Materials
Dopamine hydrochloride, disulfiram, rhodamine123, phloretin, and N-acetyl-L-cysteine were obtained from Aladdin Industrial Corporation (Shanghai, China). Hydrogen peroxide (30%), ethanol (AR, ≥ 95%), and ammonia solution (AR, 25–28%) were purchased from Sinopharm Chemical Regent Co., Ltd (Shanghai, China). SH-PEG-Mannose (Mn, 5000 Da) was purchased from Huateng Pharma (Hunan, China). 2’,7’-Dichlorofluorescin diacetate (DCFH-DA), calcein acetoxymethyl ester (Calcein-AM), propidium iodide (PI), and Enhanced ATP Assay Kit were purchased from Beyotime Chemical Reagent (Jiangsu, China). Lysotracker was purchased from Yeasen Biotech Co., Ltd (Shanghai, China). HMGB1 antibody and goat anti-Mouse lgG AF 488 were provided by Abmart Co., Ltd (Shanghai, China). Antibodies for flow cytometry were purchased from Biolegend, lnc (USA). In Vivo MAb anti-mouse PD-1 (CD279) was obtained from BioXcell (USA).
Preparation of DSF@CuPDA-PEGM
CuPDA nanoparticles were prepared through the oxidative polymerization and self-assembly of dopamine (DA) with Cu2+ chelation under the alkaline condition. Firstly, dopamine hydrochloride (380 mg, 2 mmol) and CuCl2·2H2O (28.4 mg, 0.17 mmol) were dissolved in 10 mL of deionized water, respectively. Then, dopamine hydrochloride and CuCl2·H2O solutions were added into a mixture of ammonium hydroxide (3 mL), ethanol (40 mL), and deionized water (90 mL) at room temperature stirring for 24 h. CuPDA nanoparticles were collected by centrifuging, washing, and freeze-drying.
SH-PEG-Mannose (5k) was modified to the surface of the CuPDA nanoparticles. Firstly, CuPDA NPs (20 mg) were dispersed in 20 mL of Tris buffer (10 mM, pH 8.5) containing SH-PEG-Mannose (20 mg). The reaction carried on at room temperature for 24 h and CuPDA–PEGM was collected via centrifugation and washing.
To encapsulate the hydrophobic DSF, CuPDA-PEGM (20Â mg) was dispersed in ethanol solution (10 mL) with DSF (10Â mg) and stirred at room temperature for 24Â h. The resulting DSF@CuPDA-PEGM was collected by centrifugation and washing with ethanol and water.
Characterization of DSF@CuPDA-PEGM
A JEM-2010Â F transmission electron microscope (TEM, JEOL, Japan) was employed for morphological characterization of nanoparticles. Element mapping was conducted on FEI-Talos F200S. X-ray photoelectron spectroscopy (XPS) spectra were obtained by ESCALAB 250Xi (Thermo Fischer). The hydrodynamic size and zeta potential of nanoparticles were measured by the Zetasizer Nano series (Nano-ZS ZEN3600, Malvern). UV-Vis absorption spectra were observed on a VICTOR Nivo Multimode Microplate Reader (PerkinElmer). CuPDA-PEGM NPs were incubated in PBS or DMEM with 10% FBS for 1Â h and 24Â h to investigate their stability, then the hydrodynamic sizes were measured.
Hydrogen peroxide-responsive degradation and copper ion release in vitro
CuPDA NPs (0.25Â mg/mL) were incubated with different amounts of H2O2 (0, 0.1, 1, 5, 10 mM) in aqueous solutions. After 72Â h, UV-vis absorption spectra of solutions from 400 to 800Â nm were measured. Cu2+ concentrations in supernatants were investigated by Inductively coupled plasma atomic emission spectrometer (ICP-AES). For different incubation times (6, 12, 24, 48, 72Â h), the absorbance of CuPDA NPs at 500Â nm was recorded.
To study the degradation behaviors, DSF@CuPDA-PEGM dispersions were incubated in the buffers mimicking different environments (pH 5.0 with 100 µM H2O2, pH 6.5 with 100 µM H2O2, and pH 7.4 with 3.6 µM H2O2) over 72 h. After incubation (6, 12, 24, 48, 72 h), the absorbance of DSF@CuPDA-PEGM dispersion at 500 nm was recorded. To test DSF release, DSF@CuPDA-PEGM (2 mg of DSF) was dispersed in the buffers (pH 7.4, 3.6 µM H2O2 or pH 6.5, 100 µM H2O2), and then dialyzed in 10 mL of corresponding buffers. At given time intervals, the release media were collected and replaced with fresh buffers. The amount of released DSF was evaluated and calculated according to the reported method [26].
