An ultrasound-responsive targeted microbubble drug delivery system, its preparation method and application

Abstract

This invention relates to the field of medical engineering technology, specifically disclosing an ultrasound-responsive targeted microbubble drug delivery system, its preparation method, and its applications. The ultrasound-responsive targeted microbubble drug delivery system comprises Var7-modified manganese porphyrin / nigiricin co-loaded microbubbles (vMnp-Nig MBs), which consists of a microbubble interface formed by DSPC, DSPE-PEG2000-Var7, and manganese porphyrin lipids, nigiricin loaded within the microbubbles, and perfluoropropane gas encapsulated within the microbubbles. vMnp-Nig MBs, combined with ultrasound, enable visualized delivery to the tumor site. Nigiricin initiates pyroptosis, and the manganese porphyrin-mediated sonodynamic effect amplifies the pyroptosis cascade, enhancing DAMPs release and further promoting antigen presentation and T-cell-mediated anti-tumor immune activation. This invention transforms TNBC treatment from simple local tumor reduction to a comprehensive treatment modality that combines immune remodeling and metastasis inhibition, providing a new treatment strategy to address the problems of insufficient immune response, high risk of recurrence and metastasis, and lack of effective targeted therapy in TNBC.

Description

Technical Field

[0001] This invention relates to the field of medical engineering technology, specifically to an ultrasound-responsive targeted microbubble drug delivery system, its preparation method, and its application. Background Technology

[0002] Triple-negative breast cancer (TNBC) lacks stable and effective molecularly targeted therapies due to the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HGF-2) expression. Although immune checkpoint blockade (ICB) therapy has been gradually incorporated into clinical treatment, its benefit rate in the overall population remains limited. The combination strategy of chemotherapy-based therapy with immunosuppressive therapy remains the mainstream clinical approach. This situation reflects the main difficulty in treating TNBC: short-term tumor burden reduction is difficult to translate into long-term anti-tumor immune memory, resulting in a high frequency of recurrence and metastasis. Existing research indicates that traditional chemotherapy primarily works by inducing apoptosis, a death pattern typically characterized by low inflammation, making it difficult to trigger the full release of damage-associated molecular patterns (DAMPs), leading to low antigen presentation efficiency and inhibited T cell activation. Therefore, for TNBC with severe immune escape, shifting the treatment strategy from simple cytotoxic killing to a model that emphasizes both direct tumor reduction and immune cascade activation holds promise for overcoming the current treatment challenges.

[0003] Compared to traditional chemotherapy, sonodynamic therapy (SDT) offers better tissue penetration, spatial controllability, and non-invasiveness, making it suitable for local treatment of solid tumors and modulation of the immune microenvironment. Recent studies have begun utilizing nano-sonic sensitizers to induce pyroptosis and synergize it with immunotherapy to amplify anti-tumor effects. However, current strategies primarily focus on direct pyroptosis induction mediated by sonosensitizers, which can lead to insufficient pyroptosis initiation and limited amplification with the inflammatory cascade.

[0004] Unlike apoptosis, pyroptosis is a highly inflammatory programmed cell death process. However, the in vivo application of existing pyroptosis induction strategies is still limited by insufficient tumor selectivity and delivery barriers in solid tumors. The former easily leads to off-target inflammation and systemic toxicity, while the latter weakens drug penetration, local accumulation, and cellular uptake in tumors due to the dense extracellular matrix and barrier microenvironment. Therefore, how to achieve precise delivery of pyroptosis inducers at the tumor site and further amplify the pyroptosis process through exogenous stimulation has become the key to improving the therapeutic efficacy and safety of this strategy.

[0005] Ultrasound-targeted microbubble destruction (UTMD) technology, which triggers controlled drug release via ultrasound, has become an effective means of drug delivery in recent years. As a clinically mature ultrasound imaging platform, microbubbles not only enable real-time visualization and localization but also disintegrate locally under ultrasound, thus achieving spatiotemporally controlled drug release. Simultaneously, the mechanical and cavitation effects associated with UTMD can enhance local vascular and cell membrane permeability, promoting the penetration of therapeutic molecules into deeper tumors and improving uptake efficiency. Recent studies and reviews generally agree that UTMD has unique advantages in local tumor delivery, integrated diagnosis and treatment, and reduced systemic toxicity. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing an ultrasound-responsive targeted microbubble drug delivery system, its preparation method, and its applications.

[0007] To achieve the first objective mentioned above, the technical solution adopted by the present invention is as follows:

[0008] An ultrasound-responsive targeted microbubble drug delivery system, wherein the ultrasound-responsive targeted microbubble drug delivery system comprises Var7-modified manganese porphyrin / nigibacin co-loaded microbubbles vMnp-Nig MBs, including a microbubble interface formed by DSPC, DSPE-PEG2000-Var7 and manganese porphyrin lipids, nigibacin, and perfluoropropane gas encapsulated inside the microbubbles.

[0009] In the ultrasound-responsive targeted microbubble drug delivery system described above, the average particle size of the vMnp-NigMBs is further 2-3 μm.

[0010] In the ultrasound-responsive targeted microbubble drug delivery system described above, the microbubbles further have a hollow structure and a microbubble membrane thickness of 100-250 nm.

[0011] In the ultrasound-responsive targeted microbubble drug delivery system described above, specifically, the manganese porphyrin lipid is prepared by reacting pyrophyllite lipid with manganese chloride and sodium acetate.

[0012] In the ultrasound-responsive targeted microbubble drug delivery system described above, the microbubbles operate at a frequency of 1 MHz, a duty cycle of 20%, and an acoustic intensity of 1 W / cm². 2 After 30 seconds of ultrasonic treatment, at least 96% of the microbubbles ruptured.

[0013] To achieve the second objective mentioned above, the technical solution adopted by the present invention is as follows:

[0014] The preparation method of the ultrasound-responsive targeted microbubble drug delivery system described above includes the following steps: 1) mixing DSPC, DSPE-PEG2000-Var7, manganese porphyrin lipid and Nigerian styraxin to obtain an organic phase; 2) injecting the organic phase into the aqueous phase under ultrasonic water bath conditions, dialysis to remove organic reagents, and obtaining vMnp-Nig NPs; 3) mixing vMnp-Nig NPs with 1,2-propanediol and glycerol, loading into a container, filling with perfluoropropane gas, sealing, and oscillating to form vMnp-Nig MBs.

[0015] In the preparation method described above, preferably, the molar ratio of DSPC, DSPE-PEG2000-Var7 and manganese porphyrin lipid is 75~87:2~5:14~18.

[0016] In the preparation method described above, preferably, the total mass ratio of DSPC, DSPE-PEG2000-Var7 and manganese porphyrin lipids to Nigerian mycotoxin is 180~220:1.

[0017] In the preparation method described above, preferably, the manganese porphyrin lipid is prepared by reacting pyrophyllite lipid with manganese chloride and sodium acetate; the molar ratio of the pyrophyllite lipid to manganese chloride and sodium acetate is 0.5~1.5:3~8:15~25. Further, the reaction is carried out under water bath conditions of 50~70°C.

[0018] In the preparation method described above, the organic phase further includes a solvent for dissolving DSPC, DSPE-PEG2000-Var7, and manganese porphyrin lipids. Preferably, the volume ratio of the organic phase to the aqueous phase is 1:6~9. Dialysis is performed using a dialysis bag with a molecular weight cutoff of 8000~12000 Da. The volume ratio of vMnp-Nig NPs, 1,2-propanediol, and glycerol is 0.5~1.0:0.2~0.3:0.2~0.3.

[0019] To achieve the third objective mentioned above, the technical solution adopted by the present invention is as follows:

[0020] Use of any of the above-described ultrasound-responsive targeted microbubble drug delivery systems in the preparation of medicaments for combined ultrasound therapy of triple-negative breast cancer. Further, the medicaments for treating triple-negative breast cancer also include anti-PD-L1 (aPD-L1).

[0021] This invention utilizes nigrain as a pyroptosis initiation module to construct a Var7-modified manganese porphyrin / nigrain co-loaded microbubble delivery platform (vMnp-Nig MBs) with responsiveness to the acidic tumor microenvironment and ultrasound triggering. Under ultra-UTMD, vMnp-Nig MBs can undergo micro-nano transformation, achieving spatiotemporally controlled drug release and deep tumor penetration. Nigrain activates the NLRP3-Caspase-1-GSDMD pyroptosis pathway; Mnp, as a pyroptosis amplification module, generates ROS and amplifies the inflammatory cascade under ultrasound. vMnp-Nig MBs combined with ultrasound-induced pyroptosis further promotes dendritic cell activation and antigen presentation, increasing CD8+. + T cell activation alleviates the immunosuppressive state of the tumor microenvironment by reducing immunosuppressive cell populations. Combined with aPD-L1 therapy, it can significantly inhibit tumor growth, reduce lung metastasis, and prolong survival, providing a new treatment strategy for reshaping the tumor immune microenvironment of triple-negative breast cancer and improving the efficacy of immune checkpoint blockade therapy. Attached Figure Description

[0022] Appendix Figure 1 A schematic diagram illustrating the construction and therapeutic mechanism of vMnp-Nig MBs.

[0023] Appendix Figure 2 Characterization of the in-situ micron-to-nanoscale conversion process of vMnp-Nig MBs. (A) Schematic diagram of the preparation process of vMnp-Nig MBs and its micron-to-nanoscale conversion via UTMD-induced fragmentation; (B) Optical microscope image of vMnp-Nig MBs (scale bar: 10 μm); (C) Particle size distribution of vMnp-Nig MBs; (D) Cryo-scanning electron microscope image of vMnp-Nig MBs (scale bar: 5 μm); (E, F) Changes in average particle size and concentration of vMnp-Nig MBs during storage, indicating good storage stability (n = 4); (G) UV-Vis absorption spectra of free Mnp and vMnp-Nig NPs; (H) Fluorescence emission spectra of different particles (500–800 nm) under 650 nm excitation conditions; (I) Fluorescence emission spectra of vMnp-Nig MBs at 1 MHz, 20% duty cycle, and 1 W / cm². 2(n = 3) Changes in microbubble concentration after different ultrasonic treatment times under ultrasonic conditions; (J) Transmission electron microscopy images of vMnp-NigMBs nanoparticles after UTMD treatment (scale bar: 50 nm); (K) Particle size distribution of vMnp-Nig NPs generated after microbubble rupture; (L) Zeta potential analysis of vMnp-Nig NPs, Mnp-Nig NPs, and nanoparticles obtained by UTMD conversion of microbubbles prepared from these two types of nanoparticles (n = 3); (M) In vitro cumulative release curves of Nig in vMnp-Nig MBs with and without UTMD conditions (n ​​= 3).

[0024] Appendix Figure 3 Characterization of ROS generation capacity and catalase-like activity of vMnp-Nig NPs / MBs. (A) At 1 MHz, 1 W / cm 2 (a) Changes in SOSG fluorescence intensity induced by different concentrations (5, 15, and 25 μg / mL) of vMnp-Nig NPs under 20% duty cycle ultrasonic irradiation; (b) At 1 MHz and 1 W / cm 2 Under ultrasound conditions, 25 μg / mL vMnp-Nig NPs induced changes in SOSG fluorescence intensity at different duty cycles (10%, 20%, and 50%); (C) ddH2O, vMnp-Nig NPs, vMnp NPs, and vMnp-Nig NPs+US were used to precipitate SOSG fluorescence at 1 MHz and 1 W / cm². 2 (a) Comparison of relative SOSG fluorescence intensity after 2 min of ultrasonic irradiation with a 20% duty cycle; (d) When TEMP is used as a spin trapping agent, vMnp-Nig MBs, vMnp-Nig MBs+UTMD and vMnp-Nig MBs+UTMD+US generate singlet oxygen ( 1 Electron spin resonance (ESR) spectral analysis of O2; (E)vMnp-Nig MBs obtained by DMPO with or without US irradiation to capture hydroxyl radicals ( . ESR spectra of (OH); (F) DMPO capture obtained by vMnp-Nig MBs with or without US irradiation. . Superoxide anion radical (O2• -(G) ESR spectra of ddH2O, vNigNPs, vMnpNPs, and vMnp-NigNPs for consuming H2O2; (H) Quantitative analysis of the H2O2 consumption capacity of vMnp-NigNPs at different concentrations (0, 100, 250, and 500 μg / mL); (I) Comparison of the increase in dissolved oxygen (ΔO2) produced by vNigNPs, vMnp-NigMBs, vMnpMBs, and vMnp-NigNPs catalyzing H2O2; (J) Time-dependent curves of oxygen production by vMnp-NigNPs at different concentrations (0, 100, 250, and 500 μg / mL) in the presence of H2O2. All data are expressed as mean ± standard deviation (mean ± SD, n=3).

