Biology subjects

Radeghieri, A.

Publications and source records attributed to Radeghieri, A..

5 recordsLinked to original sources

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↗

bioRxiv2024

Biogenic nanoparticles from liquid and solid matrices: biochemical and biophysical properties of Extracellular Vesicles-enriched samples from human plasma and skeletal muscle tissue.

AimStudies on extracellular vesicles (EVs) focused on samples enriched from liquid matrices, such as cell culture media and blood. Recent research highlights the roles of EVs derived from the extracellular matrix of solid tissues, and how investigating these specific EVs offers insights into their microenvironment and potential biological influences on surrounding cells. This study presents a shared method to separate and compare EV enriched from solid (human skeletal muscle biopsy) and liquid (human plasma) matrices, addressing technical challenges and minimizing biases in separation techniques. MethodsPlasma and skeletal muscle-EVs were obtained combining serial centrifugation steps and discontinuous sucrose density gradient. EVs characterization employed advanced analytical techniques such as Western Blot, Colorimetric Nanoplasmonic assay, Atomic Force Miscoscopy, Nanoparticle Tracking Analysis and Dynamic Light Scattering to focus on biomolecular composition, nanomechanical properties, particle yield, size distribution, and colloidal stability. ResultsThe analysis revealed distinct differences between skeletal muscle-EVs and plasma-EVs including: molecular composition, physical and nanomechanical properties, as well as particle size distribution. Skeletal muscle-EVs exhibited unique colloidal behavior compared to their plasma counterparts, suggesting tissue-specific features that may influence their biological activity and stability. ConclusionsThe findings demonstrate that EVs from skeletal muscle tissue possess unique biochemical and biophysical characteristics when compared to those derived from plasma. These differences reflect their diverse biological origins and microenvironments. Understanding these distinctions could advance the development of EV-based diagnostic tools, particularly for muscular disorders, and broaden our knowledge of EV roles across various tissue contexts. HIGHLIGHTSO_LIShared protocol to enrich and compare extracellular vesicles (EVs) from both solid (skeletal muscle biopsy) and liquid (plasma) human samples, reducing methodological bias across matrices. C_LIO_LISkeletal muscle-derived EVs differ significantly from plasma EVs in biochemical and biophysical properties such as molecular composition, size distribution, nanomechanical properties, and colloidal stability. C_LIO_LIEV heterogeneity across microenvironments emerged as a key biological feature, highlighting their potential for specific diagnostic applications. C_LI

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↗

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↗