Impact of Sample Preparation Methods on Single-Cell X-ray Microscopy and Light Elemental Analysis Evaluated by Combined Low Energy X-ray Fluorescence, STXM and AFM.

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.

Triple negative breast cancer-derived small extracellular vesicles as modulator of biomechanics in target cells.

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.

UV Resonance Raman explores protein structural modification upon fibrillation and ligand interaction.

Amyloids are proteinaceous deposits considered an underlying pathological hallmark of several degenerative diseases. The mechanism of amyloid formation and its inhibition still represent challenging issues, especially when protein structure cannot be investigated by classical biophysical techniques as for the intrinsically disordered proteins (IDPs). In this view, the need to find an alternative way for providing molecular and structural information regarding IDPs prompted us to set a novel, to our knowledge, approach focused on UV Resonance Raman (UVRR) spectroscopy. To test its applicability, we study the fibrillation of hen-egg white lysozyme (HEWL) and insulin as well as their interaction with resveratrol, employing also intrinsic fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and atomic force microscopy (AFM). The increasing of the ß-sheet structure content at the end of protein fibrillation probed by FTIR occurs simultaneously with a major solvent exposure of tryptophan (Trp) and tyrosine (Tyr) residues of HEWL and insulin, respectively, as revealed by UVRR and intrinsic fluorescence spectroscopy. However, because the latter technique is successfully used when proteins naturally contain Trp residues, it shows poor performances in the case of insulin, and the information regarding its tertiary structure is exclusively provided by UVRR spectroscopy. The presence of an increased concentration of resveratrol induces mild changes in the secondary structure of both protein fibrils while remodeling HEWL fibril length and promoting the formation of amorphous aggregates in the case of insulin. Although the intrinsic fluorescence spectra of proteins are hidden by resveratrol signal, UVRR Trp and Tyr bands are resonantly enhanced, showing a good sensitivity to the presence of resveratrol and marking a modification in the noncovalent interactions in which they are involved. Our findings demonstrate that UVRR is successfully employed in the study of aggregation-prone proteins and of their interaction with ligands, especially in the case of Trp-lacking proteins.

Tomography of a seeded free-electron laser focal spot: qualitative and quantitative comparison of two reconstruction methods for spot size characterization.

Performing experiments at free-electron lasers (FELs) requires an exhaustive knowledge of the pulse temporal and spectral profile, as well as the focal spot shape and size. Operating FELs in the extreme ultraviolet (EUV) and soft X-ray (SXR) spectral regions calls for designing ad-hoc optical layouts to transport and characterize the EUV/SXR beam, as well as tailoring its spatial dimensions at the focal plane down to sizes in the few micrometers range. At the FERMI FEL (Trieste, Italy) this task is carried out by the Photon Analysis Delivery and Reduction System (PADReS). In particular, to meet the different experimental requests on the focal spot shape and size, a proper tuning of the optical systems is required, and this should be monitored by means of dedicated techniques. Here, we present and compare two reconstruction methods for spot characterization: single-shot imprints captured via ablation on a poly(methyl methacrylate) sample (PMMA) and pulse profiles retrieved by means of a Hartmann wavefront sensor (WFS). By recording complementary datasets at and nearby the focal plane, we exploit the tomography of the pulse profile along the beam propagation axis, as well as a qualitative and quantitative comparison between these two reconstruction methods.

Cell-stiffness and morphological architectural patterns in clinical samples of high grade serous ovarian cancers.

High grade serous ovarian carcinoma (HGSOC) is recognized as the most frequent type of ovarian cancer and the main cause of ovarian cancer related deaths worldwide. Although homologous recombination deficiency testing has been adopted in the clinical workflow, morphological analysis remains the main diagnostic tool. In this study Atomic Force Microscopy (AFM) was tested in standard hematoxylin and eosin (H&E) stained sections to investigate the biomechanical properties of different architectural growing patterns of HGSOC. Our results showed that AFM was able to discriminate HGSOC morphological growing patterns as well as patients' stage. Micropapillary pattern, which has been associated to poor outcome, had lower Young's moduli. In addition stage IV HGSOC was significantly softer than stage III cancers. Based on our results, AFM analysis could represent an additional tool in HGSOC morphological diagnosis as the biomechanical proprieties of HGSOC were quantitatively associated to tumor staging and architectural pattern.

