Photothermal therapy and radiotherapy of Bi₂S₃-GNRs hybrid nanoparticles in treatment of breast cancer

Abstract

Radiotherapy (RT) is one of the main methods of breast cancer treatment that can be used alone or together with other therapy methods. Also, photothermal therapy (PTT) has attracted attention as another modality in tumor treatment. Recently, combination therapy has been proposed as a new alternative approach to increase the effectiveness of cancer therapy. On the other side, the investigations on the use of metal-based hybrid nanoparticles (MHNs) simultaneously as a radiosensitizer and a photothermal agent in the case of combination of RT and PTT have been increased to enhance the efficiency of cancer treatment too. In this research, Bi₂S₃ NPs, gold nanorods (GNRs) and their hybrid (GNRs-Bi₂S₃ as new MHNs) were synthesized. Then, the quality control tests were confirmed the successful synthesis of NPs and GNRs-Bi₂S₃ hybrid. Next, the likely enhanced effect of combination of RT (at two X-ray doses of 2 and 6 Gy) and PTT (at 808 nm wavelength with 1500 mW power) using of Bi₂S₃-GNRs hybrid (at 3 concentrations of 50, 100 and 200 µg/ml) was investigated for breast cancer therapy in comparison with alone Bi₂S₃ NPs or GNRs in single therapy of RT or PTT. The cell viability test showed a significant reduction at all tested concentrations of Bi₂S₃-GNRs hybrid after single therapy method of RT or PTT. The synthesized hybrid was even more effective in case of combination therapy method compared to use of each of modalities or NPs alone especially at concentration of 200 µg/ml and X-ray dose of 6 Gy. In summary, our novel hybrid showed enhanced effectiveness in killing of breast cancerous cells as MHNs in combination therapy method of RT and PTT.

Introduction

Although significant progress has been made in recent years in the field of early diagnosis and timely treatment for breast cancer, this disease is still the most common cause of death among women¹. Radiotherapy is one of the main methods of treatment in breast cancer, which can be used alone or together with other treatment methods such as surgery, chemotherapy and hormone therapy etc². Although RT has advantages in cancer treatment, it has limitations such as inducing harm to nearby normal tissues and leading to severe side effects¹. To overcome these limitations, some strategies have been investigated to increase the effectiveness of RT. One of these ways is the use of radiation sensitizers². Radiosensitizers are compounds that, if administered at the same time of RT, have the ability to increase the lethal effect of radiation in tumor cells. These compounds seem to stabilize the free radicals produced by radiation damages at the molecular level³. In this respect, some of nanomaterials like gold nanoparticles (GNPs) and bismuth sulfide NPs with high Z and X-ray attenuation coefficient can play a key role in tumor therapy⁴. In one of the first researches investigating the sensitization effect of GNPs, Henfield et al., found a significant difference in mammary tumor growth rate between mice treated with X-ray radiation alone and those administered with GNPs followed by radiation⁵. In this respect, GNPs is one of the metal NPs we used as an X-ray radiation sensitizer in this study. Bismuth sulfide NPs has long been used as a dose-enhancing agent in RT⁶. So, it was another metal NP we used as a radiosensitizer in this project. Xiaju Cheng et al. used Bi₂S₃ NPs to enhance radiation therapy effects in 2017 in mouse model with breast tumor. This research showed that Bi₂S₃ NPs could boost the effectiveness of radiation therapy by enhancing anti-tumor efficacy⁷.

On the other hand, PTT has attracted considerable attention as another treatment modality with controllable and non-invasive properties in tumor treatment in recent years⁸. Photothermal conversion agents are used to generate heat for thermal ablation of cancerous cells when exposed to laser radiation in NIR region (700–900 nm). A large number of near infrared (NIR) absorbing agents were used in PTT again including GNPs and bismuth NPs^9,10. These absorbent agents have shown great success in killing of cancerous cells in vitro and in vivo. PTT using GNPs as a conversion agent was first reported by Pitsillides et al. in 2003¹¹. They used GNPs combined with a visible pulsed laser to destroy T lymphocyte cells successfully. The El-Sayed group further studied PTT of malignant epithelial cells (HSC and HOC cell lines) using GNRs. They showed that increased uptake of nanorods by cancerous cells reduces the energy required for cell destruction by about half compared to nonmalignant cells¹². As it was mentioned, bismuth is another NP reported as a photothermal agent in PTT method. Nanoparticles based on bismuth like Bi₂S₃, due to high stability, high surface area and strong NIR absorption can act as a photothermal conversion agent^6,13.