In vitro cellular uptake
Rhodamine123 was loaded on CuPDA-PEG or CuPDA-PEGM to explore in vitro cellular uptake. For Rh123@CuPDA-PEG and Rh123@CuPDA-PEGM synthesis, 3 mg Rhodamine123 was dissolved in 500 µL ethanol, which was later added into CuPDA-PEG or CuPDA-PEGM suspensions (10 mg/mL). After stirring overnight, Rh123@CuPDA-PEG and Rh123@CuPDA-PEGM were dialyzed in water for two days and freeze-dried for use.
To investigate GLUT1-mediated cellular uptake, HCT116 cells were seeded in confocal dishes (5 × 104 cells/well). After 24 h, cell media were replaced with fresh media containing Rh123@CuPDA-PEG or Rh123@CuPDA-PEGM (1.25 µg/mL). For the phloretin inhibition group, the cells were pretreated with phloretin for 12 h before exposure to Rh123@CuPDA-PEGM. For the mannose competition group, the cells were exposure to Rh123@CuPDA-PEGM in the media containing mannose (4.5 g/L) for 4 h. After treatment for 4 h, the cells were stained with Hoechst 33,342, and then observed on a confocal. For flow cytometry analysis, HCT116 cells (2 × 105 cells per well) were cultured in a 6-well plate for 24 h, and treated as mentioned above before flow cytometry analysis.
In vitro cytotoxicity and apoptosis assay
HCT116, HUVEC, L929, CT26, NCM460, and 3T3 cells were seeded in the DMEM medium with 10% fetal bovine serum (FBS) at 37 °C in an incubator with 5% CO2. HCT116 L/OHP cells were cultured in RPMI-1640 medium with 10% FBS at 37 °C under the atmosphere of 5% CO2.
The cytotoxicity of different drug formulations (DSF, CuPDA-PEGM, DSF@CuPDA-PEG, DSF@CuPDA-PEGM) towards HCT116 cells, HCT116 L/OHP cells, HUVEC cells, L929 cells, Hela cells, MCF-7 cells and A549 cells were measured by a CCK-8 kit (Vazyme). Briefly, cells (6 × 103 cells per well) were cultured in 96-well plates for 24 h. Then, the cell culture media were replaced with media containing different formulations (0–5 µg/mL DSF, 0–50 µg/mL CuPDA-PEGM). After incubating for 24 h, the cell viability was evaluated by a CCK-8 assay according to the manufacturer’s instructions.
Cell apoptosis assay was conducted through an Annexin V/PI kit. First, HCT116 or HUVEC cells (2 × 105 cells per well) were cultured in a 6-well plate for 24 h. DSF (2.5 µg/mL), CuPDA-PEGM (25 µg/mL), DSF@CuPDA-PEG (2.5 µg/mL DSF), DSF@CuPDA-PEGM (2.5 µg/mL DSF) were applied to treat these cells. After 24 h, the cells were washed, collected, and stained with Annexin V and PI for flow cytometry analysis.
For live/dead staining, HCT116 or HUVEC cells (1 × 105 cells per well) were cultured in a 12-well plate and incubated for 24 h. The cells were treated as mentioned above. After that, the media were discarded and the cells were stained with Calcein-AM and PI for 30 min before observation by a fluorescence microscope.
The mechanisms of in vitro cell cytotoxicity
For intracellular ROS levels detection, HCT116 cells (1 × 105 cells per well) were seeded in a 12-well plate and incubated for 24 h, followed by treatment with DSF@CuPDA-PEGM (1.25 µg/mL) for different times (1 h, 2 h, 4 h, 8 h, 24 h). DCFH-DA probe (10 µM) was used to indicate the cellular ROS of the treated cells.
To alter the ROS content in HCT116 cells, H2O2 and N-acetyl-L-cysteine (NAC) were applied. HCT116 cells (1 × 105 cells per well) were seeded in a 12-well plate and incubated overnight. Then the culture media were replaced with fresh medium containing 0.2 mM H2O2 or 0.5 mM NAC and incubated for 4 h. Afterward, the cells were treated with serum-free media containing DCFH-DA (10 µM) and incubated for 20 min before imaging using a fluorescence microscope. For flow cytometry analysis, HCT116 cells (2 × 105 cells per well) were cultured in a 6-well plate overnight. Cells were treated with the same procedure as mentioned above, and then the cells were collected for flow cytometry analysis.
To investigate the impact of intracellular ROS on DSF@CuPDA-PEGM’s selective killing ability, cells (6 × 103 cells per well) cultured in a 96-well plate were pretreated with 0.2 mM H2O2 or 0.1 mM NAC, then cells were incubated with DSF@CuPDA-PEGM (DSF, 0–5 µg/mL) for 24 h. Afterward, the cell viability was measured by a CCK-8 assay.
In order to explore the cell death mechanisms induced by DSF@CuPDA-PEGM, HCT116 cells were pretreated with Z-VAD-FMK (30 µM) or rotenone (100 nM), and then exposed to DSF@CuPDA-PEGM (DSF, 0–5 µg/mL). After incubation for 24 h, the cell viability was evaluated by a CCK-8 assay.
