Introduction

Current studies have shown that gut microbiota dysbiosis is associated with the regulation of the occurrence and development of various tumours, such as colorectal cancer,1 breast cancer2 and hepatocellular carcinoma.3 Changes in gut microbiota diversity may have carcinogenic effects, and a reduction in gut microbiota diversity significantly reduces the immune function of patients with tumours.4 Many microbial metabolites have significant impacts on anticancer immunity.5 Moreover, the gut microbiota plays an important role in the metabolism of chemotherapy drugs, which affects antitumour efficacy.6 Therefore, a deeper exploration of the effects of chemotherapy on the gut microbiota could provide feasible solutions for antitumour treatment.

In recent years, curcumin has made progress in the treatment of tumours. One of the main antitumour mechanisms of curcumin is to promote apoptosis by regulating reactive oxygen species.7,8 Recent studies have shown that curcumin has a positive effect on the gut microbiota. Curcumin plays a role in preventing and combating cancer by increasing the abundance of beneficial microbes and decreasing the abundance of pathogenic microbes.9,10 However, curcumin is insoluble in water. The bioavailability of curcumin is very poor, making it difficult to exert its curative effect.11 Therefore, improving the solubility of curcumin is an urgent problem to be solved.

Currently, nanotechnology is a promising strategy for drug delivery and cancer treatment.12,13 Nanocarriers have unique advantages in improving drug delivery, bioavailability and targeting. Research has shown that nanocarriers can overcome the hydrophobicity of curcumin.14,15 Nanoselenium has attracted widespread attention because of its high biocompatibility, surface modification ability and low toxicity.16 However, nanoselenium is usually unstable and tends to aggregate into inactive black precipitates. Therefore, developing new methods to improve the stability of nanoselenium has become the focus of current research. Chemical methods are among the main methods used to prepare selenium nanoparticles. Chemical synthesis methods often consume many chemical reagents and have high costs and high toxicity. The use of plant-based nanoselenium provides a strategy to solve these problems.

Considering that curcumin has excellent reducing properties, it can be used as a reducing and capping agent in the preparation of nanoselenium. To obtain well-dispersed curcumin-modified selenium (Cur/Se) nanoparticles, Tween 80 was added as a stabilizer. In this study, Cur/Se nanoparticles were successfully prepared via a one-step method with excellent water dispersion. The physicochemical and biological properties were studied. The antitumour activity of the Cur/Se nanoparticles was evaluated in vitro and in vivo. This study preliminarily investigated the antitumour mechanism of nanoparticles. 16S rRNA Illumina high-throughput sequencing technology was used to investigate the effects of Cur/Se nanoparticles on the gut microbiota. This study provides strategies through which nanoparticles exert antitumour effects by regulating the gut microbiota.

Materials and Methods Materials

Curcumin (Cur), 2.2-diphenyl-1-picrylhydrazyl (DPPH), 2.2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and potassium persulfate (K2S2O8) were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Sodium selenite, Tween 80, salicylic acid, iron (II) sulfate heptahydrate and hydrogen peroxide were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). CCK-8 reagent was purchased from Beyotime Biotechnology, Inc. (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Aldrich China Inc. (Shanghai, China).

Synthesis and Characterization of Cur/Se Nanoparticles

Cur was dissolved in dimethyl sulfoxide solution. Sodium selenite was dissolved in a Tween 80 aqueous solution. The solutions were subsequently mixed and stirred for 3 h at room temperature. The products were transferred into a dialysis bag and dialyzed against distilled water for 2 d (MWCO = 1000 Da). The obtained Cur/Se nanoparticles were characterized via UV-Vis spectrophotometry (Hitachi UV-3310, Japan), transmission electron microscopy (TEM; Thermo Fisher FEI Talos F200x, USA), dynamic light scattering (Malvern NANO-ZS, UK), inductively coupled plasma‒mass spectrometry (ICP‒MS; Thermo Fisher ICAP‒Qc, USA), and Fourier transform infrared spectroscopy (FTIR, Nicolet 5700, USA).

Cur Release

The drug release behaviour of Cur and Cur/Se nanoparticles was detected at pH 7.4 and 5.5 at 37 °C via a previously described method with some modifications.17 The same concentrations of free Cur and Cur/Se nanoparticle solutions were placed in a dialysis bag (MWCO = 1000 Da), immersed in phosphate buffer solution (PBS, pH 7.4 and 5.5) containing 0.1% Tween 80, and shaken (100 rpm) at 37 °C. The samples were collected, and the release media were supplemented at specified time points. The Cur concentration of the samples was detected via a UV-Vis spectrophotometer.18,19

DPPH Radical Scavenging Activity Assay

The DPPH radical scavenging capacity of the samples was determined via a previously described method with some modifications.20 Briefly, DPPH ethanol solution (0.1mM, 2 mL) was mixed with the same drug concentration of Cur or Cur/Se NPs solution (2 mL) and incubated at room temperature in the dark for 30 min. Then, the absorbance of the reaction mixture was measured at 517 nm.

