A Novel Multimodal NIR-II Nanoprobe for the Detection of Metastatic Lymph Nodes and Targeting Chemo-Photothermal Therapy in Oral Squamous Cell Carcinoma

Abstract

Current surgical treatment for oral squamous cell carcinoma (OSCC) must be as precise as possible to fully resect tumors and preserve functional tissues. Thus, it is urgent to develop efficient fluorescent probes to clearly identify tumor delineation, as well as metastatic lymph nodes. Chemo-photothermal therapy combination attracted a growing attention to increase anti-tumor effect in various types of cancer, including OSCC. In the present study, we designed a multimodal NIR-II probe that involves combining photothermal therapy with chemotherapy, imaging OSCC tumors and detecting metastatic lymph nodes. Methods: In this study, we synthesized a novel near infrared (NIR)-II probe named TQTPA [4,4'-((6,7-bis(4-(hexyloxy)phenyl)-[1,2,5]thiadiazolo [3,4-g]quinoxaline-4,9-diyl)bis(thiophene-5,2-diyl))bis(N,N-diphenylaniline)] via the Suzuki reaction and prepared multimodal nanoparticles (NPs) loading TQTPA and cis-dichlorodiammine platinum (CDDP) (HT@CDDP) by hyaluronic acid. The characteristics of the NPs, including their photothermal and imaging capabilities were investigated in vitro and in vivo. Their anti-tumor efficacy was evaluated using orthotopic, tongue tumor-bearing, nude mice. Results: The NPs possessed good stability and water solubility and were pH/hyaluronidase sensitive. The good tissue penetration quality and active targeting ability enabled the NPs to draw the outline of orthotopic tongue tumors and metastatic lymph nodes as small as 1 mm in nude mice by IR-808 under NIR exposure. In vitro and in vivo experiments validated the biocompatibility and low systematic toxicity of the NPs. At the same time, the NPs acted as multimodal therapy agents, combining photothermal therapy with chemotherapy. Conclusion: With a good imaging capability and anti-tumor efficacy, our NPs successfully outlined orthotopic tongue tumors and metastatic lymph nodes as well as enabled chemo-photothermal therapy combination. Our study established a solid foundation for the application of new clinical diagnosis and treatment patterns in the future.

Keywords: NIR-II imaging; active targeting; chemo-photothermal therapy; metastatic lymph node detection.

Figure 1

The schematic illustration of the preparation and application of HT@CDDP NPs. (A) A schematic illustration of the preparation of HT@CDDP NPs. (B) NIR-II imaging/PTT applications of HT@CDDP NPs in orthotopic OSCC tumors.

Figure 2

Synthesis procedures for TQTPA.

Figure 3

Characterization of HT@CDDP NPs. (A) The hydrodynamic radius (Rh) of HT@CDDP NPs in PBS determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM) image of HT@CDDP NPs. Scale bars represent 200 nm. (B) The absorption and fluorescence emission spectra of HT@CDDP NPs. (C) NIR-II imaging and (D) signal intensities of HT@CDDP NPs covered by chicken breast tissues with varying thicknesses. The results represent mean ± SE (n=3).

Figure 4

The release profile and photothermal property of NPs. (A) The cumulative drug release profile of HT@CDDP NPs at different pH values and in the case of hyaluronidase. The results represent mean ± SD (n=3). (B) The heating curves of HT@CDDP NPs at different concentrations by an 808-nm laser at a power density of 1.5 W cm^-2. (C) The heating curves of HT@CDDP NPs (300 μg mL^-1 TQTPA) by an 808-nm laser at different radiant power values. (D) The heating-cooling curve of HT@CDDP NPs (300 μg mL^-1 TQTPA) by an 808-nm laser at a power density of 1.5 W cm^-2.

Figure 5

Cellular uptake and cytotoxity of NPs. (A) NIR-II imaging and (B) signal intensities of HSC3 and SCC4 cell lines with and without anti-CD44 antibody blocking after incubating with HT@CDDP NPs at different time points. (C) Cell viability, determined using a CCK8 assay, of T@CDDP and HT@CDDP NPs at different concentrations of CDDP with or without irradiation in HSC3 and (D) SCC4 cell lines. The results represent mean ± SE (n=3). *p<0.05, **p<0.01, **** p < 0.0001, ANOVA analysis.

Figure 6

Cell apoptosis of HSC3 cells treated with different media and examined by Annexin V-FITC/PI staining kit and flow cytometry.

Figure 7

NIR-I/NIR-II images of HSC3 orthotopic tumor-bearing mice (n = 3 per group). The images were acquired immediately, 4 h, 10 h, 20 h, 24 h, and 30 h after tail vein injection of ICG and HT@CDDP NPs, with or without excess HA blocking. Red circles indicate tumors. Green and blue circles indicate two lymph nodes. (NIR-I: 808 nm excitation, 830 LP and 300 ms; NIR-II: 1064 LP and 2000 ms).

Figure 8

The fluorescence intensity of tumor, metastatic lymph nodes and dissected organs. (A) The NIR-II SBR (signal intensity-to- -background ratio) of tumors in the HT@CDDP and HA blocking groups at different time points. Data shown here are mean ± SE (n=3). (B) NIR-II imaging of dissected organs, tumor tissues, and lymph nodes at 30 h postinjection. (C) The NIR-II signal intensity of lymph nodes at different time points. The results represent mean ± SE (n=3). (D) HE and CK staining of LN#1 and LN#2.

Figure 9

The photothermal property in vivo and anti-tumor ability of NPs. (A) Heat map of the tumors in vivo after injecting HT@CDDP and HT. The range min-max represents 24.2℃ -54.3 ℃. (B) The in vivo temperature curves. The results represent mean ± SE (n=4). (C) In vivo anti-tumor efficacy is detected by tumor volumes and the results are expressed as the mean ± SE (n = 4), *P < 0.05, **P < 0.01. (D) Mice tumor photographs.

Figure 10

HE and IHC staining of dissected tumors. HE, Ki67, and Bcl2 staining of tumors for evaluating anti-tumor effect as well as the effect of inhibiting tumor proliferation and promoting cell apoptosis.