[0025] Appendix Figure 4 To assess cellular uptake, hypoxia relief, and ROS generation. (A) Time-dependent uptake of Cy5-vMnp-Nig MBs by 4T1 cells after UTMD treatment; (B) Comparison of cellular uptake efficiency of Cy5-vMnp-Nig NPs and Cy5-Mnp-Nig NPs; (C) Comparison of cellular uptake efficiency of vMnp-Nig NPs and vMnp-Nig MBs + UTMD treatment; (D) Confocal laser scanning microscopy fluorescence images (scale bar: 200 μm) of 4T1 tumor cell spheres after co-incubation with Cy5-Mnp-Nig NPs, Cy5-vMnp-Nig NPs, Cy5-vMnp-Nig MBs, and Cy5-vMnp-Nig MBs + UTMD; (E, F) Imaging of 60 μm and 80 μm of tumor cell spheres in (D). The fluorescence intensity of the yellow line region at a depth of μm was analyzed; (G) Fluorescence images of [Ru(dpp)3]Cl2 stained in hypoxic cells under different treatment conditions (scale bar: 100 μm); (H) Semi-quantitative analysis of the fluorescence intensity of [Ru(dpp)3]Cl2 in (G); (I) Intracellular DCFH-DA fluorescence imaging results after different treatments under normoxic and hypoxic conditions (scale bar: 200 μm); (J, K) Semi-quantitative analysis of the fluorescence intensity of DCFH-DA in (I); (L) Fluorescence images of Rhodamine 123 (RH-123) in 4T1 cells after different treatments (scale bar: 50 μm); (M) Semi-quantitative analysis of the fluorescence intensity of RH-123 in (L). All data are expressed as mean ± standard deviation (mean ± SD, n = 3).

[0026] Appendix Figure 5For in vitro cytotoxicity evaluation: (A) Effects of different concentrations of vMnp MBs, vMnp-Nig MBs, and Nig on HUVEC cell viability; (B) Effects of different concentrations of vMnp-Nig MBs on cell viability under ultrasound irradiation; (C) Comparison of cell viability among different treatment groups; (D) Calcein-AM / PI double-staining fluorescence images of cells in different treatment groups (scale bar: 200 μm); (E) Annexin V-FITC / PI double-staining flow cytometry analysis of cells in different treatment groups; (F) Transwell migration assay images of cells in different treatment groups (scale bar: 200 μm); (G) Quantitative analysis of the proportion of early and late apoptotic cells in (E); (H) Quantitative analysis of the relative number of migrating cells in (F); (I) Analysis of scratch healing rate of cells in different treatment groups. All data are expressed as mean ± standard deviation (n = 3).

[0027] Appendix Figure 6 vMnp-Nig MBs+US induces pyroptosis and triggers immunogenic cell death (ICD). (A) Transmission electron microscopy images of ultrastructural changes in 4T1 cells in different treatment groups (scale bar: 2 μm, 500 nm); (B) Immunofluorescence staining images of γ-H2AX in cells in different treatment groups and quantitative analysis of the corresponding mean fluorescence intensity (MFI) (scale bar: 10 μm); (C) Western blot analysis of the expression levels of NLRP3, Caspase-1, cleaved Caspase-1, GSDMD, and GSDMD-N proteins in cells in different treatment groups; (D) Quantitative analysis of the expression level of cleaved Caspase-1 protein in (C); (E) Quantitative analysis of the expression level of GSDMD-N protein in (C); (F) Immunofluorescence images of calreticulin (CRT) exposure on the cell surface in different treatment groups (scale bar: 50 μm); (G) Quantitative analysis of the fluorescence intensity of CRT in (F); (H) Quantitative analysis of the release level of HMGB1 in cells in different treatment groups; (I) Quantitative analysis of the release level of lactate dehydrogenase (LDH) in cells in different treatment groups. All data are expressed as mean ± standard deviation (n = 3).

[0028] Appendix Figure 7Transcriptome sequencing analysis of 4T1 cells after different treatments (n = 4). (A) Venn diagram showing the intersection of differentially expressed genes among the control group, vNig MBs+US group, and vMnp-Nig MBs+US group; (B) Volcano diagram analysis of differentially expressed genes between vNig MBs+US group and control group, vMnp-Nig MBs+US group and control group, and vMnp-Nig MBs+US group and vNig MBs+US group; (C) Heatmap showing the expression profiles of representative differentially expressed genes in the control group, vNig MBs+US group, and vMnp-Nig MBs+US group; (DF) KEGG enrichment analysis showing signaling pathways with significantly enriched differentially expressed genes in the vNig MBs+US group compared to the control group, the vMnp-Nig MBs+US group compared to the control group, and the vMnp-Nig MBs+US group compared to the vNig MBs+US group; (GJ) Gene set enrichment analysis (GSEA) results showed that, compared to the vNig MBs+US group, vMnp-Nig MBs+US group had significantly richer differentially expressed genes. The NOD-like receptor, Toll-like receptor, NF-κB signaling pathway, and cytosolic DNA-sensing pathway were significantly enriched in the MBs+US group.

[0029] Appendix Figure 8 Evaluation of the ultrasound, fluorescence, photoacoustic, and magnetic resonance imaging performance of vMnp-Nig MBs. (A) In vitro, vMnp-Nig MBs diluted 100-fold (3.7 × 10⁻⁶) 6(a) Ultrasound imaging images of 4T1 tumor-bearing mice after intravenous injection of vMnp-NigMBs; (b) In vivo ultrasound imaging results of 4T1 tumor-bearing mice after intravenous injection of Cy5.5-vMnp-Nig MBs with or without UTMD treatment; (c) In vivo fluorescence imaging images of tumor-bearing mice after intravenous injection of Cy5.5-vMnp-Nig MBs with or without UTMD treatment; (d) Quantitative analysis of fluorescence intensity changes in tumor sites at different time points in (c); (e) Ex vivo fluorescence imaging results of major organs and tumor tissues 48 h after intravenous injection of vMnp-Nig MBs with or without UTMD treatment; (f) Quantitative analysis of fluorescence distribution in each tissue in (e). Data are presented as mean ± standard deviation (n = 3); (G) Ex vivo fluorescence imaging images of tumor tissues in the vMnp-Nig MBs group and the vMnp-Nig MBs+UTMD group; (H) Evaluation of changes in tumor oxygen saturation (SO2) in the control group and the vMnp-Nig MBs+UTMD group by in vivo photoacoustic imaging (PAI); (I) In vitro magnetic resonance imaging (MRI) results of vMnp-Nig NPs with different Mn concentrations; (J) Linear fitting curves between 1 / T2 and Mn concentration in vMnp-Nig NPs; (K) Linear fitting curves between 1 / T1 and Mn concentration in vMnp-Nig NPs; (L) In vivo T2-weighted imaging (T2WI) and T1-weighted imaging (T1WI) results of vMnp-Nig MBs with and without UTMD treatment; (M) Quantitative analysis of T2WI signal intensity in (L); (N) Quantitative analysis of T1WI signal intensity in (L).

[0030] Appendix Figure 9To evaluate the in vivo therapeutic efficacy of vMnp-Nig MBs combined with aPD-L1 for 4T1 tumors. (A) Schematic diagram of the in vivo treatment protocol; (B) Representative photographs of tumor tissues from mice in different treatment groups on day 20 post-treatment; (C) Tumor growth curves of mice in different treatment groups (n = 5); (D) Changes in body weight of mice in different treatment groups over time (n = 5); (E) Survival curves of mice in each treatment group; (F) Hematoxylin-eosin (H&E) staining, Ki67 immunohistochemical staining, and TUNEL staining results of tumor tissues from different treatment groups (scale bar: 50 μm); (G) Immunofluorescence staining images of γ-H2AX, HIF-1α, cleaved Caspase-1, and GSDMD-N in tumor tissues from different treatment groups (scale bar: 50 μm); (H) Immunofluorescence staining images of CD11c / CD3, CD8 / GZMB, and CD86 / CD206 in tumor tissues from different treatment groups (scale bar: 50 μm). (μm); (I) Quantitative analysis of MFI of CD11c, CD3, CD8, GZMB, CD86 and CD206 signals in tumor tissue sections in (H). All data are expressed as mean ± standard deviation (n = 3).

[0031] Appendix Figure 10 To enhance anti-tumor immunity and inhibit lung metastasis in 4T1 tumor-bearing mice by combining vMnp-Nig MBs with aPD-L1. (A) Schematic diagram of the in vivo treatment regimen; (B) Mature dendritic cells (CD11c) in the tumor draining lymph nodes of 4T1 tumor-bearing mice. + CD80 + CD86 + (C) Flow cytometry analysis and quantitative analysis of CD4 in tumor tissue; + T cells (using CD3) + (D) Flow cytometry analysis and quantitative analysis of cells (gated); CD8+ in tumor tissue + T cells (using CD3) + (E) Flow cytometry analysis and quantitative analysis of cells (gated); (E) Tumor-infiltrating regulatory T cells (Tregs, CD3+) + CD4 + FoxP3 + CD25 + Flow cytometry analysis and quantitative analysis of (F) mature DCs (CD11c) in 4T1 tumor tissue; + CD80 + CD86 + Quantitative analysis of the proportion of M1 and M2 macrophages in tumor tissue; (G) Quantitative analysis of the proportion of M1 and M2 macrophages in tumor tissue (M1:CD11b + F4 / 80+ CD86 + M2: CD11b + F4 / 80 + CD206 + (H) Tumor-infiltrating neutrophils (CD45) + CD11b + Ly6G + Quantitative analysis by flow cytometry of (I) tumor-infiltrating monocyte-like myeloid-derived suppressor cells (M-MDSCs, CD45) + CD11b + Ly6C + Quantitative analysis by flow cytometry. Data are expressed as mean ± standard deviation (n = 3); (J) H&E staining results of lung tissue sections in lung metastasis experiments of 4T1 tumor-bearing mice in different treatment groups (scale bar: 100 μm).

[0032] Appendix Figure 11 The synthesis of manganese porphyrin lipids is achieved through the chelation of PL and Mn. (a) High-resolution mass spectrometry (HRMS) of PL; (b) HRMS of manganese porphyrin lipids; (c) UV-Vis absorption spectra of PL and manganese porphyrin lipids; (d) Fluorescence spectra of PL and manganese porphyrin lipids.

[0033] Appendix Figure 12 Characterization of DSPE-PEG2000-Var7. (a) Electrospray ionization mass spectra (ESI-MS) of Var7 peptide; (b) HRMS of DSPE-PEG2000-Mal; (c) HRMS of DSPE-PEG2000-Var7.

[0034] Appendix Figure 13 Confirmation of the molecular weight of Nig-Cy5. (a) HRMS of Nig-Cy5; (b) Theoretical molecular weight and elemental composition analysis of Nig-Cy5.

[0035] Appendix Figure 14 Encapsulation efficiency and drug loading of vMnp-Nig NPs.

[0036] Appendix Figure 15 Optical microscope image of vMnp-Nig MBs after UTMD processing. Ultrasonic processing conditions: operating frequency 1MHz, duty cycle 20%, sound intensity 1W / cm². 2 Processing time: 30s; Scale bar: 50μm.

[0037] Appendix Figure 16 (a) Figure 4 Quantitative analysis of Cy5 by MFI in A. (b) Figure 4Quantitative analysis of Cy5 by MFI in C. (c) Figure 4 Quantitative analysis of Cy5 in E by MFI.

[0038] Appendix Figure 17 Confocal laser scanning microscopy was used to evaluate the uptake of Cy5-Mnp-Nig NPs with and without Var7 modification by 4T1 cells at 1, 2, 4 and 6 h (scale bar = 20 μm).

[0039] Appendix Figure 18 : Optical microscopic images of scratch healing experiment on cell migration ability in different treatment groups (scale bar = 100 μm).

[0040] Appendix Figure 19 Optical microscopic images of 4T1 cells after different treatments (scale bar = 20 μm).

[0041] Appendix Figure 20 Quantitative analysis of the relative expression levels of NLRP3 in different treatment groups.

[0042] Appendix Figure 21 Immunofluorescence images of HMGB1 staining in 4T1 cells after different treatments (scale bar = 20 μm).

[0043] Appendix Figure 22 Blood was incubated with double-distilled water (positive control), physiological saline (negative control), and different concentrations of vMnp-Nig MBs, and hemolysis was observed. Data are expressed as mean ± standard deviation (SD) (n = 3).

[0044] Appendix Figure 23 Routine hematological analyses were performed on healthy BALB / c mice before intravenous injection of vMnp-Nig MBs and on days 1, 3, 7, and 15 after injection (n = 3).

[0045] Appendix Figure 24 Serum biochemical analysis of mice in different treatment groups. a, Blood urea nitrogen (BUN); b, Creatinine (CR); c, Alanine aminotransferase (ALT); d, Aspartate aminotransferase (AST) (n = 3).

[0046] Appendix Figure 25Representative microscopic images of major organs stained with H&E on day 15 in different treatment groups (scale bar = 50 μm). G1: control, G2: US, G3: vMnp-Nig MBs, G4: vMnp MBs+US, G5: aPD-L1, G6: vMnp-Nig NPs+US, G7: vMnp-Nig MBs+US, G8: vMnp-Nig MBs+US+ aPD-L1.

