Advancing colorectal cancer therapy with biosynthesized cobalt oxide nanoparticles: a study on their antioxidant, antibacterial, and anticancer efficacy

Background: Colorectal cancer (CRC) ranks as the third most common cancer globally and the second leading cause of cancer-related mortality. Traditional chemotherapy, while effective, often results in significant side effects, highlighting the need for more efficient cancer therapies. Recent advancements in nanotechnology have led to the development of strategies that aim to minimize toxicity to normal cells by more precise targeting of cancer cells. In this context, cobalt oxide nanoparticles (Co 3 O 4 NPs) have shown promising anticancer potential. Our study focuses on evaluating the anti-oxidant, antibacterial, and anticancer properties of Co 3 O 4 NPs synthesized using Vibrio sp. VLC, a bioluminescent bacterium. Results: XRD and FTIR analyses confirmed the successful synthesis of Co 3 O 4 NPs, which displayed spherical morphology with an average diameter of 60 nm. The nano-particles demonstrated significant antioxidant and antibacterial activities. The MTT assay indicated that the NPs caused dose-and time-dependent toxicity against CT26 cells, while exhibiting relatively lower toxicity towards normal cells. In vivo experiments further confirmed the significant tumor suppressive effects in BALB/c mice, with minimal side effects on the liver, spleen, and kidney tissues compared to the widespread toxicity of cisplatin. Conclusion: This study verifies the successful synthesis of Co 3 O 4 NPs and their potent antioxidant, antibacterial, and anticancer activities. The biosynthesized Co 3 O 4 NPs represent a promising targeted method for CRC therapy. However, further research is needed to elucidate their mechanism of action and also their application in the clinical phase.


Background
Colorectal cancer (CRC) remains a significant global health challenge due to its high incidence and mortality rates.Despite advancements in the traditional treatment modalities, such as chemotherapy and radiation therapy, the quest for more effective and less toxic therapeutic options persists.In this context, nanotechnology emerges as a revolutionary approach, offering promising strategies for CRC treatment.Nanoparticles, due to their unique physicochemical properties, provide a versatile platform for cancer therapy, enabling targeted drug delivery, reduced systemic toxicity, and enhanced therapeutic efficacy.Nanoparticles can be engineered to selectively target cancer cells, facilitating the delivery of therapeutic agents and enabling image-guided treatments (Bhatia et al. 2022).Recent studies have highlighted the significant potential of nanoparticles in CRC treatment; for instance, the targeted delivery of chemotherapeutic agents using nanoparticles has been shown to increase drug accumulation in tumor cells, thereby enhancing antitumor activity while reducing the side effects (Huang et al. 2021).Additionally, nanoparticles have been employed as carriers for genes and RNA molecules, offering a novel method to gene therapy for CRC (Abrishami et al. 2024b;Lombardo et al. 2019).
The application of metal oxide nanoparticles, including Co 3 O 4 NPs, has received considerable attention in cancer research due to their unique properties, such as magnetic responsiveness and photocatalytic activity, which can be exploited for therapeutic purposes (Iravani and Varma 2020).Cobalt salt exhibits strong cytotoxic effects on various cell types, but when transformed into cobalt oxide nanoparticles, it demonstrates selective toxicity towards cancer cells.These nanoparticles hold immense potential for diverse biological applications owing to their small size, large surface area, and high reactivity (Huang et al. 2021;Iravani and Varma 2020;Yu et al. 2022).A variety of methods, including physical, chemical, and biological approaches, have been employed to synthesize Co 3 O 4 NPs.Recent advancements in synthesis techniques have expanded the potential applications of cobalt oxide nanoparticles, particularly in cancer therapy (Huang et al. 2021;Kalam et al. 2012).While there has been limited research on the utilization of biological materials such as bacteria, fungi, and plant extracts for the synthesis of cobalt oxide nanoparticles, it is essential to consider the desired size, shape, and surface chemistry of the nanoparticles when selecting the appropriate technique (Huang et al. 2021).Co 3 O 4 NPs have shown great promise in cancer treatment due to their ability to induce oxidative stress in cancer cells, leading to cell death through apoptosis or necrosis (Shete et al. 2022).These nanoparticles have demonstrated high toxicity towards cancer cells while maintaining a reasonable level of safety for normal cells (Das and Saikia 2023).Furthermore, the incorporation of targeting ligands into Co 3 O 4 NPs can enhance their selectivity towards cancerous cells (Lombardo et al. 2019).Additionally, Co 3 O 4 NPs have been utilized in combination with chemotherapy drugs to enhance drug delivery and therapeutic efficacy (Huang et al. 2021).
The biosynthesis of nanoparticles presents an eco-friendly and cost-effective alternative to conventional synthesis methods, leveraging biological entities like bacteria, fungi, and plant extracts.This green synthesis method not only aligns with the principles of sustainability but also enhances the biocompatibility and therapeutic efficacy of nanoparticles (Das and Saikia 2023).
The use of bacteria in synthesizing nanoparticles, known as biological synthesis, offers several advantages.Firstly, bacteria play a crucial role in reducing metal ions, facilitating the synthesis process.Additionally, they act as capping agents, by controlling the size and shape of nanoparticles, which leads to the enhancement of their stability.Moreover, biological nanoparticles often exhibit excellent biocompatibility due to their organic coating, which make them suitable for biomedical applications.Their biological origin also contributes to their eco-friendliness.Overall, leveraging bacteria in nanoparticle synthesis not only improves efficiency but also enhances the biocompatibility and stability of the resulting nanoparticles (Huang et al. 2021;Iravani and Varma 2020;Jang et al. 2015).
Moreover, recent advancements in nanotechnology have underscored the significance of plant materials not only in the synthesis and stabilization of metallic nanoparticles but also in their application for cancer therapeutics.Plant extracts, rich in phytochemicals, offer a green and efficient method for the synthesis of nanoparticles, acting as both reducing and capping agents that confer stability and biocompatibility to the nanoparticles (Kavaz et al. 2018(Kavaz et al. , 2019;;Umar et al. 2023aUmar et al. , 2023b)).
To date, there have been limited studies on the environmentally friendly synthesis of Co 3 O 4 NPs using bacteria alone, with only a few reports available worldwide (Huang et al. 2021).Furthermore, no study has investigated the potential anticancer effects of biologically synthesized Co 3 O 4 NPs on CT26 CRC cells in both in vitro and in vivo settings.In this study, Co 3 O 4 NPs were biologically produced using Vibrio luminous bacteria.After characterizing their properties, the nanoparticles were assessed for their antioxidant and antibacterial activities as well as their cytotoxic effects on CT26 mouse CRC cells both in vitro and in vivo.

Bacteria and cell culture
Nanoparticles were synthesized using the luminescent bacterium Vibrio sp.VLC, which was cultured in sea water complex (SWC) medium for this study.
The CT26 cell line, derived from mouse colorectal cancer, and the NIH/3T3 cell line, derived from mouse embryonic fibroblasts, were obtained from Ferdowsi University of Mashhad (FUM).The CT26 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), while the NIH/3T3 cells were cultured in Dulbecco's modified Eagle's medium-high glucose (DMEM-HG).Both cell lines were maintained in a humidified environment with 5% CO 2 at 37 °C.All experimental protocols involving the use of CT26 and NIH/3T3 cell lines were approved by the institutional review board and the ethics committee of FUM, under the approval number IR.UM.REC.1401.01.

Animals
Male BALB/c mice, aged 6 to 8 weeks, were bred and housed in the animal facility at the university.The animals were maintained in a climate-controlled environment with a 12 h light/dark cycle, a temperature of approximately 25 °C, and a humidity level of around 55%.The animal studies were approved by the ethics committee at FUM.