ABTS Radical Scavenging Activity Assay

The ABTS radical scavenging capacity of the samples was determined via a previously described method with some modifications.21 Briefly, the same amounts of ABTS (7.4 mmol/L) and K2S2O8 (2.6 mmol/L) were mixed at room temperature and reacted in the dark for 16 h. Then, the mixture was diluted to obtain an absorbance of 0.70 at 734 nm. The diluted ABTS reagent (3 mL) was mixed with different sample solutions (1 mL) and incubated at room temperature in the dark for 20 min. The absorbance of the reaction mixture was measured at 734 nm.

The following Formula (1) was used to calculate the DPPH and ABTS radical scavenging activities of Cur and Cur/Se NPs.

(1)

where A0 and A1 are the absorbance of the DPPH or ABTS radical solution without and with Cur or Cur/Se NPs solution, respectively, and A2 is the absorbance of the Cur or Cur/Se NPs samples.

Antitumour Activity of Cur/Se Nanoparticles in vitro

The human cervical cancer cell line HeLa and the mouse sarcoma S180 tumour cell line S180 and the human hepatocyte cell line L02 were purchased from ATCC, and the cells were cultured with DMEM. The cytotoxicity of Cur/Se nanoparticles to HeLa tumour cells was detected via the MTT assay. HeLa tumour cells at the logarithmic growth stage were digested with trypsin, and culture medium was added to prepare a cell suspension. The cells were subsequently seeded into 96-well plates in 100 μL of culture medium and cultured overnight. Cur and Cur/Se nanoparticle solutions were prepared with the same concentration gradient of Cur (0.5 μg/mL, 5 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, and 250 μg/mL). The cells were cultured with 25 μL of the above solutions for 24 h or 48 h. Then, 31.5 μL of MTT solution (5 mg/mL, in PBS) was added to each well and incubated for another 4 h. Afterward, the culture medium was removed, and 200 μL of DMSO was added. The absorbance of each well was detected by using a microplate reader at 570 nm. The cytotoxicity of Cur/Se nanoparticles to L02 cells was detected via the MTT assay as mentioned above.

The cytotoxicity of the Cur/Se nanoparticles to S180 tumour cells was detected via a CCK-8 assay. S180 tumour cell suspensions were prepared and seeded into 96-well plates in 80 μL of culture medium. Culture medium without S180 tumour cells was used as a blank control. Cur and Cur/Se nanoparticle solutions were prepared with the same concentration gradient of Cur (0.5 μg/mL, 5 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, and 250 μg/mL). The cells were cultured with 20 μL of the above solutions for 24 h or 48 h. Then, 10 μL of CCK-8 solution was added to each well and incubated for another 4 h. The absorbance of each well was detected by using a microplate reader at 450 nm.

Intracellular Distribution of Cur/Se Nanoparticles

A suspension of HeLa tumour cells was prepared via the above method and seeded into a 6-well plate at a density of 2 × 105/mL. After incubation for 24 h, the tumour cells were cocultured with Cur or Cur/Se nanoparticle solutions (Cur, 2 μg/mL) for predetermined durations. After washing with PBS three times, the fluorescence was observed by a fluorescence microscope (Nikon ECLIPSE Ti-S, Japan).

Cellular Uptake of Cur/Se Nanoparticles

A suspension of HeLa tumour cells was prepared via the above method and seeded into a 12-well plate at a density of 1 × 105/mL. After incubation for 24 h, the tumour cells were cocultured with Cur or Cur/Se nanoparticle solutions (Cur, 2 μg/mL) for predetermined durations. After being washed with PBS three times, the cells were collected and resuspended in PBS for fluorescence analysis via flow cytometry (BD AccuriTM C6 Plus, USA). At least 1×104 cells were analysed via flow cytometry for each sample.

Intracellular Reactive Oxygen Species (ROS) Detection

Intracellular ROS generation was detected via 2´,7´-dichlorofluorescein-diacetate (DCFH-DA). HeLa cells were cocultured with Cur or Cur/Se nanoparticle solutions (Cur, 2 μg/mL) in 12-well plates for 4 h. The cells were washed with PBS and incubated in DCFH-DA working solution (10 μM, 1 mL per well) at 37 °C in the dark for 30 min. Then, the cells were washed with PBS three times. The cells were collected and resuspended in PBS for analysis of intracellular ROS via flow cytometry. At least 1×104 cells were analysed via flow cytometry for each sample.

