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Enhancement of antitumor immunotherapy using mitochondria-targeted cancer cell membrane-biomimetic MOF-mediated sonodynamic therapy and checkpoint blockade immunotherapy | Journal of Nanobiotechnology



H2TCPP (379077), ZrOCl2·8H2O (224316), triphenylphosphonium (TPP, 157945), N, N-dimethylformamide (DMF, 227056), and Benzoic acid (242381) were from Sigma-Aldrich (St Louis, MO). (3-Carboxypropyl)triphenylphosphonium bromide (TPP, D051213) was from energy chemical. Imiquimod (R837, CAS No.: 99011-02-6) was from MCE. Anti-CTLA-4 (BE0164, USA) was from Bioxcell. Antibodies utilized for flow cytometry were from Biolegend.

Cell culture

Murine 4T1 and 4T1-Luci cancer cell lines were from the American Type Culture Collection (ATCC) and were grown in RPMI-1640 (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and penicillin/streptomycin (Beyotime, Shanghai, China). Cells were cultured in a 37 °C humidified 5% CO2 incubator. Cell viability was monitored based on Trypan blue exclusion.

4T1 cell membrane isolation

A Membrane Protein Extraction Kit (Beyotime, Shanghai, China) was used to prepare 4T1 cell membranes. Briefly, 1 mL of membrane protein extraction buffer solution supplemented with PMSF (1 mM) was used to suspend 5 × 107 4T1 cells, which were then incubated for 10–15 min in an ice bath before being frozen to − 80 °C and then thawed to room temperature. The freeze–thaw process was repeated three times, after which samples were centrifuged for 15 min at 3500 rpm at 4 °C. Supernatants were then centrifuged for 30 min at 14,000 rpm at 4 °C, with precipitates being collected and stored at − 80 °C.

Zr-TCPP(TPP)/R837@M nanoparticle synthesis

Initially, H2 TCPP (100 mg, 0.13 mmol), ZrOCl2·8H2O (300 mg, 0.93 mmol), and benzoic acid (2.9 g, 24 mmol) were dissolved in 100 mL of DMF and continuously stirred (300 rpm) for 5 h at 90 °C. Nanoparticles (NPs) were then collected by centrifuging this mixture for 10 min at 10,000 rpm, after which they were washed three times in DMF. Next, Zr-TCPP (75 mg) was suspended in methanol (20 mL), followed by sonication for 5 min. In addition, 4-carboxybutyl-TPP (35 mg) was dissolved in MeOH (20 mL), with the mixture then being supplemented with triethylamine (20 mL). These solutions were then combined together overnight at room temperature with constant stirring. NPs were then isolated via centrifugation, rinsed two times with methanol to remove free TPP, and dried overnight at room temperature under vacuum.

R837 loading was achieved via physical absorption. Briefly, R837 was loaded into the mitochondria-targeting MOF probe to yield Zr-TCPP(TPP)/R837, with the loading of these particles then being assessed via ultraviolet–visible (UV–Vis) spectrophotometry.

Zr-TCPP(TPP)/R837@M construction was achieved using a mini extruder (Avanti Polar Lipids). Briefly, the 4T1 cell membranes and the Zr-TCPP(TPP)/R837 were dissolved at 2 mg/mL in distilled water, followed by sonication for 15 min. The mini extruder was then used to extrude these cell membranes a minimum of 10 ties using a polycarbonate porous membrane (400 nm). Subsequently, 0.5 mL of Zr-TCPP(TPP)/R837 (2 mg/mL) was added to pretreated 4T1 cell membranes, with the solution again being extruded a minimum of 10 times, this time using a smaller polycarbonate porous membrane (200 nm). After extrusion, samples were collected via centrifugation for 10 min at 6000 rpm at 4 °C, after which the free membrane was removed by washing three times using water.

