Iron-based nanoparticles for ultrasound enhancement and their application in sonodynamic therapy of solid tumors

Abstract

This invention provides iron-based nanoparticles for ultrasound sensitization and their application in sonodynamic therapy of solid tumors. The nanoparticles have a core-shell structure, with the core consisting of dihydroporphyrin e6 (Ce6) molecules and Fe. 3+ The ions are formed through coordination self-assembly, with a shell consisting of cell membrane nanovesicles derived from engineered probiotics (such as E. coli Nissle 1917) and overexpressing catalase on their surface. These nanoparticles can target and accumulate in the solid tumor microenvironment, improving hypoxia through catalytic oxygen production, inducing ferroptosis in tumor cells, and significantly sensitizing ultrasound-mediated sonodynamic therapy. They can also be combined with PD-1 monoclonal antibodies to produce synergistic immunotherapeutic effects, effectively inhibiting the growth of various solid tumors such as renal cell carcinoma and melanoma.

Description

Technical Field

[0001] This invention relates to the field of iron-based nanoparticles for ultrasound sensitization, and particularly to an iron-based nanoparticle for ultrasound sensitization and its application in sonodynamic therapy of solid tumors. Background Technology

[0002] Renal cell carcinoma (RCC) is a common malignant tumor of the urinary system, posing a significant threat to human health and life. Early-stage RCC often presents with no specific symptoms, resulting in distant metastases in approximately 30% of patients at initial diagnosis. In recent years, with the continuous availability of novel targeted and immunotherapeutic drugs and the standardization of multidisciplinary treatment models, the 5-year survival rate of RCC has improved. However, chemotherapy and targeted immunotherapy drugs still face limitations in application, such as off-target toxicity from systemic administration, drug resistance, and adverse drug reactions, necessitating the development of novel local control and anti-tumor strategies.

[0003] Sonodynamic therapy (SDT) is an emerging ultrasound treatment modality with advantages such as deep tissue penetration, high spatial precision, and non-invasiveness and radiation-free operation, enabling local treatment of deep tumors through non-invasive procedures. Sonosensitizers are the core component of SDT, capable of generating reactive oxygen species (ROS) such as singlet oxygen through ultrasound excitation, thereby killing tumor cells. Dihydroporphyrin e6 (Ce6) is a commonly used sonosensitizer, which can generate a large amount of ROS under ultrasound, promoting tumor cell apoptosis. However, free Ce6 has a small molecular weight, rapid metabolism, and low tumor accumulation, making it difficult to achieve efficient solid tumor targeting and ultrasound-sensitizing effects when administered intravenously. Drug nanoparticle coating and delivery technologies hold promise for improving the targeting of Ce6, enhancing the efficacy of sonodynamic therapy by enriching sonosensitizing molecules in the tumor microenvironment. Traditional nanoparticles mainly rely on enhanced penetration and retention effects (EPR effect) to achieve tumor targeting, but this effect is highly heterogeneous and inefficient in solid tumors, making it difficult to meet the needs of clinical translation. Furthermore, studies have shown that renal cell carcinoma cells are highly sensitive to ferroptosis (an iron-dependent lipid peroxidation-induced cell death). Ferroplasmosis inducers (such as Erastin) can significantly promote ROS accumulation and abnormal expression of key regulatory factors (such as GPX4) in renal cell carcinoma cells, indicating that the ferroptosis pathway has potential in the treatment of renal cell carcinoma. However, existing ferroptosis induction strategies still lack targeting and synergy, and are prone to off-target effects. In summary, the current treatment of solid tumors faces the following technical problems: (1) Existing chemotherapy and targeted immunotherapy drugs have limitations in terms of off-target toxicity and drug resistance; (2) The targeted delivery efficiency of sonosensitive agents (such as Ce6) in sonodynamic therapy is low, affecting the treatment effect; (3) Traditional nanoparticles rely on the EPR effect and have insufficient targeting; (4) Ferroplasmosis induction strategies lack precise regulation adapted to the tumor microenvironment.

[0004] Therefore, in order to solve the above-mentioned technical problems, it is necessary to develop a multifunctional nanoparticle system that can achieve efficient tumor targeting and integrate sonodynamic sensitization and ferroptosis induction functions to improve the treatment effect of solid tumors. Summary of the Invention

[0005] The purpose of this invention is to provide an iron-based nanoparticle for ultrasound sensitization and its application in sonodynamic therapy of solid tumors. The nanoparticle can be targeted and enriched in the solid tumor microenvironment, improve hypoxia by catalyzing oxygen production, induce iron death in tumor cells, and significantly sensitize ultrasound-mediated sonodynamic therapy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the present invention, an iron-based nanoparticle for ultrasound sensitization is provided, wherein the iron-based nanoparticle for ultrasound sensitization has a core-shell structure and comprises: Core layer: composed of Ce6 molecules and Fe 3+ Nanoparticle cores formed by ion self-assembly through coordination; Shell: A cell membrane nanovesicle that encloses the core of the nanoparticle, wherein... Cell membrane nanovesicles are derived from engineered probiotic exosomes, and their surface expresses catalase.

