Encapsulation of msc‐derived extracellular vesicles in biomaterials for effective drug delivery to the ocular surface

Corresponding Authors: Liam Grover and Thomas Ritter l.m.grover@bham.ac.uk and thomas.ritter@universityofgalway.ieIntroduction: Extracellular vesicles (EVs) are particles secreted from many cell types, containing lipids, proteins, and nucleic acids. Mesenchymal stromal cell‐derived EVs (MSC‐EVs) demonstrate similar therapeutic effects as their origin cells such as immunomodulation and tissue regeneration, while addressing safety concerns and other challenges associated to cell‐therapy (Rani et al. 2015, Wang et al. 2023). Delivering MSC‐EVs to the site of injury in a controlled and sustained manner may augment MSC‐EVs therapeutic efficacy. In this context, gellan gum fluid gel appears as a promising delivery system for MSC‐EVs which can extend the residence time of therapeutics on the cornea due to its shear‐dependent viscoelasticity, mucoadhesive feature and potential to bind to water. The solid‐liquid‐solid phase transition properties of the gellan gum fluid gel overcome the main challenges of commercial eyedrops like rapid clearance through lid overflow and drainage through the nasolacrimal duct (Chouhan et al. 2019). The aim of this study was to encapsulate MSC‐EVs in a gellan gum fluid gel and to investigate its release in‐vitro.Method: Human bone marrow MSCs were isolated, cultured in xeno‐free media, and characterized for surface biomarker expression using flow cytometry. MSC‐EVs were isolated from culture supernatants by size exclusion chromatography and further characterized following MISEV guidelines for size distribution, zeta potential, morphology, and surface biomarkers profile, respectively. Gellan gum fluid gels were created by cooling a solution of low acyl gellan gum in a shear field, with sodium chloride as crosslinker under a constant mechanical separation. The gellan gum fluid gel was further characterized using shear and dynamic oscillatory techniques, measuring the material viscosity and viscoelasticity. MSC‐EVs were encapsulated in the gellan gum fluid gel, followed by the analysis of their release profile from the gellan gum fluid gel at 0, 1, 2, 3, 4, 5, and 6hr.Results: MSCs showed positive surface biomarker expression for CD90, CD73, and CD44, and negative for CD45, CD11b, and HLA‐DR. MSC‐EVs had spherical bilayer morphology with a size of approximately 83 nm and displayed a slight negative surface charge. MSC‐EVs were positive for surface biomarkers CD9, CD63, and CD81 analysed by flow cytometry. The Gellan gum fluid gels were shown to act as solids at rest, improving retention on the eye, but as fluids when a force is applied, easing application and distribution on the ocular surface through blinking. At lower strains, the gellan gum fluid gel viscoelasticity showed that the G’ (storage modulus) was higher than G” (viscous modulus) states in the linear viscoelastic region. Conversely, in higher strains the G’ declined to reach lower values than for the G”, resulting into a liquid phase system.The cumulative release profile of the MSC‐EVs from the gellan gum fluid gels showed a controlled release profile through 6 hr investigation.Conclusion: Altogether, these results evidenced that the physical and release properties of the MSC‐EV laden gellan gum fluid gels demonstrate their potential as ocular healing materials, necessitating investigation of the therapeutic efficacy of the released‐MSC‐EVs in 2D and 3D in‐vitro studies of corneal injury.ReferencesChouhan G, Moakes RJA, Esmaeili M, et al. (2019): A self‐healing hydrogel eye drop for the sustained delivery of decorin to prevent corneal scarring. Biomaterials 210: 41–50.Rani S, Ryan AE, Griffin MD & Ritter T (2015): Mesenchymal Stem Cell‐derived Extracellular Vesicles: Toward Cell‐free Therapeutic Applications. Molecular Therapy 23: 812–823.Wang J, Donohoe E, Canning A, Moosavizadeh S, Buckley F, Brennan MÁ, Ryan AE & Ritter T (2023): Immunomodulatory function of licensed human bone marrow mesenchymal stromal cell‐derived apoptotic bodies. International Immunopharmacology 125: 111096.