Materials. Acrylonitrile (AN) was purchased from Qinghongfu Technology Co., Ltd. (Beijing, China) and purified by atmospheric distillation before use. Acrylamide (AAm), 4,4'-azobis (4-cyanovaleric acid) (ACVA), dimethyl sulfoxide (DMSO) and azelaic acid were provided by Aladdin (Shanghai, China). The amino polyethylene glycol amine (H2N-PEG-NH2) (Mw=5kDa) was purchased from ToYongBio Tech.Inc. (Shanghai, China). Nα,Nα-Bis (carboxymethyl)-L-lysine (NTA) was obtained from Energy Chemical (Shanghai, China). Doxorubicin hydrochloride and indocyanine green (ICG) were brought from Meilun Biotechnology Co., Ltd. (Dalian, China). SCH 58261 was purchased from TCI (Tokyo, Japan). Nile red was obtained from Aladdin (Shanghai, China). Recombinant mouse E-selectin Fc chimera (ES) was from R&D Systems (Minneapolis, USA). 5′-(N-ethylcarboxamido)adenosine (NECA) was bought from ApexBio Technology LLC (Houston, USA). RPMI 1640 medium and fetal bovine serum (FBS) obtained from Sigma (St. Louis, MO, USA) and Sijiqing Biological Engineering Materials Co. Ltd. (Hangzhou, China), respectively. The ELISA kits were all purchased from Meimian industrial Co., Ltd. (Jiangsu, China).
Cell culture and animals. The murine 4T1 breast cancer cells and Luc-4T1 (luciferase-expressing mouse breast carcinoma) cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS and penicillin/streptomycin (100 U/mL of each) and maintained in the cell incubator (37℃ and 5% CO2). The cells were regularly split using trypsin/EDTA. For the hyperthermia treated groups, the cells were placed in the cell incubator (43℃ and 5% CO2, 30min) immediately after adding the test agents, followed by incubation at 37℃ for pre-set time period.
Balb/c mice (female, 6 to 8 weeks old, 18-20 g) were purchased from Slack Laboratory Animal Co., Ltd (Shanghai, China). All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals with the approval of the Scientific Investigation Board of Zhejiang University, Hangzhou, China.
Synthesis and characterization of NTA-PEG-p-(AAm-co-AN). Firstly, p-(AAm-co-AN) with a UCST of 43℃ was synthesized by solution copolymerization of AN and AAm initiated by ACVA. Briefly, 10.95g (150mmol) of AAm was weighed into a 500-mL three-necked flask and dissolved in 170mL of anhydrous DMSO. Subsequently, 2.55g (50mmol) of AN was added. Nitrogen was pumped for 1 h to remove the oxygen from the system. After that, 30mL of separately degassed anhydrous DMSO containing 0.519g (1.853mmol) of ACVA was dropped into the system through a constant pressure dropping funnel. Then placed the flask into a water bath which had been preheated to 65°C. The reaction mixture was subsequently polymerized for 8h under nitrogen protection and rapidly cooled to room temperature in an ice bath. The product was precipitated in 10-fold excess volume of methanol. The precipitate was then washed thrice with methanol and dried in a vacuum oven at 70°C for 24h.
Next the H2N-PEG-NH2 was introduced to p-(AAm-co-AN) through the chemical reaction between one of the amine groups in H2N-PEG-NH2 and the carboxyl groups of p-(AAm-co-AN). Briefly, 500mg (0.1mmol) of p-(AAm-co-AN) was weighed into a 50-mL flask and dissolved in 10mL of DMSO, to which 95mg (0.5mmol) of EDC and 57mg (0.5mmol) of NHS was added and stirred at room temperature for 4h. Subsequently, the mixture solution was added dropwise to 10mL DMSO containing 500mg (0.2mmol) of H2N-PEG-NH2 (Mw=5kDa) at 50°C. The reaction mixture was stirred for 48h and then dialysis against deionized water with a dialysis membrane (MWCO: 8~14kDa) for 48h, followed by lyophilization and the PEG-p-(AAm-co-AN) was obtained.
