Scientific Papers

GM-CSF augmented the photothermal immunotherapeutic outcome of self-driving gold nanoparticles against a mouse CT-26 colon tumor model | Biomaterials Research

Reagents and biological materials

Chloroauric acid (HAuCl4) was purchased from Xiangbo Biotechnology Co. (Guangzhou, China). Dopamine hydrochloride, trisodium citrate, thiazolyl blue bromide (MTT), DMSO, and the Calcein/PI cell activity and cytotoxicity assay kits were provided by Beyotime Biotechnology (Shanghai, China). Fetal bovine serum (FBS), DMEM, mouse TNF-α, INF-γ, IL-6, CRT, HMGB1 and ATP ELISA kits were purchased from Xinkesheng Biotechnology Co. Ltd. (Luzhou, China). PE anti-mouse CD11c antibody, APC anti-mouse CD86 antibody, and Annexin V-FITC/PI apoptosis assay kit were from Becton, Dickinson Co. Ltd.

The CT-26, 4T1, A549 and AML-12 cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). B. infantis (GIMI.207) was purchased from Guangdong Microbial Culture Collection Center (Guangzhou, China). Male BALB/c mice weighing 16–18 g (6 weeks old) were purchased from Tengxin Bill Laboratory Animal Sales Co. Ltd. (Chongqing, China). All animal experiments were approved by the ethical and scientific committee of the Animal Care and Treatment Committee of Southwest Medical University (SWMU20220026).

Preparation and characterization of Au-NPs, PAu-NPs, and Bif@PAu-NPs

The sodium citrate reduction method was used to prepare Au-NPs [48]. Briefly, 100 mL of 0.01% chloroauric acid was brought to boiling and then mixed with 4 mL of 1% trisodium citrate dihydrate. The mixture was heated until it turned a burgundy color, and then cooled to room temperature. After centrifugation at 10,000 rpm for 10 min, the resulting Au-NPs were freeze-dried in a vacuum and stored at 4 °C. The morphology of the Au-NPs was observed using transmission electron microscopy (TEM, FEI Tecnai G2 F30 USA). Particle size and zeta potential were measured using dynamic light scattering (DLS, NanoBrook90 plus Zeta, Brookhaven, NY) at 25 °C.

PDA coated Au-NPs (PAu-NPs) were prepared through oxidative self-polymerization [49]. To elaborate, 50 mg of Au-NPs and 20 mg of dopamine hydrochloride were dissolved in 50 mL of Tris-HCl buffer (10 mM, pH = 8.5), and stirred in the dark for 6 h. The solution was then centrifuged at 15,000 rpm for 10 min to obtain PAu-NPs, and their morphology, particle size and zeta potential were characterized.

To create Bif@PAu-NPs hybrids, a 1 mg/mL suspension of PAu-NPs was incubated with Bif (2 × 107 CFU/mL) at 37 °C for 4 h. The mixture was then centrifuged at 2500 rpm for 5 min, and the precipitate was washed twice with PBS (pH = 7.4) to obtain Bif@PAu-NPs hybrids. Hybrids labeled with Nile Red (NR) (Bif@PDA-NR-NPs) were prepared in a similar manner. The morphology and energy spectra of the Bif@PAu-NPs were observed using a scanning electron microscope (SEM, SU8020 Hitachi, Japan), and their elemental distribution was evaluated using high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM, FEI Tecnai G2 F30 USA). Physical phase was assessed using X-ray diffraction (BRUCKER D8 ADVANCE Germany) and thermogravimetric analysis (TGA, NETZSCH STA 449 F5/F3 Jupiter Germany). UV absorption spectra of Au-NPs, PAu-NPs, Bif, Bif@PAu-NPs were also analyzed. To evaluate the impact of PAu-NPs on bacterial growth, freshly prepared Bif@PAu-NPs and Bif were incubated for 48 h in an anaerobic environment, and the number of viable bacterial cells was counted.

The in vitro stability of the Bif@PAu-NPs hybrids was investigated by incubating them in an acidic solution (pH = 6.5) with a high concentration of reductive glutathione (GSH, 10 mM) for 4 h to simulate the tumor microenvironment. The incubation in a GSH-free solution at a pH of 7.4 was used as a control. After centrifugation, the UV absorbance of the supernatants was measured using a UV-Vis spectrophotometer (UV-5800PC, Shanghai Metash Instruments Co. Ltd., Shanghai, China).