However, despite huge investments to develop new cancer treatment methods, only limited success has been achieved with current clinical treatment options due to the complexity, diversity, and heterogeneity of tumors. To overcome these limitations, combination therapy employing two or more treatment modalities simultaneously, has emerged as an alternative strategy. On the other hand, recent studies have explored the suitable use of MHNs to enhance the effectiveness of cancer therapy in combination methods¹⁴. By integrating NPs with diverse therapeutic approaches, more effective treatments can be achieved^15–17. Because, many studies performed up to now demonstrated that both GNPs and bismuth can be used either as a radiosensitizer or as a photothermal agent, we hybridized GNPs in rod shape with Bi₂S₃ NPs in spherical shape for making a novel MHNs (GNRs-Bi₂S₃ hybrid), simultaneously as a photothermal agent in PTT method and X-ray radiation sensitizer agent in RT method for breast cancer treatment. In summary, we combined two important modalities of RT and PTT using the new designed hybrid of GNRs and Bi₂S₃ NPs for the first time to develop a more effective procedure for killing of breast cancer cells more effectively and less harmfully in a very detailed comparison study.

Materials and methods

Materials

Bismuth (III) nitrate pentahydrate (Bi(NO₃)₃), bovine serum albumin (BSA), ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and phosphate buffer saline (PBS) were purchased from Sigma Aldrich, USA, Nitric acid (HNO₃), silver nitrate (AgNO₃), chloroauric acid hydrate (HAuCl₄), sodium borohydride (NaBH₄), dimethyl sulfoxide (DMSO), cetyltrimethylammonium bromide (CTAB) and methylthiazol tetrazolium (MTT), trypan blue, streptomycin, penicillin and trypsin-EDTA were purchased from MERC, Germany. Mouse breast carcinoma cell line (4T1) was purchased from Pasteur institute, Tehran, Iran. FBS and DMEM medium were purchased from Gibco, USA.

Bi₂S₃-BSA NPs synthesis

This was achieved in a facile and mild way by using bovine serum albumin (BSA) as reducer and stabilizer. 250 mg of BSA was dissolved in 8 ml of water and 50 mmol of Bi(NO₃)₃ in 1 ml of 2 mol HNO₃ was slowly added to BSA while stirring, and after 30 min, 2 mol NaOH was added to the solution to reach the pH to 12. Then, the solution was stirred for 12 h until its color turned from pale yellow to black that indicated the formation of bismuth sulfide NPs coated with BSA. Finally, the solution was dialyzed against distilled water for 48 h for purification¹³.

GNRs synthesis

GNRs were synthesized using seed-mediated growth method¹⁸. At first, 250 µl of 1 mmol HAuCl₄ was added to 7.5 ml of 0.2 mol CTAB. Then, 600 µl of 10 mmol NaBH₄ was added to the solution and the reaction was allowed to continue for 2 h in a bain-marie at 30 °C until the color of solution turned to pale brownish golden (seed solution). Then, in order to prepare the growth solution, 200 µl of 1 mmol HAuCl₄ was added to 4.7 ml of 0.2 mol CTAB and 40 µl of 4 mmol AgNO₃. Then, 32 µl of 78.8 mmol ascorbic acid was added to the above solution and stirred slowly until the color of solution became transparent. In next step, 21 µl of the seed solution was added to the growth solution and the resulting solution was incubated for 2 h to form GNPs in rod shape.

Synthesis of Bi₂S₃-GNRs hybrid

Firstly, the solution of synthesized GNRs was centrifuged at 1400 rpm for 10 min in a microtube to remove CTAB. Then, the supernatant was poured off and 2 ml of deionized water was added to the remaining sediment. This process was repeated three times to remove CTAB completely. Next, the GNRs solution was sonicated to dissolve the NPs completely. Then, Bi₂S₃-BSA NPs solution was added dropwise to the GNRs solution to cover the surface of NPs under sonication condition to make Bi₂S₃-GNRs hybrid¹⁷.