Then the NTA was grafted onto PEG-p-(AAm-co-AN) with azelaic acid as the linker. Briefly, 19mg (100μmol) of azelaic acid was dissolved in 10mL of DMSO, to which 20mg (100μmol) of EDC and 11.5mg (100μmol) of NHS was added and stirred at room temperature for 10h to activate one of the carboxyl groups of azelaic acid. Subsequently, 500mg (33.5μmol) of PEG-p-(AAm-co-AN) was dissolved in 10mL of DMSO and added dropwise into above mixture solution, 67μmol of triethylamine was also supplemented. The reaction mixture was stirred for 17h at room temperature and then dialysis against deionized water with a dialysis membrane (MWCO: 3.5kDa) for 48h, followed by lyophilization to afford the carboxyl-containing PEG-p-(AAm-co-AN). Next, 420mg (28μmol) of carboxyl-containing PEG-p-(AAm-co-AN) was dissolved in 10mL of DMSO, 54mg (280μmol) of EDC and 32.5mg (280μmol) of NHS was added and stirred at room temperature for 4h. Then 147mg (560μmol) of NTA and 1.12mmol of triethylamine were dissolved in 10mL of DMSO/H2O mixed solution (DMSO:H2O=3:2), added dropwise into above solution and reacted at room temperature for 24h. After dialysis against deionized water with a dialysis membrane (MWCO: 3.5kDa) for 48h and lyophilization, the final product NTA-PEG-p-(AAm-co-AN) was afforded.
The 1H-NMR spectra of the polymers were obtained using an NMR spectrometer (AC-80, BrukerBioSpin, Germany). p-(AAm-co-AN), PEG, PEG-p-(AAm-co-AN) and NTA-PEG-p-(AAm-co-AN) were dissolved in DMSO-d6 at concentrations of 20mg/mL. The molecular weights of p-(AAm-co-AN) and PEG-p-(AAm-co-AN) were analyzed using gel permeation chromatography (GPC) with DMSO as an eluent. PLgel MIXED-C columns (particle size: 5mm; dimensions: 7.5mm × 300mm) that had been calibrated with narrow dextran monodisperse standards were employed with a differential refractive index detector. The flow rate was 0.6mL/min. Dispersed the polymers in water at a concentration of 2mg/mL to facilitate the determination of UCST value, the optical transmittance of polymer solutions at different temperature was measured at 637nm using an ultraviolet-visible spectrophotometer (UV-2401, Shimadzu, Japan). The UCST value of p-(AAm-co-AN) was determined at the temperature when the optical transmittance became constant. The critical micelle concentration (CMC) of NTA-PEG-p-(AAm-co-AN) was determined using fluorescence spectroscopy and pyrene as a probe. Pyrene was first dissolved in acetone at a concentration of 0.0012mg/mL and added into tubes. Following evaporation of the acetone at 50°C, 5 mL of polymer solutions at different concentrations ranging from 2 to 1000μg/mL were added. After the solution was treated with water bath ultrasonication for 30 min, the emission spectra were recorded on a fluorescence spectrophotometer (F-2500, Hitachi High-Technologies Co., Japan) at room temperature. The excitation wavelength was 336 nm, and the slit widths were set at 10 nm (excitation) and 2.5 nm (emission). The pyrene emission was monitored over a wavelength range of 360-450 nm. From the pyrene emission spectra, the intensity ratio of the first peak (I1, 374 nm) to the third peak (I3, 384 nm) was analysed and used to calculate the CMC.
Thermal sensitivity of blank micelles. The NTA-PEG-p-(AAm-co-AN) was dispersed in water at a concentration of 0.5mg/mL, followed by 30 rounds of probe-type ultrasonic treatment (pulsed every 2s for a 3s duration, 400W). After stirring at 25°C for 0.5h, the blank micelles solution was obtained. The blank micelles solution was quartered and incubated at different temperature (25, 37, 43, 50°C) for 0.5h, dropped onto the preheated copper grids and dry at corresponding temperature. Subsequently, the morphologies of blank micelles at different temperature were observed by TEM.
