Nickel 2 nitrate hexahydrate

Materials: 2-methylimidazole (MIM, 99.0%), cobalt nitrate hexahydrate (Co(NO₃)₂·6H₂O, 98.0%), nickel(II) nitrate hexahydrate ((Ni(NO₃)₂·6H₂O, 98.0%), and sodium hexachloroplatinate(IV) hexahydrate (Na₂PtCl₆·6H₂O, 99.0%) were purchased from Sigma-Aldrich. Hoechst 33342, ThiolTrace Violet, 2,7-dichlorofluorescein diacetate (DCFH-DA), and Liperfluo were purchased from Dojindo. BODIPYTM 581/591 C11, Tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and Dulbecco's Modified Eagle medium (RPMI-1640) were purchased from Thermo Fisher Scientific. Cell Counting Kit-8 (CCK-8), CFDA SE Cell Proliferation and Cell Tracking Kit, Cell Cycle and Apoptosis Analysis Kit, Calcein-AM/PI Double Stain Kit, Rhod-2, AM, Cell Permeant, ER-Tracker Green, MitoTracker Green FM, and LysoTracker Green DND-26 were acquired from Yeasen, China. GSH and GSSG Assay Kits, Total Antioxidant Capacity Assay Kit using the ABTS technique, CellTiter-Lumi Luminescent 3D Cell Viability Assay Kit, BeyoClic EdU Cell Proliferation Kit with Alexa Fluor 647, Mitochondrial Permeability Transition Pore Assay Kit (MPTP Assay Kit), Mito-Tracker Deep Red FM, Intracellular Iron Colorimetric Assay Kit, and Lipid Peroxidation MDA Assay Kit were purchased from Beyotime, China. The anti-gamma γ-H2A.X, cGAS, STING, p-STING, TBK1, p-TBK1, p65, p-p65, IRF3, and p-IRF3 antibody were purchased from Abcam, and the GPX4, XBP1S, LC-3 antibody were obtained from Proteintech. BV510-anti-mouse CD45, PE/CY7-anti-mouse CD80, PE-anti-mouse CD86, APC-anti mouse CD11c, BV421-anti-mouse CD44, PE-CY7-anti-mouse CD26L, BV605-anti-mouse CD11b, BV421-anti-mouse Foxp3, PE-anti-mouse F4/80, FITC-anti-mouse CD3, APC-anti-mouse CD8a and PE-anti-mouse CD8a antibody were purchased from BioLegend. RMP1-14 and DiR were purchased from MCE.
Synthesis of ZIF-67. Co(NO₃)₂·6H₂O (0.3 mmol, 87.3 mg) was dissolved in methanol (4 mL), denoted as solution A. 2-methylimidazole (1.8 mmol, 147.8 mg) was dissolved in methanol (4 mL), denoted as solution B. After sonication for 5 min, solution B was added to solution A. The mixture was allowed to stand for 3 h. The precipitated product was collected by centrifugation (8000 rpm) and washed three times with methanol.
Synthesis of Pt-ZIF-67. During the cation exchange process, 10 mg of ZIF-67 nanocrystals were dispersed in 5 mL of deionized water. Then 1.1 mg (2 mmol L-1) of sodium hexachloroplatinate (IV) hydrate (Na₂PtCl₆·6H₂O) was dissolved in 1 mL of deionized water and slowly added to the ZIF-67 solution under stirring conditions. The reaction was quenched after 3 h by centrifuging the suspension. The Pt-ZIF-67 precipitate was collected and washed twice with deionized water and three times with ethanol. Pt-ZIF-67 nanocrystals were obtained by drying the precipitate in a vacuum oven overnight at 60 °C.
Synthesis of Pt-CoNi LDH. First, 12 mg of Pt-ZIF-67 was dispersed in 20 mL of ethanol, designated as solution A. Then 20 mL of an ethanol solution containing 24 mg of Ni(NO₃)₂·6H₂O was prepared and denoted as B. Solution B was quickly added to solution A. The resulting mixture was stirred at room temperature for 10 min, and then heated to 80 °C for 5 min. The product was centrifuged and washed several times with methanol and dried at 40 °C overnight.
Erythrocyte Membrane Extraction. Erythrocyte membranes were isolated using differential centrifugation. Briefly, fresh blood was collected from BALB/c mice and centrifuged at 1000 × g for 5 min at 4 °C to remove the buffy coat and plasma. The resulting RBCs were washed twice with 1×PBS and then suspended in 0.25×PBS to induce lysis through hypotonic treatment. Subsequent centrifugation at 15,000 × g for 7 min removed hemoglobin and other cytoplasmic components, yielding a pink precipitate. The RBC membranes were then collected, and their protein concentrations were quantified using a BCA assay (Thermo Fisher Scientific Inc.).