Quality control tests of synthesized NPs

The tests of UV-vis spectroscopy (F-2500, Hitachi, Japan) was applied to record the spectra of synthesized GNRs, Bi₂S₃ NPs and Bi₂S₃-GNRs hybrid in the wavelength range of 200 to 900 nm. X-ray diffraction (XRD) (XRD; a Bruker AXS.

model D8 Advance difractometer) by using a Cu-Kα1 radiation source, λ = 0.15406 nm. Te XRD data in 2θ ranging from 10° to 80° were collected with a scanning step size of 0.02°) technique was used to examine the general crystal structure of GNRs, Bi₂S₃ NPs and the successful formation of Bi₂S₃-GNRs hybrid. Fourier transform infrared (FTIR) (Vector 22, Bruker, Germany) technique was used for the record of spectra of synthesized NPs at the frequency between 400 and 4000 cm¹⁻. The microscopic images of GNRs, Bi₂S₃ and Bi₂S₃-GNRs hybrid were gained by a transmission electron microscopy (TEM) (Ziess Em-900 Germany).

Cell cytotoxicity test

The 4T1 cells (4T1 cell line (mouse breast cancer cell line) were purchased from Pasteur Institute cell bank in Tehran.) were seeded in 96-well plates at the density of 1.5 × 104 cells per well in 100 µl of complete DMEM culture medium and incubated for 24 h. Then, the plates were split into the three groups for the different trials including: a group of 3 plates for photothermal therapy (using Bi2S3, GNRs and Bi2S3-GNRs hybrid each one plate), 2 groups of 3 plates for 2 and 6 Gy radiotherapy (using Bi2S3, GNRs and Bi2S3-GNRs hybrid each two plates) and 2 groups of 3 plates for combination of RT (2 and 6 Gy) and PTT (using Bi2S3, GNRs and Bi2S3-GNRs hybrid each two plates). In this study, we used 6 groups of 4T1 cells as the controls for comparison: one group without any NPs and any X-ray or laser radiations (just 4T1 cells), two groups just for 4T1 cells plus X-ray or NIR laser radiations and 3 groups each containing just for 4T1 cells plus Bi2S3, GNRs or Bi2S3-GNR hybrid alone (no X-ray or NIR laser radiations). Finally, the MTT assay was used to determine the killing capability of PTT, RT and simultaneous application of both methods (combination therapy) using GNRs, Bi2S3 and Bi2S3-GNRs hybrid were investigated as following:

Photothermal therapy

The first 3 plates were labeled for treating with PTT modality using Bi₂S₃ NPs, GNRs and Bi₂S₃-GNRs hybrid, individually. 100 µl of Bi₂S₃ NPs were added to the wells of first plate at various concentrations of 50, 150, and 200 µg/ml. Then, the plate was incubated at 37 °C for 5 h. The old cultivation medium was emptied; the cells were washed twice by PBS and a new culture medium was added to each well. Then, the plate was exposed to infra-red laser radiation (IR Laser Diode, Hamerz-Rad Company, Tehran, Iran) at 808 nm wavelength with 1500 mW power for 2 min on each well¹⁹. Next, the cells were kept at a 37 °C incubator for 24 h. Finally, the cell viability percentage was evaluated by measuring the absorbance proportion in the cells of sample group to the control group using MTT method. The same procedure was repeated for the second and third plates using GNRs and Bi₂S₃-GNRs hybrid, respectively.

Radiotherapy

There were 2 groups of 3 plates for investigating the effect of doses of 2 and 6 Gy X-ray radiation at the various concentrations of 50, 150, and 200 µg/ml of Bi₂S₃ NPs, GNRs or Bi₂S₃-GNRs hybrid. 100 µl of Bi₂S₃ was added to each well and subjected to 2 and 6 Gy doses of X-ray radiation (each dose one plate) with an energy of 6 megavolts (Siemens, model Oncor, Germany). The gantry angle was set at 180 degrees and a 1.6 cm thick slab was placed under the plates to adjust the maximum depth on the cells. To ensure complete scattering conditions, several slabs were placed on the plate. 200 and 600 monitor units were used for 2 and 6 Gy radiation, respectively. Then, the cells were incubated for 24 h at 37 °C. Next, the MTT assay was performed and the cell viability percentage was gained by measuring the absorbance proportion in the cells of sample group to control group. Then, the same procedure was repeated for the second and third plates containing GNRs and Bi₂S₃-GNRs hybrid, respectively.

