Materials

Glutathione (GSH), N-hydroxysuccinimide (NHS), tetrachloroauric (III) acid (HAuCl4), and 1-ethyl-3-(3-dimethylaminopropyl) carboxylate (EDC) were purchased from Sigma-Aldrich. Isopropyl alcohol, acetone, silver nitrate (AgNO3), and hydrazine hydrate (N2H4·H2O) were purchased from Sinopharm Chemical Reagent Co., LTD (Beijing, China).

Synthesis of AgNDs

To synthesize AgNDs, 0.082 6 g AgNO3 and 0.163 5 g GSH were added to 15 mL deionized water, and the solution was stirred for 30 min till a cloudy gel was obtained. The pH of the Ag-GSH gel was adjusted to 6-7 using 1 mol/L NaOH, and 1 mL N2H4·H2O was added. After stirring in the dark for 48 h, excess isopropyl alcohol was added to the mixture, followed by centrifugation, after which the supernatant was removed. The AgNDs were finally obtained by redissolving the precipitate in 9 mL deionized water.

Synthesis of AuNDs

To synthesize Au NDs, 250 μL of a 50 mol/L HAuCl4 solution and 500 μL AgNDs were sequentially added to 12 mL deionized water. The pH of the solution was modified to a range of 7 to 8 by adding 1 mol/L NaOH. After heating at 80 °C for 4 h, the solution was centrifuged at 8 700 r/min for 13 minutes, and the supernatant was mixed with isopropanol. The precipitate was collected by centrifuging at 6 500  r/min for 10 min, and then redispersed in 8 mL deionized water to obtain the AuNDs solution.

Synthesis of Au-HN-1

To synthesize Au-HN-1, 4 mg NHS and 12 mg EDC were added to 2 mL AuNDs solution and stirred for 20 min. Following addition of 100 μL HN-1 (5 mg/mL), the mixture was left undisturbed for 12 h and then dialyzed in a dialysis bag with a molecular weight cutoff of 3 500 Da for another12 h to obtain the Au-HN-1 solution.

Characterization of Au-HN-1

Transmission electron microscopy (TEM) imaging was conducted using the JEOL TECNAI F20 instrument set to an operating voltage of 200 kV. The UV-Visible (UV-Vis) absorption spectrum was recorded using Lambda 800 UV-Vis spectrophotometer. Fourier transform infrared (FTIR) spectroscopy was performed over the range of 500 to 4 000 cm−1 using Nicolet Avatar 360 FTIR spectrophotometer. Laser scanning confocal microscopy was performed with the Olympus Fluoview FV1000 confocal microscope. X-ray photoelectron spectra (XPS) analysis was performed using the VG ESCALAB MKII spectrometer, with excitation provided by Mg Kα radiation at 1253.6 eV. Fluorescence properties were assessed with Shimadzu RF-5301 PC fluorescence spectrometer. Infrared thermal imaging was performed with FLIR T420 infrared thermal imaging camera.

Cell culture

Human cell lines of TSCC (SCC9, CAL 27, CRL-1623), non-small cell lung cancer (A549), hepatocarcinoma (HCC-LM3), gastric cancer (MKN-45) and breast cancer (SK-BR-3), and the human embryonic kidney cell line (HEK 293 T) were gained from ATCC and Shanghai Zhong Qiao Xin Zhou Biotechnology. The cells were cultured in DMEM/F12(Gibco, Grand Island, NY,USA), Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY,USA)), or RPMI 1640 (Gibco, Grand Island, NY,USA) containing 10% fetal bovine serum (Gibco, Grand Island, NY,USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY,USA) at 37 °C and 5% CO2.

