Leaves of Moringa oleifera were subjected to sequential extraction using absolute ethanol, 50% ethanol, and deionized water. Briefly, 25 g of dried leaf powder was extracted with 250 mL of absolute ethanol (1:10 w/v), sonicated for 30 minutes, and centrifuged at 6,000 rpm for 15 minutes. The extraction was repeated twice, and the pooled supernatants were filtered and concentrated using a rotary evaporator at 60˚C. The extract was stored at 4˚C. The remaining pellet was re-extracted sequentially with 50% ethanol and deionized water using the same procedure. The final aqueous extract was lyophilized and stored at 4˚C for further use.

Green synthesis of MO-AgNPs was carried out by mixing 10 mL of 1% (w/v) aqueous extract with 90 mL of 169.88 μg/mL silver nitrate (AgNO₃) solution. The reaction mixture was incubated at 60˚C for 8 h under continuous agitation. Nanoparticle formation was indicated by a color change from yellowish to dark brown. The synthesized nanoparticles were collected by centrifugation at 6,000 rpm for 30 minutes, washed three times with deionized water, freeze-dried, and stored at 4˚C until use [6].

Basic physicochemical characterization of the synthesized nanoparticles was performed using ultraviolet-visible (UV-Vis) spectroscopy, dynamic light scattering (DLS), and zeta potential analysis. As the DLS and zeta potential data have been previously published in our earlier work [7], UV-Vis spectroscopy data are included in Figure S1.

To investigate the anti-invasive potential of MO-AgNPs as a targeted anti-tumor therapy, this study assessed their ability to inhibit the invasion of the EC line (Ea.hy926) through a Matrigel^® barrier (Corning, USA) [8, 9]. Matrigel, diluted in Dulbecco’s modified eagle medium (DMEM) at a 1:1 ratio, was dispensed into a 96-well plate and allowed to solidify for 1 h. Subsequently, 5,000 cells per well were seeded and treated with one of the following: 0.05% DMSO:0.05% deionized water (negative control), 15.11 μg/mL quercetin (Genochem World, Spain) as a positive control, or MO-AgNPs at concentrations of 12, 6, 3, and 1.5 μg/mL.

The plates were incubated for 24 h at 37˚C in a humidified atmosphere containing 5% CO₂. Following incubation, the upper medium containing non-invaded cells was gently removed, and wells were washed with phosphate buffered saline (PBS). The number of invaded cells remaining in the lower matrix was quantified using an inverted microscope at 40× magnification [10].

The degree of invasion inhibition was calculated by averaging cell counts from multiple microscopic fields and applying the following formula:

C e l l   i n v a s i o n   i n h i b i t i o n   ( % ) = 1 - A v e r a g e   c e l l s   i n v a d i n g   M a t r i g e l   i n   t r e a t e d   g r o u p × 100 A v e r a g e   c e l l s   i n v a d i n g   M a t r i g e l   i n   u n t r e a t e d   g r o u p

Three independent experiments were conducted, and the results were presented as mean ± standard deviation (SD).

This assay was conducted to evaluate the inhibitory effects of MO-AgNPs on the ability of Ea.hy926 cells to form capillary-like structures on Matrigel^® [11, 12]. Ea.hy926 cells were first cultured in flasks for 48 h. After this incubation period, the culture medium was replaced with fresh DMEM containing either 0.05% DMSO:0.05% deionized water (negative control), 15.11 μg/mL quercetin (positive control), or MO-AgNPs at concentrations of 12, 6, 3, and 1.5 μg/mL.

Following 24 h of treatment, Matrigel diluted 1:1 with DMEM was added to each well of a 48-well plate and allowed to solidify for one hour. Cells from the treated flasks were then counted, and 100,000 cells were resuspended in fresh DMEM and seeded into each well. The formation of tube-like structures was monitored at two-hour intervals under an inverted microscope, with typical tube formation observed approximately two and a half hours post-seeding. Images were captured at 40× magnification using an Olympus CKX41 digital camera (Japan).

The percentage of inhibition of tube formation was calculated using the following formula:

T u b e   f o r m a t i o n   i n h i b i t i o n   ( % ) = 1 - A v e r a g e   o f   e n c l o s e d   a r e a   i n   t r e a t e d   g r o u p × 100 A v e r a g e   o f   e n c l o s e d   a r e a   i n   u n t r e a t e d   g r o u p

The ex vivo rat aortic ring assay was employed to assess the anti-angiogenic potential of MO-AgNPs. This 3D assay is widely regarded as one of the most effective models for studying angiogenesis. Modifications were applied to the standard protocols originally described by Rahman et al. [13], Aplin and Nicosia [14, 15] to optimize the procedure for this study.

