E. ang L. was collected during its blossoming phase in April, 2021, from a cultivated region on Istiqlal Street, Amman, Jordan. The identification of the plant was verified by Professor Barakat Abu Irmaileh (Faculty of Agriculture, The University of Jordan, Amman, Jordan). A voucher specimen (no. ELEA-1FMJ) was deposited in the Department of Pharmaceutical Sciences, School of Pharmacy, The University of Jordan. Following collection, the leaves were passively air-dried in the shade for ~1 week until a consistent weight was observed. Once dried, the leaves were ground to achieve a fine consistency. This powdered form was then utilized to prepare the aqueous extract.

Leaves of E. ang L. were carefully rinsed with deionized water, then dried at 30˚C in a contamination-free setting. A total of ~200 g dried leaves were finely ground into a powdery consistency using a pestle and mortar. This powdered material was combined with 500 ml deionized water. The mixture was subjected to reflux for 4 h in an oil bath at 90˚C. After the reflux period, the solution was allowed to return to room temperature and filtered through Whatman No.1 filter paper to remove any solid residues. The resulting extract of E. ang L. was used as a reducing agent in NP synthesis.

The aqueous extract was mixed with an equivalent volume of 1 mM iron (III) chloride hexahydrate (FeCl₃•6H₂O) and 1 mM zinc acetate [Zn(OAc)₂]. This mixture was stirred for ~1 h and was then exposed to ultrasonic waves in an ultrasonic bath for 30 min at room temperature. pH of the reaction mixture was maintained at 10-12 using a NaOH solution. NPs were separated by centrifugation at 7,500 x g for 10-15 min at room temperature, and dried in an oven at 80˚C for 4 h. NPs were washed multiple times with water until a neutral pH was achieved, as previously described (24).

Metal salt solutions (1 mM) (silver nitrate and zinc acetate) were combined and heated to 80˚C for ~30 min. Following this, 50 ml E. ang L. extract was gradually added using a burette until the total volume reached 90 ml. This blend was stirred continuously at 80˚C for 1 h. After stirring, the solution was allowed to settle, leading to the formation of a precipitate. This precipitate underwent sonication in an ultrasonic chamber for 30 min, as previously described (25). The pH was then adjusted to 10-12 using a NaOH solution. The NPs were isolated by centrifugation at 7,500 x g for 10-15 min at room temperature. NPs were dried in an oven set at 80˚C for 4 h and the remaining solid was washed several times with water until a neutral pH was reached, as previously described (29).

The composition of the particles was confirmed via Fourier-transform infrared spectroscopy (FTIR) analysis. The crystallinity of the bimetallic NPs was investigated using x-ray diffraction (XRD) analysis, covering a range of 25-65˚.

Crystallographic structure of the prepared NPs was determined by powder X-ray diffraction pattern. The infrared (IR) spectrum was acquired using a Perkin Elmer RX-FTIR spectrometer with potassium bromide disc and scanning range of 4,000-300 cm^-1.

Cell lines were sourced from the American Type Culture Collection (ATCC), including the breast cancer cell line MCF-7 (cat. no. ATCC® HTB-22TM) and a standard dermal fibroblast cell line (BJ; cat. no. ATCC® CRL-2522). The cell lines were cultured in RPMI-1640 medium (HyClone; Cytiva) in vented 75 cm² culture flasks. The medium was supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco, Thermo Fisher Scientific) antibiotics (100 U/ml penicillin and 100 g/ml streptomycin), 2 mM L-glutamine and 25 µM HEPES. These flasks were kept in an atmosphere of 5% CO₂ and 95% air and a set temperature of 37˚C. All cell experiments were performed in a class II biological safety cabinet to ensure sterility. MCF-7 breast cancer and the standard fibroblast cells were seeded in 96-well plates at densities of 7,000 and 9,000 cells/well, respectively, and were then incubated at 37˚C overnight adhere to the plate well surface.

The cytotoxicity of bimetallic NPs on MCF-7 and fibroblast cells was evaluated by MTT assay, as previously described (30). A total of 7,000 MCF-7 cells were seeded in duplicate in each well of a 96-well plate and incubated for 24 h at 37˚C. A total of 10 mg/ml bimetallic E. ang-Fe₂ZnO₄ and Fe₂ZnO₄ NPs and 10 mM aqueous leaf extract of E. ang L. was dissolved in 1% dimethyl sulfoxide (DMSO). Seven distinct concentrations, ranging from 200, 100, 50, 25, 12.5, 6.25 and 3.125 µg/ml, were tested. After 72 h incubation at 37˚C, the viability of the cells was assessed. Doxorubicin was used as a positive control. Spectrophotometric measurements were employed to ascertain the antiproliferative activity across varied concentrations of E. ang-Fe₂ZnO₄. The absorbance of these solutions was determined at 570 nm. Dose-response curves were analyzed by regression analysis using sigmoidal curves [log(concentration) vs. normalized absorbance]. The half-maximal inhibitory concentration (IC₅₀) was determined with GraphPad Prism software version 8.0.0 (La Jolla, CA).

