Bovine serum albumin and folic acid-modified aurum nanoparticles loaded with paclitaxel and curcumin enhance radiotherapy sensitization for esophageal cancer

Abstract Background Nanocarrier systems have been used in the study of esophageal cancer (EC) and other diseases, with significant advantages in improving the non-targeted and nonspecific toxicity of traditional formulations. Some chemotherapeutic drugs and high atomic number nanomaterials have sensitization effects on ionizing radiation and can be used as chemoradiation sensitizers. Methods Aurum (Au) nanoparticles were modified by bovine serum albumin (BSA) and folic acid (FA), and were core-loaded with paclitaxel (PTX) and curcumin (CUR). The basic characteristics of FA-BSA-Au@PTX/CUR nanomedicines were evaluated by transmission electron microscopy, Fourier transform infrared spectroscopy, and Malvern Zetasizer. The encapsulation and release of drugs were monitored by ultraviolet–visible spectrophotometry (UV–Vis). The biological toxicity and radiotherapy sensitization effect of FA-BSA-Au@PTX/CUR were observed by cell viability, colony formation, cell apoptosis, cell cycle distribution, and γ-H2AX analysis experiments. Results The prepared nanomedicines showed good stability and spherical morphology. The results of cell uptake and cell viability detection revealed that FA-BSA-Au@PTX/CUR could specifically target EC cell KYSE150 and exert a certain inhibitory effect on proliferation, with no obvious toxicity on healthy cells Het-1A. In addition, the results of the colony formation experiment, cell apoptosis detection, cell cycle distribution, and γ-H2AX analysis showed that compared with X-rays alone, FA-BSA-Au@PTX/CUR combined with X-rays exhibited relatively stronger radiotherapy sensitization and anti-tumor activity. Conclusions FA-BSA-Au@PTX/CUR could target EC cancer cells and act as a safe and effective radiotherapy sensitizer to improve the radiotherapy efficacy of EC. Graphical Abstract


