Natural Baicalein-Rich Fraction as Radiosensitizer in Combination with Bismuth Oxide Nanoparticles and Cisplatin for Clinical Radiotherapy

Purpose Chemotherapy has been used in conjunction with radiation therapy to improve the treatment outcomes of cancers. Cisplatin (Cis) is a standard treatment that has been used as a chemotherapeutic drug in medical settings. However, the possibility of complications constrains the treatment due to the exposure of healthy organs to unnecessary radiation and the drugs’ toxicities. As a result, researchers have been looking for non-toxic chemotherapeutic agents which can be used as radiosensitizers, possibly produced from natural derivatives and nano sized materials. Methods BRF, Cis, and BiONPs were irradiated individually and in combinations with 6 MV of photon beam and 6 MeV of electron beams with 0 to 10 Gy radiation doses on MCF-7, MDA-MB-231, and NIH/3T3 cell lines. Then, the experimental sensitization enhancement ratios (SER) of each treatment obtained were compared to the theoretical dose enhancement factor (DEF). The interactions within the BRF-BiONPs (BB) and BRF-Cis-BiONPs (BCB) combinations were also estimated using the Combination Index (CI). Results BRF induced radiosensitization in all cells under 6 MV photon beam (SER of 1.06 to 1.35), and MDA-MB-231 cells only under 6 MeV electron beam (SER = 1.20). The highest SER values for BiONPs and Cis were obtained from MCF-7 cells under a 6 MeV electron beam (SER of 1.50 and 2.24, respectively). The theoretical DEFs were generally lower than the experimental SERs. Based on the SER and CI relationships, it was estimated that BB and BCB therapy methods interacted in either a synergistic or additive manner. Conclusion The BRF is found to induce relatively less radiosensitization effects compared to the BiONPs and Cis. The BB and BCB combinations have shown better effects with potential for becoming competently suitable radiosensitizers in breast cancer therapies.


Introduction
Radiotherapy (RT) is one of the cancer therapies along with surgery, chemotherapy, immunotherapy, hormone therapy, targeted therapy, stem cell transplant, and precision medicine, as specified by the International Agency for Research on Cancer (IARC) and the National Cancer Institute (NCI). 1,2 There are currently around 7600 RT centers across the world. 3 The International Atomic Energy Agency (IAEA) also stated that RT could be provided alone or in conjunction with chemotherapy and after surgery in cancer treatment programs. 3 Breast cancer accounted for up to 2 million new cancer cases worldwide in 2018, roughly matching the number of lung cancers, followed by colorectal, prostate, and stomach cancers. 1,4 Breast cancer is the principal source of death in This publication is the first to present the radiosensitization impact of each BRF, BiONPs, and Cis under photon and electron beam radiotherapy. Subsequently, the theoretical dose enhancement factors (DEFs) of BB and BCB combinations were compared to the experimental radiosensitization effects from our previous work, 41 and interactions among each component were predicted. This work aims to perceive if combinations of prospective radiosensitizers were more successful than a single radiosensitizer after clinical external beam RT, their comparisons with the theoretical dose enhancement, and their interactions in breast cancer and normal fibroblast cells.

Preparation of Baicalein-Rich Fraction (BRF)
The BRF was extracted and fractionated from OI plant leaves as reported by the previous literature. 13 The OI plant leaves had been verified with voucher number PIIUM 0276. 13 The IO leaves were dried, ground, and subjected to the Soxhlet extraction with methanol solvent (Merck, USA). The extracts were then fractionated using thin-layer chromatography (TLC, Merck, USA) with n-hexane: ethyl acetate (HmbG, Germany) as the mobile phase with 50:50 ratios. Bands from the leaves extract were detected, air-dried, and scraped on TLC sheets that paralleled the band of standard baicalein compound (Sigma-Aldrich, USA). The scraps were eluted with a few mL of methanol and dried at room temperature. The dried powder was given the name BRF since it contained only 75% of the baicalein component. 13 Before being employed in subsequent investigations, the BRF powder was diluted with dimethyl sulfoxide (DMSO) solvent as the primary stock and kept at −20°C. The flow of the BRF extraction and fractionation is depicted in Figure 1.