On the Formation and Stability of Chitosan/Hyaluronan-Based Complex Coacervates.

This contribution is aimed at extending our previous findings on the formation and stability of chitosan/hyaluronan-based complex coacervates. Colloids are herewith formed by harnessing electrostatic interactions between the two polyelectrolytes. The presence of tiny amounts of the multivalent anion tripolyphosphate (TPP) in the protocol synthesis serves as an adjuvant "point-like" cross-linker for chitosan. Hydrochloride chitosans at different viscosity average molar mass, , in the range 10,000-400,000 g/mol, and fraction of acetylated units, FA, (0.16, 0.46 and 0.63) were selected to fabricate a large library of formulations. Concepts such as coacervate size, surface charge and homogeneity in relation to chitosan variables are herein disclosed. The stability of coacervates in Phosphate Buffered Saline (PBS) was verified by means of scattering techniques, i.e., Dynamic Light Scattering (DLS) and Small-Angle X-ray Scattering (SAXS). The conclusions from this set of experiments are the following: (i) a subtle equilibrium between chitosan FA and does exist in ensuring colloidal stability; (ii) once diluted in PBS, osmotic swelling-driven forces trigger the enlargement of the polymeric mesh with an ensuing increase of coacervate size and porosity.

Functionalizing gold with single strand DNA: novel insight into optical properties via combined spectroscopic ellipsometry and nanolithography measurements.

We have studied the self-assembly of 22-base oligonucleotides bound by a short alkyl thiol linker (C6-ssDNA) on flat Au films. The self-assembled monolayer (SAM) was modified by addition of a spacer (mercaptohexanol, MCH). Molecular depositions were monitored in situ by spectroscopic ellipsometry (SE). SAMs were characterized in a liquid environment by coupling SE (difference spectra method) with Atomic Force Microscope (AFM) measurements. We exploited the biofilm thickness obtained by AFM nanolithography and imaging to solve the refractive index/thickness correlation in optical measurements on ultrathin molecular layers. The combined SE/AFM analysis provided reliable estimates of the thickness and the refractive index of the biofilm in the NIR region (650-1300 nm) and revealed new aspects of DNA molecular organization: exposure to MCH leads to an increase of both film thickness and refractive index, which points to a reorganization of C6-ssDNA film. We show that the contribution of the thiol/Au interface has to be included in the optical model to obtain a more reliable determination of the refractive index of the biofilm in a liquid. The careful, correlative characterization of the mixed C6-ssDNA/MCH SAM represents a key step towards the optimization of a robust detection scheme based on helix-helix hybridization.

Multi-technique microscopy investigation on bacterial biofilm matrices: a study on Klebsiella pneumoniae clinical strains.

Biofilms are communities of bacteria living embedded in a highly hydrated matrix composed of polysaccharides, proteins, and extracellular DNA. This life style confers numerous advantages to bacteria including protection against external threats. However, they also contribute to increase bacterial resistance against antimicrobials, an issue particularly relevant in dangerous infections. Due to the complexity of the matrix, few information is present in the literature on details of its architecture including the spatial distribution of the macromolecular components which might give hints on the way the biofilm scaffold is built up by bacteria. In this study, we investigated the possibility to combine well-established microbiological procedures with advanced microscopies to get information on composition and distribution of the macromolecular components of biofilm matrices. To this, confocal microscopy, diffraction-limited infrared (IR) spectral imaging, and atomic force microscopy (AFM) were used to explore biofilm produced by a clinical strain of Klebsiella pneumoniae. IR imaging permitted to have clues on how the biofilm grows and spreads on surfaces, and the local distribution of the components within it. Through the analysis of the pure component spectra, it was possible to assess the chemical and structural composition of the saccaridic matrix, confirming the data obtained by NMR. It was also possible to follow the time course of biofilm from 6 up to 48 h when the biofilm grew into a 3-dimensional multi-layered structure, characteristic of colonies of bacteria linked together by a complex matrix. In addition, nanoFTIR and AFM investigations allowed the estimation of biofilm growth in the vertical direction and the morphological analysis of bacterial colonies at different time points and the evaluation of the chemical composition at the nanoscale.