Introduction
Esophageal cancer (EC) is the seventh most common cancer and the sixth leading cause of cancer-related deaths worldwide.According to the 2020 Global Burden of Disease (GBD) statistics, EC is responsible for one out of every 18 cancer deaths (Sung et al. 2021).EC is frequently caused by multiple factors, including dietary habits, carcinogens, and genetic factors.The main clinical characteristic of EC patients is gradually worsening dysphagia, which can ultimately result in multiple organ failure and death due to starvation (Uhlenhopp et al. 2020).Although combination therapy strategies have greatly improved the prognosis of EC patients, the 5-year survival rate remains relatively low, and traditional anticancer drugs have limitations such as poor targeting, high toxicity and side effects, and short halflife, which cannot benefit all EC patients effectively (Cao et al. 2022).Evidence suggests that compared with traditional formulations, nanomedicines have unparalleled advantages in cancer treatment.The clinical translation of nanomedicine may provide better treatment options for EC patients (Zhan et al. 2020;Pavitra et al. 2021).
Currently, various nanocarrier systems have been developed to reduce the toxic side effects of traditional chemotherapy.Simultaneously, some high atomic number (Z) nanomaterials (such as noble metals) have also been used as radiation sensitizers, which can enhance the efficacy of radiotherapy (Schuemann et al. 2016).Aurum (Au) nanoparticles (NPs) have good chemical stability and biocompatibility as noble metal materials.They can not only concentrate the energy of ionizing radiation at the tumor site, showing a dose accumulation effect of radiation, but also produce more cytotoxic secondary charged particles, thus maximizing X-ray damage to the lesion (Schuemann et al. 2016;Song et al. 2017).In the study by Hainfeld et al. (2004), mice with tumors were injected with Au NPs and received radiotherapy, and most of these particles were cleared from the body through the kidneys without causing toxicity in mice.
Au NPs possess good surface modifiability and can be further used to reduce NP toxicity and improve biocompatibility in vivo by coating with specific protective molecules.Bovine serum albumin (BSA) is a common surface modifier for metal NPs (Chu et al. 2019), and BSA-coated Au NPs (BSA-Au) have been widely used for tumor monitoring and treatment (Zu et al. 2016;Wei et al. 2022).Murawala et al. (2014), for example, prepared BSA-Au NPs loaded with methotrexate (MTX) and demonstrated that BSA-Au-MTX had a more significant inhibitory effect on breast cancer cells than an equivalent dose of free MTX.Moreover, due to the high expression of folate receptors (FRs) on cancer cells, folic acid (FA) can be conjugated to Au NPs to improve the targeting effect of nanomedicines on cancer cells (Narmani et al. 2019).In the study by Sinai Kunde and Wairkar (2022), the antiestrogen drug, raloxifene hydrochloride (RLX), is loaded into BSA-NPs and further surface modified with FA, with the results of cell experiments showing that this complex nanomedicine has a better therapeutic effect on breast cancer than free RLX and RLX-BSA-NPs without FA modification.
Paclitaxel (PTX) is a chemotherapeutic agent extracted from the bark of the Pacific yew that can induce cell arrest in the G2/M phase by promoting microtubule aggregation and assembly, thereby exerting an anti-tumor effect (Safran and Rathore 2002).The G2/M phase is the most sensitive stage of the cell cycle to ionizing radiation, so drugs that can induce G2/M phase arrest are potential radiosensitizers.PTX has been shown to make various human cell lines radiosensitive (Sikov and Safran 1997).Moreover, as single-drug therapy frequently results in adverse reactions and poor patient compliance, some traditional Chinese medicines are often combined to alleviate side effects and improve therapeutic efficacy.Curcumin (CUR) is the most representative traditional Chinese medicine, extracted from turmeric and known for its anti-inflammatory and anti-tumor properties.The excellent pharmacological activity of CUR has been proven to reduce the side effects of PTX (Giordano and Tommonaro 2019;Ashrafizadeh et al. 2020).We speculated that the combination of CUR and PTX had the potential to improve EC treatment.
In summary, we constructed a novel nanomedicine (FA-BSA-Au@PTX/CUR) to improve the efficacy of EC radiotherapy by loading PTX and CUR onto BSA and FAmodified Au NPs (Graphical abstract).We prepared the composite nanomedicine using a simple and low-toxicity method and systematically characterized its morphology, particle size, drug loading, and release.We also investigated the cellular uptake, cytotoxicity, and radio-sensitizing effects of FA-BSA-Au@PTX/CUR in vitro.From the research results, the nanomedicine prepared in this study not only exhibited good targeting to EC cells but also showed significant toxicity and radio-sensitizing effects on EC cells, demonstrating the potential of nanomedicines in combination therapy and providing a corresponding basis for the clinical translation of nanomedicines.

Preparation of BSA-Au@PTX/CUR
Two milligrams of PTX and 2 mg of CUR were added to 10 mL of BSA (5 mg/mL) and sonicated to dissolve.The solution was stirred at room temperature for 5 min, then 5 mL of a 10 mM HAuCl4 solution was added and stirred for 10 min.One milliliter of 1 M NaOH was added dropwise to the solution to adjust the pH to 12, and the solution was then stirred at 70 � C for 1 h.After the reaction, the material was centrifuged, washed, and freeze-dried for storage.

Preparation of FA-BSA-Au@PTX/CUR
Ten milligrams of FA was dissolved in 3 mL of DMSO, followed by the addition of 2 mg of EDC and 3 mg of NHS.The mixture was stirred in the dark at room temperature for 2 h to activate the FA.Then, 20 mL of BSA-Au@PTX/ CUR (1 mg/mL) was mixed with the FA solution and stirred in the dark overnight.The material was centrifuged, washed, and freeze-dried for storage.

Material characterization
The microscopic morphology of the materials was observed by transmission electron microscopy (TEM, JEM-2100, JEOL, Akishima, Japan).In short, a small number of samples were dissolved in ethanol, and 1-2 drops of samples were dropped into the copper mesh.After drying, the samples were placed under TEM and photos were taken.
The particle size distribution and zeta potential of the materials were analyzed using a Malvern Zetasizer Nano-ZS90 (Malvern, Malvern, UK).We first prepared about 5 mL of a sample with an appropriate concentration, then took about 2/3 volume of the solution from the colorimetric dish for zeta potential measurement, and dropped 1-2 mL of the solution into the DLS measuring dish to determine the particle size distribution.
The various functional groups of the materials were characterized by Fourier transform infrared spectroscopy (FTIR, Nicolet iS50, Thermo Scientific, Waltham, MA).The specific step was to collect an appropriate number of samples as well as potassium bromide for grinding.After grinding, the sample was placed in the tablet press for extrusion, and finally, the press plate was detected in the spectrometer.
The drug encapsulation and release were detected using a ultraviolet-visible (UV-Vis) spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan).Briefly, we used a UV spectrophotometer to detect the absorbance of OD 420 nm and OD 227 nm in centrifugal collected supernatant or drug release solution (approximately 2/3 volume of the cuvette).

