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Patients affected by glioma frequently suffer of epileptic discharges, however the causes of brain tumor-related epilepsy (BTRE) are still not completely understood. We investigated the mechanisms underlying BTRE by analyzing the effects of exosomes released by U87 glioma cells and by patient-derived glioma cells. Rat hippocampal neurons incubated for 24 h with these exosomes exhibited increased spontaneous firing, while their resting membrane potential shifted positively by 10-15 mV. Voltage clamp recordings demonstrated that the activation of the Na+ current shifted towards more hyperpolarized voltages by 10-15 mV. To understand the factors inducing hyperexcitability we focused on exosomal cytokines. Western Blot and ELISA assays show that TNF-α is present inside glioma-derived exosomes. Remarkably, incubation with TNF-α fully mimicked the phenotype induced by exosomes, with neurons firing continuously, while their resting membrane potential shifted positively. RT-PCR revealed that both exosomes and TNF-α induced over-expression of the voltage-gated Na channel Nav1.6, a low-threshold Na+ channel responsible for hyperexcitability. When neurons were preincubated with Infliximab, a specific TNF-α inhibitor, the hyperexcitability induced by exosomes and TNF-α were drastically reduced. We propose that Infliximab, an FDA approved drug to treat rheumatoid arthritis, could ameliorate the conditions of glioma patients suffering of BTRE.
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The main proteases Mpro are a group of highly conserved cysteine hydrolases in ß-coronaviruses. They have been demonstrated to play an unavoidable role in viral replication, and consequently they have been suggested as key targets for treating coronavirus-caused infectious diseases, mainly from the COVID-19 epidemic. Since the most functional form for Mpro enzymatic activity is associated to its homodimer, compounds inhibiting dimerization should also inhibit catalytic activity. We show how PIR-SEIRA (Plasmonic Internal Reflection-Surface Enhanced InfraRed Absorption) spectroscopy can be a noteworthy technique to study proteins subtle structural variations associated to inhibitor binding. Nanoantennas arrays can selectively confine and enhance electromagnetic field via localized plasmonic resonances, thus promoting ultrasensitive detection of biomolecules in close proximity of nanoantenna arrays and enabling the effective investigation of protein monolayers. By adopting this approach, reflection measurements conducted under back illumination of nanoantennas allow to probe anchored protein monolayers, with minimum contribution of environmental buffer molecules. PIR-SEIRA spectroscopy on Mpro was carried out by ad hoc designed devices, resonating in the spectral region of Amide I and Amide II bands. We evaluated here the structure of anchored monomers and dimers in different buffered environment and in presence of a newly designed Mpro inhibitor. Experimental results show that dimerization is not associated to relevant backbone rearrangements of the protein at secondary structure level, and even if the compound inhibits the dimerization, it is not effective at breaking preformed dimers.
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Introduction: Neuroinflammation is a hallmark of multiple neurodegenerative diseases, shared by all pathological processes which primarily impact on neurons, including Central Nervous System (CNS) injuries. In reactive CNS, activated glia releases extracellular vesicles (EVs), nanosized membranous particles known to play a key role in intercellular communication. EVs mediate neuroinflammatory responses and might exacerbate tissue deterioration, ultimately influencing neurodegenerative disease progression. Methods: We treated spinal cord organotypic slices with LPS, a ligand extensively used to induce sEVs release, to mimic mild inflammatory conditions. We combine atomic force microscopy (AFM), nanoparticle tracking (NTA) and western blot (WB) analysis to validate the isolation and characterisation of sEVs. We further use immunofluorescence and confocal microscopy with live calcium imaging by GCaMP6f reporter to compare glial reactivity to treatments with sEVs when isolated from resting and LPS treated organ slices. Results: In our study, we focus on CNS released small EVs (sEVs) and their impact on the biology of inflammatory environment. We address sEVs local signalling within the CNS tissue, in particular their involvement in inflammation spreading mechanism(s). sEVs are harvested from mouse organotypic spinal cord cultures, an in vitro model which features 3D complexity and retains spinal cord resident cells. By confocal microscopy and live calcium imaging we monitor glial responses in naïve spinal slices when exposed to sEVs isolated from resting and LPS treated organ slices. Discussion: We show that sEVs, only when released during LPS neuroinflammation, recruit naïve astrocytes in the neuroinflammation cycle and we propose that such recruitment be mediated by EVs hemichannel (HC) permeability.