In vitro Antibacterial Study

Bacterial suspensions at logarithmic growth stage with OD = 0.5 were diluted 100 times. The bacterial suspensions were subsequently seeded into 96-well plates in 80 μL of culture medium. Cur and Cur/Se nanoparticle solutions were prepared at the same concentration gradient of curcumin (0.5 μg/mL, 5 μg/mL, 25 μg/mL, 125 μg/mL, 250 μg/mL, and 500 μg/mL). The bacteria were cultured with 20 μL of the above solutions for 17 h. The absorbance of each well was detected by using a microplate reader at 600 nm.

Antitumour Activity of Cur/Se Nanoparticles in vivo

An ICR male mouse was intraperitoneally inoculated with 1.0×106 S180 cells. On Day 7, S180 ascites were extracted under aseptic conditions and diluted 1:3 with aseptic saline. Five-week-old ICR male mice were inoculated with 0.1 mL of tumour cell suspension subcutaneously in the right axillary area. Therefore, S180 tumour-bearing ICR mouse models were constructed. When the tumour volume increased to 50–100 mm3, the S180 tumour-bearing mice were randomly divided into 3 groups: the control group, Cur group and Cur/Se nanoparticle group (n = 6). The antitumour experiment was carried out according to previous methods with some modifications.18 Cur and Cur/Se nanoparticle solutions were orally administered daily at a dose of 10 mg/kg Cur for 10 days. Body weight and tumour volume were measured every two days. At the end of treatment, the tumours were harvested and weighed. The heart, liver, spleen, lung and kidney were collected for haematoxylin and eosin (HE) staining. After the mice were killed, the abdominal cavity was opened quickly, and the intestinal tissue was removed. The faecal matter in the ileocecum of each mouse was rapidly collected and stored in a 1.5 mL sterile EP tube at −80 °C. 16S rRNA sequencing was used to analyse the structural composition and diversity of the gut microbiota.

Statistical Analysis

Statistical analysis was performed via Microsoft Excel software and GraphPad Prism software. All the experiments were repeated at least three times. After confirming that the values followed a normal distribution, a two-tailed Student’s t-test was applied to determine the significant differences. The data are presented as the means ± standard errors, and p < 0.05 was considered statistically significant.

The 16S rRNA sequencing work of the gut microbiota was completed in cooperation with Shanghai Majorbio Biopharm Technology Co., Ltd. (Shanghai, China). The data were analysed on the online platform of the Majorbio Cloud Platform. The experimental results are presented as the means ± standard errors, and p < 0.05 was considered statistically significant.

Results Synthesis and Characterization of Cur/Se Nanoparticles

The detail of the synthesis of Cur/Se nanoparticles is shown in Scheme 1. The Cur/Se nanoparticles were prepared by reducing sodium selenite with curcumin in one step. Curcumin was applied as a reducing and capping agent, and Tween 80 was used as a stabilizer. A diagram of the preparation of Cur/Se nanoparticles is shown in Figure 1a. An orange‒red aqueous solution of Cur/Se nanoparticles was obtained, as shown in Figure 1a. The Cur/Se nanoparticles showed good water dispersion. A total of 25 mL of Cur/Se nanoparticle solution was prepared, and the curcumin concentration of the Cur/Se nanoparticles was 350 μg/mL. The ICP‒MS results revealed that the Se concentration was 1.6 μg/mL.

Scheme 1 Schematic illustration of the curcumin-modified selenium nanoparticle preparation. Curcumin is a reducing and capping agent. Tween 80 is a stabilizer, and sodium selenite is the selenium source.

Figure 1 Preparation and characterization of Cur/Se nanoparticles. (a) Preparation of Cur/Se nanoparticles. (b) TEM image of Cur/Se nanoparticles. (c) Hydrodynamic diameters of Cur/Se nanoparticles detected by dynamic light scattering. (d) UV-Vis spectra of Cur and Cur/Se nanoparticles. (e) FTIR spectra of Cur, Tween 80, the mixture of curcumin and Tween 80 (Cur + Tween 80), and Cur/Se nanoparticles.

A TEM image of the nanoparticles is shown in Figure 1b. The morphology of the nanoparticles was nearly spherical with a regular shape and a diameter of 5–10 nm. The Cur/Se nanoparticles clearly exhibited lattice fringes, and the selected area of the electron diffraction results revealed that the nanoparticles had a polycrystalline structure. The hydrodynamic size of the Cur/Se nanoparticles was 6.86 nm, as detected by dynamic light scattering (Figure 1c), which was in accordance with the TEM results.

