Massimo Bottini

The concept of the “biomolecular corona” (BC) was first introduced to describe the spontaneous absorption of molecules, mainly proteins, onto the surface of synthetic nanoparticles upon exposure to biological fluids. More recently, the same term has been used to describe biomolecules that dynamically interact with the surface of extracellular vesicles (EVs) during their biogenesis, secretion, or upon exposure to intracellular or extracellular milieus, including biological fluids. The EV biomolecular corona is a key determinant of EV identity, shaping their cellular uptake, biodistribution, and functional outcomes in both physiological and pathological contexts. EVs, in contrast to synthetic nanoparticles, introduce distinct challenges and perspectives due to their biological origin, innate heterogeneity, and complex combination of physicochemical and biological properties. To address this evolving topic, the International Society for Extracellular Vesicles (ISEV) organized a dedicated workshop in Brescia, Italy, on 27th to 28th February, 2025. The event gathered selected participants from academia and industry, with expertise spanning synthetic nanomaterials, EVs, biophysics, and translational nanomedicine. The program included keynote lectures, oral presentations, posters, and a series of interactive roundtables structured around fundamentals of the biomolecular corona, EV corona biogenesis and composition, analytical and separation tools and clinical and diagnostic potential applications. This report summarizes the key scientific insights, methodological challenges, conceptual debates, and future perspectives that emerged from the workshop.

Membrane blebbing is a dynamic cellular phenomenon that plays a key role in numerous biological processes such as migration, apoptosis, cytokinesis, and extracellular vesicle (EV) formation. This phenomenon is traditionally observed via high‐resolution imaging techniques on fixed samples, since it is difficult to quantify and monitor on live cells. This aspect limits our understanding of their temporal evolution and mechanical behavior. In this work, we present and validate a novel approach based on Atomic Force Microscopy (AFM) force spectroscopy combined with a custom data analysis pipeline that is capable to detect and map bleb‐ and EV‐like structures directly on live cells. The method leverages the mechanical signatures embedded in force‐distance (FD) curves—specifically, the presence of breakthrough points, which indicate the rupture or collapse of soft, vesicular structures. A custom MATLAB algorithm was developed to automatically identify and spatially map FD curves exhibiting EV‐like mechanical behavior, based on signal smoothing, inflection point analysis, and user‐defined force thresholds. This algorithm was at first validated against isolated EVs in liquid and then applied to live cultures of MDA‐MB‐231 breast cancer cells, monitored over 3, 7, 10, and 14 days to follow the temporal changes in membrane activity. This method enables the quantitative mapping of vesicle‐ and bleb‐associated curves on the cell surface, highlighting their spatial distribution. A progressive increase in the number of EV‐like events was observed up to Day 10, followed by a decline at Day 14—suggesting a dynamic remodeling of the membrane during cell culture maturation or stress. Moreover, they exhibit a preferential localization at the cell periphery. To validate the correspondence between EV‐like force signatures and blebs, standard AFM high‐resolution images were acquired on the same cells fixed and then analyzed using a Circular Hough Transform‐based algorithm to detect and count blebs based on their circular geometry. The time trends obtained from both techniques showed good agreement, confirming the robustness of the force spectroscopy‐based detection on live samples. This approach represents a significant advancement in the use of AFM for live‐cell analysis. By extracting meaningful biophysical information from FD curves beyond standard elasticity metrics, we demonstrate that AFM spectroscopy can serve as a high‐content, non‐invasive tool for the detection of nanoscale membrane phenomena.

Oral delivery of insulin holds great promise for improving patient compliance. However, the harsh gastrointestinal environment, the low permeability of the intestinal epithelium, and hepatic clearance of foreign particles remain key challenges in this area. Here, we report the site-specific adaptive milk-derived nanovesicles (MiNVs) capable of overcoming intestinal and hepatic barriers for oral insulin delivery. These MiNVs could bind natural IgG on their surface, enabling FcRn-mediated transcytosis by evading the lysosomal degradation pathway. Upon reaching the liver, MiNVs responded to the elevated levels of biothiols in the hepatic microenvironment, triggering site-specific insulin release. In type 1 diabetic rats, the oral bioavailability reached 20.4%, which is about 20-fold higher than that of free insulin. Notably, MiNVs showed effective glycemic control over long-term treatment in type 1 diabetic rat and minipig models. By integrating transepithelial transport and liver-specific responsiveness, the site-specific adaptability of MiNVs supports their promise in oral insulin administration.