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Amiodarone repositioning in cancer treatment is promising, however toxicity limits seem to arise, constraining its exploitability. Notably, amiodarone has been investigated for the treatment of ovarian cancer, a tumour known for metastasizing within the peritoneal cavity. This is associated with an increase of fatty acid oxidation, which strongly depends on CPT1A, a transport protein which has been found overexpressed in ovarian cancer. Amiodarone is an inhibitor of CPT1A but its role still has to be explored. Therefore, in the present study, amiodarone was tested on ovarian cancer cell lines with a focus on lipid alteration, confirming its activity. Moreover, considering that drug delivery systems could lower drug side effects, microfluidics was employed for the development of drug delivery systems of amiodarone obtaining simultaneously liposomes with a high payload and amiodarone particles. Prior to amiodarone loading, microfluidics production was optimized in term of temperature and flow rate ratio. Moreover, stability over time of particles was evaluated. In vitro tests confirmed the efficacy of the drug delivery systems.
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Amiodarona , Nanopartículas , Neoplasias Ováricas , Humanos , Femenino , Amiodarona/farmacología , Amiodarona/uso terapéutico , Reposicionamiento de Medicamentos , Microfluídica , Liposomas/uso terapéutico , Sistemas de Liberación de Medicamentos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patologíaRESUMEN
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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This study compares the impact of two isolation methods, ultracentrifugation (UC) and size exclusion chromatography (SEC), on small extracellular vesicles (sEVs) from primary human cardiac mesenchymal-derived progenitor cells (CPCs). sEV_UC and sEV_SEC exhibit similar size, marker expression, and miRNA cargo, but sEV_UC contains notably higher total protein levels. In vitro assays show that sEV_UC, despite an equal particle count, induces more robust ERK phosphorylation, cytoprotection, and proliferation in iPS-derived cardiomyocytes (iPS-CMs) compared to sEV_SEC. sEV_UC also contains elevated periostin (POSTN) protein levels, resulting in enhanced focal adhesion kinase (FAK) phosphorylation in iPS-CMs. Importantly, this effect persists with treatment with soluble free-sEV protein fraction from SEC (Prote_SEC), indicating that free proteins like POSTN in sEV_UC enhance FAK phosphorylation. In vivo, sEV contamination with soluble proteins doesn't affect cardiac targeting or FAK phosphorylation, underscoring the intrinsic tissue targeting properties of sEV. These findings emphasize the need for standardized sEV isolation methods, as the choice of method can impact experimental outcomes, particularly in vitro.
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Carcinoma , Neoplasias del Plexo Coroideo , Vesículas Extracelulares , Humanos , Proteína-Tirosina Quinasas de Adhesión Focal , Cromatografía en GelRESUMEN
Biofilm formation, or microfouling, is a basic strategy of bacteria to colonise a surface and may happen on surfaces of any nature whenever bacteria are present. Biofilms are hard to eradicate due to the matrix in which the bacteria reside, consisting of strong, adhesive and adaptive self-produced polymers such as eDNA and functional amyloids. Targeting a biofilm matrix may be a promising strategy to prevent biofilm formation. Here, femtosecond laser irradiation was used to modify the stainless steel surface in order to introduce either conical spike or conical groove textures. The resulting topography consists of hierarchical nano-microstructures which substantially increase roughness. The biofilms of two model bacterial strains, P. aeruginosa PA01 and S. aureus ATCC29423, formed on such nanotextured metal surfaces, were considerably modified due to a substantial reduction in amyloid production and due to changes in eDNA surface adhesion, leading to significant reduction in biofilm biomass. Altering the topography of the metal surface, therefore, radically diminishes biofilm development solely by altering biofilm architecture. At the same time, growth and colonisation of the surface by eukaryotic adipose tissue-derived stem cells were apparently enhanced, leading to possible further advantages in controlling eukaryotic growth while suppressing prokaryotic contamination. The obtained results are important for developing anti-bacterial surfaces for numerous applications.
