Red Blood Cell-Mimic Nanocatalyst Triggering Radical Storm to Augment Cancer Immunotherapy

Highlights A red blood cell-mimic nanocatalyst with photodynamic/chemodynamic-like, catalase-like and glutathione peroxidase-like activities was developed to boost radical storms for tumor eradication. Combined with the Tim-3 immune checkpoint blockade, such radical therapy can systematically evoke a robust systemic antitumor immune response to eliminate residual cancer cells. Supplementary Information The online version contains supplementary material available at 10.1007/s40820-022-00801-z.


S1.2 Instruments
The morphologies of nanoparticles were observed using transmission electron microscopy (TEM, FEI Company, Hillsboro, OR). The UV-vis absorbance and fluorescence spectrum were measured by a Spectra Max M5 microplate reader (Molecular Devices, USA) and a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Malaysia), respectively. The 670 nm laser (Diode Laser System, BWT Beijng Ltd.) was used to induce photodynamic therapy (PDT). Coomassie blue staining was observed under a ChemiDoc™ MP imaging system. The images of cell uptake were obtained by a CLSM (Zeiss LSM780). Live and death assay and ROS generation assay were observed by a fluorescence microscopy (Zeiss Axio Vert.A1, Germany). The mice imaging was performed on the UniNano-NIR Ⅱ system. The frequencies of DC maturation and T cell infiltration were measured by a flow cytometry (BD FACSVerse TM ).

S1.3 Cell Culture
The human hepatocellular carcinoma line Hep3B cells, human normal liver cell line LO2 cells, murine hepatocellular carcinoma line Hepa1-6 cells and murine normal liver cell line CL2 cells were obtained from ATCC (Manassas, VA) and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) at the humidified incubator with 5% CO2.

S1.4 Synthesis of Pt Nanoparticles
The Pt nanoparticles were synthesized according to the previous literature. Briefly, 50.75 mg of H2PtCl6⋅6H2O and 222 mg of poly(vinylpyrrolidone) (PVP) were added into 20 mL of ethylene glycol. The above homogeneous solution was heated to 180 °C and kept at this temperature for 20 minutes, then cooled down to room temperature. Subsequently, the assynthesized PVP-protected Pt nanoparticles were precipitated in acetone and centrifugated at 10,000 rpm for 10 minutes. After being washed with acetone and hexane three times to remove excess free PVP, the Pt nanoparticles were re-dispersed in ethanol for further usage.

S1.5 Synthesis of FTP
30 mg of FeCl3· 6H2O dissolved in 10 mL DMF and then added with 2 mg of Pt nanoparticles, 10 mg of TCPP and 280 mg of benzoic acid. After stirred for 10 min, the mixture was heated to 90 °C and further reacted for 5 h. The crude product was collected and washed with DMF by centrifugation at 10,000 rpm for 15 min. Finally, the as-prepared MOFs were stored in ethanol for further usage.

S1.6 Preparation of Red Blood Cell Membranes (RBCMs)
Blood collected from mice was used to prepare the RBC membrane fragments. Briefly, the collected fresh blood was centrifuged at 3,000 rpm for 10 min at 4 °C to remove the plasma. Then the obtained RBCs were further washed twice using cold PBS, and subsequently suspended in 0.25×PBS for hemolysis treatment for 4 h. The released hemoglobin was removed by centrifugation at 12,000 rpm for 10 min. Next, the obtained light pink pellet was washed with cold PBS for three times and stored at -80 °C for further usage. The RBC membrane proteins were quantified by BCA protein assay kit.

S1.7 Preparation of RBCMs-Coated FTP (FTP@RBCM)
The harvested RBCMs were dispersed in PBS and conducted a strong ultrasonic bath for 1 min at a power of 950 W to form uniform RBC vesicles. Next, FTPs were vigorously mixed with as-prepared RBC vesicles at the mass ratio of 5:1 (TCPP in FTPs to total protein in RBCM protein) in 4 mL PBS, and then treated in ultrasonic bath at 4 °C with a moderate power for 10 min. Subsequently, the resulting FTP@RBCM was collected by centrifugation at 13,000 rpm for 15 min.

S1.8 Coomassie Blue Staining
The samples of RBCM, FTP and FTP@RBCM were heated to 100 °C for 10 min in the loading buffer of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the protein components of different samples were separated by 10% SDS-PAGE. Subsequently, the SDS-PAGE gel was stained with Coomassie blue for 30 min, then washed with the destaining solution (40% ethanol, 10% acetic acid and 50% deionized water) for 3 h and finally imaged by a ChemiDoc TM MP imaging system.