Preparation of Bismuth Oxide Nanoparticles (BiONPs)
The hydrothermal technique is employed to synthesize the BiONPs, as previously described and in the literature. [44][45][46] The synthesis began with the dilution and stirring of bismuth (III) nitrate pentahydrate (Bi[NO 3 ] 3 .5H 2 O, Sigma Aldrich, USA) and sodium sulfate (Na 2 SO 4, Sigma Aldrich, USA) in distilled water. The yellow precipitates were centrifuged in a 50 mL tube with deionized water after chilling for 10 minutes at room temperature, then washed twice with ethanol. Finally, the precipitates were filtered and dried in an incubator at 80°C on filter paper. The BiONPs were rod-shaped,  with an average diameter of 60 nm, as characterized in the prior research. 44,45 Before being employed in future investigations, the BiONPs powder was diluted with DMSO solvent as the primary stock and kept at room temperature.

Preparation of Cisplatin (Cis)
Cisplatin (PtCl 2 (NH 3 ) 2 , Tokyo Chemical Industry, Japan) was diluted to a concentration of 33.33 mMol/L (mM) in a 1.5 mL tube with DMSO solvent as the primary stock. This concentration was further diluted to achieve the required concentration.

Cytotoxicity Assay
According to our earlier studies, the cytotoxicity tests for the BRF natural compound were conducted. 47

Cell Samples Irradiation
The samples were irradiated at the Nuclear Medicine, Radiotherapy and Oncology Department, Hospital Universiti Sains Malaysia. The irradiations of megavoltage photon and electron beam were conducted using Siemens Primus medical linear accelerator (Siemens Medical Systems, USA). Table 1 shows the results of our investigation into the impacts of six therapy components. The concentrations of each component were chosen based on our previous cytotoxicity experiments. The concentration of BRF (0.76 µg/mL) was 3856 chosen based on the abovementioned cytotoxicity assay. For the 60 nm size of BiONPs, a cytotoxicity experiment revealed that 0.5 mM is a safe dose, and Cis concentration was chosen based on its respective IC 25 value of 1.30 µM, as reported in our previous research. 40 The IC 25 was used instead of the IC 50 to limit the side effects of each BRF and Cis and emphasize the combined effects in the future study. 49 The treatment components were co-cultured with the cells for one hour before irradiation, with a total sample suspension volume of 150 µL in PCR tubes. Triplicate samples were taken for each of the treatment components. Gafchromic TM EBT3 films (Ashland Specialty Ingredients, USA), which were placed beneath the cell sample tubes, were used to verify radiation dosages.

Megavoltage Photon Beam Therapy
The cells were suspended in the 200 µL of PCR tubes at a 1×10 5 cells/mL concentration before getting treated upon each treatment element. Before being exposed to a 6 MV photon beam, the cell samples were put between a superflab bolus (CIVCO ® Radiotherapy, USA) and a 10 cm water equivalent phantom (Nuclear Associates Inc., USA) for backscatter, with a depth of maximum dosage (d max ) of 1.5 cm for a field size of 10 cm x 10 cm. The radiation doses varied from 0 to 10 Gy, with a source-to-surface distance (SSD) of 100 cm.

Megavoltage Electron Beam Therapy
Cell suspensions were prepared in 200 µL PCR tubes at a density of 1×10 5 cells/mL before being treated in triplicates with each treatment component. For a 10 cm × 10 cm applicator, the tubes were arranged with an SSD of 100 cm and a d max of 1.4 cm. The samples were subjected to a 6 MeV electron beam with a 10 cm water equivalent phantom for backscatter and radiation doses ranging from 0 to 10 Gy.

Post-Irradiation Clonogenic Assay
All cell suspensions were seeded onto 6-well plates without washing away the treatment components after the irradiations, with 1.5 mL of new media added to each well. MCF-7 and NIH/3T3 cells were incubated for five days, while MDA-MB-231 cells were incubated for ten days to ensure that the cell colonies developed appropriately, based on the control cells' growth efficiency. After that, the cells in the well plate were gently rinsed, fixed for 30 minutes with cold methanol, and stained for 1 hour with 1% of crystal violet. Then, the plates were rinsed again and air-dried. A flatbed scanner (Epson SeikoCorp, Japan) was used to scan the cell colonies. The picture of colony formation in the wells was processed using ImageJ software with the ColonyArea plugin developed by Guzman et al 50 to calculate the proportion of the area covered by dyed cells. The ratio of colony formation after radiation exposure to the control colonies that were not exposed to radiation was used to compute survival fractions.