Synthesis of RmPLH. Erythrocyte membranes were prepared by sonicating the Erythrocyte membranes and Pt-CoNi LDH at a 1:4 ratio in a bath sonicator for 6 min. The resulting mixture was then sequentially extruded through polycarbonate membranes with pore sizes of 1 μm, and 400 nm (Whatman, UK) using an Avanti Polar Lipids mini extruder.
Analysis and Characterization:A scanning electron microscope SEM (MAIA 3 LMH, Czech Republic) was used to study the morphology of the samples. High-resolution TEM (HRTEM) images were acquired usingan FEI Tecnai F20 microscope operating at 200 kV. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and energy-dispersive X-ray spectroscopy (EDS) elemental mapping images were obtained at 200 kV by using a JEOL JEM-NeoAEM-200F TEM. X-ray diffraction (XRD) patterns were measured using a Rigaku SmartLab diffractometer with Cu Kα radiation. X-ray photoelectron spectroscopy (XPS) was performed using a PHI 5000 VersaProbe III system. Fourier transform infrared (FTIR) spectra were recorded on a Thermo Scientific (Nicolet iS10, USA) spectrometer. The hydration kinetic diameter (Dh) and zeta potential were determined using dynamic light scattering (DLS; Brookhaven Instruments Omni).
Analysis of Cellular Uptake: 4T1 cells were initially cultured in a confocal dish until they reached 80% confluence. We primarily adsorb RBITC onto PLH through physical adsorption, followed by encapsulation with a red blood cell membrane. We take the required amount of RBITC at a ratio of 1mg PLH to 0.01mg RBITC, and dissolve RBITC in DMSO solution at a ratio of 1mg RBITC to 1mL DMSO. The dissolved RBITC solution is then added to the PLH solution and stirred continuously. Under the influence of physical adsorption forces, RBITC adsorbs onto PLH. After stirring for 24 hours, the liquid is collected and centrifuged, and thoroughly washed until the supernatant after centrifugation is colorless and transparent, leaving the PLH-RBITC precipitate for further use. Subsequently, the cells were treated with TRITC-labelled RmPLH to induce phagocytosis. The cells were analyzed at designated intervals throughout the incubation period. The phagocytic rate of RmPLH in 4T1 cells was assessed by nuclear staining with Hoechst 33342, followed by observation CLSM. Upon reaching 80% confluence in the confocal plate, 4T1 cells were exposed to TRITC-labeled RmPLH and allowed to co-localize for 12 Green fluorescent probes were employed to visualize and image various cellular organelles, including the ER, mitochondria, and lysosomes, for subsequent analysis of their co-localization. These probes ncluded ER-Tracker Green (BODIPY FL Glibenclamide) for ER visualization, Mito-Tracker Green (Benzoxazolium) for mitochondria, and LysoSensor Green DND-189 for lysosomal visualization. CLSM was utilized to quantify cellular uptake rates, while TEM was employed to investigate the intracellular localization and distribution of RmPLH within 4T1 cells. The 4T1 cells were seeded in 6-well plates at a density of 1×10⁵ cells/well and cultured for 24 h. Fresh medium containing PLH and RmPLH was added to the plates, and the cells were co-incubated for 1 and 4 h, respectively. After incubation, the excess medium was removed, and the cells were rinsed three times with ice-cold PBS. Subsequently, the cells were fixed in 4% paraformaldehyde and stained with DAPI for 30 min to enable laser confocal cell imaging. The cells were observed and photographed using CLSM at various time points.
In vitro cytotoxicity evaluation: 4T1 cells were sourced from the American Type Culture Collection, and were cultured in high glucose RPMI-1640 supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). Cell viability was assessed using the CCK-8 assay (Yeason Biotechnology, China) following the manufacturer's instructions. Briefly, 96-well plates were seeded overnight with 5×10³ 4T1 cells. After treatment, the cells in the US group were subjected to US exposure at 1 W/cm² for 2 min. Subsequently, cell viability was quantified by measuring optical density (OD) at 450 nm using a SpectraMax microplate reader(Model 680; Bio-Rad Laboratories, Inc, Tokyo, Japan). For subsequent experiments, 4T1 cells were seeded in 6-well plates and treated with RmPLH for 12h after reaching 80% confluence. Cell viability was further assessed using Calcein-AM/PI staining. Following RmPLH treatment, the cells were subjected to various interventions, including ultrasonic therapy(1 W/cm², 2 min). After an additional 4-hour incubation period, cells were stained with Calcein-AM/PI for 25 min at room temperature in the dark, washed with PBS, and subsequently analyzed using CLSM. The concentration of RmPLH employed in these experiments was 20 μg/mL.