S1.10 ROS Detection
Singlet oxygen sensor green (SOSG) agent as a detection probe was used to monitor reactive oxygen species (ROS) generation. 1 µL of SOSG (5 mM) methanol solution was added into 1 mL FTP solution (TCPP: 20 µg). After irradiation under 670 nm laser (50 mW cm -2 ) for different times (0, 30, 60, 90, 120, 150, 180, and 240 s), the fluorescence spectrum from 500 to 750 nm was recorded under 480 nm wavelength excitation. The ROS generation efficiency was further analyzed by calculating Ft/F0 at 530 nm, where F0 refers to the initial fluorescence intensity, while Ft refers to the fluorescence intensity after irradiation at different times.
To investigate the role of self-compensation of O2 based on catalase-like reactivity of FTP on ROS generation. 1 mL of FTP (TCPP: 20 µg) solution containing 10 mM H2O2 and 5 µM SOSG probe was mixed and co-incubated for 5 min. Then, the mixture was irradiated under 670 nm laser (50 mW cm -2 ) for 1 or 2 min. Finally, the ROS generation ability was evaluated as the above methods.

S1.11 Detection of GSH Depletion
Briefly, FTP (TCPP: 20 µg) were added into a certain concentration of GSH solution. After incubation for 2, 4 or 6 h, the mixture was centrifugated at 13,000 rpm for 10 min. Then, the supernatant was collected and detected by a Reduced GSH Assay Kit (Solarbio life science, BC1175) according to manufacturer's instruction through a Spectra Max M5 microplate reader.

S1.12 Fe 2+ Detection
The o-phenanthroline can react with Fe 2+ to form orange complexes, while its complexes with Fe 3+ are colorless, which can be used to detect the transformation of Fe 3+ into Fe 2+ . Simply, FTP (TCPP: 20 µg) was incubated with GSH solution (100 mM) under oscillation for 6 h. Then 50 µL of o-phenanthroline solution (1 mg mL -1 ) was added into the above mixture. The color changes with different treatments could be observed and taken photos finally.

S1.13 OH Generation
FTP (TCPP: 20 µg) was incubated with GSH solution (100 mM) under oscillation for 6 h. Then, the mixture was centrifugated at 13,000 rpm for 10 min, and subsequently the obtained precipitation was added to acetic acid buffer solution (1 mL, 0.1 M, pH 5.0) containing TMB (1 mM) and various concentrations of H2O2. After co-incubation for different time intervals, the absorbance at 652 nm was measured using a Spectra Max M5 microplate reader.
Terephthalic acid (TPA) was also used to investigate ·OH generation. Briefly, the 5 mM TPA solution containing 5 mM sodium hydroxide (NaOH) was prepared. Then, the precipitation obtained as aforementioned procedures and 10 mM H2O2 were added to the mixture (2 mL, pH 6.5). After incubation for 20 min, the fluorescence spectrum from 350 to 600 nm was recorded under 312 nm wavelength excitation by a Cary Eclipse fluorescence spectrophotometer.

S1.14 Cellular Internalization
FTP (TCPP: 400 µg) was dispersed in an aqueous solution containing 20 µM of Cy5. After oscillation for 2 h, the precipitates (Cy5 loaded FTP) were obtained by centrifugation at 13,000 rpm for 10 min and subsequently washed with PBS to remove free Cy5. The Cy5 labelled FTP was further coated with RBCMs or RBCMs pre-treated under 80 °C for 10 min (defined as h-RBCM) to obtain Cy5 labeled FTP@RBCM or FTP@h-RBCM. Subsequently, different cells were seeded at the density of 2 × 10 5 cells and then incubated for 24 h at standard condition. Then the cells were co-cultured with Cy5 labeled FTP@RBCM (TCPP: 80 µg mL -1 ) for 4 or 8 h. Afterward, the cells washed with PBS, fixed with 4% paraformaldehyde, stained with DAPI and finally observed under CLSM (Zeiss LSM780).