Cell Survival Analysis and Radiosensitization Effects Measurement
The cell survivals were evaluated using Equation 1 to get the cell colonies' survival fraction (SF).
The SF for samples with and without therapies were then plotted and fitted using OriginLab Pro 2018 software to the LQ model, 41,51 a gold standard for fitting survival curves in clinical RT. Finally, means and SEM of triple samples are used in expressing the data.
The sensitization enhancement ratio (SER), which demonstrated amplification of radiation effects by each treatment in cells, was used to quantify the experimental radiosensitization effects from the cell survival curves. The SER was computed by dividing the dosage that causes a 50% cell survival fraction (D 50 ) for control cells by D 50

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Theoretical Dose Enhancement Factor Calculation The theoretical dose enhancement factors (DEF) referred to the predicted monoenergetic dose enhancement values that could be computed using several formulas. 52,53 The DEF values utilized in this investigation were calculated using the ratio of the mass-energy absorption coefficient μ en ρ h i E of the BiONPs, Cis, and BRF to water, as given in Equation 3.

DEF ¼
The Z eff of BRF, Cis, and BiONPs had been calculated in our previous research, which are 6, 62, and 78, respectively. 40,54 These Z eff values are approximate to carbon, samarium, and platinum metals, respectively.
Only photon emitters such as radionuclides, LINAC photon beams, and diagnostic x-rays can be calculated theoretically. The electron, on the other hand, is a charged particle. Therefore, only the theoretical DEF for two sources, the HDR brachytherapy with an average energy of 0.38 MeV and the 6 MV photon beam with an effective photon energy of 2.0 MeV, was examined in this work concerning each treatment component. [55][56][57][58] Combination Treatments Analysis of Synergism/Antagonism Various approaches are utilized to analyze the interactions in the combination of several agents or stressors, including those based on the median-effect principle and its mass-action law, Bliss Independence, Loewe Additivity, directional classification system, Constant-Dose Method, and Constant-Efficacy Method. [59][60][61][62] According to prior research on CRT, the interaction of different anticancer agent combinations could be established using the CompuSyn program (ComboSyn, Inc., Paramus, USA). [63][64][65][66][67][68][69][70] The Combination Index (CI) analysis was founded on Chou-CI Talalay's Theorem based on a computer analytical simulation of the median-effect principle and its massaction law. 61,71 Many radiosensitization studies used this approach since it is simple, inexpensive, and primarily produces quantitative data. [63][64][65]67,69,[72][73][74] The software was loaded with doses (radiation doses, Gy) and effects (SF) for each individual and combination therapy. The software then generated the data for CI values for each radiation dose. Finally, the average CI values were calculated.

Cell Viability of BRF
The cell survival curves after 48 hours of BRF treatment are shown in Figure 2. The BRF was observed to be highly toxic to MCF-7 and MDA-MB-231 breast cancer cells but demonstrate less toxicity to NIH/3T3 normal cells. The IC 50 values for MCF-7, MDA-MB-231, and NIH/3T3 were determined using cell viability curves which resulting in values of 11.99, 26.53, and 80.53 µg/mL, respectively. Meanwhile, the IC 25 values calculated for MCF-7, MDA-MB-231, and NIH/3T3 were 0.76, 3.99, and 32.58 µg/mL, respectively. Therefore, the lowest dose of 0.76 µg/mL from IC 25 was used for the following tests. This value is only 0.007% differed from the lowest IC 25 value found in our prior work. 47 In this study, the BRF from the OI plant was evaluated as an alternative to the commercial anticancer drug as a lower cytotoxic radiosensitizer. Based on previous published research regarding the BRF, there are more works on the OI plant extracts in comparison to pure baicalein compound. Therefore, some discussions will be consociated with the OI extracts and baicalein compound. The IC 50 value is the gold standard to speculate about the anticancer effect of plant extracts. A treatment agent with an IC 50 value of less than 30 µg/mL could be considered an anticancer agent. 48 The BRF showed high toxicity on the breast cancer cells MCF-7 (11.99 µg/mL) and MDA-MB-231 (26.53 µg/mL) cells, while not toxic to NIH/3T3 normal cells (80.53 µg/mL). The results are supported by another study on the anticancer properties of BRF towards HeLa and SiHa cells which exhibited IC 50

