Supplies and reagents
The reagents used included QUANTI-Luc™ (InvivoGen), DAPI-containing anti-fade medium (P36962, Thermo Fisher Scientific), Gelstain (GS101, Transgen), Trans2K Plus DNA Marker (BM111, Transgen), Direct-zol RNA Miniprep Plus Package (R2070, ZYMO RESEARCH), PrimeScriptTM RT reagent Package with gDNA Eraser (RR047A, Takara), DreamTaq polymerase (EP0701, Thermo Scientific), GeneJET PCR purification package (K0701, Thermo Scientific), 50 × Tris–acetate-EDTA (TAE, ST716, Beyotime Biotechnology), Radio Immunoprecipitation Assay buffer (RIPA, PC101, Epizyme), phosphatase (GRF102; Epizyme), protease inhibitors (GRF101, Epizyme), TRIzol Reagent (15,596,026, Invitrogen). PierceTM BCA Protein Assay Package (23,225, Thermo Scientific), SDS-PAGE Pattern Buffer (P0015F, Beyotime Biotechnology), Multicolor Prestained Protein Ladder (WJ103, Epizyme), BD Pharmingen™ Leukocyte Activation Cocktail (550,583, BD Pharmingen), Fixation/Permeabilization Resolution Package with BD GolgiPlug (555,028, BD Pharmingen), Foxp3/Transcription Issue Staining Buffer Set (00–5523-00, Thermo Scientific), Collagenase I (C10130, Sigma), Hyaluronidase (H3506, Sigma), 25 μg/mL DNase I (DN25-100, Sigma), Triethanolamine (TEA, V900257, Sigma), Hexadecyl trimethyl ammonium bromide(CTAB, H5882, Sigma), Sodium salicylate (NaSal, 54–21-7, Sigma), Tetraethyl orthosilicate (TEOS, 78–10-4, Sigma), Bis[3-(triethoxysilyl)propyl] tetrasulfide (BTES, 40,372–72-3, Sigma-Aldrich), 3-Aminopropyl triethoxysilane (APTES, 919–30-2, Sigma-Aldrich), sulfo-Cyanine5 NHS ester (43,320, Lumiprobe), DreamTaq DNA Polymerase (EP0705, Thermo Scientific), GeneJET PCR Purification Package (K0701, Thermo Scientific), H-151 (HY-112693, MedChemExpress), Lipofectamine 2000 Transfection Reagent (11,668,019, Thermo Scientific), Simulated physique fluid (SBF, R24165, Shanghai yuanye Bio-Know-how).
Antibodies
Anti-PD-L1 (ab213480, Abcam), anti-GAPDH antibody (2118, CST), anti-Phospho-TBK1/NAK antibody (5483, CST), HRP-conjugated secondary antibodies (7074, CST), anti-PD-L1(BE0101, BioXcell), anti-mouse CD16/32 antibody (553,141, BD Pharmingen), Fixable Viability Stain (565,388, BD Pharmingen), anti-CD80-BV421(562,611, BD Pharmingen), anti-CD86-PE-Cy7(560,582, BD Pharmingen), anti-mouse PD-1-PE (135,206, BioLegend), anti-mouse LAG3-PE-Cy7 (125,226, BioLegend), anti-mouse TIM3-BV421 (119,723, BioLegend), anti-mouse FOXP3-BV421 (126,419, BioLegend), anti-mouse Ki67-PE-CY7 (652,426, BioLegend), anti-mouse PD-L1-PE (124,308, BioLegend), anti-mouse CD11b-FITC (101,206, BioLegend), anti-mouse Ly6G-PE (551,461, BD Pharmingen), anti-mouse Ly6C-PE-Cy7 (128,018, BioLegend), anti-mouse F4/80-BV421 (123,132, BioLegend), anti-mouse CD206-APC (141,708, BioLegend), anti-mouse TNFα-PE (554,419, BD Pharmingen), anti-mouse CD3-BV605 (100,237, BioLegend), anti-mouse CD11C-APC (117,310, BioLegend), anti-mouse MHC-II-BV421 (107,632, BioLegend), anti-mouse NK1.1-PE-Cy7 (552,878, BD Pharmingen), anti-mouse CD80-PE (552,769, BD Pharmingen).
Cells
MC38, B16-F10, 4T1, MDA-MB-231, and A375 cells had been acquired from ATCC. Panc02 cells had been obtained from the Cell Financial institution of the Shanghai Institutes for Organic Sciences, Chinese language Academy of Sciences (SIBS, CAS). RAW-Lucia ISG cells with an interferon regulatory issue (IRF)-inducible Lucia luciferase reporter assemble had been acquired from Invivogen. Mouse dendritic cells 2.4 (DC2.4) had been kindly offered by Dr. Jing Qian from Jiangsu Academy of Agricultural Sciences (JAAS). All cell traces had been routinely examined utilizing a mycoplasma contamination package (R&D) and cultured within the indicated medium following the producer’s instruction and supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin. All cells had been saved at 37 °C with 5% CO2.