The UV-Vis spectra of the Cur and Cur/Se nanoparticles presented characteristic maximum absorption peaks at 432 nm with no redshift or blueshift phenomenon (Figure 1d). The nanometreization of Cur does not affect its UV-Vis absorption.

The FTIR spectra of the Cur and Cur/Se nanoparticles are shown in Figure 1e. The characteristic absorption peaks at 3514 cm−1, 1628 cm−1, and 1153 cm−1 corresponded to the stretching vibrations of the phenolic hydroxyl group, mixed C=O and C=C, and C-O-C of Cur, respectively. The bending vibration of C=C (1600 cm−1) and out-of-plane deformation vibration (856 and 744 cm−1) of the benzene ring were observed in the FTIR spectrum of Cur. These characteristic absorption peaks were observed in the FTIR spectrum of the Cur/Se nanoparticles. The results showed that the structure of curcumin in the Cur/Se nanoparticles did not obviously change. The Cur/Se nanoparticles presented the same characteristic peaks as the mixture of curcumin and Tween 80. Owing to the low content of selenium, the FTIR spectra did not reveal interactions between curcumin and nanoselenium. The stability of the Cur/Se nanoparticles was dependent mainly on physical interactions with Tween 80.

Cur Release

The release behaviour of the samples at different pH values is shown in Figure 2a and b. Within 72 h, the amount of drug released from free Cur was approximately 56% at pH 7.4. The amount of drug released from the Cur/Se nanoparticles was approximately 49%. As the pH decreased to 5.5, the amount of drug released from free Cur or Cur/Se nanoparticles increased to approximately 96% and 66%, respectively. The drug release rate of the Cur/Se nanoparticles was slower than that of free Cur, which showed sustained release behaviour. The pH of the release medium could affect the release behaviour of Cur from the samples. At pH 5.5, both the free Cur and Cur/Se nanoparticles resulted in faster drug release than at pH 7.4. The formation of Cur/Se nanoparticles did not affect the pH-sensitive release behaviour of curcumin. These results implied that the drug release behaviour from Cur/Se nanoparticles occurred in a pH-dependent and sustained manner.

Figure 2 Drug release behaviour of Cur and Cur/Se nanoparticles at (a) pH 7.4 and (b) pH 5.5. (c) Schematic illustration of the DPPH and ABTS radical scavenging process. (d) DPPH radical scavenging rates of curcumin and Cur/Se nanoparticles. (e) ABTS radical scavenging rates of curcumin and Cur/Se nanoparticles. The results are expressed as the means ± SDs (n = 3). **P < 0.01. *P < 0.05.

Antioxidant Activities

DPPH and ABTS radicals are widely used to test the antioxidant activity of antioxidant substances. Both Cur and selenium are reported to possess antioxidant activity. Thus the antioxidant ability of Cur/Se nanoparticles was tested via DPPH and ABTS reagents (Figure 2c). The DPPH radical scavenging rates of Cur and Cur/Se nanoparticles are shown in Figure 2d. The DPPH radical scavenging rates of curcumin at concentrations of 2 μg/mL, 5 μg/mL and 10 μg/mL were 9.1 ± 1.0%, 19.2 ± 1.1% and 31.4 ± 1.2%, respectively. For the same concentration of Cur/Se nanoparticles, the DPPH radical scavenging rates were 13.9 ± 1.4%, 26.6 ± 0.9% and 40.9 ± 1.0%, respectively. Both Cur and Cur/Se nanoparticles exhibited concentration-dependent DPPH radical scavenging activity. The DPPH radical scavenging activity was significantly improved for the formation of Cur/Se nanoparticles. The results of the ABTS radical scavenging rates of Cur and Cur/Se nanoparticles were similar to those of DPPH (Figure 2e). The ABTS radical scavenging rates of curcumin at concentrations of 2 μg/mL, 5 μg/mL and 10 μg/mL were 12.7 ± 0.9%, 24.3 ± 1.0% and 38.4 ± 0.7%, respectively. For the same concentration of Cur/Se nanoparticles, the DPPH radical scavenging rates were 15.6 ± 0.4%, 29.3 ± 0.4% and 48.2 ± 0.6%, respectively. An increase in the curcumin concentration of Cur and Cur/Se nanoparticles was beneficial for antioxidant activity. The Cur/Se nanoparticles significantly increased the ABTS radical scavenging rates. These results implied that Cur/Se nanoparticles had better antioxidant activity than Cur.