3D cell culture:To minimize adsorption, cell culture was conducted using ultralow adsorption culture plates. Upon the formation of cellular spheres, various drug treatments were administered according to the assigned experimental groups. Following a 12-hour incubation period, images of the cellular spheres within each group were acquired, with the ultrasonic group subjected to US treatment at a previously determined power level. Subsequently, the cells were incubated for an additional 6 h, after which images were captured, and cell viability in the different treatment groups was assessed using the CellTiter-Lum Luminescence 3D Cell Viability Detection Kit.
In vitro oxidative stress:Initially, 4T1 cells were seeded in a confocal dish and allowed to proliferate until reaching a density of 90%. Subsequently, various drugs were added according to the experimental design, and the cells were incubated for 12 h. Following this incubation period, US treatment was applied to the cells, followed by an additional 6-hour incubation. Intracellular levels of ROS, mitochondrial membrane potential, mitochondrial permeability transition pore (MPTP) opening, mitochondrial calcium ion concentration, and mitochondrial mass were assessed using fluorescent probes such as DCFH-DA and JC-1, following the protocols provided by the respective manufacturers. The nuclei were stained with Hoechst 33342. Finally, the results were analyzed using CLSM or flow cytometry techniques.
Cell transwell analysis: Cells were cultured in an appropriate culture medium. Upon reaching the logarithmic growth phase, the cells were washed with PBS and digested with trypsin to prepare a single-cell suspension. The cell concentration was adjusted to 1×10⁶ cells/mL. For invasion assays, 100 µL of Matrigel was added to the upper layer of the Transwell chamber and incubated at 37 °C for 30 min to allow the Matrigel to solidify; this step was omitted for migration experiments. Subsequently, 200 µL of the serum-free cell suspension was added to the upper chamber of the Transwell apparatus, while 600 µL of complete culture medium containing 20% FBS was added to the lower chamber. The Transwell plate was then incubated at 37 °C with 5% CO₂ for 24 h. After incubation, the Transwell chamber was removed, and the cells in the upper chamber were gently washed with PBS and fixed with 4% paraformaldehyde for 15 min. Following fixation, cells were stained with 0.1% crystal violet for 30 min. Non-invading cells in the upper chamber were gently removed using a cotton swab. The invasive cells on the membrane surface of the lower chamber were observed and counted using a microscope.
Western blotting:Cell lysates were electrophoretically separated and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes. Following overnight incubation at 4 °C with primary antibodies, membranes were subjected to blocking with Tris-buffered saline (TBS) supplemented with 5% skim milk. After three washes in TBS-Tween 20 (TBST) buffer, the membranes were incubated with secondary antibodies at room temperature for 45 min. Subsequently, the membranes were washed five times in TBS buffer containing 0.1% Tween-20 before images were captured using the ChemDoc Imaging system (Bio-Rad).
Evaluation of IFN-β secretion level: 4T1 cells were seeded into 96-well plates at a density of 8×10³ cells per well and cultured in a constant temperature incubator at 37 °C with 5% CO₂ for 24 h. Subsequently, the cells were cultured for an additional 12 h after undergoing various treatments. The release of IFN-β was quantified using the cytotoxicity assay kit as per the manufacturer's instructions.
Immunofluorescence Staining:4T1 cells were cultured in 12-well plates until reaching approximately 80% confluence. Subsequently, cells were allocated into distinct groups and administered either a placebo or medication. The US group received a 2-minute US therapy session using a machine operating at 1 W/cm². After treatment, the cells were fixed with 4% paraformaldehyde overnight, followed by permeabilization with 0.2% Triton X-100 on ice for 20 min. This was followed by three 10-minute rinses in 0.01 M phosphate-buffered saline (PBS). The cells were then blocked with 2% bovine serum albumin for 1 h at room temperature, rinsed three times with PBS (10 min each), and incubated overnight at 4 °C with rabbit anti-GPX4 (1:200; Abcam) and rabbit anti-H2A.X (1:100; Abcam) primary antibodies. The following day, the cells were treated with Alexa Fluor 488 donkey anti-rabbit antibody (1:1000; Abcam) at room temperature 1 h, followed by three washes with 0.01 M PBS. After fixation in a DAPI-containing medium and three additional PBS washes, the cells were subjected to CLSM for three-dimensional reconstruction.