Mice
Feminine C57BL/6 mice, six weeks previous, had been obtained from the Shanghai Lingchang Biotechnology Co., LTD. Mice had been housed in an SPF animal facility in temperature-controlled rooms at 21 °C, with 45–65% relative humidity and 12-h mild/darkish cycles on the Shanghai Jiao Tong College Faculty of Medication. The animal experiments had been carried out utilizing protocols authorised by the Institutional Animal Care and Use Committee.
Synthesis of DMONs
0.068 g of TEA was added to 25 mL of water and stirred at 300 rpm for 0.5 h at 80 °C, after which 380 mg of CTAB and 168 mg of NaSal had been added and stirred for one more 1 h. 2 mL of TEOS and a couple of mL of BTES had been added and continued to stir at 80 °C for two h. The merchandise had been collected by centrifugation at 20,000 rpm on the finish of the response, after which the residual reactants had been eliminated by washing 3 times utilizing water and ethanol. The DMONs had been extracted with a combination answer of HCl and anhydrous ethanol (V HCl: V ethanol = 1: 9) at 80 °C for 12 h 3 times to take away the CTAB. The obtained DMONs had been dried underneath vacuum at room temperature. 100 mg of DMONs had been dispersed into 100 mL of a combination of ethanol and water (V water: V ethanol = 1: 1), and 1 mL of APTES was added and continued to stir at 80 °C for 12 h. The response was carried out for 12 h, after which DMONs-NH2 was obtained by centrifugation utilizing ethanol 3 times.
Preparation of dsDNA
Double-stranded DNA molecules had been generated by PCR amplification with DreamTaq polymerase utilizing pcDNA3.1 as a template as described beforehand [37]. The size of the PCR merchandise is 94 base pairs. The PCR merchandise had been purified utilizing a GeneJET PCR purification package.
Ahead primer: 5’-CGATGTACGGGCCAGATATACG-3’;
Reverse primer: 5’-CATATATGGGCTATGAACTAATGACC-3’.
dsDNA loading
DMONs-NH2 (Si: 100 ng/mL) was dispersed into 1 mL of dsDNA aqueous answer with totally different mass ratios of dsDNA to Si, and stirred in a single day at room temperature, after which unloaded dsDNA was eliminated by centrifugation. The dsDNA focus of the supernatant was detected by Nanodrop assay to calculate the loading capability of dsDNA.
In vitro dsDNA launch
The in vitro dsDNA launch profile of dsDNA@DMONs was studied in SBF (pH 7.4) with totally different GSH concentrations of 0, 5 and 10 mM at 37 °C with a shaking pace of 200 rpm. The focus of dsDNA launched within the supernatant at scheduled timepoints was decided utilizing Nanodrop and adopted by the substitute of buffer with contemporary SBF with corresponding GSH concentrations.
Materials characterization
Transmission electron microscopy (TEM) photographs had been acquired on a JEOL-2100F transmission electron microscope. Scanning electron microscopy (SEM) photographs and corresponding ingredient mapping scanning had been acquired on a field-emission Magellan 400 microscope (FEI Firm, USA). The quantitative evaluation of the Si ingredient was decided by inductively coupled plasma mass spectrometry (ICP-MS, NexION 2000B, PerkinElmer, US). Confocal laser scanning microscopy (CLSM) photographs had been acquired by FV1000 (SP8, Leica, US). Nitrogen adsorption–desorption isotherms had been recorded at liquid nitrogen temperature with an ASAP 2020 adsorption analyzer (Micromeritics).
IFN-I exercise reporter assay
Murine IFN-Is exercise was measured in RAW-Lucia ISG cells. Uncooked-Lucia ISG cells had been seeded at a density of 30,000 cells per nicely in a 96-well flat backside plate (Corning). After adhering to the plate, Uncooked-Lucia ISG cells had been handled with totally different concentrations of dsDNA@DMONs for twenty-four h. Subsequently, 50 μL supernatant was transferred to a 96-well opaque white plate, and 50 μL QUANTI-Luc was added to detect luciferase exercise.
Agarose gel electrophoresis
After mixing the pattern with 6 × loading dye, the pattern was loaded into the nicely of 1% agarose gel containing Gelstain, and the gel was marked with a DNA marker. 1 × TAE buffer was used because the working buffer, and the electrophoresis was carried out at a situation of 200 mA for 30 min utilizing a nucleic acid electrophoresis equipment. After electrophoresis, the gel was imaged utilizing the ChemiDoc MP gel imaging system produced by Biorad.
RNA extraction and quantitative real-time PCR
Complete RNA was extracted from cells utilizing the TRIzol Reagent and the Direct-zol RNA Miniprep Plus Package in accordance with the producer’s directions. The RNA high quality and amount had been evaluated utilizing a microvolume spectrophotometer. Reverse-transcribed with the PrimeScriptTM RT reagent Package with gDNA Eraser in accordance with the producer’s directions. RT-qPCR was carried out utilizing SYBR Inexperienced and primer pairs designed to focus on Ifnb, Cxcl10, Isg15, Tapbp, Tap1, Tap2, B2m, Psmb5 and β-actin (Primer sequences are listed in Supplementary Desk 1). The relative mRNA expression ranges had been decided utilizing the comparative CT methodology, with normalization to the housekeeping gene β-actin, and the info was analyzed as fold induction in comparison with the management pattern.