Cell Counting Kit-8 assay

Cell viability was assessed by the CCK-8 assay (Meilune, Dalian, China). Briefly, SCC9 (3×103) and HEK 293 T cells (5×103) were seeded into 96-well plates and allowed to culture for 24 h, and then incubated with varying concentrations of Au-HN-1 (0, 100, 200, 300, 400 and 500 mg/mL) for 4 h. The cells were irradiated with NIR light at the wavelength of 808 nm for varying durations (60 s, 180 s, 300 s, and 600 s; n = 3 per group), and a non-irradiated control was also included (n = 3). Complete medium containing 10% CCK-8 solution was added to each well, and the cells were then incubated for another hour. The absorbance was measured at 450 nm, and the proliferation rates were calculated for the different drug concentrations.

Apoptosis assay

SCC9 cells were treated with Au-HN-1 (100 μg/mL) or the vehicle and then exposed to 808 nm NIR light at the intensity of 2 W/cm2 for 5 min; respective non-irradiated controls were also included. After a 24 h period of cell treatment, the cells were harvested from the cell culture dishes. After rinsing twice with PBS, the cells were stained using propidium iodide and Annexin V-FITC as per the kit instructions (PI; Beyotime, China). The apoptotic cells were detected by flow cytometry (BD Biosciences, USA). Each experiment was conducted in three replicates.

Cell migration assay

The wound-healing assay was utilized to evaluate cell migration. SCC9 cells were seeded in 6-well plates at the density of 1 × 105 cells per well and incubated for 24 h. Following treatment with Au-HN-1 (100 μg/mL) and 808 nm NIR irradiation (2 W/cm2 5 min) as described above, the monolayer was scratched to create a “wound” and cultured without FBS. Images of the wound area were captured at 0, 24, and 48 hours to evaluate cell migration. The captured images are processed using ImageJ software to quantify the extent of wound closure. Each experiment was conducted in three replicates.

Cell invasion assay

Cell invasion assays were performed using SCC9 cells (1 × 104). The cells were treated with Au-HN-1 (100 μg/mL), 808 nm NIR light (2 W/cm2, 5 min), or Au-HN-1 (100 μg/mL) + 808 nm NIR light (2 W/cm2, 5 min) for 24 h. After treatment, these cells were routinely digested using pancreatic enzymes, then counted separately using cell counters, and subsequently resuscitated utilizing serum-free medium. The cells were subsequently seeded into the wells. The wells were pre-treated with Matrigel matrix (10 μL, BD Biosciences, USA) and incubated overnight in the presence of medium. The lower cell compartment was immersed in a medium containing serum. The cells seeded into the wells were incubated for 24 hours. Then the invaded cells were stained using 0.1% crystal violet dye (Solarbio, China). The stained cells are rinsed with water to eliminate any residual dye. The cells that had been stained were subsequently subjected to analysis using the ImageJ software, developed by the National Institutes of Health (NIH) in the United States. Each experiment was conducted in three replicates.

Cell targeting

The targeting ability of Au-HN-1 was examined on multiple TSCC cell lines using flow cytometry and laser confocal cell imaging. Specifically, SCC9, CAL 27, CRL-1623, HEK 293 T, HCC-LM3, SK-BR-3, and MKN-45 cells were plated in 6-well plates, allowed to incubate for 24 h, and subsequently exposed to Au-HN-1 for 6 h. Following three washes with PBS, the cells were re-suspended in buffer and subjected to flow cytometry analysis. A total of 1 × 104 cells were collected for each sample.

The cellular uptake of Au-HN-1 was further tracked by confocal microscopy. The SCC9, A549, and HCC-LM3 cells were seeded into confocal laser plates and treated with Au-HN-1 for 6 h. After washing thrice with PBS, the cells were stained with DAPI for 10 min, washed three times with PBS to eliminate any residual dye, and imaged under a confocal laser scanning microscope (Carl Zeiss AG, Germany).