[0047] Appendix Figure 26 In vitro ultrasound images of vMnp-Nig MBs at different dilution ratios, including a 50-fold dilution (7.3 × 10⁻⁶). 6 / mL), 200-fold dilution (1.8 × 10⁻⁶) 6 / mL), 400-fold dilution (9.1 × 10⁻⁶) 5 / mL) and 800-fold dilution (4.6 × 10 5 / mL).

[0048] Appendix Figure 27 (a) In vivo fluorescence imaging of 4T1 tumor-bearing mice at 0, 1, 2, 4, 6, 8, 12, 24, 36, 48, and 72 h after intravenous injection of Mnp-Nig NPs, vMnp-Nig NPs, and vMnp-Nig MBs + UTMD. (b) Semi-quantitative analysis of fluorescence intensity in tumor regions at different time points in (a) (n = 4). (c) Ex vivo fluorescence imaging of major organs (heart, liver, spleen, lung, and kidney) and tumor tissues after drug administration. (d) Semi-quantitative biodistribution analysis of fluorescence signals in different tissues in (c) (n = 4). (e) Ex vivo fluorescence imaging of tumor tissues in different treatment groups (n = 4).

[0049] Appendix Figure 28 Representative photographs of mice were taken starting on day 20 after treatment began, and recorded every 6 days thereafter until the end of the experiment, including day 20 (a) and day 26 (b). G1, Control; G2, US; G3, vMnp-Nig MBs; G4, vMnp MBs+US; G5, aPD-L1; G6, vMnp-Nig NPs+US; G7, vMnp-Nig MBs+US; and G8, vMnp-Nig MBs+US+aPD-L1. Blank boxes indicate mice that could not be imaged at the corresponding time points.

[0050] Appendix Figure 29 Tumor growth curves of individual mice after different treatments.

[0051] Appendix Figure 30 (a) CD11c in tumor-draining lymph nodes + Quantitative analysis of MHC II expression in dendritic cells. (b) CD8 in tumor tissue. + GzmB in T cells + Quantitative analysis of cell proportions.

[0052] Appendix Figure 31 The serum levels of TNF-α, IFN-γ, IL-1β, and IL-18 in 4T1 tumor-bearing mice after different treatments were detected by ELISA. Data are expressed as mean ± standard deviation (SD) (n = 3). Detailed Implementation

[0053] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0054] This invention constructs a multifunctional microbubble platform, vMnp-Nig MBs, for precision treatment of TNBC, aiming to achieve localized and precise amplification of pyroptosis efficacy. The microbubble platform serves as a delivery carrier, relying on UTMD to achieve visualization and spatiotemporally controlled release of therapeutic components at the tumor site, and leveraging cavitation effects to promote drug penetration into deeper tumor cells and cellular uptake. Var7 endows the system with responsive active targeting capabilities in the acidic tumor microenvironment, thereby further enhancing local enrichment and binding with tumor cells. Based on this, nigrain serves as a pyroptosis initiation module, providing deterministic inflammasome activation signals; manganese porphyrin serves as a multifunctional acoustic module, generating reactive oxygen species under ultrasound to amplify oxidative stress and pyroptosis-related cascade reactions, while simultaneously alleviating tumor hypoxia through catalytic oxygen production, improving the microenvironmental basis limiting sonodynamic therapy. Figure 1 In summary, the vMnp-Nig MBs combined with ultrasound constructed in this invention can achieve localized, visualized delivery to tumors. Furthermore, by initiating pyroptosis through nigericin and amplifying the pyroptosis cascade reaction through manganese porphyrin-mediated sonodynamic effects, it enhances DAMPs release, further promoting antigen presentation and T-cell-mediated anti-tumor immune activation. This transforms TNBC treatment from simple local tumor reduction into a comprehensive treatment modality that combines immune remodeling and metastasis suppression, thus providing a new treatment strategy to address the problems of insufficient immune response, high risk of recurrence and metastasis, and lack of effective targeted therapy in TNBC.

[0055] Appendix Figure 1This is a schematic diagram illustrating the construction and therapeutic mechanism of vMnp-Nig MBs of the present invention. vMnp-Nig MBs are composed of DSPC, DSPE-PEG2000-Var7, Mnp, and Nig, internally filled with C3F8 gas, enabling dual-modal ultrasound / magnetic resonance imaging (US / MRI) of TNBC. After UTMD treatment, vMnp-Nig MBs undergo a transformation from micron-scale to nanoscale and release Nig within the tumor tissue. Under ultrasound, MnP generates a large amount of ROS, amplifying oxidative stress signals and enhancing Nig-induced inflammasome activation, thereby activating the pyroptosis-related pathway NLRP3–caspase-1–GSDMD, promoting the secretion of IL-1β / IL-18 cytokines and the release of DAMPs. These processes collectively induce dendritic cell (DC) maturation, T cell activation, and immune microenvironment remodeling, thereby enhancing aPD-L1-mediated anti-tumor immune responses and inhibiting lung metastasis.

[0056] The experimental materials used in the following examples are as follows:

[0057] Distearylphosphatidylethanolamine-polyethylene glycol 2000-maleimide-Var7 (DSPE-PEG2000-Var7) was purchased from Nanjing Jietai Biotechnology Co., Ltd. (Nanjing, China). 1,2-Distearyl-sn-glycerol-3-phosphatidylcholine (DSPC), 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and pyropheophorbide-lipid (PL) were purchased from Xi'an Ruixi Biotechnology Co., Ltd. Nigericin (Nig) was purchased from Shanghai Yuanye Technology Co., Ltd. 2,7-Dichlorofluorescein diacetate (DCFH-DA) and Cell counting kit-8 (CCK8) were purchased from LABLEAD CO., Ltd. (Beijing, China). Singlet Oxygen Sensor Green (SOSG) was purchased from Thermp Fisher (Waltham, MA, USA). Calcein-AM / PI was purchased from Beyotime Biotechnology Co., Ltd. Annexin V-FITC / PI double staining kit was purchased from KGI Biotechnology Co., Ltd. (Nanjing, China). H2O2 Quantitative Assay Kit was purchased from Sangon Biotech (Shanghai, China). β-actin, Cleaved caspase 1, and GSDMD-N antibodies were purchased from Abclonal (Woburn, MA, USA), ImmunoWay (Jiangsu, China), and Affinity Biosciences (Cincinnati, OH, USA), respectively. The anti-PD-L1 antibody (aPD-L1) was purchased from BioXCell, catalog number BE0101. All solvents were purchased from Aladdin (Shanghai, China). It should be noted that the experimental material used, distearylphosphatidylethanolamine-polyethylene glycol 2000-maleimide-Var7, uses the maleimide group for coupling Var7. For convenience, the abbreviations DSPE-PEG2000-Var7 are used in this context. Figure 1 -mal is omitted.

[0058] Example 1: Preparation of an ultrasound-targeted microbubble delivery system to enhance pyroptosis and improve the immunotherapy effect of triple-negative breast cancer.

[0059] I. Experimental Methods

[0060] (1) Preparation of manganese porphyrin lipids (Mnp): PL, MnCl2 and NaOAc were dissolved in glacial acetic acid at a molar ratio of 1:5:20 and stirred in a water bath at 60℃ for about 2 h under light-protected conditions. The reaction process was monitored by thin-layer chromatography. After the reaction was completed, glacial acetic acid was removed by rotary evaporation under reduced pressure to obtain crude Mnp product. Subsequently, the obtained crude Mnp product was used to obtain self-assembled MnP NPs by ethanol injection method, and then purified by dialysis and freeze-dried to obtain the target product. The specific operation steps of ethanol injection method are as follows: the crude Mnp product was dissolved in DMSO or DMF, and the solution was added dropwise to 6 volume equivalents of ddH2O under water bath ultrasonic conditions (temperature 25±5℃, ultrasonic power 160~400W).

[0061] (2) Preparation of Nig-Cy5: First, Nig (3.36 mg, 0.00464 mmol) was dissolved in anhydrous N,N-dimethylformamide (DMF), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 1.78 mg, 0.00929 mmol) was added. The mixture was stirred at room temperature for 10 min to activate the carboxyl group. Then, N-hydroxysuccinimide (NHS, 0.64 mg, 0.00557 mmol) was added, and the mixture was stirred at room temperature for 1 h to generate the NHS active ester. Subsequently, Cyanine5 amine (Cy5-NH2, 2.7 mg, 0.00464 mmol) was added to the reaction system, and the mixture was reacted at room temperature in the dark for 12 h to allow the amino group in the Cy5 molecule to undergo a nucleophilic substitution reaction with the activated ester to form a stable amide bond. After the reaction was completed, DMF was removed by rotary evaporation under vacuum to obtain the final Nig-Cy5 coupling product. The obtained Nig-Cy5 was used only for the characterization of Nig.

[0062] (3) Cell lines and animals: Mouse breast cancer cells (4T1 cells) and umbilical vein endothelial cells (HUVEC cells) were purchased from Peking University Third Hospital (Beijing, China). 4T1 cells were cultured in RPMI 1640 medium at pH 6 with 10% fetal bovine serum and 1% penicillin / streptomycin added, while HUVEC cells were cultured in ECM medium. Cell culture was performed in an incubator at 37 ℃ and 5% CO2. BALB / c mice (6-8 weeks old) were purchased from Jicui Pharmaceutical Technology Co., Ltd. (Jiangsu, China). All in vivo experiments were approved by the ethics committee of the Cancer Hospital of the Chinese Academy of Medical Sciences.

[0063] (4) Preparation of vMnp-Nig MBs microbubbles: DSPC and DSPE-PEG2000-Var7 were dissolved in anhydrous ethanol, and manganese porphyrin lipid was dissolved in dimethyl sulfoxide (DMSO). The solutions were mixed at a molar ratio of DSPC:DSPE-PEG2000-Var7:manganese porphyrin lipid = 81:3:16 to obtain a mixture. Nigerian styracin was added to the mixture, and the mass ratio of the total mass of DSPC, DSPE-PEG2000-Var7, and manganese porphyrin lipid to Nigerian styracin was controlled at 200:1. After thorough mixing, an organic phase was obtained (the volume of the organic phase was the total volume of anhydrous ethanol and DMSO, i.e., the sum of the volumes of organic solvents used after all four components were dissolved). Subsequently, the above organic phase was injected into the aqueous phase under ultrasonic water bath conditions (temperature 25±5℃, ultrasonic power 160~400W), and the volume ratio of the organic phase to the aqueous phase was 1:6. The obtained nanoparticle dispersion was dialyzed in distilled water for 2 hours using a dialysis bag with a molecular weight cutoff of 8000-12000 Da to remove residual organic solvents, thus preparing vMnp-Nig NPs. Finally, the prepared vMnp-Nig NPs were mixed with 1,2-propanediol and glycerol at a volume ratio of 0.8:0.1:0.1 ml, placed in a vial, filled with perfluoropropane gas, and tightly sealed with a cap. The mixture was then shaken for 45 seconds to finally form vMnp-Nig MBs.

[0064] Preparation of vMnp-MBs microbubbles: Except for the absence of Nig, the remaining steps are the same as those for preparing vMnp-Nig MBs microbubbles.

[0065] Preparation of vNig-MBs microbubbles: Except for the absence of manganese porphyrin lipids, the other steps are the same as those for the preparation of vMnp-Nig MBs microbubbles.

[0066] (5) Characterization of vMnp-Nig MBs: First, the morphology of the microbubbles was observed using an optical microscope. To further observe the internal structure of the microbubbles, a high-resolution field emission scanning electron microscope was used to perform cryo-cross-sectional imaging of the microbubbles. Secondly, the changes in the concentration and particle size of the vMnp-Nig MBs in the microbubbles were detected using a Coulter Multisizer III on days 0, 1, 3, 5, and 7. The prepared vMnp-Nig MBs microbubbles were then ruptured using an ultrasonic physiotherapy device. The specific parameters were: 1 MHz frequency, 20% duty cycle, and 1 W / cm². 2 The intensity was determined by designing different blasting times; particle size and potential were observed using a dynamic light scattering analyzer; particles were negatively stained with a 2wt% uranium acetate aqueous solution as a negative staining agent, and the morphology of vMnp-Nig MBs after ultrasonic blasting was further observed by transmission electron microscopy.

[0067] (6) Encapsulation efficiency and drug loading of vMnp-Nig MBs: A series of Nig-cy5 free solutions of various concentrations were prepared, fluorescence values ​​were measured, and a standard curve was plotted. The drug-loaded vMnp-Nig NPs were placed in an ultrafiltration tube, centrifuged at 3900 r / min for 5 min, and the solution at the bottom of the ultrafiltration tube was collected. The fluorescence value was measured using a microplate reader, and the encapsulation efficiency and drug loading of the nanoparticles were calculated using an indirect method. Drug encapsulation efficiency (EE)% = mass of nigrain encapsulated in nanoparticles / actual mass of nigrain added × 100%; Drug loading (LC)% = mass of nigrain encapsulated in nanoparticles / total mass of nanoparticles × 100%.

