TITLE: Antiangiogenic research using AgNPs-FD
AUTHORS: Rudy Agung Nugroho

[DESCRIPTION]
Background: Ficus deltoidea L. Jack is a folk medicinal plant known for its pharmacological properties, including anti-inflammatory, anticancer, and anti-angiogenic. This study aimed to evaluate the anti-angiogenic properties of silver nanoparticles biosynthesized using F. deltoidealeaf extract (AgNPs-Fd).

Methods: The AgNPs-Fd were synthesized by mixing 100 mL 1 mM aqueous silver nitrate (AgNO3) and 100 mL 0.1%F. deltoidea ethanolic leaf extract. The resulting AgNPs-Fd were observed for color change and Tyndall effects. Reaction mixture color change from pale brown to reddish brown was observed at 48 h at 37℃oC. The characterization of AgNPs-Fd was completed with UV–Vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. For quantitative analysis of the vascular network in thechorio allantoic membrane(CAM) assay, AngioTool open-source software was used.

Results:The plasmon resonance peak for AgNPs-Fd at 430 nm was visible in the UV-Visible spectrum, indicating the formation of AgNPs-Fd. TheF. deltoidea extract and nanoparticles interacted well according to FTIR analysis. The AgNPs-Fd morphology of 20 nm particle sizes was observed using TEM. The chromatographic analysis of AgNPs-Fd identified potential anti-angiogenic compounds, such as phytol, stigmasterol, lupeol, and sitosterol. The angiogenic inhibition properties of AgNPs-Fd were tested using the CAM assay. The 90mg dose AgNPs-Fd treatment in CAMs demonstrated significant anti-angiogenesis, indicating effectiveness in controlling vessel formation.
Conclusion: The present study suggests that eco-friendly work and the “green” process of AgNPs-Fd is potentially applicable for nanobiotechnology in anti-angiogenic fields.

Keywords:anti-angiogenic;Ficus deltoidea; green synthesize; nanomaterials

[STEPS]
SECTION: Biosynthesis of AgNPs-Fd
1. Aqueous silver nitrate (AgNO3) (100 mL, 1 mM) was combined with 100 mL 0.1%F. deltoidealeaf extract.
SECTION: Characterization of AgNPs-Fd
5. To determine phytochemicals surrounding the AgNPs-Fd, FTIR spectroscopy (Agilent, Cary 630 model, US) was performed
SECTION: Characterization of AgNPs-Fd
4. The TEM (MIRA3 model, Czech Republic) was operated to display the surface morphology of the AgNPs-Fd. XRD was applied to evaluate the chemical characterization of AgNPs-Fd
SECTION: Characterization of AgNPs-Fd
3. The resulting AgNPs-Fd were confirmed using UV-Vis spectrophotometry (Shimadzu, UV-1280, Japan) in the range of 350–750 nm
SECTION: Biosynthesis of AgNPs-Fd
2. The pH of the solution was adjusted to 7.0, and it was subsequently incubated on a rotary shaker at 150 rpm for 48 h at 28°C
SECTION: Phytochemical analysis
6. Phytochemical analysis of the AgNPs-Fd was performed using GC–MS (HP-5MS UI, Agilent, USA) to evaluate the chemical compounds that potentially serve as anti-angiogenic.

SECTION: Phytochemical analysis
7. For this, samples of AgNPs-Fd were dissolved in ethanol in a microtube, vortexed, and centrifuged for three minutes at 9500 rpm
SECTION: Phytochemical analysis
8. The resulting supernatant was used for identification and injected into the GC–MS apparatus.
SECTION: Phytochemical analysis
9. The condition of the GC–MS is as follows: column: HP-5MS UI, gas carrier: helium UHP (He), injector temperature: 290℃, split flow: 10 ml/min, split ratio: 10; front inlet flow: 1.00 ml/min, MS transfer line temp: 230℃, ion source temp: 200℃, mass list range (amu): 40–500, purge flow: 3 ml/min, gas saver flow: 5 ml/min, and gas saver time: five minutes
SECTION: CAM Assay
10. To analyze AgNPs-Fd for anti-angiogenic properties, a CAM assay was performed following previous methods byRibatti [24], Camposano and Torre [28], and Gamallo and Espere [29]
SECTION: CAM Assay
13. Before incubation, eggshells were cleansed with 70% alcohol. The eggs were incubated in for six days at 37−39°C with 50−60% humidity
SECTION: CAM Assay
12. A basic fibroblast growth factor (bFGF-Thermo Fisher Scientific, USA) is a group of proteins secreted by tissues to regulate cell metabolism, proliferation, differentiation, and survival. The bFGF was used to induce neovascularization[30]
SECTION: CAM Assay
11. In total, 24 chicken eggs were collected in preparing for the CAM analysis and were dosed with F. deltoidea extract (Fd) or AgNPs-Fd. The doses used in the paper disk for the CAM assay were as follows: negative control (30 ng basic fibroblast growth factor (bFGF); positive control (30 µg cortisone acetate with 30 ng bFGF); treatment groups (30 ng bFGF with AgNPs-Fd at 45, 60, 75 and 90mg, respectively)
SECTION: CAM Assay
14. The boundaries of the air space, which indicated the location of the embryo, were marked on all eggshells with a pencil (1 × 1 cm)
SECTION: CAM Assay
15. Candling, with egg binoculars, was used to determine the location of the embryo. By using a povidone-iodine solution, the eggshell was cleaned at the pole containing the air space and the part above the embryo
SECTION: CAM Assay
16. Using a needle, a small hole was made in the air chamber and vacuumed until the air moved from the pole to the top of the egg.
SECTION: CAM Assay
17. Next, a window measuring 1 × 1 cm was made on the marked area using a mini drill. Paper discs for each treatment were then embedded onto the CAM through this window
SECTION: CAM Assay
18. The holes in the polar regions and 1 × 1 cm windows were sealed with paraffin film after they had been planted according to the treatment
SECTION: CAM Assay
20. After, the eggs were removed and subsequently killed by freezing for 24 h.
SECTION: CAM Assay
19. The eggs were then incubated for 48 h at 37−39°C with 50−60% humidity
SECTION: CAM Assay
21. The eggs were then opened by cutting the eggshell into two parts, beginning with the part closest to the air cavity
SECTION: CAM Assay
22. The egg contents were slowly and carefully removed, keeping the CAM attached to the shell. Each CAM was photographed and analyzed using AngioTool 0.6 software
SECTION: CAM Assay
23. AngioTool is a small, easy-to-use application that allows for the quick, hands-free and reproducible measurement of vascular networks in micrographs
SECTION: CAM Assay
24. AngioTool calculates a variety of morphological and geographical data, such as the area covered by a vascular network, the number of vessels, vessel length, vascular density, and lacunarity.
SECTION: CAM Assay
25. AngioTool also computes the "branching index" (branch points/unit area), which quantifies specimen sprouting activity[26].