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. 2023 May 23:2023:5956154.
doi: 10.1155/2023/5956154. eCollection 2023.

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

Gowtham Kumar Subbaraj  1 , Harini Elangovan  1 , Prema Chandramouli  1 , Santhosh Kumar Yasam  1 , Kirubhanand Chandrasekaran  2 , Langeswaran Kulanthaivel  3 , Sangavi Pandi  3 , Senthilkumar Subramanian  4
Affiliations

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

Gowtham Kumar Subbaraj et al. Biomed Res Int. .

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 ± 0.12% and 13.2 ± 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.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Ramachandran plot for cluster of differentiation 104 (CD104) protein.
Figure 2
Figure 2
Ramachandran plot for vimentin protein.
Figure 3
Figure 3
Interaction between troxerutin and CD104 protein.
Figure 4
Figure 4
Interaction between troxerutin and vimentin protein.
Figure 5
Figure 5
SEM results of (a) troxerutin and (b) Chi-Trox NPs.
Figure 6
Figure 6
DLS results of (a) troxerutin and (b) Chi-Trox NPs.
Figure 7
Figure 7
FT-IR results of (a) chitosan, (b) troxerutin, and (c) Chi-Trox NPs.
Figure 8
Figure 8
Chorioallantoic membrane in ova photographs showing vascular plexus after the incubation with control and treated groups (Trox and Trox-Chi NPs). (a) The photographs were taken after the treatment, incubation day-5. (b) Morphometric analysis of CAM treated with Trox and Chi-Trox NPs.
Figure 9
Figure 9
Drug release kinetic assay of Chi-Trox NPs.
Figure 10
Figure 10
Histological examinations of Chi-Trox NPs on CAM morphology: (a) control, (b) Trox, and (c) Chi-Trox NPs. lbv: large blood vessel; ae: allantoic epithelium; ce: chorionic epithelium.
Figure 11
Figure 11
Immunohistostaining of CD104 in chick embryo chorioallantoic membranes (CAM): (a) control, (b) Trox, and (c) Chi-Trox.
Figure 12
Figure 12
Immunohistostaining of vimentin in chick embryo chorioallantoic membranes (CAM): (a) control, (b) Trox, and (c) Chi-Trox. Red arrow shows expression of vimentin.
Figure 13
Figure 13
Effects of Trox and Chi-Trox NPs on the expression of FGF2 in chick chorioallantoic membrane. Comparison made between control and Trox, control and Chi-Trox NPs. Trox: troxerutin; Chi-Trox NPs: chitosan nanoparticles encapsulated with troxerutin; SD: standard deviation. The statistical analysis was done by using ANOVA and Tukey's post hoc test. ∗significant difference compared with control (p < 0.05); ∗∗significant difference compared with the control (p < 0.01); ∗∗∗significant difference compared with the control (p < 0.001); and ∗∗∗∗significant difference compared with the control (p < 0.0001).
Figure 14
Figure 14
Effects of Trox and Chi-Trox NPs on the expression of MMP in chick chorioallantoic membrane. Comparison made between control and Trox and control and Chi-Trox NPs; Trox: troxerutin; Chi-Trox NPs: chitosan nanoparticles encapsulated with troxerutin; SD: standard deviation. The statistical analysis was done by using ANOVA and Tukey's post hoc test. ∗Significant difference compared with the control (p < 0.05); ∗∗significant difference compared with the control (p < 0.01); ∗∗∗significant difference compared with the control (p < 0.001); ∗∗∗∗significant difference compared with the control (p < 0.0001).
Figure 15
Figure 15
(a) Inhibition of growth in HeLA cells in the CAM by Trox and Chi-Trox NPs. (b) Quantification of A498 cell tumor size after treatment with Trox and Chi-Trox NPs using box plot.
Figure 16
Figure 16
Histological examinations of Chi-Trox NPs on CAM morphology: (a) control, (b) Trox, and (c) Chi-Trox NPs. lbv: large blood vessel; ae: allantoic epithelium; ce: chorionic epithelium.
Figure 17
Figure 17
Immunohistostaining of CD44 in chick embryo chorioallantoic membranes (CAM): (a) control, (b) Trox, and (c) Chi-Trox.
Figure 18
Figure 18
Anticancer effects of Trox and Chi-Trox NPs on the expression of FGF2 in chick chorioallantoic membrane. Comparison made between control and Trox and control and Chi-Trox NPs; Trox: troxerutin; Chi-Trox NPs: chitosan nanoparticles encapsulated with troxerutin; SD: standard deviation. The statistical analysis was done by using ANOVA and Tukey's post hoc test. ∗Significant difference compared with the control (p < 0.05); ∗∗significant difference compared with the control (p < 0.01); ∗∗∗significant difference compared with the control (p < 0.001); ∗∗∗∗significant difference compared with the control (p < 0.0001).
Figure 19
Figure 19
Anticancer effects of Trox and Chi-Trox NPs on the expression of MMP in chick chorioallantoic membrane. Comparison made between control and Trox and control and Chi-Trox NPs. Trox: troxerutin; Chi-Trox NPs: chitosan nanoparticles encapsulated with troxerutin; SD: standard deviation. The statistical analysis was done by using ANOVA and Tukey's post hoc test. ∗Significant difference compared with the control (p < 0.05); ∗∗significant difference compared with the control (p < 0.01); ∗∗∗significant difference compared with the control (p < 0.001); ∗∗∗∗significant difference compared with the control (p < 0.0001).
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