Detection of intracellular lipid oxidation and antioxidant capacity:4T1 cells were seeded at a density of 6 × 10⁵ cells per well in 6-well dishes and treated with RmPLH at a concentration of 20 μg/mL for a duration of 12 h. Subsequently, the cells were subjected to US treatment using a machine operating at 1 W/cm2 for 2 min. Following an additional 6-hour incubation period, changes in intracellular antioxidant capacity were assessed usingthe GSH and GSSG Assay Kit as well as the Total Antioxidant Capacity Assay Kit employing the ABTS method, adhering to the guidelines provided by the reagent manufacturer. The degree of lipid peroxidation was evaluated using BODIPY C11 and Liperfluo fluorescent probes, and the mean fluorescence intensity after treatment was quantified using CLSM. Additionally, the degree of lipid peroxidation was quantitatively assessed using Lipid Peroxidation MDA Assay Kit.
In Vivo Targeting and Imaging:To create a subcutaneous tumor model, female BALB/c mice (6-8 weeks old) were obtained from the Xiamen University Experimental Animal Centre. The mice received a subcutaneous injection of 4T1 cells (1×10⁶ cells in 100 μL). When the tumor volumes reached 100 mm³, the mice were intravenously injected with 20 mg/kg of DiR-labeled RmPLH. Following an esthesia, fluorescence imaging was performed at 1, 4, 8, 12, 24, 48, 72, 96, 120, and 144 h using a PerkinElmer IVIS Lumina III instrument. Tumors and major organs were harvested 144 h after the mice were implanted with fluorescent tags, and the animals were euthanized for ex vivo fluorescence imaging.
The Efficacy of RmPLH for Cancer: To evaluate the anticancer effect of SDT on RmPLH, a subcutaneous tumor model was established in female BALB/c mice. Once the average tumor volume reached 100 mm³. The tumor-bearing mice were randomized into four treatment groups: (I) saline, (II) ultrasound (US), (III) RmPLH, and (IV) RmPLH + US. Each mouse received a tail vein injection of 100 μL of the assigned treatment, with RmPLH administered at a dosage of 20 mg/kg. 24 hours post-injection, the tumor sites in the respective group received US treatment at a frequency of 1.0 MHz, and an intensity of 1.5 W/cm², for 2 min. Throughout the experiment, the mice were weighed twice daily, and tumor dimensions (length [L] and width [W]) were measured using Vernier calipers and a microelectronic scale. The tumor volume (V) was calculated using the formula: V = L × W² / 2. After 14 days of treatment, tumor tissues and major organs were harvested from the euthanized mice. The harvested tumor tissues were subjected to H&E staining for further histopathological analysis.
Preparation of Single-Cell Suspensions from Mouse Tissue and Flow Cytometry Analysis Procedure: First, the mice were euthanized, and the target tissues, such as the spleen or lymph nodes, were harvested. The tissue was placed in an ice-cold PBS buffer and scissors and forceps were used to mince the tissue. Next, the minced tissue was transferred in to a 50 mL centrifuge tube with a 200-mesh filter. The tissue was rinsed with PBS repeatedly until it was fully dispersed into a single-cell suspension. The single-cell suspension was collected into a new centrifuge tube and centrifuged at 300 × g for 5 min. The supernatant was discarded and the cell pellet was resuspended in PBS and centrifuged again. The resuspended cell suspension was passed through a 40 µm filter specific for flow cytometry to ensure that no large cell clumps or tissue fragments were remaining. Next, the cell suspension was treated with 1 mL of red blood cell lysis buffer to remove the red blood cells. The suspension was incubated at room temperature for 5 min after which 10 mL of PBS was immediately added to stop the reaction followed by centrifugation at 300 × g for 5 min. The supernatant was discarded, and the cell pellet was resuspended in PBS. These washing steps were repeated once more. For flow cytometry staining, 100 µL of resuspended cells were added to each flow tube, with approximately 1 ×10⁶ cells per tube. An appropriate amount of the antibody mixture was added to each flow tube and incubated on ice, protected from light, for 30 min. After staining, the cells were washed twice with PBS and centrifuged at 300 × g for 5 min. Finally, the cells were resuspended in an appropriate amount of flow cytometry analysis buffer and analyzed using a flow cytometer. Data collection and interpretation were performed using FlowJo software.
In Vivo Biosafety Assessment: The obtained organs were fixed and H&E staining was applied to the tissue slices. Blood was drawn from each group, and standard blood analysis was performed.
All animal experiments were approved by the Xiamen University Ethics Committees (XMULAC20230200).
