Bismuth-functionalized probiotics for enhanced antitumor radiotherapy and immune activation

Abstract

Radiotherapy (RT) is a mainstay treatment modality for solid tumors, employing high-energy radiation to induce reactive oxygen species (ROS) generation and DNA damage. However, RT is limited by insufficient DNA damage and collateral damage to normal tissues. Developing next-generation nanoradio-sensitizers to enhance tumor radiosensitivity while sparing healthy tissues remains a significant challenge. Herein, We propose a versatile bio–nano hybrid therapeutic system (BPBR), comprising Bifidobacterium infantis, bismuth-based nanoparticles, and the toll-like receptor 7/8 agonist (Resiquimod, R848). B. infantis exhibits tumor hypoxia-targeting properties, enabling the targeted delivery of bismuth nanoparticles and R848 to the tumor site. Bismuth, a high-atomic-number metal, possesses a higher mass attenuation coefficient for X-rays, enhancing X-ray radiation energy deposition and inducing DNA damage. R848, an activator of toll-like receptor 7/8, triggers immune responses. The combination of BPBR and X-ray irradiation significantly suppressed tumor growth in mice. This versatile bio–nano hybrid therapeutic system holds considerable promise for clinical translation and provides valuable insights for the design and development of novel therapeutics.

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1. Introduction

In recent years, bacteria have played a significant role in the treatment of numerous diseases.^1–3 They also have a long research history in anti-tumor therapy, remaining one of the main research directions. For instance, at the end of the 19th century, an American surgeon used bacteria to treat sarcoma.⁴ Later, Bacillus Calmette–Guerin (BCG) was effective in treating bladder cancer.⁵ Numerous studies have demonstrated that several bacteria have entered Phase I clinical trials for cancer treatment. For example, an attenuated strain of Salmonella enterica serovar Typhimurium VNP20009 has been applied to fight melanoma and renal cancer.⁶ Bacteria not only play beneficial roles in immune regulation and homeostasis maintenance but also can target and colonize specific biological interfaces in vivo.^7–9 Solid tumors often exhibit necrotic and hypoxic regions, which can be major factors for anaerobic bacteria targeting and infiltration.^10,11 Therefore, bacteria have been widely utilized as therapeutic agents or drug carriers.

Radiotherapy (RT) is a primary treatment modality for solid tumors, and most cancer patients require this treatment.¹² Radiotherapy mainly utilizes ionizing radiation such as X-rays, γ-rays, or high-energy electron beams to target tumor tissues, causing DNA double-strand breaks or indirectly generating reactive oxygen species (ROS) through the radiolysis of water, ultimately inducing tumor cell death.¹³ Localized ionizing radiation not only damages the DNA of tumor cells but also triggers the release of tumor-associated antigens from damaged tumor cells, promoting anti-tumor immune responses, effectively combating tumor progression and recurrence.^14–16 However, due to the low mass energy absorption coefficient of soft tissues, high doses of radiation are usually required to effectively kill cancer cells, leading to damage to surrounding normal tissues and even severe side effects for patients. Therefore, the complex tumor microenvironment, radioresistance of conventional radiotherapy, and its impact on normal tissues are major obstacles in clinical cancer treatment.¹⁷ A significant proportion of patients experience tumor recurrence and metastasis after radiotherapy, necessitating the development of efficient methods to enhance the effectiveness of radiotherapy. In recent years, nanomaterials have played an indispensable role as drug delivery carriers.^18–20 Multifunctional nanomaterials as radiosensitizers have attracted significant scientific interest. Materials containing high atomic number elements exhibit a higher mass attenuation coefficient for X-rays, far exceeding soft tissues, making them effective radiosensitizers that enhance radiotherapy efficacy.^21,22 These elements can interact with low-energy photons and further release photoelectrons and Auger electrons, leading to increased ROS concentration and damage to neighboring cells.²³ Several materials containing high-Z elements, such as nanoparticles²⁴ and metal–organic frameworks,²⁵ have been shown to significantly enhance radiotherapy.

We have constructed a novel bio–nanomaterial hybrid drug system. First, porous bismuth-based nanoparticles (Bi) were synthesized, utilizing the porous structure of bismuth-based nanoparticles to adsorb toll-like receptor 7/8 agonist R848 (BR). Subsequently, using the strong adhesion of polydopamine (PDA), porous bismuth R848 was attached to the surface of Bifidobacterium infantis (Bac), successfully constructing this novel bio–nanomaterial hybrid drug system (BPBR). Bismuth (⁸³Bi) is the least toxic heavy metal element and is well tolerated by the human body (Bi³⁺ biocompatible plasma concentration is 50 μg mL⁻¹).²⁶ Bismuth (Bi)-based nanomaterials, due to Bi (Z = 83) being a high-Z element, increase the deposition of X-ray energy at the tumor site to enhance radiotherapy.²⁷ Resiquimod (R848) is an agonist of toll-like receptors 7 and 8 (TLR7/8), and R848 effectively promotes dendritic cell maturation, alleviating the immunosuppressive state of tumor-infiltrating dendritic cells.²⁸ Polydopamine exhibits strong adhesion,²⁹ acting as an adhesive to bond nanomaterials to the bacterial surface. Polydopamine nanomaterials, as commonly used drug carriers, possess good biocompatibility.³⁰

Bifidobacterium infantis is a probiotic widely used in oral treatment for digestive diseases, exhibiting good biocompatibility. Furthermore, Bifidobacterium infantis is an anaerobic bacterium with hypoxia-targeting properties, allowing it to target hypoxic regions within tumors.³¹ Therefore, we leverage the tumor-targeting ability of Bifidobacterium infantis to deliver bismuth-based nanoparticles loaded with R848 to the tumor site. Under the weakly acidic conditions of the tumor microenvironment, bismuth nanoparticles and R848 are released. Simultaneously, combined with in vitro X-ray irradiation, the radiation energy deposition of X-rays can be enhanced, thereby increasing the efficacy of radiotherapy. Additionally, the enhanced X-ray action combined with R848 can further promote the infiltration of immune cells within the tumor, improving the immunosuppressive microenvironment of the tumor, and ultimately activating anti-tumor immune responses (Scheme 1). This novel bio–nanomaterial hybrid drug system shows promising clinical application prospects and has the potential to open a new chapter in the development of novel nano-radiosensitizers.

