Modulatory effects of bisdemethoxycurcumin-conjugated silver/selenium nanoparticles on 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis

Abstract

Compared with curcumin, bisdemethoxycurcumin, a dimethoxy derivative of curcumin, is a bioactive compound with greater anti-inflammatory and anticancer effects. However, its hydrophobic nature, rapid metabolism, and poor bioavailability have limited its application in cancer therapy. This study investigates the modulatory effects of bis-demethoxycurcumin-conjugated silver/selenium nanoparticles (BDMC-AgSeNPs) on 7,12-dimethylbenz(a)anthracene-induced tumorigenesis. BDMC-conjugated bimetallic spherical Ag templates decorated with Se nanodots were fabricated and characterized via solid-state techniques. The BDMC-AgSeNPs were employed as nanocarriers, and their anticancer efficacy was evaluated. The results revealed that the viability of MCF-7 cells decreased with increasing concentrations of BDMC, AgSeNPs, and BDMC-AgSeNPs, with IC₅₀ values of 22.41, 10.20, and 8.07, respectively. In vivo, BDMC-AgSeNPs significantly decreased lactate dehydrogenase activity by 52%. In the same manner, BDMC-AgSeNPs reduced the serum malondialdehyde level by 35%. Additionally, BDMC-AgSeNPs drastically increased mammary superoxide dismutase and glutathione peroxidase activities by 52% and 47%, respectively. In contrast, mammary nitric oxide and malondialdehyde levels decreased in BDMC-AgSeNPs-rats. Immunohistochemistry showed mild expression of progesterone and human epidermal receptors in BDMC-AgSeNPs-treated rats. In addition, BDMC-AgSeNPs and AgSeNPs reduced Bcl-2-associated X-protein (BAX) levels. Histological examination revealed mammary glands with moderate proliferating ducts and fibrosis in DMBA-rats, while post-treatment with BDMC-AgSeNPs appeared to reveal normal ductal epithelial cells with stromal hyalinization foci. Overall, post-treatment with BDMC-AgSeNPs enhanced antioxidant status and apoptosis, with decreased levels of inflammatory biomarkers. In conclusion, BDMC-AgSeNPs mitigate mammary tumorigenesis by targeting cellular inflammation and apoptotic pathways.

Graphical Abstract

Introduction

Breast cancer is the most common type of cancer affecting women worldwide (Orrantia-Borunda et al. 2022). It is characterized by the uncontrolled growth of abnormal cells within the breast, leading to the formation of tumors (Mokhatri-Hesari and Montazeri 2020). If not addressed, these tumors have the potential to metastasize, spread to other parts of the body, and pose a fatal threat (Oladipo et al. 2017). In 2022, approximately 2.3 million women were diagnosed with breast cancer globally, resulting in 670,000 fatalities. Due to population expansion and aging, it is predicted that the incidence of breast cancer will increase by more than 40% by 2040, reaching around 3 million cases annually. The International Agency for Research on Cancer (IARC) reported that Nigeria had 28,380 new cases of breast cancer in 2020, accounting for 22.7% of all new cancer cases and making it the most common type of cancer in the country (Azubuike et al. 2018; Sung et al. 2020). Breast cancer can affect women across the globe, emerging at any age post-puberty, with rates notably escalating in later stages of life. One major cause of breast cancer is exposure to a chemical carcinogen known as 7,12-dimethylbenz(a) anthracene. Many mouse models have been created over time to better understand the development of breast cancer. The most common type is breast carcinoma caused by carcinogens such as 7,12-dimethylbenz[a]anthracene (DMBA). After being subjected to a single dose of DMBA during the peripubertal phase (4–10 weeks of age), 30–70% of rats typically develop mammary tumors 42–60 days later, which occasionally spread to the lungs (Sung et al. 2020). Because DMBA-induced carcinogenesis closely resembles this multistep process, it is therefore believed to be an accurate and pertinent model for investigating breast cancer.

For many decades, chemotherapy has been recognized as a treatment strategy for breast cancer; however, its deployment has been plagued with many challenges that limit its application (Oladipo et al. 2017). Several chemotherapeutic drugs have been developed and tested; rather than being effective in preventing the growth of tumors, they frequently harm healthy cells and inflict more discomfort. In addition, patients may have to cope with the high treatment cost, associated social effects, and the possibility of reoccurrence (Oyebadejo and Solomon 2019a). Given these challenges, aggressive studies have been devoted to the development of novel chemodrugs with significant improvements in their activity while simultaneously mitigating these drawbacks. One such development is focused on the investigation of novel targeted therapies based on natural products to improve therapy outcome and address the challenges (Bouabdallah et al. 2023). These natural product formulations have been implicated in the induction of autophagic apoptosis, resulting in effective anticancer treatment. For example, naturally occurring compounds such as safranal (Abdalla et al. 2022), crocin (Abdu et al. 2022), and curcumin (Ren et al. 2024) have been reported as inhibitors of cancer progression with minimal side effects.

For many years, curcuminoids have been extensively investigated for their diverse biological effects, including antiulcer (Savaringal and Sanalkumar 2018), antifibrotic (El-Tantawy and Temraz 2022), antiviral (Jennings and Parks 2020), antibacterial (Dai et al. 2022), antiprotozoal (Rai et al. 2020), and anticancer properties (Rodrigues et al. 2019). Recently, this group of compounds has become essential for cancer research and is widely acknowledged as promising agents in the battle against cancer because of their low toxicity and affordability (Khan et al. 2022). Curcumin is the primary polyphenol extracted from the rhizomes of Curcuma longa and is commonly known as turmeric (Giordano and Tommonaro 2019). Curcumin has shown therapeutic benefits across various chronic conditions, including inflammation, arthritis, metabolic syndrome, liver disease, obesity, neurodegenerative disorders, and notably, several types of cancer. Curcumin, a bioactive component in turmeric, has been extensively studied and has demonstrated anticancer effects and the ability to help prevent breast cancer (Guneydas and Topcul 2022). Moreover, bisdemethoxycurcumin (BDMC) was recently reported to exhibit anticancer properties through multiple mechanisms, including the inhibition of cell proliferation, invasion, and migration; the suppression of metastasis and tumor growth; and the induction of apoptosis in cancer cells (Aminnezhad et al. 2023). Compared with curcumin and demethoxycurcumin, bisdemethoxycurcumin has better chemical stability and anti-invasion potential, possibly because of its numerous phenolic groups (Ma et al. 2022). However, despite its relatively hydrophilic nature due to its lack of methoxy groups, bisdemethoxycurcumin still suffers greatly from limited solubility in aqueous environments, rapid metabolism, and unpredictable absorption, thus impacting its bioavailability for effective therapy (Liu et al. 2020; Sudeep et al. 2021).

