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

Abstract

Background: Hypoxia is a frequent characteristic observed in solid tumors and is strongly associated with tumor metastasis, angiogenesis, and drug resistance. While the vasculature of hypoxic tumor tissues poses obstacles to the efficient administration of conventional drugs, it may prove advantageous in sustaining hyperthermia. Photothermal therapy (PTT) offers a promising treatment strategy that utilizes the activation of photosensitizers to produce heat, thus facilitating the selective ablation of tumor tissues.

Method: To enhance the accumulation of photothermal agents in tumor tissue and improve the effectiveness of PTT, we developed a self-propelled hybrid called Bif@PAu-NPs. This hybrid consists of polydopamine (PDA)-coated gold nanoparticles (Au-NPs) loaded onto the anaerobic Bifidobacterium infantis (Bif).

Results: The Bif@PAu-NPs actively aggregated at the tumor site because the ability of Bif can target hypoxic regions, and PAu-NPs achieved precise PTT due to their high photothermal conversion efficiency (η = 67.8%). The tumor tissues were ablated by PTT, resulting in the release of antigens through immunogenic cell death (ICD), which stimulates an immune response. The inclusion of GM-CSF enhanced the immune response by recruiting dendritic cells and initiating long-term anti-tumor immunity.

Conclusion: The Bif@PAu-NPs hybrid effectively suppressed the growth of both primary tumors and re-challenged tumors. The utilization Bif@PAu-NPs in conjunction with GM-SCF exhibits great potential as a photothermal-immunotherapeutic strategy for precisely treating solid tumors. In this study, the bacterial Bif@PAu-NPs biohybrid is exploited the self-driving ability of anaerobic Bifidobacterium infantis to deliver polydopamine-modified gold nanoparticles to hypoxic region of tumor. Under irradiation with 808 nm NIR laser, the hybrid exerts precise photothermal therapy to stimulate the immune response, which is further enhanced by GM-CSF, leading to recruitment of dendritic cells and initiation of a long-term anti-tumor immunity remember to prevent tumor recurrence.

Keywords: Anaerobic bacteria; Colorectal cancer; GM-CSF; Gold nanoparticels; Immunotherapy; Photothermal therapy.

Scheme 1

Schematic illustration of the synthesis and antitumor performance of Bif@PAu-NPs

Fig. 1

Characterization of Au-NPs, PAu-NPs and Bif@PAu-NPs. A TEM images of (a) Au-NPs, (b) PAu-NPs and (c) Bif@PAu-NPs hybrids. B Average particle size of Au-NPs and PAu-NPs (n = 3). C HAADF-STEM images and elemental mapping of Bif@PAu-NPs. D XRD patterns of Au-NPs, PAu-NPs and Bif@PAu-NPs. E TGA curves of Au-NPs, PAu-NPs and Bif@PAu-NPs. F UV-Vis absorption spectra and the color (insert) of Au-NPs (a), PAu-NPs (b) and Bif@PAu-NPs (c) solutions

Fig. 2

In vitro characterization of photothermal properties. A The relative absorbance intensity of PAu-NPs solution with different concentrations at 808 nm NIR irradiation. B Heating curves of PAu-NPs at concentrations of 0, 25, 50, 100, 200 µg/mL under 808 nm laser. C Infrared thermal images of PAu-NPs solutions under 808 nm laser. D Heating curves of PAu-NPs irradiated with different laser powers (0.5, 1, 1.5, 2.0 W/cm²). E Photothermal stability of PAu-NPs (200 µg/ml, 2.0 W/cm²) in three heating-cooling cycles. F Heating and cooling curve of PAu-NPs solution under 808 nm laser irradiation (2.0 W/cm²). G Heating curves of PAu-NPs and Bif@PAu-NPs solutions under 808 nm laser irradiation (2.0 W/cm²)

Fig. 3

In vitro cellular experiments. Cellular uptake of in vitro photothermal therapy (A) Cell uptake of NR-labeled nanoparticles (scale bar 50 μm). B Schematic diagram of in vitro PTT. C Cell viability of CT-26 cells. (n = 3). D cell viability of AML-12 cells incubated with Bif@PAu-NPs for 24 h (n = 3). E Cell viability of CT-26 cells with different treatments. F Live/dead staining of CT-26 cells with Calcein AM (green, live cells), PI (red, dead cells). The white dashed line represents the boundary line of laser irradiation. Scale bar = 500 μm. G Apoptosis rate of each group. I: Control; II: Au-NPs; III: PAu-NPs; IV: Bif@PAu-NPs; V: NIR; VI: Au-NPs + NIR; VII: PAu-NPs + NIR; VIII: Bif@PAu-NPs + NIR. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