Cell Viability Assay

The antitumour activities of free Cur and Cur/Se nanoparticles were detected in HeLa and S180 tumour cells, and the IC50 values were calculated via GraphPad Prism software. The cytotoxicity of Cur and Cur/Se nanoparticles to HeLa tumour cells at 24 h is shown in Figure 3a. The Cur/Se nanoparticles showed significant cytotoxicity at a curcumin concentration of 5 μg/mL. Free Cur did not effectively inhibit the growth of HeLa tumour cells. As the concentration of free Cur increased to 50 μg/mL, the viability of HeLa tumour cells was as high as 51.1% ± 1.8%. The viability of the HeLa tumour cells significantly decreased to 13.5% ± 1.4% after treatment with the Cur/Se nanoparticles. The IC50 value of the Cur/Se nanoparticles was 10.4 μg/mL against HeLa tumour cells at 24 h. As the incubation time increased to 48 h, the cytotoxicity of the free Cur and Cur/Se nanoparticles increased (Figure 3b). The IC50 values of Cur and Cur/Se nanoparticles were 15.7 μg/mL and 4.7 μg/mL, respectively, against HeLa tumour cells at 48 h. The cytotoxicity of Cur and Cur/Se nanoparticles to S180 tumour cells is shown in Figure 3c and d. Free Cur showed no obvious cytotoxicity, with 78.2% ± 4.3% cell viability at the highest concentration of 50 μg/mL at 24 h (Figure 3c). For the Cur/Se nanoparticles, the viability of the S180 tumour cells significantly decreased to 3.8% ± 4.1%. The IC50 value of the Cur/Se nanoparticles was 26.7 μg/mL against the S180 tumour cells at 24 h. As the incubation time increased to 48 h, the cytotoxicity of the free Cur and Cur/Se nanoparticles also increased (Figure 3d). The IC50 values of Cur and Cur/Se nanoparticles were 33.0 μg/mL and 8.4 μg/mL, respectively, against S180 tumour cells at 48 h. These results implied that Cur and Cur/Se nanoparticles exhibited concentration- and time-dependent cytotoxicity. Compared with free Cur, the Cur/Se nanoparticles had a stronger effect on killing tumour cells at the same concentration and duration of treatment.

Figure 3 Cytotoxicity of Cur and Cur/Se nanoparticles to HeLa tumour cells at (a) 24 h and (b) 48 h, as detected by the MTT assay. Cytotoxicity of Cur and Cur/Se nanoparticles to S180 tumour cells at (c) 24 h and (d) 48 h, as detected by the CCK8 assay. Cytotoxicity of Cur and Cur/Se nanoparticles to L02 cells at (e) 24 h and (f) 48 h, as detected by the MTT assay. The results are expressed as the means ± SDs (n = 3). **P < 0.01. *P < 0.05.

The cytotoxicity of Cur and Cur/Se nanoparticles to L02 cells was also detected. At a concentration of 50 μg/mL, the viability of L02 cells was 56.9% ± 3.7% and 14.8% ± 1.4% after treatment with Cur and Cur/Se nanoparticles, respectively, for 24 h (Figure 3e). The IC50 value of the Cur/Se nanoparticles was 25.6 μg/mL against L02 cells at 24 h. As the incubation time increased to 48 h, the cytotoxicity of the free Cur and Cur/Se nanoparticles increased (Figure 3f). The IC50 values of Cur and Cur/Se nanoparticles were 10.5 μg/mL and 11.2 μg/mL, respectively, against L02 cells at 48 h. These results implied that the Cur and Cur/Se nanoparticles were also cytotoxic to normal cells.

Intracellular Distribution and Uptake of Cur/Se Nanoparticles

The cellular distributions of free Cur and Cur/Se nanoparticles were detected via fluorescence microscopy (Figure 4a). The fluorescence images revealed that both Cur and Cur/Se nanoparticles rapidly entered the tumour cells at 1 h. Cur and Cur/Se nanoparticles were observed in whole tumour cells, mainly in the cytoplasm. The cellular uptake of free Cur and Cur/Se nanoparticles was subsequently quantified via flow cytometry (Figure 4b). At 1 h, the relative fluorescence intensities of the Cur and Cur/Se nanoparticles were 1.30 ± 0.06 and 1.93 ± 0.02 times greater than that of the control, respectively. As the incubation time increased to 4 h, the relative fluorescence intensity of the Cur and Cur/Se nanoparticles significantly increased to 2.23 ± 0.01 and 2.42 ± 0.06 times that of the control, respectively. The intracellular accumulation of Cur and Cur/Se nanoparticles was time dependent. More curcumin was delivered into the HeLa tumour cells by the Cur/Se nanoparticles than free Cur at the same time points of 1 h and 4 h. These results indicated that the Cur/Se nanoparticles improved the accumulation of intracellular curcumin to increase its cytotoxicity.