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Several pathways activated the anticancer effects of OI extracts and baicalein on cancer cells. In MCF-7 and MDA-MB-231 breast cancer cell lines, OI extracts were initially found to induce apoptosis. [75][76][77] BRF or baicalein compound would not disturb the NIH/3T3 cells' proliferation. 13,78 Comparable to the present study, the pathways involved might include the increment of p53 genes and the decrease of cyclinD1, vascular endothelial growth factor (VEGF), CDK4 genes, and collagen-synthesis-related proteins such as α-smooth muscle actin, collagen-1, and collagen-3 gene expressions. 78 The mechanisms might also happen in the present study, which determined the low toxicity effect of the BRF. The cytoselectivity of an agent towards different cells is an excellent characteristic of a potent anticancer agent.
Finally, this study also addressed the IC 25 value of BRF as complementary to Cis results in the previous study, which also used the IC 25 value. 40 Thus, the lowest concentration of 0.76 µg/mL from IC 25 values was chosen as one of the components for the subsequent experiments. The present study only discussed the cell viability after the treatment with BRF. The cell viability after BiONPs and Cis treatments had been reported in our previous research, 40 and the results are mentioned in Table 1.  Table 2 shows all the SER values produced when each component was treated on the three cell lines.

SER of Individual Components BRF, BiONPs, and Cis
A combination of photon beam irradiation and BRF induced cellular sensitization with SER values of 1.06, 1.08, and 1.35 for MCF-7, MDA-MB-231, and NIH/3T3 cells, respectively. In electron beam, BRF treatment caused greater cell death in MDA-MB-231 cells than the control with an SER of 1.20, but MCF-7 cells (SER of 0.9) and NIH/3T3 cells (SER of 0.63) showed good cell survival with the survival curves above the control's curves. After the MCF-7, MDA-MB-231, and NIH/3T3 cell lines were treated with BRF and exposed to HDR brachytherapy, they did not show any cellular sensitization with SER values 0.96, 0.87, and 0.60, respectively. 47 Incubation This study is the first to determine the effects of baicalein-based compound BRF with photon and electron beam radiations in cancer cells. However, as previously stated, the BRF in this study only contained 75% baicalein and was not a 100% pure baicalein compound, as was the case in earlier research. [79][80][81] As a basis, most of the discussion will focus on natural active compounds in OI plants, such as its extract, baicalein, baicalin, and oroxylin A compounds.
Overall, findings showed that when irradiated with 6 MV photon beams

