In vivo antitumor therapy through AUNPs

Materials:

All chemicals were purchased from Sigma-Aldrich and used as received without further purification. Deuterated solvents were purchased from Cambridge Isotope Laboratories Inc. The HPLC grade solvents methanol and acetonitrile were purchased from Aldrich. Tetrahydrofuran (THF) and dichloromethane were dried by distillation using sodium or calcium hydride as the drying agent, respectively. All non-aqueous reactions were carried out under nitrogen atmosphere in oven-dried glassware. RPMI-1640 medium, Penicillin, streptomycin, fetal bovine serum (FBS) and trypsin were obtained from GIBCO (Thermo Fisher Scientific). Milli-Q water (18.2 MΩ) was used to prepare the buffer solutions from 10× phosphate-buffered saline (PBS) stock buffer (1st Base, Singapore). The murine melanoma cell line B16F10 and B16F10-luc cells were purchased from ATCC and Shanghai Science light Biology Science &Technology Co., Ltd, respectively. All antibodies for flow cytometry were purchased from Biolegend. All antibodies for immunofluorescence staining were purchased from Abcam. All cytokines for cell culture were purchased from PeproTech, Inc. All other biological reagents including 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and singlet oxygen sensor green (SOSG) were purchased from (Thermo Fisher Scientific).

Instruments:

NMR spectra were measured on a Bruker ARX 400 NMR spectrometer. Chemical shifts were reported in parts per million referenced to residual solvent for ¹H NMR and ¹³C NMR. The extent of reaction was monitored by thin-layer chromatography (TLC) using Merck 60 F254 pre-coated silica gel plates with fluorescent indicator UV254. After the plates were subjected to elution in the TLC chamber. Flash column chromatography was carried out using Merck silica gel (0.040-0.063). Mass spectra were recorded on Amazon X LC-MS for electrospray ionization (ESI). UV-vis absorption spectra were taken on a Shimadzu Model UV-1700 spectrometer. Photoluminescence (PL) spectra were measured on a Perkin-Elmer LS 55 spectrofluorometer. All UV and PL spectra were collected at 24 ± 1 °C. Hydrodynamic size distributions and zeta potentials were measured on a Zetasizer Nano ZS ZEN3600 analyzer (Malvern Instruments Ltd, UK) at 25 °C. CLSM imaging was carried out on confocal laser scanning microscopy platform Leica TCS SP8. A Cold Light Illuminator L-150A was used for white light irradiation. BD LSRFortessa™ cell analyzer was used for flow cytometry analysis. TEM images were obtained from a JEOL JEM-2010 transmission electron microscope with an accelerating voltage of 200 KV. 980 nm laser 980 nm laser (1 W, continuous wave, Beijing Hi-Tech Optoelectronics Co., Ltd.) was used for animal imaging and photodynamic therapy.

Synthesis of compound 1 (4,4'-(2-(4-bromophenyl)-2-phenylethene-1,1-diyl)bis(methoxybenzene)). 

In a typical experiment, 4,4'-dimethoxybenzophenone (4.84 g, 20.0 mmol) and 4-bromobenzophenone (5.20 g, 20.0 mmol) were dissolved in anhydrous tetrahydrofuran (100 mL). Active zinc power (4.48 g, 80.0 mmol) was added into the solution. The mixture was degassed and charged with nitrogen and then stirred at -78 °C. Titanium (IV) chloride (8.8 mL, 80.0 mmol) was injected dropwise within 30 min. The mixture was heated at 78 °C for 4 h after recovery to room temperature. The reaction was quenched by deionized water (100 mL). The residue was filtered with the assistance of Celite^® S and washed with ethyl acetate (100 mL × 3). The collected organic layer was washed with water (150 mL × 3), dried with sodium sulfate. The solvent was removed by rotor evaporation. The residue was purified by column chromatographic silica gel with elution of dichloromethane/hexane (V/V = 1/4). Compound 1 (3.96 g, 8.4 mmol) was obtained as a light-yellow powder in a yield of 42.1%. ¹H NMR (400 MHz, CDCl₃) δ 7.23-7.20 (m, 2H), 7.11-7.08 (m, 3H), 7.02-6.99 (m, 2H), 6.95-6.87 (m, 6H), 6.68-6.61 (m, 4H), 3.76 (s, 3H), 3.73 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 158.27, 143.80, 143.32, 140.78, 137.91, 136.03, 133.06, 132.57, 132.54, 131.36, 130.87, 127.83, 126.31, 120.02, 113.21, 113.03, 55.14, 55.10.