Preparation and characterization of E-selectin modified DOX/SCH co-loaded micelles (ES-DSM). The DOX used in the preparation of drug-loaded micelles was obtained by the reaction between DOX·HCl and two molar equivalents of triethylamine in DMSO for 24 h. Dialysis against water to precipitate the insoluble DOX, followed by centrifuging and lyophilizing to obtain DOX powder for further use. 20mg of NTA-PEG-p-(AAm-co-AN) was dispersed in 3mL of water and treated by probe ultrasound for 30 rounds, stirring at 25°C for 0.5h to form the stable blank micelles. DOX and SCH 58261 (SCH) were dissolved together in DMSO at the final concentrations of 0.8mg/mL and 0.2mg/mL, respectively. Then 1mL of DMSO solution of DOX/SCH was added dropwise to the micelles solution with constant stirring (DOX: SCH: polymer= 4:1:100). Subsequently, 3mg of NiCl2·H2O was added and the mixture was stirred at 25°C for anther 2h, followed by dialyzing against water (MWCO: 3.5 kDa) for 24h and centrifuging at 4000rpm for 10min to eliminate aggregates of non-encapsulated DOX/SCH. Ultimately, the solution of DOX/SCH co-loaded micelles (DSM) was lyophilized and stored at 4°C. E-selectin could be introduced onto the surface of DSM between the interaction of His-tag of E-selectin and Ni-NTA of polymer. Briefly, different concentrations of E-selectin (0, 0.1, 0.2, 0.5, 1, 2, 3μg/mL) were added to the DSM solution (at a polymer concentration of 1mg/mL) respectively, incubated at 37°C for 1h and further in 4°C overnight to afford the E-selectin modified DSM (ES-DSM). The preparation of DOX loaded micelles (DM and ES-DM) were the same as above, except the absence of SCH. The particle sizes and zeta potentials of DSM and ES-DSM were recorded by dynamic light scattering (DLS) (Zetasizer, 3000HS, 66 Malvern Instruments Ltd.). The morphology of ES-DSM was observed by transmission electron microscopy (TEM) (JEOL JEM-1230, Japan). The encapsulation efficiency (EE) and drug loading (DL) were determined by fluorospectro photometer (DOX: Ex=480nm, Em=560nm, Slit width=5nm; SCH: Ex=320nm, Em=385nm, Slit width=5nm). Briefly, the drug-loaded micelles were disrupted by DMSO and the total DOX and SCH contents were quantified. EE% and DL% were calculated by the following formulas:
Thermal-triggered size changes of micelles. The size changes of micelles in response to temperature were monitored by DLS. The sizes of blank micelles, DSM and ES-DSM in different temperatures (5, 25, 25, 37, 43, 50°C) were measured. The samples (at a polymer concentration of 1mg/mL) were incubated at the corresponding temperature for 5 minutes before measurement. There are three repeat groups for each sample.
Thermal-sensitive in vitro drug release behavior of ES-DSM. The DOX and SCH release profiles of ES-DSM in different temperatures were tested by dialysis method. The dialysis bags (MWCO: 3.5 kDa) containing 1mL of free DOX and SCH (DS), and ES-DSM (concentrations of DOX and SCH were 90μg/mL and 15μg/mL, respectively) were immersed into falcon tubes containing 30mL PBS (pH 7.4).The tubes were put into incubator shakers (37℃ and 43℃, respectively) and horizontally shaken at 60rpm/min. At each pre-set time point, the release media were collected and replaced with fresh PBS. The DOX and SCH contents in the release media were detected by fluorospectro photometer. Each time point was performed trice.
Leukocyte-adhering ability of ES-DSM. 200μL of DSM or ES-DSM (concentrations of DOX and SCH were 300μg/mL and 50μg/mL, respectively) was injected into the mice via the tail vein, and at 2, 8 and 24h after injection, the leukocytes of treated mice were isolated. The DOX fluorescence on the obtained leukocytes was analyzed by flow cytometry (ACEA NovoCyte, USA) and confocal laser scanning microscope (CLSM) (Leica SP8, Germany).
Thermal-sensitive drug release behavior of micelles at cellular level. Firstly, Nile red was loaded into the micelles. The preparation of Nile red-loaded micelles was the same as DSM, excepted the model drug used was Nile red instead of DOX/SCH. 4T1 cells were suspended in RPMI 1640 medium and seeded in 12-well plate at a density of 1×105 cells per well and allowed to attach overnight. Subsequently, the cells were treated with free Nile red or Nile red-loaded micelles (at a final Nile red concentration of 0.1μg/mL) and the hyperthermia treated groups were placed in the cell incubator (43℃ and 5% CO2, 30min) immediately, followed by incubation at 37℃ for 6h. After washed trice with PBS, the cells were harvested and fluorescence intensity was detected by flow cytometry. Besides, the cell fluorescence was also observed by CLSM. After incubation and washed trice with PBS, the cells were fixed and the nuclei were stained by DAPI, followed by CLSM observation.
