Supplies
DPPC, DSPE-PEG2000, and SPIO (Fe3O4, 2 mg·mL− 1, 10 nm) have been bought from Xi’an RuiXi Organic Know-how Co., Ltd. (Xi’an, China). DSPE-PEG2000-CREKA was bought from Bioworld Know-how Co., Ltd. (Shanghai, China). PFP, Calcein-AM, and propidium iodide (PI) have been acquired from Sigma-Aldrich (Shanghai, China). cGAMP was bought from APExBIO Know-how (USA). Cell Counting Package-8 (CCK-8) was bought from Dojindo (Japan). TGF-β was obtained from Abbkine (Wuhan, China). Anti-CD3+-FITC, anti-CD4+-PerCP, anti-CD8+-APC, anti-CD11c+-FITC, anti-CD80+-APC, and anti-CD86+-PE antibodies have been bought from Thermo Fisher Scientific Inc. (Shanghai, China). ELISA kits for TNF-α, IL-12, and IFN-γ have been bought from Huyu Organic Know-how Co., Ltd. (Shanghai, China). ELISA kits for IFN-β and CXCL10 have been bought from Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). FITC-fibrinogen and Cy5-albumin have been obtained from Solarbio Science & Know-how Co., Ltd. (Beijing, China). All reagents used on this work have been of analytical grade.
Synthesis of SSCOL
The STING-ODVLipo/SPIO-CREKA nanoparticle (SSCOL) was fabricated by utilizing the thin-film hydration technique. Briefly, 6 mg of DPPC, 2 mg of DSPE-PEG2000-CREKA, 2 mg of ldl cholesterol, and 300 µL SPIO have been dissolved in 10 mL of trichloromethane. Beneath a water tub at 45 °C, a skinny lipid movie was shaped by vacuum rotary evaporation. PBS buffer was added to hydrate the movie, and the flask was shaken to kind a homogeneous suspension. Beneath an ice tub, 200 µL of PFP and 1 mg of cGAMP have been added to the suspension, which was then emulsified utilizing an ultrasonic homogenizer (52 W, 3 min). The combination was subsequently stirred magnetically at room temperature to make sure full evaporation of the natural solvent. Lastly, the suspension was subjected to low-temperature centrifugation to take away free PFP, SPIO, and cGAMP, yielding the SSCOL. By following the identical process and substituting DSPE-PEG2000 for DSPE-PEG2000-CREKA, SSOL (with out CREKA) was ready.
Characterization of SSCOL
The microstructure and dispersion of the SSCOL have been characterised utilizing an optical microscope and transmission electron microscopy (TEM, Hitachi H-7600, Japan). The particle dimension distribution, zeta potential, and polymer dispersity index (PDI) have been measured utilizing dynamic gentle scattering (DLS, ZEN3600, Malvern Devices, UK) with a Malvern particle dimension analyzer. Steady monitoring of adjustments within the distribution, zeta potential, and PDI of SSCOL dispersed in fetal bovine serum (FBS) was carried out over a 6-day interval. DSPE-PEG2000-CREKA and DSPE-PEG2000 have been analyzed utilizing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The entrapment effectivity of SPIO was assessed through inductively coupled plasma optical emission spectrometry (ICP-OES, ThermoFisher, USA). The height areas of cGAMP options at numerous concentrations (10, 20, 30, 40, and 50 µg mL− 1) have been measured utilizing high-performance liquid chromatography (HPLC, Shimadzu LC-20AD, Japan). Subsequently, the cGAMP normal curve was constructed, and the entrapment effectivity of cGAMP was evaluated. The loading capability of the nanoparticles was calculated by the next formulae:
Loading capability (%) = (mass of SPIO or cGAMP/complete mass of nanoparticles) × 100%.
Photothermal conversion of SSCOL
SSCOL options at numerous concentrations have been uncovered to NIR laser irradiation (2 W·cm− 2, 10 min). Variations in temperature have been monitored by infrared thermal imaging throughout 808 nm NIR laser irradiation at variable energy intensities.