With the emergence of nanomaterials, numerous novel and innovative drug delivery systems, such as nanoparticles, hydrogels, metal‒organic frameworks, and micelles, have been constructed to incorporate poorly soluble bioactive agents to improve their solubility and bioavailability. For example, Liu and coworkers demonstrated that self-microemulsifying formulations can improve the oral bioavailability of bisdemethoxycurcumin, with approximately 70% of the drug released within 84 h, unlike the 20% released from the free drug (Liu et al. 2020). Similarly, the solubility of bisdemethoxycurcumin has been improved via the use of PLGA (Mehanny et al. 2017) and polymeric lipid nanoparticles (Wilhelm Romero et al. 2021), thus highlighting the effectiveness of nanocarrier delivery systems. Among these nanocarriers, silver/selenium nanoparticles (AgSeNPs) have garnered attention as integral constituents of cutting-edge drug carriers in cancer treatment. These nanoparticles are commonly synthesized through the interaction of reducing agents with silver and selenium ions (Ratan et al. 2020). With significantly improved physicochemical properties and synergistic plasmonic bimetallation arising from the combination of silver and selenium metal components, AgSeNPs present appropriate conditions for enhanced drug loading and stability due to their optimized surface area.

One of the main factors influencing a drug's bioavailability—the percentage of a dose that enters the bloodstream in an active form is its solubility. Poor bioavailability is frequently caused by poor solubility, leading to subtherapeutic amounts of the drugs at the target site and thus reducing its effectiveness (Zhuo et al. 2024). By incorporating drugs like bisdemethoxycurcumin into nanoparticles (usually between 1–100 nm), the large surface area of the AgSeNPs can facilitate the reduction of the drug’s particle size available for dissolution, thereby enhancing their absorption on the surface of nanoparticles. Hence, incorporating bisdemethoxycurcumin with AgSeNPs could increase its solubility and subsequent bioavailability. Although many studies have highlighted the potential of various curcumin nanoformulations in improving its antioxidant and anticancer efficacy (Kanwal et al. 2023), there is limited study on the utilization of bimetallic nanoparticles for improving bisdemethoxycurcumin bioactivity and therapeutic outcome. Besides, insights into the mechanism of action, antioxidant profile, signaling pathway, and modulatory effects on carcinogen-induced tumorigenesis remain poorly understood in disease treatment.

In this study, we explored the enhanced anticancer activity of BDMC by improving its solubility, stability, and bioavailability via the use of a novel selenium nanodot-decorated silver bimetallic nanoparticle (AgSeNPs) delivery system. The morphology, spectroscopy, crystallinity, zeta-potential, and loading efficiency of the nanocarrier were investigated. Additionally, we investigated the modulatory effect of silver/selenium nanoparticles conjugated with bisdemethoxycurcumin (BDMC-AgSeNPs) on 7,12-dimethylbenz(a)nthracene-induced mammary tumorigenesis in female Wistar rats.

Materials and methods

Chemicals and reagents

7,12-Dimethylbenz(a)anthracene and bisdemethoxycurcumin were acquired from AK Scientific (Union City, California, US) and stored at 4 °C without light. Hydrogen peroxide, reduced glutathione, O-dianisidine, 5,5’-dithios-bis-2-nitrobenzoic acid, and epinephrine were also obtained from AK Scientific (Union City, California, US). Other chemicals, including trichloroacetic acid, dithiobis-(−2-dinitrobenzoic acid), sulfosalicylic acid, sodium azide, 1-chloro-2,4-dinitrobenzene, thiobarbituric acid, sodium hydroxide, phosphoric acid, sulfanilamide, phosphoric acid, naphthylenedihydroxide, silver nitrate, and selenium, were sourced from British Drug House (BDH) Chemical Ltd., Poole, UK. Vincristine (VIN) was procured from KunleArá Pharmacy in Ibadan, Nigeria. Other chemicals utilized were of analytical grade.

Synthesis of bisdemethoxycurcumin-silver/selenium nanoparticles

AgNO₃ (40 mL) and 20 mL of Na₂SeO₃ were added to 1 g of methanol crude extract of Bridelia ferruginea. The solution was stirred for 5 min via a magnetic stirrer, and the pH was adjusted to 7.0. After neutralization, 10 mL of BDMC was added to the solution, mixed for 1 min, and then left undisturbed for 1 h to form a precipitate. Thereafter, the mixture was stirred for another 15 min and then spun in a centrifuge for 30 min at 4000 rpm. The clear liquid was discarded; the residue was mixed with distilled water, poured into filter paper, and rewashed three times with distilled water. The diluted ethanol (70%) was added after washing to neutralize the ions that escaped washing. The residue remaining on the filter paper was oven-dried for 30 min and then ground into smaller particles. The obtained bisdemethoxycurcumin-silver/selenium powder was dissolved in phosphate buffer/normal saline for experimental use.

Characterization of bisdemethoxycurcumin-silver/selenium nanoparticles

The optical and absorption properties of the synthesized AgSe nanoparticles and their BDMC conjugates were studied via a UV‒Vis spectrophotometer (EVOLUTION One Plus, Thermo Scientific) in the range of 200–800 nm. Fourier transform infrared spectroscopy (FTIR) equipped with an ATR detector was used to characterize the synthesized BDMC-AgSeNPs. An ATR detector was installed on a PerkinElmer (Frontier FT-IR) apparatus. The range of 500–4000 cm⁻¹ was reported for the bioactive chemicals found in BDMC, as well as the BDMC–conjugated AgSeNPs and AgSeNPs. The different functional groups found in the compound and those connected to the nanoparticles were identified via the vibrational frequencies derived from the spectral analysis. Transmission electron microscopy (TEM, JEOL JEM 2100 F) at an accelerating voltage of 120 kV was used to investigate the shape and size of the AgSe nanoparticles. A selected area electron diffraction (SAED) instrument attached to the transmission electron microscope was used to analyze the diffraction pattern of the sample. The particle size distribution of the AgSeNPs and BDMC-AgSeNPs from TEM measurements was calculated using ImageJ software. A colloidal suspension of the AgSe nanoparticles was dropped onto a carbon-coated copper grid, and the sample was allowed to air dry before the measurements were performed. An X-ray diffraction instrument (PANalytical X’Pert Pro-powder diffractometer, Eindhoven, Netherlands) operating at a Cu Kα radiation source of 40 mA and 40 kV was used to analyze the crystallinity of the AgSe and its conjugate. Using the Scherrer equation, the crystallite size of the AgSe nanoparticles was determined. The change in the surface charge of the AgSe nanoparticles before and after BDMC conjugation was studied via a Malvern Nano ZS Zetasizer (Malvern Ltd., UK). The loading efficiency and loading content of BDMC by the AgSeNPs were evaluated via UV‒Vis spectrophotometry at a wavelength of 424 nm.