Then, DOX was loaded into the micelles. The free DOX and DOX-loaded micelles were added to 4T1 cells at a final DOX concentration of 4.5μg/mL. After treated with hyperthermia and 6h incubation, the cells were washed trice with PBS and fixed. After staining by DAPI, the cells were observed by CLSM.
Cytotoxicity and apoptosis. Firstly, the cytotoxicity of blank micelles was measured by MTT assay. 4T1 cells were suspended in RPMI 1640 medium and seeded in 96-well plate at a density of 1×104 cells per well and allowed to attach overnight. Then the cells were exposed to blank micelles at a series of concentrations (0, 100, 200, 400, 600, 800, 1000μg/mL) for 48 hours. The hyperthermia treated groups were placed in the 43℃ cell incubator for 30min, followed by incubation at 37℃ until 48h. Subsequently, 20μL of 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (5mg/mL) was added to each well for an additional 4 hours incubation at 37℃. After that, the medium was replaced with 100μL of DMSO to dissolve the purple formazan crystals in the bottom of the well. The plate was shaken for 30min, and the absorbance of the solution in each well was measured by microplate reader at 570nm. Cell viability was calculated in reference to negative cells without exposure to test agents. All of experiments were repeated thrice.
Subsequently, the cytotoxicity of free DOX/SCH (DS), DSM and ES-DSM combined with or without hyperthermia were determined by MTT assay. 4T1 cells were suspended in RPMI 1640 medium and seeded in 96-well plate at a density of 1×104 cells per well and allowed to attach overnight. Then the cells were exposed to DS, DSM or ES-DSM at different drug concentrations for 48hours (the concentration ratio of DOX and SCH is 6:1). The hyperthermia treated groups were immediately placed in the cell incubator which had pre-set to 43℃ for 30min after exposing to the test agents, followed by incubation at 37℃ until 48h. Cell viability was measured as described above.
Cell apoptosis induced by DS, DSM and ES-DSM combined with or without hyperthermia were investigated by flow cytometry. 4T1 cells were suspended in RPMI 1640 medium and seeded in 12-well plate at a density of 1×105 cells per well and allowed to attach overnight. Subsequently, the cells were exposed to DS, DSM or ES-DSM (concentrations of DOX and SCH were 4.5μg/mL and 0.75μg/mL, respectively) and treated with or without hyperthermia. After a 24h-incubation, cells were harvested and stained by the Annexin V-FITC/PI apoptosis detection kit (Beyotime Biotech, China) according to the manufacturer’s instructions, followed by flow cytometer analysis.
Detection of the ICD biomarkers. The exposure of DAMPs (CRT, HMGB1 and ATP) of tumor cells after different treatment were detected. Briefly, 4T1 cells were treated with DS, DSM or ES-DSM (concentrations of DOX and SCH were 4.5μg/mL and 0.75μg/mL, respectively) with or without hyperthermia. The expression of CRT was observed by their immunofluorescence via CLSM at the time of 12h (Calreticulin Rabbit Monoclonal Antibody, 1:500, Beyotime, China). Semi-quantitative analysis was performed using Image J software. After incubating for 48h, the cell culture supernatant was collected and the contents of ATP and HMGB1 were detected by corresponding ELISA kits.