Optical droplet vaporization efficiency of SSCOL
SSCOL resolution (50 µg·mL− 1) was dropped onto a glass slide and heated utilizing a heating plate. Through the heating course of, the liquid-gas section transition of PFP was monitored in actual time below an optical microscope. Subsequently, to research the impact of laser irradiation on the optical droplet vaporization (ODV) efficiency, the SSCOL (2 µg·mL− 1) was added to a 96-well plate and irradiated with an 808 nm laser (2 W·cm⁻²). The variety of microbubbles generated at totally different temperatures was instantly noticed below an optical microscope.
Drug-release profile of cGAMP from SSCOL
The two mg of SSCOL and a pair of mg of SSCL have been individually added to sealed dialysis baggage and positioned in a pre-prepared PBS sustained-release resolution (containing 30% v/v anhydrous ethanol, 0.1% v/v Tween-80, and 0.02% w/v sodium azide). The nanoparticles have been irradiated with an 808 nm laser (2 W·cm⁻², 5 min). After laser irradiation, the dialysis baggage have been incubated in a 37 °C shaker (rotation velocity 150 rpm). Through the drug launch course of, resolution samples have been collected from every group at 0, 1, 2, 4, 6, 8, 12, 24, and 48 h, and an equal quantity of contemporary resolution was added to take care of a continuing quantity. The cGAMP launch quantities from the nanoparticles have been quantified by HPLC, and the cumulative launch share of cGAMP was calculated.
In vitro cytotoxicity and biosafety of SSCOL
A regular CCK-8 protocol was used to judge the in vitro cytotoxicity of SSCOL. Endothelial cells have been seeded in 96-well plates and allowed to stick till steady. The cells have been then co-incubated with various concentrations of SSCOL (100, 200, 300, 400, 500, and 600 µg·mL− 1). After incubation, the medium was changed with contemporary cell tradition medium, and CCK-8 resolution was added to every effectively. Absorbance (OD) was measured at 490 nm utilizing an automated microplate reader, and the cell survival price was calculated based mostly on the absorbance values.
For in vivo biocompatibility, wholesome feminine BALB/c mice have been intravenously administered SSCOL (2 mg·mL− 1, 200 µL). At totally different time factors (1, 3, 7, 14, 21, 28 d), the mice have been euthanized by cervical dislocation. Blood samples have been collected for full blood counts (CBC) and biochemical evaluation. Main organs (coronary heart, liver, spleen, lung, kidney) have been harvested and glued in 4% v/v paraformaldehyde. The tissue sections have been subjected to hematoxylin − eosin (H&E) staining. Untreated mice have been used as clean controls.
Cell tradition and animal fashions
The 4T1 murine breast most cancers cell line was initially obtained from JXQ Know-how (Chongqing, China) and cultured in RPMI-1640 medium supplemented with 10% v/v FBS and 1% v/v penicillin-streptomycin below normal circumstances (5% CO2, 37 °C). Bone marrow-derived dendritic cells (BMDCs) have been generated from murine bones in response to a longtime technique. Briefly, wholesome feminine BALB/c mice (6–8 weeks outdated) have been euthanized through cervical dislocation, and their bilateral femurs and tibias have been harvested. The bone marrow cavities have been flushed with RPMI-1640 medium, and the flushing resolution was collected. Following centrifugation, the supernatant was discarded, and the pellet was resuspended in purple blood cell lysis buffer and incubated at the hours of darkness to lyse erythrocytes. The lysed cells have been washed with tradition medium and resuspended to kind a cell suspension. GM-CSF and IL-4 have been added to the cell suspension at a ratio of 1:10000 to organize DC-specific tradition medium. The BMDCs have been seeded into 6-well plates utilizing this medium and cultured at 37 °C with 5% CO2 for two days. On day 3, the supernatant was eliminated and changed with an equal quantity of contemporary DC-specific medium. On day 5, half of the supernatant was changed with an equal quantity of contemporary DC-specific medium. On day 7, the floating cells have been harvested, representing immature BMDCs.