Cell culture

Human breast carcinoma (MCF-7) and human embryonic kidney (HEK293) cell lines were obtained from the cell bank of the Toxicology Department, National Institute for Occupational Health (NIOH), Johannesburg, South Africa. Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin‒streptomycin antibiotics, and 1% glutamine (Merck, Johannesburg, South Africa) was used to maintain the cells in a CO₂ humidified incubator at 37 °C.

Cytotoxicity assay

MCF-7 and HEK293 cells were seeded at 50 μL/well in a 96-well plate (1 × 10⁵ cells/mL) and allowed to grow for 24 h. Thereafter, the cells were exposed to AgSeNPs, BDMC, and BDMC-AgSeNPs at various concentrations and incubated for an additional 24 h. The viability of the cells was determined via CellTiter-Blue reagent (Promega, WI, USA). The fluorescence values of the wells were measured via an ELISA microplate reader (Thermo Fisher Scientific, Vantaar, Finland), and the percentage viability of the triplicate results was determined.

Experimental animals

A total of 48 virgin female Wistar rats, each aged between 3 and 4 weeks and weighing from 29 to 48 g, were sourced from the experimental animal facility within the Department of Veterinary Medicine at the University of Ibadan in Nigeria. Upon arrival, they were transferred to the animal facility of the Department of Biochemistry at Dominion University Ibadan, Nigeria. They were housed in plastic cages and allowed to acclimatize for 1 week under controlled conditions: the temperature was maintained at 25 ± 3 °C, the relative humidity was 60 ± 10%, and the light/dark cycle was 12 h. The rats had unrestricted access to laboratory feed (procured from Ladokun Feeds Industry, Ibadan, Nigeria) and water throughout the study period.

Experimental design

A total of 48 virgin female rats aged 4–5 weeks were meticulously weighed and grouped into 6 distinct categories (n = 8 per group) in the following manner: Group 1 served as the control (corn oil), Group 2 received DMBA only (50 mg/kg), Group 3 received DMBA and was subsequently treated with BDMC-AgSeNPs (10 mg/kg), Group 4 was given DMBA and subsequently treated with AgSeNPs only (10 mg/kg), Group 5 received only BDMC-AgSeNPs (10 mg/kg), and Group 6 was administered DMBA and later treated with vincristine (VIN, 0.5 mg/kg). After 12 weeks of the single-dose administration of DMBA to the rats, VIN (i.p.; thrice weekly) and BDMC-AgSeNPs (oral; daily) were administered for 2 weeks. The dosage and delivery methods used complied with the studies of Adefisan et al. (2024) and Mahalunkar et al. (2019). Four weeks after DMBA administration, the animals were checked once a week to confirm the incidence of tumors via palpation. At the end of 14 weeks, the animals were killed by cervical dislocation.

Tissue preparation

Following the completion of the final treatment, the rats were subjected to overnight fasting and killed by cervical dislocation. Mammary tissues were excised, weighed, and washed with ice-cold 1.15% KCl solution. A section of the mammary gland was fixed in a 10% formalin solution for histological analysis. The remaining tissue samples were homogenized in 4 volumes of 50 mM phosphate buffer at pH 7.4 via an electronic Teflon homogenizer. The homogenates were centrifuged at 10,000 × g for 15 min at 4 °C via an ice-cold ultracentrifugation apparatus to isolate the postmitochondrial fraction (PMF) for biochemical analysis.

Serum preparation

Blood samples were obtained from the rats via ocular puncture and placed into plain centrifuge tubes. The blood was subsequently centrifuged at room temperature at a speed of 3000 × g for 10 min on a benchtop centrifuge to obtain the supernatant (serum), which was subsequently used for biochemical analyses.

Biochemical assays

Aminotransferase activity measurement

The activities of aspartate and alanine aminotransferases were assessed via the approach of Reitman and Frankel (1957), which is based on the amount of oxaloacetate hydrazone produced by 2,4-dinitrophenyl hydrazine. A diluted sample (0.1 mL) was mixed with phosphate buffer (100 mmol/L, pH 7.4), L-aspartate (100 mmol/L), and α-oxoglutarate (2 mmol/L) and incubated at 37 °C for 30 min. To stop the reaction, 5.0 mL (0.4 mol/L) of NaOH was added to the mixture after 0.5 mL (2 mmol/L) of 2,4-dinitrophenylhydrazine was added, and the mixture was left at 25 °C for 20 min. The absorbance was measured at 546 nm and compared with that of the reagent blank.

Lactate dehydrogenase activity measurement

Lactate dehydrogenase activity was assessed according to the method described by Weissharet and colleagues (2014). The R1 buffer substrate (1000 μL) and sample (20 μL) were thoroughly combined in a cuvette. Next, R2 enzyme (200 μL) was added, mixed well, and incubated at 37 °C for 90 s. After incubation, the mixture was immediately evaluated. The absorbance was recorded at 370 nm every minute for 3 min.

Protein concentration estimation

Serum and mammary tissue protein concentrations were measured quantitatively via the biuret method, with bovine serum albumin used as a standard, as outlined by Gornall and colleagues (1949), with minor adjustments. Potassium iodide was included in the biuret reagent to prevent the precipitation of Cu²⁺ ions and cuprous oxide. The postmitochondrial fractions (PMFs) of the tissue homogenates were mixed with distilled water at a 1:10 ratio to adjust the protein concentration to fall within the detection range of the biuret method. Subsequently, 1.0 mL of the diluted PMF was combined with 4 mL of biuret reagent. This mixture was incubated at room temperature for 30 min before the absorbance at 540 nm was measured, while distilled water was used as a blank. Protein values were determined via extrapolation from the calibration curve generated via bovine serum albumin as a standard and multiplied by the dilution factor (10) to obtain the actual protein concentration in the sample.