Mineralizing cells release a special class of extracellular vesicles known as matrix vesicles (MV), crucial for bone mineralization. Following their release, MV anchor to the extracellular matrix (ECM), where their highly specialized enzymatic machinery facilitates the formation of seed mineral within the MV’s lumen, subsequently releasing it onto the ECM. However, how MV propagate mineral onto the collagenous ECM remains unclear. In this study, we address these questions by exploring the “protein corona” paradigm whereby nanoparticles entering a biological milieu become cloaked by a corona of soluble proteins modifying their biological functions. We isolated native MV from the growth plates of chicken embryos. After removing the protein corona from the native MV using high ionic strength buffer, we obtained shaved MV. Reconstituted MVs were produced by incubating shaved MV with the removed protein corona constituents. Our results show that both the removal and reconstitution of protein corona significantly affect the biochemical and physicochemical properties of MV, resulting in 3 well-defined groups. Shaved MV exhibited an increase in tissue nonspecific alkaline phosphatase (TNAP) activity and a decrease in mineral deposition compared to native MV. Reconstituted MV partially recovered these functions, showing a reduction of TNAP activity and mineral deposition compared to native MV. Furthermore, changes in the protein corona affect the MV ability to anchor to the collagenous ECM, which is crucial for initiating the propagation of the mineral phase within this organic matrix. Proteomic analyses revealed changes in the protein profile of the MV resulting from the removal of the protein corona, indicating that shaved proteins were primarily related to external structural and ECM organization and catabolism. These findings underscore the role of the protein corona in modulating the mineralization capabilities of MV. Understanding these interactions could lead to new therapeutic strategies for enhancing bone repair and regeneration.

Mineralization-competent cells, including hypertrophic chondrocytes, mature osteoblasts, and osteogenic-differentiated smooth muscle cells secrete media extracellular vesicles (media vesicles) and extracellular vesicles bound to the extracellular matrix (matrix vesicles). Media vesicles are purified directly from the extracellular medium. On the other hand, matrix vesicles are purified after discarding the extracellular medium and subjecting the cells embedded in the extracellular matrix or bone or cartilage tissues to an enzymatic treatment. Several pieces of experimental evidence indicated that matrix vesicles and media vesicles isolated from the same types of mineralizing cells have distinct lipid and protein composition as well as functions. These findings support the view that matrix vesicles and media vesicles released by mineralizing cells have different functions in mineralized tissues due to their location, which is anchored to the extracellular matrix versus free-floating.

Matrix vesicles are a special class of extracellular vesicles thought to actively contribute to both physiologic and pathologic mineralization. Proteomic studies have shown that matrix vesicles possess high amounts of annexin A5, suggesting that the protein might have multiple roles at the sites of calcification. Currently, Annexin A5 is thought to promote the nucleation of apatitic minerals close to the inner leaflet of the matrix vesicles’ membrane enriched in phosphatidylserine and Ca²⁺. Herein, we aimed at unravelling a possible additional role of annexin A5 by investigating the ability of annexin A5 to adsorb on matrix-vesicle biomimetic liposomes and Langmuir monolayers made of dipalmitoylphosphatidylserine (DPPS) and dipalmitoylphosphatidylcholine (DPPC) in the absence and in the presence of Ca²⁺. Differential scanning calorimetry and dynamic light scattering measurements showed that Ca²⁺ at concentrations in the 0.5–2.0 mM range induced the aggregation of liposomes probably due to the formation of DPPS-enriched domains. However, annexin A5 avoided the aggregation of liposomes at Ca²⁺ concentrations lower than 1.0 mM. Surface pressure versus surface area isotherms showed that the adsorption of annexin A5 on the monolayers made of a mixture of DPPC and DPPS led to a reduction in the area of excess compared to the theoretical values, which confirmed that the protein favored attractive interactions among the membrane lipids. The stabilization of the lipid membranes by annexin A5 was also validated by recording the changes with time of the surface pressure. Finally, fluorescence microscopy images of lipid monolayers revealed the formation of spherical lipid-condensed domains that became unshaped and larger in the presence of annexin A5. Our data support the model that annexin A5 in matrix vesicles is recruited at the membrane sites enriched in phosphatidylserine and Ca²⁺ not only to contribute to the intraluminal mineral formation but also to stabilize the vesicles’ membrane and prevent its premature rupture.

