(W0930-05-28) Exploring the Effects of Nano-Enabled Oral Insulin and Anti-Inflammatory Agent in Mitigating Diabetic Ocular Complications in Type 2 Diabetic Rat Model

Purpose: Cataract and Retinopathy are among the two major and most common diabetes- related complications, eventually leading to preventable blindness. Given the complexity and diverse nature of diabetes, a comprehensive strategy is essential for effective management of both the disease and its associated complications. An integrated approach is needed to address underlying metabolic dysfunction and chronic low-grade inflammation, alongside achieving glycemic control. Receptor-mediated nanoscale drug delivery systems present a promising avenue for improving therapeutic outcomes by enhancing drug bioavailability and targeted delivery.  Building on prior findings from Type 1 diabetes models [1-3], this study investigates the effect of co-administration of a hypoglycemic agent, insulin (INS), early in the disease course, in combination with an anti-inflammatory agent, curcumin (CUR). Through systemic delivery of these agents via a ligand-functionalized nanoparticles platform, we aim to assess the long-term therapeutic effects on glycemic control and the potential to delay ocular pathology in a Type 2 Diabetes Mellitus (T2DM) rat model.

Methods: We synthesized PLA-PEG-based polymer (P2s) and functionalized it with gambogic acid (GA), a non-competitive ligand targeting the transferrin receptor [4]. These polymers were used to formulate insulin and curcumin-loaded nanoparticles (functionalized and non-functionalized with GA). Using GPC, 1H NMR, UV-vis, HPLC, DLS, and SEM, the polymer and the nanoparticles (NPs) were successfully characterized at various stages of synthesis. In this study, a total of 108 Sprague-Dawley rats were acclimatized for one week and then grouped into control (Group 1) and high-fat diet (HFD) groups. After two weeks on HFD, all groups except the non-diabetic obese group (Group 2) received a single intraperitoneal injection of streptozotocin (STZ, 35 mg/kg) to induce T2DM. Diabetic rats were randomized into eight treatment groups (n=12 per group). Group 3 (Diabetic) received no treatment. Group 4 received subcutaneous insulin (INS s.c.)  (1IU/kg). Groups 5 and 6 received oral gavage of nanoparticle-encapsulated insulin (40 IU), with or without a Gambogic acid ligand (nINS and nGA-INS). Group 7 received an admixture of insulin-loaded and curcumin-loaded nanoparticles orally (Combo). Group 8 received only curcumin nanoparticles (10mg/Kg) (nGA-CUR) via oral gavage, and Group 9 received empty nanoparticles (VOID). After 20 weeks, rats were euthanized for further analysis.

Results: Our initial analysis suggests that insulin-loaded nanoparticles perform similarly to insulin injections in maintaining glucose homeostasis, particularly in managing overnight blood glucose levels, as shown by 24-hour glucose plots (Figure 1a). Notably, the INS-loaded nanoparticles reduced the risk of hypoglycemia compared to conventional insulin, which often caused glucose levels to drop below 100 mg/dL after subcutaneous insulin administration (Figure 1b). Terminal blood analyses revealed a notable reduction in neutrophils in the combination therapy group compared to untreated diabetics (Figure 1c). Additionally, platelet count abnormalities observed in diabetic animals were stabilized with the combination nanoparticle treatment. (Figure 1d). Ocular assessments showed cataract development and elevated intraocular pressure (IOP) in diabetic rats. However, combination therapy delayed cataract onset and stabilized IOP levels (Figure 2a and b). Furthermore, analysis of the retina indicated that Glial Fibrillary Acidic Protein (GFAP) expression by activated microglia was markedly reduced in groups receiving insulin nanoparticles (Groups 5 and 6), with the most pronounced effect seen in the combination group. (Figure 3a and b).