Combination of photothermal therapy and radiotherapy

The combined effect of RT and PTT was investigated on killing of breast cancer cells using Bi₂S₃ NPs, GNRs and Bi₂S₃-GNRs hybrid. To perform this, the 6 plates with 4T1 cells were treated as follows: 1th plate: GNRs + RT (2 Gy) + PTT, 2nd plate: Bi₂S₃ + RT (2 Gy) + PTT, and 3rd plate: GNRs-Bi₂S₃ + RT (2 Gy) + PTT. The same procedure was exactly repeated with the next 3 plates but at the dose of 6 Gy instead of 2 Gy. To perform the assay, after 24 h of cell incubation, the nanoparticles were added to the plates at the concentrations of 50, 150, and 200 µg/ml. Then, after 5 h of incubation, the plates were exposed to the NIR laser (1.5 W/cm2) for 2 m. Then, the plates were transferred to the radiotherapy center and were exposed to X-ray radiation in two separate groups of 3 with doses of 2 and 6 Gy. Finally, the MTT assay was used to investigate the cell viability as mentioned above.

Statistics

Data analyses were performed using Graphpad Prism version 8.0.2. (GraphPad Software, San Diego, CA, USA), Descriptive statistics for study variables were expressed as mean, standard deviation, minimum and maximum. Tukey’s multiple comparisons test was used for comparison of means between and among different subgroups of study. P < 0.001 was considered statistically significant.

Results and discussion

Characterization of synthesized NPs

The successful synthesis of Bi₂S₃ NPs, GNRs and Bi₂S₃-GNRs hybrid and their characterization including size and morphology were assessed using various techniques, including UV-visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, and transmission electron microscopy (TEM).

UV-vis spectroscopy

The spectrum of Bi₂S₃ NPs showed that the majority of the absorption peak was below 300 nm, with the highest point at 218 nm that revealed the successful synthesis of Bi₂S₃ NPs (Fig. 1, blue line)¹⁷. As shown in Fig. 1 (red line), the synthesized GNRs have two surface plasmon resonance peaks. The strong peak at 755 nm was due to the longitudinal electron oscillations of the nanorods, while the smaller peak at 520 nm was due to the electron oscillations that pass through the width of nanorods²⁰. The absorption spectrum of Bi₂S₃-GNRs hybrid not only showed a Bi₂S₃ peak with a shift at 220 nm, but also showed a clear peak at 747 nm corresponding to the longitudinal electron oscillations of GNRs that demonstrated successful hybridization (Fig. 1, green line).

Fig. 1.

Absorption spectrum curve of synthesized GNRs (red line), Bi₂S₃ NPs (blue line) and Bi₂S₃-GNRs hybrid hybrid (green line).

XRD

The result related to the determination of the crystal structure of Bi₂S₃ NPs was shown in Fig. 2, blue line. The sharp peaks are visible on the graph, with the most prominent ones located at lower angles. The crystal structure of Bi₂S₃ NPs was confirmed according to JCPDS No. 79-2384. The XRD pattern of synthesized GNRs typically showed several reflections corresponding to the face-centered cubic crystal structure of Au (red line). The peak positions for GNRs were approximately: 37.87 (corresponding to 111) and 43.43 (corresponding to 200)²¹. The XRD pattern for Bi₂S₃-GNRs hybrid revealed several sharp peaks (green line). The peaks around 28° and 55° align with the characteristic angles for Bi₂S₃ and the peaks around 38° and 44° align with the characteristic angles for gold in the sample demonstrated successful hybridization.

Fig. 2.

XRD patterns of Bi₂S₃ NPs (blue line), GNRs (red line) and Bi₂S₃-GNRs hybrid (green line) confirmed the crystal structure of prepared NPs. XRD pattern of Bi₂S₃-GNRs hybrid revealed sharp peaks corresponding to Bi₂S₃ and GNRs presence in the sample.