All experimental protocols have been authorized by the Animal Ethics Committee of the Second Affiliated Hospital of Chongqing Medical College (Coren Trial(265), 2021) and performed on the Laboratory Animal Middle of Chongqing Medical College. Feminine BALB/c mice (aged 6–8 weeks) have been obtained from Enswell Biotechnology Ltd. (Chongqing, China). To determine a 4T1 orthotopic murine breast most cancers mannequin, 4T1 cells within the logarithmic progress section have been diluted with sterile PBS (2 × 106 cells per mouse). The cell suspension was then slowly injected into the bilateral mammary fats pads of BALB/c mice to simulate major and metastatic lesions. Tumor dimension was monitored each 1–2 days utilizing calipers. When the tumor quantity reached roughly 60 mm³, the mice have been used for therapeutic research; when the tumor quantity reached roughly 100 mm³, the mice have been used for imaging research. In in vivo research, mice have been anesthetized intraperitoneally with 1% pentobarbital sodium, and have been euthanized by cervical dislocation.
Photoacoustic/ultrasound dual-modal imaging
To judge the ultrasound imaging efficiency in vitro, the nanoparticle options from every group (2 mg·mL− 1, 200 µL) have been added to the wells of a 3% w/v agarose gel and subsequently irradiated with an 808 nm laser (2 W/cm², 10 min). Instantly following irradiation, ultrasound photos of every group have been acquired utilizing the MyLab90 ultrasound diagnostic system (Esaote, Italy), and the depth values of the ultrasound echo alerts within the areas of curiosity (ROIs) have been quantitatively analyzed. Moreover, ultrasound photos have been collected at numerous time factors post-irradiation (4, 6, 8, and 10 min) for additional evaluation. To judge the photoacoustic (PA) imaging efficiency in vitro, SSCOL options (2 mg·mL− 1, 200 µL) have been added to the wells of a 3% w/v agarose gel. Utilizing the Vevo LAZR PAI machine (VisualSonics, Toronto, Canada), PA scans have been carried out throughout the excitation wavelength vary of 680–970 nm to find out the excitation wavelength comparable to the utmost PA sign depth of the nanoparticles. This optimum excitation wavelength (700 nm) was then used for subsequent PA imaging experiments. Beneath this optimized excitation wavelength, in vitro PA photos of the totally different nanoparticle options from every group have been acquired, and the PA sign intensities of every group have been quantitatively measured.
To judge the in vivo photoacoustic/ultrasound dual-modal imaging efficiency of SSCOL, a unilateral orthotopic most cancers mannequin was employed. Following intravenous injection through tail vein of SSCOL into tumor-bearing mice (200 µL per mouse), PA photos of the tumor have been acquired at numerous time factors earlier than and after injection (2, 4, 6, 24, and 48 h). The PA sign depth throughout the tumor area was quantitatively measured for every group to find out the optimum time level comparable to one of the best PA imaging impact. This optimum time level was then used because the timing for subsequent focused PTT. The tumor was irradiated with an 808 nm laser (2 W·cm− 2, 10 min), whereas concurrently buying ultrasound photos. The ultrasound echo sign depth within the tumor area was quantified.
The expression of fibronectin within the tumor
In vitro, 4T1 cells have been routinely cultured in 6-well plates. As soon as the cells had stably adhered, TGF-β was added for co-incubation. Within the management group, 4T1 cells have been cultured below similar circumstances with out TGF-β therapy for a similar period. The mesenchymal phenotype transformation of every group of 4T1 cells was noticed utilizing an optical microscope, and the expression ranges of fibronectin (Fn) within the two teams of 4T1 breast most cancers cell samples have been quantified by RT-PCR.
Within the in vivo experiments, a unilateral orthotopic tumor was employed. Tumor and main organ tissues (liver, kidney, mind) from tumor-bearing mice have been collected. These tissues have been homogenized in RIPA lysis buffer containing protease inhibitors at 4℃ after which centrifuged. The supernatants of every pattern have been collected, and rabbit anti-mouse fibronectin polyclonal antibody was added to the lysates. Western blot evaluation was carried out to detect the expression ranges of fibronectin in each tumor tissues and main organ tissues.
In vitro and in vivo photothermal therapeutic results of SSCOL
In vitro, 4T1 cells have been seeded in 96-well plates and co-incubated with nanoparticles from every experimental group. Following this, the cells have been uncovered to laser irradiation at an depth of two W·cm− 2 for five min. Cell viability was subsequently assessed via the CCK-8 assay. 4T1 cells have been routinely cultured in confocal microscopy-compatible dishes. After therapy with every group’s nanoparticles, Calcein-AM and PI dyes have been added to the respective samples, and cell apoptosis and necrosis have been evaluated utilizing confocal laser scanning microscopy (CLSM). Moreover, 4T1 cells have been additionally seeded in 24-well plates. Publish-treatment, the cells have been trypsinized with 0.25% trypsin, labeled with Annexin V-FITC and PI, and analyzed for apoptosis through move cytometry.
