For the antibacterial studies, Gram-negative bacteria were selected as the target pathogens. Specifically, Shigella flexneri (ATCC12022) (Philpott et al., 2000), Salmonella choleraesuis (ATCC10708) (Chiu et al., 2004), and Escherichia coli (ATCC352118) (Kaper et al., 2004) were chosen as representative human pathogens. These bacteria were cultured in Nutrient broth (NB) at 37°C ± 2°C for an overnight incubation period (Rather et al., 2023). Subsequently, during the exponential growth phase, the bacterial cells were harvested. To prepare the cells for further antibacterial analysis, they were washed with PBS and suspended in the same buffer. The concentration of the bacterial suspension was adjusted to 1 × 10⁶ CFU/mL, ensuring a standardized inoculum size for the subsequent antibacterial assays. This standardized approach enabled accurate and consistent assessment of the antibacterial activity of the Zinc Chromium vanadate nanoparticles against these specific Gram-negative bacteria.

The broth dilution technique was used to evaluate the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the synthesized nanomaterial VCrZnO4 using the same method described elsewhere (Wijesundara et al., 2021; Rather et al., 2022a). Samples were sonicated and diluted with Mueller Hinton broth (MHB) to obtain homogenized broth suspension at 16, 8, 4, and 2 mg/mL. The adjusted inoculum was added to the prepared broth solution at 1 × 10⁶ CFU/mL. Bacteria were incubated at 37°C ± 2 °C for 18–20 h. Control was taken as untreated bacteria under the same conditions. After incubation, antibacterial activity was determined by obtaining the MIC and MBC. Briefly, the aliquot of incubated bacterial suspension was streaked onto freshly prepared MHA plates, which were then incubated at 37°C overnight. Then, the plates were observed, and the MIC was taken as the concentration of the nanomaterial at which the 70%–90% growth is inhibited, while the MBC was taken as the concentration at which no cell or less than 3 cells were found (Rehman et al., 2019).

Morphological changes in bacteria treated with VCrZnO4 were studied using scanning electron microscopy (SEM) (Rather et al., 2023). The representative concentration obtained as MIC was used to treat S. flexneri, S. cholerasuis, and E. coli, while untreated bacteria were used as a control. The cells were washed multiple times in PBS and then centrifuged to remove the residual medium. After that, the cells were fixed with 2.5% glutaraldehyde for 6 h, followed by fixation with 1% osmium tetroxide for 1 h. Next, the cells were dehydrated using varying concentrations of ethanol solution and again washed and suspended in PBS. Finally, the cells were placed on a stub and dried in a desiccator to complete the drying process. After gold coating, the samples were analyzed under SEM at 20 kV using a scanning electron microscope (Rehman et al., 2020).

Human colorectal cancer cells were obtained from American Type Cell Culture ATCC, United States, and were used to study the viability of cancer cells mediated by nanoparticles VCrZnO4 (Alzahrani et al., 2022; Rather et al., 2022b). HEK-293 cells were also used as a control to ensure the effectiveness of the study. First, 96 well plates containing special DMEM media were seeded in a CO2 incubator and kept for 24 h. To conduct the MTT assay, the nanocomposites VCrZnO4 were added to each well containing HCT-116, HELA, and HEK-293 cells in concentrations ranging from 2.0 μg/mL to 75 μg/mL for 48 h. The control wells were not treated with nanoparticles VCrZnO4. After treatment, the cells were exposed to MTT (5.0 mg/mL) for 4 h, and the media was replaced with DMSO. The optical density (OD) of the cells was then measured at 570 nm using a Plate Reader supplied by Bio-Tek Instruments, United States. The percentage of cell viability was calculated based on OD, and the data obtained from triplicates were statistically evaluated using GraphPad Version 6.0 Prism Software United States. Mean ± standard deviation values were presented in the graph (Khan et al., 2018).

To examine the impact of nanocomposite VCrZnO4 on the nucleus of cancer HCT-116 and HELA cells, we stained cells with DAPI staining post-48-h treatment of nanoparticles VCrZnO4 with 60 μg/mL. In brief, cells were treated with paraformaldehyde and labeled with DAPI dye. The blue, fluorescent staining was examined under a Confocal Scanning Microscope (Zeiss, Germany) (Zeiss, Germany) (Khan et al., 2018).

Determination of the antibacterial activity of Zinc Chromium vanadate VCrZnO4 through the broth dilution method obtained the growth inhibition of all the bacteria tested. The antibacterial activities were examined using three gram-negative bacteria (S. flexneri, S. cholerasuis, and E. coli). Bacteria were treated with different concentrations of VCrZnO4. It was observed that with increasing nanomaterial concentration, the MIC/MBC was reduced, indicating the effect of nanomaterial on the tested bacteria. As shown in Figure 6, the MIC/MBC for S. flexneri, S. cholerasuis, and E. col was 1/2, 2/4, and 16/16 mg/mL, respectively. This indicated variation in the inhibitory effect. The presented results summarize the effect on bacteria growth when treated with VCrZnO4 compared to the untreated bacteria grown under the same conditions on the gram-negative bacteria. However, E. coli had a higher MBC and MBC than S. flexneri and S. cholerasuis. The nanomaterial overall showed the inhibition of growth with all three gram-negative bacteria, mostly water-dwelling organisms responsible for many enteric infections.