Statistical Analysis: All values are expressed as mean ± SD, and the significance of the data is based on Student's t-test, one-way analysis of variance (ANOVA), and two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

Materials: 2-methylimidazole (MIM, 99.0%), cobalt nitrate hexahydrate (Co(NO₃)₂·6H₂O, 98.0%), nickel(II) nitrate hexahydrate ((Ni(NO₃)₂·6H₂O, 98.0%), and sodium hexachloroplatinate(IV) hexahydrate (Na₂PtCl₆·6H₂O, 99.0%) were purchased from Sigma-Aldrich. Hoechst 33342, ThiolTrace Violet, 2,7-dichlorofluorescein diacetate (DCFH-DA), and Liperfluo were purchased from Dojindo. BODIPYTM 581/591 C11, Tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and Dulbecco's Modified Eagle medium (RPMI-1640) were purchased from Thermo Fisher Scientific. Cell Counting Kit-8 (CCK-8), CFDA SE Cell Proliferation and Cell Tracking Kit, Cell Cycle and Apoptosis Analysis Kit, Calcein-AM/PI Double Stain Kit, Rhod-2, AM, Cell Permeant, ER-Tracker Green, MitoTracker Green FM, and LysoTracker Green DND-26 were acquired from Yeasen, China. GSH and GSSG Assay Kits, Total Antioxidant Capacity Assay Kit using the ABTS technique, CellTiter-Lumi Luminescent 3D Cell Viability Assay Kit, BeyoClic EdU Cell Proliferation Kit with Alexa Fluor 647, Mitochondrial Permeability Transition Pore Assay Kit (MPTP Assay Kit), Mito-Tracker Deep Red FM, Intracellular Iron Colorimetric Assay Kit, and Lipid Peroxidation MDA Assay Kit were purchased from Beyotime, China. The anti-gamma γ-H2A.X, cGAS, STING, p-STING, TBK1, p-TBK1, p65, p-p65, IRF3, and p-IRF3 antibody were purchased from Abcam, and the GPX4, XBP1S, LC-3 antibody were obtained from Proteintech. BV510-anti-mouse CD45, PE/CY7-anti-mouse CD80, PE-anti-mouse CD86, APC-anti mouse CD11c, BV421-anti-mouse CD44, PE-CY7-anti-mouse CD26L, BV605-anti-mouse CD11b, BV421-anti-mouse Foxp3, PE-anti-mouse F4/80, FITC-anti-mouse CD3, APC-anti-mouse CD8a and PE-anti-mouse CD8a antibody were purchased from BioLegend. RMP1-14 and DiR were purchased from MCE.
Synthesis of ZIF-67. Co(NO₃)₂·6H₂O (0.3 mmol, 87.3 mg) was dissolved in methanol (4 mL), denoted as solution A. 2-methylimidazole (1.8 mmol, 147.8 mg) was dissolved in methanol (4 mL), denoted as solution B. After sonication for 5 min, solution B was added to solution A. The mixture was allowed to stand for 3 h. The precipitated product was collected by centrifugation (8000 rpm) and washed three times with methanol.
Synthesis of Pt-ZIF-67. During the cation exchange process, 10 mg of ZIF-67 nanocrystals were dispersed in 5 mL of deionized water. Then 1.1 mg (2 mmol L-1) of sodium hexachloroplatinate (IV) hydrate (Na₂PtCl₆·6H₂O) was dissolved in 1 mL of deionized water and slowly added to the ZIF-67 solution under stirring conditions. The reaction was quenched after 3 h by centrifuging the suspension. The Pt-ZIF-67 precipitate was collected and washed twice with deionized water and three times with ethanol. Pt-ZIF-67 nanocrystals were obtained by drying the precipitate in a vacuum oven overnight at 60 °C.
Synthesis of Pt-CoNi LDH. First, 12 mg of Pt-ZIF-67 was dispersed in 20 mL of ethanol, designated as solution A. Then 20 mL of an ethanol solution containing 24 mg of Ni(NO₃)₂·6H₂O was prepared and denoted as B. Solution B was quickly added to solution A. The resulting mixture was stirred at room temperature for 10 min, and then heated to 80 °C for 5 min. The product was centrifuged and washed several times with methanol and dried at 40 °C overnight.
Erythrocyte Membrane Extraction. Erythrocyte membranes were isolated using differential centrifugation. Briefly, fresh blood was collected from BALB/c mice and centrifuged at 1000 × g for 5 min at 4 °C to remove the buffy coat and plasma. The resulting RBCs were washed twice with 1×PBS and then suspended in 0.25×PBS to induce lysis through hypotonic treatment. Subsequent centrifugation at 15,000 × g for 7 min removed hemoglobin and other cytoplasmic components, yielding a pink precipitate. The RBC membranes were then collected, and their protein concentrations were quantified using a BCA assay (Thermo Fisher Scientific Inc.).
Synthesis of RmPLH. Erythrocyte membranes were prepared by sonicating the Erythrocyte membranes and Pt-CoNi LDH at a 1:4 ratio in a bath sonicator for 6 min. The resulting mixture was then sequentially extruded through polycarbonate membranes with pore sizes of 1 μm, and 400 nm (Whatman, UK) using an Avanti Polar Lipids mini extruder.
