| Background:Glaucoma filtration surgery(GFS) is a major treatment for glaucoma that may decrease the intraocular pressure(IOP) by draining the aqueous humor to the subconjunctival space, forming a bleb; subconjunctival scarring of the filtering bleb is the most important cause of the surgery failure. Although applications of mitomycin-C(MMC) or 5-flurouracil(5-FU) have been demonstrated to inhibit scarring and improve GFS outcomes in large prospective randomised trials, sight-threatening complications greatly hinder their clinical utility. Thus, new anti-fibrotic agents capable of inhibiting postoperative scarring are urgently needed.Ample evidence supports that expression of TGF-β activate proliferation, synthesis, and migration of extracellular matrix proteins by human Tenon fibroblasts(HTF). Studies have indicated that TGF-β may be an effective strategy to reduce scarring by impairing TGF-β activity through neutralisation with antibodies, inactivation by proteoglycans-like decorin, and blockage of function by exogenous receptors. Despite development of a number of compounds served as therapeutics, few have had clinical success. The compound’s poor activity in vivo is most often attributed to their low bioavailability, the extent and rate at which a drug reaches at target tissue. In addition, bioavailability of traditional ocular drug delivery systems, such as eye drops, is poor due to the rapid loss and low corneal penetration of drugs. Thus, re-administration is often required, which can lead to decreased patient compliance. Our previous study revealed that nucleic acid aptamers targeting TGF-β receptor II(TβRII) were isolated and developed in vitro by systematic evolution of ligands by exponential enrichment(SELEX). We showed in vitro that aptamer S58 in HTF could inhibit TGF-β2-induced myofibroblast transdifferentiation, and a novel chitosan nanoparticle-S58 complexes was synthesised to preserve and prolong the efficacy of S58 for aptamer delivery in vitro.In the past decades, chitosan-based drug delivery systems in gel forms have been developed. As a natural polymer, chitosan thermo-sensitive hydrogels have especially gained great interest in pharmaceutics due to their non-toxicity, biocompatibility and biodegradability. Chitosan-based hydrogels are also promising biomaterials that can provide sustained, local delivery of agents, such as proteins, peptides and oligonucleotides, for therapeutic applications. Specific examples can be observed in cancer therapeutics, subcutaneous release or oral delivery. The uses of chitosan-based hydrogels in wound dressing applications have also been widely studied. Studies have demonstrated that chitosan can inhibit proliferation of keloid fibroblasts. Therefore, chitosan-based gels could be considered as an ocular drug delivery system for GFS.In this study, we suggest aptamer S58-conjugated chitosan-based hydrogel(CS/S58) as a type of injectable agent to obtain a controlled delivery system in vivo. Histological assessments were conducted following GFS to investigate wound healing and the inflammatory response to the CS or CS/S58 hydrogel.Objective:This study was designed to assay effect of TβRII-binding aptamers on TGF-β-induced transdifferentiation of fibroblasts and investigate whether application of chitosan gel with or without aptamer S58 improved bleb survival in a rat GFS model and compared its effectiveness with the currently used postoperative anti-scarring agent MMC.Methods:1. Human Tenon fibroblasts(HTFs) were treated with TGF-β2 with or without aptamer S58/68, or aptamer S58/68. Western blot was performed to detect the expression of α-smooth muscle actin(α-SMA) and p Smad2. α-SMA subcellular distribution and F-actin were evaluated by confocal immunofluorescence microscopy. Cell contractility was assessed through collagen gel contraction assays.2. Aptamer S58-conjugated chitosan-based hydrogel(CS/S58) as a type of injectable agent was developed to obtain a controlled delivery system. Aptamer S58 release rate from the CS/S58 gel were investigated in vitro.3. The effect of MMC, TGF-β2, CS or CS/S58 gel on wound healing were investigated in a rat GFS model by detecting scar-related factors and the involved inflammatory response. Slit lamp examination and anterior segment optical coherence tomography(AS-OCT) were performed on subconjunctival blebs. The levels of collagen I and α-SMA were detected by immunohistochemistry and western