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Sensitization increases by BRF for 6 MV photon beam as reported in this investigation are found to be consistent with the previous studies on substances derived from OI plant extracts such as baicalin and oroxylin A. 82,83 The joined combination of baicalin compound and 6 MV photon beam irradiation sensitized CNE-2R nasopharyngeal cancer cells, whereas SER values of 1.14 to 1.25 were achieved from TE13 and ECA109 esophageal carcinoma cell lines treated with oroxylin A and X-rays. 82,83 When HeLa cervical cells with 10 µg/mL OI leaves extract were bombarded with a 6 MV photon beam, Rahman et al reported an SER of 5.00. 12 In comparison, larger extract concentrations, different cell types, or the combinatorial actions of the baicalein compound with numerous other constituents within the OI extracts might explain why the OI extracts yielded a higher SER value than the baicalin compound. 12,82,83 Furthermore, a baicalein investigation on lung cancer cells demonstrated cellular apoptosis and autophagy via AMPK expression. 84 The stability of the Sestrins (SESN)-2 gene after irradiation stimulated AMPK signaling in MCF-7 cells, and it was initiated in dermal fibroblast cells following baicalin treatment. 85,86 However, following irradiation, AMPK expression in MDA-MB-231 cells had various effects, including inhibiting apoptosis, inducing G1 cell cycle arrest, and promoting cell growth. 87,88 The contradicting effects of AMPK pathways in different cell lines might be the causal Further investigation utilizing immunohistochemistry, Western blotting, or PCR must be performed following the therapy to corroborate this hypothesis.
Nonetheless, natural extracts and phytochemicals have antioxidative characteristics, resulting in radioprotection. The results of an in vivo study on OI extracts irradiated with a 60 Co source with 1.25 MeV energy 89 were comparable and confirmed the radioprotective advantages of BRF under 192 Ir of HDR brachytherapy in this work. Furthermore, overexpression of AMPK genes established the radioprotective effects of baicalin compounds on skin fibroblast cells against UV A and B radiations from ROS and apoptosis. 86,90 Previous research corroborated the current findings, indicating that the BRF may perform its job as an antioxidant to overcome irradiation-induced oxidative stress, which was generated by the low energy delivered by the HDR brachytherapy. Thus, it was hypothesized that the natural agent BRF might cause radiosensitization to a lesser extent than BiONPs and Cis due to its anti-radical qualities. The presence of BiONPs alone during RT increased the radiosensitization impact of the radiation (SER of 1.03 to 1.67), lowering cancer cell survival. The SER values for the BiONPs in the MCF-7 cells had declined following the irradiation sources; HDR brachytherapy, electron, and photon. The effect of irradiation sources on SER values for BiONPs in both MDA-MB-231 and NIH/3T3 cell lines, on the other hand, followed a similar order: electron > photon >  25 More interestingly, the SER values of BiONPs radiosensitization on MCF-7 cells in this investigation were greater than in other studies using other metal NPs under 6 MV photon beam and 6 MeV electron beam radiation energies. 93,94 Using similar MDA-MB-231 cell line, the current work on BiONPs obtained lower SER values (1.03 and 1.20 for photon and electron beams, respectively) compared to the research on gold NPs resulting in a DEF of 1.25 to 1.29 in MDA-MB-231 cells at different photon radiations. 49,95 The data might point to BiONPs being less effective than gold NPs in the triple-negative breast cancer model.
In comparison, the SER value generated by NIH/3T3 normal cells in the presence of BiONPs for HDR brachytherapy (0.77) was lower than that of photon and electron beams. These data conflicted with previous research on selenium NPs on NIH/3T3 cells which discovered a significant 85% cell survival following an 8 Gy dose of X-rays. 96 This finding validated the safe usage of individual BiONPs for HDR brachytherapy only in the future. BiONPs are a novel element in nano-research as a potential radiosensitizer, and no in vivo studies have been conducted. Nonetheless, a few studies on the radiosensitizing effects of bismuth complexes have been discovered in mice. [97][98][99] If the in vivo investigation was carried out, it was predicted that the BiONPs would improve the radiosensitization impact of the X-ray and electron beams on the mice.
Meanwhile The interactions between Cis and electrons may cause the cleavage of the chlorine atom of the Cis molecule and the bonding of the product to the cellular DNA, 100 resulting in the high sensitization enhancement by Cis for electron beam therapy. For example, when treated with Cis and 10 KeV, a DNA double-strand breakage is reported to have an enhancement factor of 1.30. 101 Low energy electrons were used in the investigations on Cis and platinum-complex medicines. 100,101 However, energy ranging from a few MeV to a few KeV might initiate the same processes in cells. 101 Thus, the interactions may be extended to the current work, which employed high-energy ionizing radiation (6 MeV) on MCF-7 and MDA-MB-231 cells.
Moreover, the SER values of BRF, BiONPs, and Cis obtained in the present study could be compared to the intracellular ROS generations due to the presence of these individual components under 192 Ir HDR brachytherapy, photon and electron beams in our past research. 41 Previously, it was found that Cis could induce the highest ROS generation in each clinical beam, followed by the BiONPs and BRF, 41 similar to the SER values in this study. Therefore, these comparisons of the ROS production and the SER values could further confirm that ROS induction is one factor in the radiosensitization effects in cancer cells.
Even though Cis is a good radiosensitizer, a few experiments on kidney cells demonstrated that it causes nephrotoxicity. 102,103 As a result, the investigation, as mentioned earlier, on BRF and BiONPs as an alternative to low-toxic radiosensitizer derived from natural plant and metal NPs were also conducted in this current study. Then, the BRF, BiONPs, and Cis were mixed to form two other treatment combinations, BRF-BiONPs (BB) and BRF-Cis-BiONPs (BCB), as stated in Table 1.
The SER values of the BB and BCB had been mentioned and discussed in our previous published work. 41 It was verified that BB combinations could cause radiosensitization for the MCF-7, MDA-MB-231, and NIH/3T3 cells under all photon and electron beams, as well as brachytherapy (SER of 1.18 to 1.80) except for NIH/3T3 normal cells for the brachytherapy (SER of 0.97). 41  lines for the three clinical RT beams. 41 Subsequently, in the present study, the SER values of BB and BCB combinations were compared to the theoretical DEFs.

