Materials
All the chemicals used in our studies were sourced from Sigma-Aldrich unless otherwise stated. NHS-PEG-MAL was purchased from JenKem Technology CO., Ltd. c(RGDyC) peptide was obtained from Chinese peptide company. Losartan was obtained from APExBIO (Catalog No.B1072/114798-26-4). Oxaliplatin was obtained from Aladdin (Catalog No.61825-94-3). Fibrinogen and thrombin were abtained from Solarbio (Catalog No.T8021 and Catalog No. F8051). In vivo anti-mouse PDL1 antibody purchased from BioXell (B7-H1, catalogue No. BE0101).
Cell lines and laboratory animals
The 4T1 and MCF-7/ADR cell lines were originally obtained from the American Type Culture Collection (ATCC). All cells were cultured in RMPI 1640 medium (Solarbio) containing 1% penicillin (HyClone), 1% streptomycin (HyClone) and 10% fetal bovine serum (FBS, Sigma). Female BALB/c mice (4–6 weeks) were purchased from Shanghai Sakas Biotechnology Co., Ltd. Mice were housed in a pathogen-free SPF room at 20 ± 3 °C, 40–70% relative humidity, and a 12-h light/dark cycle. All animal experiments were performed in accordance with national Ministry of Health guidelines and protocols approved by the Experimental Animal Center of Shanghai Tenth People’s Hospital.
Synthesis of FeOX NPs
FeMOF nanoparticles were synthesized by a solvothermal reaction. Generally, FeCl3-6H2O (2250 mg) was dissolved in 15 ml DMF and 2-aminoterephthalic acid (450 mg) was dissolved in 15 mL DMF and dissolved by sonicate, then FeCl3-6H2O was added dropwise to 2-aminoterephthalic acid, stirred for 30 min, in The FeMOF NPs were obtained in a teflon lined autoclave, after centrifugation at 120 °C, 24 h. Then 13,000 rpm for 15 min, washed twice with ethanol and deionised water, respectively, and finally redispersed in ethanol (10 mL). FeMOF (4 mg) and NHS-PEG-MAL (50 mg) were mixed in MES buffer pH = 6.0 and reacted for 2 h at room temperature, followed by centrifugation for surface grafting of RGD peptides. The c(RGDyC) peptide (4 mg mL− 1) was then added to the FeMOF suspension and further reacted for 2 h. The RGD-grafted FeMOF NPs were centrifuged and dispersed in ethanol (10 mL) containing free OX (15 µg) and rotary evaporated at 30 °C. The finished product was washed with deionised water and then collected for further analysis and application, and the loading efficiency of OX within FeMOF was determined by high performance liquid chromatography (HPLC).
Characterizations
The morphology and elemental composition of the nanoparticles were observed by transmission electron microscopy (TEM, FEI Tecnai G2 F30). Particle size and zeta potential were measured using a Zetasizer Nano ZS90 laser particle size analyzer. The valence state of Fe, N, C, O were analyzed by X-ray photoelectron spectroscopy (XPS, Thermo scaleb 250Xi). X-ray diffraction (XRD) was performed in Riraku D/MAX-2550 V medium under the influence of Cu-Ka radiation.
Degradation assay of FeMOF NPs in vitro
20 mg of FeMOF was placed into 10 mL of phosphate buffer solution (pH 7.4; pH 6.4, with or without 20 mM GSH) and agitated at 37 °C. At designated time points (0, 2, 4, 8, 12, and 24 h), the mixture was centrifuged, and 1 mL of the supernatant was harvested. The cumulative release of iron ions was quantified utilizing ICP-MS.
Formation of Fibrin Gel
Dissolve thrombin (500 IU/ml) in deionized water or PBS (comprising 26 mg/L NaCl and 7 mg/L CaCl2). Dissolve 50 mg/ml fibrinogen solution in deionized water or PBS and sonicate for 3 min. Then, heat the fibrinogen solution at 37 °C for 30 min to dissolve the required amount of FeOX or LOS in the fibrinogen solution. Mix thrombin and fibrinogen solutions (with or without drug) in a 1:2 volume ratio using a dual syringe to form the fibrin gel in situ.
