Synergistic Combination of Letrozole and Berberine in Ascorbic Acid-Stabilized AuNPs: A Promising Solution for Breast Cancer

Breast cancer is a deadly disease that affects countless women worldwide. The most conventional treatments for breast cancer, such as the administration of anticancer medications such as letrozole (LTZ), pose significant barriers due to the non-selective delivery and low bioavailability of cytotoxic drugs leading to serious adverse effects and multidrug resistance (MDR). Addressing these obstacles requires an innovative approach, and we propose a combined strategy that synergistically incorporates LTZ with berberine (BBR) into stabilised AuNPs coated with ascorbic acid (AA), known as LTZ-BBR@AA-AuNPs. The LTZ-BBR@AA-AuNPs, a novel combined drug delivery system, were carefully designed to maximise the entrapment of both LTZ and BBR. The resulting spherical nanoparticles exhibited remarkable efficiency in trapping these two compounds, with rates of 58% and 54%, respectively. In particular, the average hydrodynamic diameter of these nanoparticles was determined to be 81.23 ± 4.0 nm with a PDI value of only 0.286, indicating excellent uniformity between them. Furthermore, their zeta potential was observed to be −14.5 mV, suggesting high stability even under physiological conditions. The release profiles showed that after being incubated for about 24 h at pH levels ranging from acidic (pH = 5) to basic (pH = 7), the percentage released for both drugs ranged from 56–72%. This sustained and controlled drug release can reduce any negative side effects while improving therapeutic efficacy when administered directly to cancer. MDA-MB-231 cells treated with LTZ-BBR@AA-AuNPs for 48 h exhibited IC50 values of 2.04 ± 0.011 μg/mL, indicating potent cytotoxicity against cells. Furthermore, the nanoparticles demonstrated excellent stability throughout the duration of the treatment.


Introduction
Breast cancer is the most common malignancy in women worldwide, and its prevalence is increasing year by year [1]. Breast cancer is caused by the malignant growth of epithelial cells that line the excretory ducts or lobules of the breast. Despite significant advances in early detection and treatment methods, breast cancer is still considered a leading cause of mortality in women [2]. Over many years, breast cancer has posed a significant threat to several generations. The number of breast cancer cases (18.1 million shown that berberine, or BBR, is a natural alkaloid extracted from the rhizome of the Cupid plant, which is known to be an effective anticancer agent. It now exhibits various pharmacological effects, including antibacterial, anticholinergic, antihypertensive, antiinflammatory and antioxidant properties [26]. Recent research has shown that nanoparticles loaded with anticancer drugs are a superior technology for the delivery of drugs with higher efficacy [1,27]. One technique is the use of metallic nanoparticles, representing a new approach to cancer treatment. In this method, gold nanoparticles (AuNPs) are injected and bound to tumour sites [28]. Due to their excellent compatibility with the biological system, small size, low toxicity, easy surface adaptation, and controllable drug release, AuNPs are among the inorganic nanoparticles most commonly used in biomedicine [29]. Because they have non-celldamaging, biocompatible, immunologically safe, and adaptable surface functions, they can be used for drug delivery [30]. To enhance the stability of AuNPs, ascorbic acid was incorporated into the method. During the synthesis process, it functions as a stabilizer and plays a role in reducing AuNPs and protecting them from oxidation. The effectiveness and potential degradation or aggregation of AuNPs can be prevented while also enhancing their stability and integrity through the incorporation of ascorbic acid [31,32].
With the goal of reducing the toxicity associated with letrozole (LTZ), a novel approach was pursued. The strategy involved the combination of LTZ with berberine (BBR), a bioactive anticancer agent derived from plants. By doing so, the cytotoxic effects of LTZ could be mitigated. Additionally, instead of using conventional nanoparticles, metal nanocarriers were employed for sequential distribution of the compounds through conjugation and encapsulation. The investigation focused on evaluating the cytotoxicity of the nanocomplex containing LTZ and BBR in MDA-MB breast cancer cells at varying doses.