Synthesis of cobalt oxide nanoparticles
For the biological synthesis of cobalt oxide nanoparticles, a bacterial lysis solution of Vibrio sp.VLC was prepared.The bacteria were cultured in a liquid SWC medium and allowed to grow for 48 h.The resulting culture was centrifuged at 5000g for 15 min at 4 °C to separate the cells and eliminate the bacterial residues.The obtained sediment was washed with distilled water and subsequently homogenized in an additional 150 ml distilled water, and incubated at room temperature for 24 h.Subsequently, the suspension was centrifuged at 15 °C for 20 min to separate the supernatant, which was utilized for nanoparticle production.In this experiment, 100 ml bacterial cell lysis extract was mixed with 50 ml Co(NO 3 ) 2 .6H 2 O (a ratio of 2:1 and 0.15 mol/l), and the mixture was then incubated in a warm water bath for 90 min.
The suspension containing the purple precipitate was subjected to centrifugation at 16,500g for 10 min at 4 °C to initiate the separation and purification processes.In order to clean the sediment, it was washed with ethanol and distilled water.Subsequently, the precipitate was subjected to overnight heating at 60 °C, followed by annealing at 500 and 600 °C for 2 h.These steps were conducted to enhance the purity and crystalline structure of the nanoparticles (Abdurakhmonov et al. 2022;Bechelany et al. 2010).
The yield of Co 3 O 4 NPs production was calculated by weighing the final nanoparticles (after annealing at 600 °C for 2 h) obtained from a known volume of the reaction mixture (Eq. 1) (Riddin et al. 2006).
The exact concentration of nanoparticles in the stock solution, prepared for biological activity assays, was determined by dissolving a known mass of nanoparticles after annealing at 600 °C for 2 h in a defined volume of solvent (DMEM-HG culture medium), resulting in a stock solution with a concentration expressed in mg/ml.The concentration was calculated using the Eq. 2.
Additionally, dynamic light scattering (DLS) and zeta potential analysis were utilized to investigate the size distribution and stability of the nanoparticles in suspension, further informing the effective concentration of nanoparticles in biological interactions (Kavitha et al. 2017;Cuello et al. 2020).The integration of these methodologies ensures a rigorous and reproducible method to nanoparticle synthesis, characterization, and application in biological systems.Through careful calculation of the yield and its concentration, this study may contribute valuable data to the field of nanomaterials research, offering insights into the scalability and biological efficacy of biosynthesized Co 3 O 4 nanoparticles.

XRD and FTIR
The crystalline structure and the effect of annealing on decreasing the impurity rate of Co 3 O 4 NPs were confirmed through high-resolution X-ray diffraction (XRD) and Fourier-transform infrared (FTIR) analysis.XRD was performed using a Cu Kα radiation source operating at 40 kV and 40 mA.The analysis was conducted on powders of Co 3 O 4 NPs, which were synthesized with a concentration of 0.15 mol/l, both before and after annealing at 500 and 600 °C.The crystal size was calculated using the Debye-Scherrer equation (Eq.3).
In the Debye-Scherrer equation, D represents the crystallite size, λ is the wavelength of the X-ray radiation, K is a constant (commonly taken as 0.89), θ is the Bragg angle of the peak, and β is the full width at half maximum (FWHM) after subtracting instrumental broadening (Yang et al. 2010;Stanjek and Häusler 2004).Moreover, the biosynthetic mechanism of Co 3 O 4 NPs was investigated using FTIR analysis to explore potential interactions between Co ions and biological molecules in both annealed and unheated (1) Yield(%) = ( Mass of heated nanoparticles(g) Total mass of reactants used(g) ) × 100 (2) Concentration(mg/ml) = Mass of nanoparticles(mg) Volume of solvent(ml) (3) samples.In this study, the biosynthesis method was successfully examined by analyzing the powder of nanoparticles before and after annealing at 600 °C (Thermo Nicolet AVA-TAR 370 FTIR, USA).

FE-SEM, EDX, and elemental mapping
In order to examine the size, surface structure, elements, and the percentage of cobalt relative to oxygen, the nanoparticle powder synthesized with a concentration of 0.15 mol/l and annealed at 600 °C was analyzed using a field emission-scanning electron microscope (FE-SEM), energy-dispersive X-ray spectroscopy (EDX), and elemental mapping.In order to prepare the sample, the Co 3 O 4 NPs powder was coated with a layer of gold, and then evaluated (FE-SEM ZEISS Sigma 300, Germany).The specific elemental compounds present in the sample and their relative abundance were identified using the EDX method.Additionally, elemental mapping was conducted on electron microscope images to determine the ratio of certain elements present in the samples (Scimeca et al. 2018).

TEM, HR-TEM, AFM, DLS, and zeta potential
The biosynthesized Co 3 O 4 NPs were characterized for their size, shape, and morphology using transmission electron microscopy (TEM), high resolution-transmission electron microscopy (HR-TEM), atomic force microscopy (AFM), and DLS techniques.To prepare the samples, Co 3 O 4 NPs powder was ultrasonicated in ethanol and then sprinkled over carbon-coated gold TEM grids and carbon-coated HR-TEM grids.The grids were allowed to dry prior to measurements.TEM imaging was conducted using an accelerating voltage of 100 kV (TEM Philips EM 208S, Netherlands), while HR-TEM imaging was performed at 200 kV (HR-TEM FEI TEC9G20, USA) (Chen et al. 2014).The AFM analysis was carried out on a stock solution of Co 3 O 4 NPs that were annealed at 600 °C and dispersed in DMEM-HG + FBS (1:1 ratio) (Bibi et al. 2017).Furthermore, DLS was employed to determine the average size of nanoparticles after annealing at 600 °C in different media, including RPMI 1640, DMEM-HG, and distilled water.In order to prepare the sample, Co 3 O 4 NPs powder was ultrasonicated in different media and then subjected to analysis (Cordouan 3 vasco, France) (Jang et al. 2015).The zeta potential of cobalt oxide nanoparticles at neutral pH in RPMI 1640 (+ FBS, ratio 1:1), DMEM-HG (+ FBS, 1:1 ratio), and distilled water was also investigated using the Zeta Compact Potential Analyzer device at the central laboratory of Ferdowsi University of Mashhad.

Preparation of cobalt oxide nanoparticle stock solutions
Co 3 O 4 NPs were weighed and suspended in the DMEM-HG + FBS (1:1 ratio), at a concentration of 10 mg/ml.The suspension was stirred continuously overnight to achieve a thorough mixing, followed by sonication for 30 min to ensure a homogeneous dispersion of the nanoparticles.The resulting stock solution can be stored in a dark place at 4 °C until use.Momen Eslamiehei et al. Cancer Nanotechnology (2024) 15:22

Antioxidant activity
In this study, the antioxidant properties of the nanoparticles were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical reduction method.DPPH is an unstable free radical that can be stabilized by accepting an electron (Brand-Williams et al. 1995;Zhu et al. 2011).The absorbance of the DPPH stock solution was measured at 517 nm.Methanol was added to the mixture until the absorbance value reached 0.98.As a positive control, vitamin C, a natural antioxidant, was used at concentrations ranging from 3.9 to 500 μg/ml.Co 3 O 4 NPs were prepared at a concentration of 1000 μg/ml, and a series of dilutions were made in a 96-well plate.Following this step, 500 μl of the mixture of ethanol and DPPH was added to each well.The plate was then incubated in the dark at room temperature for 30 min.After that, the absorbance at 517 nm was measured using a spectrophotometer (Unico, Germany), and the data were collected with three repetitions for each concentration.The antioxidant activity of different concentrations of nanoparticles was compared to that of vitamin C using Eq. 4.
In this equation, AC represents the absorption of the control, and AS represents the absorption of the sample (Ghadi et al. 2018).