Tissue engineering offers attractive strategies to develop three-dimensional scaffolds mimicking the complex hierarchical structure of the native bone. The bone is formed by cells incorporated in a molecularly organized extracellular matrix made of an inorganic phase, called biological apatite, and an organic phase mainly made of collagen and noncollagenous macromolecules. Although many strategies have been developed to replicate the complexity of bone at the nanoscale in vitro, a critical challenge has been to control the orchestrated process of mineralization promoted by bone cells in vivo and replicate the anatomical and biological properties of native bone. In this study, we used type I collagen to fabricate mineralized scaffolds mimicking the microenvironment of the native bone. The sulfated polysaccharide κ-carrageenan was added to the scaffolds to fulfill the role of noncollagenous macromolecules in the organization and mineralization of the bone matrix and cell adhesion. Scanning electron microscopy images of the surface of the collagen/κ-carrageenan scaffolds showed the presence of a dense and uniform network of intertwined fibrils, while images of the scaffolds' lateral sides showed the presence of collagen fibrils with a parallel alignment, which is characteristic of dense connective tissues. MC3T3-E1 osteoblasts were cultured in the collagen scaffolds and were viable after up to 7 days of culture, both in the absence and in the presence of κ-carrageenan. The presence of κ-carrageenan in the collagen scaffolds stimulated the maturation of the cells to a mineralizing phenotype, as suggested by the increased expression of key genes related to bone mineralization, including alkaline phosphatase (Alp), bone sialoprotein (Bsp), osteocalcin (Oc), and osteopontin (Opn), as well as the ability to mineralize the extracellular matrix after 14 and 21 days of culture. Taken together, the results described in this study shed light on the potential use of collagen/κ-carrageenan scaffolds to study the role of the structural organization of bone-mimetic synthetic matrices in cell function.

Matrix vesicles (MVs) contain the whole machinery necessary to initiate apatite formation in their lumen. We suspected that, in addition to tissue-nonspecific alkaline phosphatase (TNAP), Na,K,-ATPase (NKA) could be involved in supplying phopshate (P_i) in the early stages of MV-mediated mineralization. MVs were extracted from the growth plate cartilage of chicken embryos. Their average mean diameters were determined by Dynamic Light Scattering (DLS) (212 ± 19 nm) and by Atomic Force Microcopy (AFM) (180 ± 85 nm). The MVs had a specific activity for TNAP of 9.2 ± 4.6 U·mg⁻¹ confirming that the MVs were mineralization competent. The ability to hydrolyze ATP was assayed by a colorimetric method and by ³¹P NMR with and without Levamisole and SBI-425 (two TNAP inhibitors), ouabain (an NKA inhibitor), and ARL-67156 (an NTPDase1, NTPDase3 and Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) competitive inhibitor). The mineralization profile served to monitor the formation of precipitated calcium phosphate complexes, while IR spectroscopy allowed the identification of apatite. Proteoliposomes containing NKA with either dipalmitoylphosphatidylcholine (DPPC) or a mixture of 1:1 of DPPC and dipalmitoylphosphatidylethanolamine (DPPE) served to verify if the proteoliposomes were able to initiate mineral formation. Around 69–72% of the total ATP hydrolysis by MVs was inhibited by 5 mM Levamisole, which indicated that TNAP was the main enzyme hydrolyzing ATP. The addition of 0.1 mM of ARL-67156 inhibited 8–13.7% of the total ATP hydrolysis in MVs, suggesting that NTPDase1, NTPDase3, and/or NPP1 could also participate in ATP hydrolysis. Ouabain (3 mM) inhibited 3–8% of the total ATP hydrolysis by MVs, suggesting that NKA contributed only a small percentage of the total ATP hydrolysis. MVs induced mineralization via ATP hydrolysis that was significantly inhibited by Levamisole and also by cleaving TNAP from MVs, confirming that TNAP is the main enzyme hydrolyzing this substrate, while the addition of either ARL-6715 or ouabain had a lesser effect on mineralization. DPPC:DPPE (1:1)-NKA liposome in the presence of a nucleator (PS-CPLX) was more efficient in mineralizing compared with a DPPC-NKA liposome due to a better orientation of the NKA active site. Both types of proteoliposomes were able to induce apatite formation, as evidenced by the presence of the 1040 cm⁻¹ band. Taken together, the findings indicated that the hydrolysis of ATP was dominated by TNAP and other phosphatases present in MVs, while only 3–8% of the total hydrolysis of ATP could be attributed to NKA. It was hypothesized that the loss of Na/K asymmetry in MVs could be caused by a complete depletion of ATP inside MVs, impairing the maintenance of symmetry by NKA. Our study carried out on NKA-liposomes confirmed that NKA could contribute to mineral formation inside MVs, which might complement the known action of PHOSPHO1 in the MV lumen.

