Extracellular Vesicle-Derived Bioactive Molecules for Corneal and Ocular Surface Regeneration

Abstract

Cell-based therapies emerge as potential treatment options for various debilitating diseases. Preclinical research and clinical studies involving cells increased exponentially in the past decade. In addition to cell-based approaches, the use of extracellular vesicles (EVs), which are released by nearly all cell types, emerged as a promising cell-free alternative. Those approaches are also being explored in the field of ophthalmology. Several clinical trials involving EVs are underway to develop potential treatments for advanced ocular surface diseases, including corneal disorders, injuries, and dry eye disease. The cargo carried by EVs has been shown to include a diverse array of functional molecules such as transcription factors, cytokines, growth factors, mRNA, tRNA, rRNA, miRNA, and fragments of dsDNA. While the molecular composition of EVs is already well characterised, the specific activity of these molecules upon delivery to recipient cells remains poorly understood. In this review, we summarise recent studies investigating the bioactive molecules within EVs shown to influence or modulate cellular activity on the ocular surface. Among these, various miRNAs have most commonly been identified as therapeutic agents targeting distinct molecular pathways. The EVs studied were predominantly derived from various mesenchymal stem cells.

Keywords: bioactive molecules; cornea; dry eye disease; extracellular vesicles; mesenchymal stem cells; miRNA; ophthalmology.

Figure 1

Extracellular vesicle composition: Presented are the main groups of molecules expressed on the surface of EVs. The internal cargo of the EV consists of DNA, various RNAs, amino acids, and proteins, which are more accurately presented in Table 1. Created in BioRender (2025). Abbreviations: (extracellular vesicles (EVs).

Figure 2

Schematic representation of an experimental study design to analyse the molecular basis of EVs’ therapeutic effect on corneal wound healing according to Liu, 2022 [41]. (a) MSC- or other cell-derived EVs are collected from conditioned media and isolated using methods such as ultracentrifugation. (b) Identity, purity, and quality of isolated EVs is confirmed with various methods, including different light scattering techniques, electron microscopy, and Western blotting. (c) To evaluate the potential therapeutic effect of EVs on tissue regeneration, hCECs are treated with EVs. Uptake and proliferation assays are performed to assess their effects on cellular internalisation, migration, and growth. (d) Total RNA from EVs and hCECs is extracted for molecular cargo identification, which could be responsible for the studied therapeutic effect. (e) To test whether a specific molecule is responsible for the therapeutic effect of EVs on hCECs, its gene is knocked out in MSCs. EVs are then isolated from these modified cells. Their effects on hCEC healing are compared to EVs from unmodified (wild-type) MSCs. (f) To evaluate the therapeutic relevance, EVs are tested in an animal rodent corneal injury in vivo model. Created in BioRender (2025). Abbreviations: mesechymal stem cells (MSC), extracellular vesicles (EVs), nanoparticle tracking analysis (NTA), and human corneal epithelial cells (hCEC).

Figure 3

Key EV-derived molecules, their corneal cellular targets, and resultant therapeutic effects during wound healing across the epithelium, stroma, and endothelium. Created in BioRender (2025). Abbreviations: phosphatase and tensin homolog (PTEN), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), programmed cell death protein 4 (PDCD4), homeodomain-interacting protein kinase 2 (HIPK2), cellular tumour antigen p53 (p53), SMAD family member 3 (Smad3), translocation-associated membrane protein 2 (TRAM2), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), and insulin-like growth factor (IGF).

Figure 4

EV-derived bioactive bolecules, their cellular targets, and therapeutic effects in the pathophysiology of dry eye disease. Created in BioRender (2025). Abbreviations: interleukin-1 receptor-associated kinase 1 (IRAK1), TAK1-binding protein 2 (TAB2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), signal transducer and activator of transcription 3 (Stat3), and F-box/WD repeat domain-containing 7 (Fbxw7).