Analysis and Characterization:A scanning electron microscope SEM (MAIA 3 LMH, Czech Republic) was used to study the morphology of the samples. High-resolution TEM (HRTEM) images were acquired usingan FEI Tecnai F20 microscope operating at 200 kV. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and energy-dispersive X-ray spectroscopy (EDS) elemental mapping images were obtained at 200 kV by using a JEOL JEM-NeoAEM-200F TEM. X-ray diffraction (XRD) patterns were measured using a Rigaku SmartLab diffractometer with Cu Kα radiation. X-ray photoelectron spectroscopy (XPS) was performed using a PHI 5000 VersaProbe III system. Fourier transform infrared (FTIR) spectra were recorded on a Thermo Scientific (Nicolet iS10, USA) spectrometer. The hydration kinetic diameter (Dh) and zeta potential were determined using dynamic light scattering (DLS; Brookhaven Instruments Omni).
Analysis of Cellular Uptake: 4T1 cells were initially cultured in a confocal dish until they reached 80% confluence. We primarily adsorb RBITC onto PLH through physical adsorption, followed by encapsulation with a red blood cell membrane. We take the required amount of RBITC at a ratio of 1mg PLH to 0.01mg RBITC, and dissolve RBITC in DMSO solution at a ratio of 1mg RBITC to 1mL DMSO. The dissolved RBITC solution is then added to the PLH solution and stirred continuously. Under the influence of physical adsorption forces, RBITC adsorbs onto PLH. After stirring for 24 hours, the liquid is collected and centrifuged, and thoroughly washed until the supernatant after centrifugation is colorless and transparent, leaving the PLH-RBITC precipitate for further use. Subsequently, the cells were treated with TRITC-labelled RmPLH to induce phagocytosis. The cells were analyzed at designated intervals throughout the incubation period. The phagocytic rate of RmPLH in 4T1 cells was assessed by nuclear staining with Hoechst 33342, followed by observation CLSM. Upon reaching 80% confluence in the confocal plate, 4T1 cells were exposed to TRITC-labeled RmPLH and allowed to co-localize for 12 Green fluorescent probes were employed to visualize and image various cellular organelles, including the ER, mitochondria, and lysosomes, for subsequent analysis of their co-localization. These probes ncluded ER-Tracker Green (BODIPY FL Glibenclamide) for ER visualization, Mito-Tracker Green (Benzoxazolium) for mitochondria, and LysoSensor Green DND-189 for lysosomal visualization. CLSM was utilized to quantify cellular uptake rates, while TEM was employed to investigate the intracellular localization and distribution of RmPLH within 4T1 cells. The 4T1 cells were seeded in 6-well plates at a density of 1×10⁵ cells/well and cultured for 24 h. Fresh medium containing PLH and RmPLH was added to the plates, and the cells were co-incubated for 1 and 4 h, respectively. After incubation, the excess medium was removed, and the cells were rinsed three times with ice-cold PBS. Subsequently, the cells were fixed in 4% paraformaldehyde and stained with DAPI for 30 min to enable laser confocal cell imaging. The cells were observed and photographed using CLSM at various time points.
In vitro cytotoxicity evaluation: 4T1 cells were sourced from the American Type Culture Collection, and were cultured in high glucose RPMI-1640 supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). Cell viability was assessed using the CCK-8 assay (Yeason Biotechnology, China) following the manufacturer's instructions. Briefly, 96-well plates were seeded overnight with 5×10³ 4T1 cells. After treatment, the cells in the US group were subjected to US exposure at 1 W/cm² for 2 min. Subsequently, cell viability was quantified by measuring optical density (OD) at 450 nm using a SpectraMax microplate reader(Model 680; Bio-Rad Laboratories, Inc, Tokyo, Japan). For subsequent experiments, 4T1 cells were seeded in 6-well plates and treated with RmPLH for 12h after reaching 80% confluence. Cell viability was further assessed using Calcein-AM/PI staining. Following RmPLH treatment, the cells were subjected to various interventions, including ultrasonic therapy(1 W/cm², 2 min). After an additional 4-hour incubation period, cells were stained with Calcein-AM/PI for 25 min at room temperature in the dark, washed with PBS, and subsequently analyzed using CLSM. The concentration of RmPLH employed in these experiments was 20 μg/mL.
3D cell culture:To minimize adsorption, cell culture was conducted using ultralow adsorption culture plates. Upon the formation of cellular spheres, various drug treatments were administered according to the assigned experimental groups. Following a 12-hour incubation period, images of the cellular spheres within each group were acquired, with the ultrasonic group subjected to US treatment at a previously determined power level. Subsequently, the cells were incubated for an additional 6 h, after which images were captured, and cell viability in the different treatment groups was assessed using the CellTiter-Lum Luminescence 3D Cell Viability Detection Kit.