Figure 4 (a) Intracellular fluorescence microscopy images of HeLa tumour cells treated with Cur and Cur/Se nanoparticles detected by fluorescence microscopy. The scale bar is 50 μm. (b) Fluorescence intensity of HeLa tumour cells treated with Cur and Cur/Se nanoparticles as determined by flow cytometry. (c) DCF fluorescence intensity of HeLa tumour cells treated with Cur and Cur/Se nanoparticles was detected by flow cytometry. The results are expressed as the means ± SDs (n = 3). **P < 0.01. *P < 0.05.

ROS Level

The ROS levels of the HeLa cells after Cur and Cur/Se nanoparticle treatment are shown in Figure 4c. At a concentration of 2 μg/mL, the relative fluorescence intensity of the Cur group was 0.46 ± 0.01 times that of the control. However, Cur/Se nanoparticle treatment significantly increased the relative fluorescence intensity of DCF to 5.29 ± 0.75 times that of the control. As the curcumin concentration increased to 5 μg/mL, Cur and Cur/Se nanoparticles increased the relative fluorescence intensity of DCF to 2.15 ± 0.26 and 7.69 ± 0.31 times that of the control, respectively. The results showed that Cur could decrease the intracellular ROS level at low concentrations while increasing the intracellular ROS level at high concentrations. Compared with Cur, the Cur/Se nanoparticles significantly improved the ROS level in a concentration-dependent manner. High ROS levels are beneficial for the treatment of tumours.

In vitro Antibacterial Effect

The in vitro antimicrobial activity was investigated (Figure S1). Escherichia coli is a Gram-negative bacterium. The results showed that Cur could not inhibit the growth of Escherichia coli (Figure S1a). At the highest concentration of 100 μg/mL, free Cur treated Escherichia coli showed a viability of 85.8% ± 5.6%. However, the Cur/Se nanoparticles showed a significant stronger inhibition of Escherichia coli. At 25 μg/mL of Cur/Se nanoparticles, the viability of Escherichia coli decreased to less than 50%. And the Cur/Se nanoparticles showed concentration dependent antibacterial activity. Staphylococcus aureus is a Gram-positive bacterium. The results showed that the Cur/Se nanoparticles had a stronger inhibition of Staphylococcus aureus (Figure S1b). As the concentration of free Cur increased to 50 μg/mL, the viability of Staphylococcus aureus was as high as 53.9% ± 0.2%. While with the same treatment of Cur/Se nanoparticles, the viability of Staphylococcus aureus significantly decreased to 24.1% ± 5.8%. And at 5 μg/mL of Cur/Se nanoparticles, the viability of Staphylococcus aureus was 51.1% ± 4.8%. The results showed that the Cur/Se nanoparticles showed greater activity against harmful Escherichia coli and Staphylococcus aureus compared with Cur in vitro.

In vivo Antitumour Activity

The antitumour effects of free Cur and the Cur/Se nanoparticles are shown in Figure 5. The growth curves of the tumour volume revealed that the growth rate of the tumours treated with free Cur was similar to that of the tumours treated with water (Figure 5a). From Day 6, the mean tumour volume in the Cur group was slightly smaller than that in the control group, and there was no significant difference in the results. The tumour volume in the Cur/Se nanoparticle treatment group was significantly smaller than that in the control and Cur groups, indicating a significant inhibition of tumour growth (**p < 0.01). There was no significant difference in body weight among the groups (Figure 5b), and the survival rate of the mice in each group was 100% during the treatment. After 10 days of treatment, the S180-bearing mice were killed, and the tumours were removed and weighed (Figure 5c). The mean tumour weight was 2.00 g in the control group, 1.60 g in the curcumin group, and 0.91 g in the nanoparticle group (Figure 5d). Compared with the free curcumin-treated group, the Cur/Se nanoparticle-treated group presented the smallest tumour weight (*p < 0.05 compared with the free curcumin-treated group and **p < 0.01 compared with the control group). The tumour inhibitory rate of free curcumin was 19.76%, and that of the Cur/Se nanoparticles was 54.33%. The HE staining results are shown in Figure 5e. The cells of the heart, liver, spleen, lung and kidney in each group were arranged neatly. There was no inflammatory cell infiltration, indicating that there was no obvious tissue damage. The results showed that oral curcumin and nanoparticle therapy had good biocompatibility.

Figure 5 Anti-S180 tumour study in vivo. (a) S180 tumour growth curves. (b) Body weight curves. (c) Photograph of excised tumours. (d) Excised tumour weights on Day 10. (e) HE staining of the organs. The results are expressed as the means ± SDs (n = 6). *P < 0.05. **P < 0.01.