Scheme 1 Schematic diagram illustrating the mechanism of BPBR for anti-tumor therapy. Created with BioRender.com.

2. Materials and methods

2.1. Reagents, animals, and bacteria

Bismuth nitrate pentahydrate (Bi(NO₃)₃·5H₂O) and polyvinylpyrrolidone (PVP) were purchased from Shanghai Titan Technology Co., Ltd. Nitric acid, sodium hydroxide, ethylene glycol, and sodium borohydride were obtained from Tianjin Opusheng Chemical Co., Ltd. Antibodies for flow cytometry analysis (anti-CD45-PerCP, anti-CD3-APC/Cy7, anti-CD4-FITC, anti-CD8-PE/Cy7, anti-CD3-APC, anti-CD11c-APC, anti-CD80-FITC, anti-CD86-PE, and anti-PD-L1-PE) were acquired from BioLegend (California, USA). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Dakewe Biotech Co., Ltd (Shenzhen, China). CT26 cells were obtained from the American Type Culture Collection (ATCC, Rockville, USA). Female BALB/c mice (6–8 weeks old, 17–19 g) were purchased from Chengdu Dossy Experimental Animals Co., Ltd (Chengdu, China). All animal procedures were conducted in accordance with the guidelines of the Ethics Committee of the Animal Experiment Center of West China Hospital of Sichuan University. Infant Bifidobacterium (GIMI.207) was generously provided by the laboratory of Oncology Department, Southwest Medical University Hospital, and was originally obtained from the Strains Preservation Center of Guangzhou Institute of Microbiology, Guangdong, China. Upon receipt, the strain was immediately frozen and assigned a unique laboratory identification code.

2.2. Synthesis and characterization of bismuth-based nanoparticles (Bi)

Bismuth nitrate pentahydrate (Bi(NO₃)₃·5H₂O) was dissolved in nitric acid (HNO₃) solution, followed by the sequential addition of sodium hydroxide (NaOH), polyvinylpyrrolidone (PVP), and ethylene glycol. The mixture was homogenized and maintained at 150 °C for 3 hours, resulting in the formation of milky-white bismuth oxide (Bi₂O₃) nanoparticles. The mixture was repeatedly centrifuged and washed until odorless and the supernatant was clear. The resulting Bi₂O₃ nanoparticles were then redispersed in ultrapure water. To synthesize porous bismuth-based nanoparticles (Bi), the Bi₂O₃ nanoparticle dispersion was placed on a magnetic stirrer, and a sodium borohydride (NaBH₄) solution was slowly added dropwise. The color gradually changed from milky white to black, indicating the successful reduction of Bi₂O₃ nanoparticles to bismuth-based nanoparticles (Bi). The synthesized Bi nanoparticles were characterized using various techniques, including dynamic light scattering (DLS) for particle size analysis (Nano Brook 90Plus PALS, America), transmission electron microscopy (TEM) for morphology visualization (JEOL JEM-F200, Japan), Brunauer–Emmett–Teller (BET) analysis for specific surface area and porosity determination (Micromeritics ASAP 2460, America), X-ray diffraction (XRD) for elemental composition analysis (Rigaku Ultima IV, Japan), and X-ray photoelectron spectroscopy (XPS) for surface elemental analysis (Thermo Fisher K-Alpha+, America).

2.2.1. Encapsulation of R848 into Bi nanoparticles. A mixture of Bi nanoparticles and the TLR7 agonist, R848, was stirred overnight at room temperature. After removing the free R848 in the supernatant through high-speed washing and centrifugation, bismuth-based nanoparticles loaded with R848 (BR) were obtained. The absorbance of the supernatant at 318 nm was measured using a UV spectrophotometer to quantify the amount of unencapsulated R848, which was used to calculate the drug loading efficiency.

2.3. Construction and characterization of BPBR

The method for synthesizing the BR nanoparticles (bismuth nanoparticles loaded with R848) is detailed in the ESI.† BR nanoparticles and dopamine hydrochloride were dissolved in Tris–HCl buffer (10 mM, pH = 8.5) and stirred in the dark for 6 hours. The solution was then centrifuged and washed to obtain polydopamine-coated BR nanoparticles (PBR). To prepare the BPBR conjugate, the PBR suspension was incubated anaerobically with bacteria (Bac) at 37 °C for 4 hours. After centrifugation and washing, the suspension was resuspended in phosphate-buffered saline (PBS) to obtain the BPBR suspension, which was stored at 4 °C for later use. The elemental composition of BPBR was determined using XRD (Rigaku Ultima IV, Japan) and XPS (Thermo Fisher K-Alpha+, America). The morphology and elemental distribution of BPBR were visualized using transmission electron microscopy combined with an energy dispersive spectrometer. The as-prepared polydopamine-coated Bi and BPBR were left to stand for 24 hours, followed by centrifugation and photographic comparison between the 0-hour and 24-hour samples. The drug loading of R848 in BPBR was measured using a UV spectrophotometer. Additionally, BPBR@FITC was prepared using the same method and analyzed by flow cytometry. The release of R848 from BPBR dispersions at different pH values (5.5, 6.8, and 7.4) was measured using a UV spectrophotometer at selected time points.