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Small extracellular vesicles (sEVs) are known to play an important role in the communication between distant cells and to deliver biological information throughout the body. To date, many studies have focused on the role of sEVs characteristics such as cell origin, surface composition, and molecular cargo on the resulting uptake by the recipient cell. Yet, a full understanding of the sEV fusion process with recipient cells and in particular the role of cell membrane physical properties on the uptake are still lacking. Here we explore this problem using sEVs from a cellular model of triple-negative breast cancer fusing to a range of synthetic planar lipid bilayers both with and without cholesterol, and designed to mimic the formation of 'raft'-like nanodomains in cell membranes. Using time-resolved Atomic Force Microscopy we were able to track the sEVs interaction with the different model membranes, showing the process to be strongly dependent on the local membrane fluidity. The strongest interaction and fusion is observed over the less fluid regions, with sEVs even able to disrupt ordered domains at sufficiently high cholesterol concentration. Our findings suggest the biophysical characteristics of recipient cell membranes to be crucial for sEVs uptake regulation.
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In the present study, the intermolecular interactions between a water-insoluble phospholipid (DOPC) and water-soluble protein (myoglobin) and the interaction among themselves were investigated at the air-water interface using the Langmuir and Langmuir-Blodgett techniques. The effects of changes in physicochemical factors, like pH and temperature, on these interactions were also examined. Surface pressure-molecular area (π-A) isotherms of the DOPC monolayer at the air-water interface, with and without myoglobin (Myo) revealed the evolution of various physical properties, such as elastic, thermodynamic, and hysteric properties, in response to changes in subphase pH and temperature. With the increment of subphase pH from 5 to 7 at a fixed temperature (20 °C), the DOPC isotherm expanded, and the in-plane elasticity (CS-1) decreased, but no significant presence of hysteresis was encountered in either of the pH values. On the other hand, a diminution of temperature (from 20 to 5 °C) leads to an expansion of monolayers yielding low elasticity and significant hysteresis. The incorporation of Myo molecules within the DOPC monolayer decreased the CS-1 value of the DOPC monolayer. Such a decrement in CS-1 was also encountered while increasing the pH and decreasing the temperature (T) of the subphase in the absence of Myo. Systematic expansion of DOPC isotherm and increased hysteric area with the increase in Myo proportion were observed and the atomic force microscopy (AFM) observations suggested a strong conjugation between Myo and DOPC in the mixed monolayer. The denaturation effect of Myo molecules was studied using AFM at different temperatures. Furthermore, the Myo molecules were found to be most surface active at pH = 7, which is very close to its isoelectric point. These observations come up with the interaction mechanism between biomolecules under dynamically varied conditions.
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External and internal mechanical forces modulate cell morphology, movement, proliferation and metabolism, and represent crucial inputs for tissue homeostasis. The transcriptional regulators YAP and TAZ are important effectors of mechanical signaling and are frequently activated in solid tumors, correlating with metastasis, chemoresistance, and shorter patient survival. YAP/TAZ activity is controlled by various pathways that sense cell shape, polarity, contacts, and mechanical tension. In tumors, aberrant YAP/TAZ activation may result from cancer-related alterations of such regulatory networks. The tumor suppressor DAB2IP is a Ras-GAP and scaffold protein that negatively modulates multiple oncogenic pathways and is frequently downregulated or inactivated in solid tumors. Here, we provide evidence that DAB2IP expression is sustained by cell confluency. We also find that DAB2IP depletion in confluent cells alters their morphology, reducing cell packing while increasing cell stiffness. Finally, we find that DAB2IP depletion in confluent cells favors YAP/TAZ nuclear localization and transcriptional activity, while its ectopic expression in subconfluent cells increases YAP/TAZ retention in the cytoplasm. Together, these data suggest that DAB2IP may function as a sensor of cell interactions, contributing to dampening cellular responses to oncogenic inputs in confluent cells and that DAB2IP loss-of-function would facilitate YAP/TAZ activation in intact epithelia, accelerating oncogenic transformation.
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The scaffolding of agarose hydrogel networks depends critically on the rate of cooling (quenching) after heating. Efforts are made to understand the kinetics and evolution of biopolymer self-assembly upon cooling, but information is lacking on whether quenching might affect the final hydrogel structure and performance. Here, a material strategy for the fine modulation of quenching that involves temperature-curing steps of agarose is reported. Combining microscopy techniques, standard and advanced macro/nanomechanical tools, it is revealed that agarose accumulates on the surface when the curing temperature is set at 121 °C. The inhomogeneity can be mostly recovered when it is reduced to 42 °C. This has a drastic effect on the stiffness of the surface, but not on the viscoelasticity, roughness, and wettability. When hydrogels are strained at small/large deformations, the curing temperature has no effect on the viscoelastic response of the hydrogel bulk but does play a role in the onset of the non-linear region. Cells cultured on these hydrogels exhibit surface stiffness-sensing that affects cell adhesion, spreading, F-actin fiber tension, and assembly of vinculin-rich focal adhesions. Collectively, the results indicate that the temperature curing of agarose is an efficient strategy to produce networks with tunable mechanics and is suitable for mechanobiology studies.
