Materials
Triblock poly (ethylene oxide)-bpoly(propylene oxide)-b-poly(ethylene oxide) Pluronic F127 (EO106PO70EO106, Mav = 12,600) and dopamine hydrochloride (C8H11NO2·HCl, DA·HCl) were brought from Sigma-Aldrich. Ethanol (C2H6O) was purchased from Sinopharm Chemical Reagent Co., Ltd. 1,3,5-trimethylbenzene (C9H12), Calcium chloride, hydrogen peroxide (H2O2, 30 wt% solution in water), ammonia water (25–28%), N-2-hydroxyethyl piperazine-N′-2-ethanesulfonic acid (HEPES), Hyaluronic acid sodium salt from Streptococcus equi, Rhodamine B (RhB) and dichloride [Ru(dpp)3]Cl2 were purchased from Aladdin (Shanghai, China). The lactate assay kit was brought from Nanjing Jiancheng Bioengineering Institute. Cell Counting Kit-8 (CCK-8), the Annexin V-FITC Apoptosis Detection Kit, and the Calcium Colorimetric Assay Kit were obtained from Beyotime Biotechnology Co., Ltd. Anti-CD16/32, anti-CD45-PE, anti-CD11b-APC, anti-CD11c-APC anti-F4/80-PE/Cy7, anti-CD80-FITC, anti-CD206-PE, anti-CD86-PE, anti-MHCII-V450, anti-CD3-APC, anti-CD4-FITC, and anti-CD8-PE/Cy7 were purchased from BioLegend. ELISA kits were purchased from Dakewei Biotech Co., Ltd.
All chemicals were of analytical grade and used without further purification. Deionized water (18.2 MΩ·cm resistivity at 25 °C) was used for all experiments.
Cells and animals
The murine 4T1 breast cancer cells were cultured in DMEM containing 10% FBS and 1% penicillin–streptomycin and were incubated at 37 °C under a humidified atmosphere containing 5% CO2.
BALB/c mice (female, 5–6 weeks old) were ordered from the Shanghai Laboratory Animal Center (SLAC, Shanghai, China) and bred in a sterile, specific pathogen-free (SPF) laboratory at Tongji University. The experimental research was carried out in accordance with relevant guidelines and regulations and all animal procedures in compliance with the guidelines of the Institutional Animal Care and Use Committee of Tongji University.
Synthesis of mPDA
The synthesis of mPDA was carried out according to our previous work [28], firstly, 500 mg DA and 1000 mg Pluronic F127 were dissolved in 100 mL 50% ethanol under stirring. Then, 2 mL of TMB was added drop by drop into the solution. After 30 min of stirring, 5 mL of ammonia solution was introduced slowly with continuous stirring for 2 h at room temperature. Then the products were purified by centrifugation, washed at least three times with 50% ethanol to remove the template F127. The final separated products were suspended in water for further use.
Synthesis and characterization of CaO2@mPDA-SH, Ca(OH)2@mPDA-SH and CaO2@mPDA
0.2 mg mPDA was added in a glass vial, then 500 μL 0.5 mM CaCl2 aqueous solutionand 125 μL 0.5 mg/mL sodium hyaluronate solution were added during stirring, later 100 μL 28–30% ammonia was added and stirred, 360 μL 30% H2O2 was finally added dropwise for 30 min. The reaction mixture was subjected to centrifuge at 13,000 rpm for 15 minto discard the supernatant, the residual was then resuspended with HEPES buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) and washed twice, thus obtained CaO2@mPDA-SH dispersion in HEPES buffer. To obtain unmodified CaO2@mPDA NPs, the sodium hyaluronate solution was just replaced by deionized water in the above procedure. And the preparation of Ca(OH)2@mPDA-SH was carried out according to the above steps, without additional injection of hydrogen peroxide.
The morphology of the formed CaO2@mPDA-SH, mPDA was characterized by a transmission electron microscope (TEM, JEM-1230). The size and zeta potential were determined by dynamic light scattering (DLS, ZS90, Malvern) at 25 °C.
Stability
The stability of CaO2@mPDA-SH was evaluated by monitoring the size distribution in HEPES buffer for 7 days. Size variations were measured by DLS at 2, 4, 8, 12, 24 hour 2, 3, 4, 5, 6, 7 days, respectively.