Synthesis of compound 2 (2-(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane). 

Compound 1 (0.94 g, 2.0 mmol), bis(pinacolato)diboron (1.02 g, 4.0 mmol), 1,1'-Bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex (Pd(dppf)Cl₂.CH₂Cl₂, 33 mg, 0.04 mmol) and potassium acetate (0.76 g, 8.0 mmol) were mixed in anhydrous 1,4-dioxane (50 mL). The mixture was stirred and heated at 100 °C for 24 h under nitrogen atmosphere. After cooling to room temperature, the residue was dissolved in ethyl acetate (100 mL) and washed with water (100 mL × 3). The organic layer was dried with sodium sulfate. The solvent was removed by rotor evaporation, and the residue was purified by column chromatographic silica gel with elution of ethyl acetate/hexane (V/V = 1/20). Compound 2 (0.73 g, 1.4 mmol) was obtained as white powder in 70% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.55 (d, J = 7.2 Hz, 2H), 7.12-7.06 (m, 3H), 7.06-6.98 (m, 4H), 6.94 (d, J = 8.5 Hz, 4H), 6.63 (d, J = 8.6 Hz, 4H), 3.75 (s, 3H), 3.74 (s, 3H), 1.33 (s, 12H). ¹³C NMR (100 MHz, CDCl₃) δ 158.24, 158.21, 147.54, 144.25, 140.70, 139.22, 136.41, 136.30, 134.22, 132.72, 131.53, 130.90, 127.79, 126.22, 113.18, 113.09, 83.79, 55.20, 55.17, 25.02.

Synthesis of compound 3 ((E)-4-bromo-7-(2-(pyridin-4-yl)vinyl)benzo[c][1,2,5]thiadiazole). 

In a typical experiment, 4,7-dibromo-2,1,3-benzothiadiazole (0.58 g, 2.0 mmol), 4-vinylpyridine (0.20 g, 1.9 mmol) and palladium(II) acetate (22 mg, 0.10 mmol) were dissolved in the mixture of N,N-diisopropylethylamine (5 mL) and N,N-dimethylformamide (10 mL), and heated at 100 °C for 24 h under nitrogen atmosphere. After cooling to room temperature, the residue was dissolved in ethyl acetate (50 mL) and washed with water (100 mL × 3). The organic layer was dried with sodium sulfate. The solvent was removed by rotor evaporation, and the residue was purified by column chromatographic silica gel with elution of ethyl acetate/hexane (V/V = 1/2). Compound 3 (0.47 g, 1.5 mmol) was obtained as yellow powder in a yield of 78.9%. ¹H NMR (400 MHz, CDCl₃) δ 8.62 (d, J = 6.0 Hz, 2H), 7.95 (d, J = 16.3 Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.66 (d, J = 16.3 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.46 (d, J = 6.0 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 153.98, 152.79, 150.47, 144.45, 132.31, 131.83, 129.09, 128.65, 128.20, 121.19, 114.23.

Synthesis of compound 4 ((E)-4-(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-7-(2-(pyridin-4-yl)vinyl)benzo[c][1,2,5]thiadiazole). 

Compound 2 (518 mg, 1.0 mmol), compound 3 (285 mg, 0.90 mmol) and tetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄, 23 mg, 0.02 mmol) were dissolved in tetrahydrofuran (20 mL). Potassium carbonate (552 mg, 4.0 mmol) dissolved in deionized water (2 mL) was injected into the solution. The mixture was stirred and refluxed for 6 h under nitrogen atmosphere. After cooling to room temperature, the residue was dissolved in ethyl acetate (50 mL) and washed with water (50 mL × 3). The organic layer was dried with sodium sulfate. The solvent was removed by rotor evaporation, and the residue was purified by column chromatographic silica gel with elution of ethyl acetate/hexane (V/V = 1/2). Compound 4 (0.48 g, 0.76 mmol) was obtained as red powder in a yield of 84.8 %. ¹H NMR (400 MHz, CDCl₃) δ 8.67-8.59 (br, 2H), 7.98 (d, J = 16.4 Hz, 1H), 7.80-7.70 (m, 5H), 7.49 (d, J = 5.9 Hz, 2H), 7.22-7.17 (m, 2H), 7.14-7.09 (m, 5H), 7.06-7.02 (m, 2H), 6.99-6.95 (m, 2H), 6.70-6.63 (m, 4H), 3.75 (s, 3H), 3.74 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 158.39, 158.29, 154.00, 150.31, 144.93, 144.27, 140.97, 138.80, 136.41, 134.68, 132.80, 132.75, 131.83, 131.64, 130.66, 129.11, 128.80, 128.57, 128.15, 127.92, 127.72, 126.35, 121.21, 113.32, 113.14, 55.21. ESI-MS, m/z: [M+H]⁺ calcd 629.21, found 629.23.