In vitro drug release
The release of PTX and CUR from FA-BSA-Au@PTX/CUR was determined using a dialysis method at different pH values (5, 7.4, and 10).Ten milligrams of FA-BSA-Au@PTX/CUR was dispersed in 5 mL of solution and added to a dialysis bag.The bag was then immersed in 25 mL of PBS solution at different pH values and incubated at 37 � C with shaking (100 r/ min).At different time points, 1 mL of the dialysis solution was extracted, and an equal volume of fresh PBS was added to maintain the release under the same conditions.The content of PTX and CUR in the dialysis solution was measured by UV-vis.The following formulas were used to calculate encapsulation efficiency and loading capacity:

Cell experiment
The human EC cell line KYSE150 was purchased from Tongpai Biotechnology Co., Ltd.(Shanghai, China), and the human esophageal epithelial cell line Het-1A was purchased from BeNa Culture Collection (Kunshan, China).KYSE150 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco, Life Technologies, Carlsbad, CA), while Het-1A cells were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin.The cells were incubated at 37 � C in a humidified atmosphere containing 5% CO 2 .

Cellular uptake
Cellular uptake of the NPs was observed using a fluorescence microscope (DM4, Leica, Wetzlar, Germany).KYSE150 cells (or Het-1A cells) were seeded on cell slides at a density of 1 � 10 6 cells/mL and incubated overnight.We purchased Sulfo-Cyanine5-labeled BSA (BSA-Cy5, Qiyue Biotech, Xi'an, China) and then used BSA-Cy5 to prepare the same concentrations of BSA-Au@PTX/CUR and FA-BSA-Au@PTX/CUR NPs.After incubating NPs with cells for 4 h, the medium was discarded and cells were washed three times with PBS.PBS without NPs was used as the control.The nuclei were stained with DAPI (Sigma, St. Louis, MO) for 15 min.After that, the cells were photographed under a fluorescence microscope.

Cell viability assay
MTT assay was performed to investigate the effect of different nanomaterials on the viability of Het-1A or KYSE150 cells, with PBS as the blank control.Het-1A and KYSE150 cells were seeded in 96-well plates at a density of 5 � 10 3 cells/ well and incubated at 37 � C with 5% CO 2 for 24 h.Different concentrations (0-12 lg/mL) of drug suspensions were added to the plates to compare the cytotoxicity of PTX, CUR, PTX/ CUR (1:1), BSA-Au@PTX/CUR, and FA-BSA-Au@PTX/CUR.After incubation for 48 h, MTT solution (20 lL, 5 mg/mL) was added and further incubated for 4 h.The liquid in the wells was discarded, and DMSO (150 lL/well) was added to treat the cells.Finally, the absorbance (k ¼ 490 nm) of cell samples was measured using a microplate reader (Thermo, Waltham, MA) and recorded.KYSE150 cells were seeded in 96-well plates according to the above method and incubated overnight to determine the effect of drugs on radiosensitization.Different concentrations of drugs were added to the plates and incubated for 24 h.The solution in the 96-well plates was discarded, and an equal amount of incomplete medium was added, followed by irradiation (source skin distance (SSD): 100 cm, dose rate: 200 cGy/min) under a linear accelerator (Elekta, Stockholm, Sweden) with a single dose of 0, 2, 4, 6, and 8 Gy.After another 24 h of cell culture, MTT and DMSO solutions were added, and the absorbance at 490 nm was measured using a microplate reader.