Co-incubation of tumor cells and bone-marrow-derived DCs. The murine bone-marrow-derived DCs (SMDCs) were isolated from 6-week old Balb/c female mice according to the established protocols.1, 2 Briefly, the bone marrow of mice was collected via flushing the femurs and tibias with PBS, and red blood cells were lysed. The remaining cells were washed twice with PBS and cultured in the complete RPMI 1640 medium containing recombinant murine GM-CSF (20ng/mL) (MedChemExpress, USA) for 6 days to acquire the immature DCs. On day 7, the immature DCs were co-incubated with 4T1 cells which had been previously treated with PBS, DS, DSM or ES-DSM (concentrations of DOX and SCH were 4.5μg/mL and 0.75μg/mL, respectively) (supplemented with or without hyperthermia) 24h ago. After a 48h-co-incubation, DCs were stained with the indicated antibodies including PE-CD80, APC-CD86 (BioLegend, USA) and PE-MHC Ⅱ (ThermoFisher, USA), analyzed by flow cytometry. In addition, the cytokine levels in the supernatant of the co-incubation system including IL-12p70, IL-6 and IL-10 were detected using ELISA kits.
Besides, the immature DCs were co-incubated with 4T1 cells which had been previously treated with D (DOX alone), DS, DM, DSM, ES-DM or ES-DSM (concentrations of DOX and SCH were 4.5μg/mL and 0.75μg/mL, respectively) and supplemented with hyperthermia 24h ago. After a 48h-co-incubation with the presence of 1μM (a dose that mimics the concentration of adenosine found in the tumor microenvironment) of NECA (adenosine analog),3 DCs were stained with the indicated antibodies including PE-CD80, APC-CD86 (BioLegend, USA) and PE-MHC Ⅱ (ThermoFisher, USA), analyzed by flow cytometry. In addition, the cytokine levels in the supernatant of the co-incubation system including IL-12p70, IL-6 and IL-10 were detected using ELISA kits.
Co-incubation of tumor cells, bone-marrow-derived DCs and spleen lymphocytes. Spleen lymphocytes were extracted from the spleens of Balb/c mice using lymphocyte density gradient centrifugation with Ficoll-paque PREMIUM. The immature DCs and lymphocytes were co-incubated with 4T1 cells which had been previously treated with PBS, DS, DSM or ES-DSM (concentrations of DOX and SCH were 4.5μg/mL and 0.75μg/mL, respectively) (supplemented with or without hyperthermia) 24h ago. After a 48h-co-incubation, lymphocytes were stained with the indicated antibodies including FITC-CD3, APC-CD8, PE-CD4 and Percific Blue-Foxp3 (BioLegend, USA), analyzed by flow cytometry. In addition, the cytokine levels in the supernatant of the co-incubation system including TNF-α, IL-2 and IFN-γ were detected using ELISA kits.
Besides, the immature DCs and lymphocytes were co-incubated with 4T1 cells which had been previously treated with D, DS, DM, DSM, ES-DM or ES-DSM (concentrations of DOX and SCH were 4.5μg/mL and 0.75μg/mL, respectively) and supplemented with hyperthermia 24h ago. After a 48h-co-incubation with the presence of 1μM of NECA, lymphocytes were stained with the indicated antibodies including FITC-CD3, APC-CD8, PE-CD4 and Percific Blue-Foxp3 (BioLegend, USA), analyzed by flow cytometry. In addition, the cytokine levels in the supernatant of the co-incubation system including TNF-α, IL-2 and IFN-γ were detected using ELISA kits.
Biodistribution of DSM and ES-DSM. The orthotopic tumor models were established by subcutaneous injection of 4T1 cells (5×105) dispersed in serum-free RPMI 1640 medium into the third breast pad of Balb/c mice. Treatment began when the tumor volume reached 500 mm3. For the observation and imaging of the micelles biodistribution, ICG-loaded micelles were prepared the same as DSM, and the modification of E-selectin was the same as ES-DSM. 200μL of ICG-loaded micelles or ES-ICG-loaded micelles was injected into the mice via the tail vein and at 2, 6, 12, 24h after injection, the treated mice were anesthetized and the fluorescence images were acquired by Maestro in vivo imaging system. 24h after injection, the mice were sacrificed to harvest the main organs (heart, liver, spleen, lung, kidneys, and tumor). Fluorescence images were acquired, and the fluorescence intensity of these organs was measured ex vivo using an in vivo imaging system. The fluorescence of ICG and CD45 in tumors were analyzed by immunofluorescence.