Male Wistar rats, weighing approximately 200 g, were housed under a controlled 12 h light/dark cycle at 25 ± 2˚C with regulated humidity. All animal care and experimental procedures strictly complied with the USM Institutional Animal Care and Use Committee (IACUC) guidelines and received ethical approval under reference number USM/IACUC/2022/(137)(1230).

Rats were humanely euthanized via cervical dislocation following CO₂ inhalation to ensure death. The thoracic aorta was carefully excised and immediately immersed in M199 medium to preserve its structural integrity. The aorta was then sectioned into uniform rings and individually placed into the wells of a 48-well plate (Costar Corning, USA). After the lower matrix layer had solidified, 10 μL of thrombin (50 U/mL, Sigma-Aldrich, USA) in normal saline containing bovine serum albumin was added to each well. The plate was incubated for 1.5 h at 37˚C in a humidified incubator with 5% CO₂ to facilitate matrix polymerization and initial tissue stabilization [16].

M199 medium (Gibco/Life Technologies, USA) was used as the base growth medium. The assay was performed using two freshly prepared layers. The lower layer consisted of M199 medium mixed with fibrinogen (Merck, Germany), L-glutamine (Gibco/Life Technologies, USA), and aprotinin (Sigma-Aldrich, USA). The upper layer contained M199 medium supplemented with fetal bovine serum (FBS), ε-aminocaproic acid (Sigma-Aldrich, USA), L-glutamine, amphotericin B (Lonza Group Ltd., Switzerland), gentamicin, and penicillin-streptomycin.

After incubation, MO-AgNPs were added to the upper layer of each well at concentrations of 12, 6, 3, and 1.5 μg/mL. Suramin (100 μg/mL; Sigma-Aldrich, USA) served as the positive control, while 0.05% DMSO mixed with 0.05% deionized water was used as the negative control. The plates were then incubated at 37˚C in a 5% CO₂ atmosphere for seven days. On the fourth day, the medium in the upper layer was replaced with fresh M199 medium containing the same treatment concentrations. Neovascularization was evaluated using an inverted microscope at 40× magnification, equipped with a digital camera.

Microvessel outgrowth was measured to assess neovascularization, following the method described by Nicosia [15] and Kapoor et al. [17]. Images were analyzed using Motic Image Plus Version II software (Motic China Group Co., Ltd.). The percentage of neovascularization inhibition was calculated using the following formula:

N e o v a s u l a r i z a t i o n   i n h i b i t i o n   ( % ) = 100 × 1 - A 0 A

where A₀ is the distance of blood vessel growth in the sample, and A is the distance of blood vessel growth in the control. Results were presented as mean ± SD.

Data are presented as mean ± SD. Statistical significance was indicated by * for P-values less than 0.05 and ** for P-values less than 0.01. The non-parametric Kruskal-Wallis test, which is equivalent to ANOVA, was used to assess significant differences. All analyses were performed using SPSS version 22.0 (SPSS Inc., Chicago, IL) [19].

The Ea.hy926 and the colorectal cancer cell line HT29 were obtained from the American type culture collection (ATCC). Both cell lines were authenticated using short tandem repeat (STR) profiling prior to experimentation to confirm their identity and ensure the absence of cross-contamination with other cell lines. STR profiling was conducted by ATCC following their standard protocols. Additionally, all cell cultures used in this study were confirmed to be free from mycoplasma contamination.

Moringa oleifera leaves were sequentially extracted using ethanol, 50% ethanol, and distilled water. Among these, the aqueous extract characterized by its yellowish color and dry, flaky texture was chosen for nanoparticle synthesis due to its high solubility and rich bioactive content. For the synthesis of MO-AgNPs, the water-soluble extract was mixed with a 169.88 μg/mL AgNO₃ solution. The mixture was incubated in a water bath at 60˚C with continuous agitation for 8 h. A visible color change from clear yellow to dark brown indicated the successful reduction of Ag⁺ ions and formation of silver nanoparticles.

The successful synthesis of MO-AgNPs was confirmed using UV-Vis spectroscopy, DLS, and zeta potential analysis. UV-Vis spectroscopy showed a characteristic absorption peak at approximately 430 nm, indicating the formation of silver nanoparticles. The UV-Vis spectroscopy results are provided in Figure S1. In our previous study, we observed that DLS analysis revealed an average particle size of 57.88 nm and a polydispersity index (PDI) of 0.475, indicating moderate size uniformity. The zeta potential value was −15.9 mV, suggesting moderate colloidal stability and reduced nanoparticle aggregation. These results confirm that the green synthesis method successfully produced stable and well-dispersed nanoparticles suitable for biological applications [7].