FT-IR

Figure 3 displays the FT-IR spectra of Bi₂S₃-BSA NPs, GNRs and Bi₂S₃-GNRs hybrid. The spectrum of Bi₂S₃ NPs showed the absorption band at the wavenumbers of 1646.65 cm⁻¹ and 1550 cm⁻¹ related to BSA and wavenumber of 2374 cm⁻¹ belongs to S-H stretching of BSA(green line)²². The spectrum of GNRs exhibited characteristic absorption bands at wavenumbers of 485.90 cm⁻¹, 612.87 cm⁻¹, 1352.01 cm⁻¹, 1637.24 cm⁻¹ related to GNPs. (red line). The band at 485.90 cm⁻¹ is attributed to Au–Cl stretching vibrations, which may arise from residual used during synthesis. The peak at 612.87 cm⁻¹ is associated with Au–O or Au–S surface interactions, possibly due to stabilizing agents or trace impurities. The band at 1352.01 cm⁻¹ may correspond to C–N stretching from CTAB residues. The peak at 1637.24 cm⁻¹ is likely due to N–H bending or C = O stretching from organic stabilizers such as CTAB or BSA²¹. The FTIR spectrum of Bi₂S₃-GNRs hybrid shared absorption peaks related to both Bi₂S₃ and GNRs (blue line) that showed the successful hybridization.

Fig. 3.

FT-IR spectra of Bi₂S₃ NPs, GNRs and Bi₂S₃-GNRs hybrid. The Bi₂S₃ NPs exhibits absorption bands related to BSA and S-H (green line), while the GNRs shows characteristic absorption peaks at wavenumbers of 485.90 cm⁻¹, 612.87 cm⁻¹, 1352.01 cm⁻¹, 1637.24 cm⁻¹ (red line). The Bi₂S₃-GNRs hybrid shares absorption features from both Bi₂S₃ and GNRs (blue line).

TEM

To determine the size and morphology of the Bi₂S₃ NPs, GNRs and successful hybridization of Bi₂S₃ NPs with GNRs, TEM was used. Figure 4 shows TEM image of GNRs in rod shape with the length of 40 ± 5 nm and width of 9 ± 2 nm which was successfully synthesized via the seed-mediated growth method. The uniform dispersion and spherical shape of Bi₂S₃ -BSA with the size of 40 ± 5 nm in diameter was shown in figure. TEM image also confirmed successful hybridization of Bi₂S₃-BSA with GNRs.

Fig. 4.

TEM image of GNRs confirmed the rod shape of synthesized GNPs (a). The micrograph of Bi₂S₃-GNRs hybrids (b) showed the successful hybridization and size distribution histograms from TEM analysis (c).

PTT effect of Bi₂S_3, GNRs and Bi₂S₃₋GNRs on killing of breast cancer cells

Figure 5 shows the effect of NIR laser radiation on killing of breast cancer cells using Bi₂S₃, GNRs and Bi₂S₃-GNRs hybrid in different concentrations of 50, 100 and 200 µg/ml. The results showed that the cell viability was decreased significantly in all concentrations of Bi₂S₃-GNRs hybrid after NIR laser irradiation in comparison with the control group (just cells, no laser, no NPs) (P < 0.0001), but Bi₂S₃ and GNRs were effective only at 100 and 200 µg/ml (P < 0.0001). Furthermore, the comparison study among the NPs on their capability of killing properties, revealed the significant difference between the killing effect of Bi₂S₃ or GNRs with the hybrid at 200 µg/ml for the hybrid (P < 0.0001). Nearly similar to the first part of our work, Zhao and colleagues in 2020 conducted a study utilizing GNPs-Bi₂S₃ but in a nanoflower shape as a photothermal agent to kill Hela cells²³. They observed a significant decrease the same as the results of our PTT study in cell viability compared to GNPs or Bi₂S₃ NPs alone and their mixture. Since the cell groups treated with GNRs and Bi₂S₃-GNRs hybrid alone (just NPs with no laser) as well as cells treated with NIR laser radiation alone (no NPs) did not show any impact on killing of 4T1 cells, their effects were not mentioned in the chart due to the summarization.

