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

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.


Stability assay of siSPARC treated with H2O2
siSPARC (100 pmol/ul) was treated with 3mM H2O2 and its stability was compared with naked siSPARC as control. After the treatment, the siSPARC was analyzed using 5 % native agarose gel electrophoresis. The 5 % agarose gel was prepared by dissolving 5 g of agarose powder (Vivantis Technologies Sdn Bhd, Malaysia) in 100 ml of 1 × Tris-Acetate-EDTA (TAE) buffer (1st Base, Sinagpore). The mixture was microwave for 1-3 min until the agarose is completely dissolved. 10 ul SYBR Safe (Invitrogen, US) was then added to the agarose solution for staining of nuclei acid. The mixture was then poured into a gel tray with the well comb and allowed to set. siSPARC sample was mixed DNA Gel Loading Dye, Blue (6 ×; Thermo Fisher Scientific, US) and loaded onto the agarose gel with 1 nmol per lane.

Rheological analysis
Gtn-Tyr hydrogel was fabricated using 0.12 units/ml HRP and 3 mM H2O2 and cast into a mould. Cylinder samples with 8 mm diameter and 2 mm thickness were then cut out for rheological analysis. Storage modulus (G') of the sample was determined using rheometer MCR 501 (Anton Paar, Austria) with probe PP08/150. The measurement was performed using oscillation mode with a constant frequency of 1 Hz at 37 o C. Figure S2. Storage modulus at 1 % strain of Gtn-Tyr hydrogels with increasing zeta potential (F1 to F4). *** denote P < 0.001 and **** denote P < 0.0001.

In vitro degradation studies
5 w/v% Gtn-Tyr solution was prepared by dissolving freeze-dried precursor into 1× PBS.
HRP and H2O2 with a final concentration of 0.12 unit/ml and 3 mM respectively were added to 0.5 ml precursor solution in a 2 ml microtube to initiate the crosslinking process. The sample was allowed to set for 0.5 h before topping up with 0.5 ml collagenase type I (
The hydrogel precursor was sterilized using a 0.22µm syringe filter. Next, 0.5 ml Gtn-Tyr hydrogel was fabricated by adding HRP and H2O2 with a final concentration of 0.15 units/ml and 3 mM, respectively. The mixture was then cast onto a 24 well plate. C57Bl6/J MTFs were seeded on top of the hydrogel with a density of 2 × 10 4 cells per well. The sample was topped up with 0.5 ml culture medium and incubated at 37 o C and 5 % CO2. At day 1, 3 and 7, the cell proliferation rate was measured using PrestoBlue cell viability reagent (Life Technologies, US). LIVE/DEAD cell viability assay (LifeTechnologies, US) was used to observe live and dead cells cultured on the hydrogel at day 1, 3 and 7 using the Zeiss Axio Observer Z1 inverted microscope (Carl Zeiss, Germany). Figure S9. Graph on (a) cell proliferation of MTFs cultured on F2 hydrogel using the