The High Mobility Group A1 (HMGA1) Chromatin Architectural Factor Modulates Nuclear Stiffness in Breast Cancer Cells.

Plasticity is an essential condition for cancer cells to invade surrounding tissues. The nucleus is the most rigid cellular organelle and it undergoes substantial deformations to get through environmental constrictions. Nuclear stiffness mostly depends on the nuclear lamina and chromatin, which in turn might be affected by nuclear architectural proteins. Among these is the HMGA1 (High Mobility Group A1) protein, a factor that plays a causal role in neoplastic transformation and that is able to disentangle heterochromatic domains by H1 displacement. Here we made use of atomic force microscopy to analyze the stiffness of breast cancer cellular models in which we modulated HMGA1 expression to investigate its role in regulating nuclear plasticity. Since histone H1 is the main modulator of chromatin structure and HMGA1 is a well-established histone H1 competitor, we correlated HMGA1 expression and cellular stiffness with histone H1 expression level, post-translational modifications, and nuclear distribution. Our results showed that HMGA1 expression level correlates with nuclear stiffness, is associated to histone H1 phosphorylation status, and alters both histone H1 chromatin distribution and expression. These data suggest that HMGA1 might promote chromatin relaxation through a histone H1-mediated mechanism strongly impacting on the invasiveness of cancer cells.

Single-stranded DNA oligomer brush structure is dominated by intramolecular interactions mediated by the ion environment.

Single-stranded DNA (ssDNA) brushes, in which ssDNA oligomers are tethered to surfaces in dense monolayers, are being investigated for potential biosensing applications. The structure of the brush can affect the selectivity and the hybridization efficiency of the device. The structure is commonly thought to result from the balance of intramolecular interactions, intermolecular interactions within the monolayer, and molecule-surface interactions. Here, we test the hypothesis that ssDNA oligomer brush structure is dominated by intramolecular interactions. We use AFM to measure the height of an ssDNA brush and molecular dynamics to simulate the end-to-end distance, both as a function of ionic strength of the surrounding solution. The brush height and the molecule end-to-end distance match quantitatively, providing evidence that the brush structure is dominated by intramolecular interactions (mediated by ions). The physical basis of the intramolecular interactions is elucidated by the simulations.

Experimental setups for FEL-based four-wave mixing experiments at FERMI.

The recent advent of free-electron laser (FEL) sources is driving the scientific community to extend table-top laser research to shorter wavelengths adding elemental selectivity and chemical state specificity. Both a compact setup (mini-TIMER) and a separate instrument (EIS-TIMER) dedicated to four-wave-mixing (FWM) experiments has been designed and constructed, to be operated as a branch of the Elastic and Inelastic Scattering beamline: EIS. The FWM experiments that are planned at EIS-TIMER are based on the transient grating approach, where two crossed FEL pulses create a controlled modulation of the sample excitations while a third time-delayed pulse is used to monitor the dynamics of the excited state. This manuscript describes such experimental facilities, showing the preliminary results of the commissioning of the EIS-TIMER beamline, and discusses original experimental strategies being developed to study the dynamics of matter at the fs-nm time-length scales. In the near future such experimental tools will allow more sophisticated FEL-based FWM applications, that also include the use of multiple and multi-color FEL pulses.

Glioma-associated stem cells: a novel class of tumor-supporting cells able to predict prognosis of human low-grade gliomas.