Characterisation of fibrin gels
Fibrin gels were frozen overnight at -80℃ and then the microstructure of the gels was inspected by cryo-SEM (SU8010). The dynamic rheology of the fibrin gel was measured at 25℃ using an advanced rotational rheometer (HAAKE MARS 60) with a 20 mm cross-etched aluminum plate.
Bio-degradability and bio-compatibility of fibrin gels in vitro and in vivo
For in vitro degradation behaviour, fibrin gel was added to PBS and photo-recorded on days 0, 1, 3, 7, 14, 21 to observe gel degradation. For in vivo bio-degradable and bio-compatible behaviour, fibrin gel was injected into the subcutaneous tissue of the abdominal cavity of BALB/c mice (200 µL per sample) and executed at 0, 1, 3, 7, 14, 21 and 28 day. The bio-degradation of the fibrin gel was photo-recorded, while H&E staining of the surrounding skin was performed to observe the bio-compatibility of the tissue.
Drug release from fibrin gel
In vitro experiments were performed in neutral PBS at 37℃ for drug release assays. Fibrin gels were loaded with LOS, and samples were collected at different time points to evaluate the drug release characteristics of LOS from fibrin gels by UV-Vis spectrophotometry. To assess the in vivo drug release behavior, ICG with or without fibrin gels (free ICG and ICG@Gel) were injected into the subcutaneous tissue of the peritoneal cavity of BALB/c mice. Then monitored and quantified the fluorescence intensity at different time points utilising the IVIS imaging system.
Cellular uptake evaluation of nano formulations
MCF-7/ADR cells were inoculated at a density of 1 × 105 into confocal culture dishes and incubated overnight in medium. The medium was then replaced with fresh medium containing PBS, FITC-labelled FeMOF, FeMOF/OX and FeOX (100 µg mL−) and incubated for another 24 h. The cells will be incubated with Typan blue to quench cytoplasmic fluorescence, then washed 3 times with PBS, fixed in paraformaldehyde for 30 min at 4℃ and washed 3 times with PBS. The nuclei were stained with DAPI for 5 min. The stained cells were washed 3 times with PBS and fixed in glycerol before CLSM observed afterward.
Quantification of intracellular iron levels
MCF-7/ADR cells were inoculated at a density of 1 × 105 onto 6 well plates overnight and the previously added medium was replaced with new medium containing PBS, FeMOF, FeMOF/OX and FeOX (100 µg mL− 1) and incubation continued for 12–24 h. Subsequently, the medium was removed and washed 3 times with PBS. The cells were digested with EDTA-free trypsin, then purified 2 times by repeated centrifugation and finally lysed by adding cell lysis solution and the resulting solution was sonicated 10 min to ensure competitive cell disintegratio. The solutions obtained were used for ICP assays.
Evaluation of ICD expression
MCF-7/ADR cells were inoculated at a density of 1 × 103 in Nunclon Sphera microplate for 48 h to purchase multicellular spherical cells. The formed multicellular spherical cells were co-incubated with PBS, FeMOF, FeMOF/OX and FeOX (100 µg mL− 1) for 12 h. Then cells were washed 3 times with PBS, incubated for 1 h with HMGB1 (CST, Catalog No. 3935, diluted at 1:500), CRT (CST, D3E6, Catalog No. 12238, diluted at 1:1000) monoclonal antibodies, and stained with goat anti-rabbit IgG H&L (Alexa Fluor® 488) or goat anti-rabbit IgG H&L (Alexa Fluor® 555) for another 1 h, DAPI stained for 20 min before CLSM observed afterward.
Cell viability assay
MCF-7/ADR cells were inoculated in 96-well plates at a density of 5 × 103 cells/well and incubated for 12 h to allow complete cell wall attachment. The medium was replaced with fresh medium containing FeMOF, OX, FeMOF/OX, FeOX (0, 25, 50, 75, 100, 150, 200 µg mL− l) and incubated for 24 h before performing the CCK-8 assay.
Live/dead cell staining assay
MCF-7/ADR cells were inoculated at a density of 1 × 105 into confocal culture dishes overnight, then treated with PBS, FeMOF, OX, FeMOF/OX and FeOX (100 µg mL−). After 24 h incubation, the cells were stained with Calcein-AM and PI solutions for 30 min. Finally, the cells were washed three times with PBS and then visualised by CLSM.
