**6. Role of exosomes in corneal infection and regeneration**

Exosomes are membrane-bound extracellular vesicles (EVs) secreted from most cell types into the extracellular space, ranging from 30 to 150 nm in size. The role of exosomes in corneal diseases and their biogenesis during pathological or physiological immune responses has been documented in several studies [67–69]. Secreted exosomes could regulate immune responses as they have the potential for transferring and presenting antigenic peptides, regulating gene expression, and inducing various immune signaling pathways. Furthermore, their detection in a biological fluid can offer a window to the altered cellular or tissue states, and cell-to-cell communication in healthy and disease patients could lead to early diagnosis and potential treatment [70].

Exosomes are natural nanovesicles and are superior to synthetic nanovesicles like liposomes in terms of their stability, biocompatibility, plasma half-life, ability to enter non-accessible tissue regions, and their role in immunomodulation. Exosomes are potentially effective drug delivery vehicles, but their nonspecific targeting feature limits their use in drug delivery applications [71]. However, engineered exosomes secreted by specific cell types could overcome this problem.

Exosomes have recently shown potential in regulating therapeutic functions via cell-to-cell communications. Induced pluripotent stem cells derived exosomes (iPSCs-Exos) are being examined as a natural therapeutic nanoparticle for treating corneal epithelial defects. iPSCs-Exos have induced cell proliferation, migration, cell cycling, and apoptosis inhibition of human corneal epithelial cells (HCECs) in-vitro. Studies also showed the upregulation of cyclin A and CDK2 that induce the HCECs to enter the S phase of the cell cycle for potential regeneration. In vivo studies also revealed the accelerated healing potential of iPSCs-Exos in treating corneal epithelial defects [72]. Zhong and coworkers showed that when exosomes

derived from human umbilical cord mesenchymal stem cells (HucMSC-Exos) were combined with an autophagy activator, they positively affected the repair of a corneal injury (CI). The repair process was regulated by accelerated cell proliferation and migration while inhibiting apoptosis and inflammation by activating the AMPKmTOR-ULK1 autophagy pathway [73]. Han et al. [74] examined the possible role of exosomes in corneal wound healing and neovascularization. They showed exosomes between epithelium and stroma after epithelium debridement during the wound healing process. Exosome-like vesicles were also observed in the stroma after anterior stromal keratectomy. They were fused with keratocytes to induce myofibroblast transformation in vitro. These epithelial-derived exosomes also helped induce endothelial cell proliferation, indicating the ability of exosomes to communicate among corneal epithelial cells, keratocytes, and vascular endothelial cells [74].

As these naturally derived nanotherapeutics show emerging potential in treating corneal infection and regeneration, it would be interesting to encapsulate these particles into scaffold/hydrogel for sustained release in clinical applications. In this context, chitosan-based thermoresponsive hydrogels encapsulating exosomes derived from induced pluripotent stem cell–derived mesenchymal stem cells (iPSC-MSCs) were reported to reduce corneal scar formation and accelerated wound healing [75]. In addition, the sustained release of miRNA-containing exosomes helped regenerate the corneal epithelium and stromal layer. The exosomes prevented ECM deposition by downregulating the translocation-associated membrane protein 2 (TRAM2) gene, providing a new strategy for the clinical treatment of fibrotic corneal diseases [75]. Exosomes are paving a new path with their potential utility for early diagnosis and treatment of corneal diseases. Their incorporation into biomaterial-based hydrogels could facilitate their use as therapeutic particles for sustained release in clinical applications.
