Antiangiogenic Potential of Troxerutin and Chitosan Loaded Troxerutin on Chorioallantoic Membrane Model

Abstract

Angiogenesis is crucial to the development of cancer because it allows the transport of oxygen, nutrients, and growth factors as well as the spread of tumors to distant organs. Inhibitors of angiogenesis prevent the formation of blood vessels that allow tumor cells to shrink, rather than promote tumor growth. Chitosan acts as a carrier for many drugs, since the compound has various properties such as biodegradable, less toxicity, more stable, simple, easy to prepare, and biocompatible. The aim of the current study was to evaluate the efficacy of chitosan nanoparticles encapsulated with troxerutin (Chi-Trox NPs) against angiogenesis and cancer in ova chick chorioallantoic membrane (CAM) model. Chi-Trox NPs were synthesized using a nanoprecipitation method and were characterized by various analyses. 24 hours’ fertilized eggs (6 eggs/group) were treated with native Trox and Chi-Trox NPs for 5 days. The antiangiogenic activity was evaluated by morphometric, histopathological, immunohistochemical (CD104 and vimentin), and mRNA expression of MMP and FGF2 using RT-PCR. The anticancer activity was evaluated by histopathological, immunohistochmical (CD44), and mRNA expression of FGF2 and MMP. The synthesized chitosan NPs were successfully encapsulated with troxerutin, and the loading efficiency of chitosan NPs was found to be $86.4\pm 0.12\%$ and $13.2\pm 0.16\%$ respectively. Morphometric analysis of Chi-Trox NPs showed a considerable decrease in the number of blood vessels compared with control and native Trox. The histopathological observation of CAM confirmed that Chi-Trox NPs induce a significant reduction in inflammatory cells and the thickness of blood capillaries compared to control and native Trox. The immunohistochemical evaluation of CAM revealed Chi-Trox decreased CD104, vimentin and CD44 protein levels were compared with control and native Trox. Furthermore, the mRNA expression levels of FGF2 and MMP were significantly downregulated compared to their native forms. From the obtained results, Chi-Trox NPs possess significant inhibition of angiogenesis and can be used as therapeutic agents for cancer in the future.