2.4. In vitro anti-tumor efficacy and toxicity evaluation

2.4.1. Cell uptake. CT26 cells were seeded in 24-well plates at a density of 5 × 10³ cells per well and cultured for 24 hours. The cells were then co-incubated with complete culture medium containing Cy3, Bi@Cy3, and BPBR@Cy3 for either 1 hour or 4 hours. After incubation, the cells were stained with DAPI for 5 minutes. Finally, the slides were sealed with an anti-fluorescence quencher, and images were obtained using a laser confocal microscope. Flow cytometry was used to quantify the uptake of Bi@FITC and BPBR@FITC.

2.4.2. Cell apoptosis. CT26 cells in logarithmic growth phase were incubated with a solution containing either BPBR or Bi nanoparticles for 4 hours. The cells were then irradiated with 4 Gy of X-rays for 78 seconds and continued to incubate for 24 hours. Following incubation, cells were stained with Annexin V-FITC/PI in the dark according to the manufacturer's instructions. Finally, apoptosis in each group of cells was detected and analyzed using flow cytometry.

2.4.3. DNA damage. CT26 cells were incubated with BPBR for 4 hours, followed by exposure to X-ray irradiation (4 Gy). The cells were then incubated overnight with H₂AX antibody, followed by incubation with Alexa Fluor® 488 mouse anti-H₂AX antibody for 1 hour. Finally, cells were incubated with DAPI for 5 minutes at room temperature in the dark and imaged using a confocal microscope.

2.4.4. Live/dead staining. CT26 cells were seeded in 12-well plates and incubated for 24 hours. The medium was then replaced with fresh medium containing either BPBR or Bi nanoparticles. After 3 hours of incubation, cells were irradiated with 4 Gy of X-rays for 78 seconds and incubated at 37 °C for 24 hours. Cells were then stained with calcein AM/PI according to the kit instructions. Finally, images were taken using a fluorescence microscope and collected.

2.4.5. Cytotoxicity assay. CT26 cells were co-incubated with BPBR or Bi nanoparticles for 3 hours, with or without 4 Gy of X-ray irradiation. After 24 hours, cytotoxicity was evaluated using the CCK-8 assay.

2.4.6. Hemolysis assay. 1 mL of erythrocyte suspension (0.2% v/v) was mixed with 1 mL of PBS containing Bi nanoparticles, PBi nanoparticles, Bac, and BPBR, and incubated at 37 °C for 4 hours. Positive control was hemolyzed with deionized water, and negative control was with PBS. All samples were centrifuged at 4 °C and 3000 rpm for 5 minutes, and the absorbance of the supernatant at 540 nm was measured using a UV-Vis spectrophotometer. The hemolysis rate was calculated according to the following formula:

2.4.7. Bacterial toxicity. The same amount of BPBR and Bac were inoculated on agar plates and incubated anaerobically for 24 hours before taking pictures and counting colonies.

2.5. In vitro immune activation

2.5.1. BMDC maturation and cytokine production. Mouse bone marrow-derived dendritic cells (BMDCs) were seeded in 24-well plates at a density of 5 × 10⁵ cells per well. After 24 hours of incubation with BPBi, R848, or BPBR, the cells were harvested and blocked with goat serum for 30 minutes. Flow cytometry was then used to analyze BMDC maturation (CD11c+CD80+CD86+) by staining with anti-CD80-FITC, anti-CD86-PE, and anti-CD11c-APC antibodies. The levels of IL-6 and TNF-α in the cell supernatant were measured using an enzyme-linked immunosorbent assay (ELISA). CT26 cells were co-incubated with R848 and BPBR for 4 hours, with or without X-ray irradiation. Cell supernatants were collected and used to stimulate BMDCs seeded in 24-well plates for 24 hours. BMDCs were then co-stained with anti-CD80-FITC, anti-CD86-PE, and anti-CD11c-APC antibodies and analyzed by flow cytometry. The concentrations of IL-6 and TNF-α in the culture supernatant were also measured using an ELISA kit.

2.5.2. DCs activation and inhibition of tumor cell proliferation. Logarithmic growth phase CT26 cells were labeled with 5 μM CFSE for 20 minutes. BMDCs and CT26 cells were then co-cultured in 24-well plates at a ratio of 10 : 1. After CT26 cells adhered, BPBi, R848, or BPBR were added and co-incubated for 4 hours, with or without X-ray irradiation. The cells were blocked with goat serum for 30 minutes and incubated with CD11c flow antibodies for 30 minutes on ice. The proportion of CD11c+CFSE+ cells was detected by flow cytometry. Additionally, BPBi, R848, or BPBR were added to the co-culture system of BMDCs and CT26 cells. After incubation at 37 °C for 4 hours, the cells were irradiated with 2 Gy of X-rays for 39 seconds. The cells were then further cultured for 24 hours, and the percentage of CFSE+ CT26 live cells was evaluated using flow cytometry.

2.6. Tumor targeting of BPBR

2.6.1. In vivo imaging. Cy5.5, Bi@Cy5.5, and BPBi-Cy5.5 were administered to CT26 tumor-bearing mice via tail vein injection. In vivo fluorescence images were acquired using a small animal in vivo imaging system at 1, 6, 12, and 24 hours post-injection. All mice were anesthetized with isoflurane prior to imaging. The dose of Cy5.5 was 0.75 mg kg⁻¹ for all groups.

2.6.2. Bacterial biodistribution. To assess the biodistribution of BPBR, the compound was administered to mice on days 1, 5, and 10 post-injection. Major organs (heart, liver, spleen, lung, and kidney) and tumor tissues were harvested, homogenized in sterile water containing 0.1% Triton X-100, and serially diluted. The tissue homogenates were then incubated on solid LB agar plates at 37 °C for 24 hours, and bacterial counts were determined by flow cytometry.