Colony formation assay
KYSE150 cells were seeded in 6 cm culture dishes at an appropriate density and incubated overnight.Then, cells were treated with different concentrations (5, 10, and 15 lg/mL) of FA-BSA-Au@PTX/CUR nanodrugs.After 24 h, the culture dishes were gently washed with sterile saline to remove any remaining nanodrugs, and a fresh culture medium was added.The cells were then irradiated with 6-MV X-rays as soon as possible at single doses of 0, 2, 4, 6, and 8 Gy.Subsequently, the cells were seeded in 12-well plates at a density of 100 cells/well and incubated for 14 d.When the cell colonies became visible to the naked eye, the culture was terminated.The cells were washed twice with PBS before being fixed with 75% ethanol and stained with 5% crystal violet (MACKLIN, Shanghai, China).The number of colonies was counted after photographing with a camera.

Cell apoptosis and cell cycle analysis
KYSE150 cells (1 � 10 6 cells/well) were seeded in six-well plates and incubated overnight.Different concentrations (5, 10, and 15 lg/mL) of FA-BSA-Au@PTX/CUR solution were added to the cells, which were then replaced with fresh culture medium after 24 h and irradiated with a single dose of 6 Gy Xrays.The cells were further incubated for 24 h, and the cells and supernatant were collected by centrifugation at 1000 rpm for 2 min.The cell pellets were resuspended in 100 lL binding buffer, and the cells were stained with Annexin V-FITC/PI double staining kit (Lianke, Hangzhou, China) and incubated at room temperature for 30 min in the dark.
For cell cycle analysis, KYSE150 cells after treatment were collected, fixed with 75% ethanol at 4 � C for 2 h, and resuspended in PBS.The cells were then centrifuged at 1500 rpm for 5 min, and the cell pellets were collected and treated with PI/RNase Staining Buffer (BD Pharmingen, San Diego, CA) for 20-30 min at room temperature in the dark.The samples were detected and analyzed using a flow cytometer (Accuri C6, BD, San Diego, CA).

c-H2AX assay
We used immunofluorescence (IF) and Western blotting (WB) to detect the ionizing radiation marker c-H2AX to evaluate the radiosensitization ability of nanodrugs.For IF, KYSE150 cells were incubated with nanodrugs for 12 h, followed by irradiation with 6 Gy and further incubation for 1 h.The cells were then fixed with 4% paraformaldehyde, and incubated at room temperature with 0.1% Triton X-100 (Millipore, Billerica, MA) for 5 min.Subsequently, cells were incubated with anti-c-H2AX (1:250, ab81299) overnight at 4 � C. Following cell rinses, samples were incubated with fluorescein-labeled goat anti-rabbit IgG antibody (1:200, bs-0295G-AF555) for 1 h at room temperature.The cells were observed using a fluorescence microscope after the nuclei were stained with DAPI.
For WB, proteins were extracted from cells using RIPA lysis buffer (Thermo Scientific, Waltham, MA), and protein concentrations were detected by BCA.Equal amounts of cell lysates were separated by 10% SDS-PAGE, transferred onto PVDF membranes (Amersham, Piscataway, NJ), and sealed with 5% skim milk for 1 h at room temperature.Afterwards, membranes were incubated with anti-c-H2AX (1:250, ab81299) and anti-GAPDH antibody (1:5000, ab8227) overnight at 4 � C. Membranes were washed with PBS and incubated with horseradish peroxidase-labeled goat anti-rabbit IgG antibody (1:2000, ab6721) for 1 h at room temperature.Protein bands were observed using an ECL detection kit (Thermo Scientific, Waltham, MA).ImageJ software (v1.8.0, NIH, Bethesda, MD) was utilized to calculate the gray scale values of the bands.All antibodies were purchased from Abcam (Cambridge, UK).

Statistical analysis
All experiments were performed at least three times and data were presented as mean ± standard deviation.GraphPad Prism (v8.0, La Jolla, CA) software was used to process the experimental data in this study.The differences between the two groups were analyzed using t-test, while one-way and two-way ANOVA were used for multiplegroup comparisons.A p value of less than .05was considered statistically significant ( � p < .05).