Toxicological evaluation in vivo
Female BALB/c mice (4–6 weeks) were divided into two groups and injected with PBS or FeOX/LOS@Gel subcutaneously. On days 10 and 20, serum were collected for blood biochemistry analysis, and major organs were extracted for H&E staining.
Tumor models and treatment trials
To construct a unilateral post-chemotherapy TNBC tumor model, a density 1 × 106 4T1 cells were injected subcutaneously into the fat pad of the right breast of female BALB/c mice (4–6 weeks), tail vein doxorubicin (dose 5 mg kg− 1) was given once a week for 3–4 months. The tumor grew slowly to become larger and then gradually smaller. Thereafter, the extracted tumor was divided into subcutaneous implants of approximately 5 mm in diameter and placed into the fat pad of the right breast of female BALB/c mice (4–6 weeks).
To estimate the difference in tumor stiffness between TNBC and post-chemotherapy TNBC, ultrasound SWE imaging was performed every two days when the TNBC and post-chemotherapy TNBC, tumor volumes reached 100 mm3 and the tumor growth curve was recorded. A region of interest (ROI) of 1 mm diameter was selected from the three stiffest segments during ultrasound SWE to measure the stiffness values and mean values were calculated. The mouse SWE imaging performer was a specialist sonographer with over 5 years of clinical experience. The difference in SWE imaging stiffness was greatest when the tumor reached 800 mm3, the mice were euthanised, the tumor extracted and weighed. A portion of the isolated tumor was excised for AFM evaluation and another portion was taken for RNA sequencing.
To evaluate the sustained release of losartan reduce the solid pressure in post-chemotherapy TNBC mice, post-chemotherapy TNBC mice were randomly divided into control, single free LOS, sequential free LOS and LOS@Gel groups. When the tumor volume reached 50 mm3, each group was injected with PBS, LOS, sequential free LOS (3 times, every 2 days) or LOS@Gel (LOS = 60 mg kg− 1) peritumorally. SWE imaging was performed every two days after treatment, and the tumor size and body weight were measured. When the tumors reached 800 mm3, mice were euthanised, tumors were extracted and weighed. A portion of the isolated tumor was excised for AFM evaluation and another portion was taken for mIHC staining.
To investigated the efficacy of combination therapy in unilateral tumors, post-chemotherapy TNBC were randomly divided into control groups, FeMOF@Gel, LOS@Gel, FeOX@Gel, FeOX/LOS@Gel, and when the tumor volume reached 50 mm3, each group was injected peritumorally with PBS, FeMOF@Gel, LOS@Gel, FeOX@Gel, FeOX/LOS@ Gel (OX = 10 mg kg− 1, LOS = 60 mg kg− 1). Tumor size and body weight were measured every two days after treatment. Mice were euthanised when there were tumors that reached 1000 mm3 in size or showed signs of cachexia.
To investigated the efficacy of combination therapy in bilatera tumors, distal tumors (5 mm tumor masses) were grafted to the left side of female BALB/c mice 5 days after inoculation of the right side tumor (4–6 weeks) and randomly divided into control groups, aPDL1, LOS &FeOX@Gel, αPDL1&LOS &FeOX@Gel, and when the right tumor volume reached 50 mm3, each group was peritumorally injected on the right tumor with PBS, aPDL1, LOS &FeOX@Gel, αPDL1&LOS &FeOX@Gel (aPDL1 = 3.75 mg kg− 1, OX = 10 mg kg− 1, LOS = 60 mg kg− 1). Bilateral tumor size and mouse weight were measured every two days after treatment. Mice were euthanised when there were tumors that reached 1000 mm3 in size or showed signs of cachexia.