Synthesis of TPEBTPy ((E)-4-(2-(7-(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)vinyl)-1-ethylpyridin-1-ium bromide).        

Compound 4 (63 mg, 0.1 mmol) and bromoethane (108 mg, 1 mmol) were dissolved in N,N-dimethylformamide (2 mL). The solution was stirred and heated at 80 °C in a sealed heavy wall pressure vessel. After cooling to room temperature, the residue was dissolved in dichloromethane (20 mL) and washed with water (50 mL × 5). The organic layer was dried with sodium sulfate. The solvent was removed by rotor evaporation and the residue was purified by column chromatographic silica gel with elution of dichloromethane/methanol (V/V = 1/20). TPEBTPy (52 mg, 0.07 mmol) was obtained as dark red powder in a yield of 70.0 %. ¹H NMR (400 MHz, CDCl₃) δ 9.17 (d, J = 5.6 Hz, 2H), 8.17 (d, J = 16.0 Hz, 1H), 8.11 (d, J = 5.6 Hz, 2H), 8.00 (d, J = 16.0 Hz, 1H), 7.86 (d, J = 7.7 Hz, 1H), 7.70-7.61 (m, 3H), 7.13-7.00 (m, 7H), 6.95 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 6.59 (t, J = 9.2 Hz, 4H), 4.92-4.80 (br, 2H), 3.68 (s, 3H), 3.65 (s, 3H), 1.65 (t, J = 7.0 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 158.45, 156.58, 153.85, 153.50, 144.24, 141.21, 138.64, 137.91, 136.30, 134.02, 133.42, 132.74, 131.88, 131.62, 128.76, 127.96, 127.56, 126.41, 124.65, 113.36, 113.14, 56.41, 55.22, 17.10. ESI-MS, m/z: [M-Br]⁺ calcd 658.25, found 658.30.

Synthesis of compound 5 ((E)-4-(2-(7-(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)vinyl)-1-(3-bromopropyl)pyridin-1-ium bromide). 

In a typical experiment, 1,3-dibromopropane (404 mg, 2 mmol) was dissolved in N,N-dimethylformamide (5 mL), stirred and heated at 80 °C under a nitrogen atmosphere. Compound 4 (126 mg, 0.2 mmol) in N,N-dimethylformamide (5 mL) was added dropwise within 2 h. The mixture was further heated at 80 °C for 12 h. After cooling to room temperature, the residue was dissolved in dichloromethane (20 mL) and washed with water (50 mL × 5). The organic layer was dried with sodium sulfate. The solvent was removed by rotor evaporation, and the residue was purified by column chromatographic silica gel with elution of dichloromethane/methanol (V/V = 1/20). Compound 5 (108 mg, 0.13 mmol) was obtained as dark red powder in a yield of 65.0 %. ¹H NMR (400 MHz, CDCl₃) δ 8.84-8.66 (br, 2H), 8.29 (d, J = 15.9 Hz, 1H), 8.06 (s, 2H), 7.98 (d, J = 16.0 Hz, 1H), 7.81 (d, J = 7.3 Hz, 1H), 7.74-7.67 (m, 3H), 7.12 (d, J = 8.3 Hz, 2H), 7.07-7.00 (m, 5H), 6.95 (d, J = 8.6 Hz, 2H), 6.89 (d, J = 8.6 Hz, 2H), 6.62-6.56 (m, 4H), 4.80-4.65 (br, 2H), 3.68 (s, 3H), 3.66 (s, 3H), 3.42-3.30 (br, 2H), 2.58-2.49 (br, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 158.35, 158.22, 154.88, 153.97, 153.52, 145.62, 144.08, 141.19, 138.82, 138.51, 136.71, 136.19, 133.98, 133.61, 132.71, 132.65, 131.84, 131.52, 128.72, 127.86, 127.48, 126.44, 126.32, 126.03, 124.59, 113.23, 113.04, 59.14, 55.12, 33.10, 28.42. ESI-MS, m/z: [M-Br]⁺ calcd 750.18, found 750.19.

