Positive-charge tuned gelatin hydrogel-siSPARC injectable for siRNA anti-scarring therapy in post glaucoma filtration surgery

Timothy T Y Tan,Ying Shi Lee,Li Fong Seet,Tina T Wong,Stephanie W L Chu,Li Zhen Toh,Hiok Hong Chan,Yong Yao Chun,Zhu Li Yap

doi:10.1038/s41598-020-80542-4

Abstract

Small interfering RNA (siRNA) therapy is a promising epigenetic silencing strategy. However, its widespread adoption has been severely impeded by its ineffective delivery into the cellular environment. Here, a biocompatible injectable gelatin-based hydrogel with positive-charge tuned surface charge is presented as an effective platform for siRNA protection and delivery. We demonstrate a two-step synthesis of a gelatin-tyramine (Gtn-Tyr) hydrogel with simultaneous charge tunability and crosslinking ability. We discuss how different physiochemical properties of the hydrogel interact with siSPARC (siRNA for secreted protein, acidic and rich in cysteine), and study the positive-charge tuned gelatin hydrogel as an effective delivery platform for siSPARC in anti-fibrotic treatment. Through in vitro studies using mouse tenon fibroblasts, the positive-charge tuned Gtn-Tyr hydrogel shows sustained siSPARC cellular internalization and effective SPARC silencing with excellent biocompatibility. Similarly, the same hydrogel platform delivering siSPARC in an in vivo assessment employing a rabbit model shows an effective reduction in subconjunctival scarring in post glaucoma filtration surgery, and is non-cytotoxic compared to a commonly used anti-scarring agent, mitomycin-C. Overall, the current siRNA delivery strategy involving the positive-charge tuned gelatin hydrogel shows effective delivery of gene silencing siSPARC for anti-fibrotic treatment. The current charge tunable hydrogel delivery system is simple to fabricate and highly scalable. We believe this delivery platform has strong translational potential for effective siRNA delivery and epigenetic silencing therapy.

Highlights

Small interfering RNA therapy is a promising epigenetic silencing strategy
Seet et al demonstrated that silencing of SPARC for human tenon fibroblasts (HTF) significantly reduced pro-fibrotic genes such as collagen I and TGF-β2
We develop a simple two-step synthesis of a gelatin hydrogel delivery system that enables tunability of its surface charge properties while simultaneously introduces the crosslinking sites on the polymer’s backbone, simplifying the synthesis process while preventing Small interfering RNA (siRNA) lost during the process (Fig. 1a)

Summary

Introduction

Small interfering RNA (siRNA) therapy is a promising epigenetic silencing strategy. its widespread adoption has been severely impeded by its ineffective delivery into the cellular environment. A biocompatible injectable gelatin-based hydrogel with positive-charge tuned surface charge is presented as an effective platform for siRNA protection and delivery. We discuss how different physiochemical properties of the hydrogel interact with siSPARC (siRNA for secreted protein, acidic and rich in cysteine), and study the positive-charge tuned gelatin hydrogel as an effective delivery platform for siSPARC in anti-fibrotic treatment. Through in vitro studies using mouse tenon fibroblasts, the positive-charge tuned GtnTyr hydrogel shows sustained siSPARC cellular internalization and effective SPARC silencing with excellent biocompatibility. The current siRNA delivery strategy involving the positive-charge tuned gelatin hydrogel shows effective delivery of gene silencing siSPARC for anti-fibrotic treatment. The current charge tunable hydrogel delivery system is simple to fabricate and highly scalable We believe this delivery platform has strong translational potential for effective siRNA delivery and epigenetic silencing therapy. They typically involve multiple synthesis steps of introducing cationic molecules (usually not biocompatible) and complex crosslinking/formation of the siRNA-hydrogel system, which might result in poor siRNA encapsulation efficiency[33,34,35,36]

Methods

Results

Conclusion