Analysis of the Gut Microbiota

An increasing number of studies have shown that the gut microbiota is closely related to tumour treatment. By analysing the faecal microbiota of mice in different treatment groups, the regulatory effect of Cur/Se nanoparticles on the gut microbiota was explored. The 16S rRNA gene sequencing results revealed that the α-diversity of the gut microbiota was significantly greater in response to Cur/Se nanoparticle treatment than in response to Cur treatment and that the richness and diversity of the microbial community were significantly greater (Figure 6a). S180 tumour-bearing mice treated with Cur or Cur/Se nanoparticles presented significant changes in community composition according to principal coordinate analysis (PCoA) of β diversity (Figure 6b). Community histogram analysis revealed the relative abundance of the gut microbiota at the family level (Figure 6c), and community heatmap analysis revealed the relative abundance of the top 50 microbiota at the genus level (Figure 6d). Compared with the control treatment, Cur and Cur/Se nanoparticle treatment significantly reduced the relative abundance of harmful bacteria such as Rikenellaceae_RC9_gut_group, Enterorhabdus and Bilophila at the genus level (Figure 6e). The relative abundance of beneficial Lachnospiraceae_UCG-006 increased after treatment with Cur and Cur/Se nanoparticles (Figure 6f). Moreover, compared with Cur treatment, Cur/Se nanoparticle treatment significantly improved the relative abundance of beneficial Limosilactobacillus (Figure 6f).

Figure 6 Regulation of the gut microbiota. (a) Microbial α diversity in terms of the Sobs and Ace indices at the operational taxonomic unit (OTU) level. (b) Microbial β diversity illustrated by a PCoA plot. (c) Relative abundance of the gut microbiota at the family level. (d) Heatmap of the relative abundance of the gut microbiota at the genus level. (e) Relative abundance of harmful Rikenellaceae_RC9_gut_group, Enterorhabdus and Bilophila. (f) Relative abundances of beneficial Lachnospiraceae_UCG-006 and Limosilactobacillus. The results are expressed as the means ± SDs (n = 6). *P < 0.05.

Discussion

ROS are the main molecules produced by oxidative stress in the body and are closely related to the occurrence, development, and apoptosis of tumours.22 In normal cells, the oxidation and antioxidant systems are relatively balanced. However, the level of ROS in tumour cells is greater than that in normal cells. Therefore, tumour cells are in a state of oxidative stress and are more sensitive to ROS than normal cells.23 Thus, ROS are considered tumour-specific targets for the rational design of anticancer agents. Among the redox regulatory agents, curcumin and selenium compounds have received extensive attention because of their good chemotherapy potential.

However, curcumin is a hydrophobic drug with poor water solubility and limited permeability in the gastrointestinal mucosa.24 In addition, curcumin can be quickly eliminated from the system because of its strong gut-liver metabolism in vivo.24 Therefore, the poor bioavailability of curcumin has limited its application. Nanopreparations of curcumin provide strategies to improve the bioavailability of curcumin.25

Selenium nanoparticles are an effective form of selenium with high activity and low toxicity. Using plant extracts to synthesize selenium nanoparticles is a green and environmentally friendly method. Plant-based selenium nanoparticles are prepared by reducing selenium ions with plant extracts, such as Rosmarinus officinalis,26 peppers,27 and Punica granatum,28 among others. These natural plant extracts can not only reduce selenium ions into selenium nanoparticles but also play a stable dispersion role by attaching or wrapping the nanoparticles to prevent their agglomeration.

Therefore, we designed a simple method to prepare Cur/Se nanoparticles by reducing sodium selenite with curcumin in one step. During the preparation process, nanoparticles with excellent water solubility could be obtained by adding the extra stabilizer Tween 80. Tween 80 is a widely used surfactant with good biocompatibility.29 Tween 80 can be coated on the surface of Cur/Se nanoparticles, thereby improving their dispersibility in water. The classic method for preparing curcumin-loaded selenium nanoparticles involves two steps. Selenium nanoparticles were prepared first, and curcumin was then loaded onto the selenium nanoparticles.30 Compared with the reported curcumin-loaded selenium nanoparticles, our method is simpler and more practical. In contrast, the concentration of curcumin in the nanoparticles obtained in this study was more than 200-fold greater than that of selenium.