Assay for superoxide dismutase activity

Superoxide dismutase activity was assessed following the method described by McCord and Fridovich (1969). The assay mixture comprised 20 μL of the sample and 2.5 mL of 0.05 M carbonate buffer (pH 10.2). After incubation and equilibration, freshly prepared adrenaline (0.3 mmol/L) (0.3 mL) was added to the reaction mixture. The absorbance at 480 nm was recorded at 30-s intervals for 150 s. The quantifiable enzyme activity of SOD is expressed in units per milligram of protein.

Assay for glutathione-S-transferase activity

The activity of glutathione-S-transferase was evaluated via the method outlined by Habig and colleagues (1974), which employs 1-chloro-2,4 dinitrobenzene as a substrate. The reaction mixture consisted of 1.7 mL of phosphate buffer (pH 6.5) at a concentration of 100 mmol/L and 10 μL of 1-chloro-2,4-dinitrobenzene at a concentration of 30 mmol/L. The reaction was initiated by adding 20 μL of sample after the reaction mixture was preincubated at 37 °C for 5 min. The absorbance was continuously monitored via a spectrophotometer at 340 nm for 5 min, with the reaction mixture lacking the biocatalyst serving as a blank. The enzyme activity of glutathione S-transferase was quantified in micromoles of GSH/CDNB conjugates formed per minute per milligram of protein, utilizing an extinction coefficient of 9.61 mmol⁻¹ cm⁻¹.

Estimation of reduced glutathione levels

The concentration of reduced glutathione was measured via the procedure outlined by Moron et al. (1979). The sample (0.1 mL) was mixed with 0.9 mL of distilled water and then deproteinized with an equal volume of 4% sulfosalicylic acid. The mixture was subsequently centrifuged at 3000 rpm for 10 min. After centrifugation, 0.5 mL of the resulting supernatant was combined with 1.5 mL of dithiobis-(−2-dinitrobenzoic acid). The absorbance at 412 nm was measured via a spectrophotometer. The concentration of reduced glutathione was directly proportional to the absorbance at 412 nm, and the values are reported in mg/g tissue.

Assay for glutathione peroxidase activity

Glutathione peroxidase activity was measured following the method described by Rotruck et al. (1973), with minor adjustments. Sodium phosphate buffer (500 μL), 100 μL of 10.0 mM sodium azide, and 200 μL of 4.0 mM reduced glutathione were combined in the reaction mixture, along with 100 μL of 2.5 mM H₂O₂ and 500 μL of the test sample. Distilled water was added to bring the total quantity to 2.0 mL, and the mixture was incubated at 37 °C for 3 min. The reaction was stopped by introducing 0.5 mL of 10% trichloroacetic acid followed by centrifugation at 3000 × g for 5 min. After centrifugation, the supernatant was collected. To each sample, 1 mL of this supernatant, 2.0 mL of 0.3 M disodium hydrogen phosphate, and 1.0 mL of dithiobis-(−2-dinitrobenzoic acid) were added. Finally, the absorbance at 412 nm was measured via a spectrophotometer against a reagent blank of 1 mL distilled water. The activity of glutathione peroxidase was calculated in micromoles per milligram of protein.

Assay for myeloperoxidase activity

The activity of myeloperoxidase was evaluated via a protocol adapted from Trush et al. (1994). Briefly, a portion of O-dianisidine (200 μL) and diluted H₂O₂ (50 μL) were added to the sample (7.0 μL). The enzyme activity was quantified spectrophotometrically by measuring the change in absorbance at 470 nm over 3 min, with readings taken at 60-s intervals. One unit of MPO activity corresponds to an increase in the absorbance of 0.001/min under these conditions. The specific activity is expressed as IU/mg protein, utilizing an extinction coefficient of 11.3 mM/cm.

Estimation of nitrite level

The concentrations of NO₃− and NO₂⁻ in serum and mammary tissue, which serve as indicators of total nitric oxide, were determined via the method described by Palmer et al. (1987), which involves the Griess reaction. The samples (0.5 mL) were mixed with an equal volume of Griess reagent (1:1 mixture of 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in water and 1% sulfanilamide in 5% phosphoric acid) and incubated at room temperature for 20 min. Following incubation, the absorbance of the resulting product was measured at 550 nm via a spectrophotometer. The total nitrite concentration was determined by comparing these absorbance readings to those of a standard solution with known concentrations of sodium nitrite as a reference.

Lipid peroxidation estimation

The Buege and Aust (1978) method was employed to assess malondialdehyde levels in serum and mammary tissue PMF. This involved measuring the formation of thiobarbituric acid reactive substances (TBARS) as indicators of lipid peroxidation in the samples. The sample (0.4 mL) was combined with 1.6 mL of Tris–KCl solution and 0.5 mL of 30% trichloroacetic acid. Subsequently, 0.5 mL of 0.75% thiobarbituric acid was added to the mixture, which was then heated in a water bath at 80 °C for 45 min. After cooling, the mixture was centrifuged at 3000 × g for 15 min. The absorbance of the resulting supernatant was measured at 532 nm via a spectrophotometer, and distilled water served as the blank.

Histopathological examination of mammary tissues

Mammary tissue from each group of animals was collected and fixed in 10% formalin. The samples were dehydrated with 95% ethanol, cleared with xylene, and embedded in paraffin. The paraffin blocks were sliced into 4 μm sections, which were floated to remove wax and mounted on slides coated with egg albumin. The slides were sequentially treated with decreasing concentrations of alcohol, rinsed with water, and stained with hematoxylin and eosin. After staining, the slides were treated with ammonia, washed, air dried, and mounted with DPX. Finally, a histopathologist, blinded to the treatment groups, examined the slides under a light microscope.

Immunohistochemical analysis

The immunohistochemical staining kits used were from Abcam Chemical, Inc. (Cambridge, MA), Santa Cruz Biotechnology (TX), and DakoProduktionsvej (Glostrup, Denmark). Immunohistochemistry was performed via a modified technique based on Chakravarthi et al. (2020), which involves the binding of a primary antibody to a specific antigen (dilution of 1:100 or as directed by the manufacturer). The resulting antibody‒antigen complex was then incubated with an enzyme-conjugated secondary antibody. In the presence of both a chromogen and a substrate, the enzyme catalyzes a reaction to produce colored deposits at the antigen-binding sites. These deposits were observed under a binocular microscope. Cells showing distinct pigments in their membrane, nuclei, or cytoplasm were identified as positive based on the antigenic site and compared with external controls. To extract the antigen, the sections were heated in a citric acid solution (pH 6.0) for 15 min at 100 °C on a hot plate. ImageJ software was used to analyze and quantify the intensity of the bands.