Within the in vivo experiments, a unilateral orthotopic tumor mannequin was utilized. nanoparticle options (200 µL, 2 mg·mL− 1) from every group have been administered through tail vein injection. Twenty-four hours after injection, tumors have been irradiated with an 808 nm laser at an influence density of two W cm−² for 10 min for PTT. Floor temperature adjustments of the tumors throughout irradiation have been monitored utilizing an infrared thermal digicam (Fotri226, Shanghai, China). Following PTT, tumor tissues from every group have been harvested and subjected to H&E staining.
In vivo biodistribution of SSCOL
To evaluate tumor accumulation and biodistribution of SSCOL earlier than PPT, DiR fluorescent dye-labeled focused SSCOL and non-targeted SSOL have been ready. A unilateral orthotopic most cancers mannequin was used, with tumor-bearing mice anesthetized through isoflurane inhalation. Fluorescence photos of tumor websites have been captured utilizing an imaging system at totally different time factors post-injection (2, 4, 6, 24, and 48 h) of nanoparticles (2 mg·mL− 1, 200 µL). Fluorescence intensities at tumor websites have been quantified for each teams. At 48 h, tumor tissues and main organs (coronary heart, liver, spleen, lung, kidney) have been harvested for fluorescence depth evaluation of nanoparticles in excised tissues.
To judge tumor accumulation and biodistribution of SSCOL after PTT, a bilateral carcinoma in situ mannequin was used. Unlabeled SSCOL or SSOL (200 µL, 2 mg·mL− 1) have been injected through tail vein into tumor-bearing mice. Primarily based on in vivo PA imaging, the optimum time level for imaging was recognized, and one tumor website was irradiated with a laser (2 W cm−², 10 min). Following PTT, 200 µL of DiR-labeled SSCOL or SSOL was re-injected through tail vein. Six hours post-injection, fluorescence photos of bilateral tumors have been captured, and alerts have been quantified to check SSCOL and SSOL accumulation. Moreover, FITC-labeled fibrinogen and Cy5-labeled albumin have been injected post-PTT. Tumors have been harvested, and fluorescence of FITC-fibrinogen and Cy5-albumin was noticed ex vivo.
To additional consider the focusing on efficacy of SSCOL in the direction of the fibrin-fibronectin complicated throughout the tumor stroma, tumor-bearing mice have been injected with DiR-labeled SSCOL or SSOL (200 µL, 2 mg·mL− 1) through the tail vein. After 24 h, tumors have been harvested and subjected to immunofluorescence staining to detect fibronectin. Fibronectin was recognized utilizing an alpha-chain antibody and TYR-570 secondary antibody, and the co-localization of nanoparticles with fibronectin within the tumor microenvironment was analyzed utilizing a laser confocal microscope. Moreover, below the identical PTT circumstances as beforehand described, DiI-labeled SSCOL or SSOL have been re-injected through the tail vein. Tumors and main organs (coronary heart, liver, spleen, lung, and kidney) have been harvested to look at the distribution of DiI-labeled nanoparticles. This evaluation was additionally carried out utilizing a laser confocal microscope to judge nanoparticle accumulation in tumors and non-target organs.
In vitro and in vivo activation of the STING pathway
In in vitro experiments, immature BMDCs have been remoted from mice and seeded into 6-well plates. The SSCOL or SSOL options (1 mL, 600 µg·mL− 1) have been added to the corresponding wells for co-incubation with DCs. The expression ranges of interferon (IFNb1, IFNa1), tumor necrosis issue (TNF), interleukin (IL1b, IL6, IL12b), and chemokine (CXCL9, CXCL10) within the DCs have been quantified utilizing RT-PCR. The phosphorylation ranges of TBK1 and IRF-3 within the DCs have been assessed by Western blot evaluation.
In in vivo experiments, a unilateral orthotopic carcinoma mannequin was utilized. The SSCOL and SSOL options (200 µL, 2 mg·mL− 1) from every group have been administered to tumor-bearing mice through tail vein injection, and the tumor was subjected to laser irradiation (2 W·cm− 2, 10 min). On the second day after PTT, the blood from the attention sockets of the mice was collected. The expression ranges of IFN-β and CXCL10 within the serum have been decided utilizing an ELISA equipment.