Rat aortic ring assay was conducted using eight adult (8-10 weeks) male Wister albino rats (Rattus norvegicus; weight, 200-250 g; Animal House Unit, The University of Jordan). Rats were housed in The University of Jordan Animal House Unit at 22±1˚C under a 12/12-h light/dark cycle, and the humidity range was 50-60% with free access to food and water. Experimental procedures were approved by the scientific research committee (The Institutional Review Board of the University of Jordan) at the University of Jordan (approval no. 47-2022). All experiments were performed according to the Animal (Scientific Procedure) Act 1986 and International Association for the Study of Pain guidelines (13). Rats were euthanized using CO₂ overdose followed by decapitation. A fill rate of 30-70% of the chamber volume/min with CO₂ was used to induce rapid unconsciousness with minimal distress to the animals.

Immediately post-extraction, the aortae were submerged in a cold sterile 1% PBS solution in a Petri dish. Using a dissecting microscope for precision, extraneous connective tissue was removed. The aortae, after cleaning with PBS, were sectioned into ~1 mm thick rings using a surgical scalpel. These segments were immersed in cold PBS solution and stored on ice until use.

Each aortic ring was positioned within a 48-well plate using cold pipette tips. Rings were immersed in 25 µl low growth factor Matrigel™ (Corning, Inc.) and were placed in pairs. Following 30 min incubation at 37˚C to allow coagulation, aliquots of 250 µl, with concentrations 100, 50, 25, 12.5, 6.25 and 3.125 µg/ml, of the three different extracts diluted in RPMI-1640 were added. For the control group, DMSO was used at a concentration of 100 µg/ml. The plate was then incubated at 37˚C. On day 4, medium was refreshed. On day 5, images were captured using an inverted light microscope at 4x magnification.

The angiogenic response was quantified by measuring the extension of blood vessels from the primary ring explants using ImageJ software1.29 (National Institutes of Health). For each ring, ≥35 comparable structures, evenly spaced around the ring, were measured (26).

Data were analyzed with GraphPad Prism 8 software. The elongation distance of the emerging vessels for each ring was recorded as the mean of percentage inhibition relative to the unaltered control (n=2) (27). Vessel lengths were expressed in arbitrary units and the inhibition of vessel formation was as follows: Angiogenic inhibition=[1-(A0/A)] x100, where A0 represents vessel growth distance in treated rings and A signifies the vessel growth distance in control rings.

A total of ~1x10⁵ MCF-7 cells were seeded in a 24-well plate and incubated overnight at 37˚C in 5% CO₂ to ensure adhesion. A 10 mg/ml stock solution of E. ang-Fe₂ZnO₄ and Fe₂ZnO₄ NPs and organic extract was then prepared using DMSO. Serial dilutions were made in RPMI-1640 (HyClone; Cytiva) complete media at 200.00, 100.00, 50.00, 25.00, 12.50 and 6.25 µg/ml E. ang-Fe₂ZnO₄ NPs and organic extract. For Fe₂ZnO₄ NPs, dilutions were made to 200, 100, 50 and 25 µg/ml based on cytotoxicity and anti-angiogenic effects. DMSO concentration was <1% for all treatments. Quadruplicate samples were used for every concentration. Control wells, containing only the complete medium, were also included in each assay. Following 72 h incubation at 37˚C with the treatments, conditioned media was harvested and stored at -80˚C until use.

Secretion levels of VEGF in conditioned media from both treated and control (untreated) cells were quantified by ELISA, as previously described (31). Human VEGF (cat. no. #DY293B05; R&D Systems, Inc.) ELISA kit was used to measure the concentration of VEGF secreted in conditioned media, following the manufacturer's protocol. Concentrations were calculated using Microsoft Excel software (Microsoft Office professional plus 2016) using the standard curve equation and further analyzed using Prism 8 statistical analysis software (GraphPad Software, Inc.).