2.6.3. Drug distribution. To determine the distribution of BPBR in the body, the compound was injected into the tail vein, and the mice were euthanized after 24 hours. Heart, liver, spleen, lung, kidney, and tumor tissues were dissected and homogenized. An equal volume of extractant (hydrochloric acid : ethanol = 6 : 94) was added to each homogenate, and the mixture was incubated for 5 minutes followed by centrifugation at 10 000 rpm for 10 minutes. The supernatant was collected, and its absorbance at 318 nm was measured using a UV spectrophotometer to determine the drug concentration.

2.6.4. Bacterial localization analysis. Following tail vein injection of BPBR, highly colonized organs (liver and kidney) and tumor tissues were collected, embedded in paraffin, and sectioned. The sections were stained with Cy3-labeled anti-Bac antibody (anti-Bac@Cy3) to localize Bac, and cell nuclei were counterstained with DAPI. Fluorescence microscopy was used to observe the fluorescence intensity of each tissue, and the data were statistically analyzed.

2.6.5. Co-localization of BPBR with hypoxic regions. BPBR was administered to tumor-bearing mice via the tail vein, and tumor tissues were collected 12 hours later. Tissue sections were stained with antibodies against Bifidobacterium (anti-Bac) and anaerobic-inducible factor (Hif-2α). Cy3 and Alexa Fluor 488 were used as secondary antibodies to detect Ab and Hif-2α, respectively. Bacterial colonization sites were represented by red fluorescence, while hypoxic areas were indicated by green fluorescence. Cell nuclei were counterstained with DAPI. The distribution of Bac in the hypoxic zone was observed under a fluorescence microscope, and the fluorescence intensity was quantified.

2.7. In vivo antitumor immunotherapy efficacy of BPBR.

To evaluate the anti-tumor efficacy of BPBR, a CT26 subcutaneous tumor model was established. Mice with tumor volumes approaching ∼100 mm³ were randomly assigned to eight groups (n ≥ 3): (I) control, (II) Bac, (III) BPBR, (IV) X-ray, (V) Bac + X-ray, (VI) Bi + X-ray, (VII) BPBi + X-ray, and (VIII) BPBR + X-ray. PBS or the corresponding preparations were administered intravenously via the tail vein. The dose of R848 was 2 mg kg⁻¹. Twelve hours after treatment, tumor sites were irradiated with 6 Gy of X-rays for 116 seconds. Tumor growth and body weight were monitored daily. Tumor volume was calculated using the formula: tumor volume = 0.5 × W² × L, where W is the short diameter and L is the long diameter of the tumor. On day 15, three mice from each group were euthanized. Major organs and tumor tissues were harvested. The heart, liver, spleen, lung, kidney, and tumor were subjected to H&E staining for histological analysis. Apoptosis in the tumor was assessed using TUNEL fluorescence staining. We performed H₂AX immunofluorescence staining on the tumor tissues, major organs, and adjacent tissues of mice in the BPBR + X-ray group to assess the DNA damage in normal and tumor tissues. Immunohistochemistry was employed to analyze the expression of hypoxia-inducible factor (HIF-2α) in tumor tissues. Immunofluorescence staining was performed to examine immune infiltration in the tumor tissues, specifically for CD4 and CD8 T cells. Mouse serum was analyzed using ELISA to measure the levels of TNF-α, IFN-β, and IL-6. Drainage lymph nodes from the tumor area of three mice in each group were harvested. Extracted lymphocytes were blocked for 30 minutes, stained with fluorescently labeled antibodies (anti-CD11c-APC, anti-CD80-FITC, anti-CD86-PE) for 30 minutes, and analyzed by flow cytometry. Tumor tissues were minced and digested with 1 mg mL⁻¹ of collagenase (containing 2 U mL⁻¹ of DNase I) for 2 hours. The resulting cells were stained with fluorescently labeled flow antibodies (anti-CD45-Percp, anti-CD3-APC, anti-CD8-PE, anti-CD4-FITC) and (anti-CD11c-APC, anti-CD80-FITC, anti-CD86-PE) and analyzed by flow cytometry. Spleens were removed, milled into single-cell suspensions, stained with flow-through antibodies (anti-CD45-percp, anti-CD3-APC, anti-CD8-APC/Cy7, anti-CD62L-FITC, anti-CD44-PE), and visualized using flow cytometry.

2.8. Statistical analysis

All experimental data are presented as mean ± standard deviation (SD) with a minimum of three replicates (n ≥ 3). Significance levels and exact P values are indicated in all relevant figures. Immunofluorescence intensity was quantified using ImageJ v1.48 software. Statistical analysis of experimental data was performed using one-way ANOVA test, two-way ANOVA test, or an unpaired Student's t-test (GraphPad Prism 8.0.1) after confirming normality and equal variances. Data visualization and figure generation were conducted using Origin 2021 or GraphPad Prism 8.0.1 software.

