Biology subjects

Zendrini, A.

Publications and source records attributed to Zendrini, A..

8 recordsLinked to original sources

bioRxiv2026

Extracellular vesicles from Manila clam (Ruditapes philippinarum): tailored isolation from hemolymph and insights into water-derived vesicles

Extracellular vesicles (EVs) are evolutionarily conserved mediators of intercellular communication released by cells into biological fluids and the extracellular environment. Despite their growing relevance in biomedical and veterinary research, knowledge on EVs in marine bivalves remains limited. The aim of this study was to optimize tailored protocols for EV isolation from the hemolymph of the Manila clam (Ruditapes philippinarum) based on density gradient ultracentrifugation (dgUC) or size exclusion chromatography (SEC). EV-enriched fractions were identified through nanoparticle tracking analysis, protein quantification, transmission electron microscopy, and cryo-electron microscopy. Both methods successfully isolated small EVs (<200 nm). While dgUC yielded higher-purity preparations, SEC provided a higher recovery rate and compatibility with downstream metabolomic analyses. Metabolomics performed on SEC fractions and on hemolymph, revealed that EV-enriched fractions possessed a distinct metabolic signature including enrichment in metabolites associated with nucleotide metabolism, glycolysis, redox regulation, and energy metabolism. Furthermore, we performed a pilot investigation into the presence of EVs released into conditioned water by Manila clams. Using tangential flow filtration and ultrafiltration, EVs were successfully concentrated from water samples and characterized by nanoparticle tracking analysis, CONAN assay, atomic force microscopy, and electron microscopy. Our findings demonstrate the feasibility of isolating EVs both from Manila clam hemolymph and from conditioned water, providing the first evidence of water-derived EV recovery in aquatic animals. Although further methodological refinement is needed to improve the purity of EVs isolated from water, and additional characterization studies are required to better define the molecular composition of clam-derived EVs, these results establish a foundation for future investigations into the role of EVs in bivalve biology and their potential application as minimally invasive biomarkers for aquaculture, environmental monitoring, and ecosystem health assessment.

systems biology↗

bioRxiv2026

Isolation and preliminary characterization of extracellular vesicles from bottlenose dolphin (Tursiops truncatus) and long-finned pilot whale (Globicephala melas) blow

Cetaceans are key sentinel species for environmental health monitoring. Although sampling from free-ranging animals is challenging, the analysis of cetacean blow offers a minimally invasive approach to assess their health status. Extracellular vesicles (EVs) are cell-derived nanostructures present in biological fluids and widely studied as disease biomarkers in humans. Despite the potential for similar uses, EVs have not been studied in cetacean blow to date . This proof-of-concept study aims to assess the feasibility of the isolation and characterization of EVs from blow samples collected from five bottlenose dolphins (Tursiops truncatus) kept under human care and from a free ranging specimen of long-finned pilot whale (Globicephala melas). EVs were purified from bottlenose dolphin blows by ultracentrifugation (UC) or size exclusion chromatography (SEC) and from the long-finned pilot whale by SEC. Particle concentration and size distribution were assessed by Nanoparticle Tracking Analysis (NTA), morphology by Air-atomic force microscopy (AFM) and protein expression by Western Blotting (WB). NTA revealed a higher mean particle concentration in bottlenose dolphin EVs isolated by UC compared to SEC, while EVs isolated from the long-finned pilot whale presented a lower particle concentration. AFM confirmed the presence of EV-like particles within the typical EV size range in bottlenose dolphins EVs obtained both by SEC or UC. All EV samples were positive for CD9 and integrin-{beta} and negative to Calnexin. SEC was more sensitive to detect OmpA, a membrane protein of Gram-negative bacteria, in EVs from both species. Our pilot study demonstrates that EVs are present in cetacean blow and can be isolated and characterized. Future investigations focused on characterizing and quantifying a wider array of EV associated molecules may further the application of blow EV analysis for cetacean health assessments.

zoology↗

bioRxiv2025

Red blood Cell-derived Extracellular Vesicles as biomaterials: the opportunity of freezing-induced accelerated aging

Red blood cell-derived extracellular vesicles (RBC-EVs) are emerging as promising biomaterials for next-generation drug delivery, due to their inherent biocompatibility, immune evasion capabilities, and minimal oncogenic risk. However, their clinical translation remains limited by unresolved challenges related to heterogeneity, reproducibility, and long-term storage. This study proposes a method that leverages freezing-induced ageing for obtaining highly homogeneous RBC-EV batches, an important step towards using RBC-EVs as healthcare biomaterials and advancing their clinical translation in EV-based nanomedicine. This method was made possible thanks to the analytical support of discontinuous sucrose density gradient and high-resolution interferometric nanoparticle tracking analysis, which allowed the identification of a bimodal subpopulation distribution, in terms of vesicle size, interferometric contrast, and subpopulation profiles, in freshly prepared samples, and then tracked how long-term cold storage at -80 {degrees}C channeled this heterogeneity into a monomodal population. Finally, we evaluated the functionality of homogenized RBC-EV samples by assessing surface-associated enzymatic activity and uptake in cancer cell lines, demonstrating that freeze-thaw-induced accelerated-aging provides a viable strategy for producing RBC-EV preparations that retain membrane integrity and remain readily internalized by cells. These findings offer valuable insights into the optimization and standardization of RBC-EV handling and storage protocols, providing a foundation for their reliable integration into EV-based therapeutic applications.

cell biology↗

bioRxiv2025

Red Blood Cell-derived Extracellular Vesicles enable Cisplatin and Cetuximab Synergistic Therapy against Triple-Negative Breast Cancer

