Materials
A 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) and a DNA damage detection kit were obtained from Beyotime Biotechnology (China). 1-Ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and n-hydroxysuccinimide (sulfo-NHS) were purchased from Sigma (USA). Agar and LB broth were purchased from HuanKai Microbial (China). AuNPs were obtained from Shanghai Dibai Biotechnology Co., Ltd. (China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin solution, and trypsin containing 0.25% EDTA were purchased from Gibco (USA).
Cell lines
4T1 cells (mouse breast cancer cells) were obtained from the China Center for Type Culture Collection (CCTCC). The 4T1 cells were incubated in high-glucose DMEM containing 10% FBS and 1% penicillin‒streptomycin in a humidified incubator with 5% CO2 at 37 °C.
CAT plasmid and preparation of PCM
The E. coli MG1655 strain was purchased from Angyubio Biotechnology Co. (China). LB broth supplemented with 0.01% ampicillin was used for bacterial culture. The CAT-expressing thermosensitive plasmid (pBV220-CAT) synthesized by Sangon Biotech (China) contains the PR-PL tandem promoter and AmpR promoter. The recombinant plasmid was transformed into E. coli DH5α competent cells via a chemical transformation protocol. The pBV220-CAT plasmids were subsequently transformed chemically into the E. coli MG1655 strain to obtain PCM. The resulting PCM solution was plated on an LB solid plate with ampicillin and incubated at 37 °C for 12 h. PCM colonies were removed and amplified in LB medium at 37 °C and 220 rpm overnight. Afterward, the PCM solution was diluted 100-fold in LB medium and further grown to OD600 = 0.4–0.6 for further experiments.
Thermal-induced and ultrasound-induced CAT expression
The PCM transformed with the pBV220-CAT plasmid was incubated at 37–45 °C for a given time and further incubated for 2 hours at 37 °C. The bacterial OD was measured via a spectrophotometer. An equal number of 2 × 109 bacteria were collected, and the protein expression of CAT was analyzed via SDS‒PAGE. To evaluate the feasibility of ultrasound-induced CAT gene expression, PCM was added to a 24-well plate (1000 µl per well). Ultrasound irradiation was applied at varying acoustic intensities of 0.8 W/cm2, 1.2 W/cm2, 1.6 W/cm2, 2.0 W/cm2, and 2.4 W/cm2 with different durations to generate localized heat for gene induction, the selection of intensity referenced the work of Liu et al. [29] The fixed setting of intensity by Sonovitro ensured stability during sonication. Following the methodology of Chen et al. [28], an ON-OFF pulsed irradiation protocol was implemented to maintain consistent thermal conditions (ΔT within ± 1.0 °C over 30 min). The temperature of the PCM solution was monitored by an infrared thermal imager. In the following in vitro and in vivo experiments, we used this parameter (1.6 W/cm2, 1 MHz, 3 s ON, and 7 s OFF), which could keep the irradiated bacterial mixture at 45 °C constant. Following ultrasound irradiation, western blotting was used to confirm CAT expression in vitro.
Preparation and characterization of PCM@AuNPs
The PCM was cultured to an OD600 of 0.4–0.6 at 37 °C, collected by centrifugation (3,000 rpm, 6 min), and then suspended in PBS. The AuNPs with -COOH surface modifications were suspended in 2 mL of MES buffer (0.1 M, pH = 5.5), and EDC and sulfo-NHS were added, corresponding to an EDC: sulfo-NHS: -COOH molar ratio of 30:30:1. After incubation at room temperature for 1 h to activate the carboxyl groups of COOH-AuNPs, the COOH-AuNPs were centrifuged at 10,000 rpm for 5 min and washed with PBS to remove residual EDC and sulfo-NHS. The centrifuged precipitate was washed 3 times, added to the PCM solution, and incubated for 2 h to obtain PCM@AuNPs. After centrifugation (3,000 rpm, 6 min), the PCM@AuNPs were suspended in PBS (pH = 7.4) and stored at 4 °C.