Material characterization
After the preparation of the materials, their structure was analyzed using TEM.As shown in Figure 1(A), Au NPs modified with BSA and FA were formed into spherical nanomaterials of approximately 150 nm in size, encapsulating both PTX and CUR.Subsequently, the size and potential of BSA-Au@PTX/CUR and FA-BSA-Au@PTX/CUR were characterized using an NP size analyzer.As shown in Figure 1(B-D), the average particle size of BSA-Au@PTX/CUR and FA-BSA-Au@PTX/CUR were approximately 204 nm and 220 nm, respectively.The increased size of FA-BSA-Au@PTX/CUR was mainly due to the structural changes during the FA modification process with BSA.Furthermore, due to the FA modification, the zeta potential of BSA-Au@PTX/CUR changed from −18.7 mV to −0.02 mV, indicating successful FA modification on the surface of BSA-Au@PTX/CUR.The drug-loaded BSA-Au@PTX/CUR and FA-modified FA-BSA-Au@PTX/CUR nanomaterials were freeze-dried and characterized using FTIR (Figure 1(E)).The spectra of BSA (black) showed characteristic peaks of BSA at 1641 cm −1 and 1523 cm −1 , corresponding to the stretching vibration of the amide C═O bond and the bending vibration of the amide N-H bond, respectively.The spectra of FA (red) showed characteristic peaks of FA at 3317 cm −1 , 1686 cm −1 , 944 cm −1 , and 763 cm −1 , corresponding to the stretching vibration of the primary amine N-H bond, the stretching vibration of the amide C-O bond, the benzene ring absorption peak of FA, and the bending vibration of the aromatic C-H bond, respectively.The spectra of BSA-Au@PTX/CUR (blue) showed a smaller stretching vibration peak of S-H at 2350 cm −1 , in addition to the characteristic peaks of BSA, which confirmed the formation of Au and the connection between BSA and Au.In the spectra of FA-BSA-Au@PTX/CUR (green), all characteristic peaks of FA and BSA-Au@PTX/CUR were observed, indicating successful coupling between FA and BSA-Au@PTX/CUR.In conclusion, this study successfully prepared FA-BSA-Au@PTX/CUR nanodrugs.

In vitro drug release
A dialysis method was used to investigate the nanomaterials under different pH conditions (5, 7.4, and 10) to simulate the drug release in different environments in vivo.Based on the UV-vis measurement of absorbance and standard curve calculation, the encapsulation efficiency and loading capacity of PTX were 52.2% and 20.3%, respectively, while those of CUR were 40.2% and 12.3%, respectively (Figure 2(A,B)).Figure 2(C) shows the drug release of PTX under different pH conditions.After 72 h, the release rate of PTX reached approximately 50% at pH 5 and 7.4, while it reached 65% at pH 11.This was mainly due to the faster decomposition of BSA at alkaline pH, which resulted in the release of more drugs.The drug release trend of CUR in Figure 2(D) was similar to that of PTX, and the drug release rate of CUR reached about 23% after 72 h at pH 7.4.These results indicated that the nanodrug exhibited a good response to pH values and could stably release drugs in both neutral and acidic environments.It should be noted that although PTX and CUR are released faster in alkaline environments, drug release is not necessarily as fast as possible.The stable release can allow the drug to exert sustained effects at the site of action.

Cytotoxicity of the nanodrug
Before investigating the toxicity of FA-BSA-Au@PTX/CUR, we first conducted a cell uptake experiment to evaluate whether the nanodrug could target cancer cells and be effectively taken up by them.As depicted in Figure 3(A), after incubation with Het-1A cells, the NPs that entered Het-1A cells were relatively small due to the low expression of FA receptors in Het-1A cells, resulting in a weak red fluorescence intensity.In KYSE150 cells, compared with the blank control group, both BSA-Au@PTX/CUR and FA-BSA-Au@PTX/CUR displayed certain fluorescence, and the fluorescence intensity of the FA-BSA-Au@PTX/CUR group was much higher than that of the BSA-Au@PTX/CUR group.This was because FA could specifically recognize the FA receptor on the surface of cancer cells, mediating the phagocytosis of nanodrugs, whereas the amount of BSA-Au@PTX/ CUR without FA modification entering cancer cells was relatively low.These experimental results indicated that FA modification could promote the effective entry of nanodrugs into cancer cells.
To test the effects of different (nano)drugs on the viability of Het-1A or KYSE150 cells, we conducted an MTT assay.As shown in Figure 3(B), with the increase in drug concentration, PTX, CUR, or the synergistic effect of the two drugs all significantly affected the viability of Het-1A cells.However, BSA-Au@PTX/CUR showed low toxicity to cells, and both the PBS and FA-BSA-Au@PTX/CUR groups showed no significant toxicity to Het-1A cells, indicating that the synthesized FA-BSA-Au@PTX/CUR nanodrug was biocompatible.In KYSE150 cells, compared with the PBS control, PTX, CUR, PTX/CUR, BSA-Au@PTX/CUR, and FA-BSA-Au@PTX/CUR all inhibited the viability of KYSE150 cells with increasing drug concentrations.However, the inhibitory effect of FA-BSA-Au@PTX/CUR nanodrug was the most significant, and its anticancer effect was dose-dependent.Therefore, compared with free PTX, CUR, or PTX/CUR, the use of FA-BSA-Au@PTX/CUR nanocarrier to deliver both drugs simultaneously effectively strengthened the inhibitory effect of drugs on KYSE150 cells and avoided affecting the activity of normal cells.