Preparation and Fourier Transform Infrared Analysis of LTZ-BBR@AA-AuNPs
At room temperature, the LTZ-BBR@AA-AuNP mixture was a clear and transparent liquid with a distinct yellow hue, as shown in Figure 1. The characteristic spectra of berberine, letrozole, and ascorbic acid showed stretching of alkane groups (-CH,-CH 2 , -CH 3 ) at 2800-3000 cm −1 . The spectra of a solution of L-ascorbic acid showed that the stretching vibration of a C-C double bond was seen at 1645 cm −1 , along with the peak of an enol hydroxyl at 1384 cm −1 . The most represented berberine vibration modes were found at 1504 cm −1 (aromatic C=C vibrations), 1103 cm −1 (ring deformation and CH in-plane bending), and 1034 cm −1 (ring deformation and CH in-plane vibrations). The characteristic absorption peaks of letrozole were detected at 1270 cm −1 for C-N stretching and 2231 cm −1 for -CN stretching. The observed bands demonstrate that the gold nanoparticles contain both berberine and letrozole molecules close to each other.

Drug Capping
A UV spectrophotometer determined each drug's presence in LTZ and BBR. The results confirmed that LTZ and BBR effectively encapsulated the drugs. LTZ and BBR achieved high drug-loading efficiencies of 58% and 54%, respectively.

Shape, Particle Size, Polydispersity Index, and Zeta Potential
The TEM image depicted a magnified view of the surface of a material called LTZ-BBR@AA-AuNP, which contained nanoparticles. The particles appeared nearly spherical, and their average size was approximately 5 nm, as clearly observed in Figure 2a. The size of particles and their zeta potential play a crucial role in maximizing the effectiveness of cancer treatment. These factors significantly impact the performance of the delivery system. In a study, it was observed that the average hydrodynamic diameters of AA-AuNPs and LTZ-BBR@AA-AuNPs were measured to be 70.89 ± 3.54 nm and 81.23 ± 4.0 nm, respectively. The polydispersity index (PDI) values for LTZ-BBR@AA-AuNPs and AA-AuNPs were 0.309 and 0.286, respectively. Additionally, the zeta potential of LTZ-BBR@AA-AuNPs was determined, and the results confirmed the successful formation of nanoparticles. AA-AuNPs and LTZ-BBR@AA-AuNPs exhibited zeta potentials of −16.6 mV and −14.5 mV, respectively, as depicted in Figure 2b.

Drug Capping
A UV spectrophotometer determined each drug's presence in LTZ and BBR. The results confirmed that LTZ and BBR effectively encapsulated the drugs. LTZ and BBR achieved high drug-loading efficiencies of 58% and 54%, respectively.

Shape, Particle Size, Polydispersity Index, and Zeta Potential
The TEM image depicted a magnified view of the surface of a material called LTZ-BBR@AA-AuNP, which contained nanoparticles. The particles appeared nearly spherical, and their average size was approximately 5 nm, as clearly observed in Figure 2a. The size of particles and their zeta potential play a crucial role in maximizing the effectiveness of cancer treatment. These factors significantly impact the performance of the delivery system. In a study, it was observed that the average hydrodynamic diameters of AA-AuNPs and LTZ-BBR@AA-AuNPs were measured to be 70.89 ± 3.54 nm and 81.23 ± 4.0 nm, respectively. The polydispersity index (PDI) values for LTZ-BBR@AA-AuNPs and AA-AuNPs were 0.309 and 0.286, respectively. Additionally, the zeta potential of LTZ-BBR@AA-AuNPs was determined, and the results confirmed the successful formation of nanoparticles. AA-AuNPs and LTZ-BBR@AA-AuNPs exhibited zeta potentials of −16.6 mV and −14.5 mV, respectively, as depicted in Figure 2b.

