Development of a biomaterial releasing immunomodulatory extracellular vesicles for enhanced corneal repair

Introduction: The cornea is the dome-shaped, transparent, outermost layer of the eye, playing a crucial role in both vision and protection of intraocular structures. Corneal diseases represent a leading cause of blindness worldwide. Despite advances in available therapies, effective treatment options for severe corneal injuries remain an unmet clinical need. Mesenchymal stromal cells (MSCs) have emerged as promising candidates for corneal repair due to their anti-inflammatory, immunomodulatory, and regenerative properties. However, the clinical application of MSCs is challenged by issues such as optimal dosage and delivery, potential immunogenicity and tumorigenicity, as well as donor variability and dependency. Extracellular vesicles (EVs) are nanoscale particles secreted by virtually all cell types, including MSCs (MSC-EVs). Acting as mediators of intercellular communication, EVs deliver functional cargo, such as proteins, lipids, and nucleic acids (including miRNAs), to recipient cells, thereby recapitulating many of the therapeutic effects of their parent cells. MSC-EVs have demonstrated immunomodulatory and regenerative effects in corneal tissue, while potentially overcoming several limitations associated with cell-based therapies. Pre-conditioning or ‘licensing’ of MSCs with cytokines such as IFN-γ and TGF-β1, either individually or in combination, can enhance their immunomodulatory capacity and, consequently, the therapeutic efficacy of their EVs in tissue regeneration. Nonetheless, the systemic administration of MSC-EVs is limited by rapid clearance and off-target accumulation in organs such as the liver and lungs. Local ocular delivery, particularly via topical eye drops, also faces significant challenges, including rapid clearance and limited retention on the ocular surface due to tear turnover and blinking. Alternative delivery systems have been explored, among which fluid gels, such as gellan gum fluid gels, have shown promise for ocular application. Gellan gum fluid gels exhibit unique rheological properties, enabling easy application to the ocular surface, prolonged retention, controlled release of therapeutic agents like MSC-EVs, and gradual removal through natural blinking. In this work, we suggest cytokine-licensed MSC-EVs as a novel therapeutic for ocular diseases, which can be delivered to the ocular surface in a controlled manner employing innovative phase-transition gellan gum fluid gel system. Methods: MSC-EVs were isolated from both naïve and cytokine-licensed (IFN-γ and TGF-β1) MSC-conditioned media using size exclusion chromatography (SEC). The isolated EVs were characterized for size distribution by nanoparticle tracking analysis (NTA), morphology by transmission electron microscopy (TEM), surface and intraluminal markers by flow cytometry and western blotting, and miRNA cargo profiling. The immunomodulatory effects of MSC-EVs were assessed using macrophage and T cell functional assays. Additionally, the tissue repair potential of MSC-EVs was evaluated in human corneal epithelial (HCEpi) and human corneal keratocyte (HCK) cell cultures, as well as in ex-vivo human and bovine corneal injury models. To elucidate the mechanisms underlying MSC-EV-mediated wound healing, mRNA sequencing was performed on HCEpi cells treated with MSC-EVs during the active phase of wound repair, enabling the identification of key miRNA-mRNA regulatory interactions. Gellan gum fluid gels (FGs) were synthesized and optimized based on their rheological properties under small and large deformations. MSC-EVs were incorporated into FGs, and their distribution and release kinetics were evaluated using confocal microscopy and spectrophotometry. The bioactivity of released MSC-EVs was assessed by their uptake into HCEpi and HCK cells, in-vitro wound healing assays, and an ex-vivo bovine corneal alkali burn model. The therapeutic efficacy of FG-delivered MSC-EVs was further investigated in a pre-clinical limbal stem cell deficiency (LSCD) model, with outcomes measured in terms of corneal vascularization and wound healing. Results: Cytokine-licensed MSC-EVs exhibited a uniform size distribution of approximately 100 ± 20 nm and a characteristic lipid-bilayer, spherical morphology. These vesicles expressed canonical tetraspanin surface markers (CD9, CD63, CD81) and the intraluminal marker TSG101. MSC-EVs accelerated corneal wound healing in-vitro (HCEpi and HCK cells) and in ex-vivo human and bovine corneal injury models. Cytokine licensing altered the miRNA profile of MSC-EVs, and integrative mRNA-miRNA analysis revealed novel regulatory interactions implicated in corneal repair. Functionally, MSC-EVs derived from cytokine-licensed MSCs exhibited enhanced immunomodulatory activity, significantly suppressing pro-inflammatory macrophage activation, promoting an anti-inflammatory phenotype, and reducing the secretion of cytokines such as TNF-α and IL-1β. Moreover, cytokine-licensed MSC-EVs more effectively inhibited allogeneic T cell proliferation and increased regulatory T cell induction compared to naïve MSC-EVs. Gellan gum fluid gels demonstrated phase transition and shear-thinning properties ideal for ocular drug delivery. FGs enabled controlled release of MSC-EVs, with approximately 18% of the loaded vesicles released after 6 hours. Released MSC-EVs retained their bioactivity, as evidenced by successful cellular uptake and promotion of wound healing in-vitro and in the ex-vivo bovine corneal model. In the pre-clinical LSCD model, FG-functionalized MSC-EVs significantly enhanced corneal wound healing, although no significant effect on corneal neovascularization was observed. Conclusion: Cytokine licensing enhances the immunomodulatory and regenerative properties of MSC-EVs. These licensed MSC-EVs promote ocular surface repair across in-vitro, ex-vivo, and pre-clinical models, supporting their potential as therapeutic agents for corneal injuries. Gellan gum fluid gels provide an effective platform for the controlled delivery and sustained release of MSC-EVs, preserving their biological activity and offering a promising approach for ocular drug delivery. Collectively, the findings of this thesis suggest that cytokine-licensed MSC-EVs delivered via gellan gum fluid gels represent a novel and promising therapeutic strategy for ocular injuries, necessitating further optimization and preclinical investigation.

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University of Galway

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University of Galway Theses (PhD Theses)