3. Results

3.1. Construction and characterization of bacterial nano-hybrid BPBR

Firstly, we synthesized bismuth-based nanoparticles (Bi). Transmission electron microscopy (TEM) results showed that Bi nanoparticles were porous spherical nanoparticles formed by the aggregation of multiple small nanoparticles (Fig. 1a). TEM images of bismuth-based nanoparticles loaded with R848 (BR) are shown in Fig. 1b, where the pores of the nanoparticles become denser, and the overall nanostructure is more solid. The average particle sizes of Bi and BR were 101.63 ± 22.05 nm and 108.15 ± 26.35 nm, respectively (Fig. 1i and Fig. S1a, b, ESI†). TEM of PDA-Bi nanoparticles demonstrated the presence of a distinct polymeric film on the surface of polydopamine-coated bismuth nanoparticles (Fig. S1c, ESI†). The adsorption–desorption curve and pore size distribution of Bi are shown in Fig. S1d (ESI†), with a measured specific surface area of approximately 16.4078 m² g⁻¹ and an average pore size of 21.33 nm. The surface of Bifidobacterium is smooth and rod-shaped (Fig. 1c), while the bacteria bound to BR (BPBR) are surrounded by numerous porous spherical nanoparticles (Fig. 1d). TEM-EDS of BPBR is shown in Fig. 1e, where numerous porous spherical nanoparticles adhere to the rod-shaped bacteria, and the EDS spectrum scan results indicate strong bismuth signals (Fig. S2, ESI†). Additionally, the X-ray diffraction (XRD) results of BPBR and Bi are shown in Fig. 1f, both showing characteristic peaks (27.16, 37.95, and 39.61) that are consistent with the standard bismuth card (PDF# 44-1246). XPS results (Fig. 1g) show that BPBR and Bi contain four elements: Bi, O, C, and N: the presence of bismuth in BPBR and Bi was confirmed by two Bi(4f) peaks at 158.65 eV and 163.96 eV (Fig. 1h). The UV-Vis spectrum of BPBR (Fig. S3a, ESI†) also shows the same characteristic peak (318 nm) as the single drug R848, while the individual bacterial or Bi suspension shows no corresponding characteristic peak. Subsequently, we performed flow cytometry detection of BPBR@FITC and Bac + FITC, and the results suggested that BPBR@FITC prepared by polydopamine adhesion had higher fluorescence intensity (Fig. S3b and c, ESI†). In vitro release experiments (Fig. S4a, ESI†) showed that BPBR could better release the R848 drug in an acidic environment (PH = 5.5). The number of colonies grown in BPBR and Bac under the same conditions showed no significant difference (Fig. S4b, ESI†). After 24 hours of standing, experimental observations revealed that PDA-Bi nanoparticles maintained excellent dispersion stability in PBS, with no significant particle dissociation from BPBR being detected (Fig. S4c, ESI†).

Fig. 1 Preparation and characterization of bacterial nano-hybrid BPBR. Transmission electron microscopy (TEM) images of bismuth-based nanoparticles (Bi) (a) and R848-loaded bismuth-based nanoparticles (BR) (b). Scale bar = 200 nm. (c) TEM morphology of Bifidobacterium infantis (Bac). Scale bar = 1 μm. (d) TEM images of bacterial nano-hybrid BPBR. Scale bar = 500 nm. (e) TEM image and the corresponding element mapping of C, N, O, and Bi of the synthesized BPBR. Scale bar = 1 μm. (f) X-ray diffraction (XRD) patterns of Bi and BPBR. (g) X-ray photoelectron spectroscopy (XPS) spectra of Bi and BPBR. (h) XPS spectra of Bi 4f in Bi and BPBR. (i) Particle size of Bi and BR.

3.2. In vitro enhanced X-ray antitumor efficacy of BPBR

The relevant schematic diagram of BPBR enhancing the tumor-killing ability of X-ray is shown in Fig. 2a. Bi@Cy3 was taken up by CT26 cells after 1 hour of incubation (Fig. 2b), and significant uptake was observed after 4 hours. The uptake of Bi@FITC after 4 hours of incubation was 1.34 times that of single drug FITC, and the uptake of BPBR@FITC was like that of Bi@FITC (Fig. 2c and Fig. S5a, b, ESI†). CCK8 results are shown in Fig. 2d, where BPBR showed no significant toxicity to CT26 cells. When combined with X-ray, the cell viability was only 54.75 ± 15.55% at low concentrations and as low as 16.89 ± 3.40% at high concentrations. The apoptosis rate of CT26 induced by BPBR + X-ray was significantly higher than that of other groups (Fig. 2g), reaching 59.76 ± 0.43%, 2.14 times that of X-ray (Fig. 2e). Additionally, BPBR + X-ray had a stronger effect on causing DNA damage than X-ray (Fig. 2f). The live/dead staining results of CT26 also showed that BPBR + X-ray had a stronger ability to kill tumor cells compared to the control and X-ray groups, while there was no difference compared to Bi + X-ray (Fig. 2h and Fig. S5c, ESI†). Additionally, the hemolysis assay results of BPBR showed no difference compared to the negative control group (Fig. S6, ESI†).

Fig. 2 In vitro evaluation of the enhanced X-ray antitumor efficacy of BPBR. (a) Schematic illustration of the mechanism of BPBR-enhanced X-ray antitumor efficacy. Created with BioRender.com. (b) Confocal images of CT26 cells uptake of BPBR@Cy3. Scale bar = 20 μm. (c) Flow cytometry quantification analysis of CT26 cells uptake of BPBR@FITC. (d) In vitro viability of 4T1 cells after co-incubation with Bi or BPBR with or without X-ray irradiation (4 Gy). (e) and (g) Flow cytometric statistical analysis (e) of Annexin V-FITC/PI-stained CT26 cells and corresponding representative flow cytometric analysis images (g) with different treatments. (f) Confocal images of CT26 cancer cells with various treatments were stained with γ-H₂AX. Scale bar = 20 μm. (h) Live/dead staining of CT26 cells with calcein AM (green, live cells)/PI (red, dead cells). Scale bar = 100 μm. Data are presented as mean values ± SD (n = 3). NS: no statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001. Significance between every two groups was calculated using the unpaired two-tailed Student's t test and one-way or two-way analysis of variance (ANOVA) was used for multiple comparisons.

