Chemical compounds and reagents
Copper chloride, iron(II) chloride, manganese chloride, zinc nitrate, cobalt nitrate hexahydrate, vitamin E, Ferrostatin-1 (Fer-1), and propidium iodide (PI) had been procured from Shanghai Aladdin Biochemical Know-how Co., Ltd. 3-Methyladenine (3-MA) was received from Shanghai Maclin Biochemical Know-how Co., Ltd. Hydrochloric acid (HCl) was bought from Sinopharm. Z-VAD-FMK, the reactive oxygen species (ROS) package, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), Annexin V-FITC apoptosis package, and Calcein-AM had been obtained from Beyotime Biotechnology. Liperfluo was obtained from Dojindo. CCK-8 package was received from Sangon Biotechnology. 3,3′,5,5′-tetramethylbenzidine (TMB) answer (1%) was purchased from Solarbio. STING agonist-4 was obtained from Invivochem. PE anti-mouse CD8a antibody (Cat#100708), FITC anti-mouse CD4 antibody (Cat#116003), APC anti-mouse CD3 antibody (Cat#152306), PE anti-mouse CD86 antibody (Cat#105008), FITC anti-mouse CD80 antibody (Cat#104706), and APC anti-mouse CD11c antibody (Cat#117310) had been purchased from Biolengend. Anti-CALR antibody (A00894-1) and goat anti-rabbit IgG(H + L) (AF594 conjugated, E-AB-1060) had been obtained from Boster organic know-how and Elabscience, respectively. Dulbecco’s Modified Eagle Medium (DMEM) was bought from Hyclone. Fetal bovine serum (FBS) was received from Gibco. Penicillin-streptomycin was purchased from BasalMedia. Phosphate buffer (PBS) was purchased from Sigma. DEPE-PEG-FA and DEPE-PEG (MW 2000) had been obtained from AVANTI.
Devices
3,3′,5,5′-tetramethylbenzidine (TMB) absorbance was measured utilizing a microplate reader (Thermofisher, Multiskan Sky). Fluorescent photos had been acquired utilizing a laser scanning confocal microscope (Zeiss, LSM 780). Zeta potential and hydrodynamic measurement had been carried out by ZS90 (Zetasizer Nano, Malvern). TEM photos had been captured utilizing a Hitachi HT7700 transmission electron microscopy. Circulate cytometry evaluation was carried out utilizing FACSymphony™ A5, BD Bioscience.
OMV purification and functionalization
OMV was obtained by ultracentrifugation of DH-5α tradition supernatant. Briefly, DH-5α was cultured in Luria-Bertani medium for 16 h at 200 rpm shaking velocity at 37 °C. Then, bacterial cells had been eliminated by centrifugation at 4000 g for 20 min at 4 °C. The obtained supernatant was additional filtered by 0.45 μm membrane filters to take away giant bacterial particles. Following this, the supernatant was centrifuged at 30,000 rpm for two.5 h at 4 °C. The ensuing precipitate was resuspended in 500 µL PBS and saved at -20 °C for the next experiments. Whole protein focus was quantified utilizing a bicinchoninic acid (BCA) package, defining the focus because the OMV focus.
To kind OMV/Fe composites, 1.4 mL OMV (4.2 mg/mL) was combined with 200 µL metallic salts (with a focus of 1.2 mg/mL) for two h at 4 °C utilizing a rotary mixer. The combination was then centrifuged at 14,000 rpm for 0.5 h at 4 °C, and the ensuing precipitate was re-suspended in 1 mL PBS.
For the fabrication of OMV/SaFeFA, 6 mg OMV was first stirred with 1 mg STING agonist-4 for 8 h at 4 °C. Subsequently, FeCl2 answer (2 mg) was slowly dropped into the above combination underneath vigorous shaking. After 2 h, 3 mg DSPE-PEG-FA was added for an additional 1 h. Final, the precipitate was received underneath centrifugation at 4 °C (14000 rpm, 30 min). The ensuing OMV/SaFeFA was resuspended in PBS and saved at -20 °C.