S1.15 Hypoxic Relief
The oxygen indictor of [Ru(dpp)3]Cl2 was also used to detect intracellular oxygen level. First, the cells were seeded in a 96-well plate at the density of 2 × 10 4 cells per well and cultured for 24 h. Next, the medium was replaced with fresh medium containing FTP@RBCM or FT@RBCM (TCPP: 80 µg mL -1 ) and further incubated for 8 h. After being washed with PBS for two times, the cells were incubated for another 24 h in normoxia (20% oxygen) or hypoxia (2% oxygen). Then, 2 µM of [Ru(dpp)3]Cl2 was carefully added into the cells for 30 min incubation. Finally, the cells were washed with PBS for two times and imaged by a fluorescence microscope (Zeiss Axio Vert.A1, Germany).

S1.16 Intracellular ROS Detection
The cells were seeded in a 96-well plate at the density of 2 × 10 4 cells per well and allowed to culture for 24 h in normoxia (20% oxygen) or hypoxia (2% oxygen). Then, the medium was replaced with fresh medium containing FTP@RBCM or FT@RBCM (TCPP: 80 µg mL -1 ) and further incubated for 8 h in normoxia (20% oxygen) or hypoxia (2% oxygen). After that, the cells were washed with PBS and the medium containing 40 µM DCFH-DA (an intracellular ROS detection probe) were added for another 15 min of incubation. Next, the cells were washed twice with PBS and irradiated under a 670 nm laser (50 mW cm -2 ) for 3 min. Finally, the cells were observed under a fluorescence microscope.

S1.17 Intracellular GSH Depletion
The cells were seeded in a 6-well plate at a density of 4 × 10 5 cells and cultured for 24 h at standard condition. Then, different concentrations of FTP@RBCM were added into the cells and incubated for 8 h. After wash with PBS for three times, the cells were collected by centrifugation. Subsequently, the concentration of intracellular GSH was detected according to instruction of Reduced GSH Assay Kit.

S1.18 In Vitro CCK-8 Assay
To assess the cytotoxicity induced by · OH produced from the Fenton reaction of FTP@RBCM. The cells were seeded in the 96-well plate at a density of 2 × 10 4 cells per well and incubated for 24 h. Then, the cells were cultured with fresh medium containing different concentrations of FTP@RBCM and further incubated for another 24 or 48 h. After that, the cells were washed twice with PBS and the cell viability was measured by CCK-8 assay.
To evaluate the anti-cancer efficacy of PDT in vitro, cancer cells were seeded in the 96-well plate at a density of 2 × 10 4 cells per well and incubated for 24 h in normoxia (20% oxygen) or hypoxia (2% oxygen). Next, different concentrations of FTP@RBCM were added into the cells, which were cultured for another 8 h in normoxia or hypoxia condition. Then, the cells were irradiated under a 670 nm laser (50 mW cm -2 ) for 5 min. The cells with hypoxic treatment were sealed with 30 µL of paraffin oil to isolate the O2 in air before irradiation. After another 24 h incubation, the CCK-8 assay kit was used to measure the cell viability.

S1.19 Live/Death Cell Staining Assay
The cells with aforementioned treatment were stained with calcein AM and PI for 30 min and imaged by a fluorescence microscope. It's worth noting that the cell with hypoxic treatment need to be sealed with 30 µL of paraffin oil to isolate the O2 in air.

S1.20 CRT, HMGB1 Detection and ATP Assay
Nano-Micro Letters S5 /S19 The cells with aforementioned treatment stained with the primary antibodies of CRT or HMGB1 and corresponding secondary antibodies. Finally, the cells were analyzed by flow cytometry, or stained with DAPI and observed by CLSM (Zeiss LSM780).
The ATP concentration in cells with aforementioned treatment were detected using an ATP assay kit according to the manufacture's instruction.

S1.22 Hemolysis Test
The collected fresh mouse blood was washed twice with PBS, and then centrifugated at 3,000 rpm for 10 min to obtain red blood cells. Subsequently, the red blood cells were incubated with various concentrations of FTP@RBCM at 37 °C for 5 h. The red blood cells incubated with ddH2O and PBS were used as positive and negative control. After that, the mixture in the tube was centrifugated at 3,000 rpm for 10 min and taken photos. Besides, the supernatants were collected, and then the absorbance at 570 nm of which were measured using a Spectra Max M5 microplate reader. The hemolysis rates were calculated according the below formula: Hemolysis rate (%) = (A1-Anegative) / (Apositive-Anegative) × 100% Herein, A1 is the absorbance value of sample with different concentrations of FTP@RBCM treatment. Anegative and Apositive are respectively the absorbance values of negative and positive groups at 570 nm. The final absorbance value of each sample was the averaged value from three independent repetitions.