Antibacterial activity
The antibacterial properties of the nanoparticles were assessed against both Gramnegative Escherichia coli (E.coli) and Gram-positive Staphylococcus aureus (S. aureus) bacteria.

Measuring the minimum growth inhibitory concentration (MIC)
To measure the minimum inhibitory concentration (MIC) of the nanoparticles, 100 μl Mueller Hinton Broth culture medium with a 2 × concentration was added to each well of the first column of a 96-well plate, while in the remaining wells, 100 μl Mueller Hinton Broth with 1 × concentration was added.100 μl of the nanoparticles, in a stock suspension with the concentration of 2000 μg/ml, were added to the wells of the first column.A dilution series of the nanoparticles was then prepared in subsequent rows of the culture plate, extending up to the 10th row.Following the preparation of pure cultures of bacteria in Mueller Hinton broth, half of McFarland's standard for each bacterium was prepared.Subsequently, 10 μl of each bacterial suspension was added to the respective wells of the 96-well plate.The culture plates were then incubated at 37 °C and 150 rpm for a period of 24 h.The MIC 50 of the nanoparticles was calculated using Eq. 5, as described in previous studies (Iravani and Varma 2020;Gheidari et al. 2020;Durán et al. 2016;Hosseini-Giv et al. 2021).
In Eq. 5, the variables are defined as follows: a represents the absorbance of the wells containing different concentrations of substances with bacteria; b represents the (4) DPPH radical scavenging activity(%) = (AC − AS) AC × 100 (5) absorbance of the wells containing nutrient broth culture medium (negative control); c represents the absorbance of different concentrations of substances without bacteria (blank); and d represents the absorbance of wells containing medium with bacteria (positive control) (Arsalan et al. 2020).The data were collected with three repetitions for each concentration.The inhibition graph was generated using GraphPad Prism 8 software, and the concentration at which half of the bacterial growth was inhibited was determined as the MIC 50 .

Cytotoxic studies
To assess the cytotoxic effects of Co 3 O 4 NPs synthesized using Vibrio sp.VLC bacterial extract, both in vitro and in vivo experiments were conducted.In vitro cytotoxicity studies were performed using the MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay, while tumor measurement and histopathological analyses were employed for in vivo evaluation.

MTT assay
The cytotoxic effects of the biosynthesized Co 3 O 4 NPs were assessed against CT26 CRC cells as well as NIH/3T3 normal cell line.The cancer cells were cultured in RPMI 1640 medium containing 10% FBS, while the normal cells were cultured in DMEM-HG supplemented with 10% FBS.The CT26 and NIH/3T3 cells were seeded in 96-well plates at densities of 6 × 10 3 and 1 × 10 4 cells/well, respectively.After incubating the cells for 24 h, they were treated with different concentrations of cisplatin (ranging from 3.125 to 100 μg/ml) and Co 3 O 4 NPs (ranging from 6.25 to 800 μg/ ml) for durations of 24, 48, and 72 h.Subsequently, the MTT assay was performed in the 96-well plates.The MTT solution, prepared at a concentration of 5 mg/ml in phosphatebuffered saline (PBS), was added to each well, and the plates were incubated for 3 h.The culture medium was then replaced with dimethyl sulfoxide (DMSO), and the samples were incubated for an additional 25 min.The formation of formazan crystals, indicative of viable cells, was detected using an ELISA reader at 545 nm wavelength (Stat Fax, USA) (Iranpour et al. 2021;Hosseini-Giv et al. 2021).

In vivo experiments
To induce colorectal tumors, a subcutaneous injection of 5 × 10 5 CT26 cells was administered into the right flank of male BALB/c mice (Sato et al. 2021;Gong et al. 2021).
Once the tumor reached a volume of approximately 150 mm 3 , the animals were subjected to intraperitoneal injection of PBS (as a control), cisplatin (at a concentration of 5 mg/kg), and Co 3 O 4 NPs (at concentrations of 10 and 20 mg/kg) once every three days for a total of 16 days (6 injections in total) (Chattopadhyay et al. 2014b(Chattopadhyay et al. , 2014a;;Arghiani et al. 2014).

Measurement of tumor volume
The volume of CT26 tumors in BALB/c mice was measured from day 1 to day 19 following the injection of cisplatin and Co 3 O 4 NPs.The tumor volumes were determined using Eq.6, which involved measuring the length and width of the tumors using a caliper (Mitutoyo, Tokyo, Japan): In this equation, A and B are the length (mm) and width (mm) of the tumor, respectively (Owyang et al. 2017;Chattopadhyay et al. 2014b;Hosseini-Giv et al. 2021).

Histopathological analysis
After the completion of the injection period, mice were euthanized using CO 2 , and their organs, including liver, kidney, spleen, and tumor, were dissected and fixed in a 10% paraformaldehyde solution for histological analysis.The tissues were then processed, embedded in paraffin blocks, and sectioned into thin slices of approximately 4 μm thickness, followed by hematoxylin and eosin (H&E) staining (Sigma-Aldrich, Germany).The stained tissue sections were examined under a microscope at a magnification of 400 × to observe histological features.Descriptions of the tumor type and pathological characteristics in all tissue samples were determined by a pathologist (Dr.Shaterzadeh, Mashhad, Iran).The extent of necrosis was assessed by investigating the necrotic areas in H&Estained tumor sections, following previously established methods (Wang et al. 2021;Gong et al. 2021).

Observable detection of Co 3 O 4 NPs
The purity of the bacteria was assessed by observing their luminescence in the SWC culture medium and confirming the formation of single colonies (Fig. 1a, b).As shown in Fig. 1c-f, the color of the synthesis reaction solution changed from clear to purple, indicating the successful synthesis of nanoparticles.In the control group, which was exposed only to lysed extracts of bacteria, no color change was observed.

XRD and FTIR
The XRD and FTIR spectra of the Co 3 O 4 NPs are shown in Fig. 2. The reflections in XRD spectra after heating are consistent with the spherical phase of Co 3 O 4 NPs, as indicated by JCPDS No. 76-1802.The absence of peaks corresponding to impurity phases, demonstrates the high purity of the final products, which were calcined at 600 °C for 2 h (Fig. 2a).The peaks observed in the XRD spectrum at 2θ values of 31.92 (220), 37.46 (311), 44.72 (400), and 65.11 (404) are in accordance with the data provided by the International Center for Diffraction Analysis.The presence of protein molecules in the bacterial lysis biomass led to a shift in certain peaks, while other crystallographic contaminants may be attributed to the biomass sources.The average size of the nanoparticles was estimated to be approximately 18 nm using the Debye-Scherer equation.Further, the biosynthesized Co 3 O 4 NPs, both before and after annealing at 600 °C, were characterized using FTIR spectra in the wave number ranges of 400-4000 cm −1 .The spectra revealed the presence of various functional groups within the biosynthesized Co 3 O 4 NPs obtained from the cellular extract of Vibrio sp.VLC bacteria.In Fig. 2b, sharp peaks at 3478 cm −1 , 2927 cm −1 , 1648-1530 cm −1 , and 1067-1227-1397 cm −1 are indicative of hydroxyl groups, amino groups, amide I and II groups, and C-H 2 bonds, (6) respectively.The effects of heating and annealing were observed to reduce the presence of biological impurities during the biosynthesis of Co 3 O 4 NPs, as evidenced by a decrease in the intensity of functional group peaks and noise in the annealed sample.The formation of Co 3 O 4 NPs is indicated by a peak at 567 cm −1 in Fig. 2b and peaks at 561 cm −1 and 605 cm −1 in Fig. 2c, which correspond to the characteristic vibrations of Co-O bonds.

