In vivo therapeutic efficiency of anti-angiogenic peptide decorated PEG-b-PPS Micelles in Danio rerio (Zebrafish) Diabetic Retinopathy Model

Introduction: An αVβ3 specific peptide (GHSDVHK) bound PEG-b-PPS micelles (MCs) developed to target Diabetic Retinopathy (DR) has yielded significant anti-angiogenesis and anti-inflammatory properties in in vitro models However, an in vivo understanding of angiogenesis and toxicity has not been investigated. Therefore, this project focuses on studying in vivo toxicity and in vivo anti-angiogenesis efficacy in the zebrafish embryo DR model. We hypothesize that a diabetic retinopathy model in Danio rerio embryos will efficiently mimic the key aspects of diabetic retinopathy, allowing for comprehensive evaluation and analysis of in vivo toxicity and anti-angiogenic therapeutic efficiency of anti-integrin-PEG-b-PPS MCs.

Materials and

Methods: The DR model is established in Danio rerio embryos by growing the embryos in high glucose concentrations, including 90, 130, 150, and 160 mM. A series of experiments were performed to prove the initiation of DR, including H & E staining of retinal layers, hyaloid retinal diameter, branch measurement, and gene expression analysis to examine IL-6, TNF-α, IL-1β, VEGF, HIF-1α, insulin, insa, glucagon, and PCK-1. Apart from that, survival rate, hatching rate, and morphological abnormalities were analyzed to check for dose-dependent response. Nile red-loaded PEG-b-PPS MCs with 5% anti-integrin peptides (SDV-5, and cRGD-5 MC) were synthesized and their sizes, zeta potential, and polydispersity index were characterized using Dynamic Light Scatter (DLS) and Cryogenic Transmission Electron Microscopy (cryo-TEM). To study the biocompatibility and anti-angiogenesis efficiency, embryos from 0 dpf were treated with 130 mM, and 150 mM Glucose to initiate DR and further treated with SDV and cRGD-MCs. The survival rate, hatching rate, and morphological abnormalities in the embryos post-treatment were analyzed. The number of hyaloid retinal vessel branches and their thickness was measured post-treatment to check the anti-angiogenesis efficiency. Students t-test and One-way ANOVA were performed to study the statistical significance.

Results, Conclusions, and Discussions: Results showed that 130 mM glucose treatment initiates diabetic retinopathy in Danio rerio embryos without causing adverse effects on survivability, hatching rate, and normal morphology. While 150 mM, 160 mM glucose-treated embryos showed abnormal morphology such as yolk sac edema and bent-tail phenomenon. There was also a significant (P < 0.0001) increase in the thickness of hyaloid retinal blood vessels, nearly double compared to the control. Pro-inflammatory genes linked with DR (IL-6, TNF-α, IL-1β), angiogenesis-related genes (VEGF, HIF-1α), as well as genes associated with diabetes and glucose metabolism (insulin, insa, glucagon, and PCK-1) significantly increased in embryos treated with 130 mM Glucose compared to control. All the above results indicate that 130mM glucose-treated embryos showed an optimal DR model. The DR model embryos treated with 50 µg/ml and 100 µg/ml MCs did not affect normal morphology, survivability, or hatching rate. The results also suggest that aANGP MCs are being localized in the eye and yolk sac of DR embryo and showed a significant (P < 0.0001) reduction in the thickness and number of hyaloid-retinal blood vessels. In conclusion, 130 mM glucose treatment was effective in initiating DR in Danio rerio embryos. PEG-b-PPS-SDV 5% MCs successfully reduced angiogenesis in DR model Danio rerio embryos.

Acknowledgements (Optional): NIH R03EB032602-01 (Bijukumar), Blazer Foundation, MBT Program at UIC.