Quantitative real-time PCR

The SCC9 cells were subjected to a 24-hour treatment, followed by extraction of RNA for the purpose of gene expression analysis. Total RNA was extracted using the TRIzol reagent (Invitrogen) and reverse transcribed to cDNA using an RNA reverse transcription kit (Genesand Biotech Co., Ltd., China). Quantitative PCR (qPCR) was performed using the GS AntiQ SYBR Green Fast Mix Kit (Genesand Biotech Co., Ltd., China). The cycling conditions were as follows: 95 °C for 15 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 70 °C for 20 s. The relative gene expression levels were calculated by the 2–ΔΔCt method. Each assay was performed in triplicate. The primer sequences are listed in Table SI.

Western blotting

The protein fraction was extracted from the suitably treated SCC9 cells using a protein extraction buffer (Beyotime, China) and quantified with the BCA protein assay kit (Tiangen, China). The extracted protein should be supplemented with loading buffer and subjected to boiling for 10 minutes in order to achieve denaturation of the protein. The proteins were separated by SDS-PAGE using 10% gel and then transferred onto PVDF membranes. After blocking with 5% non-fat milk powder, the membranes were washed with TBST (0.1% Tween-20) and incubated overnight with anti-p53 (Cell Signaling Technology, 2524, USA), anti-Bcl2 (Cell Signaling Technology, 15071, USA), anti-Bax (Cell Signaling Technology, 2772, USA), anti-caspase 3 (ZEN-BIOSCIENCE, R23315, China) and anti-GAPDH (Affinity, AF7021, USA) antibodies at 4 °C. Subsequently, the membranes were incubated with HRP-conjugated affiniPure goat IgG antibodies (BOSTER, China) for 2 h. The protein bands were developed using the ECL Super Signal kit (Pierce, Thermo Fisher Scientific, USA). The gray values of the bands were analyzed for protein level analysis using ImageJ software. Each experiment was conducted in three replicates.

In vivo targeting

Female nude mice (6-8 weeks) were acquired from the Laboratory Animal Center of Jilin University (Approval number: SY202310026). All animal procedures were conducted in accordance with the guidelines of the National Regulation of China for Care and Use of Laboratory Animals. The mice were kept in a specific pathogen-free environment at 24 °C, with a relative humidity of 50%-60%, and maintained on a 12 h light/dark cycle with free access to standard rodent diet and fresh water. Throughout the experiment, all mice remained healthy and uninfected. All surgical procedures were conducted under sterile conditions. The animals that exhibited abnormal eating patterns, rapid weight loss, or cachexia were euthanized through CO2 inhalation.

The mice were inoculated with SCC9 cells (8×106 cells per animal) into their right flank to establish a tumor model. Tumor-bearing mice received intravenous injections of Au-HN-1 (100 μL of 1 mg/mL solution) or AuNDs (100 μL of 1 mg/mL solution), and imaged at specified intervals using the IVIS Lumina LT imaging system. The mice were euthanized 24 h after administering the drugs, and the hearts, livers, spleens, lungs, kidneys, and tumors were harvested and imaged.

Evaluation of in vivo anti-tumor efficacy

Female nude mice (N = 20, 6–8 weeks old) were injected with SCC9 cells (8×106 cells per animal) into their right flank to establish TSCC xenografts. The tumor-bearing mice were divided into the following groups: Control (CON), NIR (808 nm, 2 W/cm2, 5 min), AuNDs + NIR (808 nm, 2 W/cm2, 5 min), and Au-HN-1 + NIR (808 nm, 2 W/cm2, 5 min). The mice were euthanized after 12 days of treatment, and the tumors and vital organs were collected for histological analysis. The tumor dimensions were measured, and the volume (V) was calculated as a × b2/2, where a is the longer diameter and b is the shorter diameter. To evaluate the biosafety of the formulations, the mice were regularly weighed, and the major organs were histologically examined for any signs of toxicity.

Statistical analysis

Each experiment was conducted three times. Statistical evaluations were carried out using GraphPad Prism software (version 9). The groups were compared by Students’ t-test, one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test, and two-way ANOVA followed by Tukey’s multiple comparisons test. A P-value of less than 0.05 was deemed to be statistically significant.