The DSPE-PEG was escorted into ready OMV to get DSPE-PEG modified OMV (OMV-PEG) by merely co-incubation [41]. Comparable procedures had been used to acquire DSPE-PEG-FA-modified OMV (OMV/FA), Fe-anchored OMV/FA (OMV/FeFA), STING agonist-4-loaded OMV/FA (OMV/SaFA), DSPE-PEG-modified OMV (OMV-PEG), Cyanine 5 (CY5)-loaded OMV-PEG (OMV/CY5-PEG), CY5-loaded OMV/FA (OMV/CY5-PEG-FA), and CY5-loaded OMV/FeFA (OMV/CY5FeFA).
•OH detection
OMV/ions (50 µL) had been combined with 100 µL TMB and 5 µL 0.03% H2O2 options at room temperature. After 10 min, the response was terminated by 155 µL HCl (1 M), and the absorbance at 450 nm was recorded by a microplate reader.
Attribute protein of OMVs profiles
5 µg OMV or OMV/Fe had been combined with loading buffer, boiled 10 min, after which subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins of OMVs had been visualized by Coomassie good blue staining for 30 min and analyzed utilizing a gel imaging system (JS-6800).
Launch conduct assay
OMV/SaFeFA was ready and incubated in buffer options of various pH values for various durations (0, 1, 2, 4, 8, and 12 h). Subsequently, the samples had been centrifuged at 14,000 rpm for 10 min, and the supernatant was collected to evaluate the degrees of STING agonist and Fe2+ ions. The STING agonist stage was decided by measuring the UV-vis spectra absorbance at 320 nm, whereas the Fe2+ ion stage was evaluated utilizing phenanthroline spectrophotometry.
Cell tradition
MC38 and 4T1 cells had been cultured in DMEM media supplemented with 10% FBS and 1% streptomycin/penicillin and maintained in a cell incubator at 37 ℃ with 5% CO2. DC2.4 cells had been cultured in RPMI-1640 media supplemented with 10% FBS, 1% streptomycin/penicillin, and maintained in a cell incubator at 37 ℃ with 5% CO2.
Cell uptake
MC38 cells (1 × 106) had been seeded in 6-well plates and cultured in a single day. Subsequent, OMV/CY5FeFA answer (OMV: 40 µg/mL, CY5: 20 µg/mL) was co-cultured with MC38 cells for varied period (5 min, 2 h, 4 h, 8 h, and 12 h). Lastly, cells samples had been collected and analyzed utilizing circulate cytometry.
CCK-8 assay
MC38 cells (5000) had been seeded in 96-well plates and cultured in a single day. Subsequent, PBS, OMV/FA, OMV/FeFA, OMV/SaFA, and OMV/SaFeFA had been added to the cell tradition medium at concentrations of 15 µg/mL for OMV and seven.5 µg/mL for STING agonist-4. After 24 h, cell viability was assessed utilizing a CCK-8 package.
To discover the cell demise pathway, MC38 cells (5000) had been seeded in 96-well plates and cultured in a single day. Then, cell samples had been co-incubated with OMV/SaFeFA and varied inhibitors (Fer-1: 2 µM; vitamin E: 20 µM; and 3-MA: 10 µM). Subsequently, cell viability was decided utilizing a CCK-8 package.
To analyze the impact of focus on cell viability, MC38 cells (5000) had been seeded in 96-well plates and cultured in a single day. Then, the cell samples had been cultured with varied concentrations of OMV/SaFeFA (2.5, 5, 7.5, 10, 15, and 20 µg/mL, complete protein focus was outlined to be the OMV/SaFeFA focus) for twenty-four h. Following incubation, the cell viability was assessed utilizing a CCK-8 package.