Statistical analysis was performed using GraphPad Prism software, version 8.0.0. IC50 values for each tested NP were derived by fitting the observed data to a logarithmic trend line depicted on the cytotoxicity graphs (log concentration against inhibition percentage). Data are presented as the mean ± SEM. Each experiment was performed in triplicate. To assess number and length of blood vessels, images were analyzed using the ImageJ 1.29 (National Institutes of Health). Data were analyzed using one-way ANOVA followed by Bonferroni's multiple comparisons post hoc test. P<0.05 was considered to indicate a statistically significant difference.

IC₅₀ of E. ang-Fe₂ZnO₄ and metallic ZnO NPs and E. ang L. aqueous leaf extract was evaluated in MCF-7 and fibroblast cell lines using MTT assay. Cytotoxicity of E. ang-Fe₂ZnO₄ NPs and Fe₂ZnO₄ NPs in MCF-7 cells were assessed in comparison with the fibroblast cell line. Further, the cytotoxicity exhibited by E. ang-Fe₂ZnO₄ was determined in both MCF-7 and fibroblast cell lines using IC₅₀. Fe₂ZnO₄ NPs had IC₅₀ values of 3.574 µg/ml for MCF-7 cells and 61.290 µg/ml for fibroblasts (Figs. S1 and S2). Conversely, E. ang-Fe₂ZnO₄ NPs demonstrated an augmented resilience, producing an IC₅₀ value >200 for MCF-7 and 67.15 µg/ml for fibroblasts. The isolated E. ang L. aqueous extract had IC₅₀ values >200 for MCF-7 and 78.65 µg/ml for fibroblasts. Doxorubicin, a chemotherapeutic agent, had IC₅₀ values of 0.350±0.025 for MCF-7 and 7.037±2.960 µM for fibroblasts, underscoring its cytotoxic effect.

The angiogenic potential of E. ang-Fe₂ZnO₄ and Fe₂ZnO₄ NPs and the aqueous extract of E. ang L. leaf was evaluated utilizing the rat aortic ring assay. Angiogenesis was quantified by measuring the extension of vessels emanating from the primary ring (Fig. S3). A significant inhibition of micro-vessel outgrowth from the aortic rings was observed when treated with E. ang-Fe₂ZnO₄ NPs in a dose-dependent manner (Fig. 3). At a concentration of 100 µg/ml, the mean inhibition of 98.71±0.36% was noted compared with the control group. At 50.0, 25.0 and 12.5 µg/ml, the growth of new blood vessels was significantly decreased by 98.190±0.365, 96.650±10.365 and 81.680±1.090%, respectively. Moreover, the concentration 6.25 µg/ml significantly decreased the growth of new blood vessels by 47%±1.075. However, 3.125 µg/ml did not significantly inhibit micro-vessel outgrowth (5.30±0.36%).

Significant inhibition of micro-vessel outgrowth from the aortic rings was observed when treated with Fe₂ZnO₄ NPs in a dose-dependent manner (Fig. 4). At 100 µg/ml, a significant inhibition of 86.32±1.09% was noted in comparison with the control group. Moreover, concentrations of 50.0, 25.0 and 12.5 µg/ml attenuated the growth of new blood vessels by 81.300±3.102, 67.740±1.094 and 48.640±1.095%, respectively. A concentration of 3.125 µg/ml did not significantly inhibit micro-vessel outgrowth (3.741±1.095%).

Micro-vessel outgrowth from the aortic rings was observed to be significantly inhibited following exposure to E. ang L. extract at 100 µg/ml (96.38±0.73% compared with the control; Fig. 5). E. ang L. aqueous extract also significantly decreased micro-vessel outgrowth by ~94.580±1.090, 85.030±1.450 and 50.710±1.094 at concentrations of 50.0, 25.0 and 12.5 µg/ml, respectively. At 3.125 µg/ml, E. ang L. extract resulted in growth inhibition of 1.680±1.095%.

The secretion of VEGF was significantly inhibited by 200 µg/ml E. ang-Fe₂ZnO₄ NP, reducing to ~0.427 times that of the control. Also, significant 0.690 and 0.814 times suppression of VEGF protein was observed upon treatment with 50 and 25 µg/ml, respectively; other concentrations showed insignificant reduction of protein levels of VEGF (Fig. 6).

At a concentration of 200 µg/ml, VEGF secretion in response to Fe₂ZnO₄ -NPs decreased to ~0.348 times that observed in the control group, but this reduction was not statistically significant. The other concentrations also did not cause any significant change in VEGF secretion (Fig. 7).

VEGF secretion, upon exposure to 200 µg/ml organic extract, was observed to be significantly decreased by 0.366 times relative to the levels in the control. However, no other concentrations caused a significant decline in VEGF protein levels (Fig. 8).