Cells and Animal Models. CAL 27 human squamous carcinoma cells were purchased and cultured as we reported before [44]. HIOEC (normal human epithelial cells) were used as a normal control. Two mouse models including ICR mice, BALB/c nude mice were purchased as previously reported and according to this previously study, we replaced the MCF-7 breast tumor cells to CAL 27 tumor cells as previously reported to obtain the HNSCC xenografts [45]. And when the tumor size was at about 50 mm3, these xenograft mouse models were used for further studies. The inducible tissue-specific immunocompetent Tgfbr1/Pten 2cKO mice were obtained and maintained as we described before [41, 46]. All the animal studies were approved by the Institutional Animal Care and Use Committee of Wuhan University.
Materials and Reagents. Gelatin type B (225 bloom) from bovine skin, selenium (99%, powder), sulfur (99.9%, powder), glutaraldehyde solution (Grade I, 50%) were obtained from Sigma Aldrich. Trioctylphosphine (TOP, 90%) was obtained from Acros Organics. SU-8 2050 photoresist was purchased from MicroChem, USA. RTV615 Silicone Potting Compound was obtained from Momentive Performance Materials (Waterford, NY, USA). Recombinant human tissue inhibitor of metalloproteinases 2 (TIMP2) was obtianed from Sino Biological (Beijing, China).
Synthesis of Gel-N-ICG NPs, drug loading, release and nanoparticle characterization. The SGNPs were prepared via desolvation method as the previous studies [30]. The method to achieve the loading of NSC inside the gelatin NPs and the measure of the morphology, structure, ultraviolet–visible–NIR absorbance spectra, dynamic diameters of nanoparticles, the encapsulation efficiency of NSC and ICG were referred to the previous literature [39]. The way to measure the DLE and DLC of NSC also has been described in previous literature [39].
Cell proliferation assay and Annexin V/PI staining. To evaluate the cell proliferation, CCK8 assay and Annexin V/PI staining was conducted following the manufacture’s instruction, and proliferation of cells were counted by flow cytometry.
Dynamic light scattering (DLS). We used a Zetasizer Nano instrument (Zetasizer Nano ZS, Malvern Instruments Ltd., UK) with a 10-mW He-Ne laser and a thermoelectric temperature controller at the temperature of 25℃ and detection angle of 90° to measure the hydrodynamic particle size. The data were processed subsequently by the Dispersion Technology Software (Malvern Instruments Ltd. U.K.).
In Vivo Toxicity Evaluation. The potential in vivo toxicity of Gel and ICG was evaluated through i.v. injection of 200 µL PBS, PBS containing ICG, PBS containing Gel with the concentration of 5 mg ml− 1 into 15 ICR mice (n = 5), respectively. And every three days, we measured the body weights of treated mice. After the injection of solutions for 24 days, we euthanized the treated mice and collected their blood and major organs to measure the blood panel data and observed the organs sections stained with HE. The healthy mice were used as the control group.
In Vivo PTT Evaluation. 35 mice bearing SCC (n = 5) received i.v. injection of 100 µL PBS or PBS with laser (1 W cm− 2, 5 min), NSC, ICG with laser, Gel-ICG with laser, NCS with ICG with laser and Gel-N-ICG NPs plus laser at the concentration of 5 mg NSC kg− 1. During the PTT, tumor sites were measured with an IR thermographic camera (FORTRIC225, Shanghai Thermal Image Electromechanical Technology Co. Ltd, China). After the PTT, tumor volumes of mice were measured every other day. On the 28th day after treatment, the changes of body weight of mice were measured and all mice were euthanized to collect the tumors. Then, the collected tumors were sectioned into 4 µm and observed after staining using Ki-67, TUNEL.
Tgfbr1/Pten 2cKO mice were treated with a 5-day oral application of 2 mg tamoxifen every day. On the 30-day after induction, SCCs were inducted into the mice oral cavity. To further compare the tumor of 2cKO mice with the human HNSCC, representative mice were euthanized to collect the tumor. Subsequently, 35 2cKO mice (n = 5) received the i.v. injection of 100 µL PBS or PBS with laser (1 W cm− 2, 5 min), NSC, ICG with laser, Gel-ICG with laser, NCS with ICG with laser and Gel-N-ICG NPs plus laser at the concentration of 5 mg NSC kg− 1. During the PTT, IR thermographic camera was used to record the temperatures of the tumor. After the PTT, the tumor volumes of treated mice were measured every five days. At the 15th day after treatment, the changes of body weight were measured and all mice were euthanized to collect the tumor for the further experiments.
Flow cytometry analysis. Single cell suspensions from tumor site, spleens, and blood of Tgfbr1/Pten 2cKO mice with a variety of treatments were prepared. Tamoxifen inducted wide-type mice were set for flow cytometry analysis. And FITC-conjugated anti-mouse CD11b antibody, PE-conjugated anti-mouse PD-1 and Gr-1 antibody were used to label these cells. FACS of these cells was conducted via flow cytometer.
Statistical Analyses. Graph Pad Prism for Windows was used as introduced before for statistical analyses [45].