Photothermal properties of Au-NPs

Photothermal effect

To determine the optimal power and concentration of the nanoparticles for PTT, different concentrations (25, 50, 100 and 200 µg/mL) of a 1 mL aqueous solution of PAu-NPs were exposed to an 808 nm NIR laser (2 W/cm2) for 5 min. The temperature of the solution was measured every 30 s using an infrared thermographer (Fluke, Ti75+, USA). Additionally, a 200 µg/mL solution of PAu-NPs was exposed to the NIR laser at various power densities (0.5, 1, 1.5 and 2 W/cm2), and the temperatures of the solutions were recorded as described.

Photothermal stability

The aqueous solution of PAu-NPs (200 µg/mL) was continuously exposed to an 808 nm laser at 2 W/cm2 for 5 min and subsequently cooled to room temperature. The temperature was recorded every 30 s, and this cycle was repeated three times.

Photothermal conversion efficiency

The PAu-NPs solution (200 µg/mL) was irradiated with an NIR laser (808 nm, 2 W/cm2, 5 min) and the temperature was recorded. The photothermal conversion efficiency (η) was calculated using the following Eq. (1), as previously described [50]:

$$\eta =\frac{\text{h}\text{S}\left({T}_{\text{m}\text{a}\text{x}}-{T}_{ssur}\right)-{Q}_{dis}}{\text{I}\left(1-{10}^{-{A}_{808}}\right)}$$


Where Tmax is the maximum equilibrium temperature, Tsurr is the surrounding ambient temperature, Qdis is the heat loss of light absorbed by the vessel, I (W/cm2) represents the incident laser power, A808 is the absorbance of the sample at 808 nm, h (W/cm2·K) represents the heat transfer coefficient, and S (cm2) represents the surface area of the vessel. The value of hs was calculated by Eq. (2) as follows:

$${\tau }_{s}=\frac{{\text{m}}_{D}{c}_{D}}{hs}$$


Where τs is the time constant of the sample system, and mD and cD are respectively the mass (1 g) and heat capacity (4.2 J/g·°C) of the solvent.

In vitro biological assays

Cellular uptake and cytotoxicity

CT-26 cells were seeded in 6-well plates and incubated with normal saline (NS), Nile Red (NR), NR-loaded NPs (NR-NPs), or PDA-NR-NPs for 3 h. After staining with DAPI, the cells were observed under a fluorescence microscope (OLYMPUS, IX73, Japan) to assess the uptake of different particles. CT-26 and AML-12 cells (5 × 103 cells) were incubated with Au-NPs and PAu-NPs for 24 h, and then irradiated with 808 nm NIR light (2 W/cm2) for 5 min. After 24 h, 20 µL of MTT solution (5 mg/mL) and 150 µL of DMSO were added to each well, and the optical density at 490 nm was measured using a FLUOstar Omega microplate reader.

Calcein-AM/PI assay

CT-26, 4T1 and A549 cells were incubated with 0.5 mL of NS, Au-NPs or PAu-NPs (200 µg/mL) for 4 h. After irradiation with 808 nm laser (2 W/cm2) for 5 min, the cells were further incubated further for 2 h. The suitably treated cells were gently washed and stained with Calcein-AM (1 µg/mL) and pyridinium iodide (PI) (1 µg/mL) for 30 min. The viable cells emitting green fluorescence (λex = 490 nm, λem = 515 nm) and dead cells with red fluorescence (λex = 535 nm, λem = 617 nm) were counted using a fluorescence microscope (OLYMPUS, IX73, Japan).

Apoptosis assay

CT-26 cells and AML-12 cells were seeded at a density of 5 × 104 cells/well and incubated with 0.5 mL of NS, Au-NPs or PAu-NPs at a concentration of 200 µg/mL for 4 h. Some wells were exposed to an 808 nm laser at an intensity of 2 W/cm2 for 5 min. After incubation, the cells were stained with 5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI) in the dark for 15 min. Apoptosis was measured using flow cytometry (BD FACSVerse, Piscataway, NJ).

