Angiogenesis is necessary for organogenesis, proliferation, and cell differentiation in the embryonic period; it can be divided into two categories of physiology and pathology [1]. Angiogenesis plays an important role in the metastasis of cancerous tumors; thus, it can be targeted by antitumor therapies [4]. Studies indicated that the encapsulation and emulsification of vegetable oils due to the anti-cancer effects of these compounds can lead to their widespread use in inhibiting cancer cells. Studies have been conducted on the use of vegetable oils, like dill (Anethum graveolens L) [12] in the treatment of some cancers, including breast and liver types [11]. The present study examined the effects of different concentrations of dandelion essential oil nanoemulsion using the CAM method.

In preparing the nanoemulsion of the dill plant, Tween 20 and Tween 80, ethylene glycol, and distilled water were used. The nanoemulsion was prepared in the presence of ultrasonic waves or power of 200 watts for 60 minutes. In this study, 50 fertilized eggs of ROSS breed were divided into 5 groups, including control and laboratory control groups and 3 experimental groups; accordingly, they were placed in an incubator at 37°C and 65% humidity. On the second day of incubation, a window was made on the eggs. On day 8 of incubation, a gelatinous sponge (a combination of egg white albumin, isothermal agar solution, & antibiotics) was placed on the chorioallantoic membrane. In the control group, 10 μL of sterile distilled water was added to the gelatinous sponge on the chorioallantoic membrane; the laboratory control group was treated with 10 μL of normal saline, followed by 10 μL of different concentrations (12.5, 25, & 50 μg/mL) dill nanoemulsion, i.e., added to the sponge. On the 12th day, the study samples were collected by stereomicroscope. J Image software was used to evaluate the length and number of vessels. Fetal height and weight were measured using calipers and scales, respectively. The obtained data were analyzed in SPSS by one-way Analysis of Variance (ANOVA) and Least Significant Difference (LSD) test.

The morphological images of the chorioallantoic membrane in the study samples treated with different concentrations of nanoemulsion (12.5, 25, & 50 μg/mL), compared to the control and laboratory samples presented a decrease in the number of blood vessels (Figure 1). Exploring the number of blood vessels revealed no significant difference between the control and laboratory controls; however, there was a significant difference between the research samples treated with different concentrations of nanoemulsion and the controls (*P<0.05 & ***P<0.001).

Preventing the growth of cancer cells and inhibiting them can be performed by various strategies, such as inducing apoptosis or inhibiting angiogenesis. Thus, inhibiting the angiogenic process inhibits the growth and development of cancer cells. Recently, numerous anti-angiogenic drugs have been studied; however, most of them presented high toxicity and have developed drug resistance. Additionally, they failed to cross obstacles to achieve the desired goal [19]. Due to their small size, nanoemulsions can cross the blood barrier and easily be present in tumor tissues; thus, they can exert their effects through the blood vessels surrounding the tumor. They can also be designed to encapsulate different drugs and select specific targets [20]. Dehelean et al. (2011) applied nanoemulsion with antiangiogenic properties for drug delivery. They investigated the antiangiogenic effect of nanoemulsion synthesized from flaxseed oil using the CAM method. The relevant results indicated that this combination of effects on vascular mesenchyme prevents angiogenesis [21]. Another study Dehelean et al. (2013) examined the effect of betulin nanoemulsion on angiogenesis in skin carcinoma cells in mice and chicken embryo chorion membranes. The related results also supported the anti-angiogenic effects of nanoemulsions [22]. Yousefian et al. (2020) explored the anti-angiogenic effect of nanoemulsion of lemon essential oil was using the CAM method; they concluded that this compound prevents angiogenesis [23]. In the present study, the anti-angiogenic effects of dill nanoemulsion were confirmed. This study, similar to the studies mentioned above, identified a significant reduction (***P<0.001) in the number and length of blood vessels in the chorioallantoic membrane of chickens.

All experiments of this research were carried out in compliance with ethical standards and according to the approval of the Research Ethics Committee of Mashhad Branch, Islamic Azad University (Code: IR.IAU.MSHD.REC.1398.027).

This research has not received any special funding from public, commercial or non-profit sector financing organizations.

Conceptualization: Masoud Homayouni Tabrizi Methodology: Masoud Homayouni Tabrizi, Turan Ardalan. Validation: Masoud Homayouni Tabrizi, Turan Ardalan. Analysis, Research: Haleh Sadat Tavakol Afshari. Draft: Haleh Sadat Tavakol Afshari. Edited and finalized by: Masoud Homayouni Tabrizi, Turan Ardalan. Visualization, Supervision, Project Management, Financing: Masoud Homayouni Tabrizi.

The authors declared no conflicts of interest.