Materials
The medium of cell culture was purchased from Gibco Life Technologies, Inc. (Grand Island, NY, USA), including Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 medium. Fetal Bovine Serum (FBS) was obtained from Zhejiang Tianhang Biotechnology Co., Ltd. (Huzhou, China). Plasmocin was bought from InvivoGene (Toulouse, France) and penicillin/streptomycin was obtained from Biosharp (Hefei, China). Sterile 1×phosphate buffered saline was purchased from Gibco Life Technologies, Inc. (Grand Island, NY, USA). All the cytokines were purchased from Biolegend (San Diego, CA, USA), containing granulocyte-macrophage colony-stimulating factor macrophage colony-stimulating factor, interleukin-4, interleukin-13, lipopolysaccharide and interferon-γ. RSL3 was bought from Selleck (Houston, TX, USA). CT20p peptide was bought from BankPeptide Inc. (Hefei, China). Sucrose for electroporation buffer was gained from Sinopharm (China). Acetonitrile, methanol and chloroform for High Performance Liquid Chromatography (HPLC) were all purchased from Thermo Fisher Scientific (Waltham, MA, USA) and their purity was more than 99%. The fluorescence dye DiO, DiD and Rhodamine were obtained from Yeasen (Shanghai, China). H2DCFDA and PKH26 were bought from MedChemExpress (NJ, USA). FITC-Liperfluo was obtained from Dojindo (Japan) and Phen Green SK diacetate was bought from GLPBIO (USA). Radioimmunoprecipitation assay buffer and the inhibitors of protease and phosphatase were obtained from Beyotime (Shanghai, China). For western blot, primary antibodies STING, p-STING, NFκB, p-NFκB and Laminin B1 were purchased from ABclonal (Boston, MA, USA) and GAPDH, CD63, CD81 and Alix were obtained from Proteintech Group, Inc. (Chicago, IL, USA). Secondary antibodies goat anti-mouse IgG H&L-HRP conjugated and goat anti-rabbit IgG H&L-HRP conjugated were bought from Abcam (Cambridge, UK). Collagenase IV and hyaluronidase were purchased from Biosharp (Hefei, China). All the antibodies for flow cytometry and immunocyte depletion were bought from Biolegend (San Diego, CA, USA). Clodronate liposomes were purchased from FormuMax (Silicon Valley, CA, USA). PD-1 mAb for treatment was obtained from BioXell (Italy).
Cells culture
All the murine cell lines were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China), including prostate cancer cell line (RM-1), Lewis lung carcinoma cell (LLC), mammary cancer cell line (4T1), colon adenocarcinoma cell line (MC38), B16F10 melanoma cells, GL261 glioma cells, DC line (DC2.4) and monocyte cell line (RAW264.7). All the cell lines were treated with 25 µg mL− 1 Plasmocin for at least two weeks and were mycoplasma-negative as determined by MycoProbe Mycoplasma Detection Kit (R&D Systems, Minneapolis, MN, USA). RM-1, 4T1 and LLC cells were cultured in DMEM while other cells were maintained in RPMI 1640 medium. Bone marrow-derived dendritic cells and macrophages (BMDCs & BMDMs) from C57BL/6J mice were generated as previous descriptions in RPMI 1640 medium[32, 33]. All the mediums were added with 10% (v/v) FBS and 100 µg mL− 1 penicillin/streptomycin.
Preparation of RMPs
In 10-cm cell culture dishes, 6×106 RM-1 cells were planted and irradiated with a single dose of 20 Gy by 6-MV x-rays (CHIRAD 225). Next, the medium of irradiated cells was renewed by 20 mL DMEM completed medium which its microparticles had been removed via centrifugation. 72 hours later, the medium was collected, and cell debris were removed by 1000 g for 10 min and 14,000 g for 2 min. RMPs were gained from the supernatant via 14,000 g for further 60 min at 4 ℃ and washed with sterile 1×PBS for 2 times. At last, the RMPs were resuspended with 1×PBS for subsequent experiments.
RMPs encapsulated with RSL-3 and CT20p via electroporation
RSL3 was dissolved in DMSO to a concentration of 10 mg mL− 1. CT20 peptide (CT20p) was diluted by ultrapure water. RSL3 or CT20p was mixed with RMPs in a 1:2 ratio of mass in 400 mM sucrose solution. By an electroporation system (GenePulser Xcell, BioRad, USA), 400 µL mixture was electroporated in 0.2 cm cuvettes via exponential pulse (voltage: 500 V; capacitance: 125 µF).