3.3. In vitro immune activation of BPBR

Fig. 3a shows the schematic diagram of directly co-incubating BPBR with mouse bone marrow-derived dendritic cells (BMDCs). After co-incubation of BPBR with BMDCs, the measured DC maturation (CD11c+CD80+CD86+) (Fig. 3b and d), IL-6 (Fig. 3c), and TNF-α (Fig. 3e) were 1.6, 3.38, and 1.97 times that of the control group, respectively, while no difference was observed compared to R848. Fig. 3g shows the schematic diagram of co-culturing BMDCs with the supernatant after co-incubating BPBR with CT26. After co-incubation of BPBR with CT26, with or without X-ray irradiation, the cell supernatant was co-cultured with BMDCs, and the DC maturation was detected by flow cytometry. The results showed that the DC maturation of the BPBR + X-ray group was significantly higher than that of the control group (Fig. 3i and j). BP + X-ray group has some ability to promote BMDC maturation, but its effect is weaker than that of the BPBR + X-ray group. The cytokines IL-6 (Fig. 3f) and TNF-α (Fig. 3h) showed similar trends. Fig. 3k shows the schematic diagram of co-incubating BPBR with the CT26/BMDC system. After co-incubation of BPBR with the CT26/BMDC system (with or without X-ray irradiation), the phagocytosis rate of BMDCs was detected. The BPBR + X-ray group had the highest phagocytosis rate (Fig. 3l and Fig. S7, ESI†), reaching 28.78 ± 1.2%. The proliferation rate of CT26 in the BPBR + X-ray group was significantly lower than that of other groups (Fig. 3m and Fig. S8, ESI†), at 14.55 ± 2.45%.

Fig. 3 In vitro immune activation of BPBR. (a) Schematic illustration of the experimental method of BPBR stimulating BMDC. Created with BioRender.com. (b and d) Maturation of BMDC (b) and representative flow cytometry figure (d) after treatment with different treatment groups. (c) and (e) IL-6 levels (c) and TNF-α levels (e) after BMDC treatment with different treatment groups. (g) Schematic illustration of the method of BMDC treated with supernatant after BPBR treatment of CT26 cells. Created with BioRender.com. (f) and (h) IL-6 levels (f) and TNF-α levels (h) of BMDC treated with cell supernatant after different treatment of CT26 cells. (I) Control; (II) R848; (III) BPBR; (IV) R848 + X-ray; (V) BR + X-ray; (VI) BPBR + X-ray. (i) and (j) Representative flow cytometry figure (i) and DC maturation analysis result (j) of CD11c+CD80+CD86+ after BMDC treatment with CT26 cell supernatant. (I) Control; (II) R848; (III) BPBR; (IV) BP + X-ray; (V) R848 + X-ray; (VI) BR + X-ray; (VII) BPBR + X-ray. (k) and (l) Proportion of CT26 cells (CFSE+) engulfed by BMDC (k) and proliferation rate of CT26 cells (l) after treatment with different treatment groups. (m) Schematic illustration of the co-culture system of BMDC and CT26 cells treated with BPBR. Created with BioRender.com. (I) Control; (II) R848; (III) BPBR; (IV) BPBi + X-ray; (V) R848 + X-ray; (VI) BPBR + X-ray. Data are presented as mean values ± SD (n = 3). NS: no statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001. Significance between every two groups was calculated using the unpaired two-tailed Student's t test and one-way or two-way analysis of variance (ANOVA) was used for multiple comparisons.

3.4. BPBR preferentially targets and colonizes tumor hypoxic regions

The schematic diagram of BPBR tumor targeting is shown in Fig. 4a. The R848 drug distribution suggested that the BPBR group had more drug accumulation at the tumor site (Fig. 4b). In vivo imaging in mice is shown in Fig. 4c, where the tumor site of the BPBR@Cy5.5 group had the strongest fluorescence signal, significantly higher than that of the single drug Cy5.5 and Bi@Cy5.5. After 6 hours, the BPBR@Cy5.5 group showed almost no fluorescence signal in other major organs. The in vivo biodistribution of BPBR is shown in Fig. 4d, where many bacteria could still be cultured at the tumor site up to 10 days. This is 51.27 and 64.44 times higher than in the liver and kidney, respectively (Fig. 4e). However, some bacteria were cultured in the heart, liver, spleen, lung, and kidney during the early stage, but they significantly decreased on day 5. Bacterial localization slices of the liver, kidney, and tumor also suggested that strong fluorescence signals of relevant bacteria were visible at the tumor site, significantly higher than in the liver and kidney (Fig. 4f and Fig. S9, ESI†). Bacterial co-localization with the hypoxic zone of the tumor is shown in Fig. 4h, where co-localization of BPBR bacteria with the hypoxic zone was observed, and the fluorescence intensity co-localization analysis results were consistent (Fig. 4g).

Fig. 4 BPBR preferentially targets and colonizes tumor hypoxic regions. (a) Schematic illustration of BPBR preferentially targeting tumor sites. Created with BioRender.com. (b) Drug concentration distribution of R848 in mice. (c) In vivo fluorescence distribution map in mice. (d) and (e) Distribution of bacteria in mice (d) and corresponding quantitative results (e). (f) Bacterial colonization in liver, kidney and tumor sites of mice. (g) ImageJ analysis of the co-localization map of BPBR with tumor hypoxic region. (h) Co-localization of BPBR with tumor hypoxic regions. Green: hypoxic region; red: bacteria. Scale bar = 200 μm. Data are presented as mean values ± SD (n = 3). NS: no statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001. Significance between every two groups was calculated using the unpaired two-tailed Student's t test and one-way or two-way analysis of variance (ANOVA) was used for multiple comparisons.