S1.23 In Vivo Bio-Distribution
The C57BL/6 mice were purchased from China Wushi, Inc. (Shanghai, China). All animal experiments were approved by the Animal Ethics Committee of Mengchao Hepatobiliary Hospital of Fujian Medical University and were conducted according to the institutional guidelines. Hepa1-6 subcutaneous tumor model was constructed by subcutaneous injection of Hepa1-6 cells (1 × 10 6 ) in the rear right flanks of mice. The mice were fed for animal experiment when the volume of tumor reached about 50 mm 3 .
The tumor-bearing mice were intravenously injected with ICG labeled FTP or FTP@RBCM (TCPP: 5 mg kg -1 ). Then, the mice were imaged using the UniNano-NIR II system at different time points. After 48 h of injection, the mice were sacrificed, and the major organs and tumors were excised to observe the fluorescence distribution of ICG in major organs.

S1.24 In Vivo Enhanced Therapeutic Efficacy and Immune Response
he Hepa1-6 tumor-bearing mice were randomly divided into 5 groups including PBS group, PBS + L group, FT@RBCM + L group, FTP@RBCM group and FTP@RBCM + L group. Then at day 0 and 3, the mice were intravenously (i.v.) injected with different formulations with the TCPP dose of 5 mg kg -1 . After 5 h of enrichment, the mice were conducted with 670 nm laser irradiation (100 mW cm -2 ) for 10 min. The tumor volumes and mice body weights were measure every other day. The tumor volumes were calculated following the formula: V = length × width 2 / 2 At day 20, the tumors and major organs were excised from mice. And the tumors were weighed and recorded, following fixed in formalin for hematoxylin and eosin (H&E), Ki67 and TUNEL staining.
To evaluate the immune response of CRT expression and DC maturation in vivo, the tumorbearing mice were treated with different formulations as above. Next, the tumors and draining inguinal lymph nodes were excised after 3 days of treatment. The tumor tissue sections were used for immunofluorescence staining of CRT. On the other side, the lymph nodes were gently ground to collect cells, and then stained with the antibody of CD11c-APC (eBioscience™, 17-0114-82), CD80-PE (eBioscience™, 12-0801-82) and CD86-PE-Cy7 (eBioscience™, 25-0862-82) for FACS measurement.

S1.25 In Vivo Enhanced Synergistic Anti-Tumor Efficacy and Abscopal Effect
To establish the bilateral Hepa1-6 tumor model, the right flanks of mice were subcutaneously injected with 1 × 10 6 Hepa1-6 cells as the primary tumors, while the left flanks were subcutaneously injected with 5 × 10 5 cells as the distant tumors. When the volumes of primary tumors reached about 100 mm 3 while the distant tumors were reached about 50 mm 3 , the mice were randomly divided into 4 groups including PBS group, FTP@RBCM + L group, anti-Tim-3 group and FTP@RBCM + L + anti-Tim-3 group. The primary tumors of mice were intratumorally injected with 50 μL of FTP@RBCM (TCPP: 2 mg/kg). After 5 h, primary tumors were irradiated with 670 nm laser (100 mW cm -2 ) for 10 min. On day 1, 4 and 7 after irradiation, the anti-Tim-3 antibodies were i.p. injected at the dose of 100 µg per mouse. The tumor volumes in both flanks and body weights of mice were all monitored every other day. Mice were sacrificed when the tumor volume reached or exceeded 1500 mm 3 , or when the health condition of mice was impaired seriously.

S1.26 Cytokine Assay
The tumors were isolated from mice after 3 days of treatment. Then, the tumors weighed about 20 mg were homogenized in 0.5 mL of PBS containing protease inhibitor cocktail. The supernatants were obtained by centrifugation at 10,000 rpm for 15 min. Finally, the cytokines in supernatants were detected using Mouse ELISA Kit (IFN-γ, Boster, EK0375; IL-12, Boster, EK0422; Granzyme B, Boster, EK0417).

S.1.27 Statistical Analysis
Data was presented as the mean ± standard deviation (SD) or min to max, show all points as indicated. Significance was calculated using one-way analysis of variance (ANOVA) or t test as indicated. Survival rates of different groups were compared using the Log-Rank test. Prism 6 software (GraphPad) was used to perform all statistical analyses. *P < 0.05, **P < 0.01, ***P < 0.001. The P-value < 0.05 was considered as statistically significant.