In Vitro Drug Release Studies
To assess the release of the drug, each sample (2 mL) was placed in a dialysis bag made of semipermeable acetate cellulose and sealed. These bags were then submerged in 50 mL of the chosen release medium. The drug release study of LTZ-BBR@AA-AuNPs was conducted under two pH conditions: 5.0 and 7.4, representing the environment of cancer cells and physiological conditions, respectively. Figure 3a,b illustrate the release profiles

In Vitro Drug Release Studies
To assess the release of the drug, each sample (2 mL) was placed in a dialysis bag made of semipermeable acetate cellulose and sealed. These bags were then submerged in  Figure 3a,b illustrate the release profiles of LTZ and BBR at the two pH values. At pH 5.0, after 3 h, the drug release percentages were 16.45 ± 0.82% for LTZ and 11.8 ± 0.59% for BBR. After 24 h, the release percentages increased to 72.18 ± 3.69% for LTZ and 68.7 ± 3.43% for BBR. At pH 7.4, the drug release percentages were 9.38 ± 0.469% for LTZ and 8.92 ± 0.446% for BBR after 3 h. After 24 h, the release percentages were 64.3 ± 3.215% for LTZ and 56.3 ± 2.815% for BBR. The sustained release of the nanoparticles is an important characteristic, as it maintains constant therapeutic concentrations of LTZ and BBR over an extended period. This sustained release is crucial for effective treatment. (a) TEM micrograph illustrating the LTZ-BBR@AA-AuNPs' formulation; (b) particle size, polydispersity index (PDI), and zeta potential were assessed before and after drug loading.

In Vitro Drug Release Studies
To assess the release of the drug, each sample (2 mL) was placed in a dialysis bag made of semipermeable acetate cellulose and sealed. These bags were then submerged in 50 mL of the chosen release medium. The drug release study of LTZ-BBR@AA-AuNPs was conducted under two pH conditions: 5.0 and 7.4, representing the environment of cancer cells and physiological conditions, respectively. Figure 3a,b illustrate the release profiles of LTZ and BBR at the two pH values. At pH 5.0, after 3 h, the drug release percentages were 16.45 ± 0.82% for LTZ and 11.8 ± 0.59% for BBR. After 24 h, the release percentages increased to 72.18 ± 3.69% for LTZ and 68.7 ± 3.43% for BBR. At pH 7.4, the drug release percentages were 9.38 ± 0.469% for LTZ and 8.92 ± 0.446% for BBR after 3 h. After 24 h, the release percentages were 64.3 ± 3.215% for LTZ and 56.3 ± 2.815% for BBR. The sustained release of the nanoparticles is an important characteristic, as it maintains constant therapeutic concentrations of LTZ and BBR over an extended period. This sustained release is crucial for effective treatment.

Anticancer Activity
MDA-MB-231 cells underwent various treatments involving different concentrations ranging from 5-40 µg/mL. The treatments included naked LTZ, naked BBR, LTZ@AuNPs, BBR@AuNPs, and LTZ-BBR@AA-AuNPs, and they lasted for 48 h. The viability of the cells was evaluated using the MTT assay, as illustrated in Figure 4. In all treatment groups, there was an observed reduction in cell viability, which depended on both the dose and duration of treatment. Notably, the combined treatment of LTZ and BBR for 48 h resulted in significantly lower cell viability (33.7 ± 1.68%) compared to the groups treated with LTZ@AuNPs (47.62 ± 2.38%) and BBR@AuNPs (67.36 ± 3.36%) alone. Conversely, the percentages of cell viability for naked LTZ and naked BBR treatments were 51.23 ± 2.56% and 67.36 ± 3.36%, respectively. Moreover, the IC50 values for MDA-MB-231 cells treated with LTZ-BBR@AA-AuNPs after 48 h were determined to be 2.04 ± 0.011 µg/mL. These findings demonstrate the enhanced cytotoxicity and synergistic effect of the combined treatment using LTZ and BBR within the AA-AuNP formulation, highlighting its potential as an effective strategy for breast cancer treatment.