3.5. BPBR augments X-ray therapy efficacy in CT26 tumor-bearing mice

The schematic diagram of the in vivo treatment scheme in mice is shown in Fig. 5a. The tumor growth of mice in the BPBR + X-ray group was the slowest (Fig. 5b and Fig. S11, ESI†), and even showed a shrinking trend. Pictures taken from three randomly selected mice from each group on day 15 and the isolated tumors showed that the tumors in the BPBR + X-ray group were almost completely eliminated (Fig. S10a and c, ESI†). The tumor weight results of the mice suggested that the tumor weight in the BPBR + X-ray group was significantly lighter than that of other groups (Fig. 5c). Moreover, the median survival time of the BPBR + X-ray group was over 50 days, while the control group and X-ray group only had 28.5 and 31.5 days, respectively (Fig. 5d). Besides the slight weight loss in the X-ray group, there were no significant differences in the body weight of the mice in other groups (Fig. S10b, ESI†). TUNEL staining results of tumors in each group are shown in Fig. 5e, where the fluorescence signal of the BPBR + X-ray group was significantly higher than that of other groups, 14.19 and 5.84 times that of the control group and the X-ray group, respectively (Fig. 5f). HE staining results showed the most obvious cell necrosis in the BPBR + X-ray group (Fig. 5g), and no significant damage was observed in major organs in each group (Fig. S18, ESI†). The expression of Hif-2α in the BPBR + X-ray group was also the lowest (Fig. 5h and Fig. S12, ESI†). Immunofluorescence staining of H₂AX was performed in heart, liver, spleen, lung, kidney, para-tumor tissues, and tumor tissues. As shown in Fig. S13 (ESI†), only tumor tissues exhibited cellular DNA damage. The blood biochemical tests of mice in each group were basically normal (Fig. S19, ESI†).

Fig. 5 In vivo antitumor effects of BPBR combined with X-ray. (a) Flowchart of mouse treatment regimen. Created with BioRender.com. (b) Tumor volume change curves of mice in different treatment groups. Data are presented as mean values ± SD (n = 5). (c) Tumor weight of mice in each group on day 15. (d) Survival curve. (e) and (f) TUNEL staining results (e) and fluorescence quantification results (f) of tumors in each group. (g) HE staining results of tumors in each group. Scale bar = 50 μm. (h) ImageJ analysis of Hif-2α expression in tumors of each group. Data are presented as mean values ± SD (n = 3). NS: no statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001. Significance between every two groups was calculated using the unpaired two-tailed Student's t test and one-way or two-way analysis of variance (ANOVA) was used for multiple comparisons.

3.6. In vivo antitumor immunotherapy efficacy of BPBR

The in vivo immune mechanism of BPBR is shown in Fig. 6a. Flow cytometry detection of dendritic cell (DC) maturation (CD11c+CD80+CD86+) in lymph nodes draining the tumor area is shown in Fig. 6b, c, where BPBR significantly stimulated DC maturation, reaching 52.09 ± 2.17%. The BPBR + X-ray group had a higher DC maturation rate, reaching 60.43 ± 2.40%, while the control group and X-ray group only had 29.18 ± 7.87% and 37.99 ± 2.03%, respectively. Additionally, the effector memory T cells (Tem) in the spleen and immune cells at the tumor site were also detected. The Tem in the BPBR + X-ray group was as high as 53.73 ± 2.67%, 2.17 times that of the control group (Fig. 6d and Fig. S14, ESI†). Flow cytometry detection of immune cells within the tumor showed that BPBR could better recruit the infiltration of CD4+ T cells and CD8+ T cells within the tumor (Fig. 6e and Fig. S15, ESI†). When BPBR was combined with X-ray, CD4+ T cells and CD8+ T cells reached 38.68 ± 1.50% and 35.15 ± 2.58%, respectively. Moreover, the proportion of mature DCs within the tumor was also higher than that of other treatment groups (Fig. 6f and Fig. S16, ESI†), 3.11 and 5.65 times that of the control group. Immunofluorescence staining of mouse tumors suggested that the expression of CD4+ T cells (Fig. 6j and k) and CD8+ T cells (Fig. 6l and Fig. S17, ESI†) was highest in the BPBR + X-ray group. At the same time, BPBR combined with X-ray strongly promoted the release of cytokines (IFN-β, IL-6, TNF-α) (Fig. 6g–i).

Fig. 6 In vivo antitumor immunotherapy efficacy of BPBR. (a) Schematic diagram of BPBR immune activation. Created with BioRender.com. Created with BioRender.com. (b) and (c) Flow cytometry analysis of the maturation of dendritic cells (DC, CD11c+CD80+CD86+) in tumor-draining lymph nodes (TDLN) of mice in each group. (d) Flow cytometry detection of the proportion of effector memory T cells (Tem, CD3+CD8+CD62L-CD44+) in the spleen. (e) Proportion of CD4+ T cells and CD8+ T cells in the tumor. (f) Proportion of mature DCs in the tumor. Elisa detection of cytokine expression in mouse serum, including IFN-β (g), IL-6 (h) and TNF-α (i). (j) and (k) Immunofluorescence expression of CD4+ T cells in tumors of each group (j) and its fluorescence quantification (k). (l) Immunofluorescence quantification results of CD8+ T cells. Data are presented as mean values ± SD (n = 3). NS: no statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001. Significance between every two groups was calculated using the unpaired two-tailed Student's t test and one-way or two-way analysis of variance (ANOVA) was used for multiple comparisons.

4. Discussion

Radiotherapy, as an effective and clinically common cancer treatment method, can reduce the size of locally advanced tumors that are not surgically resectable, thereby prolonging patient survival.^32,33 However, excessive high-dose radiation inevitably leads to damage to normal skin and organs, limiting the development of radiotherapy. In the past decades, the successful combination of medical science and nanotechnology has propelled functional nanomaterials as clinical candidates for cancer therapeutics.³⁴ Multifunctional nanomaterials as radiosensitizers have attracted significant interest in cancer radiotherapy, where nanoparticles (NPs) containing high atomic number (Z) elements exhibit significantly higher mass energy absorption coefficients for X-rays than soft tissue.³⁵ Nanomaterials containing high-Z metallic plasmas can act as effective radiosensitizers, increasing the local absorption of incident X-ray energy in the tumor, achieving low-dose-induced irreparable DNA damage.³⁶ In recent years, high-Z element-based nanomaterials, such as gold, Bi, and Gd, have emerged as potential radiosensitizers by effectively depositing more radiation energy into the tumor region.³⁷ Bismuth, an element with a high atomic number (Z = 83), exhibits a higher maximum X-ray attenuation coefficient compared to gold, platinum, tungsten, tantalum, and gadolinium.³⁸ Bismuth-based nanoparticles possess low biotoxicity and controllable morphology and size.³⁹ Therefore, bismuth is a promising candidate nanomaterial for radiosensitizers.