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Actinas , Hidrogeles , Sefarosa/química , Hidrogeles/química , Adhesión Celular , CinéticaRESUMEN
BACKGROUND: Although X-ray fluorescence microscopy is becoming a widely used technique for single-cell analysis, sample preparation for this microscopy remains one of the main challenges in obtaining optimal conditions for the measurements in the X-ray regime. The information available to researchers on sample treatment is inadequate and unclear, sometimes leading to wasted time and jeopardizing the experiment's success. Many cell fixation methods have been described, but none of them have been systematically tested and declared the most suitable for synchrotron X-ray microscopy. METHODS: The HEC-1-A endometrial cells, human spermatozoa, and human embryonic kidney (HEK-293) cells were fixed with organic solvents and cross-linking methods: 70% ethanol, 3.7%, and 2% paraformaldehyde; in addition, HEK-293 cells were subjected to methanol/ C3H6O treatment and cryofixation. Fixation methods were compared by coupling low-energy X-ray fluorescence with scanning transmission X-ray microscopy and atomic force microscopy. RESULTS: Organic solvents lead to greater dehydration of cells, which has the most significant effect on the distribution and depletion of diffusion elements. Paraformaldehyde provides robust and reproducible data. Finally, the cryofixed cells provide the best morphology and element content results. CONCLUSION: Although cryofixation seems to be the most appropriate method as it allows for keeping cells closer to physiological conditions, it has some technical limitations. Paraformaldehyde, when used at the average concentration of 3.7%, is also an excellent alternative for X-ray microscopy.
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Rayos X , Humanos , Células HEK293 , Radiografía , Microscopía de Fuerza AtómicaRESUMEN
Parkinson's disease (PD) presents the selective loss of A9 dopaminergic (DA) neurons of Substantia Nigra pars compacta (SNpc) and the presence of intracellular aggregates called Lewy bodies. α-synuclein (α-syn) species truncated at the carboxy-terminal (C-terminal) accumulate in pathological inclusions and promote α-syn aggregation and toxicity. Haemoglobin (Hb) is the major oxygen carrier protein in erythrocytes. In addition, Hb is expressed in A9 DA neurons where it influences mitochondrial activity. Hb overexpression increases cells' vulnerability in a neurochemical model of PD in vitro and forms cytoplasmic and nucleolar aggregates upon short-term overexpression in mouse SNpc. In this study, α and ß-globin chains were co-expressed in DA cells of SNpc in vivo upon stereotaxic injections of an Adeno-Associated Virus isotype 9 (AAV9) and in DA iMN9D cells in vitro. Long-term Hb over-expression in SNpc induced the loss of about 50% of DA neurons, mild motor impairments, and deficits in recognition and spatial working memory. Hb triggered the formation of endogenous α-syn C-terminal truncated species. Similar α-syn fragments were found in vitro in DA iMN9D cells over-expressing α and ß- globins when treated with pre-formed α-syn fibrils. Our study positions Hb as a relevant player in PD pathogenesis for its ability to trigger DA cells' loss in vivo and the formation of C-terminal α-syn fragments.
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Enfermedad de Parkinson , alfa-Sinucleína , Ratones , Animales , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Neuronas Dopaminérgicas/metabolismo , Sustancia Negra/metabolismo , Enfermedad de Parkinson/metabolismo , Hemoglobinas/metabolismo , CogniciónRESUMEN
Triple-negative breast cancer (TNBC) stands out for its aggressive, fast spread, and highly metastatic behavior and for being unresponsive to the classical hormonal therapy. It is considered a disease with a poor prognosis and limited treatment options. Among the mechanisms that contribute to TNBC spreading, attention has been recently paid to small extracellular vesicles (sEVs), nano-sized vesicles that by transferring bioactive molecules to recipient cells play a crucial role in the intercellular communication among cancer, healthy cells, and tumor microenvironment. In particular, TNBC-derived sEVs have been shown to alter proliferation, metastasis, drug resistance, and biomechanical properties of target cells. To shed light on the molecular mechanisms involved in sEVs mediation of cell biomechanics, we investigated the effects of sEVs on the main subcellular players, i.e., cell membrane, cytoskeleton, and nuclear chromatin organization. Our results unveiled that TNBC-derived sEVs are able to promote the formation and elongation of cellular protrusions, soften the cell body, and induce chromatin decondensation in recipient cells. In particular, our data suggest that chromatin decondensation is the main cause of the global cell softening. The present study added new details and unveiled a novel mechanism of activity of the TNBC-derived sEVs, providing information for the efficient translation of sEVs to cancer theranostics.