Synthesis of TPEBTPy-Si ((E)-4-(2-(7-(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)vinyl)-1-(3-(dimethyl(3-(trimethoxysilyl) propyl)ammonio)propyl)pyridin-1-ium bromide). 

Compound 5 (42 mg, 0.051 mmol) and (N,N-dimethylaminopropyl)trimethoxysilane (207 mg, 1.0 mmol) were dissolved in freshly prepared anhydrous tetrahydrofuran (5 mL) and sealed in a heavy wall pressure vessel. The solution was heated at 70 °C for 12 h. Anhydrous ethyl ether (30 mL) was added to the solution for TPEBTPy-Si precipitation after cooling to room temperature. The residue was centrifuged at 12,000 g for 5 min at room temperature to collect the precipitation. The precipitation was dissolved in anhydrous dichloromethane (3 mL), precipitated by adding anhydrous ethyl ether (30 mL) and collected by centrifugation at 12,000 g for 5 min at room temperature, this procedure was repeated for three times. The precipitation was dried to get dark red TPEBTPy-Si (22 mg, 0.021) in a yield of 41.6%. TPEBTPy-Si was stocked in a glovebox. HR ESI-MS, m/z: [M-2Br+H] calcd 879.3970, found 879.3957.

Preparation of SiUNPs, AUNPs and pAUNPs:

UCNPs with a chemical composition of NaYF₄:Yb/Tm were synthesized by a coprecipitation method. In a typical synthesis of blue-emitting UCNPs, 0.278 mmol of Y(CH₃CO₂)₃, 0.12 mmol of Yb(CH₃CO₂)₃ and 0.002 mmol of Tm(CH₃CO₂)₃ lanthanide precursors were mixed with 3 mL of oleic acid and 7 mL of 1-octadecene and heated at 150°C for 1.5 h to remove any moisture content. After cooling the reaction solution to 50 °C, a methanol solution containing 1 mmol of NaOH and 1.6 mmol of NH₄F was quickly added and the reaction mixture was further stirred at 50°C for another 0.5 h. After that, the reaction was heated to 100°C under vacuum for 0.5 h to remove any low boiling point solvent. After three times of N₂ purge at 100°C, the reaction was heated to 280°C and maintained at this temperature for 2 h to allow nanoparticle growth. Once the reaction was cooled to room temperature, UCNP products were purified twice by ethanol washing under centrifugation at 6,000 rpm for 10 min, followed by ethanol re-dispersion. Purified UCNPs were dispersed into 4 mL of cyclohexane. For synthesis of UCNPs with a chemical composition of NaYF₄:Yb/Er, experimental procedure was the same as that for NaYF₄:Yb/Tm except that 0.272 mmol of Y(CH₃CO₂)₃, 0.12 mmol of Yb(CH₃CO₂)₃ and 0.008 mmol of Er(CH₃CO₂)₃ were used as lanthanide precursors.

To suppress surface quenching of luminescence, an optically-inert shell layer of NaYF₄ was grown epitaxially onto the as-synthesized UCNPs. Specifically, 0.4 mmol of Y(CH₃CO₂)₃ were mixed with 3 mL of oleic acid and 7 mL of 1-octadecene and heated at 150°C for 1.5 h. After cooling to 80 °C, all UCNP products from the previous step (in 4 mL of cyclohexane) were added to the reaction mixture. The solution was further stirred at 80°C for 1 h to evaporate the cyclohexane content. Subsequently, a methanol solution containing 1 mmol of NaOH and 1.6 mmol of NH₄F was added to the reaction solution at 50°C. After 0.5 h of continuous stirring at 50°C, the reaction was heated to 100 °C under vacuum for 0.5 h. After three rounds of N₂ purging at 100°C, the reaction temperature was ramped to 280 °C and maintained at 280 °C for 2 h. Once the reaction was cooled down to room temperature, core-shell UCNP products were washed twice with ethanol and dispersed into 4 mL of cyclohexane.

The as-synthesized UCNPs were transferred into an aqueous phase through an acid-induced ligand removal process. 4 mL of the cyclohexane was added with 4 mL of ethanol and centrifuged at 14,800 rpm for 5 min. The pellet was re-dispersed with a mixture of 4 mL of ethanol and 4 mL of HCl aqueous solution (2 M) by sonication. The acid-treated UCNPs were thereafter washed three times with ethanol to remove excess acid content. The ligand-free UCNPs were dispersed into 4 mL of DI H₂O for subsequent surface modification.