Statistical analysis

The results are presented as the mean ± standard deviation (SD) of eight animals per group. Statistical analysis of the biochemical data was conducted via one-way ANOVA followed by the post hoc Duncan’s multiple range test, performed with SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was defined as P < 0.05.

Results

Synthesis and characterization of BDMC-AgSeNPs

AgSeNPs were synthesized via a green synthetic method, followed by the adsorption of BDMC onto the surface of the formed nanoparticles. UV absorption analysis was performed for BDMC, AgSeNPs, and BDMC-AgSeNPs (Fig. 1a). The characteristic peaks of the Ag and Se nanoparticles formed at 425 nm and 275 nm, respectively, suggesting the possible formation of bimetallic nanoparticles. After BDMC conjugation, these peaks blue shifted to 403 nm and 272 nm, respectively, indicating the successful formation of BDMC-AgSeNPs. TEM analysis was carried out to investigate the formation, shape, and size of the AgSeNPs before BDMC conjugation (Fig. 1b). The TEM micrograph revealed spherical-shaped AgSeNPs decorated with ultrasmall-sized selenium nanodots throughout the surface. As shown in Fig. 1c, the selected area diffraction pattern of the AgSeNPs showed multiple rings with bright spots. Furthermore, FTIR analysis was used to confirm the interaction of BDMC with the AgSeNPs (Fig. 1d). BDMC (red line) showed distinct peaks at 3223 cm⁻¹ (OH group), 2990 and 2896 cm⁻¹ (CH aliphatic group), 1605 cm⁻¹ (C=O group) and below 1500 cm⁻¹ (fingerprint region), indicating a complex molecular structure with multiple functional groups. The characteristic peaks at 3216 cm⁻¹, 2986 cm⁻¹, and 2917 cm⁻¹, and those from the fingerprint region from BDMC, were mostly detected in the infrared spectrum of BDMC-AgSeNPs (blue line). The crystallinity of the prepared AgSeNPs and BDMC-AgSeNPs was studied using XRD analysis. As shown in Fig. 1e, diffraction peaks characteristic of AgSeNPs at 27.^2°, 31.^4°, 37.^7°, 43.^9°, 54.^2°, 64.^5°, 77.^3° and 81.^3° corresponding to the (100), (101), (111), (200), (201), (220), (311), and (301) planes, respectively, were observed. In addition, using the Scherrer equation, the average crystallite size of the AgSeNPs calculated was 42.47 nm and is consistent with the result of a previous study (El-Behery et al. 2023). Upon BDMC conjugation with the AgSeNPs, intense crystallization was observed, while the crystallinity remained unchanged in the BDMC-AgSeNPs. This could be attributed to the BDMC molecules (Fig. S1a) adsorbed onto the surface of the AgSeNPs. These results confirmed that BDMC conjugation did not alter the structural architecture of the AgSeNPs. In addition, a negative zeta-potential of − 17.8 mV was obtained for the AgSeNPs, which slightly increased to − 17.5 mV after BDMC conjugation to the AgSeNPs (Fig. S1b). The particle size from the TEM analysis (Fig. 1f) showed that the average diameter of BDMC-conjugated AgSeNPs (67.35 ± 1.41 nm) was larger than that of AgSeNPs (63.87 ± 0.27 nm) (Fig. S2). Additionally, the BDMC loading efficiency and loading content were evaluated using UV‒vis spectroscopy. The results indicated a loading efficiency of 76.7% and a loading content of 33.5% for the BDMC-AgSeNPs, indicating substantial BDMC loading onto the nanoparticles, thereby improving their therapeutic activity.

Fig. 1

Characterization of nanoparticles. a UV absorption, b TEM image of AgSeNPs. c SAED pattern of AgSeNPs. d FTIR spectra of BDMC, AgSeNPs, and BDMC-AgSeNPs. e overlayed XRD pattern of AgSeNPs and BDMC-AgSeNPs. f average particle diameter of BDMC-AgSeNPs

Effect of BDMC-AgSeNPs on cell viability

Given the morphological stability and considerable BDMC loading by the AgSeNPs, the anticancer activities of BDMC, AgSeNPs, and BDMC-AgSeNPs were evaluated. The cytotoxicity was assessed using human noncancerous embryonic kidney (HEK293) cells and human breast carcinoma (MCF7) cells. No notable cytotoxic effects were observed in HEK293 cells treated with any of the tested components at different concentrations (0, 3.125, 6.25, 12.5, 25, and 50 µg/mL) for 24 h. Only the AgSeNPs and BDMC-AgSeNPs exhibited significant cytotoxicity at the highest concentration of 100 µg/mL (Fig. 2a). However, the viability of MCF-7 cells decreased with increasing concentrations of BDMC, AgSeNPs, and BDMC-AgSeNPs under the same conditions (Fig. 2b). Table S1 shows the IC₅₀ values for BDMC, AgSeNPs, and BDMC-AgSeNPs in MCF7 and HEK293 cells after 24 h. The IC₅₀ values (22.41 and 8.07) for BDMC and BDMC-AgSeNPs were lower in MCF7 cells than in (54.88 and 58.19) in HEK293 cells.

Fig. 2

Cytotoxicity and serum parameters of nanoparticles. a, b MCF-7 and HEK293 viabilities at different concentrations. c Malondialdehyde, d nitric oxide, e serum aminotransferases, f and g lactate dehydrogenase activities of DMBA-induced rats. Values are expressed as the mean ± standard deviation of 8 animals per group. * = significantly different from control (P < 0.05); ** = significantly different from DMBA (P < 0.05)

Effects of BDMC-AgSeNPs on body weight, mammary gland weight, and biochemical indices in rats administered DMBA

Table S2 shows a considerable decrease in the weight of the animals exposed to DMBA by 29% relative to the control. In contrast, post-administration of BDMC-AgSeNPs and AgSeNPs increased the body weight of the rats by 14% and 7%, respectively, compared with that of the DMBA-treated rats. In contrast, compared with the control, DMBA significantly increased the organo-somatic weight of the mammary gland by 65%. Post-treatment with BDMC-AgSeNPs and AgSeNPs decreased the organo-somatic weight by 44 and 21%, respectively (Table S2). Compared with the control condition, the administration of DMBA caused an insignificant (P > 0.05) increase in the activities of serum alanine and aspartate aminotransferases (Fig. 2c and d). Similarly, compared with control rats, DMBA-treated rats presented drastic increases in lactate dehydrogenase activity, nitric oxide and lipid peroxidation levels by 1.3-fold, 35% and 2.3-fold, respectively (Fig. 2e–g). However, upon treatment with BDMC-AgSeNPs, there was a significant reduction in the activity of lactate dehydrogenase and the levels of nitric oxide and lipid peroxidation.

