Utilizing the dextran probe diffusion data, the mesh size of the hydrogel was estimated to be 14 and 140 nm corresponding to a collagen fiber radius of 1 and 10 nm, respectively using the Ogston-Amsden model of diffusion in hydrogel networks (Figure ). The Clague-Philips model, which includes combined effects of obstruction and hydrodynamic interactions, gave similar results (Figure ). Moreover, the empirical Dg of the encapsulated EVs was found to be lying slightly outside of the fitted Ogston-Amsden model curve (Figure ). However, if this empirical Dg is moved onto the fitted Ogston-Amsden model curve, the radius of the released-solute should have been ~60 nm (Figure ) which is in very close agreement with the minor peak observed in the NTA analysis of the labeled-EVs (Figure ). To further validate the assumption of rigid fibers in Ogston-Amsden model, a viscoelastic stress-relaxation experiment was performed which revealed no major decay in the normalized relaxation modulus over time (Figure ). This modeling revealed a physical obstruction effect exerted by the hydrogel network on EVs, resulting in the preferential release of smaller particles through diffusion (Figure ). To support this hypothesis, we tracked the EV motion both in PBS and in the hydrogel matrix and compared the results with the silica nanoparticle (SiNP) motion in the hydrogels (Supplementary movies 1, 2, 3). In Figure the distribution of the maximum distance to the start point of the particle is presented. These results show that the EVs are able to move freely in PBS while this mobility is significantly reduced when the EVs are incorporated into the hydrogel matrix. By comparing the EVs with the SiNPs, we can observe that the maximum distance from the start point are slightly smaller in the case of SiNP compared to the EVs and only one population can be observed (Figure ). On the other hand, the distribution graph of the EVs represents two populations (Figure ). These data can support the EV-release data and explain why only 36 % of the encapsulated EVs were able to be released from the hydrogel matrix through diffusion. The integration of quantitative assessment techniques and theoretical modeling enhances our understanding of EV release dynamics, and thereby facilitating the design of future ocular drug delivery platforms. In conclusion, this study presents the first comprehensive characterization of corneal epithelial extracellular vesicles (CE-EVs) and provides significant insights into their role in corneal wound healing. By elucidating the interactions between various signaling proteins within the CE-EVs, we have advanced the understanding of the corneal wound healing pathway. Our findings demonstrate the potent efficacy of CE-EVs in promoting corneal epithelium wound healing in vitro, and therefore, highlight their potential as a therapeutic tool.