Strontium acetate is applied for dental hypersensitivity treatment; however, the use of strontium carbonates for this purpose has not been described. The use of Sr-carbonate nanoparticles takes advantage of both the benefits of strontium on dentin mineralization and the abrasive properties of carbonates. Here in, we aimed to synthesize strontium carbonate and strontium-substituted calcium carbonate nanoparticles and test them as potential compounds in active dentifrices for treating dental hypersensitivity. For this, SrCO₃, Sr_0.5Ca_0.5CO₃, and CaCO₃ nanoparticles were precipitated using Na₂CO₃, SrCl₂, and/or CaCl₂ as precursors. Their morphology and crystallinity were evaluated by electron microscopy (SEM) and X-ray diffraction, respectively. The nanoparticles were added to a poly (vinyl alcohol) gel and used to brush dentin surfaces isolated from human third molars. Dentin chemical composition before and after brushing was investigated by infrared spectroscopy (FTIR) and X-ray dispersive energy spectroscopy. Dentin tubule morphology, obliteration, and resistance of the coatings to acid attack were investigated by SEM and EDS. The cytotoxicity and ability of the particles to trigger the mineralization of hDPSCs in vitro were studied. Dentin brushed with the nanoparticles was coated by a mineral layer that was also able to penetrate the tubules, while CaCO₃ remained as individual particles on the surface. FTIR bands related to carbonate groups were intensified after brushing with either SrCO₃ or Sr_0.5Ca_0.5CO₃. The shift of the phosphate-related FTIR band to a lower wavenumber indicated that strontium replaced calcium on the dentin structure after treatment. The coating promoted by SrCO₃ or Sr_0.5Ca_0.5CO₃ resisted the acid attack, while calcium and phosphorus were removed from the top of the dentin surface. The nanoparticles were not toxic to hDPSCs and elicited mineralization of the cells, as revealed by increased mineral nodule formation and enhanced expression of COL1, ALP, and RUNX2. Adding Sr_0.5Ca_0.5CO₃ as an active ingredient in dentifrices formulations may be commercially advantageous since this compound combines the well-known abrasive properties of calcium carbonate with the mineralization ability of strontium, while the final cost remains between the cost of CaCO₃ and SrCO₃. The novel Sr_0.5Ca_0.5CO₃ nanoparticles might emerge as an alternative for the treatment of dental hypersensitivity.

The biochemical machinery involved in matrix vesicles-mediated bone mineralization involves a specific set of lipids, enzymes, and proteins. Annexins, among their many functions, have been described as responsible for the formation and stabilization of the matrix vesicles′ nucleational core. However, the specific role of each member of the annexin family, especially in the presence of type-I collagen, remains to be clarified. To address this issue, in vitro mineralization was carried out using AnxA6 (in solution or associated to the proteoliposomes) in the presence or in the absence of type-I collagen, incubated with either amorphous calcium phosphate (ACP) or a phosphatidylserine-calcium phosphate complex (PS–CPLX) as nucleators. Proteoliposomes were composed of 1,2-dipalmitoylphosphatidylcholine (DPPC), 1,2-dipalmitoylphosphatidylcholine: 1,2-dipalmitoylphosphatidylserine (DPPC:DPPS), and DPPC:Cholesterol:DPPS to mimic the outer and the inner leaflet of the matrix vesicles membrane as well as to investigate the effect of the membrane fluidity. Kinetic parameters of mineralization were calculated from time-dependent turbidity curves of free Annexin A6 (AnxA6) and AnxA6-containing proteoliposomes dispersed in synthetic cartilage lymph. The chemical composition of the minerals formed was investigated by Fourier transform infrared spectroscopy (FTIR). Free AnxA6 and AnxA6-proteoliposomes in the presence of ACP were not able to propagate mineralization; however, poorly crystalline calcium phosphates were formed in the presence of PS–CPLX, supporting the role of annexin-calcium-phosphatidylserine complex in the formation and stabilization of the matrix vesicles’ nucleational core. We found that AnxA6 lacks nucleation propagation capacity when incorporated into liposomes in the presence of PS–CPLX and type-I collagen. This suggests that AnxA6 may interact either with phospholipids, forming a nucleational core, or with type-I collagen, albeit less efficiently, to induce the nucleation process.