The particle size and zeta potential of the PCM@AuNPs were determined via dynamic light scattering (DLS) analysis via a Malvern NANO ZS instrument. The UV‒vis absorption spectra of each component of the PCM@AuNPs were measured via a UV spectrophotometer. To standardize AuNPs concentration in PCM@AuNPs complexes, serial dilutions of AuNPs (0–50 µg/mL in PBS) were prepared. UV-vis absorption spectra (400–800 nm) were recorded. The absorbance at the AuNPs plasmon resonance peak (λ = 520 nm) was plotted against known concentrations to generate a linear calibration curve (R² > 0.99). For PCM@AuNPs samples, free AuNPs were separated from bacteria via centrifugation (3,000 ×g, 10 min). The supernatant absorbance at 520 nm was measured and subtracted from the total AuNPs added to calculate the bound fraction. Binding efficiency (%) was defined as:
$$\:Binding\:Efficiency=\frac{\left[AuNPs\right]total-\left[AuNPs\right]free}{\left[AuNPs\right]total}*100\%$$
PCM@AuNPs were stored in PBS at 4 °C, and their UV-vis absorption spectra were collected at 0 h and 72 h to analyze stability and potential aggregation behavior. Before transmission electron microscopy (TEM), the PCM@AuNPs samples were fixed with glutaraldehyde.
Evaluation of PCM viability post-treatment
The PCM strain was cultured to an OD₆₀₀ of 0.4–0.6 at 37 °C. Bacterial suspensions were irradiated with varying doses of radiation (0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy). After irradiation, the bacteria were further incubated at 37 °C overnight (approximately 6–8 h). The cultures were collected, appropriately diluted, and plated for colony counting. Moreover,1 mL of PCM suspension (OD₆₀₀ = 0.4–0.6) was transferred to a 24-well plate. Ultrasound irradiation was applied under varying parameters (see details in Thermal-Induced and Ultrasound-Induced CAT Expression) while maintaining the temperature at 45 °C. After 30 min of irradiation, the samples were incubated at 37 °C overnight. The OD600 of the mixture was measured to assess bacterial growth.
In vitro cytotoxicity assay of AuNPs on PCM
The PCM and the PCM@AuNPs were cocultured at 37 °C for 12 h, respectively. At different time points, the OD600 of the mixture was determined. Twelve hours later, a drop of the diluted sample was placed on a microscope slide. A microscope was used to locate the bacteria, and images of bacterial movement at different time points were recorded. The data analysis software NIS Elements D4.10.00 was used to calculate the movement rate, which was recorded as distance/time.
Tumor targeting of PCM@AuNPs
The concentration of PCM was adjusted to 1 × 107 CFU/ml. The DiR dye was added to the PCM solution (30 µl/mL) and incubated at 37 °C for 45 min. The bacterial mixture was washed with PBS until the supernatant was clear. The bacteria were injected into the tumor-bearing mice via the tail vein (1 × 107 CFU per mouse) (n = 4). At the given time points, the mice were imaged with an IVIS system (PerkinElmer, USA) to evaluate the biodistribution and tumor accumulation of bacteria. Forty-eight hours after intravenous injection, the tumor-bearing mice were sacrificed, and organs, including the heart, lung, liver, kidney, and spleen, and tumors were obtained for fluorescence signal detection via the same imaging system. Additionally, the accumulation of PCM in different organs was detected via clone counting. At 48 h after the administration of PCM@AuNPs, tumors, and organs were obtained, and the diluted grinds of each organ were cultured on LB-Agar plates for clone counting.
Evaluation of the effect of PCM@AuNPs on oxygen production
Three experimental groups were used: MG1655@AuNPs + US (M1), PCM (M2), and PCM@AuNPs + US (M3). Following ultrasound induction (1.6 W/cm², 1 MHz, 3 s ON, and 7 s OFF) for 30 min, the samples were incubated at room temperature for 6 h. The active proteins from engineered bacteria were extracted via a bacterial protein extraction kit (Sangon Biotech, China). The precise quantification and activity of CAT expressed by the PCM@AuNPs were assessed via a Catalase Elisa Kit (ZCIBIO Technology Co., Ltd., China) and a Catalase Activity Kit (Nanjing Jiancheng Bioengineering Institute, China). After quantification, different high concentrations of CAT and H2O2 were mixed in 5 mL of hydrogen trichloride buffer (0.1 M, pH = 7.52) for the reaction. A balloon was attached to one end of the test tube, and the volume of oxygen was determined by measuring the diameter of the oxygen-carrying ball. A dissolved oxygen monitor was used to reflect the real-time change in oxygen solubility directly. Which was purchased from Shanghai INESA Scientific Instruments Co., Ltd. (China)
To verify the ability of PCM@AuNPs to relieve hypoxia in tumors, the 4T1 tumor-bearing mice were divided into three groups: control, PCM@AuNPs, and PCM@AuNPs + US. The mice were i.v. Injected with 100 µL of PBS or PCM@AuNPs, and the number of bacteria was 1 × 107 CFU/100 µL. At 24 h p.i., each mouse in the PCM@AuNPs + US group was subjected to ultrasound (1.6 W/cm2, 1 MHz, 3 s ON, and 7 s OFF irradiation) for 30 min. Six hours later, the tumor tissue was collected, and western blotting was used to confirm CAT expression in vivo. The tumor tissue was collected and sacrificed for immunofluorescence staining, with DAPI for the cell nucleus and HIF-1α for hypoxia-inducible factors. Images were obtained via CLSM.