The characterization of the Cur/Se nanoparticles revealed the same UV absorption as that of curcumin. The FTIR spectra also implied that the Cur/Se nanoparticles retained their active functional groups, such as phenolic hydroxyl, β-diketone, and carbonyl groups. Therefore, the functional properties of the Cur/Se nanoparticles were not affected. Both curcumin and nanoselenium have the ability to scavenge free radicals.31,32 Under experimental conditions with abundant free radicals, the Cur/Se nanoparticles exhibited excellent antioxidant activity compared with that of curcumin. The cytotoxicity results confirmed the excellent antitumour activity of the Cur/Se nanoparticles in vitro (Scheme 2). The results of the cellular uptake experiments revealed that the Cur/Se nanoparticles could deliver more curcumin into the HeLa tumour cells. The Cur/Se nanoparticles induced more ROS production in tumour cells than free Cur at the same concentration. High-dose curcumin and selenium compounds are oxidants capable of inducing the production of ROS in tumour cells, thereby exerting their potential anticancer effects.33,34 The combination of curcumin and selenium nanoparticles enhances the antitumour activity of Cur/Se nanoparticles in vitro. For normal L02 cells, the IC50 values of Cur and Cur/Se nanoparticles were similar at 48 h, with values of 10.5 μg/mL and 11.2 μg/mL, respectively. For 10 consecutive days of treatment for S180 tumours in vivo, Cur and Cur/Se nanoparticle solutions were orally administered at a dosage of 10 mg/kg per day. After treatment, HE staining of mouse organs revealed no obvious tissue damage. The treatment scheme in this study was relatively safe. The rapid release of Cur and Cur/Se nanoparticles under acidic conditions facilitates the ability of these agents to exert their effects in the acidic tumour environment.35

Scheme 2 Schematic illustration of the antitumour activity of Cur/Se nanoparticles in vitro. The Cur/Se nanoparticles could deliver more curcumin into the tumour cells and induce high ROS production, thereby exerting potential antitumour effects.

The gut microbiota is a large and complex ecosystem that is considered a potential human organ. The gut microbiota can regulate the immune and metabolic functions of the body, thereby affecting the progression and treatment of various types of cancer. In recent years, the role of the gut microbiota in the development and treatment of tumours has become a popular research area. The gut microbiota has been reported to play an important role in the treatment of tumours via chemotherapy and immunotherapy.36,37 In this study, Cur/Se nanoparticles were constructed as a gut microbiota-regulating system. Both curcumin and Cur/Se nanoparticles significantly increased the richness and diversity of the microbial community. Rikenellaceae_RC9_gut_group has been reported to be positively correlated with cancer-promoting metabolites.38Enterorhabdus, a harmful bacterium, is reported to be increased in an osteosarcoma mouse model.39Bilophila can produce genotoxic hydrogen sulfide, which promotes the development of tumours.40 This study revealed that Cur and Cur/Se nanoparticle treatment inhibited these three harmful bacteria. Lachnospiraceae_UCG-006 is positively correlated with the activities of antioxidant enzymes.41 Both curcumin and Cur/Se nanoparticles possess good antioxidant activity and therefore significantly improve the level of Lachnospiraceae_UCG-006. Lachnospira_UCG-006 is a positive short-chain fatty acid-producing bacteria.42 Moreover, it has been reported that Lachnospiraceae_UCG-006 is also positively correlated with CD8+ T cells.43Lactobacillus can produce exopolysaccharides that help probiotics strengthen the immune system and suppress pathogenic bacteria.44 The significant increase in Lactobacillus after Cur/Se nanoparticle treatment improved the antitumour activity. Therefore, the Cur/Se nanoparticles could regulate the gut microbiota by reducing harmful bacteria and increasing beneficial bacteria, thus exerting good antitumour effects.

However, the formation mechanism of Cur/Se nanoparticles needs further exploration. The selenium content in Cur/Se nanoparticles is relatively low, and further optimization of the preparation method is needed to obtain nanoparticles with better activities. In the future, we will study how to improve the tumour targeting ability of Cur/Se nanoparticles to reduce their toxicity to normal cells.

Conclusion

This study provides a simple and green method to prepare Cur/Se nanoparticles in one step. Curcumin was used as a reducing and capping agent in the preparation of the nanoparticles. The Cur/Se nanoparticles showed excellent water dispersibility when Tween 80 was used as the stabilizer. The Cur/Se nanoparticles presented significantly higher DPPH and ABTS radical scavenging rates than did the same concentration of free curcumin. The Cur/Se nanoparticles delivered significantly more curcumin into the HeLa tumour cells and induced greater ROS production. Therefore, the Cur/Se nanoparticles had significant cytotoxicity. The Cur/Se nanoparticles improved the antitumour effect by regulating the gut microbiota in vivo.

Ethics Statement

The animal experiments were approved by the Animal Ethics Committee of Shandong University of Technology (the approval date was 17/11/2021, and the approval certification number of the study was YLX20211101). The study was conducted in accordance with the local legislation and institutional requirements.

Acknowledgments

We gratefully acknowledge the financial support of the National Natural Science Foundation of China (82104112) and the Natural Science Foundation of Shandong Province (ZR2019BH054).

Disclosure

The authors report no conflicts of interest in this work.

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