     To obtain SiUNPs, 4 mL of the ligand-free UCNPs were slowly added to 18 mL of polyvinylpyrrolidone aqueous solution (50 mg/mL). The solution was sonicated for 20 min and subsequently stirred for 1 h. After adding 80 mL of ethanol, the reaction mixture was further sonicated for 20 min and stirred for 2 h. 3.2 mL of aqueous ammonia were added to adjust the solution pH, followed by 20 min sonication. Subsequently, 80 µL of tetraethyl orthosilicate were added to the solution to initiate silica growth on the surface of UCNPs. The reaction solution was kept stirring for 12 h, and SiUNPs were purified by three rounds of ethanol washing and dispersed in DI H₂O. The final concentration of SiUNPs were 200 mg/mL.

To prepare AUNPs, 50 mg silica coated UCNPs were washed with DI water two times and then dispersed in 20 mL ethanol/water mixture (9/1). The reaction mixture was further sonicated for 20 min and stirred for 2 h. 0.8 mL of ammonium was added to adjust the solution pH, followed by 20 min sonication. Subsequently, 200 μL TPEBTPy-Si in THF (2 mg/mL) were added into the solution to coat on the surface of UCNPs through a condensation reaction. The reaction solution was kept stirring for 1 h and then were purified by three rounds of ethanol washing and dispersed in DI H₂O. For further PEG modification, mPEG₂₀₀₀-Silane were dissolved in THF (5 mg/mL) and 200 μL mPEG₂₀₀₀-Silane solution were added into the reaction solution containing AUNPs. The reaction solution was continued to stir for 2 h, and the final products were purified by three rounds of ethanol washing and dispersed in DI H₂O.

Characterization of UCNPs and AUNPs

Transmission electron microscopy (TEM) measurements were carried out on a JEOL-JEM 2010F field-emission transmission electron microscope operated at an acceleration voltage of 200 kV. The hydrodynamic sizes of UCNPs were measured by dynamic light scattering (DLS) means on Malvern Nano-ZS Particle Sizer. Upconversion luminescence spectra were recorded at room temperature with an Edinburgh FLS920 fluorescence spectrometer in conjunction with a 980-nm diode laser. To detect the ¹O₂  generation in solution, 1 mL of AUNPs solution (0.1 mg/mL) was mixed with 1 μL DMSO with SOSG. The solution was kept in the dark and irradiated by a 980 nm laser (0.6 and 0.12 W/cm²) for various times. Then the solution was collected for fluorescence emission measurements.

To determine the loading efficiency of OVA onto AUNPs, 10 mg of AUNPs and 5 mg of OVA were added into 10 mL of water with stirring for 12 h to obtain the OVA@AUNPs. Subsequently, the solution was centrifuged at 3,000 rpm for 10 min. The supernatant was collected and the concentration of OVA was quantified using BCA assay. The total OVA uptake by the AUNPs was measured by subtracting the OVA concentration in the supernatant after AUNP capture from the total OVA concentration.

Preparing tumor-associated antigen from B16F10 cells

To obtain tumor-associated antigens (TAAs), B16F10 cells were re-suspended in PBS at 37 °C at a density of 1×10⁷/mL. They were then rapidly frozen in liquid nitrogen and thawed at 37 °C (5 times×5 min), respectively. Subsequently, the cell lysates were collected and centrifuged at 200 g for 5 min to remove insoluble cellular debris. Protein concentration was measured by BCA assay, and protein solution was diluted to different concentrations for further in vitro experiments. To prepare  FITC labeled TAAs (TAAs-FITC). 2 mg of FITC in 1 mL of 20 mmol/L carbonate buffer (pH 9.5) added to a solution of TAAs (1 mg/mL, 10 mL). The solution was incubated with continuous stirring at 4 ^oC for 16 h. The reaction mixture was dialyzed against distilled water (MWCO 1000) to obtain TAAs-FITC.