[0007] Furthermore, the Ce6 molecules in the nanoparticle core are related to Fe... 3+ The molar ratio of ions is 1:1 to 1:3 (preferably 1:2). This specific ratio is accurately determined by Fourier transform infrared spectroscopy. Figure 2 (D) ensures the stability and functionality of the self-assembled structure. This ratio guarantees both the density of Ce6 as a sonication agent and provides sufficient iron ions to initiate ferrodeogenesis.

[0008] Furthermore, the engineered probiotic is Escherichia coli Nissle 1917, and the cell membrane nanovesicles are obtained by inducing catalase overexpression by adding 50-100 μM H2O2 during the culture process.

[0009] Furthermore, the iron-based nanoparticles used for ultrasound sensitization have a particle size of 120-150 nm, wherein the core layer has a particle size of 90-110 nm, and the shell layer has a thickness of 15-25 nm. The core layer has a particle size range of 90-110 nm (…). Figure 2 (AB) This allows it to effectively utilize the EPR effect of solid tumors for targeted enrichment while ensuring cellular uptake efficiency. The uniformity of particle size (polydispersity index <0.2) demonstrates the controllability and sophistication of the synthesis process. The total particle size (120-150 nm) and shell thickness (15-25 nm) of the composite nanoparticles are... This demonstrates the successful construction of the core-shell structure and ensures the long-term stability of the particles in vivo. The shell thickness directly affects its ability to protect the core and preserve the function of membrane proteins.

[0010] In a second aspect of the invention, a method for preparing the iron-based nanoparticles for ultrasound sensitization is provided, the method comprising: Step S1: Mix the iron salt solution with the Ce6 solution to form nanoparticle cores through intermolecular coordination self-assembly and π-π stacking; then obtain purified Ce6@Fe nanoparticles by centrifugation and washing. Step S2: During the culture of probiotics, H2O2 is added to induce catalase overexpression, and then cell membrane nanovesicles are isolated from the bacterial culture supernatant. Step S3: Mix the Ce6@Fe nanoparticles and the cell membrane nanovesicles at a particle number ratio of 2-3:1, and fuse them by extrusion to obtain iron-based nanoparticles for ultrasound sensitization.

[0011] Further, in step S1, the iron salt solution is an FeCl3 solution with a concentration of 15-25 mg / mL, and the Ce6 solution is a solution of dihydroporphyrin e6 molecules dissolved in dimethyl sulfoxide. The concentration range of the Ce6 solution is 15-25 mg / mL, and in this patent, the Ce6 concentration is 20 mg / mL. Furthermore, in step S2, the concentration of H2O2 added is 45-55 μM, and the culture conditions are 180-300 rpm for 12-48 h.

[0012] In a third aspect of the invention, the use of the iron-based nanoparticles in the preparation of an ultrasound-sensitized sonodynamic therapeutic agent for solid tumors is provided.

[0013] Furthermore, the solid tumor includes at least one of renal cell carcinoma, melanoma, breast cancer, and colorectal cancer. Implementation data confirms that this technology is applicable to various solid tumors (such as renal cell carcinoma, melanoma, and breast cancer), and that the preparation process is simple, stable, and has good prospects for clinical translation.

[0014] In a fourth aspect of the invention, the use of the iron-based nanoparticles in combination with PD-1 monoclonal antibodies in the preparation of drugs for treating solid tumors is provided.

[0015] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: 1. The iron-based nanoparticles for ultrasound sensitization prepared in this invention have a stable structure: (1) The iron-based nanoparticles for ultrasonic sensitization prepared by this invention have uniform particle size and high stability: the Ce6@Fe nanoparticles formed by self-assembly have a particle size controlled in the range of 95-100 nm, and the particle size increases to about 130 nm after being coated with OMV, and there is no aggregation or degradation after standing for 7 days. Figure 2 (HJ). This stability ensures the integrity of the nanoparticles during in vivo circulation, providing a fundamental guarantee for drug delivery.

[0016] (2) Highly efficient loading of active ingredients: Ce6 and Fe in the core of nanoparticles 3+ The molar ratio is stable at 1:2 ( Figure 2 D), Fe element accounts for more than 26% ( Figure 2 C), and the OMV coating layer completely preserves functional proteins such as catalase ( Figure 2 L). This structural design enables efficient loading of both sonosensitive agents and ferroptosis inducers, laying the foundation for multifunctional synergistic therapy.