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Vesículas Extracelulares , Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Comunicación Celular , Cromatina , Membrana Celular , Microambiente TumoralRESUMEN
The long-known role of cell migration in physiological and pathological contexts still requires extensive research to be fully understood, mainly because of the intricate interaction between moving cells and their surroundings. While conventional assays fail to capture this complexity, recently developed 3D platforms better reproduce the cellular micro-environment, although often requiring expensive and time-consuming imaging approaches. To overcome these limitations, we developed a novel approach based on 2D micro-patterned substrates, compatible with conventional microscopy analysis and engineered to create micro-gaps with a length of 150 µm and a lateral size increasing from 2 to 8 µm, where a curved water-air interface is created on which cells can adhere, grow, and migrate. The resulting hydrophilic/hydrophobic interfaces, variable surface curvatures, spatial confinements, and size values mimic the complex micro-environment typical of the extracellular matrix in which aggressive cancer cells proliferate and migrate. The new approach was tested with two breast cancer cell lines with different invasive properties. We observed that invasive cells (MDA-MB-231) can align along the pattern and modify both their morphology and their migration rate according to the size of the water meniscus, while non-invasive cells (MCF-7) are only slightly respondent to the surrounding micro-environment. Moreover, the selected pattern highlighted a significative matrix deposition process connected to cell migration. Although requiring further optimizations, this approach represents a promising tool to investigate cell migration in complex environments.
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Matriz Extracelular , Agua , Humanos , Agua/análisis , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Movimiento Celular , Células MCF-7RESUMEN
Seizures represent a frequent symptom in gliomas and significantly impact patient morbidity and quality of life. Although the pathogenesis of tumor-related seizures is not fully understood, accumulating evidence indicates a key role of the peritumoral microenvironment. Brain cancer cells interact with neurons by forming synapses with them and by releasing exosomes, cytokines, and other small molecules. Strong interactions among neurons often lead to the synchronization of their activity. In this paper, we used an in vitro model to investigate the role of exosomes released by glioma cell lines and by patient-derived glioma stem cells (GSCs). The addition of exosomes released by U87 glioma cells to neuronal cultures at day in vitro (DIV) 4, when neurons are not yet synchronous, induces synchronization. At DIV 7-12 neurons become highly synchronous, and the addition of the same exosomes disrupts synchrony. By combining Ca2+ imaging, electrical recordings from single neurons with patch-clamp electrodes, substrate-integrated microelectrode arrays, and immunohistochemistry, we show that synchronization and de-synchronization are caused by the combined effect of (i) the formation of new neuronal branches, associated with a higher expression of Arp3, (ii) the modification of synaptic efficiency, and (iii) a direct action of exosomes on the electrical properties of neurons, more evident at DIV 7-12 when the threshold for spike initiation is significantly reduced. At DIV 7-12 exosomes also selectively boost glutamatergic signaling by increasing the number of excitatory synapses. Remarkably, de-synchronization was also observed with exosomes released by glioma-associated stem cells (GASCs) from patients with low-grade glioma but not from patients with high-grade glioma, where a more variable outcome was observed. These results show that exosomes released from glioma modify the electrical properties of neuronal networks and that de-synchronization caused by exosomes from low-grade glioma can contribute to the neurological pathologies of patients with brain cancers.