[0068] (7) Drug release from vMnp-Nig MBs: The ability of vMnp-Nig MBs to release drugs after ultrasonic blasting was investigated by dialysis. vMnp-Nig MBs were placed in two dialysis bags (capacity cutoff: 8000-12000) and immersed in phosphate buffer (pH 7.4, containing 0.1% Tween-80). The bags were placed in a 37°C constant temperature shaker. One of the bags was sonicated at the 19th minute for 30 seconds (parameters: 1 MHz, 1 W cm⁻¹). 2 (20% duty cycle) 1 ml of medium was taken out before and after ultrasound, and 1 ml of medium was added to maintain a constant volume. Then, 1 ml of solvent was taken out at 1, 2, 4, 6, 8, 10, 12 and 24 hours respectively, and 1 ml of solvent medium was added at the same time. The extracted solution was detected by ultraviolet absorption using an enzyme-linked immunosorbent assay (ELISA) reader. The content of nigrain released into the medium was calculated, and the cumulative drug release amount as a function of time was plotted.

[0069] (8) 1 O2, •O2 - •OH detection: To investigate the generation of vMnp-Nig NPs under the action of US 1 The effect of O2, using SOSG probes 1 O2 content was tested (working concentration 5 μM). To evaluate the effects of different experimental parameters... 1 The effects of O2 generation were investigated, specifically the influence of sonication time, particle concentration, duty cycle, and treatment group on SOSG fluorescence intensity. In the experiments examining the effects of particle concentration and sonication time, Mnp-Nig NPs concentrations were set to 5, 15, and 25 μg / mL. -1 The ultrasound parameters were fixed at a frequency of 1 MHz and a wavelength of 1 W / cm². -2 With a 20% duty cycle, the fluorescence emission spectrum of SOSG under 504 nm excitation in the 510-600 nm range was measured using a fluorescence spectrophotometer every 2 min, continuously monitored for 8 min. In the duty cycle-dependent experiment, the microbubble concentration was fixed at 25 μg / mL.-1 The duty cycles were set to 10%, 20%, and 50%, respectively, while the ultrasonic frequency and power remained constant. In the intergroup comparison experiment, ddH₂O and vMnp NPs (25 μg / mL) were used. -1 )+US, vMnp-Nig NPs (25 ug mL -1 Four groups were measured (±US), with ultrasound parameters of 1 MHz and 1 W cm⁻¹. -2 20% duty cycle and 2 min. To further confirm the types of ROS, ESR combined with different spin trapping agents was used to detect ROS in the ultrasonically treated system. Singlet oxygen was detected using TEMP as a spin trapping agent. 100 μL of sample solution was mixed with 100 μL of 100 mM TEMP solution, thoroughly mixed, and reacted at room temperature in the dark for 20 min; hydroxyl radical (•OH) was detected using DMPO as a spin trapping agent. 100 μL of sample solution was mixed with 100 μL of 100 mM DMPO solution; superoxide anion (•O) was detected using DMPO as a spin trapping agent. 2- The detection of ESR also used DMPO as a spin trapping agent. 50 μL of sample solution, 50 μL of methanol, and 100 μL of 100 mM DMPO methanol solution were mixed. After thorough mixing, the mixture was loaded into capillary tubes and sealed. ESR signal acquisition and analysis were then performed.

[0070] (9) vMnp-Nig NPs consume hydrogen peroxide (H2O2) to produce oxygen: First, a standard curve was prepared based on the H2O2 concentration gradient (0, 1, 10, 20, 40, 60, 80, 100 μM). To investigate different concentrations (0, 100, 200, 500 μg / mL)... -1 The ability of Mnp to consume H2O2 and different groups (ddH2O, vNig NPs, vMnp NPs, vMnp-Nig NPs) was investigated. 50 μM H2O2 was thoroughly mixed with particles of different concentrations and groups. After reacting for 30 min, the remaining H2O2 concentration was measured. The working solution was prepared at a ratio of solution A: solution B = 1:100. The sample was added to a 96-well plate, and after adding the working solution, the reaction was carried out at room temperature for an appropriate time. The absorbance at 595 nm was read using a microplate reader, and the H2O2 concentration in the sample was calculated after fitting a standard curve. To investigate the ability of Mnp to consume H2O2 and generate O2, 30 mM H2O2 was mixed with ddH2O, vNig NPs (250 μg / mL), and vNig NPs (250 μg / mL). -1 vMnp NPs (250ug / mL) -1 After being thoroughly mixed with vMnp-Nig NPs of different concentrations, the oxygen content was measured using a dissolved oxygen meter.

[0071] (10) In vitro cell uptake: The uptake of vMnp-NigMBs in 4T1 cells was qualitatively and quantitatively analyzed using laser confocal microscopy and flow cytometry. Cy5 conjugated to nigericin was used to detect cell uptake. 4T1 cells were cultured at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / well in 12-well plates and cultured overnight in complete medium. After the cells had completely adhered to the bottom of the plate, the original medium was discarded and replaced with Cy5-conjugated vMnp-Nig MBs medium for 1, 2, 4, and 6 hours of incubation. UTMD (1 MHz, 20% duty cycle, 1 W / cm²) was performed immediately after the addition of vMnp-Nig MBs. 2 The cells were treated for 30 seconds, and after incubation, the culture medium was discarded, and the cells were collected for flow cytometry. 4T1 cells were cultured at 1 x 1030 cells per dish. 5 Cells were seeded on confocal microscopy dishes, and subsequent procedures were the same as described above. After co-incubation, the cells were washed twice with PBS, fixed with 4% paraformaldehyde, and then incubated with DAPI for 10 minutes. The staining solution was discarded, and the cells were rinsed twice with PBS. Finally, cell uptake was observed under a confocal microscope. 1x10 4 Four T1 cells were seeded in 96-well plates with low adhesion to establish a three-dimensional tumor spheroid model. After 7 days of seeding, tumor spheroids with a diameter of approximately 800 μm were obtained. The culture medium was then replaced with Cy5-conjugated vMnp-Nig MBs, Mnp-Nig NPs, vMnp-Nig NPs, and vMnp-Nig MBs+US (UTMD: 1 MHz, 20% duty cycle, 1 W / cm²). 2 Co-incubate for 30 seconds. After 12 hours of incubation, remove the culture medium, wash with PBS, and image the tumor spheres using a laser confocal microscope.

[0072] (11) Fluorescence images of intracellular ROS levels: The ROS production at the cellular level was detected using the DCFH-DA fluorescent probe. 4T1 cells in the logarithmic growth phase were seeded into 12-well plates and divided into seven groups: Control, US, vMnp-Nig MBs, vMnp-MBs+US, vNig-MBs+US, vMnp-Nig NPs+US, and vMnp-Nig MBs+US. The cells were incubated for 4 hours in serum-free medium containing the above particles. The medium was discarded, and serum-free medium containing 10 μmol / L DCFH-DA was added. After incubation for 20 minutes, the cells were subjected to appropriate sonication. The US parameters were 1 MHz and 1 W / cm². 2 The sample was treated with a 20% duty cycle for 30 seconds, washed three times with PBS, and the fluorescence generation was observed using a fluorescence microscope at a wavelength of 488 nm.

[0073] (12) Detection of the cytotoxic effects of vMnp-Nig MBs under ultrasound by CCK-8 assay:

[0074] 4T1 cells were seeded into 96-well plates at a density of 7000 cells / well and incubated overnight. The cells were then treated with media containing (1) PBS; (2) US; (3) vMnp-Nig MBs; (4) vMnp-MBs+US; (5) vNig-MBs+US; (6) vMnp-Nig NPs+US; (7) vMnp-Nig MBs+US (concentration 100ug / ml), and immediately UTMD (1 MHz, 1 W cm⁻¹) was applied. −2 (20% duty cycle, 30s), continue incubation for 4 h, then perform a second sonication treatment (1 MHz, 1 W cm⁻¹). −2 After incubation for 20 h (20% duty cycle, 5 min), the supernatant was discarded, and 10 µL of CCK8 reagent and 100 µL of serum-free medium were added. After co-incubation for 20 min, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. Similarly, 4T1 cells were seeded at 7000 cells / well in 96-well plates overnight and administered vMnp-Nig MBs at concentrations of 10, 20, 40, 60, 80, 100, and 150 μg / ml. Immediately after administration, UTMD (1 MHz, 1 W cm⁻¹) was applied. −2 Cells were subjected to a 20% duty cycle (30s) and then a second US treatment was performed 4 hours later. Cell viability was then assessed using CCK-8 assay.

[0075] 4T1 cells at 1×10 5 Cells were seeded at a density of cells / well in 12-well plates and incubated overnight. Fresh culture medium (concentration of 100ug / ml) containing different particles was added to the seven groups, and then UTMD was performed immediately. After 4 hours, the cells were sonicated again and incubated for another 20 hours. The cells were then co-stained with Calcein AM / PI for 30 minutes and observed under a fluorescence microscope.

[0076] (13) Apoptosis analysis: Cell apoptosis detection: Cells were seeded in 6-well plates, drug concentration was 100ug / ml, and after Calcein AM / PI staining, the cells were digested, washed twice with PBS, and then resuspended in 100 µL binding buffer containing 5 µL APC Annexin V. The cells were incubated at room temperature in the dark for 15 min. Before detection, 10 µL PI was added, and the cells were resuspended and incubated for 10 min. Then, an appropriate amount of binding buffer was added and flow cytometry was performed.

[0077] (14) CRT exposure and HMGB1 release: 4T1 cells were seeded in confocal dishes (seeding density of 1x10⁻¹). 5 Cells were incubated at 37°C and 5% CO2 for 24 hours to ensure complete adhesion. Cells were then treated with culture media containing PBS, vMnp-Nig MBs, vMnp-MBs+US, vNig-MBs+US, vMnp-Nig NPs+US, and vMnp-Nig MBs+US (100 μg / ml), respectively. Immediately after incubation, the cells were subjected to UTMD (1 MHz, 1 W cm⁻¹) at 1 MHz. -2 20% duty cycle, 30s), continue incubation for 4 hours, then perform a second sonication treatment (1 MHz, 1 W cm⁻¹). -2 After incubation for 20 hours (20% duty cycle, 5 min), the supernatant was discarded, and tumor cells were fixed with 4% paraformaldehyde at room temperature for 20 min. The fixative was discarded, and the cells were washed once with PBS. HMGB1 staining required 0.3% triton, and the cells were permeabilized for 20 min. Both of the subsequent staining was blocked with 5% BSA for 30 min. The treated cells were then incubated with anti-HMGB1 and anti-CRT (1:200) at 4°C for 12 h, washed once with PBS, and incubated with secondary antibody (1:500) at room temperature for 1 h. The cells were washed once with PBS, stained with DAPI for 10 min, and intracellular fluorescence was detected by confocal microscopy.

[0078] (15) In vivo fluorescence imaging of mouse tumors: First, to investigate the enrichment of Mnp in the tumor region, a 4T1 subcutaneous tumor model was constructed. When the mouse tumor volume reached approximately 200 mm... 3 At the same time, 100 µL of 3% DSPE-Cy5.5-labeled vMnp-Nig MBs (concentration: 2 mg / mL) were injected via the tail vein, followed by UTMD (1.0 MHz, 20% duty cycle, 1.0 W / cm²) in one group of mice. 2 For the first 3 minutes, the enrichment of the drug in the mouse tumor region was observed at predetermined time points (1, 2, 4, 6, 8, 12, 24, 36, and 48 hours) using the IVIS Spectrum small animal in vivo fluorescence imaging system under 680 nm excitation. Mice were sacrificed 48 hours after drug injection, and major organs and tumors were collected and imaged. Semi-quantitative analysis of the fluorescence signals was performed. Next, to observe the enrichment of nigerin in the tumor region, Cy5-vMnp-Nig MBs prepared by conjugating Cy5 to Nig were used for small animal in vivo imaging, using a method similar to the above.

[0079] (16) Magnetic resonance imaging of mouse tumors

[0080] In in vitro magnetic resonance imaging (MRI) experiments, vMnp-Nig NPs were prepared into solutions with manganese concentrations of 0.01, 0.02, 0.04, 0.08, 0.16, 0.32, and 0.4 mM, respectively, and added to EP tubes as in vitro imaging samples. T1- and T2-weighted MRI were performed on a 7.0 T small animal MRI system. T1-weighted imaging (T1WI) used the RARE sequence with the following scan parameters: repetition time (TR) 300 ms, echo time (TE) 5.01 ms, flip angle (FA) 90°, mean number of times (NEX) 6, field of view (FOV) 35 mm × 35 mm, matrix size 200 × 200, and slice thickness 1.0 mm. T2-weighted imaging (T2WI) was performed using the TurboRARE sequence with the following scanning parameters: TR 3000 ms, TE 40 ms, FA 90°, NEX 2, FOV 35 mm × 35 mm, matrix 200 × 200, and slice thickness 1.0 mm. Subsequently, the T1 and T2 relaxation times of samples at different concentrations were measured using ParaVision software, and the values ​​of 1 / T1 and 1 / T2 were calculated. The longitudinal relaxation rate (r1) and transverse relaxation rate (r2) were obtained by plotting the linear relationship curve between the reverse relaxation time (1 / T1 or 1 / T2) and the vMnp-NigNPs concentration.