Intracellular Fe2+ imaging
MC38 cells had been seeded in confocal dishes and cultured in a single day. Thereafter, the cells had been incubated with OMV/SaFeFA for varied time factors (0, 2, 4, 8, and 12 h). After incubation, the cell samples had been washed thrice with PBS after which stained with FerroOrange fluorescent probes for 30 min. The fluorescent alerts of cell samples had been noticed by a confocal laser scanning microscopy (CLSM).
ROS and •OH imaging
MC38 cells (1 × 105) had been seeded in confocal small dishes and cultured in a single day. Then, PBS, OMV/FA, OMV/FeFA, OMV/SaFA, OMV/SaFeFA, H2O2 (200 µM), and OMV/SaFeFA + H2O2 (200 µM) had been added into the cell tradition medium. The concentrations of OMV and STING agonist-4 had been 15 and seven.5 µg/mL, respectively. After 24 h, the cells had been stained with the DCFH-DA (ROS fluorescent probe) and BboxiProbe O26 (•OH fluorescent probe) based on the producer protocols and imaged by a CLSM. The fluorescent depth of the pictures was measured by an Picture J software program.
Ferroptosis examine
MC38 cells (1 × 105) had been seeded in confocal small dishes and cultured in a single day. Subsequently, PBS, OMV/FA, OMV/FeFA, OMV/SaFA, OMV/SaFeFA, and FeCl2 (30 µg/mL) had been added to the cell tradition medium. The concentrations of OMV and STING agonist-4 had been 15 and seven.5 µg/mL, respectively. Following a 24-h incubation interval, cells had been stained with the Liperfluo probe based on the producer’s protocols and imaged utilizing a CLSM.
Apoptosis evaluation
MC38 cells (1 × 106) had been seeded in 6-well plates and cultured in a single day. Then, PBS, OMV/FA, OMV/FeFA, OMV/SaFA, and OMV/SaFeFA had been added into the cell tradition medium. After 24 h, cells had been harvested and stained with Annexin V-FITC and PI. The apoptosis of cells was analyzed utilizing circulate cytometry.
CRT staining
MC38 cells (1 × 105) had been seeded in confocal small dishes and cultured in a single day. Afterward, cells had been co-incubated with PBS, OMV/FA, OMV/SaFA, OMV/FeFA, and OMV/SaFeFA for twenty-four h. Following incubation, the cells had been fastened with 4% paraformaldehyde for 30 min, after which stained with anti-CALR antibody (1: 200) for 30 min. After washing thrice with PBS, the cells had been incubated with goat anti-rabbit IgG(H + L) (AF594 conjugated, 1: 100) for 30 min. Lastly, the cells had been counterstained with DAPI and imaged utilizing a CLSM.
Western blot assay
MC38 cells (1 × 106) had been seeded in 6-well plates and cultured in a single day. Then, PBS, OMV/FA, OMV/SaFA, OMV/FeFA, and OMV/SaFeFA had been added into the cell tradition medium. After 24 h, cells samples had been collected and processed following commonplace western blot protocols.
In vitro activation of STING pathway
MC38 cells and DC 2.4 cells had been seeded in 6-wells plates and cultured in a single day. MC38 cells had been incubated with PBS, OMV/FA, OMV/FeFA, OMV/SaFA, and OMV/SaFeFA for twenty-four h. Afterward, the supernatants had been collected and incubated with DC 2.4 cells for an additional 24 h. The handled DC 2.4 cells had been then lysed in radio-immunoprecipitation assay (RIPA) buffer with protease inhibitors. For western blot evaluation, 15 µg of complete cell lysates had been combined with loading buffer, boiled for 10 min, and subjected to 10% SDS-PAGE at 80 V for 20 min adopted by 120 V 60 min. The proteins had been then transferred onto a polyvinylidene fluoride membrane, which was subsequently blocked for 1 h in 5% non-fat milk. The membrane was then incubated in a single day at 4 ℃ with an anti-STING antibody. Immunoreactive protein was visualized utilizing a beneficial dilution of conjugated secondary antibody, and the luminescent bands had been visualized by Automated Gel Imaging Analyzer, JS-6800. The focus of IFN-β within the supernatants of DC 2.4 cells was measured utilizing a business package.