BACKGROUND: Translational medicine aims at transferring advances in basic science research into new approaches for diagnosis and treatment of diseases. Low-grade gliomas (LGG) have a heterogeneous clinical behavior that can be only partially predicted employing current state-of-the-art markers, hindering the decision-making process. To deepen our comprehension on tumor heterogeneity, we dissected the mechanism of interaction between tumor cells and relevant components of the neoplastic environment, isolating, from LGG and high-grade gliomas (HGG), proliferating stem cell lines from both the glioma stroma and, where possible, the neoplasm. METHODS AND FINDINGS: We isolated glioma-associated stem cells (GASC) from LGG (n=40) and HGG (n=73). GASC showed stem cell features, anchorage-independent growth, and supported the malignant properties of both A172 cells and human glioma-stem cells, mainly through the release of exosomes. Finally, starting from GASC obtained from HGG (n=13) and LGG (n=12) we defined a score, based on the expression of 9 GASC surface markers, whose prognostic value was assayed on 40 subsequent LGG-patients. At the multivariate Cox analysis, the GASC-based score was the only independent predictor of overall survival and malignant progression free-survival. CONCLUSIONS: The microenvironment of both LGG and HGG hosts non-tumorigenic multipotent stem cells that can increase in vitro the biological aggressiveness of glioma-initiating cells through the release of exosomes. The clinical importance of this finding is supported by the strong prognostic value associated with the characteristics of GASC. This patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma.

Spectroscopic ellipsometry meets AFM nanolithography: about hydration of bio-inert oligo(ethylene glycol)-terminated self assembled monolayers on gold.

For the first time, to our knowledge, spectroscopic ellipsometry (SE) has been combined with state-of-the-art AFM differential height measurements conducted after shaving nano-lithography of ultrathin, soft-matter films for thickness determination. We investigated self-assembled monolayers of SH-(CH2)11-EGn-OH molecules on gold, where EG is ethylene glycol units and n = 3 and 6, a prototypical non-fouling system. We performed SE measurements (245-1200 nm) focusing on the changes induced by the formation of the film (difference spectra). SE measurements, analysed by simple models, confirm the formation of the S-Au interface, transparency of the SAMs and provide a sharp picture of the ability of the EG functionality to protect the surface from unspecific adsorption of proteins. A quantitative assessment of the film thickness by SE was carried out ex situ, thanks to the optical contrast between the film and the ambient, and by AFM in liquid. The cross-check between SE and AFM height measurements combined with the comparison between in-liquid and ex situ SE measurements allowed obtaining non-perturbative information about the vertical density profile of the SAM. The in-liquid SE measurements indicate a refractive index matching between the aqueous medium and the outer part of the SAM, consistent with a disordered configuration of OEG and/or the penetration of water amid the OEG strands. A critical discussion provides a detailed insight into the subtle issues and pitfalls related to the thickness determination of soft-matter films to the monolayer limit.

A DNA-based nano-immunoassay for the label-free detection of glial fibrillary acidic protein in multicell lysates.

We have developed a quantitative approach to eventually enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells. Through DNA-directed immobilization of DNA-protein conjugates we immobilized antibodies specific for a certain protein of interest, on a complementary DNA nanoarray fabricated by means of nanografting, a nanolithography technique based on atomic force microscopy (AFM). The proof of concept was realized for glial fibrillary acidic protein (GFAP), a biomarker crucial in cell's differentiation of astrocytes, and functional to grade classification of gliomas, the most common of primary malignant brain tumors. The efficiency of the nano-immuno sensing was tested by obtaining the immobilization of purified recombinant GFAP protein at different concentration in a standard solution then in a cellular lysate. A comparison of sensitivity between our technique and conventional ELISA assays is provided at the end of the paper. FROM THE CLINICAL EDITOR: This team developed a quantitative approach to enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells, demonstrating the utility of this DNA-based nano-immunoassay in the detection of GFAP.

Extracellular vesicles released by LPS-stimulated spinal organotypic slices spread neuroinflammation into naïve slices through connexin43 hemichannel opening and astrocyte aberrant calcium dynamics.

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.

Microfluidic production of amiodarone loaded nanoparticles and application in drug repositioning in ovarian cancer.

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.

Impact of Isolation Methods on Extracellular Vesicle Functionality In Vitro and In Vivo.

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.

Dimeric and monomeric conformation of SARS-CoV-2 main protease: New technical approaches based on IR radiation.

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.

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.

Lipid bilayer fluidity and degree of order regulates small EVs adsorption on model cell membrane.

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.