[0081] In in vivo magnetic resonance imaging experiments, a 7.0 T small animal magnetic resonance imaging system was used to scan tumor-bearing mice. Before imaging, mice were fixed on the small animal scanning bed under 1.5–3% isoflurane inhalation anesthesia. To verify whether vMnp-Nig MBs could image tumor-bearing 4T1 mice, mice were divided into two groups: vMnp-Nig MBs and vMnp-Nig MBs+UTMD. Each mouse was injected with 200 μL of microbubbles (2 mg / ml) via the tail vein, and MRI imaging was performed before and 4 h after administration. T2-weighted imaging (T2WI) used the TurboRARE sequence with the following scan parameters: TR 3000 ms, TE 40 ms, FA 90°, NEX 3, FOV 35 mm × 35 mm, matrix 200 × 200, and slice thickness 1.0 mm. T1-weighted imaging (T1WI) used the RARE sequence with the following scan parameters: TR 389.53 ms, TE 5.08 ms, FA 90°, NEX 8, FOV 35 mm × 35 mm, matrix 200 × 200, and slice thickness 1.0 mm.

[0082] (17) In vivo anti-tumor activity: when the tumor grows to 50mm 3Mice were randomly divided into 8 groups: G1: Control group; G2: US group; G3: vMnp-Nig MBs group; G4: vMnp MBs+US group; G5: aPD-L1 group; G6: vMnp-Nig NPs+US group; G7: vMnp-Nig MBs+US group; G8: vMnp-Nig MBs+US+aPD-L1 group. Particle therapy (400ug per mouse) was administered via tail vein on days 1, 4, and 7. For groups requiring UTMD, ultrasonic blasting (1 MHz, 1 W cm⁻¹) was performed immediately after administration for 3 min. −2 (20% duty cycle), and on days 2, 5, and 8, aPD-L1 (120ug per mouse) was injected intraperitoneally, while the tumor area was subjected to secondary ultrasound (1.5 W / cm). 2 (1 MHz, 50% duty cycle, 5 minutes). Tumor volume and weight changes in mice were recorded every two days. After the third treatment, tumors were collected for HE, Ki67, Tunel, γ-H2AX, HIF-1a, Cleaved caspase1, GSDMD-N, CD11c, CD3, CD86, CD206, CD8, and GZMB staining. The remaining mice were observed, with tumor areas photographed every 6 days to observe tumor growth, and the tumor volume was recorded when it reached 1500 mm². 3 The time points were determined, and survival curves were plotted.

[0083] (18) Activation of immunity: To further investigate the immune response in mice, 4T1 (2×10⁻⁶) was subcutaneously injected into the right back of Balb / c mice. 6 (cells / each), until the tumor grows to 50mm 3 Mice were randomly divided into 5 groups: Control; vMnpMBs+US; aPD-L1; vMnp-Nig MBs+US; and vMnp-Nig MBs+US+aPD-L1. Particle therapy (400 μg per mouse) was administered via tail vein on days 1, 4, and 7. For groups requiring UTMD, ultrasonic blasting (1 MHz, 1 Wcm²) was performed immediately after administration. −2 (20% duty cycle), and on days 2, 5, and 8, aPD-L1 (120ug per mouse) was injected intraperitoneally, while the tumor area was subjected to secondary ultrasound (1.5 W / cm). 2(1 MHz, 50% duty cycle, 5 minutes). Mice were euthanized on day 5 after the third treatment, and lymph nodes and tumor tissue from the tumor drainage area were collected, digested, and separated into single-cell suspensions, all performed on ice. Dendritic cells (DCs) in lymph nodes and tumor tissue were stained with anti-CD11c, anti-CD86, and anti-CD80 to assess DC maturation. Tumor cells were divided into two groups. The first group was first stained with Zombie stain, and then the surface of the tumor cells was stained with anti-CD45, anti-CD3, anti-CD4, anti-CD8, and anti-CD25. After cell fixation and nuclear membrane rupture, Foxp3 staining was performed to assess the proportion of Treg cells. The second part of the study involved first staining the tumor cells with Zombie stain, followed by surface staining with anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-CD11c, anti-CD80, anti-CD86, anti-CD11b, F4 / 80, anti-CD206, LY6C, and LY6G. After cell fixation and membrane rupture, GZMB staining was performed. The cell pellet was resuspended in cell staining buffer and analyzed by flow cytometry. Serum samples from each group of mice were collected to assess the levels of relevant inflammatory factors. ELISA kits were used to detect the levels of TNF-α, IFN-γ, IL-1β, and IL-18 in the serum.

[0084] (19) Statistical analysis: Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using t-tests (and nonparametric tests) and one-way / multi-way ANOVA in GraphPad Prism 10.0. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001).

[0085] II. Experimental Results

[0086] (1) Preparation and characterization of vMnp-Nig MBs

[0087] First, the constituent units of vMnp-Nig MBs were synthesized, assembled, and characterized physicochemically. The preparation of vMnp-Nig MBs was as follows: Figure 2 A. Porphyrin lipids react with MnCl2 in glacial acetic acid to yield manganese porphyrin lipids, which are amphiphilic. This reaction was analyzed by high-resolution mass spectrometry (HRMS). Figure 11 ab), ultraviolet-visible absorption spectrum (UV-vis) Figure 11 c), fluorescence spectrum ( Figure 11d) This demonstrates the successful preparation of manganese porphyrin lipids. Furthermore, to improve microbubble targeting, the pH-sensitive peptide Var7 was modified onto DSPE-PEG2000 via a thioether bond. The DSPE-PEG2000-Var7 (…) was analyzed using HRMS. Figure 12 Characterization was performed. To observe the uptake and distribution of nigrain, nigrain was linked to Cy5 via an amide bond to form Nig-Cy5. Molecular weight was confirmed using HRMS, and a precise molecular ion peak matching the theoretical value was observed. Figure 13 Microbubbles were prepared using DSPE-PEG2000-Var7, MnP, and Nig-Cy5. DSPC, DSPE-PEG2000-Var7, and MnP lipids were mixed in a molar ratio of 81:3:16, with an additional 5 μg / mg of nigrain added (nigrain was replaced with Nig-Cy5 only when characterizing nigrain uptake and distribution). This resulted in vMnp-Nig MBs, which were then analyzed using optical microscopy and a Coulter counter. Figure 2 (B, C) confirmed that the particle size of vMnp-Nig MBs was mainly distributed in the range of 2-3 μm, with a uniform distribution; cryo-scanning electron microscopy images ( Figure 2 D) confirmed that vMnp-Nig MBs are hollow structures with a thickness of approximately 100–250 nm, providing structural assurance for loading hydrophobic drugs. Regarding stability, the system remained constant in particle size and average concentration during a 7-day monitoring period when stored at 4°C. Figure 2 (E, F), and the microbubble size is consistent with the results of optical microscopy and cryo-electron microscopy, demonstrating excellent stability. Ultraviolet absorption spectroscopy shows ( Figure 2 G)vMnp-Nig NPs exhibit characteristic absorption peaks similar to Mnp at 370 nm and 680 nm, and fluorescence spectroscopy analysis shows that ( Figure 2 H) Under 650nm laser excitation, cy5-vMnp-Nig NPs exhibit the same characteristic emission peak at 680nm as cy5-Nig. The appearance of this characteristic peak confirms that Mnp and cy5-Nig are successfully encapsulated. In addition, the encapsulation efficiency of cy5-Nig is 97.08±0.14%. Figure 14 This lays the material basis for its biological effects. To verify that vMnp-Nig MBs can respond to ultrasonic explosions, experiments were conducted at 1 MHz, 20% duty cycle, and 1 W / cm². 2 Under ultrasound, the concentration of vMnp-Nig MBs decreased rapidly over time, and ultrasound could induce approximately 96% of the microbubbles to burst within 30 seconds. Figure 2 I), microbubbles are almost invisible under an optical microscope ( Figure 15 The burst microbubbles transform into small nanoparticles, which, according to transmission electron microscopy (TEM), have a particle size of approximately 20 nm. Figure 2 J), hydrated particle size approximately 80 nm ( Figure 2 K). Further potential analysis of the nanoparticles before and after the explosion revealed that the ultrasonic process did not significantly affect their original potential. Figure 2 L); and after successfully introducing DSPE-PE2000-Var7, the Zeta potential of the nanoparticles changed from -19.0±0.8 mV to -25.4±0.6 mV. This significant negative potential shift strongly demonstrates that the negatively charged Var7 targeting peptide has successfully bound to the microbubble surface. Drug release experiment ( Figure 2 M) clearly demonstrated the advantages of UTMD. Compared with the group without UTMD, UTMD significantly induced the release of Nig, increasing the cumulative release rate from 40% to 76% within 12 hours. In summary, this system successfully loaded Mnp, Var7, and Niger into vMnp-Nig MBs, exhibiting good stability and drug release capability.

[0088] (2) Generation of ROS and O2 in vitro

[0089] MnP in microbubbles endows vMnp-Nig NPs / MBs with the ability to generate ROS and catalyze the production of oxygen from hydrogen peroxide under ultrasound. First, the ability of vMnp-Nig NPs to generate singlet oxygen under ultrasound (US) induction was evaluated using a SOSG fluorescent probe. The results showed that at 1 MHz and 1 W / cm², the ability of vMnp-Nig NPs to generate singlet oxygen under ultrasound (US) induction was significantly enhanced. 2 Under a 20% duty cycle, the SOSG fluorescence intensity induced by vMnp-Nig NPs gradually increased with increasing sonication time, and showed a significant concentration dependence. Figure 3 A). At a fixed concentration, as the ultrasound duty cycle increased from 10% to 50%, SOSG fluorescence further increased ( Figure 3 B); The above results indicate that the generation of singlet oxygen is positively correlated with sonication time, sample concentration, and sonication duty cycle. In contrast, almost no fluorescence enhancement was observed in ddH2O and vMnp-Nig NPs alone without sonication. Figure 3 (C) This confirmed that the generation of ROS in vitro was entirely dependent on Mnp-responsive ultrasound. To further identify the types of ROS generated by ultrasound irradiation, we used ESR technology to detect the generation of different types of reactive oxygen species. For example... Figure 3 As shown in Figure D, vMnp-Nig MBs produced a typical 1:1:1 triplet characteristic peak under ultrasonic driving, and its signal intensity was significantly higher than that of the vMnp-Nig MBs group alone, confirming its ability to generate... 1 The O2 capacity was consistent with the SOSG probe detection results. In the aqueous system, a clear 1:2:2:1 quadruple characteristic peak was observed in the ultrasonically triggered vMnp-Nig MBs group. Figure 3 E), confirming hydroxyl radicals ( . The generation of superoxide anion (OH). . O 2- It is extremely unstable in aqueous solution, so a methanol system was used as the solvent for capture, as shown in Figure 3F. Under these conditions, significant [unclear] was observed. . O2-characteristic signal. The above results indicate that vMnp-Nig MBs can generate various types of ROS under ultrasonic irradiation.

[0090] Mnp in vMnp-Nig NPs also exhibited excellent catalase-like activity. As shown in Figure 3G, significant H2O2 degradation was observed in the system containing only Mnp, while the control group without Mnp showed almost no catalytic activity, confirming that Mnp possesses catalase-like activity. With increasing concentration gradient of vMnp-Nig NPs, the relative concentration of H2O2 in the system showed a non-linear decrease (…). Figure 3 H). Dissolved oxygen meter monitoring results confirmed ( Figure 3 After adding Mnp-containing nanoparticles, the oxygen content in the system increased rapidly. The oxygen production of the 500 ug / mL group within 70 minutes was significantly better than that of the low concentration group (100 ug / mL) and the control group without Mnp particles. This in-situ oxygen production can not only directly alleviate hypoxia at the tumor site, but also provide sufficient substrate for ROS production.

[0091] (3) In vitro cell uptake, vMnp-Nig MBs to alleviate 4T1 cell hypoxia and ultrasound-induced intracellular ROS production

[0092] To systematically evaluate the uptake and delivery efficiency of vMnp-Nig MBs by 4T1 cells, Nig-Cy5 was used to replace nigrain in the preparation of microvesicles for characterization. Flow cytometry results showed that after co-incubation of Cy5-vMnp-Nig MBs with 4T1 cells for 0–6 h, the fluorescence peak gradually shifted to the right over time, reaching a plateau at 4 h. Figure 4 A, Figure 16 a). Compared with the unmodified Var7 group, the Var7-modified Mnp-Nig NPs group showed a stronger fluorescence signal in 4T1 cells ( Figure 4 B, Figure 16 b). Confocal results further confirmed that the fluorescence signal of the Var7 modified group gradually increased with prolonged incubation time, and was mainly localized in the cytoplasm rather than being limited to the cell membrane. Figure 17 After UTMD treatment, the flow cytometry fluorescence peaks of the Cy5-vMnp-Nig MBs group shifted further to the right, and the MFI significantly increased. Figure 4 C, Figure 16c), indicating that the targeting peptide and UTMD can effectively improve intracellular delivery efficiency. Subsequently, the ability of cy5-vMnp-Nig MBs to penetrate the extracellular matrix of 4T1 cells after UTMD was evaluated using the MCTS model. The results showed that a strong Cy5 fluorescence signal could still be detected in the vMnp-Nig MBs+US group at 80 μm from the tumor spheroid surface, while the vMnp-Nig NPs group showed significant fluorescence at 60 μm, but overall it was weaker than the vMnp-Nig MBs+US group at the same depth. Figure 4 (D–F). In contrast, the vMnp-Nig MBs and Mnp-Nig NPs groups showed weak fluorescence only at the edge of the tumor spheres. These results indicate that Var7-mediated active targeting, as well as the mechanical and cavitation effects induced by UTMD, significantly enhance the penetration of nanoparticles into tumor spheres and their uptake and enrichment by tumor cells, thus facilitating the overcoming of solid tumor delivery barriers.