In vivo antitumor study. 5×105 of 4T1 cells were orthotopically injected into one of the breast pads of Balb/c mice. After one week, the mice were randomly sorted into 8 groups (6 mice per group) to respectively receive one of the following treatments once every 3 days: Saline, Saline+MW, DS+MW, DSM+MW, ES-DSM, ES-DM+MW, ES-DSM+MW, ES-DSM+MW+anti-CD8, for 4 times of treatment. 3mg/kg DOX and 0.5mg/kg SCH per dose was used in the treatment and at 24h post-i.v. injection of the test agents, the mild microwave (MW) was applied locally for 30min. The microwave probe was positioned 1cm away from the fixed animal and oriented towards the tumor. The anti-CD8 antibody (BioXcell, USA) was intraperitoneal (i.p.) injected to deplete the CD8+ T cells on the days of -3 and treated every 3 days until the end of monitoring. The body weight and tumor volume were monitored every 2 days and the survival time was monitored. The tumor volume was calculated using the formula: a2×b/2, in which a and b represent the smallest and largest diameters of the corresponding tumor, respectively.
At the end of monitoring on day 23, the mice were sacrificed and main organs (heart, liver, spleen, lung, kidney, and tumor) were harvested and fixed in 4% paraformaldehyde, embedded in paraffin, cut into 5μm slices and stained with H&E, then examined under a light microscope. The apoptosis of tumor tissue also be studied by immunofluorescence of TUNEL staining. To demonstrate the ICD of tumor tissues, CRT and HMGB1 levels were studied by immunohistochemistry. To examine the immune response, the infiltration of CD8+ T cells and Tregs (Foxp3) in tumors were analyzed by immunofluorescence, while the infiltration of active T cells (CD69) and perforin were studied by immunohistochemistry. T cells (CD3+, CD8+ and CD4+) in PBMC, spleen and tumor were isolated and analyzed using flow cytometry. The CD3+CD4+Foxp3+ T cells in tumor and CD3+CD8+CD44+ T cells in spleen and tumor were evaluated by flow cytometry. Levels of TNF-α, IFN-γ and IL-2 in serum, spleen and tumor were examined using the ELISA kits. DCs (CD11c+, CD80+ and CD86+) isolated from tumor and sentinel lymph node (SLN) were also analyzed by flow cytometry.
A lung metastatic model of breast cancer was also stablished to further investigate the treatment efficacy on metastatic cancer. Initially, the orthotopical breast tumor bearing mice was established by injecting 5×105 of 4T1 cells. 6 days later, 1×105 of Luc-4T1 cells were injected intravenously. Then, the mice were randomly sorted into 5 groups (3 mice per group) to respectively receive one of the following treatments once every 3 days: Saline+MW, DS+MW, DSM+MW, ES-DM+MW, ES-DSM+MW, for 4 times of treatment. 3mg/kg DOX and 0.5mg/kg SCH per dose was used in the treatment and the MW was applied at 24h post-i.v. injection of the test agent. The growth of pulmonary metastasis tumors was monitored by IVIS Spectrum imaging system (PerkinElmer, USA) after intraperitoneal injection of D-luciferin (15mg/mL, 200 μL). At the end of monitoring on day 20, the mice were sacrificed and the fluorescence images of lungs were acquired.
Tumor recurrence and re-challenge study were further invested. The orthotopical breast tumor bearing mice was established as mentioned above and received different treatments. After 4 times of treatment, 90% of the primary tumor was removed surgically on day 12, and the tumor bed was further monitored and the volume of recurrence tumor was calculated every 2 days. Simultaneously, 5×105 of 4T1 cells were inoculated into the breast pads on the other side of mice on day12. The re-challenged tumor was also monitored every 2 days. At the end of monitoring on day 30, the mice were sacrificed and re-challenged tumor was collected to analyze the infiltration of CD8+ T cells and Tregs (Foxp3) by immunofluorescence.
Statistical Analysis. Statistical calculations were performed using Prism 7 software (GraphPad). Data were expressed as the mean and SD. The abbreviation ns means no significant difference. Differences were statistically evaluated by Student’s t test. The differences were considered to be statistically significant for a p value of <0.05 (*p<0.05, **p<0.01, ***p<0.001). To analyze the survival time of mice, Kaplan-Meier survival curves were generated, and Log-rank Mantel–Cox tests were performed. P values of < 0.05 were considered significant (*p<0.05, **p<0.01, ***p<0.001).