The remarkable versatility of nanoparticles (NPs) as antibacterial agents cannot be overstated. These tiny warriors have been shown to combat a wide range of Gram-positive and Gram-negative bacteria with unparalleled efficiency. For example, previously, ZnO NPs have been observed wielding their antimicrobial prowess against Staphylococcus aureus. At the same time, the valiant Ag NPs demonstrate a concentration-dependent capacity to vanquish foes such as Escherichia coli and Pseudomonas aeruginosa (Ramalingam et al., 2016). The remarkable antimicrobial properties of these microscopic wonders are a testament to modern science and technology.

Several studies have assessed the antibacterial properties of ZnO-Nps using the broth dilution method against a range of bacterial strains, including Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae (Yamamoto, 2001; Raghupathi et al., 2011; Sirelkhatim et al., 2015; Ramalingam et al., 2016; Fakhroueian et al., 2019). The observed results indicate that direct contact of ZnO-NPs with cell walls leads to the destruction of bacterial cell integrity (Adams et al., 2006; Brayner et al., 2006; Zhang et al., 2007; Shu et al., 2017), release of antimicrobial ions primarily Zn2+ ions (Kasemets et al., 2009; Li et al., 2011), and the formation of ROS (Sawai et al., 1998; Zhang et al., 2008; Jalal et al., 2010). The antibacterial activity of nanoparticles has been attributed to their unique structural and chemical properties, including its high surface area, porous structure, and metal ions that can interact with bacterial cell membranes and disrupt their function. The exact mechanism of the antibacterial action of nanoparticles is still under investigation. However, these findings suggest these nanoparticles have potential as a new antibacterial agent and could be further explored for their applications in medicine and healthcare.

The concentration chosen for this study was the MIC concentration of the nanomaterial for each strain. With the aid of a control experiment, i.e., untreated bacteria, the treated S. flexneri, S. cholerasuis, and E. coli showed the most abnormal appearance, as seen in Figure 7. The control cells displayed normal shape with intact cell surfaces (Figure 7A.). The bacteria displayed deformations ranging from mild to severe after being treated with Zinc Chromium vanadate VCrZnO4. Observations made in Figure 7B exhibit a remarkable distortion of S. flexneri cells, including severe disruption of the cell membrane. S. cholerasuis and E. coli also displayed comparable aberrations, where the once regular rod-shaped morphology was significantly compromised, manifesting in irregular shape and size, coupled with marked damage to the cellular surface. A possible mechanism for inhibiting bacterial proliferation is the attachment of nanomaterial to the bacterial surface after it encounters the nanomaterial (Shu et al., 2017). By attaching nanomaterial to the surface of the bacterial cell, membrane integrity is compromised, ultimately leading to cell death. Reactive oxygen species (ROS) generated by nanomaterial attachment and bacterial cells are also considered crucial in bacterial cell morphogenesis.

Several studies have shown that when bacteria are treated with zinc oxide nanoparticles, it can cause damage to their cell walls and membranes, leading to changes in their morphology and, ultimately, cell death. For instance, a study by Li et al. (2010) investigated the effect of zinc oxide nanoparticles on the growth of Escherichia coli bacteria (Li et al., 2011). The researchers found that even at low concentrations, zinc oxide nanoparticles could inhibit the growth of the bacteria, and at higher concentrations, it caused significant damage to the bacterial cells, including changes in their shape and size. While Zinc Chromium vanadate s has shown promising results as an antimicrobial agent, it is important to note that its use may negatively affect other organisms, including beneficial bacteria and humans. The Zinc Chromium vanadate nanoparticles could be toxic to certain types of beneficial bacteria found in the gut, potentially leading to disruptions in the microbiome and associated health problems (Yoo et al., 2021). Therefore, while Zinc Chromium vanadate shows promise as an antimicrobial agent, further research is needed to fully understand its potential benefits and risks, as well as its mechanisms of action and potential applications in medicine and industry.

The impact of nanoparticles VCrZnO₄ on HCT-116 and HELA cells was examined. Post 48 h; we found a significant decrease in cancer cells post-treatment of nanoparticles VCrZnO4 in both HCT-116 and HELA cells (Figure 8 ). While the treatment of nanoparticles VCrZnO showed better and stronger inhibitory action on HELA cells than on HCT-116 cells (Figure 8). The treatment of nanoparticles VCrZnO4 on HEK-293 cells showed a decline in the normal cell population. However, the treatment of nanoparticles produced less cytotoxicity on normal HEK-293 cells than on HCT-116 and HeLa cells (Figure 8).