Ecto-CRT and ecto-HSP-70 staining of B16F10 tumor cells

After the B16F10 cells were incubated with 5 μg/mL of AUNPs (based on TPEBTPy) and 6 μg/mL of Ce6 for 12 h at 37 ^oC, respectively. Subsequently, the cells were washed with PBS and irradiated by 980 nm laser (0.6 W/cm²) for 3 min. After 12 h, the cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min. The fixed cells were incubated with anti-calreticulin antibody (1:200 dilution) and anti-HSP-70 antibody for 12 h at 4 ^oC, followed by staining with Alexa Fluor 633-conjugated secondary antibody (1:200 dilution) for 2 h at room temperature. The ecto-CRT and ecto-HSP-70 expressions of cells were detected using CLSM with 633-nm excitation and signal acquisition in the range from 640 to 670 nm. To eliminate photothermal effect, cell experiment was conducted in an ice bath condition.

BMDCs isolation and stimulation by AUNPs

To evaluate the immune-stimulation effect of AUNPs plus low-power of NIR irradiation, BMDCs were isolated from C57BL/6 mice bone marrow and cultured in RPMI-1640 medium with GM-CSF (20 ng/mL) and IL-4 (10 ng/mL) at 37 °C. After 7 days, immature DCs were obtained and further incubated with AUNPs (5 μg/mL, based on TPEBTPy) for 12 h, followed by different doses of 980 laser irradiation for 5 min. In a control group, AUNPs loaded DCs were pretreated with N-acetylcysteine (NAC, a ROS scavenger) for 1 h to eliminate ROS generation from AUNPs after light treatment. After 24 h, the expression levels of CD80 and CD86 on CD11c⁺ BMDCs were measured by flow cytometry using LSRFortessa Cell Analyzer (BD Biosciences). Meanwhile, TNF-α in the supernatant was measured by ELISA assay (eBioscience) according to the manufacturer instructions. 

To assess the cytotoxicity of AUNPs, BMDCs were seeded in 96 well plates at a density of 10⁵ cells in 200 μL per well and were further incubated with different concentrations of NPs for 12 h, followed by 980 nm laser irradiation for 5 min. To assess the effect of tumor cell ablation of AUNPs, B16F10 cells were seeded in 96-well plates at a density of  2×10⁴ cells in 200 μL per well and further incubated with nanoparticles of different concentration for 12 h, followed by 980 nm laser irradiation for 5 min. After light treatment, MTT (40 μL, 0.5 mg/mL) was added into a medium for 3 h. The media was removed, and DMSO (100 μL) was added into each well and gently shaken for 10 min at room temperature. The absorbance of MTT at 550 nm was measured by using a SpectraMax M5 Microplate Reader. Cell viability was measured by the ratio of the absorbance of the cells incubated with different nanoparticles to the cells incubated with normal culture medium. 

To evaluate the lysosome rupture, BMDCs were incubated with TAAs-FITC loaded AUNPs (5 μg/mL, based on TPEBTPy) for 6 h, followed by were stained with 50 nM LysoTracker 633 (Thermo Fisher) and Hoechst 33342. DCs were irradiated by a low power of 980 nm laser (0.12 W/cm²) for 3 min to induce ruptured lysosome. Subsequently, the intracellular localization of TAA and AUNPs in BMDCs were imaged using a confocal laser scanning microscopy (TCS SP5II, Leica). In addition, BMDCs (1 × 10⁶ /mL) were treated with AUNPs (5 μg/mL, based on TPEBTPy) for 12 h and then were incubated with acridine orange (Thermo Fisher, 20,000×) for 30 min, followed by a low power of 980 nm laser (0.12 W/cm²) irradiation for 3 min. Fluorescence signals of acridine orange labeled DCs was analyzed through flow cytometry at 488 nm (excitation). BMDCs with lysosome rupture indicates the increase of fluorescence signals.

To intracellular ROS detection, DCs were cultured in a small confocal dish or a 6-well plate overnight and added AUNPs with different concentrations. After incubating for 6 h, cells were washed with PBS and ROS indicator, 2,7-dichlorodihydrofluorescein diacetate (DCFHDA, 1 µM), was added and incubated for 15 min at 37 °C. After washed with PBS, DCs were irradiated with a 980 nm laser for 3 min. Subsequently, the cells were imaged by a CLSM (Olympus, FV1000).