[0093] The ability of each treatment group to alleviate hypoxia in 4T1 cells was further evaluated using the [Ru(dpp)3]Cl2 oxygen-sensitive probe. Since the fluorescence intensity of this probe is negatively correlated with oxygen concentration, the strongest red fluorescence in the hypoxic control group indicated that the cells were in a state of significant hypoxia. Figure 4 (G, H). In contrast, all treatment groups containing Mnp showed varying degrees of fluorescence signal decrease, suggesting that Mnp can alleviate intracellular hypoxia by decomposing endogenous H2O2 and generating O2 through catalase-like activity. Among them, the fluorescence decrease was most significant in the vMnp MBs+US, vMnp-Nig NPs+US, and vMnp-Nig MBs+US groups, indicating that the Mnp-mediated oxygen production effect is an important reason for improving the hypoxic tumor microenvironment and also provides more favorable conditions for subsequent ROS generation.

[0094] Mitochondrial dysfunction is closely related to cell death. To investigate the effects of vMnp-Nig MBs+US treatment on cellular oxidative stress and mitochondrial function under normoxic and hypoxic conditions, we used the DCFH-DA probe to detect ROS levels and observed changes in mitochondrial membrane potential using RH-123 staining. The results showed that ( Figure 4 Under normoxic conditions, the green fluorescence of the Control and US groups was weak, while vMnp MBs+US and vNig MBs+US showed varying degrees of enhancement. Although the overall ROS level of each group was slightly lower under hypoxic conditions than under normoxic conditions, the vMnp-Nig MBs+US group exhibited the strongest fluorescence. These results indicate that the combination of manganese porphyrin and nigericin can significantly amplify the ROS generation effect. RH-123 dye can selectively accumulate in mitochondria based on the potential difference between the inner and outer mitochondrial membranes; the higher the mitochondrial membrane potential, the stronger the green fluorescence. The results show that (…). Figure 4The strongest fluorescence was observed in the L, M), Control, and US groups, indicating intact mitochondrial structure. The fluorescence of vMnp MBs+US and vNig MBs+US was weaker, especially in the vMnp-Nig MBs+US and vMnp-Nig MBs+US groups, with the lowest fluorescence in the vMnp-Nig MBs+US group. This suggests that vMnp-Nig MBs can cause mitochondrial damage and dysfunction under ultrasound. In summary, the large accumulation of intracellular ROS is consistent with mitochondrial depolarization, suggesting that vMnp-Nig MBs+US can further disrupt mitochondrial homeostasis through strong oxidative stress. This may be due to the efficient generation of reactive oxygen species by manganese porphyrin under ultrasound and the synergistic effect of nigericin disrupting mitochondrial ion homeostasis and weakening mitochondrial function.

[0095] (4) The killing effect of vMnp-Nig MBs on 4T1 cells and the ability to inhibit 4T1 migration and invasion under ultrasound.

[0096] We used the CCK-8 assay to detect the biocompatibility of vMnp-Nig MBs and their killing effect on 4T1 tumor cells. We first evaluated the biocompatibility of vMnp MBs, vMnp-Nig MBs and free Nig with HUVEC cells. Figure 5 Results showed that free Nigericin exhibited significant inherent toxicity, with cell viability decreasing sharply with increasing concentration; however, encapsulation with vMnp MBs resulted in significantly higher cell viability than the free group without ultrasound triggering, demonstrating that microbubble encapsulation effectively reduces off-target toxicity of the drug. Subsequently, we investigated the cytotoxic effect of vMnp-Nig MBs+US on 4T1 cells (…). Figure 5 (B) The results showed a concentration-dependent effect; as the concentration increased to 150 ug / ml, cell viability decreased to approximately 27%. Comparisons between different groups revealed ( Figure 5 C) Simple ultrasound or drug-loaded microbubbles alone had little effect on cells, while the killing effect of vMnp-Nig MBs under ultrasound triggering was significantly better than that of the vMnp-Nig NPs group. These results indicate that encapsulating Nigerian cytosine within vMnp MBs effectively reduces toxicity to normal cells and exhibits good biocompatibility; furthermore, under ultrasound, the cavitation effect generated by UTMDs enhanced the drug uptake efficiency of 4T1 cells, ultimately significantly amplifying the killing effect on 4T1 tumor cells.

[0097] Calcein-AM / PI fluorescence double staining of live and dead cells further visually validated the above results. Figure 5D). In the control and US groups, strong green fluorescence, representing viable cells, was predominant, with almost no red fluorescence, indicating minimal cell damage. However, in the vMnp-Nig NPs+US or vMnp-Nig MBs+US groups, red fluorescent bright spots increased, while green fluorescence was significantly less and dimmer, replaced by dense red fluorescence. This result is highly consistent with the quantitative trend of CCK-8, strongly demonstrating that under ultrasound-mediated treatment, vMnp-Nig MBs can exert a very strong killing effect on 4T1 cells, causing massive cell death. Subsequently, Annexin V-FITC / PI was used to detect apoptosis in 4T1 cells treated with different methods. Figure 5 E and Figure 5 (G) The results showed that the apoptosis rate of vMnp-Nig MBs was at a low level. After the addition of ultrasound intervention, the total apoptosis rate of the vMnp-Nig MBs+US group was approximately 84.3%, significantly higher than that of the vMnp-Nig NPs+US group (approximately 73.6%). This result indirectly indicates that UTMD can enhance the killing effect on 4T1 cells by promoting the local release of drugs from vMnp-Nig MBs, increasing cell membrane permeability and drug delivery efficiency. Quadrant analysis revealed that the proportion of late apoptosis in both the vMnp-Nig NPs+US group and the vMnp-Nig MBs+US treatment group was higher than 50%. This indicates that the combination of the two drugs can not only significantly induce apoptosis in 4T1 cells, but also promote more cells to enter the late apoptosis stage.

[0098] The effects of different treatments on the invasion and migration abilities of 4T1 cells were evaluated using Transwell invasion and scratch assays. Results showed that compared to the control, US, vMnp-Nig MBs, vMnp-MBs+US, and vNig-MBs+US groups, the invasion and migration abilities of cells treated with vMnp-Nig MBs+US and vMnp-Nig MBs+US were significantly weakened, with the vMnp-Nig MBs+US group showing the most significant inhibitory effect. Transwell crystal violet staining results (…) Figure 5 F) It can be seen that this group has the fewest transmembrane cells, and quantitative analysis ( Figure 5 H) showed that its relative number of metastatic cells was only 27 ± 3.0% of that in the control group; at the same time, the scratch test ( Figure 18 Wound closure was significantly impaired in the study, and quantitative analysis of the healing rate was conducted. Figure 5 I) showed a positivity rate of only 14.6 ± 4.7%, significantly lower than other treatment groups. These results indicate that, under ultrasound triggering, vMnp-Nig MBs+US can maximally inhibit the ability of 4T1 cells to penetrate the matrix and migrate laterally, weakening their invasive and migratory potential, providing a basis for subsequent in vivo anti-metastasis studies.

[0099] (5) vMnp-Nig MBs combined with ultrasound induce pyroptosis in TNBC cells and trigger immunogenic cell death.

[0100] Cell morphology of different treatment groups was observed using an optical microscope. Figure 19 Compared with the Control and US groups, the vMnp-Nig MBs, vMnp MBs+US, or vNig MBs+US treatment groups showed only limited changes in cell morphology. However, after vMnp-Nig MBs+US treatment, the cells showed obvious membrane bubbling, cell swelling, and disruption of cell membrane integrity, consistent with typical pyroptosis characteristics. Scanning electron microscopy results further confirmed this phenomenon. Figure 6 A), the above results suggest that sonodynamic therapy can amplify pyroptosis-like cell death induced by nigericin. Both vNig MBs and ultrasound-treated vMnp MBs caused DNA damage in 4T1 cells, and the vMnp-Nig MBs+US group showed the strongest γ-H2AX fluorescence signal, proving that it caused the most severe DNA damage to 4T1 cells. Figure 6 B). We further performed Western blot (WB) to detect the expression of pyroptosis-related proteins in 4T1 cells after different treatments, and the results showed that ( Figure 6 CE and Figure 20 Compared with the control group, vMnp-Nig MBs+US treatment significantly upregulated the expression levels of NLRP3, Cleaved-Caspase-1, and GSDMD-N. Caspase-1 is a key executive protein following NLRP3 activation, while the N-terminal structure formed by GSDMD cleavage can create pores in the cell membrane, releasing cellular contents. This demonstrates that this system can induce classical pyroptosis through the NLRP3-Caspase-1-GSDMD pathway.

[0101] Pyroptosis not only directly leads to lytic death of tumor cells, but also involves the release of large amounts of DAMPs, thereby promoting the activation of ICD-related processes. Therefore, we further investigated ICD-related biomarkers. Immunofluorescence results showed ( Figure 6 F and Figure 6 (G) Compared with the vNig MBs + US group, the vMnp-Nig MBs + US group showed a significant increase in CRT exposure on the cell membrane surface, suggesting that this treatment can generate a stronger "eat-me" signal and promote dendritic cell recognition of tumor cells. Simultaneously, after vMnp-Nig MBs + US treatment, the release levels of HMGB1 in the cytoplasm of 4T1 cells and LDH in the culture supernatant were significantly increased. Figure 6 H, Figure 6 I and Figure 21HMGB1, as an important DAMP, promotes dendritic cell maturation and enhances antigen presentation capacity, while LDH release reflects disruption of cell membrane integrity. In summary, vMnp-Nig MBs, formed by introducing manganese porphyrin into vNig MBs, can induce more intense pyroptosis under ultrasound and further amplify ICD-related immunostimulatory signals by promoting DAMP release. This is expected to activate a stronger local tumor immune response, reverse the immunosuppressive tumor microenvironment, and ultimately improve the efficacy of antitumor immunotherapy.

[0102] (6) RNA sequencing analysis

[0103] To further elucidate the molecular basis of this process, we performed RNA-seq analysis on 4T1 cells treated with PBS, vNig MBs+US, and vMnp-Nig MBs+US. The results showed that both vNig MBs and vMnp-Nig MBs significantly remodeled the gene expression profile of 4T1 cells under sonication. Further comparison revealed that, compared to the vNig MBs+US group, the vMnp-Nig MBs+US group induced a wider range of transcriptional changes, with 1320 genes significantly upregulated and 461 genes significantly downregulated. Figure 7 A–C). KEGG analysis showed that differentially expressed genes were mainly enriched in inflammation and innate immune-related pathways (Figure 7D–F). Further GSEA results showed that, compared with the vNig MBs+US group, the vMnp-Nig MBs+US group was significantly enriched in the NOD-like receptor, Toll-like receptor, NF-kappa B, and Cytosolic DNA-sensing pathways. Figure 7 (G–J). Furthermore, high expression of Caspase-1 was observed in the NOD-like receptor signaling pathway, further supporting the potential involvement of inflammasome-related pyroptosis. These results are consistent with the aforementioned evidence related to pyroptosis and ICD, indicating that Mnp-mediated SDT not only amplifies pyroptosis induced by nigericin at the executive level but also amplifies pro-inflammatory and immunostimulatory signals at the transcriptomic level, laying a solid molecular foundation for subsequent induction of potent anti-tumor immune remodeling and reversal of the immunosuppressive microenvironment.

[0104] (7) Biosafety assessment of vMnp-Nig MBs

[0105] Before conducting in vivo experiments, the blood compatibility of this microbubble system was evaluated through an in vitro hemolysis experiment. For example... Figure 22As shown in Figure a, under different concentrations (6.25-200 ug / ml), no obvious erythrocyte lysis was observed after co-incubation of vMnp-Nig MBs with erythrocytes. The supernatant remained nearly colorless after centrifugation, with only the positive control group showing obvious red hemolysis. Quantitative results showed ( Figure 22 (b) The hemolysis rate remained below 5% at all experimental concentrations, far below the safety threshold for blood compatibility of biomaterials, indicating that the microbubble system has good blood compatibility. Subsequently, its systemic toxicity was further evaluated in mice. Blood routine test results ( Figure 23 The results showed that blood samples were collected at 1, 3, 7, and 15 days after drug administration, and no significant differences were observed in red blood cells, white blood cells, platelets, and related blood cell indicators in mice compared to before drug administration, indicating that the nanobubble system did not significantly interfere with the hematopoietic system. Venous blood was collected from different groups of mice, and blood biochemical tests were performed 15 days after treatment. Figure 24 The levels of alanine aminotransferase and aspartate aminotransferase, which reflect liver function, and blood urea nitrogen and serum creatinine, which reflect kidney function, were all maintained within the normal physiological range, and no obvious liver or kidney dysfunction was observed. Further H&E staining of major organs (…) Figure 25 The results showed that the heart, liver, spleen, lungs and kidneys of mice in each treatment group maintained normal tissue structure, confirming that the microbubble system has good tissue compatibility in vivo.