Theoretical DEF Estimations
The experimental SER values derived from cell survival curves may be compared to theoretical DEF values for monoenergetic photon beams. Figure 6A depicts the typical fluctuations in the theoretical DEF generated by all treatment components versus radiation energy ranging from 0.001 to 20 MeV. At 0.05 MeV, the BCB component produced a comparable DEF value of 8.58. As a result, the maximum DEFs for BiONPs, Cis, and BB, respectively, are 4.51 at 0.04 MeV, 5.70 at 0.06 MeV, and 4.47 at 0.04 MeV. When reaching 0.1 MeV, the DEF values rapidly decrease. The BRF, on the other hand, did not reveal any peak of the theoretical DEF value. The DEF is affected by the beam energy and the Z eff , as displayed in Table 3. Figure 6B shows a zoomed-in graph of the theoretical DEF values for HDR brachytherapy at 0.38 MeV. At 0.38 MeV energy, the DEF values were steadily falling. Figure 6C shows a zoomed-in graph of the expected DEF values for  Table 3 organizes the theoretical DEF values of 0.38 and 2.0 MeV obtained for each treatment component. The BCB combination was expected to provide the most substantial radiosensitization effects at 0.38 MeV, with a DEF of 1.122, followed by BiONPs alone, BB combination, and Cis alone. The BRF alone yields the smallest DEF value of 0.992 at 0.38 MeV. However, at 2.0 MeV, all the DEF values for the six treatment components were less than one.
Comparisons conducted for the estimated theoretical DEF values and the investigated experimental SER values for HDR brachytherapy are illustrated in Figure 7. Again, there is a large disparity discerned in the BCB effects. In MDA-MB-231 cells, the SER values of the BCB (1.31) treatments are higher than each theoretical DEF value (1.12) but lower than the SER values in NIH/3T3 cells (2.04).
Meanwhile, the experimental SER by BB treatment was most excellent in MCF-7 cells (1.76), followed by the SER for MDA-MB-231 cells (1.18). In general, the SER values of the BB combination were greater than the predicted DEF value (1.07), except for NIH/3T3 cells. The same pattern by cell line types may be seen in the individual Cis therapy results. The DEF of Cis treatment (1.05) fell within the experimental SER values (0.66 to 1.22).
In addition, following the individual BiONPs treatment, the experimental SER in MCF-7 cells (1.67) was larger than the theoretical DEF (1.08). The impacts of BiONPs on MDA-MB-231 and NIH/3T3 cell lines (SER of 1.08 and 0.77, respectively) were lower than their predicted DEF values.
Finally, the experimental SER values of BRF therapy in all cell lines for the brachytherapy are lower than the theoretical DEF (0.99). In the presence of BRF, the SER values of all three cell lines fell below the theoretical DEF levels. Figure 8 depicts the differences in the DEF and SER values of the five treatment components in the three cell lines for photon beam radiation. According to the results, the experimental SER values in the MCF-7, MDA-MB-231, and NIH/ 3T3 cells exceeded the theoretical DEF values.
The computed DEF values for all treatment components were somewhat higher for the HDR brachytherapy, which produced less energy than the photon beam. It is consistent with previous research that found a somewhat larger dose enhancement from radionuclide sources and low-energy X-ray beams than external megavoltage-ranged beam sources. 53,93,104 In particular, the SER values of BRF for HDR brachytherapy are lower than the DEF value, but the SER values were higher than the DEF value for photon beam therapy. The DEF values of BRF were calculated from the theoretical Z eff of the BRF, which approximates a carbon element. 54 This divergence between SER values and DEF values of BRF suggested that, biologically, natural compounds could not fully mimic the actions of the carbon element. The different effects between BRF and carbon could be related to research on carbon fiber implants which found that carbon could neither increase nor decrease the radiation dose in radiotherapy. 105 Besides, the physical reaction interaction between the BRF and other components in the experiments could be less or more than the predicted reaction in the simulations.