blotting.Results:1. TGF-β2 stimulated α-SMA and F-actin expression in HTF, and α-SMA incorporation into stress fibers with a concentration-dependent response, with peak activities at 2-5 ng/ml. TGF-β2 also stimulated p Smad2 expression and HTF mediated collagen contraction.2. Aptamer S58( 20 n M) significantly inhibited α-SMA expression and phosphorylation and nuclear translocation of Smad2 induced by TGF-β2. Aptamer S58 also suppressed TGF-β2-induced cell contraction. But we failed to find any effect of aptamer S68 on TGF-β2 activity in HTF.3. The incorporated aptamer S58 complexes could be released in a sustained manner from chitosan/β-GP(CS/β-GP)thermo-sensitive hydrogel for at least 2 weeks, with gradual decrease in release rate with time. Approximately 53 % of the loaded S58 was released in the first 5 days, and 84±6.0% was released at 28 days.4. The mean bleb size on day 28 is expressed as a percentage of the size at 3 days post-surgery. The bleb size of CS/S58 gel-treated group was 36 ± 9 % on day 28 compared with 17 ± 7 % in the control group. The MMC treated-group achieved 30 ± 7 %, the CS-treated group achieved 28 ± 8 %, and TGF-treated group achieved 15 ± 5 %. The mean percentage IOP of the control, TGF-β2, MMC, CS and CS/S58 treated group was 95 ± 14 %, 103 ± 12 %, 84 ± 21 %, 78 ± 18 % and 72 ± 20 % respectively.5. A typical inflammatory response to TGF-β2 treatment was observed after GFS 1 w, thick strands of fibrous material, which predominantly consisted of collagen fibres, were observed in the subconjunctival space overlying the sclera in the control and TGF-β2-treated eyes. Fibroblast infiltration and newly formed blood vessels could also be visualised. In contrast, the MMC-treated group showed that fibroblast infiltration was mostly adjunct to the conjunctival epithelium and exhibited loosely arranged episclera with outer dense collagen fibres. Karyopyknosis and TUNEL-positive cells were observed in the subconjunctival tissue of MMC-treated eyes.6. CS/GP gel was applied under the conjunctiva during surgery. There was no significant differences were observed in the amount of collagen I and α-SMA proteins between the CS gel and MMC-treated eyes in the rat GFS model. In addition the CS/GP gel did not accelerate ECM production by fibroblasts in the conjunctiva in rats compared with the control. In rat GFS models, a-SMA and collagen I expression were significantly reduced in the conjunctiva in CS/S58 gel-treated eyes with prolonged bleb survival compared with the CS group.Conclusions:1. TGF-β2 induced a response that is concentration-dependent, with different functions being maximally stimulated at different concentrations in HTFs. Our study revealed that aptamer S58 inhibited TGF-β2-induced transdifferentiation of fibroblasts to myofibroblasts(MFs) in HTFs. Aptamer S58 inhibited TGF-β2-induced phosphorylation and nuclear translocation of Smad2. The result indicates that aptamer S58 inhibited TGF-β2 activity partially via Smad signal transduction pathway.2. The accumulation of collagen fibres and proliferation of α-SMA-positive fibroblasts in the subconjunctival space were observed in TGF-β-treated rat eyes with elevated IOP and limited bleb sizes. The results confirmed that bleb failure in glaucoma filtration surgery was primarily due to the excessive accumulation of collagen and ECM organisation in the subconjunctival space, and high levels of TGF-β activity were associated with scarring. MMC may prolong the filtration bleb survival time through inhibiting the proliferation of fibroblasts and inducing apoptosis in conjunctival lesions.3. The pronounced increase in filtration efficiency was associated with thinner fibres, and a loosely organised subconjunctival matrix was observed in CS/S58 gel-treated eyes. The levels of collagen I and α-SMA were down-regulated and conjunctival fibroblast proliferation and the inflammation response were also suppressed. These results indicated that the CS/S58 gel may have a relatively inhibitory effect on TGF-β and that the anti-scarring effect may have resulted from the sustained release of aptamer S58 from the hydrogel. This study provides evidence that combined chitosan and aptamer S58 offers superior anti-fibrotic effect over mono-therapy in a rat model of glaucoma filtration surgery. The study also provides an opportunity for further developments in anti-fibrotic drug therapy. |
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