In vitro antitumor and radiosensitizing effects
To evaluate the in vitro antitumor and radiosensitizing effects of FA-BSA-Au@PTX/CUR, we studied the effects of different concentrations (0, 5, 10, 15, and 20 lg/mL) of FA-BSA-Au@PTX/CUR and different doses (0, 2, 4, 6, and 8 Gy) of X-ray radiation on cell viability.As illustrated in Figure 4(A), cell viability gradually decreased with increasing radiation dose and FA-BSA-Au@PTX/CUR concentration.Furthermore, we used a colony formation assay to explore the effect of FA-BSA-Au@PTX/CUR on KYSE150 cells.As presented in Figure 4(B), compared with the X-ray alone group, the combination of FA-BSA-Au@PTX/CUR and radiation showed a decreased colony formation, and the inhibitory effect on colonies was more significant at higher concentrations, indicating that cell survival rate decreased with increasing radiation dose and FA-BSA-Au@PTX/CUR concentration.Additionally, we found that FA-BSA-Au@PTX/CUR could significantly enhance the killing ability of X-rays.Cell apoptosis and cell cycle distribution were important indicators for evaluating the antitumor effect of nanodrugs in vitro.As shown in Figure 4(C), as the FA-BSA-Au@PTX/CUR concentration and radiation dose increased, so did the apoptosis rate.The difference in cell cycle distribution among the groups was mainly manifested as an increase in the percentage of G2/M phase cells with increasing NP concentration and radiation dose, with no significant difference observed in the S phase (Figure 4(D)).
In addition, we used IF and WB to detect the cH2AX levels of cells treated with different concentrations of FA-BSA-Au@PTX/CUR under 6 Gy X-ray radiation conditions to evaluate the degree of DNA damage.As plotted in Figure 5, compared with X-ray alone, the addition of FA-BSA-Au@PTX/CUR nanodrug resulted in more severe DNA damage, which increased with increasing drug concentration, indicating that the nanodrug made KYSE150 cells more sensitive to ionizing radiation.These results suggested that FA-BSA-Au@PTX/CUR may be an effective radiosensitizer for the in vitro treatment of EC.