The anti-invasive effect of MO-AgNPs on Ea.hy926 was assessed using concentrations of 12, 6, 3, and 1.5 μg/mL over a 24 h period. Quercetin (15.11 μg/mL) served as the positive control, while a 0.05% DMSO:0.05% deionized water solution was used as the negative control. The results revealed a significant reduction in the number of cells invading the Matrigel^® matrix in all MO-AgNP-treated groups compared to the untreated control (Figures 1 and 2). The number of invaded cells per mm² was 34.67 ± 1.25, 36.33 ± 1.25, 45.33 ± 1.25, and 57.33 ± 1.25 for the 12, 6, 3, and 1.5 μg/mL MO-AgNP treatments, respectively. In comparison, quercetin treatment resulted in 39.67 ± 1.25 cells/mm², while the negative control showed 91.67 ± 2.05 cells/mm². The calculated invasion inhibition rates for MO-AgNPs were 62.10%, 60.40%, 50.50%, and 37.50% at 12, 6, 3, and 1.5 μg/mL, respectively. Notably, the 12 μg/mL MO-AgNP treatment demonstrated a comparable inhibitory effect (62.10%) to that of quercetin (56.70%) (Figure 3).

The ability of Ea.hy926 to form tube-like structures, an essential step in angiogenesis, was evaluated in the presence of MO-AgNPs. Treatment with MO-AgNPs at concentrations of 12 and 6 μg/mL resulted in nearly 100% inhibition of tube formation compared to the negative control. These inhibitory effects were also comparable to those observed with the positive control, quercetin (15.11 μg/mL). According to Almatroodi et al. [20], quercetin at low concentrations (15.11 μg/mL) exhibits significant antiangiogenic activity, supporting its suitability as a positive control in this study.

Images were captured after 2.5 h of incubation. No closed tubular structures were observed in the MO-AgNP-treated groups during this period. Interestingly, untreated ECs aggregated and formed a prominent tube-like structure by 2.5 h (Figure 4). In contrast, MO-AgNP-treated cells showed complete inhibition of tube formation at all tested concentrations.

The ex vivo rat aortic ring assay was conducted to assess the anti-angiogenic properties of MO-AgNPs. At a concentration of 12 μg/mL, MO-AgNPs exhibited the highest level of neovascularization inhibition, achieving 83.824 ± 0.081%, which surpassed the inhibition observed with the positive control, suramin (100 μg/mL), at 70.927 ± 0.703% (Figure 5). The inhibition rates for MO-AgNPs at concentrations of 6, 3, and 1.5 μg/mL were 72.065 ± 0.657%, 23.321 ± 0.563%, and 13.599 ± 0.755%, respectively (Table 1, Figure 6). Statistical analysis revealed that MO-AgNPs treatments at 12 and 6 μg/mL significantly inhibited neovascularization compared to the negative control (P-value < 0.01). These findings demonstrate that MO-AgNPs exert strong anti-angiogenic effects in a dose-dependent manner, highlighting their potential as therapeutic agents for angiogenesis-related diseases.

To address the need for a more complex analysis of tumor endothelial interactions, a multicellular 3D spheroid model was employed. This method simulates the physical interaction between ECs and tumor cells. In this study, the effect of MO-AgNPs at concentrations of 1.5, 3, 6, and 12 μg/mL on spheroid growth was evaluated over 14 days. Comparisons were made with a positive control (quercetin at 15.11 μg/mL) and a negative control (0.05% DMSO and 0.05% deionized water). As shown in Figure 7, spheroids were observed under an inverted microscope at 40× magnification. By day 14, a significant reduction in spheroid size was recorded in the groups treated with 6 μg/mL, 12 μg/mL MO-AgNPs, and 15.11 μg/mL quercetin (Figure 8). In contrast, the treatments at 3 and 1.5 μg/mL did not result in significant changes at any time point. Table 2 shows that the spheroid size on day 14 was reduced to 17 ± 0.158% with MO-AgNPs (12 μg/mL) and 14 ± 0.417% with quercetin. The corresponding growth inhibition rates were 83 ± 0.16% and 86 ± 0.42%, respectively (Figure 9). These findings highlight the strong inhibitory effect of MO-AgNPs at higher concentrations on tumor spheroid expansion in co-culture models.