Effects of BDMC-AgSeNPs on markers of enzymatic and nonenzymatic antioxidant status and inflammation in DMBA-treated rats

The administration of DMBA significantly elevated the activity of mammary myeloperoxidase and the levels of nitric oxide and malondialdehyde by 6.3-fold, 13% and 10%, respectively, relative to those in the controls (Fig. 3a–c). Interestingly, post-treatment with BDMC-AgSeNPs significantly reduced the levels of these inflammatory biomarkers. Moreover, the reduced glutathione levels and activities of glutathione-S-transferase, glutathione peroxidase and superoxide dismutase in mammary tissue were significantly (P < 0.05) decreased following DMBA administration compared with those in the controls, whereas post-treatment with BDMC-AgSeNPs restored the enzymatic activities (Fig. 3d–g). Similarly, post-treatment with BDMC-AgSeNPs and VIN in DMBA-treated rats increased the levels of reduced glutathione in the mammary gland.

Fig. 3

Antioxidant status, inflammation indices and oxidative stress markers. a Myeloperoxidase, b mammary nitric oxide, c malondialdehyde, d glutathione peroxidase, e reduced glutathione, f glutathione-S-transferase and g superoxide dismutase activities of DMBA-induced rats. Values are expressed as the mean ± standard deviation of 8 animals per group. * = significantly different from control (P < 0.05); ** = significantly different from DMBA (P < 0.05)

Effects of BDMC-AgSeNPs on the expression of hormone receptors and apoptosis in DMBA-treated rats

Immunochemical analysis of mammary gland sections revealed strong expression of estrogen receptor and human epidermal receptor-2 in the DMBA-treated rats compared with the control rats (Figs. 4 and 5). Similarly, compared with control rats, DMBA-treated rats presented weak expression of progesterone receptors (Fig. 6). In addition, the level of Bcl-2 associated X (Bax) was slightly reduced after the administration of DMBA (Fig. 7), whereas the level of B-cell lymphoma-2 (Bcl-2) drastically increased following DMBA administration compared with that in the control (Fig. 8). However, post-treatment with BDMC-AgSeNPs and VIN significantly reduced the expression of estrogen receptor and human epidermal receptor-2. In addition, post-treatment with BDMC-AgSeNPs increased Bax and reduced Bcl-2 levels (Fig. 7).

Fig. 4

Expression of estrogen receptor induced by BDMC-AgSeNPs and VIN in DMBA-rats. Immunohistological analysis assessed by DAB staining after 2 weeks post-treatment. Photograph taken at 400X with scale bar 50 µm

Fig. 5

Expression of human epidermal receptor-2 induced by BDMC-AgSeNPs and VIN in DMBA-rats. Immunohistological analysis assessed by DAB staining after 2 weeks post-treatment. Photograph taken at 400X with scale bar 50 µm

Fig. 6

Expression of progesterone receptor induced by BDMC-AgSeNPs and VIN in DMBA-rats. Immunohistological analysis assessed by DAB staining after 2 weeks post-treatment. Photograph taken at 400X with scale bar 50 µm

Fig. 7

Expression of Bcl-2 associated X-protein (BAX) induced by BDMC-AgSeNPs and VIN in DMBA-rats. Immunohistological analysis of apoptotic marker assessed by DAB staining after 2 weeks post-treatment. Photograph taken at 400X with scale bar 50 µm

Fig. 8

Expression of apoptosis markers induced by BDMC-AgSeNPs and VIN on B cell lymphoma-2 in DMBA-rats. Immunohistological analysis assessed by DAB staining after 2 weeks post-treatment. Photograph taken at 400X with scale bar 50 µm

Effect of BDMC-AgSeNPs on the cellular structure of mammary glands in DMBA-treated rats

The mammary glands from the control group presented an epithelial lining with a normal architecture and a normal stroma consisting of normal mammary adipose and fibrous connective tissues. The presence of mammary glands with moderately proliferating ducts and moderate fibrosis was confirmed by histological examination of mammary glands from DMBA-treated rats (Fig. 9). However, post-treatment with BDMC-AgSeNPs resulted in nonproliferating cells with ducts lined by inner columnar epithelium hyperplasia and outer myoepithelium, whereas post-treatment with VIN resulted in nonproliferating ducts with high deposits of fibrosis.

Fig. 9

Antitumor efficacy of BDMC-AgSeNPs on mammary gland and tumors in DMBA-rats. Histological analysis assessed by HE staining after 2 weeks post-treatment. Photograph taken at 400X with scale bar 50 µm

Effects of post-treatment with BDMC-AgSeNPs on mammary tumor growth in DMBA-treated rats

The presence of mammary tumors was confirmed by macroscopic and histological investigations following 12 weeks of DMBA administration (Fig. 10a). Interestingly, the tumorigenic effects of DMBA were significantly attenuated after 2 weeks of treatment with BDMC-AgSeNPs. Post-treatment with VIN and BDMC-AgSeNPs substantially reduced the tumor volume in the DMBA-treated rats. In particular, the [DMBA + BDMC-AgSeNPs] mammary tumor appears to exhibit normal epithelial cells with stromal hyalinization foci and no signs of atypia or malignancy (black arrow) (Fig. 10a). Furthermore, the [DMBA + AgSeNPs]-mammary tumor showed a clearly defined tumor edge as well as inflammatory cell infiltration (blue arrow), whereas [DMBA + VIN] revealed ductal epithelial cell hyperplasia with spindle cells exhibiting large nuclei, vacuolation (light blue) and highly fibrotic stroma (Fig. 10a).

