Subconjunctivally Implanted Hydrogels for Sustained Insulin Release to Reduce Retinal Cell Apoptosis in Diabetic Rats.

Diabetic retinopathy (DR) affects 14% of the 29 million Americans with diabetes aged 20 to 74 years.1 Currently laser surgery and vitrectomy are used to treat advanced DR, but they are destructive and do not address the underlying biology of DR.2 Inhibition of VEGF reduces macular edema,3 but does not support the integrity or loss of retinal neurons that die by apoptosis.4–10 Specifically, neurons (photoreceptors, bipolar cells, horizontal cells, amacrine cells, and ganglion cells) and glia (astrocytes, Muller cells, and microglial cells) comprising the retina are impaired in DR. To target the neuronal degeneration in DR, insulin has been investigated as a potential therapeutic to rescue retinal neurons from apoptosis.11–13 Our previous work showed that the retina exhibits high basal activity of the insulin receptor/phosphoinositide 3-kinase/Akt signaling system,14 and short-term experimental diabetes in rats reduces this activity.11 Systemic or intravitreally injected insulin restores prosurvival insulin receptor and Akt kinases and rescues retinal neuronal cells from apoptotic cell death,12,15 and hyperglycemia abrogates this effect.12,16 Moreover, intensive systemic insulin therapy reduces the risk of retinopathy onset and progression, and retinopathy is associated with insulin resistance.17,18 Together these results demonstrate that insulin receptor stimulation may have therapeutic implications for DR. Insulin has a short half-life in plasma, and its long-acting analogues such as insulin glargine and insulin degludec have durations of action only up to 24 hours.19,20 The risk of hypoglycemia also limits the ability of patients to take enough insulin systemically to minimize the risk of retinopathy.21 Intravitreal injection of insulin can be an effective route to deliver drugs to the retina compared with topical and subconjunctival routes, but intravitreal injection is invasive, and can rarely cause endophthalmitis.22,23 Eye drops are the most commonly used delivery form in ocular drug treatment, but they are neither patient compliance nor efficient for the treatment of diseases of the posterior segment of the eye.24 Factors that limit the usefulness of eye drops include corneal and conjunctival barriers, and the rapid and extensive precorneal tear loss due to drainage and tear fluid turnover.25 The subconjunctival space external to the sclera has received increasing attention as a minimally invasive and effective route for delivering drugs to the retina, obviating the risks of intraocular surgery and systemic side effects.26–28 Subconjunctivally injected very low-dose insulin activates prosurvival insulin receptor signaling pathways in the treated eye without lowering blood glucose or affecting the opposite eye.29,30 Taken together, controlled and sustained release of biologically active therapeutics to activate the retinal insulin receptor via a subconjunctival route may be clinically beneficial. We previously reported the design of subconjunctivally implantable hydrogels for insulin delivery to the retina.31 We demonstrated that the hydrogels are nontoxic in vitro to retinal neuronal cells and in vivo in rats, and capable of release insulin for more than 1 week in vitro. In this work, we improved the release property of the hydrogels and evaluated their potential for managing DR via subconjunctival implantation. The hydrogels were synthesized via UV polymerization with a slight modification from our previous report31 in terms of the solvent used, and the average repeating lactate units (DP) in the cross-linkable chains of the macromers used. The ex vivo bioactivity of the insulin released from the hydrogels was tested on freshly extracted retinas from rats using immunoprecipitation and immunoblotting to measure insulin receptor (IR) tyrosine and Akt phosphorylation. The biosafety of the hydrogels was evaluated in rats 2 months post implantation using hematoxylin-eosin (H&E) staining. The release of insulin from the hydrogels was studied both in vitro using PBS (pH 7.4) medium, and in vivo using confocal microscopy and radioimmunoassay (RIA) kit after 1 week subconjunctival implantation in rats. The effect of the subconjunctivally implanted hydrogels on the blood glucose level of rats was monitored. The in vivo bioactivity of insulin released from the subconjunctivally implanted hydrogels was investigated in diabetic rats by using the DNA fragmentation method.