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

Abstract

Purpose: Diabetic retinopathy (DR) is a leading cause of blindness in diabetic patients that involves early-onset retinal cell loss. Here, we report our recent work using subconjunctivally implantable hydrogels for sustained insulin release to the retina to prevent retinal degeneration.

Methods: The hydrogels are synthesized by UV photopolymerization of N-isopropylacrylamide and a dextran macromer containing oligolactate-(2-hydroxyetheyl methacrylate) units. Insulin was loaded into the hydrogels during the synthesis. The ex vivo bioactivity of insulin released from the hydrogels was tested on fresh rat retinas using immunoprecipitation and immunoblotting to measure insulin receptor tyrosine and Akt phosphorylation. The biosafety and the effect on the blood glucose of the hydrogels were evaluated in rats 2 months after subconjunctival implantation. The release of insulin from the hydrogels was studied both in vitro in PBS (pH 7.4), and in vivo using confocal microscopy and RIA kit. The in vivo bioactivity of the released insulin was investigated in diabetic rats using DNA fragmentation method.

Results: The hydrogels could load insulin with approximately 98% encapsulation efficiency and continuously release FITC-insulin in PBS (pH = 7.4) at 37°C for at least 5 months depending on their composition. Insulin lispro released from the hydrogels was biologically active by increasing insulin receptor tyrosine and Akt serine phosphorylation of ex vivo retinas. In vivo studies showed normal retinal histology 2 months post subconjunctival implantation. Insulin released from subconjunctivally implanted hydrogels could be detected in the retina by using confocal microscopy and RIA kit for 1 week. The implanted hydrogels with insulin lispro did not change the blood glucose level of normal and diabetic rats, but significantly reduced the DNA fragmentation of diabetic retinas for 1 week.

Conclusions: The developed hydrogels have great potential to sustain release of insulin to the retina via subconjunctival implantation to minimize DR without the risk of hypoglycemia.

Figure 1

Scheme of Dex-lactateHEMA macromer (DP = n here).

Figure 2

Cumulative release of FITC-insulin from poly(NIPAAm-co-Dex-lactateHEMA) hydrogels G-8-1 (⧫), G-6-3 (•), and G-4-5 (▴) in vitro at 37°C (n = 4).

Figure 3

Effects of insulin lispro released from G-6-3 hydrogels on the IRß tyrosine and Akt phosphorylation in rat retinas ex vivo. The IR from retina was immunoprecipitated and analyzed for tyrosine phosphorylation (PY) of the IR and Akt kinase activity against time during 30-minute incubation time. (A) Representative PY and IRß blots and fold increase of PY/IRß ratios; and (B) representative Akt^ser473 and Akt^total blots and fold increase of Akt^ser473/Akt^total ratios. (○) control, (△) blank hydrogels, (⋄) hydrogels G-6-3, and (□) 100 nM insulin-positive control. For all the rations, time 0 was set to be 1. n = 4 per group per time point; **P < 0.01 from time 0 by ANOVA and Bonferronni post hoc test.

Figure 4

Retinal histology of rat receiving subconjuctival implantation of blank G-6-3 hydrogels (H&E staining, ×40). Hydrogel-implanted eyes were not associated with an increased polymorphonuclear infiltrate or any morphological change during 2-month implantation. n = 4.

Figure 5

Confocal images of insulin in the retinas of rat eyes receiving subconjuctival implantation of FITC-insulin–loaded G-6-3 hydrogels for 1 month. Retinal sections 10-μm thick were used for imaging. Shown are representative photomicrographs after 1-day, 1-week, and 1-month implantation. In each vertical panel, the top, middle, and bottom sections were the retinas treated with nothing, blank hydrogels, and FITC-insulin loaded hydrogels at each time point. The blue color came from the DAPI-stained nuclei and the green color came from the FITC-labeled insulin. The white bar represents 100 μm. n = 4 per group per time point.

Figure 6

Blood glucose level in normal control rats (×), and normal rats subconjunctivally implanted with blank G-6-3 hydrogels (△), and insulin lispro-loaded G-6-3 hydrogels (□) for 0 (preimplantation), 1 day, 1 week, and 1 and 2 months. n = 4 per group per time point.

Figure 7

Effects of subconjunctivally implanted insulin lispro-loaded G-6-3 hydrogels on DNA fragmentation in the diabetic retina. Streptozotocin-induced diabetic rats (8 weeks) were subconjunctivally implanted with insulin lispro-loaded hydrogels or blank-hydrogels, and normal and diabetic rats without implantations were used as controls. Retinas were extracted 2 days or 1 week later and used for DNA fragmentation ELISA. “ctl” for normal rats (n = 8), “D” for diabetic rats (n = 4), “L” for insulin- loaded hydrogels (n = 6), and “B” for blank hydrogels (n = 4). **P < 0.01 from ctl by ANOVA and Student-Newman-Keuls (SNK) post hoc test and n.s. for not significant.