Hemolysis assay

To evaluate hemolysis, 1 mL of erythrocyte suspension (2%, v/v) was mixed with 1 mL of PAu-NPs, Bif or Bif@PAu-NPs. Double distilled water and saline were used as positive and negative controls, respectively. All samples were incubated at 37 °C for 4 h and then centrifuged at 3000 rpm for 5 min. The optical density (OD) of the supernatant was measured at 540 nm using a UV-Vis spectrophotometer (UV-5800PC, Shanghai Metash Instruments Co. Ltd., Shanghai, China). The hemolysis rate was calculated according to the following Eq. (3)

$$\text{Hemolysis Rate }\left({\%}\right)=\frac{OD\text{ value of experimental group }-OD\text{ value of saline group }}{\text{ OD value of positive control group }-OD\text{ value of saline group }}\times 100{\%}$$


Evaluation of the hypoxia tropism of Bif

To verify the targeting ability of Bif to hypoxic environment, bacterial migration was assessed using transwell chambers. A suspension of Bif (200 µL, 5 × 107 CFU/mL) was seeded into the upper chamber of the transwell insert, while the lower chamber was filled with 400 µL mixture of glucose (0.4 mg/mL), glucose oxidase (0.5 kU) and catalase (0.5 kU). Glucose oxidation depletes oxygen and produces hydrogen peroxide, which is then quenched by catalase, creating an artificial hypoxic environment in vitro. The control wells maintained a normoxic condition in the bottom chamber. After 2 h of incubation, the number of bacteria that migrated to the bottom chamber was counted.

In vivo evaluation of anti-tumor efficacy

CT-26 cells (0.2 mL, 5 × 106) were subcutaneously implanted into the right leg of male Balb/c mice. When tumor volume reached 50–80 mm3, the mice were randomly divided into six groups as follows (n = 6 per group): NS, Bif + NIR + GM-CSF, PAu-NPs + NIR + GM-CSF, Bif@PAu-NPs + GM-CSF, Bif@PAu-NPs + NIR and Bif@PAu-NPs + NIR + GM-CSF groups (Bif: 2 × 107 CFU/mL, PAu-NPs: 2.5 mg/kg, GM-CSF:1.5 mg/kg). The mice were intravenously injected with 100 µL of the drugs and irradiated with NIR laser (808 nm, 2 W/cm2, 5 min) 24 h after injection. The temperature was monitored and controlled at approximately 45 °C using an infrared thermography instrument (Fluke, Ti75+, USA). GM-CSF was intravenously injected 24 h after laser irradiation. Tumor volumes were measured during treatment. At the end of the treatment, the mice were euthanized and the tumor tissues were imaged and processed for TUNEL staining, hematoxylin-eosin (HE) staining and immunohistochemistry (CD4 and CD8). Serum samples were collected for ELISA (CRT and HMGB1).

In vivo biodistribution of Bif

The CT-26 tumor-bearing mice were intravenously injected with Bif@PAu-NPs (0.1 mL) once the tumor volume reached 50 cm3. The mice were euthanized on days 1, 4, 7, and 14, and the tumors along with the major organs (heart, liver, spleen, lung, and kidney) were harvested. The tissue samples were homogenized in sterile water with 0.1% Triton X-100, spread onto LB agar plates, and incubated anaerobically at 37 °C for 48 h. The number of Bif@PAu-NPs was then counted.

Tumor-bearing mice were intravenously injected with Bif@PAu-NPs or Bif (100 µL, 2 × 107 CFU/mL), and euthanized 24 h later. The tumor tissues were harvested, embedded in paraffin, and sectioned. The sections were incubated overnight with primary antibodies anti-Bif (1:25) and anti-HIF-1α (1:100) primary antibodies at 4 °C. Then secondary antibodies (goat anti-mouse FITC-conjugated (1:300) and goat anti-rabbit Cy3-conjugated (1:400)) were applied for 50 min at room temperature in the dark. DAPI solution was used for counterstaining for 10 min. The slides were observed under a fluorescent microscope and the distribution of Bif was statistically analyzed.