FE-SEM, EDX, and elemental mapping
FE-SEM analysis was employed to examine the size and morphology of the biosynthesized Co 3 O 4 NPs.Additionally, the EDX analysis was conducted on the Co 3 O 4 NPs synthesized using Vibrio sp.VLC bacteria extract prior to the calcination process to assess the presence of biological compounds in the bacterial extract.According to FE-SEM micrographs, the Co 3 O 4 NPs exhibited a spherical shape, with an average size of 65-67 nm (Fig. 3a-c).The EDX analysis confirmed the presence of cobalt and oxygen

TEM, HR-TEM, AFM, DLS and zeta potential
To gain further insight into the particle size, shape, topography, surface, and average size of Co 3 O 4 NPs produced using Vibrio sp.VLC bacteria extract, TEM, HR-TEM, AFM, DLS and zeta potential analyses were conducted (Fig. 4).The TEM micrographs revealed that the nanoparticles had a spherical morphology with an estimated size ranging from 32 to 69 nm (Fig. 4a, b).In order to achieve more precise measurements of the nanoparticles, high-resolution electron microscope images were also captured.
The HR-TEM images indicated that the Co 3 O 4 NPs had an approximate size within the range of 19 to 41 nm, while maintaining a spherical structure (Fig. 4c-e).The AFM micrographs also indicated that the nanoparticles had a predominantly spherical shape.The images revealed variations in height, as observed by the gradual transition from light to dark regions.Based on this study, the average height reported for Co 3 O 4 NPs produced by the biological method was in the approximate range of 4.5 to 10 nm (Fig. 4f-i).Co 3 O 4 NPs exhibited average sizes of 65 nm, 54 nm, and 270 nm when suspended in RPMI 1640, DMEM-HG, and water, respectively, based on DLS analysis (Fig. 4j-l).The nanoparticles demonstrated a monodisperse distribution in RPMI 1640 and DMEM-HG culture media, as indicated by the low polydispersity index (PDI).The zeta potential of Co 3 O 4 NPs was measured in RPMI 1640, DMEM-HG, and distilled water, followed by visual examination of sedimentation rate after 24 h (Fig. 4m).The stability of cobalt oxide nanoparticles in these solvents, in combination with FBS, was also examined and the zeta potential results are mentioned in Fig. 4m.Accordingly, the zeta potential for nanoparticles increased in the presence of FBS.The stability of cobalt oxide nanoparticles in DMEM-HG + FBS (at a 1:1 ratio) had the highest level after 24 h.

Antioxidant and antibacterial activity
In this study, the biological applications of Co 3 O 4 NPs, including their antioxidant and antibacterial properties, were investigated.In order to evaluate the antioxidant properties, the ability of Co 3 O 4 NPs to inhibit the DPPH radical was examined across 8 different concentrations.For comparative purposes, vitamin C (VC), a natural and potent antioxidant, was also included in the experiment.The highest rate of DPPH radical scavenging was observed at the concentration of 500 µg/ml for both Co 3 O 4 NPs and vitamin C, resulting in approximately 51% and 78% inhibition, respectively.Furthermore, an increase in the concentrations of both nanoparticles and vitamin C led to a corresponding enhancement in DPPH inhibition (Fig. 5a).In addition, the inhibitory potential of Co 3 O 4 NPs at concentrations of 1000, 500, 250, 125, 62.5, 31.12,15.62, and 7.81 µg/ml against two pathogenic bacteria, namely E. coli and S. aureus, was investigated.Accordingly, the half-minimum inhibitory concentration (MIC 50 ) values for E. coli and S. aureus were determined to be 83 and 314 µg/ml, respectively (Fig. 5b).The percentage of bacterial growth inhibition was assessed across various nanoparticle concentrations as shown in Table 1.

Investigating the cytotoxicity of Co 3 O 4 NPs and cisplatin on CT26 and NIH/3T3 cell lines
The impact of nanoparticles on cell viability was assessed by the MTT assay.Cisplatin was used as a control to better evaluate the cytotoxicity of the nanoparticles.The cell viability measurements were performed at 24, 48, and 72 h post-treatment.The findings indicated significant inhibitory effects of both nanoparticles and cisplatin on the growth of cancer cells.The inhibition rate of CT26 cells showed a direct correlation with two factors: the concentration of the compounds and the duration of the treatment.The IC 50 values of Co 3 O 4 NPs at 24, 48, and 72 h were determined to be 301.8,56.1, and 13.1 µg/ml, respectively.Moreover, the IC 50 values of cisplatin were found to be 39.8, 8.3, and 4.6 µg/ml at the corresponding time points (Fig. 6a, b).
The cytotoxicity induced with Co 3 O 4 NPs and cisplatin on the NIH/3T3 cells was also investigated in this study.The IC 50 values of nanoparticles at 24, 48, and 72 h were 5356, 1498, and 291 µg/ml, respectively, whereas those for cisplatin were determined as 53.5, 10.1, and 5 µg/ml, respectively (Fig. 6c, d).

IC 50 comparison of Co 3 O 4 NPs on cancerous vs normal cells
IC 50 values of Co 3 O 4 NPs were investigated on both CT26 CRC cells and NIH/3T3 normal cells at 24, 48, and 72 h time intervals to assess the anti-cancer properties of the biosynthesized Co 3 O 4 NPs (Fig. 6e).At 24 h, the IC 50 value of nanoparticles was 301.8 µg/ml for CT26 cells and 5356 µg/ml for NIH/3T3 cells, indicating a significant difference in the cytotoxic impact of Co 3 O 4 NPs between cancerous and normal cells.After 48 h, the IC 50 values decreased to 56.1 µg/ml for CT26 cells and 1498 µg/ ml for NIH/3T3 cells, and after 72 h, further dropped to 13.1 µg/ml and 291 µg/ml, respectively.These results indicate cell specific toxicity of Co 3 O 4 NPs as they exhibited much lower inhibitory effects on the viability of NIH/3T3 cells.

Investigating the antitumor activity and the side effects of Co 3 O 4 NPs in vivo
In order to investigate the in vivo antitumor activity of Co 3 O 4 NPs synthesized through the biological method, BALB/c mice bearing CT26 tumors were intraperitoneally injected with 10 and 20 mg/kg Co 3 O 4 NPs dispersed in PBS at 3 day intervals.Throughout the 19 day treatment period, the tumor volume was monitored (Fig. 7a).Additionally, a comparison was made between the tumor volumes at the end of the treatment period (Fig. 7b).The results demonstrated that both doses of Co 3 O 4 NPs effectively suppressed tumor development.Notably, the administration of Co 3 O 4 NPs at a dose of 20 mg/kg exhibited a comparable effect on tumor volume to that of cisplatin medication.The comparison of tumor volumes at the end of the treatment period revealed a significant inhibition of tumor growth in the groups treated with different concentrations of nanoparticles and cisplatin.

Histopathology evaluation
Based on the investigation conducted on tumor tissue sections obtained from mice subjected to various component concentrations, it was observed that all tissue sections exhibited malignant tumor cells of varying sizes (Fig. 7c-f ).In the groups treated with cisplatin and 10 mg/kg of Co 3 O 4 NPs, a substantial presence of necrotic cells was observed.Additionally, the group treated with 20 mg/kg Co 3 O 4 NPs displayed tumor cells with disorder and different sizes associated with necrosis.In the PBS-treated group, these phenomena were not prominently observed, and the tissues exhibited cells with enlarged and irregular nuclei.Thus, it can be inferred that the tumor tissue displayed an active nature and, to a certain extent, remained unaltered.
The group treated with cisplatin exhibited a notable presence of necrosis and tissue death in liver cells, along with hepatic congestion.Conversely, in the groups treated with 10 mg/kg and 20 mg/kg Co 3 O 4 NPs, a minimal extent of necrosis and a minor degree of hepatic congestion were observed.In contrast, no tissue damage was observed in the group treated with PBS (Fig. 7g-j).
The histological examination of kidney tissue from mice with cisplatin revealed the presence of necrosis and structural alterations in the renal tubules.Additionally, the glomeruli displayed signs of contraction and compression, and the kidney tissue exhibited significant swelling.In contrast, while a few instances of renal congestion were observed in the groups treated with different doses of Co 3 O 4 NPs, none of these cases was evident in the PBS-treated group (Fig. 7k-o).
Based on the histological examination of spleen tissue from mice administered with the respective compounds, the group treated with cisplatin displayed notable structural changes and necrosis in both the white and red pulps of the spleen.In contrast, the groups treated with two different concentrations of Co 3 O 4 NPs exhibited only minor structural alterations in the white and red pulps of the spleen, while no damage was detected in the PBS-treated group.The administration of PBS demonstrated its expected non-toxicity, as assessed in the investigation of potential side effects on all the tissues (Fig. 7p-s).