High Performance Liquid Chromatography (HPLC)
HPLC analysis was conducted using a LC-2030C Plus instrument (Shimadzu, Japan). The separation was implemented with a ShimNex C18 chromatographic column (4.6×250 mm, 5 µm, 100 A, Shimadzu, Japan). Three times volume of acetonitrile was mixed with RMPs and then added chloroform (1:2, v/v). After vortexed, the mixture was centrifuged at 10,000 g for 5 min and the lower layer was extracted for measurement. As the standard solution, 10 mg RSL3 was dissolved in acetonitrile and chloroform in a ratio the same as RMPs. All the samples were filtered through a 0.45 µm polytetrafluoroethylene filter. The components were separated and eluted by mobile phase eluted (A: methanol; B: acetonitrile) in the column at 25 ℃ with a flow rate of 1 mL min− 1. An ultraviolet wavelength (254 nm) was selected for the detection of RSL3.
Quantification of RMPs
The quantification of RMPs were determined by their protein concentrations. Radioimmunoprecipitation assay buffer were applied to lyse RMPs at 4°C for half an hour. Then, the lysis was centrifuged at 12,000 g for 30 min at 4°C and the supernatant was transferred into a new centrifuge tube for protein concentration measurement by BCA Protein Assay Kit (Thermo Fisher Scientific).
Characterization of RMPs Size and Transmission electron microscopy (TEM) Determination
One milliliter of 30 ng mL− 1 RMPs were taken for the measurement of the particle size and polydispersity index by Malvern laser particle size analyzer (Zetasizer Nano ZSP). For further identification of the sizes and morphology of RMPs were washed by ddH2O, deposited on copper mesh and then observed by TEM (HT7700-SS/FEI Tecnai G20 TWIN).
Cell viability measurement
All the cells to evaluate viability were planted into 96-well plates (5000 cells per well). After 24 hours growing, distinct RMPs were treated with the cells for further 48 hours. A cell counting kit-8 (CCK-8) assay kit (Meilunbio, Dalian, Chian) was used to measure cell viability.
In vitro cellular uptake assay
To evaluate the cellular uptake of RMPs, different cell lines were planted into 6-well plates and incubated with DiO pre-dyed MPs for 3 or 6 hours. The cells were collected, washed by PBS and analyzed through flow cytometry (Beckman CytoFLEX S, USA).
Identification of the colocalization of CT20p and mitochondrion
RMPs with CT20p-Rhodamine B and Rhodamine B were added into the cells which were sequently incubated with 100 nM MitoTracker Green® FM at 25 ℃ for 30 minutes. Confocal laser scanning microscopy (Carl Zeiss LSM710) was used to observe the colocalization of CT20p and MitoTracker Green® FM (standing for mitochondrion) whose Pearson correlation coefficient was calculated by image J software.
Analysis of cell apoptosis and ferroptosis
Cells were cultured in 24-well plates (30,000 cells per well) and then incubated with MPs, RSL3@MPs, CT20p@MPs and RC@RMPs for 24 hours. The cells were harvested for relevant measurement. Apoptosis was evaluated by Annexin V-Alexa Fluor 488/7-AAD apoptosis detection kit (yeasen, China). The protocol complied with the instruction of the kit. To determine mitochondrial membrane potential, JC-1 assay kit (yeasen, China) was applied. The method to dye and measure cell apoptosis and mitochondrial membrane potential obeyed the instruction of the kits. To evaluate total ROS, lipid ROS and Fe2+ level, the cells were respectively dyed with H2DCFDA (10 µM), FITC-Liperfluo (5 µM) and Phen Green SK diacetate (10 µM) in 1 mL PBS for 30 min at 37°C in a cell culture incubator. Wased by PBS twice, the cells were resuspended with 200 µL PBS and analyzed via flow cytometry.
Western blotting
All the MPs and cells that be detected were lysed by RIPA buffer with the inhibitor of protease and phosphatase at 4°C for 30 minutes, and then centrifuged at at 12,000 g for 30 min at 4°C. The mass of the sample loading was adjusted to the same according to their protein concentrations that were detected by BCA Protein Assay Kit. The samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane after boiled for 5 minutes. The membranes block by 5% not-fat milk at room temperature for 1 hour and incubated with related primary antibodies at 4°C overnight. With serveral wash by Tris-buffered saline with 0.05% Tween-20, secondary antibodies incubated with the membranes at room temperature for 1 hour. NcmECL Ultra (P10100, NCM Biotech) was applied for chemiluminescent exposure of the blot.