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Furthermore, the experimental SERs calculated in MDA-MB-231 cells for the HDR brachytherapy in the presence of BRF and BiONPs are lower than predicted. It is supported by the fact that the MDA-MB-231 cells were shown to be radioresistant. 106,107 The radioresistance of cells was also impacted by the up-and down-regulation of proapoptotic genes such as FADD, DAPK1, CASP8, BAG-4, CASP10, NOD1, and TNFSF10. 108 However, the experimental SER of Cis,

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BB, and BCB treatment components in MDA-MB-231 cells were more than 1, indicating that the radiosensitization effect was a factor in cell death. The physical interactions between BRF, BiONPs, Cis, and radiations might reduce radioresistance and promote radiosensitization to MDA-MB-231 cells.
The calculated DEF would be formed when 5 mg/mL of high-Z (20 to 90) NPs were irradiated with HDR brachytherapy, and 6 MV of photon beam was roughly 1.005 to 1.035 and 1.004 to 1.007, respectively. 53 Meanwhile, a Monte Carlo analysis on the water with 3% Cis for brachytherapy yielded a DEF of 1.07, and a Geant4 simulation with 12 mM of Cis for 6 MV energy yielded a DEF of 1.0047 to 1.0141. 109,110 The current study's theoretical DEF for the individual BiONPs and Cis differed from the studies described above. However, it could be considered consistent because the difference was less than 10%. 53 The variance in DEF might be attributed to differences in component concentration and target location depth. 24,110 Meanwhile, when the BCB combination was present during brachytherapy, the experimental SER in NIH/3T3 normal cells was greater than the theoretical DEF, demonstrating that radiosensitization's physical, chemical, and biological routes might influence the healthy tissues surrounding the cancer sites. Nonetheless, these toxicity effects may be as minimal as the side effects of concurrent CRT in clinical research. Another brachytherapy and Cis clinical investigation discovered a few acceptable hematological and renal effects. 111 In comparison to individual brachytherapy treatment, there were greater vaginal, rectal, and bladder toxicities after a few weeks of chemobrachytherapy treatment. 112 The gap between theoretical DEF and experimental SER might be caused by a few factors, including experimental conditions and setup variables, as well as the exclusion of experimental variable circumstances in theoretical simulation. 113