Discussion
In the realm of specific cancer treatment, Co 3 O 4 NPs have garnered considerable attention among various other nanoparticles.The biocompatibility of nanoparticles holds significant importance in biological applications, underscoring the need to carefully select an appropriate preparation method for obtaining biocompatible Co 3 O 4 NPs.Presently, there is a limited body of research regarding the lethal concentrations of these nanoparticles on normal cells.While cobalt salts exhibit potent cytotoxic effects on all cell types, the utilization of Co 3 O 4 NPs renders them specifically toxic to cancer cells.Utilizing biological sources for the synthesis of Co 3 O 4 NPs has proved to be a safe, straightforward, and environmentally friendly approach (Caires et al. 2010;Huang et al. 2021;Iravani and Varma 2020;Hafeez et al. 2020;Govindasamy et al. 2022;Goswami et al. 2020).
In this study, a biological method utilizing the cell extract of Vibrio sp.VLC was employed for the synthesis of Co 3 O 4 NPs.Among the three tested concentrations of Co(NO 3 ) 2 •6H 2 O salt (0.10, 0.15, and 0.20 mol/l), the 0.20 mol/l concentration proved to be more favorable for nanoparticle synthesis.This concentration exhibited superior efficiency while ensuring a size below 100 nm.The addition of the salt to the bacterial lysate extract resulted in the formation of cloudy compounds and violet-colored sediments, confirming the successful synthesis of nanoparticles.Similar research employed a Gram-positive bacterium (Bacillus subtilis) to biochemically produce Co 3 O 4 NPs by introducing CoCl 2 •6H 2 O salt into the bacterial medium.The addition of NaBH 4 as a reducing agent led to the initial color change and formation of sediments, indicating the progress of the synthesis process (Kalam et al. 2012).Additionally, Arsalan et al. reported the successful synthesis of Co 3 O 4 NPs based on the color change observed when combining a pink cobalt chloride solution with a plant extract at concentrations of 0.10 and mol/l (Arsalan et al. 2020).As mentioned by Sanità et al., biocompatibility is an important factor in the development of biosynthesized inorganic nanoparticles for biomedical applications.Various parameters influence the biocompatibility of a material, including its composition, intended use, desired biological response, and the environments it will within the body.Surface modification strategies are crucial in enhancing the biocompatibility and cellular internalization of nanoparticles.Additionally, the introduction of pH-sensitive or heat-sensitive bonds has been explored to develop nanoplatforms for controlled drug release, specifically triggered by the acidity of tumor environment or external heat sources.The identification of Si, S, P, and Al in our environment of biosynthesized Co 3 O 4 NPs underscores the biological synthesis method and the importance of thorough characterization in understanding the composition and potential influences on the nanoparticles properties and biocompatibility (Sanità et al. 2020;Saravanan et al. 2020;Abrishami et al. 2024a;Kamil Mohammad Al-Mosawi et al. 2023).
As argued by Choi & Lee, microbial cells play a significant role in the biosynthesis of inorganic nanoparticles, offering a green and sustainable alternative to conventional chemical and physical synthesis methods.These biological entities utilize various metabolic and physiological processes to reduce metal ions to their nanoparticle forms.The ability of these microorganisms to bind and reduce metal ions is attributed to the presence of specific proteins, enzymes, or biochemical pathways that facilitate the reduction process.These biosynthesized nanoparticles are of great interest for environmental remediation due to their enhanced reactivity, surface area, and the possibility of functionalization to target specific pollutants like heavy metals or organic contaminants.The biosynthetic method not only offers a more environmentally friendly approach of producing nanoparticles but also introduces the potential for creating novel nanoparticles with unique properties that might be difficult to achieve through other synthesis methods (Choi and Lee 2020;Saravanan et al. 2020).
Recent advancements in the field have focused on the preparation of biocompatible inorganic nanoparticles, bioconjugation strategies for target-specific nanoprobes, and the development of sensitive tumor imaging for diagnosis.These efforts underscore the complexity of nanoparticle behavior in vivo, influenced by their physicochemical properties and the interactions at the nano-bio interface.Based on the results of in vivo research, the biosynthesized Co 3 O 4 NPs exhibited targeted damage to cancerous cells within tumor tissue, with the lowest level of damage observed in other tissues.The challenges ahead involve better understanding these interactions and designing nanoparticles that can safely and effectively be used in clinical settings (Jiao et al. 2018;Saravanan et al. 2020).