In vitro oxidative stress:Initially, 4T1 cells were seeded in a confocal dish and allowed to proliferate until reaching a density of 90%. Subsequently, various drugs were added according to the experimental design, and the cells were incubated for 12 h. Following this incubation period, US treatment was applied to the cells, followed by an additional 6-hour incubation. Intracellular levels of ROS, mitochondrial membrane potential, mitochondrial permeability transition pore (MPTP) opening, mitochondrial calcium ion concentration, and mitochondrial mass were assessed using fluorescent probes such as DCFH-DA and JC-1, following the protocols provided by the respective manufacturers. The nuclei were stained with Hoechst 33342. Finally, the results were analyzed using CLSM or flow cytometry techniques.
Cell transwell analysis: Cells were cultured in an appropriate culture medium. Upon reaching the logarithmic growth phase, the cells were washed with PBS and digested with trypsin to prepare a single-cell suspension. The cell concentration was adjusted to 1×10⁶ cells/mL. For invasion assays, 100 µL of Matrigel was added to the upper layer of the Transwell chamber and incubated at 37 °C for 30 min to allow the Matrigel to solidify; this step was omitted for migration experiments. Subsequently, 200 µL of the serum-free cell suspension was added to the upper chamber of the Transwell apparatus, while 600 µL of complete culture medium containing 20% FBS was added to the lower chamber. The Transwell plate was then incubated at 37 °C with 5% CO₂ for 24 h. After incubation, the Transwell chamber was removed, and the cells in the upper chamber were gently washed with PBS and fixed with 4% paraformaldehyde for 15 min. Following fixation, cells were stained with 0.1% crystal violet for 30 min. Non-invading cells in the upper chamber were gently removed using a cotton swab. The invasive cells on the membrane surface of the lower chamber were observed and counted using a microscope.
Western blotting:Cell lysates were electrophoretically separated and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes. Following overnight incubation at 4 °C with primary antibodies, membranes were subjected to blocking with Tris-buffered saline (TBS) supplemented with 5% skim milk. After three washes in TBS-Tween 20 (TBST) buffer, the membranes were incubated with secondary antibodies at room temperature for 45 min. Subsequently, the membranes were washed five times in TBS buffer containing 0.1% Tween-20 before images were captured using the ChemDoc Imaging system (Bio-Rad).
Evaluation of IFN-β secretion level: 4T1 cells were seeded into 96-well plates at a density of 8×10³ cells per well and cultured in a constant temperature incubator at 37 °C with 5% CO₂ for 24 h. Subsequently, the cells were cultured for an additional 12 h after undergoing various treatments. The release of IFN-β was quantified using the cytotoxicity assay kit as per the manufacturer's instructions.
Immunofluorescence Staining:4T1 cells were cultured in 12-well plates until reaching approximately 80% confluence. Subsequently, cells were allocated into distinct groups and administered either a placebo or medication. The US group received a 2-minute US therapy session using a machine operating at 1 W/cm². After treatment, the cells were fixed with 4% paraformaldehyde overnight, followed by permeabilization with 0.2% Triton X-100 on ice for 20 min. This was followed by three 10-minute rinses in 0.01 M phosphate-buffered saline (PBS). The cells were then blocked with 2% bovine serum albumin for 1 h at room temperature, rinsed three times with PBS (10 min each), and incubated overnight at 4 °C with rabbit anti-GPX4 (1:200; Abcam) and rabbit anti-H2A.X (1:100; Abcam) primary antibodies. The following day, the cells were treated with Alexa Fluor 488 donkey anti-rabbit antibody (1:1000; Abcam) at room temperature 1 h, followed by three washes with 0.01 M PBS. After fixation in a DAPI-containing medium and three additional PBS washes, the cells were subjected to CLSM for three-dimensional reconstruction.
Detection of intracellular lipid oxidation and antioxidant capacity:4T1 cells were seeded at a density of 6 × 10⁵ cells per well in 6-well dishes and treated with RmPLH at a concentration of 20 μg/mL for a duration of 12 h. Subsequently, the cells were subjected to US treatment using a machine operating at 1 W/cm2 for 2 min. Following an additional 6-hour incubation period, changes in intracellular antioxidant capacity were assessed usingthe GSH and GSSG Assay Kit as well as the Total Antioxidant Capacity Assay Kit employing the ABTS method, adhering to the guidelines provided by the reagent manufacturer. The degree of lipid peroxidation was evaluated using BODIPY C11 and Liperfluo fluorescent probes, and the mean fluorescence intensity after treatment was quantified using CLSM. Additionally, the degree of lipid peroxidation was quantitatively assessed using Lipid Peroxidation MDA Assay Kit.