Combinatorial Treatment Analysis for BB and BCB Combinations
The effects of the combination therapies were examined using a computational analytical simulation to determine the potential interaction. The CompuSyn program was used to simulate the radiation doses and SF values of the therapy components in breast cancer cells and normal fibroblast cells to assess the synergistic, additive, and antagonistic effects of the BiONPs, Cis, or BRF for the BB and BCB combinations. Based on the CI values, the combined therapy can be regarded as synergistic (CI < 0.9), additive (CI of 0.9 to 1.2), or antagonistic (CI > 1.2). The summary of mean CI values quantified from the BB and BCB combination treatments for photon and electron beams, as well as brachytherapy, are shown in Table 4, in comparison to the SER values obtained from our previous work. 41 BB and BCB combinations demonstrated synergistic interactions (CI < 0.9) in MCF-7 breast cancer cells and NIH/ 3T3 normal cells under irradiation of 6 MV photon beam with the CI average of 0.58 to 0.82. However, the average CI value of MDA-MB-231 cells reflected the overall additive effects of both the BB (CI = 0.98) and the BCB (CI = 1.02) treatments when paired with the photon beam. For each BB and BCB treatment combination, the additive interactions calculated in MDA-MB-231 cells would result in the lowest SER levels compared to other cells. The results indicated that photon irradiation with BB and BCB combination had greatly sensitized other cells, especially the MCF-7 cells, through synergistic and additive interactions, resulting in high SER values for each combination.
In the 6 MeV electron beam irradiation, the mean CI values in MCF-7 cells of BB and BCB therapies indicated additive effects (CI of 1.06 to 1.10). However, the CI values suggested synergism in MDA-MB-231 cells. Meanwhile, in NIH/3T3 normal cells, BB combination was estimated to induce synergism (CI = 0.72), in contrast to BCB combination, which might undergo a minor antagonistic interaction (CI = 1.33). From the estimation in our previous work, 41 its SER value of 2.06 contrasted with the weak antagonism effect. However, the detailed unpublished raw data expressed that only the lowest irradiation dose of 1.0 Gy elicited a strong antagonistic reaction. In comparison, higher irradiation doses (2.0 to 10 Gy) had elicited additive and synergistic reactions. As a result, larger doses of irradiation may significantly impact the SER value of BCB combination treatment on NIH/3T3 normal cells.
The current investigation discovered that all cell lines treated with BB and BCB combinations showed synergisms during HDR brachytherapy. The mean CI values of the BB and BCB treatments suggested synergism (CI of 0.59 and 0.64, respectively). In MDA-MB-231 and NIH/3T3 cells, the average CI values of BB and BCB treatment varied from 0.42 to 0.76, also indicating synergistic effects. This result conflicted with the SER of BB for HDR brachytherapy, which shows the SER of 0.97 as if there was no enhancement in the NIH/3T3 cells. 41 Nonetheless, according to another concept of modeling of interaction evaluation between two stressors, 62 Table 2. Thus, it is hypothesized that the BB combination may have additive interaction. However, the reaction was unable to overcome the antioxidant capacity of the BRF, resulting in an SER value of 0.97.
Furthermore, the CI values of the BB combination in all cell lines from all RT sources indicated that there was no antagonistic interaction but only additive and synergistic interactions. This statement demonstrates that BRF may produce radiosensitization when combined with other components while being a natural molecule. This conclusion is corroborated by examining C-phycocyanin natural chemical and X-rays on colon cancer cells, which revealed synergistic effects and a DEF ranging from 1.05 to 1.63. 32 Besides, due to the synergistic interaction of the BRF, BiONPs, and Cis, large SER values of BCB combinations for all RT sources were induced. Furthermore, Cis, coupled with other natural chemicals, such as Ephedra alata and Sterculia quadrifida, displayed synergism in 4T1 and T47D breast cell lines without irradiation, involving caspase-7 and caspase-3 activation, PARP inhibition, and anti-apoptotic Bcl2 protein downregulation. 114,115 The combination of nanofibers and two natural compounds of curcumin and chrysin on T47D breast cancer cells resulted in synergistic responses, including the regulation of caspase-7, caspase-3, p53, Bax, Bcl2, Cyclin D1, and human telomerase reverse transcriptase genes. 71 Generally, the CI values of all treatment combinations for HDR brachytherapy were discovered to have synergistic interactions in all cell lines. For photon beam RT, the combination of BB and BCB had additive effects on MDA-MB-231 cells. In the case of electron beam RT, antagonistic interactions were found in NIH/3T3 normal cells for the BCB combination. In contrast, additive responses were found in MCF-7 cells for both BB and BCB combinations.

Conclusion
The BRF has shown a modest capability to produce radiosensitization effects than the BiONPs and Cis due to its antioxidative qualities. Most of the experimental SER values were higher than the theoretical DEF values, demonstrating the involvement of chemical and biological mechanisms instead of just physical interactions. The radiosensitization effects in BCB combinations were two-fold larger than in other individual BRF, Cis, or BiONPs and BB combination treatments. Radiosensitization effects on breast cancer cells indicate significant impact while small impact on normal cells were observed. Based on the SER and CI associations, it can be estimated that the BB and BCB therapeutic approaches had either synergistic or additive interactions, which indicates the possible potential as radiosensitizers for breast cancer treatments.