Discussion
EC is a common malignancy that threatens human health, and clinical treatments mainly involve comprehensive approaches, such as surgery, radiotherapy, and chemotherapy.However, traditional chemotherapy drugs may have drawbacks such as non-selectivity and poor utilization efficiency, and radiotherapy usually requires high doses of  ionizing radiation, which can cause varying degrees of toxic side effects in patients.With the advancement of nanomedicine, people attempt to use nanomaterials to deliver drugs, so as to enhance drug targeting and bioavailability while reducing toxicity, and some chemotherapy drugs and high-Z elements can be used as radiosensitizers to improve the efficacy of radiotherapy.Based on this background, this study designed and prepared a new type of nanodrug, FA-BSA-Au@PTX/CUR, to target deliver both PTX and CUR drugs, and enhance cancer cell sensitivity to ionizing radiation, thus improving the treatment of EC patients.
The radiosensitizing properties of PTX and Au NPs have previously been reported.As a chemotherapy drug, PTX can prevent the formation of the spindle during mitosis by microtubules, causing cells to arrest in the G2/M phase, which is the most sensitive stage of the cell cycle to ionizing radiation; therefore, PTX can also be used as a radiosensitizer (Sikov and Safran 1997;Safran and Rathore 2002).Many studies have found a polyphenol compound CUR that can be used in combination and has a significant additional effect in neutralizing the toxicity of PTX in healthy cells (Lee et al. 2020).CUR has long been commonly used in the treatment of diseases due to its multi-potent biological activity and potential anticancer properties (Lee et al. 2013;Doello et al. 2018).Multiple studies have shown that CUR is not toxic to healthy cells, and that CUR at low or high concentration has different effects, but the concentration should be chosen based on the study objective (Loo et al. 2022).Herein, we chose PTX in collaboration with CUR to treat EC and achieved quite good results.In addition, the outcomes of cell viability experiments in this study are consistent with the results of Nguyen et al.'s study (Nguyen et al. 2022), in which they prepared a nano-gel carrier loading PTX and CUR through a hydrophobic core and verified that the drug was indeed more effective in inhibiting cancer cells in vitro than free PTX.The difference lies in that the modified Au NPs in this study not only simultaneously loaded PTX and CUR but also improved cancer cell targeting and radiosensitization.
Au NPs, as a high-Z element material, can accumulate ionizing radiation at the tumor site, allowing the same dose of X-rays to have a better effect on lesion damage (Schuemann et al. 2016;Song et al. 2017).For example, in Mochizuki et al.'s study, Au NPs (OS/Au) are surface functionalized with organic silicon (OS) NPs to synthesize effective Au materials as X-ray sensitizers, which can synergize with radiotherapy to inhibit the proliferation of mouse breast cancer cells and induce cell death (Mochizuki et al. 2022).In recent years, many studies have been carried out to apply different Au-based nanomaterials to tumor therapy, and after modification, these NPs exhibit good biocompatibility in circulation and can be metabolized by the liver and kidneys (Hainfeld et al. 2004).In this study, BSA and FA were used to modify Au NPs.According to the results of cell uptake and cell viability tests, FA-BSA-Au@PTX/CUR was mainly taken up by KYSE150 cells rather than normal Het-1A cells.Moreover, the impact of the nanodrug on the survival of normal cells was weak, only exerting a toxic effect on cancer cells.Compared with using a single X-ray, after applying ionizing radiation, FA-BSA-Au@PTX/CUR inhibited the proliferation of KYSE150 cells, promoted cell apoptosis and DNA damage, and significantly inhibited the cell cycle at the G2/M phase.These effects became more significant with the increase of FA-BSA-Au@PTX/CUR concentration, indicating that the FA-BSA-Au@PTX/CUR prepared in this study was a safe and effective radiosensitizer, which may synergize with radiotherapy to improve treatment efficacy.
In summary, this study successfully prepared the FA-BSA-Au@PTX/CUR composite nanodrug based on Au NPs and systematically characterized its morphology, particle size, stability, etc.The drug showed good biocompatibility and low toxicity to normal cells after FA and BSA modifications.In vitro experiments demonstrated that FA-BSA-Au@PTX/CUR, when used in combination with X-rays, could effectively increase the radiotherapy damage to cancer cells.However, there are still some limitations to the current experiments, such as the lack of pharmacokinetic research animal model verification, etc.In the future, we will further track the distribution and metabolism of nanodrugs in the body and explore more accurate and efficient drug concentration effects.Overall, our study provides a safe and feasible radiosensitizer to promote radiotherapy for EC, and we hope that this research can accelerate the clinical application of nanodrugs.

Figure 2 .
Figure 2. Release of PTX and CUR in FA-BSA-Au@PTX/CUR.(A, B) Standard curves of PTX and CUR by UV-vis spectroscopy; (C, D) release profiles of PTX and CUR from FA-BSA-Au@PTX/CUR at different pH values (5, 7.4, and 11).

Figure 4 .
Figure 4.In vitro anti-tumor and radiosensitization effects of FA-BSA-Au@PTX/CUR.(A) Cell viability of KYSE150 cells treated with different concentrations of FA-BSA-Au@PTX/CUR in combination with different doses of 6 MV X-rays; (B) colony formation of cells treated with X-rays alone or in combination with different concentrations of FA-BSA-Au@PTX/CUR ( � p < .05,�� p < .01);(C, D) cell apoptosis and cell cycle distribution of different treatment groups.