In Vivo Targeting and Imaging:To create a subcutaneous tumor model, female BALB/c mice (6-8 weeks old) were obtained from the Xiamen University Experimental Animal Centre. The mice received a subcutaneous injection of 4T1 cells (1×10⁶ cells in 100 μL). When the tumor volumes reached 100 mm³, the mice were intravenously injected with 20 mg/kg of DiR-labeled RmPLH. Following an esthesia, fluorescence imaging was performed at 1, 4, 8, 12, 24, 48, 72, 96, 120, and 144 h using a PerkinElmer IVIS Lumina III instrument. Tumors and major organs were harvested 144 h after the mice were implanted with fluorescent tags, and the animals were euthanized for ex vivo fluorescence imaging.
The Efficacy of RmPLH for Cancer: To evaluate the anticancer effect of SDT on RmPLH, a subcutaneous tumor model was established in female BALB/c mice. Once the average tumor volume reached 100 mm³. The tumor-bearing mice were randomized into four treatment groups: (I) saline, (II) ultrasound (US), (III) RmPLH, and (IV) RmPLH + US. Each mouse received a tail vein injection of 100 μL of the assigned treatment, with RmPLH administered at a dosage of 20 mg/kg. 24 hours post-injection, the tumor sites in the respective group received US treatment at a frequency of 1.0 MHz, and an intensity of 1.5 W/cm², for 2 min. Throughout the experiment, the mice were weighed twice daily, and tumor dimensions (length [L] and width [W]) were measured using Vernier calipers and a microelectronic scale. The tumor volume (V) was calculated using the formula: V = L × W² / 2. After 14 days of treatment, tumor tissues and major organs were harvested from the euthanized mice. The harvested tumor tissues were subjected to H&E staining for further histopathological analysis.
Preparation of Single-Cell Suspensions from Mouse Tissue and Flow Cytometry Analysis Procedure: First, the mice were euthanized, and the target tissues, such as the spleen or lymph nodes, were harvested. The tissue was placed in an ice-cold PBS buffer and scissors and forceps were used to mince the tissue. Next, the minced tissue was transferred in to a 50 mL centrifuge tube with a 200-mesh filter. The tissue was rinsed with PBS repeatedly until it was fully dispersed into a single-cell suspension. The single-cell suspension was collected into a new centrifuge tube and centrifuged at 300 × g for 5 min. The supernatant was discarded and the cell pellet was resuspended in PBS and centrifuged again. The resuspended cell suspension was passed through a 40 µm filter specific for flow cytometry to ensure that no large cell clumps or tissue fragments were remaining. Next, the cell suspension was treated with 1 mL of red blood cell lysis buffer to remove the red blood cells. The suspension was incubated at room temperature for 5 min after which 10 mL of PBS was immediately added to stop the reaction followed by centrifugation at 300 × g for 5 min. The supernatant was discarded, and the cell pellet was resuspended in PBS. These washing steps were repeated once more. For flow cytometry staining, 100 µL of resuspended cells were added to each flow tube, with approximately 1 ×10⁶ cells per tube. An appropriate amount of the antibody mixture was added to each flow tube and incubated on ice, protected from light, for 30 min. After staining, the cells were washed twice with PBS and centrifuged at 300 × g for 5 min. Finally, the cells were resuspended in an appropriate amount of flow cytometry analysis buffer and analyzed using a flow cytometer. Data collection and interpretation were performed using FlowJo software.
In Vivo Biosafety Assessment: The obtained organs were fixed and H&E staining was applied to the tissue slices. Blood was drawn from each group, and standard blood analysis was performed.
All animal experiments were approved by the Xiamen University Ethics Committees (XMULAC20230200).
Statistical Analysis: All values are expressed as mean ± SD, and the significance of the data is based on Student's t-test, one-way analysis of variance (ANOVA), and two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

Commercially available food‐grade corn starch (Maizena by Unilever plc) was obtained from a local supermarket. Analytical grade glycerol plasticizer (>99.5%), glacial acetic acid (>99%), iron (III) nitrate nonahydrate (Fe(NO₃)₃·9H₂O), chromium (III) nitrate nonahydrate (Cr(NO₃)₃·9H₂O), nickel (II) nitrate hexahydrate (Ni(NO₃)₂·6H₂O), cobalt (II) nitrate hexahydrate (Co(NO₃)₂·6H₂O), copper (II) nitrate trihydrate (Cu(NO₃)₂·3H₂O), iron (III) chloride (FeCl₃) (97%) and methylene blue dye were purchased from Sigma‐Aldrich.