Conclusion: This study highlights the potential of a dual oral nanoparticle-based therapeutic strategy using an admixture of two separate nanoparticle suspensions loaded with INS and CUR, to effectively manage T2DM and its complications. The INS nanoparticles achieved glycemic control comparable to subcutaneous insulin and significantly reduced the risk of hypoglycemia, a common limitation of traditional insulin injections. Additionally, the insulin nanoparticles were more effective in managing overnight fluctuations by providing long-term glycemic control, likely due to their sustained release kinetics. Efficient management of nocturnal fluctuations, particularly in the combination group, may help reduce cardiovascular risks, a leading cause of overnight mortality in diabetic patients. Overall, the oral delivery of insulin via nanoparticles addresses key challenges in diabetes management, including injection-related non-compliance, hypoglycemia risk, and long-term cardiovascular complications. Altered neutrophil and platelet counts reflect underlying inflammation and burden of vascular risk, both playing a key role in the development of diabetic retinopathy and cataract. Stabilization of these counts, especially seen with the combination therapy but not with the commercial insulin injections or with any of the single agent nano-formulations, indicates a complementary action of the dual agents in the management of disease progression and its complications. Delayed cataract formation along with stabilized intraocular pressure and significantly decreased GFAP expression in the combination group suggest ocular protective effects.  These findings, more pronounced in the combination group than in the individual treatment groups, indicate that curcumin may complement insulin's activity at the molecular level, potentially enhancing insulin signaling across the retinal barrier. Further studies are needed to explore the molecular interplay between these agents and determine whether their combination contributes to improved therapeutic efficacy through complementary mechanisms.

References: [1] Ganugula, R., Nuthalapati, N. K., Dwivedi, S., Zou, D., Arora, M., Friend, R., Sheikh-Hamad, D., Basu, R., & Kumar, M. N. V. R. (2023). Nanocurcumin combined with insulin alleviates diabetic kidney disease through P38/P53 signaling axis. Journal of controlled release: official journal of the Controlled Release Society, 353, 621–633.
[2] Ganugula, R., Arora, M., Dwivedi, S., Chandrashekar, D. S., Varambally, S., Scott, E. M., & Kumar, M. N. V. R. (2023). Systemic anti-inflammatory therapy aided by curcumin-laden double-headed nanoparticles combined with injectable long-acting insulin in a rodent model of diabetes eye disease. ACS nano, 17(7), 6857–6874.
[3] Dwivedi, S., Gottipati, A., Ganugula, R., Arora, M., Friend, R., Osburne, R., Rodrigues-Hoffman, A., Basu, R., Pan, H. L., & Kumar, M. N. V. R. (2022). Oral Nanocurcumin Alone or in Combination with Insulin Alleviates STZ-Induced Diabetic Neuropathy in Rats. Molecular pharmaceutics, 19(12), 4612–4624.
[4] Ganugula, R., Arora, M., Saini, P., Guada, M., & Kumar, M. N. V. R. (2017). Next Generation Precision-Polyesters Enabling Optimization of Ligand-Receptor Stoichiometry for Modular Drug Delivery. Journal of the American Chemical Society, 139(21), 7203–7216.

Acknowledgements: This work is supported by NIH-R01DK125372-01


Figure 1: Effect of treatment on blood glucose and Blood count in T2DM rats. Random blood glucose levels were measured at a) 24 hours, b) 1-hour post-insulin dosing using the AlphaTrack blood glucose monitoring system, designed for rats, mice, and dogs. Terminal blood samples were analyzed using the Zoetis VETSCAN HM5, and the total percentage of c) neutrophils and d) platelets was calculated. Data are presented as mean ± SEM, with statistical analysis performed using a Two-way ANOVA and student’s t-test and a Welch's t-test (*p < 0.05, **p < 0.001, ***p < 0.001, ****p < 0.0001 for comparisons with the diabetic group (Group 3)).


Figure 2. Treatment effects on cataract progression and intraocular pressures in STZ-induced diabetic rats. a) Representative lens images in the terminal 20th week; b) Cataract scoring at the 20th week revealed statistically significant differences between the long-acting insulin group and the combination therapy group, with the latter achieving superior outcomes. c) Intraocular pressure (IOP) was significantly reduced in the combination therapy group but not in the insulin-alone group. (Icare tonolab tonometer was used for measurement of rat ocular pressure). Data are presented as mean ± SEM, with statistical analysis performed using a One-way ANOVA (*p < 0.05, **p < 0.001, ***p < 0.001, ****p < 0.0001 for comparisons with the diabetic group (Group 3)).


Figure 3. Treatment effects on diabetic retinopathy in STZ-induced diabetic rats. a) Representative images (scale bar 50µm) of retinal cross sections stained for GFAP (green), images were obtained using an Axioscan 7 microscope (Zeiss); b) Quantification of GFAP mean fluorescence area/ 12,500µm2 per retinal image (n = 5-6 animals/group) using ImageJ software. Data are presented as mean ± SEM, with statistical analysis performed using a One-way ANOVA (*p < 0.05, **p < 0.001, ***p < 0.001, ****p < 0.0001 for comparisons with diabetic group (Group 3)).