Fig. 10

Histological analysis of BDMC-AgSeNPs and VIN on mammary tumors in DMBA-rats. a Antitumor efficacy assessed by HE staining after 2 weeks post-treatment (green arrow: lymphocytic infiltration, blue arrow: infiltration of inflammatory cells, grey arrow: vacuolation, black arrow: stromal hyalinization). Photograph taken at 400X with scale bar 50 µm. b Photographs of DMBA, DMBA + AgSeNPs, DMBA + VIN, and DMBA + BDMC-AgSeNPs groups 2 weeks post-treatment

Discussion

Research on the fabrication of nanocarriers for the controlled release and stabilization of poorly water-soluble drugs is crucial for enhancing their treatment efficacy. Regardless of the challenges of nanoparticle-based delivery systems, the use of nanoparticles still holds future promise. Despite the many options available as drug nanocarriers, silver/selenium nanoparticles were selected for this study because of their high surface area, optimized plasmonics, and biocompatibility (Olawale et al. 2021). In addition, silver/selenium nanoparticles can form complexes with hydrophobic drugs, thereby improving their solubility and enhancing their anticancer efficacy (Malinga et al. 2021). A green approach was used to fabricate AgSeNPs from plant extract. It has been reported that nanoparticles' size, shape, and surface capping have a major effect on their physicochemical properties (Mariadoss et al. 2019). In this study, the precursor salts were reduced to nanoparticles and then stabilized using Bridelia ferruginea extract. Watersoluble phytochemicals found in B. ferruginea extract are appropriate for effective capping and reduction of metals. We anticipate that the abundant phytochemicals in the extract may facilitate subsequent drug conjugation for delivery applications. To control the formation of the AgSeNPs, the reduction of Ag⁺ and SeO₃²⁻ was achieved via a simple method. Despite the mixing of Ag⁺ and SeO₃²⁻ at the same time before the addition of the plant extract, simultaneous co-reduction of the two ions was anticipated during the formation of the AgSeNPs. However, it seems that the reduction of Ag⁺ was more favorable than that of SeO₃²⁻ ions. Therefore, we propose the following mechanism for the formation of the synthesized AgSeNPs: from the TEM micrograph, it appears that Ag⁺ initially formed spherical-shaped Ag nanoparticles as template seeds, followed by the fast deposition of ultrasmall Se nanodots on their surfaces. This is not surprising considering that the reduction potential of SeO₃²⁻ (1.150 V) is much greater than that of Ag⁺ (0.799 V). After being exposed to strongly reducing phytochemicals from the plant extract (phenols, flavonoids, and terpenoids), the electrons required for the reduction and subsequent nucleation of Ag ions can be rapidly released.

One major indication of successful interactions and relationships between two entities in nanoparticle characterization is related to changes in their physicochemical properties (Oladipo et al. 2023). These physicochemical changes between nanoparticle‒drug conjugates, where interactions between biomolecules and nanoparticles result in noticeable shifts in vibrational frequency, surface plasmon resonance, surface charge, fluorescence quenching, etc., suggest chemical interactions via various mechanisms. The observed shifts in the UV plasmonic peaks as well as those in the FTIR peaks before and after BDMC conjugation with the AgSeNPs could be attributed to the interactions between the individual metal ions and BDMC during the formation of BDMC-AgSeNPs. In addition, the slight increase in the negative charge of the BDMC-AgSeNPs could indicate possible coverage of the AgSeNPs by the BDMC molecules after incorporation. These observations agree with the reported sensitivity of the surface properties of nanoparticles after modification/functionalization and drug linkage (Ibrahim et al. 2023). The XRD results indicate that the intensity of the BDMC-AgSeNPs was greater than that of the unloaded AgSeNPs. While the structural properties of BDMC include strong and numerous characteristic 2Ɵ peaks from 5–42°, its conjugation to AgSeNPs indicates a highly crystalline structure with no broadening of the peaks. The present results highlighted a strong binding interaction between BDMC and the AgSeNPs, which is consistent with the literature (Ahmad et al. 2024).

In this study, BDMC-AgSeNPs demonstrated greater anticancer activity than free BDMC did, especially in breast cancer cells. In addition, the low IC₅₀ value indicates increased lethality of the BDMC-AgSeNPs against MCF7 cells. Although both AgSeNPs and BDMC-AgSeNPs showed significant cytotoxicity in MCF7 cells, the BDMC-AgSeNPs demonstrated enhanced anticancer activity. This could be attributed to several factors, including inherent Ag ion toxicity from the AgSeNPs under an acidic tumor environment, improved BDMC bioavailability, solubility, and its release from the AgSeNPs, as well as improved cell uptake by the MCF7 cells (Mehanny et al. 2017). In addition, the BDMC-AgSeNPs were nontoxic to normal cells and demonstrated a cell-specific response, which is crucial for cancer therapy applications.

Owing to their diverse structures and favorable pharmacological and molecular properties, natural products and their derivatives have shown significant promise as chemotherapeutic agents (Nelson et al. 2022). Several studies have reported the link between tissue wasting and cancer. Martínez and colleagues (2020) reported that a decrease in body weight is often associated with characteristics of human carcinogenesis and has been consistently observed in chemically induced mammary tumorigenesis. Similarly, tissue wasting is frequently observed in cancer patients and can develop at any stage of the illness (Khorasanchi et al. 2024). Monitoring changes in body weight, relative body weight, and the organo-somatic index is crucial for assessing and evaluating the harmful effects of drugs in experimental animals (Oyebadejo and Solomon 2019b). In this study, the body weights of the animals were monitored weekly, and the rats exposed to DMBA alone lost a substantial amount of weight, most likely as a result of altered metabolism and decreased appetite. Body weight was restored upon post-treatment with the bisdemethoxycurcumin-silver/selenium nanoparticles. These results suggest that bisdemethoxycurcumin-silver/selenium nanoparticles could be effective in increasing the body weight of rats by improving appetite, enhancing metabolism, and reducing tumor size. This observation is in line with the studies of Glory and Thiruvengada (2012), who reported that rats exposed to N-nitrosodiethylamine had a sharp reduction in body weight.

The ratio of aspartate to alanine aminotransferase, which is used to evaluate liver damage, has been associated with various chronic diseases and increased mortality (Chen et al. 2024). When investigating the effects of bisdemethoxycurcumin-silver/selenium nanoparticles on DMBA-induced mammary tumors, it is important to assess their integrity or alterations in organs such as the liver. The results of this investigation indicated that the integrity of the liver cells of rats exposed to DMBA may be compromised due to a modest increase in the activities of aminotransferases in the blood. Previous studies have shown that certain solid tumors express high levels of nitric oxide and lactate dehydrogenase, key components of nutrient exchange between the tumor and stroma (Forkasiewicz et al. 2020). The substantial increase in serum nitric oxide and malondialdehyde (MDA) levels along with a sharp increase in lactate dehydrogenase activity demonstrated that DMBA caused tissue damage, probably due to excessive generation of reactive oxygen species. Nevertheless, the level of MDA and activity of this enzyme were reduced by bisdemethoxycurcumin-silver/selenium nanoparticles.

