ECM Regulates Surface-Based EV Delivery by Altering Cellular Adhesion and EV Uptake

Introduction: : Extracellular vesicles (EVs) are cell-secreted nano-sized vesicles that play a key role in cell-to-cell communication by transporting nucleic acids, lipids, and proteins. EVs derived from adipose stem cells (ASC) can induce osteogenic, adipogenic, myogenic, and chondrogenic differentiation, promote angiogenesis, induce bone regeneration, and modulate the immune system [1][2]. EVs are also known to directly interact with extracellular matrix (ECM), and these interactions can regulate EV uptake. However, the specific interactions that EVs make with ECM and the mechanism for regulation EV uptake remains unclear. Here, we develop a surface-based EV delivery system that can be used to test EV-ECM binding and its impact on cellular behavior and EV uptake. Integrins serve as a key regulator in cell-material interactions [3]. Modifying substrates with ECM components can regulate both EV and cell interactions, affecting cellular processes such as adhesion, morphology, migration, and mechanotransduction [4][5]. Here, we tested substrate coatings composed of Collagen I (Col I), fibronectin (FN), laminin (Lam), hyaluronic acid (HA), and poly-L-lysine (PLL) and their impact on the EV binding and surface-based cellular uptake of EVs.

Materials and

Methods: : ECM coatings were formed on 12 mm glass coverslips by incubating 100 µg/mL solutions for 2 hours at 37°C, then overnight at 4°C. EVs were isolated from primary ASCs using 16% PEG incubation and ultracentrifugation at 127,000 rpm. The EVs were labeled with 80µM carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) dye by incubating for 2 hours at 37℃ in an atmosphere of 5% CO2. EVs binding was characterized by incubating labeled EVs (5 x 108 particles/ml) on different ECM coatings for overnight at 37°C and by measuring fluorescence via microplate reader. NIH 3T3 cells were cultured in DMEM supplemented with 10% FBS and 1% pen-strep. For focal adhesion and cell spread analysis, 10,000 cells/well were seeded onto ECM coatings with/without surface-adsorbed EVs and incubated for 24 hours and then fixed with 4% PFA, and stained with Anti-paxillin antibody [Y113], Alexa-548 phalloidin, and DAPI and then imaged at 63x. The focal adhesion area was analyzed using focal adhesion analysis server (FAAS) [6] and cell spread area was analyzed using ImageJ software. For EVs uptake, cells were seeded onto ECM coatings with surface-adsorbed EVs and incubated for 12 and 24 hours and then fixed with 4% PFA, mounted with DAPI then imaged at 63x. The EVs uptake was assessed via mean fluorescence intensity using ImageJ.

Results, Conclusions, and Discussions:: Successful adsorption of ECM coatings was observed onto glass where LAM showed the highest thickness, mass and elastic modulus. ECM coatings showed significant impact on the EV binding where COL coatings showed the highest and HA coatings showed the lowest EV binding (Figure 1A). EVs express integrins on their surfaces, facilitating binding to extracellular matrix (ECM) components such as fibronectin, collagen, and laminin [7] [8]. Similarly, ECM coatings showed significant impact on the focal adhesion formation of NIH 3T3 cells where COL and FN coatings showed significantly highest focal adhesion area (Figure 1B, E). In addition, significant impact of surface bound EVs on focal adhesion area was observed, particularly on PLL and LAM coatings, as well as on uncoated glass (Figure 1B, E). Although, there is no significant impact, an increase in the cell spread area was observed on the coatings with surface bound EVs. EVs bound to the surface express integrins and ECM mimicking ligands including fibronectin which might lead to stronger and more stable focal adhesion formation. Next, the surface bound EV uptake on NIH 3T3 cells resulted in the significant impact of ECM coatings on the uptake of EVs where PLL coatings showed significantly higher EVs uptake (Figure 1D, F). This result indicates that in addition to specific integrin-mediated interactions, non-specific interaction might also play important role in the uptake of EVs, especially through macropinocytosis.

Thus, this study highlights the critical role of ECM coatings in modulating the surface interaction, cellular uptake, and bioactivity of EVs. Laminin exhibited the highest adsorption characteristics, while collagen I coatings facilitated the greatest EV binding and focal adhesion formation. Surface-bound EVs further enhanced focal adhesion areas, likely due to integrin and ECM-mimicking ligand interactions. EVs uptake was significantly enhanced on PLL-coated surfaces, suggesting that both specific (integrin-mediated) and non-specific interactions, including macropinocytosis, contribute to EV internalization. Future work will focus on the detailed study of EVs uptake on ECM coatings via live imaging and studying the effect of surface bound EVs on immunomodulation and angiogenesis effect on different ECM coatings.

Acknowledgements and/or References (Optional):: Financial support for this project was provided by the NINDS (R01NS125016).