Fig. 5.

The PTT effect of NIR laser radiation at 808 nm wavelength with 1500 mw power for 2 m on killing of breast cancer cells using Bi₂S₃, GNRs and Bi₂S₃-GNRs hybrid in different concentrations of 50, 100 and 200 µg/ml. The cell viability significantly decreased in all concentration of Bi₂S₃-GNRs hybrid after IR laser radiation indicated significant difference with control group (P < 0.0001). considerable difference was observed between the effect of Bi₂S₃, GNRs and the hybrid at 200 µg/ml (P < 0.0001).

X-ray radiation effect of Bi₂S_3, GNRs and Bi₂S₃₋GNRs hybrid on killing of breast cancer cells

In this study, the X-ray radiation effect at two doses of 2 and 6 Gy using of Bi₂S₃, GNRs and Bi₂S₃₋GNRs hybrid as the radiation sensitizers on survival rate of 4T1 cells was investigated in vitro. Figure 6a illustrates the impact of prepared nanoparticles (NPs) on 4T1 cells after irradiation with 2 Gy radiation dose at concentrations of 50, 100, and 200 µg/ml. The findings revealed a significant difference between the cells treated with synthesized NPs plus 2 Gy radiation in comparison with the cells treated just with X-ray radiation specially at 100 and 200 µg/ml (p < 0.0001), but the results indicated no significant difference between Bi₂S₃, GNRs and Bi₂S₃₋GNRs hybrid at any concentration (ns). Figure 6b presents the outcome of the same test under the 6 Gy X-ray dose. Similarly, the cell viability was significantly decreased in the case of using the NPs as the radiosentisizers compared to 6 Gy radiation alone especially for Bi₂S₃₋GNRs hybrid (p < 0.0001). The analysis study among the NPs showed a significant difference for the hybrid compared to Bi₂S₃ or GNRs at 200 µg/ml concentration (p < 0.0001). Because after incubation of 4T1 cells with the prepared NPs (without X-ray radiation), no observable cytotoxicity was detected on cells at three used concentrations, so their effects on the cell viability were not mentioned in the related charts (Fig. 6a and b) for summary.

Fig. 6.

The study examined the effects of Bi₂S₃, GNRs and Bi₂S₃-GNRs hybrid on 4T1 cells under 2 Gy and 6 Gy radiation doses. Significant differences were found when using 100–200 µg/ml of all NPs, particularly hybrid NPs, which showed notable differences from Bi₂S₃ at 200 µg/ml. Under 6 Gy irradiation, cell viability decreased significantly with 100–200 µg/ml of hybrid NPs, and similar results were seen with 200 µg/ml of Bi₂S₃ and GNRs. Bi₂S₃-GNRs hybrid at 200 µg/ml were significantly more effective as radiosensitizers compared to Bi₂S₃ and GNRs. No significant differences were observed at concentrations below 200 µg/ml.

Enhancing X-ray radiation damages using GNPs and Bi₂S₃ NPs as the radiosentisizers have been examined in many researches. Abhari et al. used Bi₂S₃ and GNPs both in spherical shape (in core-shell morphology) for enhancing radio sensitization against 4T1 breast cancer cells¹⁷. Similarly, they reported that when the cells were exposed to X-ray radiation at the presence of GNPs, Bi₂S₃ NPs or Au-Bi₂S₃ heterodimer, it can be concluded to more damages compared to cells irradiated alone especially for their designed heterodimer at 6 Gy dose.