In vivotargeting ability of Bif@PAu-NPs

To synthesize Bif@PAu-NPs labeled with indocyanine green(ICG), a Bif@PAu-NPs solution (5 mL, 1 mg/mL) was incubated with 5 mg of ICG at room temperature in the dark for 24 h. The labeled Bif@PAu-NPs were then centrifuged and washed with saline for further use. CT-26 tumor-bearing mice were randomly divided into 4 groups (n = 3) and injected with free ICG (1 mg/kg), Bif@ICG (1 mg/kg, 2 × 107 CFU/mL), PAu-NPs/ICG (1 mg/kg) or Bif@PAu-NPs@ICG (1 mg/kg, 2 × 107 CFU/mL). The mice were imaged at 6, 12, 24 and 48 h after injection using a multimodal small animal live imaging system (ThermoFisher FXPRO USA). After the last time point, the mice were euthanized. Subsequently, the tumors and major organs were harvested for ex vivo imaging.

To determine the biodistribution of Au-NPs, CT-26 tumor-bearing mice were randomly divided into 3 groups (n = 5) and injected with 100 µL of PAu-NPs (50 µg/kg), Bif (2 × 107 CFU/mL) or Bif@PAu-NPs (50 µg/kg, 2 × 107 CFU/mL). After 24 h, the mice were euthanized. Subsequently, the tumor tissues and major organs were weighed, and then digested in aqua regia at 80 °C. The gold content in the tissue samples was measured using inductively coupled plasma mass spectrometry (ICP-MS) (Agilent ICPMS7800 USA).

Microscopic PET/CT scanning

Whole-body microPET/CT scans (Siemens Germany) were conducted to evaluate the early response of the mice to the different formulations. Three mice were chosen randomly from each group 48 h after the final treatment. After fasting for 6 h, 200–250 µCi of 18 F-FDG was injected via the tail vein. Thirty minutes later, the mice were anesthetized using isoflurane and scanned using parameters of 80 kV, 500 mA, and 1.5 mm slice collimation. The region of interest (ROI) on PET/CT images was manually delineated, and the maximum normalized uptake value (SUVmax) and mean uptake value (SUVmean) were calculated.

In vivo anti-tumor immune assay

Freshly isolated tumor tissues were homogenized into single-cell suspensions, which were then stained with anti-CD3 (FITC), anti-CD45 (FITC), anti-CD8 (APC), anti-CD4 (PE), anti-CD11b (Percp), anti-CD11c (PE) and anti-CD86 (APC) antibodies according to the manufacturer’s protocol (BD Pharmingen). The stained cells were analyzed using flow cytometry (BD FACSAria USA). Serum samples were diluted, and the levels of TNF-α, IFN-γ, IL-6 and ATP were measured using specific ELISA kits according to the manufacturer’s instructions.

In vivo validation of long-term immunological memory

To evaluate long-term immunological memory, primary tumors were established in the right leg using the method described above. Once the tumor reached a size of 100 mm3, the mice were randomly divided into 4 groups and treated with NS, Bif@PAu-NPs, Bif@PAu-NPs + NIR and Bif@PAu-NPs + GM-CSF + NIR. After 60 days of treatment, the mice were reinoculated with 5 × 106 CT-26 cells in the left leg to establish a secondary tumor. Body weight and tumor volume were measured every two days after reinoculation.

In vivo biocompatibility of Bif@PAu-NPs

Healthy Kunming mice were injected intravenously with NS and Bif@PAu-NPs (50 mg/kg) three times every 2 days. After 14 days, blood samples were collected to measure blood routine indices and biochemical indicators such as red blood cells (RBC), white blood cells (WBC), platelets (PLT), hemoglobin (HGB), mean red blood cell hemoglobin concentration (MCHC), red blood cell-specific volume (HCT), mean hemoglobin (MCH), mean hematocrit (MCV), neutrophil count (NEU), glutamate transaminase (ALT), aspartate transaminase (AST), urea (UREA), glucose (GLU), albumin (ALB), and total cholesterol (TC).

Statistical analysis

All data were expressed as the mean ± standard deviation (SD) of three independent experiments. The data were evaluated using the Student’s t-test unless otherwise stated. Survival curves were plotted using the Kaplan-Meier method, and survival times and 95% confidence intervals were compared using the log-rank test. Statistical analysis was performed using GraphPad Prism 9 software. A significance level of P < 0.05 was considered statistically significant.

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