In vitro detection of reactive oxygen species and DNA damage
To detect the generation of ROS and DNA damage induced by PCM@AuNPs during radiation in vitro. 4T1 cells were seeded into 24-well plates overnight at a density of 1 × 104 per well. After that, the cells were divided into seven groups: the control group without any treatment (G1) and the other groups were irradiated by X-ray at 6 Gy after being treated with PBS (G2), PCM (G3), AuNPs (G4), MG1655@AuNPs + US (G5), PCM@AuNPs (G6), or PCM@AuNPs + US (G7) at 37 °C with 5% CO2 for 4 h. Subsequently, DCFH-DA, γ-H2AX, and the DNA ladder assay were used for ROS or DNA damage detection, respectively. Fluorescence imaging was conducted using a fluorescence microscope (EVOS M5000, America), and fluorescence intensity was quantified with ImageJ. Gel imaging was conducted under UV light using a ChemiDoc MP Imaging System (Bio-Rad, USA).
In vitro detection of the MMP
To assess the intracellular MMP. 4T1 cells were cocultured with the same treatment described above in 24-well plates. Then, 1 mL of JC-1 was added to the cells for coculture at 1 h after irradiation, and the cells were observed under a fluorescence microscope.
In vitro colony formation assay
4T1 cells (1,000/well) were seeded in 6-well plates and incubated with the different treatments mentioned above for 6 h. The samples were subsequently irradiated with γ-rays at a dose of 6 Gy. Next, the cells were continuously cultured with fresh drug-free medium in an incubator for another 5‒8 days. The fixed colonies were then stained with crystal violet (0.25% ethanol) to subsequently count the number of colonies and evaluate the colony inhibition ability of each treatment.
Assessment of cell apoptosis
Cell apoptosis was determined with an Annexin V-FITC apoptosis detection kit. First, 4T1 cells at a density of 2 × 105 were seeded in 6-well plates. After the treatment described above, the cells were trypsinized and resuspended in 500 µL of binding buffer. Five microliters of Annexin V-FITC (20 µg/mL) and 5 µL of PI (50 µg/mL) were continuously added to the above buffers and then incubated for 15 min at room temperature in the dark. Finally, the degree of cell apoptosis in each group was analyzed via flow cytometry.
Cell cytotoxicity assay
4T1 cells were plated onto 96-well plates at a density of 1 × 104 cells per well at 37 °C in a 5% CO2 incubator. Following the previous grouping. After the addition of 10 µL of Cell Counting Kit-8 to each well, the mixture was incubated for 2 h in a cell incubator. Cell proliferation was measured by measuring the optical density (OD) at 450 nm via a microplate reader.
In vivo antitumor assay
The 4T1 tumor-bearing mice with an approximate tumor volume of 100 mm3 were randomly divided into five groups (n = 4): the control group (T1), RT group (T2), AuNPs + RT (T3), PCM + RT + US (T4), and PCM@AuNPs + RT + US (T5) groups. A total of 1 × 107 PCM in 100 µl of PBS was injected intravenously into tumor-bearing mice in the T4 and T5 groups. The T1 group was injected with 100 µl of PBS, and the concentration of AuNPs was 50 µg/ml. After 24 h of bacterial injection, the mice were irradiated with ultrasound (1.6 W/cm2, 1 MHz, 3 s ON, and 7 s OFF irradiation times) to induce PCM for 30 min. Each group received a dose of 6 Gy of γ-ray irradiation on the third day, with a treatment cycle of 4 days, and the tumor volume and mouse weight were recorded every 2 days. The formula for calculating tumor volume was as follows: volume = (tumor length × tumor width2)/2. After the mice died, the tumors were collected for H&E staining and TUNEL assays.
Metastasis treatment
For lung metastasis treatment, the treated mice (n = 4) were euthanized on the 21st day by carbon dioxide asphyxiation, and their lungs were collected. The number of metastatic nodules in the lungs and the lung weight were recorded. The lung slices were then stained with H&E to observe the metastatic foci.
Statistical analysis
The statistical analyses of the experimental data were conducted via GraphPad Prism software version 9.5.0. The data set was subjected to one-way analysis of variance (ANOVA) for statistical evaluation, and the P values were determined via a two-tailed unpaired heteroscedastic t-test, with significance indicated by *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