[0106] (8) In vivo imaging

[0107] Based on the ability of contrast-enhanced ultrasound (VEME) to visualize tumor internal structures in real time and dynamically monitor drug accumulation in the tumor region, and considering that UTMD can significantly enhance drug penetration and tumor accumulation by breaking down local tumor biological barriers, we first systematically evaluated the contrast-enhanced ultrasound capabilities and stability of the constructed vMnp-Nig MBs. In in vitro experiments, a latex tube was used to simulate a blood vessel model, and microbubble suspensions of different dilutions were injected into the tube for ultrasound imaging. Figure 8 A and Figure 26 As shown, B-mode and contrast-enhanced ultrasound imaging were performed at different dilution ratios (50×, 100×, 200×, 400×, 800×), and signal changes were recorded within 0–9 min. The results indicate that at a 100-fold dilution (concentration approximately 3.7 × 10⁻⁶), B-mode and contrast-enhanced ultrasound imaging were performed, and signal changes were recorded within 0–9 min. 6 mL -1 At a dilution factor of 4.6 × 10⁻⁶, microbubbles produce the strongest and most uniform echo signal; as the dilution factor increases, the ultrasonic signal gradually weakens, but reaches its maximum at 800 × (4.6 × 10⁻⁶). 5 mL -1Significant contrast enhancement was still detected at a concentration of 9 min, indicating that the microbubble system maintains good ultrasound imaging capabilities over a wide concentration range. Furthermore, the echo signal showed minimal change during the 9-min observation period, demonstrating good structural stability of the microbubbles in solution, providing a reliable basis for subsequent UTMD-triggered drug release. We evaluated the in vivo ultrasound imaging capabilities of vMnp-Nig MBs. Figure 8 As shown in Figure B, almost no ultrasound signal was observed in the tumor area before injection; however, approximately 30 seconds after injection, a significantly enhanced echo signal rapidly appeared in the tumor area, with a bright contrast signal almost filling the entire tumor region, indicating that vMnp-Nig MBs can quickly reach and accumulate in the tumor vascular system. Furthermore, this enhanced signal persisted for more than 5 minutes, demonstrating the good acoustic stability of the microbubble system in vivo and providing a sufficient time window for subsequent UTMD. Based on this, to verify whether UTMD could trigger microbubble rupture, tumor-bearing 4T1 mice were injected with vMnp-Nig MBs and immediately subjected to UTMD treatment (1 MHz, 20% duty cycle, 1 W cm⁻¹). -2 The study used ultrasound imaging to continuously monitor signal changes in the tumor region. Results showed that the contrast enhancement signal in the tumor region rapidly decreased after UTMD treatment. Figure 8 (B) indicates that the microbubbles ruptured effectively within the tumor blood vessels, a process accompanied by in-situ micro-nano transformation of the microbubbles at the tumor site.

[0108] The distribution of Cy5.5 fluorescently labeled vMnp-Nig MBs in tumor-bearing mice after UTMD was traced using in vivo fluorescence imaging. Figure 8 As shown in CD, fluorescence signals of a certain degree were detectable in the tumor areas of mice 1 hour after injection. With prolonged time, the fluorescence signal in the tumor area of ​​the vMnp-Nig MBs+UTMD group significantly increased, peaking at 4 hours and maintaining a high level from 6 to 36 hours, while the signal in the microbubble group without UTMD treatment was generally weak. To further evaluate the tissue distribution of the carrier in vivo, mice were sacrificed 36 hours after injection, and major organs and tumor tissues were collected for ex vivo fluorescence imaging. Figure 8As shown in the EG diagram, compared with the vMnp-Nig MBs group without UTMD treatment, the vMnp-Nig MBs+UTMD group exhibited stronger fluorescence signals in tumor tissues, while the signal differences in major organs such as the heart, liver, spleen, lungs, and kidneys were relatively small. These results indicate that ultrasound-induced microbubble rupture can effectively promote the specific accumulation of the carrier in the tumor region. Furthermore, to further track the distribution of nigrain in vivo, nigrain was conjugated with Cy5 to construct cy5-vMnp-Nig MBs for in vivo fluorescence imaging. The results showed that the overall fluorescence signal in the tumor site showed a trend of vMnp-Nig MBs+UTMD > vMnp-Nig NPs > Mnp-Nig NPs (…). Figure 27 ab). The vMnp-Nig NPs group showed stronger fluorescence than the Mnp-Nig NPs group, indicating that Var7 modification enhances the active targeting and enrichment ability of the formulation at the tumor site; while the vMnp-Nig MBs+UTMD group showed a further significant increase in fluorescence, indicating that UTMD can further improve the drug delivery efficiency in tumor tissue on the basis of Var7 targeting. Ex vivo fluorescence imaging was performed on major organs and tumor tissues 72 h after injection, and the results also showed that the vMnp-Nig MBs+UTMD group had the strongest fluorescence signal at the tumor site ( Figure 27 (ce), consistent with in vivo imaging results. These results indicate that Var7 endows the system with the ability to actively recognize the acidic tumor microenvironment, while UTMD promotes the penetration, retention, and release of nanoparticles in the tumor locality through micro-nano transformation, cavitation effect, and acoustic pore effect induced by microbubble bursting. The synergistic effect of the two ultimately achieves more efficient tumor-targeted delivery.

[0109] Photoacoustic imaging can visually display the distribution of blood vessels and the distribution of blood oxygen saturation (SO2) in the 4T1 tumor region. Figure 8 (H), where blue represents hypoxic areas and red represents oxygen-rich areas. The results showed that the control group exhibited a distinct blue color, reflecting low blood oxygen saturation, indicating a hypoxic state in the tumor region. In contrast, the vMnp-Nig MBs+UTMD group showed a shift towards a purplish-red hue in the tumor region, indicating a significant increase in SO2. This result suggests that vMnp-Nig MBs accumulates in the tumor region through the UTMD-mediated EPR effect and Var7-mediated active targeting, and can catalyze the production of O2 from H2O2 within the tumor, thus alleviating hypoxia in the tumor region.

[0110] The introduction of manganese porphyrin significantly enhances the contrast of the system in magnetic resonance imaging (MRI). The MRI performance of vMnp-Nig NPs was systematically evaluated using a 7.0 T small animal MRI system. As the manganese concentration in the system gradually increases, the color of the vMnp-Nig NPs solution gradually deepens (…). Figure 8 I). Further evaluation of its relaxation characteristics was conducted using in vitro magnetic resonance relaxation experiments. By plotting the relationship between relaxation rate and manganese concentration, it was found that both the transverse relaxation rate R2 and the longitudinal relaxation rate R1 showed a good linear correlation with manganese concentration. Figure 8 J, 8K). Linear fitting results showed that the transverse relaxation r2 of this system was approximately 150.3 mM⁻¹ s⁻¹, and the longitudinal relaxation r1 was approximately 7.37 mM⁻¹ s⁻¹, indicating that vMnp-Nig NPs have strong transverse and longitudinal relaxation capabilities. Based on the in vitro results, its in vivo MRI imaging capability was further evaluated in a 4T1 tumor-bearing mouse model. After tail vein injection of vMnp-Nig MBs into mice, they were treated with UTMD, and MRI scans were performed before and after administration. Figure 8 The results showed that vMnp-Nig MBs did not significantly change T1WI and T2WI signals before and after drug administration. After UTMD treatment, T2WI signals decreased and T1WI signals increased. Figure 8 (M, 8N). This result demonstrates that vMnp-NigMBs, triggered by UTMD, not only achieve efficient delivery but also possess excellent MRI imaging capabilities, providing a reliable basis for visual monitoring and efficacy evaluation during tumor treatment.

[0111] (9) In vivo therapeutic effects of vMnp-Nig MBs

[0112] vMnp-Nig MBs combined with ultrasound can enhance local immune activation and reverse the immunosuppressive tumor microenvironment by inducing more intense pyroptosis, promoting DAMPs release, and alleviating tumor hypoxia, thereby enhancing the anti-tumor efficacy of aPD-L1. To evaluate the in vivo therapeutic effect of vMnp-Nig MBs combined with aPDL-1 on 4T1 tumors, a 4T1 tumor-bearing mouse model was established and... Figure 9 Treatment was administered according to the timeline shown in A. Treatment continued until the tumor reached approximately 50 mm in size. 3 Following drug administration and UTMD, mice underwent aPD-L1 administration and ultrasound therapy on the second day after drug administration. Tumor volume changes were continuously monitored throughout the treatment process, and mouse survival was recorded. To more directly observe tumor progression and survival status in different treatment groups, in vivo photography of mice began on day 20 of treatment and was recorded every 6 days until the end of the experiment. Representative photographs are shown below. Figure 28 .like Figure 9 As shown in Figure B, there were significant differences in tumor volume among the groups of mice on day 20 after treatment. Compared with the control group, treatment with vMnpMBs+US or aPD-L1 alone showed only limited tumor-suppressing effects. Tumor growth was significantly inhibited after treatment with vMnp-Nig MBs+US, and the tumor volume of mice treated with vMnp-Nig MBs+US combined with aPD-L1 was further reduced. Further quantitative results of tumor volume ( Figure 9 C and Figure 29 This was also confirmed. Meanwhile, the body weight of mice in each group remained stable throughout the treatment period (Figure 9D), indicating that the treatment strategy has good in vivo safety. Survival analysis results ( Figure 9 E) Further, it was shown that mice treated with vMnp-Nig MBs+US had significantly prolonged survival, with the group treated with the combined aPD-L1 therapy exhibiting the longest survival. To investigate the anti-tumor mechanism of vMnp-Nig MBs+US combined with aPD-L1 therapy, histological and immunofluorescence analyses were performed on tumor tissues. Figure 9 As shown in Figure F, H&E staining results revealed that, compared to the Control and US groups, all treatment groups exhibited varying degrees of antitumor effects. In contrast, the vMnp-Nig MBs+US group showed significantly enhanced tissue destruction, weakened Ki67 signaling, and increased positivity for dUTP nick-end labeling mediated by terminal deoxynucleotidyl transferase. Further mechanistic analysis indicated that the vMnp-Nig MBs+US group showed enhanced γ-H2AX signaling, decreased HIF-1α expression, and significantly elevated levels of Cleaved caspase-1 and GSDMD-N in tumor tissue. Compared to aPD-L1 or vMnp MBs+US alone, this strategy exacerbated DNA damage, alleviated tumor hypoxia, and amplified pyroptosis in vivo. Figure 9 G). Further immune-related sections showed ( Figure 9 (H and I), vMnp-Nig MBs+US can promote the expression of CD11c, CD3, CD8, and GZMB in tumor tissue, accompanied by upregulation of CD86 and downregulation of CD206, indicating that it can not only enhance dendritic cell recruitment and T cell infiltration, but also promote the transformation of tumor-associated macrophages to an immune-activated phenotype, thereby facilitating the remodeling of the immunosuppressive tumor microenvironment. Based on this, the above changes are further enhanced when combined with aPD-L1. The vMnp-Nig MBs+US+aPD-L1 group showed the most significant tumor tissue destruction, pyroptosis activation, and immune cell infiltration, suggesting that vMnp-Nig MBs+US can effectively enhance the therapeutic effect of ICB by inducing DNA damage and pyroptosis, activating anti-tumor immunity, and improving the tumor microenvironment.

[0113] (10) vMnp-Nig MBs promote anti-tumor immunity

[0114] To systematically evaluate the anti-tumor immune activation effect of vMnp-Nig MBs+US combined with aPD-L1 treatment, a 4T1 tumor-bearing mouse model was first established and then... Figure 10 Treatment was administered according to the timeline shown in Figure A. Five days after treatment, the tumor-draining lymph nodes, tumor tissue immune cell composition, and serum cytokine levels of tumor-bearing mice were analyzed. Flow cytometry results showed that compared with the Control, vMnp MBs+US, and aPD-L1 groups, the proportion of mature DCs in the tumor local area and draining lymph nodes was increased in the vMnp-Nig MBs+US group (9% and 30.8%, respectively). The vMnp-Nig MBs+US+aPD-L1 combined treatment group further increased to the highest level, at 12.8% and 33.8%, respectively. Figure 10 B, F and Figure 30 (a) suggests that this treatment strategy can more effectively promote antigen presentation and initial immune activation in the tumor region.