Estimation of Intracellular ROS by ELISA
To determine the levels of reactive oxygen species in MDA-MB-231 cells, ELISA assays were used. The results are shown in Figure 5. The research found that the control group had noticeably lower levels of ROS compared to all other treatment groups, which included naked LTZ and BBR, as well as various combinations using AuNPs such as LTZ@AuNPs, BBR@AuNPs, and combinational therapies of LTZ-BBR@AA-AuNPs. There was a fluctuation in the increase of ROS levels between various treatment groups, compared to the control. The increase in potency was evident in all formulations. LTZ use resulted in a 1.6-fold increase, while BBR showed a 2.01-fold increase when used to aid cellular Pharmaceuticals 2023, 16, 1099 6 of 14 uptake or protect against degradation/metabolism. LTZ@AuNPs showed a relatively modest 2.7-fold change, while BBR@AuNPs had an approximately 2-fold improvement compared to their naked counterparts (p < 0.05). However, optimal efficacy was achieved by combining LTZ and BBR with AA-AuNP conjugates, resulting in markedly enhanced performance displaying the greatest change at 3.06-fold (p < 0.05).
BBR@AuNPs, and LTZ-BBR@AA-AuNPs, and they lasted for 48 h. The viability of the cells was evaluated using the MTT assay, as illustrated in Figure 4. In all treatment groups, there was an observed reduction in cell viability, which depended on both the dose and duration of treatment. Notably, the combined treatment of LTZ and BBR for 48 h resulted in significantly lower cell viability (33.7 ± 1.68%) compared to the groups treated with LTZ@AuNPs (47.62 ± 2.38%) and BBR@AuNPs (67.36 ± 3.36%) alone. Conversely, the percentages of cell viability for naked LTZ and naked BBR treatments were 51.23 ± 2.56% and 67.36 ± 3.36%, respectively. Moreover, the IC50 values for MDA-MB-231 cells treated with LTZ-BBR@AA-AuNPs after 48 h were determined to be 2.04 ± 0.011 µg/mL. These findings demonstrate the enhanced cytotoxicity and synergistic effect of the combined treatment using LTZ and BBR within the AA-AuNP formulation, highlighting its potential as an effective strategy for breast cancer treatment.

Estimation of Intracellular ROS by ELISA
To determine the levels of reactive oxygen species in MDA-MB-231 cells, ELISA assays were used. The results are shown in Figure 5. The research found that the control group had noticeably lower levels of ROS compared to all other treatment groups, which included naked LTZ and BBR, as well as various combinations using AuNPs such as LTZ@AuNPs, BBR@AuNPs, and combinational therapies of LTZ-BBR@AA-AuNPs. There was a fluctuation in the increase of ROS levels between various treatment groups, compared to the control. The increase in potency was evident in all formulations. LTZ use resulted in a 1.6-fold increase, while BBR showed a 2.01-fold increase when used to aid cellular uptake or protect against degradation/metabolism. LTZ@AuNPs showed a relatively modest 2.7-fold change, while BBR@AuNPs had an approximately 2-fold improvement compared to their naked counterparts (p < 0.05). However, optimal efficacy was achieved by combining LTZ and BBR with AA-AuNP conjugates, resulting in markedly enhanced performance displaying the greatest change at 3.06-fold (p < 0,05). The rise in ROS levels seen among all the groups treated indicates that the given substances or nanoparticles caused oxidative stress within MDA-MB-231 cell types. The results suggest that the use of LTZ and BBR alone or in conjunction with AuNPs (LTZ@AuNPs and BBR@AuNPs) may disrupt the cellular redox balance, leading to increased production of reactive oxygen species. Furthermore, when LTZ-BBR@AA-AuNPs were administered in combination, the increase in ROS levels was significantly higher than those of each treatment alone or that of the control group. This finding suggests a possible synergistic action between these agents.
Understanding the impact of delivered substances or formulations on the cellular oxidative state is critical [33]. Elevated levels of reactive oxygen species can lead to various consequences, including damage to cellular components, modification of signalling pathways, and the initiation of cell death mechanisms such as apoptosis and necrosis [34,35]. The significance of the research lies in comprehending these results, as they help to develop new therapeutic approaches that improve conditions associated with oxidative stress while mitigating any adverse effects associated with increased ROS production.