Characterization of biosynthesized Co 3 O 4 NPs
The XRD analysis revealed distinct 2θ values of 20. 05 (111), 31.19 (220), 36.56 (311), and 44.29 (400), which coincided with the JCPDS pattern No. 073-1701 for spherical Co 3 O 4 NPs.This agreement confirms the successful synthesis of Co 3 O 4 NPs.Notably, the absence of specific peaks in the XRD spectrum of nanoparticles indicates their amorphous nature and the presence of various impurities.Conversely, the sharp peaks observed in the XRD spectrum of the sample annealed at 600 °C, compared to the weaker peaks for the sample heated at 500 °C, indicated a reduction in impurities and the crystallization of the nanoparticles.Previous studies also examined XRD results to confirm the production and assessed the crystallization of Co 3 O 4 NPs by evaluating the similarity and absence of additional peaks when compared to registered cases (Yang et al. 2015;Das and Saikia 2023;Bechelany et al. 2010;Vijayanandan and Balakrishnan 2018;Bibi et al. 2017;Kumar et al. 2008).In order to confirm the successful synthesis of Co 3 O 4 NPs using the Vibrio extract, the FTIR spectra of both unheated and heated samples were compared with similar studies.The unheated sample exhibited prominent peaks in the wave number region associated with amino, amide, and hydrocarbon compounds.These peaks can be attributed to the presence of enzyme residues and proteins in the bacterial extract, which strongly supports the biological production of the nanoparticles (Vijayanandan and Balakrishnan 2018;Eid 2022;Farhadi et al. 2013;Naumann et al. 1991).
The EDX analysis revealed that the biologically synthesized sample had an elemental composition consisting of cobalt and oxygen, which accounted for more than 70% of the total composition.This high percentage signifies the high purity of the produced nanoparticles.Additionally, a small percentage of phosphorus, carbon, and sulfur elements was detected, indicating the presence of biological compounds during the production process.In a similar study conducted by Akhlaghi et al., green Co 3 O 4 NPs were successfully produced with an acceptable purity percentage of cobalt and oxygen elements, reaching up to approximately 60% of the total composition (Akhlaghi et al. 2020).
To examine the size and shape characteristics of cobalt oxide NPs, multiple imaging techniques were employed including TEM, HR-TEM, FE-SEM, AFM, and DLS.Analyzing the micrographs revealed that Co 3 O 4 NPs exhibited a spherical morphology, with sizes ranging from 20 to 70 nm (less than 100 nm).These findings are consistent with recent research, which also reported similar observations regarding the shape and size of Co 3 O 4 NPs (Rousta et al. 2019;Abbasi et al. 2020;Bibi et al. 2017;Ajarem et al. 2021;Mehrafza et al. 2022;Vijayanandan and Balakrishnan 2018;Jang et al. 2015).
The physical properties of Co 3 O 4 nanoparticles, specifically size, polydispersity index (PDI), and zeta potential, greatly influence their biological activities.The DLS analysis revealed that the nanoparticles exhibit a narrow size distribution, which is required for their interaction with biological systems, where smaller particles are known to penetrate cell membranes more efficiently, potentially enhancing their anticancer activity.Moreover, the zeta potential values obtained for these nanoparticles in various media indicate a stable colloidal system, which is vital for maintaining the dispersion of nanoparticles in biological environments.The negative zeta potential values, especially in the presence of FBS, suggest that the nanoparticles are likely to repel each other, preventing aggregation (AlSalhi et al. 2024).
The integration of DLS and zeta potential results into our biological findings allows us to hypothesize the mechanisms through which these characteristics enhance the effectiveness of Co 3 O 4 NPs.The optimized size and stable dispersion contribute to the nanoparticles ability to induce oxidative stress within cancer cells effectively, without causing significant harm to normal cells.This selective toxicity towards cancer cells can be attributed to the nanoparticles enhanced penetration and retention within the tumor tissues, a phenomenon often referred to as the enhanced permeability and retention (EPR) effect.Furthermore, the surface charge indicated by the zeta potential might influence the interaction of nanoparticles with cell membranes, potentially affecting the cellular uptake and the subsequent intracellular pathways activated by the nanoparticles.This could explain the observed variations in antioxidant, antibacterial, and anticancer activities, as the interaction dynamics between nanoparticles and cellular components are crucial determinants of their biological effects.DLS and zeta potential analyses conducted in this study revealed that our nanoparticles exhibit a highly uniform size distribution with average diameters of 65 nm in RPMI 1640, 54 nm in DMEM-HG,, and an unusually large size of 270 nm in distilled water, alongside stable zeta potential values indicative of colloidal stability in various media, which was enhanced by FBS (Kavitha et al. 2017).
Saravanan et al. discussed the impact of secondary metabolite conjugation on the biological activity of inorganic nanoparticles, focusing on modulating their effects for therapeutic use.Conjugating bioactive compounds from microorganisms, fungi, or plants with nanoparticles enhances their biological activity and specificity.This process can improve therapeutic potential, by increased solubility and bioavailability when attached to gold nanoparticles, enhancing their anti-cancer efficacy.Selecting appropriate natural resources for nanoparticle synthesis is crucial, especially for targeting specific therapeutic outcomes, allowing for the design of nanoparticles with enhanced activity, like those with anticancer properties.This method of integrating secondary metabolites with specific therapeutic profiles into nanoparticle design fosters the development of targeted and effective nanotherapeutics, leveraging natural compound benefits for targeted treatment while minimizing side effects (Saravanan et al. 2021).
Furthermore, biological methods for fabricating metal nanoparticles are increasingly favored over traditional physical and chemical techniques due to their alignment with green chemistry and sustainability principles.These methods are energy-efficient, often requiring only ambient conditions, and produce biocompatible nanoparticles without the need for harmful chemicals or extensive modifications.The biological methods not only avoid toxic by-products but also offer enhanced stability and dispersion of nanoparticles through natural capping agents.Furthermore, biological synthesis is cost-effective, scalable, and typically involves simpler, single-step processes, making it suitable for large-scale production.While acknowledging that each synthesis method has its pros and cons based on the application needs, the emphasis on sustainability, safety, and biocompatibility continues to drive the preference for biological approaches in nanoparticle synthesis (Samadian et al. 2020;Vahidi et al. 2021).

The influence of characteristic parameters on the biological interactions of nanomaterials
Samadian et al. emphasized the significant impact of various nanoparticle properties (such as shape, size, surface charge, and chemical composition) on their biological behavior and therapeutic potential.The size of nanoparticles not only determines their cellular uptake efficiency but also their distribution within the body, including the ability to cross the biological barriers such as the blood-brain barrier.The geometry of nanoparticles affects their internalization by cells and their subsequent intracellular fate.The shape also influences the ability of nanoparticles to navigate through the extracellular matrix and their interactions with cell membranes, potentially affecting their ability to reach target sites within the body.Nanoparticles in the range of 1-100 nm can take advantage of unique size-dependent properties not seen in their bulk material counterparts (Albanese et al. 2012;Samadian et al. 2020).
The dissolution and agglomeration behaviors of nanoparticles further dictate their bioavailability and therapeutic effectiveness.Faster-dissolving nanoparticles may release therapeutic ions more rapidly, leading to quicker biological responses.However, this can also mean a higher potential for toxicity if the released ions exceed safe concentration thresholds.The solubility of nanoparticles is influenced by factors such as pH, ionic strength of the solution, and the presence of biomolecules, which can either facilitate or inhibit dissolution.Agglomeration can significantly reduce the surface area available for interaction with biological molecules, potentially diminishing the reactivity and efficacy of nanoparticles.Moreover, larger agglomerates might be less efficiently taken up by cells and may be more readily cleared from the body, impacting the delivery efficiency of the therapeutic nanoparticles (Samadian et al. 2020;Godymchuk et al. 2022;Avramescu et al. 2022).Moreover, specific surface area and surface properties, including morphology, energy, coating, and charge, are crucial for enhancing interactions with biological systems, requiring meticulous design to enhance efficacy while minimizing adverse reactions (Samadian et al. 2020;Joudeh and Linke 2022).
As pointed out by Hu et al., the topography of the surface of a nanoparticle, including its roughness or smoothness, can also influence protein absorption patterns and cellular interactions.Rees et al. argue that coating nanoparticles with biocompatible materials (e.g., polyethylene glycol, dextran, or lipids) can significantly reduce unspecific protein adsorption, minimizing recognition by the immune system and prolonging circulation time in the bloodstream.These coatings can also be functionalized with targeting ligands or antibodies to achieve specific binding to target cells or tissues, improving the efficacy of drug delivery or imaging.This comprehensive understanding underscores the importance of tailoring nanoparticle properties to harness their full potential in medical applications (Hu et al. 2022;Rees et al. 2019;Samadian et al. 2020).

Antioxidant and antibacterial activity
The antioxidant activity of Co 3 O 4 NPs was investigated by assessing their ability to reduce the DPPH free radical.As a comparative reference, vitamin C, a potent natural antioxidant, was used as a positive control.At the highest concentration tested (500 µg/ ml), both Co 3 O 4 nanoparticles and vitamin C exhibited significant reductions in DPPH, with values of 51.24% and 77.54%, respectively.These findings indicate that the antioxidant activity strengthens with increasing the nanoparticle concentration.
Based on the results of the MIC test, the concentrations of Co 3 O 4 NPs utilized in this study exhibited antibacterial effects and inhibited the growth of both E. coli and S. aureus bacteria.Notably, the inhibition observed on E. coli was stronger compared to S. aureus.This difference in susceptibility could potentially be attributed to the variations in cell wall structures between Gram-negative and Gram-positive bacteria, with Gramnegative bacteria possessing relatively thinner cell walls (Moradpoor et al. 2019).Moreover, the unique properties of Co 3 O 4 NPs, such as their ability to generate and release oxygen radicals, can induce greater stress specifically on Gram-negative bacteria (Abbasi et al. 2020;Gupta et al. 2020;Azam et al. 2012).The MIC 50 values of Co 3 O 4 NPs synthesized using the biological method were determined to be 83 µg/ml for E. coli and 314 µg/ ml for S. aureus bacteria.Other studies have also demonstrated that the antioxidant and antibacterial properties of Co 3 O 4 nanoparticles enhance as their concentrations increase (Ajarem et al. 2021;Brand-Williams et al. 1995;Khalil et al. 2020;Khan et al. 2015;Hou et al. 2020).