[0115] To further evaluate treatment-induced systemic inflammation, the levels of inflammation-related cytokines in mouse serum were detected using ELISA. The results showed that, compared with other groups, the levels of TNF-α, IFN-γ, IL-1β, and IL-18 in the serum of mice in the vMnp-Nig MBs+US+aPD-L1 group were all elevated (…). Figure 31 Among them, IL-1β and IL-18 are key cytokines released by inflammasome activation during pyroptosis, and their elevation further demonstrates the occurrence of pyroptosis-related inflammatory responses; while the elevation of IFN-γ and TNF-α reflects enhanced anti-tumor immune activity. Under the dual action of mature DC cells and inflammatory factors, CD4 in the vMnp-Nig MBs+US group... + T and CD8 + The proportion of T cells increased significantly, to 26.8% and 26.1% respectively, after combined aPD-L1 therapy, CD4... + T and CD8 + The proportion of T further increased, increasing by 2.9 times and 3.1 times respectively compared to the control group. Figure 10 CD). The above results indicate that vMnp-Nig MBs+US can effectively amplify innate and adaptive anti-tumor immune responses in synergistically with aPD-L1 by inducing pyroptosis-related inflammation activation, promoting DC maturation, and enhancing T cell infiltration.

[0116] In addition to promoting the activation of effector immune cells, this therapeutic strategy also significantly remodeled the tumor immunosuppressive microenvironment. Flow cytometry analysis showed that regulatory T cells (Tregs, CD4+) were significantly increased in the vMnp-Nig MBs+US+aPD-L1 group. + CD25 + FoxP3 + ), neutrophils (CD45) + CD11b + Ly6G + ) and mononuclear myeloid suppressor cells (M-MDSCs, CD45) + CD11b + Ly6C + The proportion of ) decreased significantly ( Figure 10 E and Figure 10 These cell populations typically exert immunosuppressive effects in the tumor microenvironment; their reduction is beneficial for overcoming immune tolerance and enhancing antitumor immune responses. Simultaneously, tumor-associated macrophages (TAMs) exhibit significant phenotypic reprogramming, with M1 macrophages (CD11b) showing increased activity. + F4 / 80 + CD86 + The proportion of CD11b macrophages increased, while the proportion of M2 macrophages (CD11b) increased. + F4 / 80 + CD206 + The proportion decreases, thereby significantly increasing the M1 / M2 ratio. Figure 10 (G) M1 macrophages can secrete a variety of pro-inflammatory factors and enhance antigen presentation; therefore, this polarization process helps to further amplify the anti-tumor immune response. This phenomenon may be related to the release of pyroptosis-induced inflammatory factors and ROS-related oxidative stress, which can drive macrophages to transform into a pro-inflammatory phenotype. In summary, vMnp-Nig MBs+US can not only promote DC maturation and T cell infiltration by inducing pyroptosis-related inflammatory responses, but also significantly improve the tumor immunosuppressive microenvironment by reducing the proportion of immunosuppressive cells and promoting TAMs to M1 polarization. On this basis, combined with aPD-L1, it can further amplify innate and adaptive anti-tumor immune responses, thereby significantly enhancing the therapeutic effect of ICB.

[0117] To further observe the long-term anti-tumor immune response effect of vMnp-Nig MBs+US combined with aPD-L1 treatment, a lung metastasis model was constructed, and the inhibitory effect of different treatments on tumor metastasis was evaluated by H&E staining of lung tissue. The results showed that a large number of obvious metastatic nodules were visible in the lung tissue of the control group mice, a small number of metastatic lesions were visible in the vMnp-Nig MBs+US group, while almost no metastatic lesions were visible in the lung tissue of the vMnp-Nig MBs+US+aPD-L1 group. Figure 10(J). This result indicates that the combined treatment strategy not only effectively inhibits in situ tumor growth but also significantly reduces the risk of distant metastasis and prolongs survival. Considering that tumor metastasis is often closely related to immune escape, the above-mentioned metastasis-inhibiting effect is likely due to the enhanced systemic anti-tumor immune response and the surveillance function of continuously activated cytotoxic T cells.

[0118] III. Conclusion

[0119] In summary, this invention constructs a multifunctional microbubble vMnp-Nig MBs with dual-modal ultrasound / MRI imaging capabilities, providing a new strategy for precise intervention and sustained therapeutic response in TNBC. This system endows the tumor's acidic microenvironment with responsive targeting capabilities through Var7 and utilizes UTMD to achieve spatiotemporally controlled release and deep delivery of therapeutic components to the tumor site, thereby improving the lesion utilization efficiency of pyroptosis inducers and sonosensitive components. More importantly, vMnp-Nig MBs construct a programmed pyroptosis amplification mode, where Nig provides a clear inflammasome activation signal, and manganese porphyrin further enhances oxidative stress and amplifies the pyroptosis-related inflammatory cascade by acting as a sonosensitive agent and CAT-like, while alleviating tumor hypoxia through oxygen production. This achieves direct tumor killing and immune activation, significantly enhancing the pyroptosis effect and tumor immune microenvironment remodeling, and further improving the responsiveness of TNBC to PD-L1 blockade therapy. Overall, this invention proposes a programmed pyroptosis treatment strategy that integrates precise delivery, sonodynamic enhancement, microenvironment regulation, and diagnostic and therapeutic monitoring, providing a new approach for immune sensitization and integrated diagnostic and therapeutic intervention of TNBC.

[0120] Example 2: Preparation of an ultrasound-responsive targeted microbubble delivery system

[0121] Preparation of manganese porphyrin lipids: PL, MnCl2, and NaOAc were dissolved in glacial acetic acid at a molar ratio of 0.5:8:15, and stirred in a water bath at 70°C for approximately 1.5 h under light-protected conditions. The reaction progress was monitored using thin-layer chromatography. After the reaction was complete, glacial acetic acid was removed by rotary evaporation under reduced pressure to obtain crude Mnp product. Subsequently, the obtained crude Mnp product was used to obtain self-assembled MnP NPs via ethanol injection, followed by dialysis purification and freeze-drying to obtain the target product. The specific steps of the ethanol injection method are as follows: the crude Mnp product was dissolved in DMSO or DMF, and the solution was added dropwise to 5 volume equivalents of ddH2O under ultrasonic conditions in a water bath (temperature 25±5°C, ultrasonic power 160~400W).

[0122] Preparation of vMnp-Nig MBs microbubbles: DSPC and DSPE-PEG2000-Var7 were dissolved in anhydrous ethanol, and manganese porphyrin lipid was dissolved in dimethyl sulfoxide. The solutions were mixed at a molar ratio of DSPC:DSPE-PEG2000-Var7:manganese porphyrin lipid = 75:5:14 to obtain a mixture. Nigerian styracin was added to the mixture, and the mass ratio of the total mass of DSPC, DSPE-PEG2000-Var7, and manganese porphyrin lipid to Nigerian styracin was controlled at 220:1. After thorough mixing, an organic phase was obtained (the volume of the organic phase was the total volume of anhydrous ethanol and DMSO, i.e., the sum of the volumes of organic solvents used after all four components were dissolved). Subsequently, the above organic phase was injected into the aqueous phase under ultrasonic water bath conditions (temperature 25±5℃, ultrasonic power 160~400W), with a volume ratio of organic phase to aqueous phase of 1:4. The obtained nanoparticle dispersion was dialyzed in distilled water for 2 hours using a dialysis bag with a molecular weight cutoff of 8000-12000 Da to remove residual organic solvents, thus preparing vMnp-Nig NPs. Finally, the prepared vMnp-Nig NPs were mixed with 1,2-propanediol and glycerol at a volume ratio of 1.0:0.3:0.3 ml, placed in a vial, filled with perfluoropropane gas, and tightly sealed with a cap. The mixture was then shaken for 50 seconds to finally form vMnp-Nig MBs.

[0123] Example 3: Preparation of an ultrasound-responsive targeted microbubble delivery system

[0124] Preparation of manganese porphyrin lipids: PL, MnCl2, and NaOAc were dissolved in glacial acetic acid at a molar ratio of 1.5:3:25 and stirred in a 50°C water bath for approximately 2.5 h under light-protected conditions. The reaction progress was monitored using thin-layer chromatography. After the reaction was complete, glacial acetic acid was removed by rotary evaporation under reduced pressure to obtain crude Mnp product. Subsequently, the obtained crude Mnp product was used to obtain self-assembled MnP NPs via ethanol injection, followed by dialysis purification and freeze-drying to obtain the target product. The specific steps of the ethanol injection method are as follows: the crude Mnp product was dissolved in DMSO or DMF, and the solution was added dropwise to 6 volume equivalents of ddH2O under ultrasonic conditions in a water bath (temperature 25±5°C, ultrasonic power 160~400W).

[0125] Preparation of vMnp-Nig MBs microbubbles: DSPC and DSPE-PEG2000-Var7 were dissolved in anhydrous ethanol, and manganese porphyrin lipid was dissolved in dimethyl sulfoxide. The solutions were mixed at a molar ratio of DSPC:DSPE-PEG2000-Var7:manganese porphyrin lipid = 87:2:18 to obtain a mixture. Nigerian mycotoxin was added to this mixture, and the mass ratio of the total mass of DSPC, DSPE-PEG2000-Var7, and manganese porphyrin lipid to Nigerian mycotoxin was controlled at 180:1. After thorough mixing, an organic phase was obtained (the volume of the organic phase was the total volume of anhydrous ethanol and DMSO, i.e., the sum of the volumes of organic solvents used after all four components were dissolved). Subsequently, the above organic phase was injected into the aqueous phase under ultrasonic water bath conditions (temperature 25±5℃, ultrasonic power 160~400W), with a volume ratio of organic phase to aqueous phase of 1:6. The obtained nanoparticle dispersion was dialyzed in distilled water for 2 hours using a dialysis bag with a molecular weight cutoff of 8000-12000 Da to remove residual organic solvents, thus preparing vMnp-Nig NPs. Finally, the prepared vMnp-Nig NPs were mixed with 1,2-propanediol and glycerol at a volume ratio of 0.5:0.2:0.2 ml, placed in a vial, filled with perfluoropropane gas, and tightly sealed with a cap. The mixture was then shaken for 40 seconds to finally form vMnp-Nig MBs.

[0126] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. An ultrasound-responsive targeted microbubble drug delivery system, characterized in that, The ultrasound-responsive targeted microbubble drug delivery system is Var7-modified manganese porphyrin / nigiacin co-loaded microbubbles vMnp-Nig MBs, which includes a microbubble interface formed by DSPC, DSPE-PEG2000-Var7 and manganese porphyrin lipids, nigiacin (Nig), and perfluoropropane gas (C3F8) encapsulated inside the microbubble.

2. The ultrasound-responsive targeted microbubble drug delivery system according to claim 1, characterized in that, The average particle size of vMnp-NigMBs is 2~3 μm.

3. The ultrasound-responsive targeted microbubble drug delivery system according to claim 1, characterized in that, The microbubbles have a hollow structure and the thickness of the microbubble membrane is 100~250 nm.

4. The ultrasound-responsive targeted microbubble drug delivery system according to claim 1, characterized in that, The manganese porphyrin lipid was prepared by reacting pyrophyllite lipid with manganese chloride and sodium acetate.

5. The ultrasound-responsive targeted microbubble drug delivery system according to claim 4, characterized in that, The microbubbles were set at a frequency of 1 MHz, a duty cycle of 20%, and a sound intensity of 1 W / cm². 2 After 30 seconds of ultrasonic treatment, at least 96% of the microbubbles were burst.

6. A method for preparing the ultrasound-responsive targeted microbubble drug delivery system according to any one of claims 1-5, characterized in that, The steps include: 1) Mixing DSPC, DSPE-PEG2000-Var7, manganese porphyrin lipids and Nigerian styraxin to obtain an organic phase; 2) Injecting the organic phase into the aqueous phase under ultrasonic water bath conditions, dialysis to remove the organic reagents, and obtaining vMnp-Nig NPs. 3) vMnp-Nig NPs are mixed with 1,2-propanediol and glycerol, placed in a container, filled with perfluoropropane gas, sealed, and shaken to form vMnp-Nig MBs.

7. The preparation method according to claim 6, characterized in that, The molar ratio of DSPC, DSPE-PEG2000-Var7 and manganese porphyrin lipids is 75~87:2~5:14~18.

8. The preparation method according to claim 6, characterized in that, The total mass ratio of DSPC, DSPE-PEG2000-Var7 and manganese porphyrin lipids to Nigerian styraxin was 180-220:

1.

9. The preparation method according to claim 6, characterized in that, The organic phase also contains solvents for dissolving DSPC, DSPE-PEG2000-Var7, and manganese porphyrin lipids, with a volume ratio of organic phase to aqueous phase of 1:4~8; dialysis is performed using dialysis bags with a molecular weight cutoff of 8000~12000 Da; the volume ratio of vMnp-Nig NPs, 1,2-propanediol, and glycerol is 0.5~1.0:0.2~0.3:0.2~0.

3.

10. Use of the ultrasound-responsive targeted microbubble drug delivery system of any one of claims 1-5 in the preparation of a medicament for combined ultrasound therapy of triple-negative breast cancer.

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