Cell Cycle Analysis by Flow Cytometry
To comprehensively assess the cytotoxic effect of naked LTZ, naked BBR, LTZ@AuNPs, BBR@AuNPs, and their combination of LTZ-BBR@AA-AuNPs (in DMSO)  The rise in ROS levels seen among all the groups treated indicates that the given substances or nanoparticles caused oxidative stress within MDA-MB-231 cell types. The results suggest that the use of LTZ and BBR alone or in conjunction with AuNPs (LTZ@AuNPs and BBR@AuNPs) may disrupt the cellular redox balance, leading to increased production of reactive oxygen species. Furthermore, when LTZ-BBR@AA-AuNPs were administered in combination, the increase in ROS levels was significantly higher than those of each treatment alone or that of the control group. This finding suggests a possible synergistic action between these agents.
Understanding the impact of delivered substances or formulations on the cellular oxidative state is critical [33]. Elevated levels of reactive oxygen species can lead to various consequences, including damage to cellular components, modification of signalling pathways, and the initiation of cell death mechanisms such as apoptosis and necrosis [34,35]. The significance of the research lies in comprehending these results, as they help to develop new therapeutic approaches that improve conditions associated with oxidative stress while mitigating any adverse effects associated with increased ROS production.

Cell Cycle Analysis by Flow Cytometry
To comprehensively assess the cytotoxic effect of naked LTZ, naked BBR, LTZ@AuNPs, BBR@AuNPs, and their combination of LTZ-BBR@AA-AuNPs (in DMSO) on MDA-MB-231 cell growth, we have utilised a highly sensitive flow cytometry technique in conjunction with annexin V/PI staining. To determine the optimal concentrations of treatments, extensive analysis was conducted on their IC 50 values. Upon careful examination of the data obtained from flow cytometry analysis and annexin V/PI staining, a significant increase in apoptosis and necrosis was observed upon treatment with LTZ, BBR, LTZ@AuNPs, BBR@AuNPs, and their combination. These findings showed that these treatments have not only been effective but have also induced an encouraging outcome compared to untreated cells ( Figure 6). In light of these results, it seems that the therapies administered had a notable effect in increasing programmed cell death within the MDA-MB-231 cell line. These findings provide valuable insights into the mechanism of action of the treatments and contribute to our understanding of their potential as anticancer agents.

Stability Study
We conducted a stability study on LTZ-BBR@AA-AuNPs to evaluate their colour, transparency, and efficiency of drug capping for BBR and LTZ over a period of 30 days, as reported in Table 1. Throughout the study, the nanoparticles' colour remained yellow, indicating no substantial alterations or deterioration. The nanoparticles' transparency was unaffected. During the investigation, BBR capping on nanoparticles showed no variance in drug-capping effectiveness. Initially, at day 0, the drug-capping efficiency for BBR was 54.33 ± 0.47%. After 15 days, it had slightly increased to 54.87 ± 0.17%, and by the end of the 30-day period, it had reached 56.14 ± 0.65%. These findings suggest that the drug capping of BBR on the LTZ-BBR@AA-AuNP formulation remained stable throughout the

Stability Study
We conducted a stability study on LTZ-BBR@AA-AuNPs to evaluate their colour, transparency, and efficiency of drug capping for BBR and LTZ over a period of 30 days, as reported in Table 1. Throughout the study, the nanoparticles' colour remained yellow, indicating no substantial alterations or deterioration. The nanoparticles' transparency was unaffected. During the investigation, BBR capping on nanoparticles showed no variance in drug-capping effectiveness. Initially, at day 0, the drug-capping efficiency for BBR was 54.33 ± 0.47%. After 15 days, it had slightly increased to 54.87 ± 0.17%, and by the end of the 30-day period, it had reached 56.14 ± 0.65%. These findings suggest that the drug capping of BBR on the LTZ-BBR@AA-AuNP formulation remained stable throughout the study. Similarly, the drug-capping efficiency for LTZ on the nanoparticles demonstrated relative stability. At day 0, the drug-capping efficiency for LTZ was 58.13 ± 2.28%. After 15 days, there had been a slight decrease to 56.65 ± 1.06%, but this variation fell within an acceptable range. By the end of the 30-day period, the drug-capping efficiency for LTZ had increased to 59.54 ± 0.33%. These results suggest that the LTZ capping on the LTZ-BBR@AA-AuNP formulation remained stable or even improved over time. Overall, the stability study indicated consistent colour, transparency, and drug-capping efficiencies for both BBR and LTZ in the LTZ-BBR@AA-AuNP formulation throughout the 30-day period.