In vitro and in vivo studies
The use of chemotherapy drugs, including cisplatin, has become a common approach for treating different types of cancers.However, studies have indicated that cisplatin can heighten the susceptibility of mitochondria to reactive oxygen species (ROS), resulting in oxidative damage to normal cells.Prolonged exposure to oxidative stress can lead to organ dysfunction, particularly affecting the kidney, liver, and spleen.Consequently, it is of paramount importance to enhance treatment efficacy and minimize the long-term side effects associated with the use of cisplatin in cancer therapy (Ertugrul et al. 2020;Mishra et al. 2017;Taléns-Visconti et al. 2022).
The cytotoxicity of Co 3 O 4 NPs was assessed by the MTT assay at 24, 48, and 72 h following the exposure of CT26 and NIH/3T3 cells to the nanoparticles.The cytotoxicity of Co 3 O 4 NPs on the NIH/3T3 cells exhibited an enhanced trend with increasing concentrations, but did not induce a significant toxicity.In line with previous research findings, these nanoparticles demonstrated lower toxicity towards normal cells.However, Co 3 O 4 NPs exerted distinct lethal effects on CT26 CRC cells.The results revealed an inverse relationship between CT26 cells viability and the duration and dosage of Co 3 O 4 nanoparticles.The IC 50 values of Co 3 O 4 nanoparticles on the CT26 cells were determined to be 301.8,56.1, and 13.1 µg/ml after 24, 48, and 72 h of exposure, respectively.This research underscores the potential cytotoxicity of Co 3 O 4 NPs towards CT26 CRC cells.By comparing the IC 50 values of Co 3 O 4 NPs on CT26 and NIH/3T3 cell lines, it can be inferred that these nanoparticles exhibited minimal toxicity towards normal cells at the comparable concentrations and exposure durations.Additionally, the IC 50 values of cisplatin further validated its high toxicity on both the CT26 CRC and NIH/3T3 normal cells.There were no significant changes in the survival percentages of the two cell lines after 48 and 72 h of cisplatin treatment.The selective toxicity of nanoparticles on CRC cells compared to normal cells was demonstrated by comparing the survival percentages of CT26 and NIH/3T3 cells treated with Co 3 O 4 NPs and cisplatin at the equivalent concentrations of both substances after 24, 48, and 72 h of treatment.Khan et al. conducted a similar study, and investigated the cytotoxicity of Co 3 O 4 NPs, synthesized using a semi-biological method, on human CRC cells (HT-29 and SWC620).
Their findings revealed that these nanoparticles exhibited a favorable cytotoxic profile compared to doxorubicin, and induced minimal cytotoxicity on normal cells.This highlights their potential as a toxic agent specifically targeting cancerous cells (Khan et al. 2015).Moreover, an examination of the effects of Co 3 O 4 NPs, produced using biological compounds, was performed on human leukemia cells.The study showed that these NPs exhibited a controlled toxic effect on cancerous cells, while maintaining a higher cell viability percentage in normal cells.These findings further support the notion of the selective toxicity of Co 3 O 4 NPs towards cancerous cells compared to normal cells (Chattopadhyay et al. 2014a).Co 3 O 4 NPs exert cell damage through two main mechanisms.Firstly, the presence of Co 2+ ions leads to DNA damage and fragmentation by binding to the nucleosome.Secondly, these nanoparticles play a crucial role in inducing apoptosis through the generation of ROS (Faisal et al. 2016;Bhatia et al. 2022;He et al. 2018;Tripathi et al. 2019;Iqbal et al. 2020;Chattopadhyay et al. 2014b;Ajarem et al. 2021).The reductive property of biological Co 3 O 4 NPs is more pronounced in an acidic environment compared to a neutral environment.These nanoparticles exhibit a remarkable ability to penetrate cancerous cells in large quantities, facilitated by the higher concentration of phospholipids in the membrane of cancerous cells.Consequently, this leads to a substantial release of Co 2+ ions and a heightened formation of ROS within cancerous cells, surpassing that observed in normal cells.This process increases the production of ROS and causes cell apoptosis by increasing the expression of pro-apoptotic proteins like P53, Caspase 3, and Caspase 8. Our research indicates that Co 3 O 4 NPs synthesized using Vibrio sp.VLC exhibit comparable high selective toxicity against cancerous cells, in alignment with findings from earlier studies on nanoparticles synthesized via biological methods (Iqbal et al. 2020;Huang et al. 2021;Ajarem et al. 2021;Khan et al. 2015;Chattopadhyay et al. 2014b).
Our study meticulously investigated the dose-dependent effects of Co 3 O 4 NPs on tumor growth and tissue morphology, revealing significant insights into their therapeutic potential.Administering Co 3 O 4 NPs at doses of 10 and 20 mg/kg to BALB/c mice bearing CT26 tumors led to a notable suppression of tumor development, illustrating a clear dose-dependent efficacy.Particularly, the 20 mg/kg dosage of Co 3 O 4 NPs demonstrated a tumor volume reduction comparable to that achieved with cisplatin treatment, a conventional chemotherapy drug, underscoring the potent anticancer properties of Co 3 O 4 NPs.Histopathological evaluation further substantiated these findings, as tissue sections from treated mice showcased varying degrees of malignant cell necrosis, dependent on the NPs dosage.The higher dose (20 mg/kg) resulted in more pronounced necrotic changes within tumor cells, indicative of increased therapeutic efficacy.Comparatively, the histopathology results mirror findings from similar nanoparticle research, such as the study of Kavaz, where metal oxide nanoparticles also exhibited dose-dependent anticancer activities through mechanisms like ROS generation and DNA damage.Similarly, Nakhaeipour et al. noted a dose-dependent cytotoxic effect of biogenic CuO NPs, highlighting their safer production and use.This underscores the consistency of Co 3 O 4 NPs effects across different experimental frameworks and supports their potential utility in targeted cancer therapy.Furthermore, the minimal histological alterations observed in non-tumoral tissues treated with Co 3 O 4 NP, especially compared to widespread tissue damage of cisplatin, highlight the superior safety profile of these nanoparticles (Kavaz et al. 2021;Chattopadhyay et al. 2014b;Nakhaeepour et al. 2019).Comparing the tumor volumes of mice at the end of the treatment period demonstrated a significant inhibition of tumor growth in the groups treated with various concentrations of Co 3 O 4 NPs and cisplatin.Specifically, cisplatin resulted in a notable reduction and nearly complete elimination of the induced mouse colorectal tumor.Moreover, the concentration of 10 mg/ kg Co 3 O 4 NPs effectively impeded the growth of the mouse colorectal tumor, in contrast to the control group treated with PBS.These findings highlight the potential of 20 mg/ kg Co 3 O 4 NPs as a promising alternative to cisplatin, while also indicating the inhibitory effect of 10 mg/kg Co 3 O 4 NPs on tumor growth.The method for administration of nanoparticles varies depending on their characteristics such as nature, size, and shape.Due to the potential hemolytic activity associated with high concentrations of Co 3 O 4 NPs, intraperitoneal or subcutaneous injections have been utilized in previous studies (Chattopadhyay et al. 2014a(Chattopadhyay et al. , 2013;;Ajarem et al. 2021;Huang et al. 2021).Our histological data analysis demonstrated that cisplatin, aside from its potent anticancer properties, induced significant tissue damage in the liver, kidney, and spleen.In contrast, the application of Co 3 O 4 NPs at both concentrations did not cause tissue damage in the spleen and exhibited minimal damage in liver and kidney tissues (Gong et al. 2021;Ertugrul et al. 2020).The presence of necrosis in liver cells and in the white and red pulps of the spleen after cisplatin treatment align with the cytotoxic effects.The minimal extent of necrosis and hepatic congestion, observed in the groups treated with Co 3 O 4 NPs suggests a potentially lower cytotoxic effect compared to cisplatin.Additionally, the absence of renal congestion in the PBS-treated group and the presence of minimal renal congestion in the groups treated with Co 3 O 4 NPs suggest a potential difference in renal toxicity between the treatments.The structural alterations in renal tubules, glomerular contraction, compression, and significant swelling of kidney tissue after cisplatin treatment align with similar findings after exposure to ZnO NPs and cisplatin.Moreover, the disorder and different sizes of tumor cells associated with necrosis in the group treated with 20 mg/kg Co 3 O 4 NPs align with the concentration-dependent effect observed in the Kavaz et al. research, where higher concentrations of ZnO NPs induced morphological alterations in tissues (Kavaz et al. 2021).
Furthermore, in the context of exploring sustainable methods for nanoparticle synthesis, the findings of this study on Co 3 O 4 nanoparticles synthesized via biological routes contribute to a broader understanding of green nanotechnology.The capability of biological approaches to fabricate diverse metal-based nanoparticles, was also demonstrated across various studies, in the synthesis of zinc oxide nanoparticles (Ahmed et al. 2021), zirconium nanoparticles (Golnaraghi Ghomi et al. 2019), and tellurium nanoparticles (Vahidi et al. 2021).