CD8⁺ T cells proliferation assay

MHC I antigen presentation effect of AUNPs on BMDCs was evaluated by CD8⁺ T cell proliferation assay. C57BL/6 mice were intraperitoneally injected with 50 μg TAAs and 20 μg Poly I:C by once a week for three weeks. After 3 weeks, CD8⁺ T cells were isolated and purified from spleen using a CD8⁺ T cell isolation kit (Miltenyi Biotec, Inc.). Meanwhile, immature BMDCs were incubated with TAAs or TAAs-AUNPs  (5 μg/mL, based on TPEBTPy) for 12 h, followed by a low power of 980 nm (0.12 W/cm²) laser irradiation for 5 min. After 24 h, purified CD8⁺ T cells were treated with 5, 6- carboxyfluorescein diacetate succinimidyl ester (CFSE, Thermo Fisher, 3000×) and then co-cultured with TAAs or TAAs@AUNP-treated BMDCs at the ratio of T cells to BMDCs at 5:1, respectively. After 3 days, cell suspensions were stained with APC anti-CD8⁺ antibodies and the proliferation of CD8⁺ T cells was further evaluated by flow cytometry, which indicates MHC I antigen presentation of DCs. The data are presented as the percentage of CFSE^+low cells in the CD8⁺ cells.

In vivo biodistribution of AUNPs after PDT

To track the in vivo distribution of AUNPs, Maestro in vivo imaging system (CRi, Inc., Woburn, USA) using a 980 nm laser as the excitation source was utilized to obtain AUNPs signals from mice. B16F10 tumor-bearing mice (n = 3) were intravenously injected with 30 μL of AUNPs or pAUNPs (0.5 mg/mL, based on TPEBTPy), respectively. At 6 hours post-injection, the mice were anesthetized with 2% (v/v) isoflurane and the tumors were irradiated with 980 nm NIR laser (0.6 W/cm²) for 10 min (with 1 min interval for each minute to avoid heating effect). Subsequently, the mice were immediately imaged with a Maestro in vivo fluorescence imaging system (CRi, Inc., Woburn, USA). The autofluorescence was removed by spectral unmixing software and fluorescence signals were quantified by the Maestro system (Emission filter: 850 nm short-pass; Acquisition setting: 600 to 900 in 10 nm steps; Exposure time: 10 s). In addition, the tumor-bearing mice were also sacrificed at 24 h post-irradiation, and the DLNs in different groups were collected, imaged and quantified by the Maestro system. (Excitation filter: 455 nm; Emission filter: 672 nm longpass; Acquisition setting: 500 to 900 in 10 nm steps; Exposure time: 300 ms).

In vivo anti-tumor therapy through AUNPs

All animal studies were performed in compliance with the guidelines set by the Tianjin Committee of Use and Care of Laboratory Animals and the overall project protocols were approved by the Animal Ethics Committee of Nankai University. 6-week-old C57BL/6 mice (obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China)) were used to establish the B16F10 tumor-bearing mouse model. B16F10 or luciferase-transgenic B16F10 (luc-B16F10) cells (1 × 10⁶ cells per mouse) suspended in 30 μL of saline were injected subcutaneously into the right flank region of the mouse. After 10 days, AUNPs, pAUNPs or PBS was intratumorally injected into the mice on day 0 and 1 (n = 5, per group) (30 μL, 0.5 mg/mL, based on TPEBTPy). At 6 hours after each injection, the mice were anesthetized with 2% (v/v) isoflurane, and the tumors were irradiated with a 980-nm laser (0.6 W/cm²) for 10 min (with 1 min interval for each minute to avoid heating effect). After PDT, draining lymph nodes (DLNs) were imaged and determined by CRi Maestro in-vivo imaging system. DLN regions were irradiated by 980 nm laser on day 1, 2, 3 and 4 at a power of 0.12 W/cm² for 10 min (with 1 min interval for each 2 minute/4 times a day). When using low-power light to treat lymph nodes, the PDT treated tumor region was covered by a surgical cloth to avoid laser exposure to neighboring tumors.

The abscopal effect of AUNPs was evaluated through the bilateral B16F10 tumor-bearing mouse model. The tumor was built by subcutaneous injection of B16F10 or luciferase-transgenic B16F10 (luc-B16F10) cells (1 × 10⁶ cells per mouse) into the right flank region. Meanwhile, B16F10 or luc-B16F10 cells (4 × 10⁵ cells per mouse) were injected into the left flank region of the same mouse. After 10 days, AUNPs, pAUNPs or PBS was intratumorally injected into the right tumor of mice on day 0 and 1 (n = 5, per group) (30 μL, 0.5 mg/mL, based on TPEBTPy). At 6 hours post-injection, the mice were anesthetized with 2% (v/v) isoflurane, and the tumors were irradiated with 980 nm NIR laser (0.6 W/cm²). The αPD-1 (clone, RMP1-14) blocking antibody (10 mg/kg) was intraperitoneally injected into the animals on days 1, 2, 3 and 4. After PDT, right DLNs of mice were imaged and determined by CRi Maestro in-vivo imaging system and DLN regions were irradiated by 980 nm laser on day 1, 2, 3 and 4 at a power of 0.12 W/cm². 