Discussion
Breast cancer remains one of the deadliest malignancies affecting women worldwide. Aromatase inhibitors, such as LTZ, treat hormone-positive breast cancer in postmenopausal women. The drug has gained popularity because it has an excellent efficacy and safety profile compared to tamoxifen [36]. Like many other drugs, LTZ is poorly soluble in water, is rapidly metabolised, and has several undesirable side effects [37,38]. It is widely known that successful cancer treatment involves the direct administration of particular doses of anticancer medications to cancer cells without damaging nearby normal cells. Using chemotherapeutics and other phytochemicals/naturally occurring bioactive substances is a very modern and innovative approach to cancer therapy [39][40][41][42].
An optimal drug delivery system (DDS) capable of conveying many medications and discriminating cancer cells from normal tissues is urgently needed to meet this goal. The current research aims to develop a co-delivery nano system for LTZ and BBR by employing AA-AuNPs as carriers. This system should be able to effectively be internalised into breast cancer cells to have a synergistic impact. The concentrations of LTZ and BBR in the formulation affected the physiological characteristics, including the amounts of entrapped drugs. The experimental findings revealed that the concentrations of LTZ and BBR in AA-AuNP medication were so high that they reached 58 ± 2.9% and 54 ± 2.77%, respectively.
In vitro release research suggested the possibility of developing new LTZ and BBR formulations for 24-h delivery, maybe as injections. LTZ and BBR were shown to have comparable release characteristics, with rapid initial release followed by sustained release over 24 h. The rapid initial release of LTZ and BBR is attributable to drug surface adsorption on the surface or in the outer shell. At a pH of 5.0, it was found that 72.18 ± 3.69% of LTZ, and 68.7 ± 3.43% of BBR had been released after 24 h. At a pH of 7.4, the drug release after 24 h was 64.3 ± 3.215% for LTZ and 56.3 ± 2.815% for BBR.
The results of cell culture experiments indicated that LTZ-BBR@AA-AuNPs were much more cytotoxic than other drugs and free drugs when applied in vitro against MDA-MB-231 cancer cells. The 50% inhibitory concentration (IC 50 ) was 2.04 ± 0.011 µg/mL.
ROS promote cellular damage, apoptotic injuries, and cell death in cell lines [43]. ROS have been implicated in inflammation, ageing, angiogenesis, and cancer conditions. The increased level of ROS in MDA-MB-231 cells may be attributed to a decreased level of estrogen, potentially resulting from the antioxidant effect [44][45][46]. These nanoparticles are reactive to thiols, leading to antioxidant depletion and to cell death by apoptosis in malignant cells.
Berberine is a plant alkaloid with potent antioxidant and anti-inflammatory activities and is known to promote apoptosis, which is the ultimate objective in cancer treatment [47,48]. Previous research reported its effect on the reduction in cell viability of various cell lines, including MCF-7 and MDA-MB-231 breast cancer cells [49,50]. The results demonstrated an increase in apoptosis compared to the normal control due to increased ROS and enhanced the effect of oncogenes such as caspase 3 and Bax. The flow cytometry results showed that BBR prevented MDA-MB-231 cell growth in phases G1 and S, which is consistent with previous reports [48][49][50]. Previously, combining natural products and LTZ with lower doses highly influenced cell apoptosis by inducing mitochondrial apoptosis [51,52].
Overall, the prepared nano formulation seems extremely promising for treating cancers other than breast cancer. Our results suggest that concomitant treatment of LTZ-BBR increases the efficacy of chemotherapeutic agents with low BBR concentrations, especially in chemo-resistant malignancies.