Our results suggest that VCrZnO4 nanoparticles exhibit significant cytotoxicity against HCT-116 and HELA cells. Many studies have shown that nanomaterials and biomaterials produce anticancer activities. For example, ZnO nanoparticles showed a dynamic cytotoxic effect in cervical carcinoma cells, and the impact of ZnO nanoparticles on cancer cells was due to the induction of apoptosis through increased intracellular ROS levels (Pandurangan et al., 2016). In another study, the effect of ZnO nanoparticles was studied against small-cell lung cancer where ZnO nanoparticles induced cancer cell death by activating reactive oxygen species and DNA leakage from nuclei and also observed the upregulation of apoptotic gene expression (Tanino et al., 2020). In another study, thymoquinone-conjugated ZnO nanoparticles mediated cytotoxicity and anticancer action on breast cancer cells (Banupriya et al., 2022). ZnO nanoparticles conjugated with N-succinyl chitosan showed strong anticancer activities on breast cancer cells (MDA-MB-231) by inducing apoptotic action (Ghaffari et al., 2020). The treatment of ZnO nanoparticles containing Oxaliplatin via polymerization showed strong anticancer action on colorectal cancer cells, as examined in both in vitro studies and in vivo mouse models (Alavi et al., 2023). In addition, the treatment of pure ZnO nanoparticles and doped with molybdenum and graphene oxide nanoparticles showed anticancer activity on the human colon (HCT116) and breast (MCF7) cancer cells, and anticancer action was mediated through reactive oxygen species, p53, and the caspase-3 pathway (Ahamed et al., 2022). Applying chromium oxide (CrO) nanoparticles also exhibited anticancer activity on MCF-7 cells, HepG2, and HUH-7 cancer cells (Jermy et al., 2019; Li et al., 2020). Besides, there have been numerous other studies that have shown that both nanomaterials and biomaterials can produce anticancer activities. These materials can be used in various ways to help prevent, diagnose, and treat cancer, and they have shown promising results in preclinical and clinical studies (Connor et al., 2005). Researchers used gold nanoparticles to target and destroy tumor cells in mice (Peng et al., 2019). The result underpins that the nanoparticles could deliver a drug directly to the tumor cells, significantly reducing tumor growth and increasing survival rates in the mice (Peng et al., 2019). The use of hydrogel nanoparticles could be used to deliver a drug directly to the tumor cells, resulting in significantly reduced tumor growth and increased survival rates in mice (Biondi et al., 2015; Nolan et al., 2018; Rafieian et al., 2019; Jiang et al., 2020; Nadtoka et al., 2020; Stojkovska et al., 2020; Clasky et al., 2021; Sulaiman et al., 2021; Nunes et al., 2022).

In order to better understand the cause of cancer cell death, we examined both HCT-116 and HELA cells using DAPI (4′,6-diamidino-2-phenylindole), a blue-fluorescent DNA stain used to identify apoptosis. Figures 9A, B depict that administering VCrZnO4 NPs decreases HCT-116 colon cancer cell death. Similarly; Figures 9B, D displayed a reduction in cell death for HELA cervical cancer cells. Notably, post-treatment with VCrZnO4 nanoparticles, we observed chromatin condensation and the presence of apoptotic bodies in HCT-166 cells (Figures 9B,D). In contract the nuclei in the control group exhibited no discernible structural or aesthetic changes (Figures 9A,C).

In Figure 9B, VCrZnO4 nanoparticles are shown in response to colon cancer cells (HCT-116), with Figure 9A representing the control cells with normal morphology and intact and healthy cells; and 8b representing the responses after 48 h of treatment (60 ug/mL). Figures 9D–F show the impact of nanoparticles VCrZnO4 on HELA cancer cells, with Figure 9B depicting the control cells with normal morphology and intact structure. Arrows in the figures highlight observed phenomena such as chromatin condensation, nuclear augmentation, and the formation of apoptotic bodies. In addition, IC₅₀ values have also been calculated and are shown in Table 1.

Of note, nanoparticles have also shown promising potential in inducing apoptotic death of cancer cells. Apoptosis induction in cancer cells is a promising approach for cancer therapy as it selectively targets and eliminates cancer cells while sparing normal cells. Many studies suggest nanocomposites as a promising approach to kill cancer cells. Gold nanoparticles decorated with polypyrrole induced apoptotic death in breast cancer cells. The study investigated the potential use of polypyrrole-coated gold nanoparticles as a photothermal therapy for breast cancer cells (Sun et al., 2018). The researchers found that the nanoparticles could selectively target breast cancer cells and induce apoptotic cell death upon exposure to near-infrared laser irradiation. The study suggested that polypyrrole-coated gold nanoparticles could be a promising and effective therapy for breast cancer. Further, zinc oxide nanoparticles also induced apoptotic death in prostate cancer cells by activating the caspase pathway. In addition, silver nanoparticles coated with graphene oxide or reduced graphene oxide could be a potential therapeutic agent for lung cancer (Yadav et al., 2022). These results have shown promising potential in inducing apoptosis of cancer cells, which could be a selective and practical approach for cancer therapy. Further research is needed to evaluate their safety and efficacy in clinical settings.