Targeting ability of Bif@PAu-NPs hybrids. A Representative photographs of bacterial growth in heart, liver, spleen, lung, kidney and tumor tissues on days 1, 4, 7 and 14 after Bif@PAu-NPs injection in CT-26 tumor-bearing mice. B Number of Bif@PAu-NPs colonization in liver, kidney and tumor. C Schematic diagram of in vitro hypoxia model using Transwell Chambers. D Bacteria number in Transwell chambers. E Fluorescence images of tumors. (Red: hypoxic zone, green: Bif, scale bar = 20 μm). F Fluorescence intensity of Bif@PAu-NPs and hypoxic zone along the yellow line). (***P < 0.001, ****P < 0.0001)

Fig. 5

In vivo evaluation of antitumor efficacy. A Schematic diagram of the treatment procedure. B Representative photographs of CT-26 tumor-bearing mice on day 14 of post-treatment (n = 3). C Representative photographs of isolated tumors (n = 3). D Tumor weights. E Tumor volume (n = 6). F Change of body weight (n = 6). G Survival curves of mice (n = 6). H Immunofluorescence images of CD4, CD8, CRT and HMGB1, HE staining of tumors. scale bar = 50 μm. (*P < 0.05, ****P < 0.0001). I: Control; II: Bif + NIR + GM-CSF; III: PAu-NPs + NIR + GM-CSF; IV: Bif@PAu-NPs + GM-CSF; V: Bif@PAu-NPs + NIR; VI: Bif@PAu-NPs + NIR + GM-CSF

Fig. 6

In vivo evaluation of early response to treatments and anti-tumor mechanism. A 18 F-FDG PET/CT images of mice in each group, upper: cross-sectional images, lower: coronal images, red circles indicate the tumor sites. SUVmean (B) and SUVmax (C) in each group. ATP secretion (D), CRT exposure (E) and the release of HMGB1(F) in tumor tissues detected by ELISA. Pro-inflammatory cytokine including IL-6 (G), IFN-γ (H) and TNF-α (I) analyzed by ELISA. Apoptosis of tumor tissues stained with DAPI and TUNEL(J), scale bar = 50 μm (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). I: Control II: Bif + NIR + GM-CSF. III: PAu-NPs + NIR + GM-CSF. IV: Bif@PAu-NPs + GM-CSF. V: Bif@PAu-NPs + NIR. VI: Bif@PAu-NPs + NIR + GM-CSF.

Fig. 7

In vivo distribution the Bif@PAu-NPs hybrid and its anti-tumor immune response. A Fluorescence images of CT-26 tumor-bearing mice after intravenous injection of free ICG, PAu/ICG, ICG@Bif and Bif@PAu@ICG. The right panel shows in vitro fluorescence photos of isolated organs and tumors on 48 h after injection. (H: heart, Li: liver; S: spleen; Lu: lung, K: kidney, T: tumor). B Au content in tumors. C Au content in tumor and major organs of mice after Bif@PAu-NPs injection. D Percentage of mature DCs in spleen. E Percentage of mature DCs in tumor. F The ratio of CD8/CD4 in spleen. G Infrared thermography images of mice irradiated with NIR laser for 5 min. H The temperature change in each group (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Groups. I: Control. II: Bif + NIR + GM-CSF. III: PAu-NPs + NIR + GM-CSF. IV: Bif@PAu-NPs + GM-CSF. V: Bif@PAu-NPs + NIR. VI: Bif@PAu-NPs + NIR + GM-CSF.

Fig. 8

In vivo evaluation of long-term immunological memory by tumor rechallenge. A Schematic diagram of experimental design. B Weight fluctuations during treatment (n = 3). C Tumor volume of second tumors (n = 3). (D) Survival curves of mice (n = 6). Levels of pro-inflammatory cytokines including IFN-γ (E), IL-6 (F) and TNF-α (G). (*P < 0.05, ****P < 0.0001). a: Control, b: Bif@PAu-NPs, c: Bif@PAu-NPs + NIR, d: Bif@PAu-NPs + NIR + GM-CSF.