Hemolysis evaluation of CaO2@ mPDA-SH
The hemolysis ratio of CaO2@ mPDA-SH was estimated on the 5% red blood cells (RBCs). Nanoparticles were dispersed with PBS (pH 7.4) into CaO2@mPDA-SH solutions (500 μL) of concentration from 25 μg mL−1 to 1000 μg mL−1, and then mixed with RBC suspension (500 μL). PBS (500 μL) and water (500 μL) mixed with equal volume of RBC suspension to act as the negative and positive controls, respectively. The above samples were kept at 37 °C for 3 h with subsequent centrifugation (3000 rpm, 1 min). The absorbance of the supernatants in different groups was measured at 540 nm by UV–visible spectroscopy (Varian, Ltd., Hong Kong). And hemolysis ratio was calculated according to the following equation (A denotes the absorbance of different groups):
$${\text{Hemolysis}}\;{\text{ratio}}\;(\% ) \, = \frac{{{\text{A}}^{{{\text{sample}}}} - {\text{A}}^{{{\text{negative}}}} }}{{{\text{A}}^{{{\text{positive}}}} - {\text{A}}^{{{\text{negative}}}} }} \times 100\%$$
In vitro cytotoxicity of CaO2@mPDA-SH
The murine 4T1 breast cancer cells were inoculated on 96-well plates (1 × 104 cells per well) with cell culture medium DMEM for 24 h. Afterward, the DMEM was replaced by samples (CaO2@mPDA-SH, mPDA) dispersion in DMEM with different concentration gradients (0, 10, 20, 50, 100, 200, and 400 μg mL−1, respectively), after 24 h incubation, the medium was discarded and CCK-8 test was performed to assess the cell viability.
Cellular uptake
Firstly, mPDA nanoparticles were labeled with RhB. Specifically, mPDA (3.0 mg) and RhB with mass ratio is of 10:1 were mixed in sodium bicarbonate solution (0.1 M) and stirred at 300 rpm in the dark. After 12 h, the RhB-labeled nanoparticles were centrifuged (13,500 rpm, 15 min) and washed with deionized water for at least three times to obtain RhB-mPDA. RhB-labeled CaO2@mPDA-SH nanosystems were also obtained via the above similar method. For the evaluation of cellular uptake, 4T1 cells were seeded in 24-well microplates (1 × 105 cells per well). After 24 h incubation, the cells were incubated with fresh medium composed of RhB-mPDA (100 µg mL−1) for 1, 2, 4, 5, and 7 h, respectively. In addition, cytochalasin D (10 × 10–6 M) was used for investigating the cell phagocytosis. After washing with PBS for 3 times, the RhB fluorescence was determined by flow cytometry (FACSVerse, BD).
Cell apoptosis assay
4T1 cells were incubated in a 6-well plate (1 × 106 cells well−1) for 24 h and treated with samples (CaO2@mPDA-SH, mPDA) of different concentration gradients (0, 10, 20, 50, 100, 200, and 400 μg mL−1, respectively). After 12 h cultivation, the culture medium was swilled with PBS several times and cells were digested by trypsin without EDTA. Cells were then subjected to flow cytometry to detect apoptosis cells stained with annexin V-FITC/PI.
In vitro cellular uptake of CaO2@mPDA-SH
The cellular uptake behavior of CaO2@mPDA-SH in 4T1 cells was quantitatively analyzed by flow cytometry (Guava easyCyte) and visualized by fluorescent microscopy (Lionheart FX automated live cell imager, BioTek). Briefly, the 4T1 cells were seeded in a 24-well plate (2 × 105 cells·well−1) for 24 h and incubated with different samples (CaO2@mPDA-SH and mPDA) for 1, 2, and 4 h. The 4T1 cells were collected and quantified by flow cytometry. Besides, the cells incubated with the CaO2@mPDA-SH were fixed with 4% paraformaldehyde solution and stained with DAPI for fluorescence microscope observation.
In vitro lactic acid reduction
4T1 cells were seeded into the 48-well plate (2 × 104 cells·well−1) for 24 h. Then, the cell supernatant was displaced with the fresh culture medium containing different formulations of mPDA, CaO2@mPDA, Ca(OH)2@mPDA-SH, and CaO2@mPDA-SH nanoparticles. After 4 h, cell supernatant samples of cells with different treatments were collected, and lactate content was detected using the Lactic Acid Assay Kit.