Biodistribution
All mouse experiments had been carried out in accordance with protocols accepted by the Animal Experimental Ethics Committee of the Fujian Regular College (No. IACUC-20220005). C57/BL6 and BALB/c mice (4–6 weeks) had been sourced from Beijing Hua Fu Kang Biotechnology Co., ltd. 9 MC38 tumor-bearing C57/BL6 mice had been randomly divided into 3 teams. Mice had been i.v. injected with CY5, OMV/CY5-PEG, and, OMV/CY5-PEG-FA, with CY5 and OMV doses set at 50 µg and 60 µg, respectively. Then, the fluorescent alerts of mice had been recorded at totally different time factors (20 min, 2 h, 4 h, 8 h, 10 h, and 24 h) utilizing an IVIS system. After 24 h, mice had been euthanized, and the fluorescent alerts of tumors and main organs had been recorded.
For the 4T1 breast tumor mannequin, 9 tumor-bearing BALB/c mice had been randomly allotted into 3 teams. These mice obtained i.v. injection of CY5, OMV/CY5-PEG, and, OMV/CY5-PEG-FA. At 24 h post-injection, mice had been euthanized, and the fluorescent alerts of tumors and main organs had been recorded. Fluorescence photos of tumor slices had been captured utilizing a CLSM.
In vivo remedy
C57/BL6 mice obtained an injection of 1 × 106 MC38 cells in the appropriate hind leg flank. Ten days later, the MC38 tumor-bearing mice had been randomly divided into 5 teams (n = 4) and that i.v injected with PBS, OMV/FA, OMV/SaFA, OMV/FeFA, and OMV/SaFeFA on days 0 and 4. Tumor measurement and physique weight had been measured each two days all through the remedy interval, with tumor volumes calculated utilizing the method: V= (size × width2)/2. On day 16, mice had been euthanized, and the tumors had been collected. Tumors had been weighted, sliced, and analyzed for H&E, TUNEL staining, and immunohistochemical staining of IFN-γ, SLC7A11, GPX4, NCOA4, and FTH1. Serum was collected for IFN-γ detection utilizing a business package. As well as, the long-term survival charges had been evaluated by the identical procedures apart from monitoring 58-day interval.
Biosafety
PBS, OMV/FA, OMV/FeFA, OMV/SaFA, and OMV/SaFeFA had been administered by way of the tail vein of C57/BL6 mice. After a 16-day remedy, blood samples from C57/BL6 mice had been collected for biochemical evaluation. The parameters measured included blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP), and gamma glutamyl transpeptidase (γ-GT), utilizing assay kits from Solarbio (BC1555, BC1535, BC2145, BC1565, and BC1225). Subsequently, mice had been sacrificed, and the main organs (coronary heart, liver, spleen, lung, and kidney) had been harvested and subjected to H&E staining for histological evaluation.
Immune response evaluation
MC38 bearing tumor-bearing mice had been randomly divided into 5 teams (n = 3) and that i.v injected with PBS, OMV/FA, OMV/SaFA, OMV/FeFA, and OMV/SaFeFA on days 0 and 4. After 16-day remedy, mice had been euthanized, and tumors had been collected and made to single-cell suspensions by 40 μm nylon mesh filters. Subsequently, the cell samples had been stained with designated fluorescent antibodies and analyzed utilizing circulate cytometry.
Statistical evaluation
Quantitative information had been introduced as imply ± commonplace error of the imply (SEM). All experiments had been repeated thrice or extra. Statistics had been analyzed by one-way ANOVA, adopted by Dunnett’s a number of comparisons take a look at utilizing GraphPad Prism 8.0. Statistical significance was denoted by an asterisk. P values of 0.05 or much less had been thought-about statistically vital (ns: not vital, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).
