Ocular delivery of antisense oligonucleotides using colloidal Carriers: improving the wound repair after corneal surgery

Abstract

Background and Aim: Clinical outcomes of refractive surgeries are variable due to individual wound healing responses, but might be improved by effective delivery of anti-inflammatory agents. Knockdown of connexin proteins, using an antisense oligonucleotide (AsON) approach, has been shown to significantly reduce the inflammatory response and increase the rate of wound closure after corneal laser surgery. The challenge remains to find delivery systems that are easy to apply, but can still effectively deliver the AsONs to the target site. This thesis aimed to evaluate the efficacy of several in-situ gelling formulations to deliver Cx43 AsONs to the wounded tissues of a rat corneal scrape wound model. Methods: Formulations were characterised in terms of their rheological behaviour, microstructure and spreading ability. They were then evaluated for their irritation potential, precorneal retention and ability to control the release of the model hydrophilic drug pilocarpine hydrochloride both in vitro and in vivo. The stability of the AsONs in these formulations was assessed using Fluorescence Resonance Energy Transfer. Finally, formulations containing the stable AsONs were applied to a rat corneal scrape wound model and penetration depth, wound size after 12 hours and cellular dynamics underlying the wound healing response were analysed. Results and Discussion: Systems based on gellan gum, xanthan gum, carrageenan and alginate underwent sol-to-gel phase transition upon addition of the cations present in tear fluid. All tested systems exhibited favourable contact angles and were found to be non-irritant. Systems based on gellan gum, xanthan gum and carrageenan showed the longest ocular retention and exhibited the slowest release characteristics both in vitro and in vivo. AsONs were found to be stable in all formulations apart from the chitosan system, where precipitation occurred. This formulation also exhibited the slowest wound healing rate due to induction of a pro-inflammatory response. Conversely, delivery of the AsONs by gellan gum and carrageenan formulations resulted in significant reduction in wound size, inflammatory response and Cx43 levels. Conclusion: In-situ gelling systems based on gellan gum and carrageenan are able to successfully deliver Cx43 AsONs to the wounded tissues and therefore improve the healing response after corneal surgery.