Combination effect of RT with PTT using Bi₂S₃, GNRs and Bi₂S₃₋GNRs on killing of breast cancer cells

Finally, to accomplish the goal of this study, the combination effect of RT with PTT using of Bi₂S₃, GNRs and Bi₂S₃₋GNRs hybrid simultaneously as the radiosensitizers as well as photothermal agens on survival rate of 4T1 cells was investigated. Figure 7a illustrates the lethal effect of combining RT at 2 Gy dose with PTT using Bi₂S₃ NPs, GNRs and Bi₂S₃-GNRs hybrid at the concentrations of 50, 100, and 200 µg/ml on breast cancer cells. Compared to the cells treated with X-ray plus NIR laser radiations (without any of the synthesized NPs), the hybrid showed significant difference at concentrations of 100 and 200 µg/ml after combination therapy (p < 0.0001), while the same survey for Bi₂S₃ or GNRs alone indicated that these NPs were effective only at 200 µg/ml (p < 0.0001). A significant decrease in cell viability was observed for hybrid NPs at 200 µg/ml compared to either Bi₂S₃ NPs or GNRs using combination therapy modality, but no differences in cell viability was seen at concentrations of 50–100 µg/ml (p < 0.0001). Figure 7b displays the results of the above test conducted for 6 Gy X-ray plus NIR laser irradiations. As the chart shows, all synthesized NPs including the hybrid were more effective in killing of breast cancer cells especially at concentrations of 100 µg/ml (p < 0.001) and 200 µg/ml (p < 0.0001) in comparison with the cells treated just with X-ray plus NIR laser radiations (no NPs). The comparison study between Bi₂S₃, GNRs and Bi₂S₃-GNRs indicated that the hybrid was significantly more effective than the other two NPs at all concentrations used in this research when the combination therapy was being used (p < 0.0001). Because after incubation of 4T1 cells with the prepared NPs (without X-ray and NIR laser radiations), no observable cytotoxicity was detected on the cells at three used concentrations, so their effects on the cell viability were not mentioned in the related charts for summary.

Fig. 7.

The study examined the lethal effects of combining 2 and 6 Gy RT with PTT using Bi₂S₃ NPs, GNRs, and Bi₂S₃-GNR hybrid on breast cancer cells. Combination therapy of 2 Gy revealed that hybrid NPs showed significant effectiveness at 100 and 200 µg/ml compared to the X-ray plus laser group without NPs. Bi₂S₃ and GNRs were only effective at 200 µg/ml. No differences in cell viability were observed among all NPs at 50–100 µg/ml, but hybrid NPs at 200 µg/ml significantly decreased cell viability compared to Bi₂S₃ or GNRs.Under 6 Gy X-ray plus laser irradiation, hybrid NPs were effective at 100 and 200 µg/ml. Bi₂S₃ and GNRs were only effective at 200 µg/ml. No differences were found among Bi₂S₃, GNRs, and the hybrid at 50 and 100 µg/ml, but hybrid NPs were significantly more effective at 200 µg/ml.

We could not find any research so comprehensive and general like ours in the field of combination therapy method using Bi₂S₃-GNRs hybrid (Bi NPs in spherical shape) and (GNPs in rod shape) both simultaneously as a radiosensitizer and a photothermal agent to kill the breast cancer cells. In an approximate similar study, Xin Wang et al. investigated the combination effect of RT and PTT using Au-Bi₂S₃ hetero nanostructure (gold in nanocrystal shape and Bi₂S₃ NPs in rod shape) and reported the enhanced effect of combination therapy in comparison with single RT or PTT. They revealed that the heat generated by PTT can increase the sensitivity of cancer cells to radiation, while the free radicals generated during RT can enhance the thermal damage caused by PTT²⁴.

Conclusion

In this study we developed a strategy to highlight the promising role of combination of two therapy methods of RT and PTT using Bi₂S₃-GNRs hybrid (our novel synthesized nanostructure) simultaneously as a radiosensitizer as well as a photothermal agent to enhance the therapeutic outcomes of breast cancer treatment. The results showed that the new designed hybrid was significantly more effective than alone Bi₂S₃ NPs or GNRs in the case of using of combination therapy procedure especially at the concentration of 200 µg/ml and X-ray dose of 6 Gy. The future work must be conducted to use other shapes of Bi₂S₃ NPs and GNPs, other configurations, other concentrations, other X-ray doses or laser parameters etc. to optimize the killing efficacy of combination therapy modality.

Author contributions

Author contribution statement Faranak Saghatchi: Methodology; Farshid Babapour Mofrad: Supervision; Hossein Danafar: Supervision; Elham Saeedzadeh: Formal analysis; Mojtaba Salouti: Formal analysis.

Data availability

Data availabilityThe datasets used during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.