Nanoparticle characterization

Transmission electron microscopy (TEM) and high-resolution TEM (HR-TEM) were utilized to assess Zr-TCPP(TPP)/R837@M structural characteristics. X-ray diffractometry was used to assess NP structural performance. The elemental composition and valence states of Zr-TCPP(TPP)/R837@M were verified via X-ray photoelectron spectroscopy (XPS). Zr-TCPP(TPP)/R837 and Zr-TCPP(TPP)/R837@M zeta potential and dynamic light scattering (DLS) characteristics were assessed with a particle analyzer (Nano-ZS, Malvern, England) at room temperature. A UV–vis spectrophotometer (Tecan Spark, Tecan, Switzerland)was used to assess Zr-TCPP(TPP) @M, R837, and Zr-TCPP(TPP)/R837@M UV–vis absorption spectra. Zr-TCPP(TPP)/R837@M NP crystal structure properties were established through X-ray powder diffraction analyses.

Cell membrane proteins were characterized via 12% SDS-PAGE separation followed by staining with Coomassie Brilliant Blue for 1 h and multiple rounds of destaining.

Cellular uptake and efflux analyses

Assessment of Zr-TCPP(TPP)/R837@M subcellular localization

4T1 cells were incubated for 8 h with Zr-TCPP(TPP)/R837@M (50 mg/L), after which cells were rinsed using PBS and stained with MitoTracker Green (M7514, Invitrogen, USA) and Hoechst 33342 (Invitrogen, H1398, US) prior to fluorescent analysis via CLSM.

Cellular uptake assays

Zr-TCPP(TPP)/R837@M NP uptake was measured via flow cytometry (Beckman CytoFLEX; Beckman Coulter, Inc.). Briefly, 4T1 cells (3 × 105/well in 2 mL) were added to 6-well plates for 24 h, after which media was exchanged for media supplemented with Zr-TCPP(TPP)/R837@M (20 µg/mL). Following incubation for 4, 8, 12, or 24 h, cells were rinsed using PBS and assessed via flow cytometry (EX = 405 nm, EM = 780 nm).

Homologous targeting analyses

Four different tumor cell types (4T1, MDA-MB-468, Hepa1-6, and Bxpc-3) were plated at 2 × 104/well in 200 μL of media in 6-well chambered coverglass (Corning, USA). Zr-TCPP(TPP)/R837 and Zr-TCPP(TPP)/R837@M were then incubated with these cell lines for 8 h, after which they were harvested using trypsin, digested twice with PBS, and examined via flow cytometry.

Evaluation of the 1O2 Generation Ability of Zr-TCPP(TPP)/R837@M

A fluorescence spectrometer and CLSM were utilized to assess 1O2 generation via a singlet oxygen sensor green (SOSG) assay (MA0326, Meilune, China) and DCFH-DA (Meilune, MB4682, China), respectively. For fluorescence spectrometry, Zr-TCPP(TPP)/R837@M was combined with SOSG (5 μM) in PBS, and the resultant SOSG + Zr-TCPP(TPP)/R837@M preparation (0.1 mg/mL) was sonicated under US irradiation (1 MHz, 1 W/cm2, duty cycle 50%, Sonicator 740, Mettler Electronics, USA) for a range of time periods (0, 10 s, 30 s, 1 min, 2 min, and 3 min). SOSG fluorescence was then measured at respective excitation and emission wavelengths of 504 and 525 nm (Tecan Spark, Tecan, Switzerland).

In CLSM analyses, 4T1 cells were treated for 8 h with Zr-TCPP(TPP)/R837@M (50 mg/L), followed by the addition of DCFH-DA (200 µL, 10 µM). Cells were then incubated for 30 min, washed three times with PBS, and subjected to US stimulation (1 MHz, 1 W/cm2, duty cycle 50%, 1 min). After being fixed for 15 min in 4% paraformaldehyde at room temperature, cells were washed thrice with PBS, stained for 5 min with DAPI, and washed three more times with PBS. A CLSM (Leica Microsystems, Wetzlar, Germany) was then employed to measure ROS generation.