Materials
Letrozole powder (HPLC purity 99.12%) was kindly provided by Hetero drugs Pvt.

Preparation of Ascorbic Acid Solution (AA)
The 1% ascorbic acid (AA) stock solution was prepared by dissolving 1 g of AA in a mixture of 50 mL of MilliQ water and 50 mL of ethanol. The solution was shaken at room temperature for 30 min before centrifugation at 5000 rpm. The supernatant obtained was collected and used for the experiment.

Preparation of Gold Chloride Solution
A total of 3.96 mg gold chloride (HAuCl 4 .3H 2 O) was dissolved in 0.5 mL of MilliQ water and 0.5 mL of ethanol to prepare a 10 mM solution.

Synthesis and Optimization of Gold Nanoparticles (AuNPs)
The synthesis of stabilised colloidal AuNPs was performed according to the previous method, with a slide modification in the process [53]. At a temperature of 80 • C ± 1 • C, 100 µL of HAuCl 4 solution at a concentration of 10 mM was added to 5 mL of AA solution at a concentration of 1 wt% with stirring. After five hours, the previously colourless liquid had changed to a bright ruby red. This confirmed the AuNP synthesis.

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Capping of BBR and LTZ separately A solution of 5 mg BBR in 0.5 mL ethanol was prepared. It was added to 5 mL of AA-AuNPs, which were stirred at room temperature for 24 h.
Then, 5 mg of LTZ was mixed with 0.5 mL of ethanol. It was added to 5 mL of AA -AuNPs and kept at room temperature for 24 h with constant stirring. No precipitation of the active ingredient was observed.

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Capping of BBR and LTZ altogether

Morphology Study
The shape of the LTZ-BBR@AA-AuNPs was investigated by high-resolution transmission electron microscopy (HR-TEM, JEOL 2100 HRTEM, Republic of Korea). To capture a sample drop, a copper grid that had been coated with carbon was used. A 2% uranyl acetate stain was applied after air drying at room temperature.

In Vitro Drug Release Rtudies
Dialysis bag dissolutions were conducted at 37 • C in phosphate-buffered saline (PBS, pH 7.4) and sodium acetate buffer (pH 5.0) to determine the in vitro release of LTZ-BBR from LTZ-BBR@AA-AuNPs. The dialysis membrane had a molecular weight cutoff (MWCO) of 12000 Da. In addition, 500 mL of release media was prepared using pH 7.4 and pH 5.0 buffer solutions. At predefined intervals during the course of 24 h, 2 mL samples were collected and then diluted with fresh medium. A UV-Vis spectrophotometer was used to measure both LTZ (240 nm) and BBR (422 nm) emissions.

Anticancer Activity of Test Compounds In Vitro by MTT Assay
The MDA-MB-231 TNBC cell line is a good example of the epithelial-mesenchymal transition (EMT) linked to breast cancer metastasis [54]. Cell viability in the MDA-MB-231 cell line was evaluated using the MTT assay of LTZ-BBR@AA-AuNPs. Cells were subcultured every two days in a 5% CO 2 and 18-20% O 2 atmosphere at 37 • C in a CO 2 incubator with a DMEM medium containing high glucose content, 10% FBS, and 1% antibiotic solution. Without test media, 200 µL of cell suspension was seeded on a 96-well plate at the desired cell density (20,000 cells per well). After 24 h of culture, the corresponding test materials were added at the indicated concentrations. The plates were kept in an incubator for 48 h at 37 • C with 5% CO 2 . After the incubation time, the plates were removed from the incubator, and MTT reagent was introduced to them. The plates were wrapped in aluminium foil to prevent exposure to light. Again, they were placed in the incubator for 3 h. The MTT reagent was then discarded. Following that, 100 µL of DMSO was added to the mixture. To enhance dissolution, a gyro-shaker was used to provide gentle agitation. We used a 570 nm wavelength spectrophotometer to measure the absorbance. The percentage of viable cells can be determined using the following formula: % Cell viability = (A570 nm treated cells) (A570 nm untreated cells) × 100 (2) The IC 50 value was determined using a linear regression equation, i.e., y = mx + c. Here, y = 50, and the m and c values were derived from the viability graph.