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Neoplasias Encefálicas , Exosomas , Glioma , Neoplasias Encefálicas/patología , Exosomas/metabolismo , Glioma/patología , Humanos , Neuronas/patología , Calidad de Vida , Convulsiones/metabolismo , Microambiente TumoralRESUMEN
Extracellular vesicle (EV) mediated communication has recently been proposed as one of the pivotal routes in the development of cancer metastasis. EVs are nano-sized vesicles swapped between cells, carrying a biologically active content that can promote tumor-induced immune suppression, metastasis and angiogenesis. Thus, EVs constitute a potential target in cancer therapy. However, their role in triggering the premetastatic niche and in tumor spreading is still unclear. Here, we focused on the EV ability to modulate the biomechanical properties of target cells, known to play a crucial role in metastatic spreading. To this purpose, we isolated and thoroughly characterized triple-negative breast cancer (TNBC)-derived small EVs. We then evaluated variations in the mechanical properties (cell stiffness, cytoskeleton/nuclear/morphology and Yap activity rearrangements) of non-metastatic breast cancer MCF7 cells upon EV treatment. Our results suggest that TNBC-derived small EVs are able to directly modify MCF7 cells by inducing a decrease in cell stiffness, rearrangements in cytoskeleton, focal adhesions and nuclear/cellular morphology, and an increase in Yap downstream gene expression. Testing the biomechanical response of cells after EV addition might represent a new functional assay in metastatic cancer framework that can be exploited for future application both in diagnosis and in therapy.
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Vesículas Extracelulares , Neoplasias de la Mama Triple Negativas , Fenómenos Biomecánicos , Vesículas Extracelulares/metabolismo , Humanos , Células MCF-7 , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
The use of infliximab has completely changed the therapeutic landscape in inflammatory bowel disease. However, despite its proven efficacy to induce and maintain clinical remission, increasing evidence suggests that treatment failure may be associated with inadequate drug blood concentrations. The introduction of biosensors based on different nanostructured materials for the rapid quantification of drugs has been proposed for therapeutic drug monitoring. This study aimed to apply atomic force microscopy (AFM)-based nanoassay for the measurement of infliximab concentration in serum samples of healthy donors and pediatric IBD patients. This assay measured the height signal variation of a nanostructured gold surface covered with a self-assembled monolayer of alkanethiols. Inside this monolayer, we embedded the DNA conjugated with a tumor necrosis factor able to recognize the drug. The system was initially fine-tuned by testing known infliximab concentrations (0, 20, 30, 40, and 50 nM) in buffer and then spiking the same concentrations of infliximab into the sera of healthy donors, followed by testing pediatric IBD patients. A good correlation between height variation and drug concentration was found in the buffer in both healthy donors and pediatric IBD patients (p-value < 0.05), demonstrating the promising use of AFM nanoassay in TDM.
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Extracellular vesicles (EVs) are receiving increasing attention for their role in spreading both beneficial and harmful information during cell-cell communication. The complexity and heterogeneity of the origin of EVs make integrated molecular, structural, and functional studies extremely challenging but necessary at the same time. In fact, a comprehensive interdisciplinary approach is needed to correlate the features of EVs, target cells/organs, and the pathophysiological outcomes exerted by the EVs' actions. Based on these premises, after introducing a brief state-of-the-art outline on the current analytical approaches exploited to characterize EVs, this review aims to highlight the effectiveness of those studies where authors put in correlation the diverse EV data collected from different points of view. Although these examples are still just a few, they still represent an excellent starting point to be taken as a reference in the perspective for improving the correlation among EV-related clinical aspects. Of course, to fully reach this goal, several points need to be further improved and developed. Undoubtedly, new avenues in diagnostic, prognostic, and therapeutic applications by EVs will be initiated by integrative strategies, combining biophysical approaches, high-throughput omics technologies, and computational models.
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Extracellular vesicles (EVs)-mediated communication relies not only on the delivery of complex molecular cargoes as lipids, proteins, genetic material, and metabolites to their target cells but also on the modification of the cell surface local properties induced by the eventual fusion of EVs' membranes with the cells' plasma membrane. Here we applied scanning calorimetry to study the phase transition of single phospholipid (DMPC) monolamellar vesicles, investigating the thermodynamical effects caused by the fusion of doping amounts of mesenchymal stem cells-derived EVs. Specifically, we studied EVs-induced consequences on the lipids distributed in the differently curved membrane leaflets, having different density and order. The effect of EV components was found to be not homogeneous in the two leaflets, the inner (more disordered one) being mainly affected. Fusion resulted in phospholipid membrane flattening associated with lipid ordering, while the transition cooperativity, linked to membrane domains' coexistence during the transition process, was decreased. Our results open new horizons for the investigation of the peculiar effects of EVs of different origins on target cell membrane properties and functionality.