Conclusion
In this study, we explored the use of Co 3 O 4 NPs synthesized using Vibrio sp.VLC for CRC therapy.Through methods like XRD, EDX, and FTIR analyses, we established their spherical morphology and analyzed their physicochemical properties.Our investigations into the effects of Co 3 O 4 NPs on CRC cells and normal NIH/3T3 cells revealed a notable anticancer activity against CRC cells, suggesting a potential for therapeutic applications.The NPs were also found to possess antioxidant and antibacterial properties and to selectively induce apoptosis in CT26 CRC cells.
Compared with cisplatin, a standard medicine in cancer treatment, biosynthesized Co 3 O 4 NPs showed a promising tumor growth inhibition in a mouse model with less toxicity towards vital organs.This outcome underscores the potential of Co 3 O 4 NPs to reduce the side effects commonly associated with traditional chemotherapy, though it is crucial to note that these findings are preliminary.
The synthesis method utilizing Vibrio sp.VLC highlights an area of interest for nanoparticle fabrication, suggesting a direction for future research in safer and possibly more targeted cancer treatments.Yet, it is important to proceed with further studies to understand the mechanisms through which Co 3 O 4 NPs exert their effects and to verify their safety and effectiveness in more complex biological models.
Our findings contribute to the growing body of research on nanotechnology in cancer therapy.They underscore the importance of continued exploration in this area, particularly for treatments that aim to be less toxic and more targeted.Future research should focus on enhancing nanoparticle delivery mechanisms and conducting comprehensive clinical trials to assess the practical benefits of Co 3 O 4 NPs in colorectal cancer therapy.

Fig. 1
Fig. 1 Vibrio sp.VLC pure culture and visual observation of sediment formation of Co 3 O 4 NPs. a Yellow colonies formed by Vibrio sp.VLC on SWC medium in the presence of light.b Yellow colonies formed by Vibrio sp.VLC in the absence of light.c, e Before mixing bacterial cell extract with Co(NO 3 ) 2 .6H 2 O. d After 90 min of mixing bacterial cell extract with Co(NO 3 ) 2 .6H 2 O and incubation in a hot water bath.f 1 h after the end of synthesis.The cloudy and violet-color sediments in pictures (d, f) represent cobalt oxide nanoparticles

Fig. 3
Fig. 3 FE-SEM micrographs, EDX spectrum, and elemental mapping micrographs of Co 3 O 4 NPs.a, b, and c FE-SEM micrographs of biosynthesized Co 3 O 4 NPs at different magnifications.d, e Types of elements, their weights, and atomic percentages in the sample of Co 3 O 4 NPs produced using the biological method.f-h Elemental mapping image and abundance distribution of cobalt and oxygen elements in an electron microscope image.(f) Cobalt (Co) element, (g) Oxygen (O) element, and (h) cobalt oxide (CoO).The Co 3 O 4 NPs exhibit a spherical shape and have an approximate size of 60 nm.They contain cobalt (Co) and oxygen (O) as major elements

Fig. 4
Fig. 4 Morphological analysis, AFM images, and particle size distribution of Co 3 O 4 NPs.a, b Transmission electron microscopy (TEM) and (c-e) high resolution-transmission electron microscopy (HR-TEM) images of the NPs.f, h Represent 2D images, while (g, i) represent 3D images of Co 3 O 4 NPs; f, g are on a scale of 0.8 µm, and (h, i) are on a scale of 2 µm.j-l Particle size distribution pattern in RPMI 1640, DMEM-HG, and water medium with the PDI of 0.22, 0.24, and 0.92, respectively.m Zeta potential of Co 3 O 4 NPs in different solutions.Biosynthesized Co 3 O 4 NPs are spherical particles ranging in size from 19 to 69 nm and their heights are reported to range from 4.5 to 10 nm

Fig. 5
Fig. 5 Evaluation of antioxidant and antibacterial properties of Co 3 O 4 NPs. a DPPH scavenging results for various concentrations of vitamin C and Co 3 O 4 NPs.The DPPH inhibition was correlated with the concentrations of both vitamin C and Co 3 O 4 NPs.The results are indicated as mean ± SD (****p < 0.0001).b Investigating the antibacterial properties of Co 3 O 4 NPs on E. coli and S. aureus bacteria.The growth of both pathogenic bacteria was inhibited at all different concentrations of Co 3 O 4 NPs.The results are indicated as mean ± SEM and the data are collected with 3 repetitions for each concentration

Fig. 6
Fig. 6 Evaluating the cytotoxic effects of Co 3 O 4 NPs synthesized using Viobrio sp VLC, and cisplatin on different cell lines.a, c Viability rate of CT26 and NIH/3T3 cells after exposing to different concentration of Co 3 O 4 NPs, respectively.b, d Viability rate of CT26 and NIH/3T3 cells after exposing to different concentration of cisplatin, respectively.e Comparison between IC 50 values of Co 3 O 4 NPs on CT26 and NIH/3T3 cells at 24, 48, and 72 h.The results are indicated as mean ± SD (****p < 0.0001).The results demonstrated that the rate of inhibition and the percentage of cell viability decreased with increasing concentration and duration.Both Co 3 O 4 NPs and cisplatin had strong inhibitory effects on CT26 cells, but Co 3 O 4 NPs had a much smaller inhibitory effect on NIH/3T3 cells and were much less cytotoxic.

Fig. 7
Fig. 7 In vivo and histopathological examination of the effects of Co 3 O 4 NPs. a The average tumor volume variations after treatment with Co 3 O 4 NPs (at 10 and 20 mg/kg concentrations), cisplatin (at 5 mg/kg concentration), and PBS on different days of treatment.The data are provided as mean ± SEM (*** p < 0.001).b Comparison between tumor volumes in different groups on the final day of the experiment.The values are provided as mean ± SD (**** p < 0.0001).c-s Histopathological analysis of mice tumor tissues along with different organs (liver, kidney, and spleen) after 19 days exposure to Co 3 O 4 NPs (at 10 and 20 mg/ kg concentrations), cisplatin (at 5 mg/kg concentration), and PBS, (n = 5).PBS was demonstrated to be non-toxic, in all investigated tissues.Treatment with cisplatin and Co 3 O 4 NPs at the concentration of 20 mg/ kg led to the reduction and almost elimination of the induced colorectal tumors in mice, and in general, Co 3 O 4 NPs caused less side effects in all the examined organs compared to the cisplatin.N indicates necrosis, W.P indicates white pulp, and R.P indicates red pulp.Scale bars represent 200 µm