In vitro and in vivo DC maturation and associated cytokine secretion
A Transwell system was used to co-culture 4T1 cells and BMDCs. Briefly, 4T1 cells have been seeded within the higher chamber till adherent after which incubated with SSCOL, SCOL (with out cGAMP), SCOL with free cGAMP and free cGAMP options (SSCOL = 600 µg·mL− 1, SCOL = 600 µg·mL− 1, cGAMP = 16 µg·mL− 1), respectively. After 8 h of incubation, the medium was changed with contemporary medium, and the 4T1 cells have been irradiated with a laser at 2 W·cm−2 for five min. Subsequently, the handled 4T1 cells have been transferred to the decrease chamber of the plates containing DCs for co-culture. After 24 h, DCs have been collected and stained with anti-CD11c FITC, anti-CD80 APC, and anti-CD86 PE antibodies. The proportion of CD11c⁺CD80⁺CD86⁺ DCs was analyzed by move cytometry. Moreover, the degrees of cytokines (IFN-γ, TNF-α, and IL-12) within the DC suspension have been quantified utilizing an ELISA equipment in response to the usual protocol.
In vivo anti-tumor immunity
The 4T1 cells have been subcutaneously inoculated into left mammary pads of every BALB/c feminine mouse as the first tumor (1st tumor). An equal quantity of 4T1 cells was injected into the correct mammary pad as a distant tumor (2nd tumor) on the seventh days after inoculation of the first tumor. The mice have been randomly divided into six teams: management, SCOL (with out cGAMP), free cGAMP, SCOL + Laser, SCOL + free cGAMP + Laser, and SSCOL + Laser (SSCOL = 600 µg·mL− 1, SCOL = 600 µg·mL− 1, cGAMP = 16 µg·mL− 1). As soon as the tumor quantity reached roughly 50 mm³, mice in every group obtained injections of various options. Laser therapy teams underwent laser irradiation (2 W·cm− 2, 10 min) on the first tumor at 24 h post-injection. In every group, the identical therapy for the first tumors was repeated on the third and sixth days following the preliminary remedy.
The maturation standing of DCs and the activation standing of CTLs have been decided by move cytometry. On day 7 post-treatment, tumor tissues, inguinal draining lymph nodes, and spleens have been harvested from every group and processed into single-cell suspensions. The suspensions have been then stained with anti-CD11c FITC, anti-CD80 APC, and anti-CD86 PE antibodies to judge the expression ranges of co-stimulatory molecules CD11c+, CD80+, and CD86+ on DCs. Moreover, the single-cell suspensions of tumors and spleens have been stained with anti-CD3 FITC, anti-CD4 PerCP, and anti-CD8a APC. The serum concentrations of IFN-γ, TNF-α, and IL-12 in tumor-bearing mice in every group have been detected by utilizing an ELISA equipment. Moreover, tumor tissues have been harvested and subjected to immunofluorescence staining utilizing anti-CD8 FITC and anti-IFN-γ Cy3 antibodies. Co-expression of the fluorescent markers was analyzed below a laser confocal microscope to evaluate IFN-γ secretion and CD8⁺ T cell infiltration throughout the tumors.
In vivo anti-tumor efficacy
Through the therapy interval, mouse physique weight was recorded each different day, and the lengthy and brief diameters of bilateral tumors have been measured. Tumor quantity was calculated utilizing the method: [(width)² × length]/2. On day 19 of therapy, mice have been euthanized through cervical dislocation. The first tumor tissues from every group have been harvested for H&E staining and terminal deoxynucleotidyl transferase dUTP nick finish labeling (TUNEL) assay to evaluate tumor cell necrosis. The metastatic tumor tissues have been collected for proliferating cell nuclear antigen (PCNA) staining to judge tumor cell proliferation.
Statistical evaluation
Knowledge are introduced as imply ± SD. Comparisons between teams have been carried out utilizing Scholar’s t-test, one-way ANOVA, or two-way ANOVA, as acceptable. Statistical significance was outlined as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All experiments have been carried out independently no less than 3 times.