In vitro hypoxia improvement
1 × 106 4T1 cells were seeded in a 6-well plate and incubated overnight and then treated with mPDA or CaO2@mPDA-SH for 4 h, respectively. After different treatments, cells were stained with DAPI and [Ru(dpp)3]Cl2 for visualizing the nuclei and hypoxia under a fluorescence microscope.
In vivo imaging
The tumor-bearing mice were injected with Ce6-labeled nanoparticles (CaO2@mPDA-SH-Ce6) via the tail vein (5 mg/kg). The biodistribution of nanoparticles was monitored using an IVIS imaging system (Caliper PerkinElmer, Hopkinton, USA) at the predetermined time points. At the experiment endpoint, the mice were sacrificed, and the tumor and organs were harvested for ex vivo imaging. The pharmacokinetics study was also executed. CaO2@mPDA-SH-Ce6 were injected by tail vein injection with the same dose for each mice. The mice were anesthetized and 0.1 mL of blood was collected by retro-orbital bleeding at predetermined time points. The plasma was obtained by centrifugation (5000 rpm, 10 min) and the Ce6 concentration was measured by a fluorescence spectrophotometer (F-4600, HITA-CHI, Japan).
In vivo antitumor efficacy and immune response evaluation
The orthotopic breast cancer mouse model was established first. 4T1 cells (2 × 106 cells per mouse) were implanted into the breast region of female BALB/c mice. When the tumor volume reached 50 mm3, tumor-bearing mice were randomly divided into 5 groups (5 mice in each group). In the following 15-day observation period, each group was i.v. injection one of the followings of 100 µL on the 1st, 3rd, 5th day, respectively: (i) saline; (ii) mPDA (50 mg/kg, dispersed in HEPES buffer); (iii) CaO2@mPDA NPs (50 mg/kg, dispersed in HEPES buffer); (iv) Ca(OH)2@mPDA-SH NPs (50 mg/kg, dispersed in HEPES buffer); (v) CaO2@mPDA-SH NPs (50 mg/kg, dispersed in HEPES buffer). Body weight and tumor volume were measured every 2 days. And the tumor volume was calculated according to the equation of V = W·S2/2 (where W and S represented the longest diameter and the shortest diameter, respectively). Tumor inhibition ratio was defined as (VPBS − VT)/VPBS (where Vt represented the final tumor volume of other treatment group.
At the end of the treatment, all mice were sacrificed. Tumors, draining lymph nodes (DLNs) and spleen were collected for flow cytometry and immunofluorescence staining. Tumor tissue was mainly used to analyze the expression level of T cell infiltration (CD3+CD8+) and tumor-associated macrophage (M1 and M2), while the lymph node was mainly utilized to analyze DC maturation and spleen was used to analyze the relative abundance of CD8+ and CD4+ T cell subsets. Simultaneously, the main organs (heart, liver, spleen, lung and kidney) were collected for hematoxylin and eosin (H&E) analysis.
Intratumor lactic acid content test
100 mg of tumor samples, collected 15 days after treatment, were homogenized in PBS. Following the centrifugation of tissue homogenates, the supernatant of tumor tissues was collected and detected via the Lactic Acid assay Kit.
In vivo anti-metastasis and anti-angiogenesis efficacy
4T1 cells (2 × 106 cells per mouse) were implanted into the breast region of female BALB/c mice. When the tumor volume reached 50 mm3, tumor-bearing mice were randomly divided into the same 5 groups (5 mice in each group), each group was administered nanoparticles intravenously on the 1st, 4th, 7th, 11th day, respectively. The dosages were consistent with the above. After the therapy for 28 days, all the mice suffered mercy killing. The tumors were harvested, Paraffin-embedded, deparaffinized and hydrated followed by antigen retrieval for immunohistochemistry staining against CD31 and VEGF. The lung was also harvested, fixed in 4% paraformaldehyde, sectioned, and stained with H&E for evaluating the anti-metastasis efficacy.
Statistical analysis
All data in the present study were presented as mean ± S.D. All animal studies were performed after randomization. Using unpaired student’s t-test to appraise statistically significant discrepancies between two groups. One-way analysis of variance with Bonferroni tests for multiple group comparison. Significant differences were indicated by *P < 0.05, **P < 0.01, and ***P < 0.001.