Mice
Male C57BL/6J mice (aged 6–8 week, weighted 18–20 g) were purchased from SHULAIBAO Biotech. All mice were kept in micro-isolator cages, and the experimental protocols were approved by the Hubei Provincial Animal Care and Use Committee and were in compliance with the experimental guidelines of the Animal Experimentation Ethics Committee of Huazhong Agricultural University.
In vivo cellular internalization assay
To identify RMPs uptake by cells in tumor in vivo, we intratumorally injected 100 µL PKH26 marked RMPs to mice with RM-1 tumor burden. 24 hours later, the mice were sacrificed and the tumors were digested into single cell for flow cytometry analysis before they were incubated with antibodies of CD45 (clone S18009F), CD3 (clone 17A2), B220 (clone RA3-6B2), CD11b (clone M1/70), Ly6G (clone S19018G), F480 (clone BM8), CD11c (clone N418), MHCII (clone M5/114.15.2) and NK1.1 (clone PK136). Besides, some tumor tissues were fixed, dehydrated and sectioned into frozen sections which were going to stain by related antibodies and observed via confocal laser scanning microscopy. To image the distribution of RMPs, RC@RMPs were stained with DiD, the 540/20 nm excitation filter and 620/20 nm emission filter were used and the exposure time was 15 s.
Subcutaneously implanted prostate tumor model and treatment with RMPs
RM-1 tumor cells (1×106 cells in 100 µL PBS) were subcutaneously implanted into right back. Five days after tumor inoculation, mice with uniform tumor volume were randomly divided into 7 groups including control group, PD-1 mAb group, RMPs group, RSL3@RMPs group, CT20p@RMPs group, RC@RMPs group and RC@RMPs combined with PD-1 mAb group, and were treated with corresponding RMPs (intratumoral injection with 100 µg in 100 µL PBS) and PD-1 mAb (10 mg/kg, intraperitoneal injection) at 6, 8, 10, 12 and 14 days after grouping. Vernier caliper was applied to measure the length (L) and width (W) of subcutaneous tumors every other day. The volume of tumor was calculated by the the formula V = (L × W2) / 2. Mice were sacrificed when the tumor volume reached 1000 mm3.
Detection of immunocytes in tumor and draining lymph nodes (dLNs)
RM-1 tumors from mice were digested into single cell by cutting into small pieces and incubating with Collagenase IV (0.32 mg mL− 1) and hyaluronidase (0.5 mg mL− 1) for 1 hours at 37°C. The tumor cells were filtered through 70 µm cell strainer after lysis of RBCs. All the samples were blocked Fc receptors followed by incubating with detection antibodies containing CD3 (clone 17A2), CD8 (clone SK1), CD69 (clone H1.2F3), CD4 (clone GK1.5), PD1 (clone 29F.1A12), TOX (clone 6E6D03), TCF1 (clone 7F11A10), CD11c (clone N418), CD11b (clone M1/70), CD86 (clone GL-1), MHCII (clone M5/114.15.2), CD44 (clone IM7), CD62L (clone MEL-14) and Zombie NIR™, and then measured via flow cytometry.
Cytokines detection
RM-1 tumors from mice were weighted and grinded into homogenate. The supernatant was collected by 6000 g centrifugation for 20 minutes at 4 ℃. The LEGENDplex Mouse Cytokine Release Syndrome Panel (13-plex) with VBottom Plate (purchased from Biolegend) was used for cytokine detection.
Immune cell depletion
T helper cells and CTLs were depleted by CD4 (clone GK1.5) and CD8 (clone 2.43) antibodies respectively. One day before treatment, 200 µg antibodies were intraperitoneal injected into mice for 5 times at 2-day intervals. Macrophages were depleted by clodronate liposomes. One day before treatment, 200 µL clodronate liposomes were intravenously injected into mice for 5 times at 3-day intervals. Neutrophils were depleted by Ly6G (clone 1A8) antibody. One day before treatment, 200 µg antibodies were intraperitoneal injected into mice for 5 times at 2-day intervals.
Statistical analysis
All the data were analyzed by Prism software (GraphPad Prism 6.0 software). The log-rank (Mantel-Cox) test was applied to compare survival rates between groups.Kaplan-Meier analysis was used to analyze tumor growth, and comparisons of three or more groups were calculated by one-way analysis of variance (ANOVA). Two-tailed unpaired t test or the Mann-Whitney U test was performed to determine the significance of two groups. P values of < 0.05 were determined statistically significant. Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001 and ns stands for no significant.