BackgroundTriple-negative breast cancer is an aggressive breast cancer subtype characterized by the absence of human epidermal growth factor receptor 2, estrogen and progesterone receptors, limiting targeted therapy options. Cisplatin, a chemotherapeutic agent, induces DNA damage and exhibits some efficacy against triple-negative breast cancer, but its effectiveness is often reduced by chemoresistance and systemic toxicity. A very promising strategy to augment cisplatin treatment can be based on combining it with the biologic Cetuximab, an epidermal growth factor receptor inhibitor, which boosts cisplatin efficacy by inducing ferroptosis. ResultsTo optimize this strategy in a biocompatible and precise manner, we developed a nanoplatform based on red blood cell-derived extracellular vesicles for the combined delivery of Cetuximab and cisplatin, enabling immune evasion, and the possibility of autologous personalization and GMP-compliant production. Owing to their DNA-free lumen and lack of EGFR, RBC-EVs preserve cisplatin activity and prevent interference with cetuximab. This formulation enhances cisplatins cytotoxicity by up to 50%, as shown in vitro and in patient-derived organoids. It effectively reduces chemoresistance by downregulating hypoxia-related genes and promoting ferroptosis, additionally, it improves cisplatins cytotoxic effects while reducing hemotoxicity compared to the administration of free cisplatin. ConclusionsThese findings highlight the potential of red blood cell-derived extracellular vesicles as a biocompatible delivery system enabling combined therapy and offering a promising strategy to overcome current limitations in TNBC treatment.

cell biology↗

bioRxiv2023

Addressing heterogeneity in direct analysis of Extracellular Vesicles and analogues using Membrane-Sensing Peptides as Pan-Affinity Probes

Extracellular vesicles (EVs), crucial mediators of cell-to-cell communication, hold immense potential for diagnostic applications due to their ability to enrich protein biomarkers in body fluids. However, challenges in isolating EVs from complex biological specimens hinder their widespread use. In this frame, integrated isolation-and-analysis workflows are the go-to strategy, most of which see the prevalence of immunoaffinity methods. Yet, the high heterogeneity of EVs poses challenges, as proposed ubiquitous markers are less homogenously prevalent than believed, raising concerns about the reliability of downstream biomarker discovery programs. This issue extends to the burgeoning field of engineered EV-mimetics and bio-nanoparticles, where conventional immune-affinity methods may lack applicability. Addressing these challenges, we introduce the use Membrane Sensing Peptides (MSP) as "universal" affinity ligands for both EVs and EV-analogues. Employing a streamlined process integrating on-bead capture and vesicle phenotyping through Single Molecule Array (SiMoA) technology, we showcase the application of MSP ligands in the integrated analysis of circulating EVs in blood derivatives, eliminating the need for prior EV isolation. Demonstrating the possible clinical translation of MSP technology, we directly detect an EV-associated epitope signature in serum and plasma samples, demonstrating its potential for distinguishing patients with myocardial infarction versus stable angina. At last, notably, MSP exhibits a unique capability to enable the analysis of tetraspanin-lacking Red Blood Cell derived EVs (RBC-EVs). Overall, unlike traditional antibody-based methods, MSP probes work agnostically, overcoming limitations associated with surface protein abundance or scarcity. This highlights the potential of MSP in advancing EV analysis for clinical diagnostics and beyond. Of note, this represents also the first-ever peptide-based application in SiMoA technology.

biochemistry↗

bioRxiv2023

A decision-making tool to navigate through Extracellular Vesicle research and product development

Due to their intercellular communication properties and their involvement in a wide range of biological processes, extracellular vesicles (EVs) are increasingly being studied and exploited for different applications. Nevertheless, their complex nature and heterogeneity, as well as the challenges related to their isolation, purification and characterization procedures, require cautious assessment of the quality and quantity parameters to monitor. This translates into a multitude of choices and putative solutions that lie in front of any EV researcher, in both research and translational environments, resembling a labyrinth with multiple paths to cross and, possibly, more than one exit. In this respect, decision-making tools might represent our modern Ariadnes string to follow not to get lost or distracted along the journey, to choose the shorter and best-fit-to-source EV application(s) and vice versa. Here, we present the implementation of a multi-criteria EV decision-making grid (EV-DMG) as a novel, customizable, efficient and easy-to-use tool to support responsible EV research and innovation. By identifying and weighting key assessment criteria for comparing distinct EV-based preparations and/or processes, our EV-DMG may assist any EV community member in making informed, transparent and reproducible decisions regarding the EV sources and/or samples to be managed, as well as the most suitable production and/or analytical pipelines to be adopted for targeting a defined aim or application.

cell biology↗

bioRxiv2022

Compositional profiling of extracellular vesicles/lipoproteins 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 a 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 characterized 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. To the best of our knowledge, these results represent the first systematic single-particle mechanical investigation of lipoproteins. The described approach is label-free, single-step and relatively quick to perform. Importantly, it can be used to analyze 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.

biophysics↗

bioRxiv2021

Antithrombin glycoforms from type II antithrombin deficient patients selectively adsorb onto the surface of plasma extracellular vesicles

Antithrombin (AT) is a glycoprotein produced by the liver and a principal antagonist of active clotting proteases. A deficit in AT function leads to AT qualitative deficiency, challenging to diagnose. Here we report that active AT may travel physiosorbed on the surface of plasma extracellular vesicles (EVs), contributing to form the "EV-protein corona". The corona is enriched in specific AT glycoforms, thus suggesting glycosylation to play a key role in AT partitioning between EVs and plasma. Differences in AT glycoform composition of the corona of EVs separated from plasma of healthy and AT qualitative deficiency-affected subjects were also noticed. This suggests deconstructing the plasma into its nanostructured components, as extracellular vesicles, could help to unravel pathophysiological mechanisms otherwise undiscovered.

biochemistry↗