Evaluation of ICD induction

CRT exposure was measured by immunofluorescence staining and flow cytometry. Briefly, 4T1 cells were cultured with different treatments for 24 h, incubated with anti-Calreticulin antibody (ab92516, Abcam), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil, C1036, Beyotime) and Alexa Fluor® 488 Conjugate (4412S, CST), then tested by CLSM and flow cytometry. HMGB1 release was accessed through immunofluorescence staining and ELISA. 4T1 cells were cultured with different treatments for 48 h, incubated with anti-HMGB1 antibody (ab18256, Abcam), Alexa Fluor® 488 Conjugate (4412S, CST), and DAPI Staining Solution (C1006, Beyotime), then tested by CLSM, the cell supernatants were collected for HMGB1 release (ARG81351, arigo) and ATP release (S0027, Beyotime).

In vitro cytotoxicity assay

Cellular cytotoxicity was assessed via CCK-8 assay (BS350B, Biosharp). Briefly, 4T1 cells were added to 96-well plates for 12 h, followed by treatment for 24 h with Zr-TCPP(TPP)/R837@M at a range of doses (0, 12.5, 25, 50, 100, 200 μg/mL). CCK-8 assays were then conducted, with absorbance being assessed at 450 nm via microplate reader (Tecan, Switzerland).

Tumor targeting and tissue distribution

Female Balb/c mice (Shanghai SLAC Laboratory Animal Co, Ltd.) were utilized in all animal studies, which were approved by the Animal Laboratory of the Second Affiliated Hospital of Zhejiang University (permit no. 2021-118). A murine tumor model was established by subcutaneously implanting 4T1 cells in the breast pads of these mice when they were 6–8 weeks old, followed by intravenous treatment with 200 μL of Zr-TCPP(TPP)/R837@M or Zr-TCPP(TPP)/R837 (5 mg/kg) as appropriate. Tissue distributions in these animals were assessed by euthanizing them at appropriate time points and collecting tumors and major organs (heart, lungs, kidney, liver, spleen). An IVIS Spectrum imaging system (PerkinElmer, USA) was then utilized to conduct a real-time region of interest (ROI) fluorescence intensity analysis for Zr-TCPP(TPP)/R837@M at 0, 4, 8, 12, 20, 24, 36 and 48 h.

Combination of SDT and ICB antitumor treatment

Primary model tumors were established by subcutaneously implanting 5 × 105 4T1 cells suspended in PBS into the right mammary fat pads of Balb/c mice, followed 6 days later by the implantation of the same number of tumor cells into the left mammary pads of these same animals to establish a bilateral tumor model. Tumor-bearing mice were then randomized into the following treatment groups: (1) PBS, (2) US, (3) Zr-TCPP(TPP) @M + US (4) Zr-TCPP(TPP) @M + US + anti- CTLA-4, (5) Zr-TCPP(TPP)/R837 @M + US, (6) Zr-TCPP(TPP) @M + US + anti- CTLA-4, and (7) Zr-TCPP@M + US + anti-CTLA-4. All US irradiation was performed at 24 h post-injection using identical treatment settings (3 MHz, 1.5 W/cm2, duty cycle 50%, Sonicator 740, Mettler Electronics, USA). For combination ICB treatment, mice were injected with anti-CTLA-4 (50 μg/mouse) on days 1, 3, and 5. Tumor volumes were measured as follows: (width2 × length)/2. At the experimental endpoint, mice were euthanized and tumors were collected and imaged. Mice were additionally euthanized when tumors were larger than 1000 mm3 as per standard protocols.

An orthotopic 4T1 tumor model was established by implanting mice with 4T1-Luci cells (5 × 105) in the mammary fat pad, as above, followed by intravenous tail vein administration of additional 4T1-Luci cells (1 × 105) 6 days later. Seven days after incubation of the orthotopic tumor, orthotopic tumors were ~ 50 mm3 in size, primary tumors were subjected to surgical treatment with or without anti-CTLA-4 administration. An appropriate substrate was injected into mice before bioluminescent imaging was conducted with an IVIS spectrum system.