Estimation of Intracellular Reactive Oxygen Species (ROS)
The MDA-MB-231 cell line was seeded in DMEM supplemented with 10% v/v FBS and 1% penicillin/streptomycin in 96-well plates at a density of 1.2 × 10 4 (37 • C, 5% CO 2 ) in order to determine the amount of intracellular ROS using the enzyme-linked immunosorbent assay (ELISA). After removing the medium, 0.1 mL/well of each of the treatments and reference solutions was applied to each well of the experiment (n = 4). The treatments (naked LTZ, naked BBR, LTZ@AuNPs, BBR@AuNPs, and their LTZ-BBR combination LTZ-BBR@AA-AuNPs) dispersed in DMEM were added at a specified concentration (50% of the specified IC 50 in the cytotoxicity study mentioned above) and left for 48 h, while the control wells were untreated. This was performed so that the results could be compared with those of the cytotoxicity study. The ROS ELISA assay was estimated according to the method given by Mekkawy et al. [55]. Using a calibration curve made from standard solutions, we were able to calculate the ROS level by measuring the absorbance at 450 nm and comparing it with that of the control and samples we examined.

Cell Cycle Analysis by Flow Cytometry
Apoptosis was quantitatively analysed by flow cytometry after cells were stained with annexin V and propidium iodide (PI). In 6-well plates, MDA-MB-231 cells were seeded at a density of 1 × 10 5 cells per well, and the plates were left to incubate overnight. After that, the cells were placed into a 24 h incubator while being treated with DMSO, LTZ, BBR, LTZ@AuNPs, BBR@AuNPs, and LTZ-BBR@AA-AuNPs. In the cytotoxicity study, treatments were applied at 50% of the indicated concentration of IC 50 . The cells were then rinsed, trypsinised, and washed with DMEM media; they were captured in falcon tubes in which the corresponding supernatant was obtained, and the cell pellets were washed two times with PBS. Subsequently, 100 µL of cell suspension was transferred to a FACS tube and combined with 5 µL of annexin V-FITC and 5 µL of PI. The tubes were slightly yet thoroughly mixed before being incubated for 30 min at room temperature in the dark. Subsequently, within 1 h, 400 µL of the binding buffer had been added to the FACS tube, and the tube was processed through the FACS machine.

Stability Studies
Short-term physical stability was evaluated to determine how temperature affects LTZ-BBR@AA-AuNPs. The 30-day stability of LTZ-BBR@AA-AuNPs was evaluated at a temperature of 25 • C. The physical stability of LTZ-BBR@AA-AuNPs was evaluated by measuring the colour, encapsulation, and transparency.

Conclusions
There is an evident enthusiasm among researchers to develop a unified platform that combines nanoagents and nanocarriers, aiming to gain a comprehensive understanding of the bioavailability and effectiveness of molecular-based anticancer agents. The objective goes beyond creating mere new treatments, focusing on novel strategies capable of overcoming the resistance encountered with traditional chemotherapy approaches. A promising illustration of this approach is the successful fabrication of LTZ-BBR@AA-AuNPs for breast cancer treatment. By encapsulating anticancer drugs or herbal compounds within nanoparticles, several advantageous outcomes have been observed, including improved drug release, enhanced penetration into cancer cells, and facilitated endocytosis. These mechanisms enable a synergistic approach to cancer treatment, promoting higher efficacy in patient recovery while concurrently monitoring the potential toxicity associated with the administration of natural or synthetic anticancer drugs. Collectively, it is evident that the medical community persists in its quest to develop increasingly sophisticated methodologies in the battle against one of the most devastating diseases affecting humanity.