In this study, we selected bismuth-based nanomaterials as the primary structure for radiosensitizers. We first constructed porous bismuth nanospheres (denoted as Bi) with an average pore size of 21.33 nm. The prepared bismuth-based nanoparticles exhibit excellent drug loading capacity and can effectively load Resiquimod (R848). Resiquimod (R848) is a small molecule immunomodulator belonging to the TLR7/8 agonist family. Upon binding to TLR7/8, R848 releases various immunoregulatory cytokines, including interleukin 6 (IL-6), interleukin 12 (IL-12), and interferon α (IFNα), thereby triggering a series of signaling pathways, leading to the activation of antigen-presenting cells (APCs) and polarization of T cell responses.^39,40 Bi loaded with R848 (denoted as BR) possesses both X-ray sensitization and immune activation capabilities. In vitro antitumor efficacy experiments demonstrate that Bi combined with X-ray exhibits a potent antitumor effect, significantly enhancing the efficacy of X-ray alone. The addition of R848 in the drug system promotes BMDC maturation, with no significant difference in immune effects compared to free R848 drug. However, our experimental results show that bismuth-based nanoparticles alone cannot achieve reliable tumor targeting. This might be attributed to the unique physiological structure of the tumor microenvironment, such as dense stroma, heterogeneous vascular leakage, and hypoxic conditions, leading to reduced nanoparticle-mediated tumor targeting efficiency.^41–43

Nanoparticle transport primarily relies on passive accumulation at the tumor site through systemic circulation, lacking active driving forces for deep tumor penetration.^44,45 Recent research has explored new strategies for cancer therapy using bacteria. Bacteriotherapy has emerged as a promising antitumor strategy, exhibiting encouraging results in promoting tumor regression and inhibiting cancer cell metastasis, both when used alone and in combination with conventional anticancer therapies.⁴⁶ The biocompatibility of bacteriotherapy has also been widely verified.⁴⁷ Due to the prolonged hypoxic conditions in the tumor microenvironment, many anaerobic bacteria have been observed to selectively localize and grow at tumor sites, including Escherichia coli, Salmonella spp., and Bifidobacterium spp., demonstrating their colonization ability at the tumor site.⁴⁸ In this study, we selected Bifidobacterium as the bacterial carrier, attaching BR to the surface of Bifidobacterium through the adhesive properties of PDA, thereby constructing a novel bio–nanomaterial hybrid drug system (BPBR) with tumor targeting capabilities. Our studies on the in vivo distribution of BPBR in mice show that BPBR exhibits good tumor targeting, and the amount of drug distributed in the tumor site is significantly higher than in major organs like the liver and kidneys. Our research also indicates that BPBR can be significantly localized in the hypoxic regions of tumors. Therefore, the targeting of BPBR is primarily derived from the anaerobic tendency of Bifidobacterium. Furthermore, we evaluated the killing activity and immune effects of BPBR at the cellular level. Our findings demonstrate that BPBR retains the radiation sensitization ability of Bi and the immune stimulation activity of R848. Animal experiments confirm that this system can significantly enhance the tumor-killing ability of X-ray, leading to excellent antitumor efficacy and prolonging the median survival time of mice. Moreover, BPBR activates the immune response in tumor-bearing mice, promoting the maturation of dendritic cells in tumor-draining lymph nodes and increasing the infiltration of immune cells in the tumor, inducing robust immune memory effects. In conclusion, this multifunctional novel bio–nanomaterial hybrid drug system not only enhances the tumor-killing ability of X-ray but also brings good antitumor immune effects, demonstrating its great potential in tumor therapy.

5. Conclusions

In summary, we have designed and constructed a bacteria-based radiosensitizer, BPBR, consisting of infant Bifidobacterium conjugated with bismuth-based nanoparticles loaded with R848. BPBR specifically targets tumor sites and releases R848 bismuth-based nanoparticles through acid-responsive mechanisms. The combination of BPBR and X-ray enhances the tumor-killing effect of X-ray and further activates anti-tumor immune responses, demonstrating effective tumor suppression in a mouse model of colon cancer. This multifunctional bio-active nano-hybrid drug system exhibited excellent and precise anti-tumor activity both in vitro and in vivo, verifying its promising potential for clinical applications. This bio–nano hybrid radiosensitizer, BPBR, provides insights and research foundations for the application of biological and nanomaterials in the biomedical field. We anticipate that this innovative bacterial strategy can be further modified for enhanced multifunctionality and broader applicability, potentially enabling its widespread use in treating various malignant tumors and achieving diverse anti-tumor effects.

Author contributions

Susu Xiao and Gang Guo: conceptualization, methodology and writing – original draft. Yuanxiang Wang and Shulin Pan: investigation, methodology, data curation. Rangrang Fan and Min Mu: performed the animal experiments. Hui Li, Chenqian Feng and Bo Chen: performed the radiotherapy experiments. Bo Han and Wei Yu: Supervision and Funding acquisition. Nianyong Chen and Gang Guo: Supervision Project administration and Writing – review & editing.

Conflicts of interest

The authors declare no conflict of interest.

Data availability

All relevant data are within the manuscript and its ESI.† And detailed data are available from the corresponding author upon reasonable request.

Acknowledgements

This work was financially supported by National Natural Sciences Foundation of China (31971308), Science and Technology Plan Project of Shihezi University (2023AB047), National S&T Major Project (2019ZX09301-147) and Sichuan Science and Technology Program (2022YFS0007).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5tb00825e

‡ S. Xiao, Y. Wang and S. Pan contributed equally to this work.

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