Critical biological molecules have been reported to be oxidized as a result of oxidative stress that lasts longer than expected because of an imbalance in the body's defense mechanism that favors oxidants over antioxidants, which can lead to unchecked cell proliferation and abnormal signal transduction during tumor development (Hajam et al. 2022). Although reactive oxygen species are important for cellular processes such as growth and proliferation, excessive reactive oxygen species can damage crucial macromolecules. Cells contain antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, catalase, and glucocorticoid receptor, which help to neutralize oxidative damage (Cecerska-Heryć et al. 2021). This study confirmed that DMBA drastically depleted both enzymatic and nonenzymatic antioxidant levels in the mammary tissues of rats. However, post-treatment with silver/selenium-bisdemethoxycurcumin nanoparticles restored the antioxidant status, highlighting their effective antioxidative properties.

Reactive oxygen species drive the oxidative modification of lipids, a process known as lipid peroxidation. This can alter membrane fluidity and permeability, disrupt cellular metabolic functions, and cause cellular damage. Malondialdehyde, a key byproduct of lipid peroxidation, is capable of crosslinking DNA, proteins, and nucleotides (Wang et al. 2023). The increase in malondialdehyde levels observed in this study is consistent with the findings of Akhouri et al. (2020), who reported increased malondialdehyde in DMBA-exposed rats due to the induction of oxidative stress in mammary tissues. In the present study, malondialdehyde drastically decreased upon the administration of the bisdemethoxycurcumin-silver/selenium nanoparticles, which further confirms the antioxidative capacity of the bisdemethoxycurcumin-conjugated nanoparticles.

Inflammation is a key characteristic and hallmark of cancer development and progression (Wu et al. 2022). Recent studies have linked nitric oxide, a critical signaling molecule and inflammatory mediator synthesized from L-arginine by nitric oxide synthase, to the process of tumorigenesis (Drehmer et al. 2022; Adefisan-Adeoye et al. 2024). The present study revealed that DMBA administration increased nitric oxide levels and myeloperoxidase activity, indicating increased inflammation in the serum and mammary tissues of the rats. However, post-treatment with the bisdemethoxycurcumin-silver/selenium nanoparticles significantly reduced the inflammation indices. These results are in agreement with the findings of Kosemani et al. (2022), who reported that inflammatory markers (nitric oxide and COX-2) were elevated in DMBA-administered rats.

The two main pathways leading to apoptosis are the intrinsic (mitochondrial) pathway and the extrinsic (death receptor) pathway (Wang et al. 2018), both of which involve numerous essential proteins that are abnormally expressed in apoptosis-related diseases such as cancer. According to several studies, the downregulation of Bax, a proapoptotic member of the Bcl-2 family that promotes apoptosis, prevents apoptosis, whereas the upregulation of Bcl-2, an antiapoptotic member of the Bcl-2 family, prevents apoptotic cell death (Hafezi and Rahmani 2021; Wang and Zhang 2017). Our findings revealed that DMBA-treated rats presented weak expression of Bax, indicating very low levels of proapoptotic signaling. However, post-treatment with bisdemethoxycurcumin-silver/selenium nanoparticles and VIN resulted in increased expression of Bax, suggesting an increase in proapoptotic signals and potential induction of apoptotic pathways. In contrast, Bcl-2 was strongly expressed in DMBA-treated rats, which promoted the survival of malignant cells. Post-treatment with the nanoparticles and VIN led to a reduction in Bcl-2 expression, indicating an increase in proapoptotic activity. These findings suggest that the nanoparticles and VIN may promote apoptosis in cancer cells by downregulating antiapoptotic protein levels. These observations are in agreement with the studies of Wang et al. (2017), who reported that DMBA decreased the expression of Bax in rats, whereas treatment with quercetin increased BAX activity.

Breast cancer involves uncontrolled cell growth and metastasis, and key proteins, such as tumor suppressor protein (p53), human epidermal receptor-2, estrogen receptor, progesterone receptor and others, such as the thymidine marker index and Ki-67 index, are used to define this disease clinically. These markers offer insights into proliferation and tumor subtypes in breast cancer diagnosis (Gul et al. 2022). Evidence from epidemiological studies, animal studies and experimental models conducted in cellular models indicates that hormones such as estrogen and progesterone signaling through their receptors may play pivotal roles in the initiation, progression, and clinical prognosis of not only breast cancer but also other types of cancers (Mahalunkar et al. 2019; Adefisan et al. 2022; Kim et al. 2021; Belachew and Sewasew 2021). This study revealed that the progesterone receptor and human epidermal receptor-2 were overexpressed in DMBA-exposed rats. In this study, post-treatment with these nanoparticles significantly reduced the levels of these hormonal receptors, indicating effective suppression of tumorigenic processes in the rats.

Histological analysis of mammary glands from DMBA-treated rats revealed moderate proliferating ducts and mild fibrosis; however, after treatment with BDMC-AgSeNPs, normal ductal epithelial cells with stromal hyalinization foci appeared. The mammary gland shows no signs of malignancy or atypia and no visible mitotic figures, suggesting low-grade adenocarcinoma. These results clearly suggest that BDMC-AgSeNPs were able to mitigate the chemical tumorigenesis triggered by DMBA. These histological findings further corroborate our biochemical results.

Conclusion

This study reports a novel BDMC-bimetallic AgSeNPs nanoformulation with improved BDMC loading and excellent anticancer performance. The AgSeNPs nanocarrier was synthesized via a green method followed by the conjugation of more hydrophobic BDMC, resulting in high loading efficiency and improved drug solubility. From the TEM image, the fabricated AgSeNPs were spherical in morphology and decorated by ultrasmall selenium nanodots on the surface, and subsequent BDMC conjugation was confirmed by UV‒vis, FTIR, XRD, and zeta-potential. As expected, we observed that BDMC-AgSeNPs improved antioxidant levels, reduced inflammation, induced apoptosis, elicited antitumour effects, and restored the cytoarchitecture of the mammary gland in DMBA-administered rats. Overall, this study offers valuable insights into the use of bisdemethoxycurcumin-silver/selenium nanoparticles as potential anticancer agents in modulating tumorigenesis.