Immunological memory was assessed by subcutaneously implanting 1 × 106 4T1 cells in PBS into the right mammary fat pad of individual female Balb/c mice. After these primary tumors had grown to ~ 50 mm3, they were surgically removed. After 40 days, mice were implanted with a secondary tumor and were intravenously injected with anti-CTLA-4 at appropriate time points (50 μg/mouse).

In vivo immune response analysis

A total of 30 orthotopic tumor-bearing mice were selected and randomly assigned to the control, R837, Zr-TCPP(TPP)/R837@M, Zr-TCPP@M + US, Zr-TCPP(TPP)/R837@M + US, and Zr-TCPP/R837@M + US groups. Following treatment, samples of tumors and tumor-draining lymph nodes (DLNs) were collected and homogenized to produce single-cell suspensions. Cells in blood and tumor samples were stained using the following reagents prior to flow cytometry analysis: Zombie NIR™ Fixable Viability Kit (423105, Biolegend, UK), Brilliant Violet 605™ anti-mouse CD45 (103139, Biolegend, UK), PE/Cyanine7 anti-mouse CD3 (100220, Biolegend, UK), Brilliant Violet 421™ anti-mouse CD4 (100438, Biolegend, UK), Alexa Fluor® 647 anti-mouse Foxp3 (126408, Biolegend, UK).

Cells collected from DLNs were stained with the following reagents prior to flow cytometry analysis: Zombie Aqua™ Fixable Viability Kit (423101, Biolegend, UK), Brilliant Violet 421™ anti-mouse CD11c (117329, Biolegend, UK), PE/Cyanine7 anti-mouse CD80 (104733, Biolegend, UK), Brilliant Violet 785™ anti-mouse CD86 (105043, Biolegend, UK).

Immune cell infiltration in secondary tumors was assessed by collecting these cells, preparing a single-cell suspension as above, and staining them with the following reagents prior to flow cytometry analysis: Zombie NIR™ Fixable Viability Kit (423105, Biolegend, UK), Brilliant Violet 605™ anti-mouse CD45 (103139, Biolegend, UK), PE/Cyanine7 anti-mouse CD3 (100220, Biolegend, UK), Brilliant Violet 421™ anti-mouse CD4 (100438, Biolegend, UK), Brilliant Violet 785™ anti-mouse CD8a (100750, Biolegend, UK), Alexa Fluor® 647 anti-mouse Foxp3 (126408, Biolegend, UK).

Memory T cells were analyzed in single-cell suspensions prepared from homogenized lymph nodes stained with the following reagents prior to flow cytometry analysis: Zombie NIR™ Fixable Viability Kit (423105, Biolegend, UK), PE/Cyanine7 anti-mouse CD3 (100220, Biolegend, UK), Brilliant Violet 421™ anti-mouse CD4 (100438, Biolegend, UK), Brilliant Violet 510™ anti-mouse CD8a (100752, Biolegend, UK), APC anti-mouse CD62L (104412, Biolegend, UK) and PE anti-mouse/human CD44 (103008, Biolegend, UK). Central memory and effector memory T cells (TCM and TEM) exhibited respective CD3+CD8+CD62L+CD44+ and CD3+CD8+CD62LCD44+ staining profiles. All antibodies used for these staining assays were diluted 1:100.

Cytokine detection

Samples of murine serum were collected and diluted to appropriate concentrations. ELISA kits were used to measure levels of tumor necrosis factor (SEKM-0034, Solarbio), IL-12p40 (SEKM-0012, Solarbio), and IL-6 (1210602, Dakewe biotech) in these samples.

Statistical analysis

Data are means ± standard deviation and were assessed using one-way ANOVAs with Tukey’s post-hoc correction for multiple testing or Dunnett’s multiple comparisons test. ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.


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