SiO2NPs induces spontaneous clustering of AuNPs, whose extent depends on the coating integrity. Remarkably, we discovered a linear correlation between the membrane coverage and a spectral descriptor for the AuNPs' plasmonic resonance, spanning a wide range of coating yields. These results provide a fast and cost-effective assay to monitor the compatibilization of NPs with biological environments, essential for bench tests and scale-up. In addition, we introduce a robust and scalable method to prepare SiO2NPs/AuNPs hybrids through spontaneous self-assembly, with a high-fidelity structural control mediated by a lipid bilayer.

Synthesis and Characterization of a Biocompatible Nanoplatform Based on Silica-Embedded SPIONs Functionalized with Polydopamine.

Superparamagnetic iron oxide nanoparticles (SPIONs) have gained increasing interest in nanomedicine, but most of those that have entered the clinical trials have been withdrawn due to toxicity concerns. Therefore, there is an urgent need to design low-risk and biocompatible SPION formulations. In this work, we present an original safe-by-design nanoplatform made of silica nanoparticles loaded with SPIONs and decorated with polydopamine (SPIONs@SiO2-PDA) and the study of its biocompatibility performance by an ad hoc thorough in vitro to in vivo nanotoxicological methodology. The results indicate that the SPIONs@SiO2-PDA have excellent colloidal stability in serum-supplemented culture media, even after long-term (24 h) exposure, showing no cytotoxic or genotoxic effects in vitro and ex vivo. Physiological responses, evaluated in vivo using Caenorhabditis elegans as the animal model, showed no impact on fertility and embryonic viability, induction of an oxidative stress response, and a mild impact on animal locomotion. These tests indicate that the synergistic combination of the silica matrix and PDA coating we developed effectively protects the SPIONs, providing enhanced colloidal stability and excellent biocompatibility.

Particle profiling of EV-lipoprotein mixtures by AFM nanomechanical imaging.

The widely overlapping physicochemical properties of lipoproteins (LPs) and extracellular vesicles (EVs) represents one of the main obstacles for the isolation and characterization of these pervasive biogenic lipid nanoparticles. We herein present the application of an atomic force microscopy (AFM)-based quantitative morphometry assay to the rapid nanomechanical screening of mixed LPs and EVs samples. The method can determine the diameter and the mechanical stiffness of hundreds of individual nanometric objects within few hours. The obtained diameters are in quantitative accord with those measured via cryo-electron microscopy (cryo-EM); the assignment of specific nanomechanical readout to each object enables the simultaneous discrimination of co-isolated EVs and LPs even if they have overlapping size distributions. EVs and all classes of LPs are shown to be characterised by specific combinations of diameter and stiffness, thus making it possible to estimate their relative abundance in EV/LP mixed samples in terms of stoichiometric ratio, surface area and volume. As a side finding, we show how the mechanical behaviour of specific LP classes is correlated to distinctive structural features revealed by cryo-EM. The described approach is label-free, single-step and relatively quick to perform. Importantly, it can be used to analyse samples which prove very challenging to assess with several established techniques due to ensemble-averaging, low sensibility to small particles, or both, thus providing a very useful tool for quickly assessing the purity of EV/LP isolates including plasma- and serum-derived preparations.

Role of nanomechanics in canonical and noncanonical pro-angiogenic ligand/VEGF receptor-2 activation.

Vascular endothelial growth factor receptor-2 (VEGFR2) is an endothelial cell receptor that plays a pivotal role in physiologic and pathologic angiogenesis and is a therapeutic target for angiogenesis-dependent diseases, including cancer. By leveraging on a dedicated nanomechanical biosensor, we investigated the nanoscale mechanical phenomena intertwined with VEGFR2 surface recognition by its prototypic ligand VEGF-A and its noncanonical ligand gremlin. We found that the two ligands bind the immobilized extracellular domain of VEGFR2 (sVEGFR2) with comparable binding affinity. Nevertheless, they interact with sVEGFR2 with different binding kinetics and drive different in-plane piconewton intermolecular forces, suggesting that the binding of VEGF-A or gremlin induces different conformational changes in sVEGFR2. These behaviors can be effectively described in terms of a different "nanomechanical affinity" of the two ligands for sVEGFR2, about 16-fold higher for VEGF-A with respect to gremlin. Such nanomechanical differences affect the biological activity driven by the two angiogenic factors in endothelial cells, as evidenced by a more rapid VEGFR2 clustering and a more potent mitogenic response triggered by VEGF-A in respect to gremlin. Together, these data point to surface intermolecular interactions on cell membrane between activated receptors as a key modulator of the intracellular signaling cascade.

Nanomechanical recognition of N-methylammonium salts.

Turning molecular recognition into an effective mechanical response is critical for many applications ranging from molecular motors and responsive materials to sensors. Herein, we demonstrate how the energy of the molecular recognition between a supramolecular host and small alkylammonium salts can be harnessed to perform a nanomechanical task in a univocal way. Nanomechanical Si microcantilevers (MCs) functionalized by a film of tetra-phosphonate cavitands were employed to screen as guests the compounds of the butylammonium chloride series 1-4, which comprises a range of low molecular weight (LMW) molecules (molecular mass < 150 Da) that differ from each other by one or a few N-methyl groups (molecular mass 15 Da). The cavitand surface recognition of each individual guest drove a specific MC bending (from a few to several tens of nanometer), disclosing a direct, label-free, and real-time mean to sort them. The complexation preferences of tetraphosphonate cavitands toward ammonium chloride guests 1-4 were independently assessed by isothermal titration calorimetry. Both direct and displacement binding experiments concurred to define the following binding order in the alkylammonium series: 2 > 3 ≈ 1 ≫ 4. This trend is consistent with the number of interactions established by each guest with the host. The complementary ITC experiments showed that the host-guest complexation affinity in solution is transferred to the MC bending. These findings were benchmarked by implementing cavitand-functionalized MCs to discriminate sarcosine from glycine in water.

On the surface-to-bulk partition of proteins in extracellular vesicles.

Nanomaterials are characterized by an extremely large surface-to-volume ratio. Extracellular Vesicles (EVs) - which have been recently recognized as the universal agent of intercellular communication, being involved in many physiological and pathological processes and interkingdom biochemical communication - are nanoparticles, but this key aspect has never been rationally addressed. Here we report the first attempt to quantify the membrane-to-lumen partition of proteins in EVs. A semi-quantitative model based on available well-established compositional and microstructural data is formulated. The model allows for the estimation of the overall protein content of an EV as well as of the partition between membrane (surface) associated and lumen (bulk) contained proteins as a function of the EV size and shape. It further identifies 180 nm as a switch diameter, below which EVs result composed of more membrane than luminal proteins. At larger diameters the partition is reversed, reaching predominance of luminal proteins (> 80 %) in large EVs (diameter > 800 nm). The model is successfully tested to analyze and describe a real preparation composed of subpopulations of small EVs (diameter < 200 nm), including exosomes and ectosomes, and large EVs including large oncosomes (diameter > 1000 nm) from human prostate cancer cells. These findings provide the basis for a better colloidal description of EV samples, might help to understand the stoichiometry of proteins in distinct EV sub-populations, and will improve the design and interpretation of experiments, including EV engineering and dosing in-vitro and in-vivo.