In vitro ICD effect
To assess the ICD effect of DSF@CuPDA-PEGM, ICD-related DAMPs (HMGB1 and ATP) were evaluated in CT26 cells in vitro. CT26 cells were treated with free DSF, CuPDA-PEGM, DSF@CuPDA-PEG, and DSF@CuPDA-PEGM (2.5 µg/mL of DSF), respectively. After 4 h, the cells were washed, fixed, and stained with anti-HMGB1 primary antibody, followed by Alexa Fluor 488 secondary antibody according to the manufacturer’s protocol.
After CT26 cells were incubated with these drug formulations for 5 h, the media were collected and centrifuged at 1000 rpm for 5 min. ATP contents in the supernatants were tested using an Enhanced ATP Assay Kit according to the manufacturer’s instructions.
To evaluate DCs maturation, the culture media of CT26 cells were collected and centrifuged after treatment with free DSF, CuPDA-PEGM, DSF@CuPDA-PEG, and DSF@CuPDA-PEGM for 24Â h. Then, DC2.4 cells were treated with these condition media for 24Â h. Finally, the DCs were collected and stained with anti-CD11c-BV421 and anti-CD86-PE antibodies. The proportion of mature DCs was determined by flow cytometry.
Animals
Female BALB/c nude mice (6 weeks) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Female BALB/c mice (6–8 weeks) were provided by Liaoning Changsheng Biotechnology Co., Ltd. (Benxin, China). All animal experiments were performed according to the protocols approved by the Animal Care and Use Committee of Huazhong University of Science and Technology (Wuhan, China).
In vivo antitumor effect
To evaluate the in vivo therapeutic effects of DSF@CuPDA-PEGM, subcutaneous HCT116 tumor-bearing models in BALB/c nude mice were used. HCT116 cells (1.2 × 106 cells) were injected into the right infra-axillary dermis subcutaneously to develop models. Tumor-bearing mice were randomly divided into 5 groups (n = 6): (1) PBS control, (2) free DSF, (3) CuPDA-PEGM, (4) DSF@CuPDA-PEG, (5) DSF@CuPDA-PEGM. The mice were intravenously injected with 200 µL of the above formulations every three days for 4 times since the tumor volume reached approximately 100 mm3. The dosages of DSF and CuPDA-PEGM were 3.75 mg/kg and 37.5 mg/kg, respectively. Body weight and tumor volume were monitored every two days. Tumor volume was calculated according to the following formula: V = 1/2 × width2 × length. Mice were sacrificed at day 18, major organs were collected and stained with hematoxylin and eosin, while the tumors were weighed and performed with TdT-mediated dUTP nick-end labeling (TUNEL) staining.
In vitro biocompatibility
Hemolysis assay was performed to evaluate the biocompatibility of CuPDA-PEGM. Red blood cells suspension (900 µL, 2%) was incubated with 100 µL CuPDA-PEGM (0–100 µg/mL) under vibration for 4 h at 37 °C. Then, the mixtures were centrifuged at 3000 rpm for 10 min and the supernatants were measured at 545 nm. PBS and Triton X-100 were applied as the negative and positive controls.
The cytotoxicity of CuPDA-PEGM NPs (0–100 µg/mL) in noncancerous cell lines (L929, NCM460, and 3T3) was evaluated by a CCK-8 kit according to the methods mentioned above.
In vivo antitumor efficacy by DSF@CuPDA-PEGM/αPD-1 combination
When CT26 tumor volumes reached around 80 mm3, mice were treated with PBS, αPD-1, DSF@CuPDA-PEGM, DSF@CuPDA-PEGM/αPD-1, respectively (n = 6, 3.75 mg/kg of DSF, 100 µg of αPD-1 per mouse). The body weight and tumor volumes of mice were monitored every day. On day 12, mice were sacrificed and their tumors were harvested for staining with CD8 antibody. Cells with red fluorescence around blue nuclei were regarded as CD8+ T cells.
To investigate the immune responses of mice, CT26 tumor-bearing BALB/c mice were divided into 4 groups randomly (n = 4). DSF@CuPDA-PEGM (DSF: 3.75 mg/kg) were intravenously injected two times on day 0 and day 3, and αPD-1 was intraperitoneally administered on day 1 and day 4 at a dose of 100 µg/mouse. Mice were sacrificed on day 5. Their tumors and tumor-draining lymph nodes were collected for flow cytometry analysis. The tumors were pulverized and the collected cells were washed with 1640 medium followed by staining with specific antibodies including CD4-BV605, and CD8a-APC. The lymph nodes were obtained and collected for CD11c-FITC and CD86-PE staining. The cells were finally analyzed by flow cytometry.