To evaluate the therapeutic efficacy, tumor growth was monitored by the IVIS Spectrum Imaging System (PerkinElmer Ltd.). Briefly, D-luciferin (150 mg/kg) was intraperitoneally injected into the mice. The mice were then imaged, and then the bioluminescence signals were quantified in units of maximum photons per second per square centimeter per steradian. The tumor size was measured with a caliper every two days. Tumor volumes were calculated as follows: (width² × length)/2.

To establish a tumor re-challenge model, on day 34 after the first treatment of the unilateral tumor model, mice were re-challenged by intravenous injection of B16F10 cells suspension (100 μL, 5×10⁵) into C57BL/6 mouse. Subsequently, tumor growth in the lung was monitored by the IVIS Spectrum Imaging System and lung tissue were collected for flow cytometry and histological analysis on different time points.

Flow cytometry analysis

At designated time points, DLNs, lungs, spleens or tumor tissues were collected, ground and filtered through nylon mesh filters (40 μm) and washed with PBS containing 1% FBS, respectively. The single-cell suspensions were blocked with anti-CD16/32 (Biolegend) for 30 min on ice and then stained with fluorescence-labelled anti-mouse CD45 (30-F11), CD3e (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD11b (M1/70), Ly-6G/Ly-6C (RB6-8C5), F4/80 (BM8), CD62L (MEL-14) and CD44 (IM7) (all from BioLegend, dilution: 1: 100) for 30 min on ice. For DCs characterization, DCs suspensions were blocked with anti-CD16/32 (Biolegend) for 30 min on ice and then stained with fluorescence-labeled anti-mouse CD11c (N418), CD80 (16-10A1), CD86 (GL-1) for 30 min on ice. For intracellular protein staining, the samples were blocked with anti-CD16/32 (Biolegend) for 30 min on ice and then stained with anti-mouse CD4 or CD8 for 30 min on ice, respectively. Subsequently, cells were fixed, permeabilized and stained with anti-mouse Foxp3 (FJK-16s) or IFN-γ (XMG1.2) at room temperature for 30 min, respectively. Finally, all samples were washed with PBS containing 1% FBS two times. All the flow cytometric analysis was conducted using a LSRFortessa Cell Analyzer (BD Biosciences), and data analysis was carried out using FlowJo software (www.flowjo.com; Tree Star).

Histological Analysis

Mice in different groups were sacrificed, and different tissues, including tumors, were excised and fixed in 4% paraformaldehyde. A part of tumors and DLNs were embedded, frozen and sectioned at a thickness of 8 μm. All tissue section was treated according to standard manufacturer's instructions and stained with different primary antibodies: rabbit anti-CD11c (1:200), rat anti-F4/80 (1:200, Abcam), rabbit anti-CD11b (1:1000), rabbit anti-Ly-6G/Ly-6C, rabbit anti-CD4 (1:200), rabbit anti-Foxp3 (1:200), rabbit anti-CD206 (1:200) and rabbit anti-PD-L1 (1:200), followed by stained with fluorescently labeled secondary antibodies (Alexa 488-conjugated goat anti-rat IgG (H + L) (1:200, Thermo Fisher Scientific) and Alexa 633-conjugated goat anti-rabbit (H+L) (1:200, Thermo Fisher Scientific)), respectively. The nuclei were stained with DAPI containing mounting solution (Dapi Fluoromount G, Southern Biotech). All the slices were finally imaged by a confocal microscope (Lecia, SP8). The other parts of tumor tissues and other organs were fixed in 4% paraformaldehyde, then embedded into paraffin, and subsequently sliced at a thickness of 5 μm. Slices were stained with hematoxylin and eosin (H&E) and imaged by optical microscopy and assessed by three independent pathologists.

Statistical Analysis

Quantitative data were presented as mean ± standard error of the mean (SEM). ANOVA analysis was used for multiple comparisons, and Student's t-test was used for two-group comparisons. The differences in survival in each group were analyzed by the Kaplan–Meier method, and the P-value was determined by the log-rank test. All statistical analyses were carried out using GraphPad Prism 5.0. (P-value: *P < 0.05, **P < 0.01, ***P < 0.001).

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