Flow cytometry analysis
The tumor tissues were individually digested into suspensions of single cells and were initially incubated at a temperature of 4 °C for a duration of 15 min with anti-CD16/32 (eBioscience, FRC-4G8, Catalog No. MFCR00) monoclonal antibody to prevent any non-specific binding. Subsequently, the cells were stained using diluted fluorescent antibodies that were chromium-coupled. The antibodies utilized in these experiments included CD45-eF506 (eBioscience, 30-F11, Catalog No. 69-0451-82), CD4-FITC (eBioscience, GK1.5, Catalog No. 1-0041-82), CD8-Percpcy5.5 (eBioscience, 53 − 6.7, the Catalog No. 45-0081-82), Foxp3-PE (eBioscience, N418, Catalog No. 12-0114-82). Additional reagents, such as the erythrocyte lysis buffer (Catalog No. 0-4300-54), intracellular fixation/perm buffer set (Catalog No. 88824-00), and Foxp3 Transcription Factor Staining Buffer Set (Catalog No. 0-552300), were obtained from Thermo. For each experiment, the antibodies were appropriately diluted to achieve a working concentration of 0.2 µg. Finally, the stained cells were filtered and subjected to detection using FACS FCM (BD, Fortessa X20). The resulting data were then analyzed using Flowjo software (TreeStar, 10.6.2).
Immunohistochemistry (IHC)
To estimate the difference in tumor stiffness between TNBC and PC-TNBC, when the tumor reached 800 mm3, the mice were euthanised, the tumor extracted and a portion of the isolated tumor was excised for IHC staining. The tumor tissue samples were immersed in a solution of 4% (v/v) paraformaldehyde in PBS for a duration of 10 min at room temperature to ensure fixation. After overnight incubation with primary antibodies α-SMA-Cy3™ (Sigma-Aldrich, Catalog No. C6198, diluted at 1:200), collagen-Ι (abcam; Catalog No. ab270993, diluted at 1:500) at 4 °C, the sections were exposed to 0.3% H2O2 in TBS for 15 min at room temperature. And then the sections were incubated with secondary antibodies diluted 1:500 in TBS containing 10% FBS for 1 h at room temperature. Sections were then incubated with 3,3’-diaminobenzidine (DAB) to generate signal and counterstained with hematoxylin. After dehydration and mounting, tissue sections were scanned using a Leica SCN400 slide scanner.
Immunofluorescence staining
Tumors were isolated and collected from mice, and tumor sections were stained according to the instructions of the Fluorescent Immunohistochemical Staining Kit (Absin, Catalog No.abs50013). Antibodies involved in the experiments included α-SMA-Cy3™ (Sigma-Aldrich, Catalog No. C6198, diluted at 1:200), collagen-Ι (abcam; Catalog No. ab270993, diluted at 1:500), CD31 (abcam, Catalog No.ab28364, diluted at 1:100), HMGB1 (CST, Catalog No. 3935, diluted at 1:100), calreticulin (CST, D3E6, Catalog No. 12238, diluted at 1:500), Ki67 (abcam; Catalog No. ab15580, diluted at 1:5000), Foxp3 (abcam, Catalog No. ab215206, diluted at 1:200) CD8 (abcam, Catalog No. ab251596, diluted at 1:5000). Cell nuclei were stained with DAPI prior to sealing and all sections were scanned with a fluorescence scanning camera (KFBIO, KF-TB-400).
Cytokine assay
Mouse serum samples were collected and isolated. Serum TNF-α (Invitrogen, catalog number BMS607-3) and IFN-γ (Invitrogen, catalog number BMS607-3) were analyzed using an ELISA kit and according to the supplier’s protocol.
Clinical samples
84 patients with TNBC were recruited from the Shanghai Tenth People’s Hospital in Shanghai, and SWE imaging and pathological data were collected from January 2018 to December 2021 for patients before treatment or after surgical resection. This retrospective study was conducted with the informed consent of the patients and approved by the Ethics Commission of the Shanghai Tenth People’s Hospital (ChiCTR2000035381).
Statistical analysis
The quantitative data were expressed as mean ± SD. Statistical significance was calculated by GraphPad Prism (version 8.0.2) using unpaired student’s t-test or one-way analysis of variance (ANOVA) when comparing two or multiple groups, respectively. The survival curve was analyzed by the Log-rank test. All flow cytometry data were analyzed using the FlowJo™ software package (version 10.5.2). P values less than 0.05 were considered significant, *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001.