Immunofluorescence evaluation
Immunofluorescence and confocal evaluation had been carried out as described earlier than [38]. Sulfo-Cy5.5 NHS ester inventory answer was ready by including 1 mL of DMSO to 1 mg of sulfo-Cy5.5 NHS ester powder. Then 100 μL of sulfo-Cy5.5 NHS ester inventory answer was added to 1 mL of DMONs-NH2 and incubated in a single day on a shaker shielded from mild. Afterward, the DMONs-Cy5.5 was obtained by centrifugation two instances. B16-F10 or DC2.4 cells had been cultured on glass-bottomed wells (150,680, Nunc) and handled with DMONs-NH2 for 30 min, 2 h or 6 h. After that, cells had been mounted in 4% PFA in PBS for 10 min, washed twice with PBS, after which stained with DAPI for 10 min. No less than eight consultant photographs had been taken for every pattern utilizing a Leica TCS SP8 confocal laser scanning microscope (CLSM).
Protein extraction and immunoblotting
For western blotting experiments, cells had been cultured with indicated therapies adopted by being lysed with RIPA buffer supplemented with phosphatase and protease inhibitors and centrifuged for 15 min at 14,000 g in 4 °C. The protein focus was decided by the PierceTM BCA Protein Assay Package, and the protein samples had been denatured utilizing SDS-PAGE buffer by heating at 100 °C for five min. Subsequently, Equal protein quantities of samples had been separated on 10% PAGE gels with a Multicolor Prestained Protein Ladder after which transferred to nitrocellulose membranes (Millipore) by the Trans-Blot Turbo Switch System (Bio-Rad). The membranes had been blocked in 5% BSA in TBST for 1 h at room temperature earlier than incubation with major antibodies at 4 °C in a single day. The membranes had been washed with TBST 3 times and incubated with HRP-conjugated secondary antibodies for two h at room temperature. Protein photographs had been captured with the Tanon 5200 Sequence Totally Automated Chemiluminescence/Fluorescence Picture Evaluation System.
In vivo tumor fashions
For in vivo tumor fashions, 1 × 106 B16-F10 cells in 100 μL DMEM per mouse had been used and injected subcutaneously into the flank of mice. The tumor quantity was monitored each different day by measuring the size (a) and width (b), and the tumor quantity was calculated to be 1/2a × b2. Mouse physique weight was monitored through the therapeutic interval. The mice in every group had been intratumorally (i.t.) administrated with 50 µL/dose PBS, DMONs, dsDNA or dsDNA@DMONs for a complete of 4 doses when the tumor quantity was about 60 mm3. For the anti-PD-L1(Clone:10F.9G2) antibody, mice had been administered with intraperitoneal injections of anti-PD-L1 antibody (q3d, 100 μg per mouse, a complete of three doses). The research endpoint for max tumor quantity was roughly 2000 mm3.
Move cytometry evaluation of the tumor microenvironment
Tumor tissues had been minced and enzymatically digested with 2 mg/mL collagenase I supplemented with 1 mg/mL hyaluronidase and 25 μg/mL DNase I for 30 min at 37 °C, to amass a single-cell suspension. The dissociated cell suspensions had been handed via a 70 μm filter, counted, and resuspended in 100 μL PBS to succeed in a cell density of 5 × 106 cells per pattern. Then the cells had been blocked with anti-mouse CD16/32 antibody for 30 min. After washing with FACS buffer twice, cells had been stained with Fixable Viability Stain for 15 min on ice in the dead of night. After washing, cells had been stained with antibody combine (already titrated antibody concentrations) for 30 min on ice in the dead of night. After washing, cells had been resuspended with 300 μL FACS buffer. For Ki67 and FOXP3 staining, cells had been handled with eBioscience™ Foxp3 / Transcription Issue Staining Buffer Set by following the directions after which stained with titrated antibodies. After washing, cells had been resuspended with 300 μL FACS buffer. For intranuclear TNFα staining, cells had been stimulated with Leukocyte Activation Cocktail in accordance with the directions earlier than permeabilization with Fixation/Permeabilization Resolution Package, after which stained with titrated anti-TNFα antibody. Information had been acquired with a FACSAriaTM III circulation cytometer and analyzed by FlowJo software program.
H&E staining
Mice had been euthanized on the remaining time level. The kidney, liver, spleen, coronary heart and lung tissues had been eviscerated, mounted in 4% formaldehyde and reduce into 7-μm formalin-fixed paraffin-embedding (FFPE) slides. The FFPE slides had been then stained with hematoxylin and eosin after which photographs had been taken on an Olympus IX73 microscope.