[0017] 2. Significant anti-tumor effects in vitro: (1) The ultrasound-sensitizing effect is outstanding: In the 786-0 renal cell carcinoma model, OMV-Ce6@Fe nanoparticles combined with ultrasound treatment increased the cell proliferation inhibition rate to 70-80%. Figure 2 B), significantly better than the group using nanoparticles alone or the ultrasound treatment group (inhibition rate only 30-40%). This indicates that nanoparticles effectively enhance the sensitivity of sonodynamic therapy. (2) The mechanism of ferroptosis induction is clear: nanoparticle treatment leads to a 40-60% decrease in intracellular GSH levels, a 2-3 fold increase in MDA content, and a 3-5 fold increase in ROS levels. Figure 4 AC). The ferroptosis-specific inhibitor Fer-1 can reverse this effect ( Figure 5 AC) confirmed that it exerts its lethal effect through ferrodeogenesis.

[0018] (3) Mechanism synergy: Nanoparticles exert synergistic effects through three mechanisms: (1) OMV catalase consumes H2O2 to produce oxygen, improving the hypoxic microenvironment; (2) Fe 3+ / Fe 2+ Transformation induces ferroptosis; (3) Ce6 molecules enhance ultrasound to generate ROS. This multi-mechanism synergy breaks through the limitations of single therapy.

[0019] 3. Enhanced tumor targeting: DIR labeling experiments showed that the enrichment of OMV-encapsulated nanoparticles at tumor sites was 3-5 times that of normal tissues. Figure 6 (AB) confirmed that engineered OMV modification significantly improved the tumor targeting efficiency of nanoparticles. 4. Significant synergistic therapeutic effect: In the Renca renal cell carcinoma model, OMV-Ce6@Fe combined with PD-1 monoclonal antibody resulted in a tumor growth inhibition rate of over 80% and a 50% extension of mouse survival. Figure 7 AC); In the 786-0 model, combined ultrasound therapy almost completely suppressed tumor growth ( Figure 8 (BD). This synergistic effect, where 1+1 is far greater than 2, highlights the therapeutic advantages of this invention. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The following diagram illustrates the preparation process and mechanism of the iron-based nanoparticles for ultrasound sensitization according to the present invention, where A is a flow chart and B is a mechanism diagram.

[0022] Figure 2 This study describes the synthesis of iron-based nanoparticles and their characterization, including morphology, particle size, and zeta potential. Figure A: Transmission electron microscopy (TEM) image; Figure B: Dynamic light scattering (DLS) analysis of the hydrodynamic size distribution of Ce6@Fe nanoparticles; Figure C: Energy dispersive X-ray spectroscopy (EDX) scanning results; Figure D: Fourier transform infrared spectroscopy (FTIR) detection of Ce6 and Ce6@Fe nanoparticles; Figure E: Particle size distribution of engineered probiotic-derived exosomes (OMV); Figure F: TEM image of OMV-Ce6@Fe composite nanoparticles; Figure G: Mapping scan showing the distribution of Fe and O elements in OMV-Ce6@Fe; Figure H: DLS comparison of particle size of Ce6@Fe and OMV-Ce6@Fe; Figure I: Zeta potential analysis; Figure J: Particle size stability test after standing for 1-7 days; Figure K: Electron spin resonance (ESR) detection of the ability of different nanoparticles to generate ·OH under hydrogen peroxide conditions; Figure L: SDS-PAGE electrophoresis verification that OMV and OMV-Ce6@Fe retain functional membrane proteins.

[0023] Figure 3To enhance apoptosis induced by OMV-Ce6@Fe NPs in renal cell carcinoma cells and inhibit their growth and metastasis, the following figures were used: Figure A: CCK-8 assay showing the inhibitory effect of different concentrations of nanoparticles on cell proliferation; Figure B: Cell proliferation inhibition rate after combined ultrasound treatment; Figure C: Live / dead cell death statistical analysis showing ultrasound-enhanced nanoparticle-induced renal cell carcinoma cell death (green fluorescent markers indicate live cells, red fluorescent markers indicate dead cells); Figures D and E: Transwell migration assay; Figure F: Apoptosis detection kit verifying the effect of ultrasound-enhanced apoptosis promotion by nanoparticles.

[0024] Figure 4 OMV-Ce6@Fe NPs-sensitized ultrasound-mediated ferroptosis and mitochondrial damage in renal cell carcinoma cells. Figure A shows that GSH / GSSG assays revealed that the nanoparticles reduced reduced glutathione levels by 40-60%. Figure B: MDA content measurement confirmed a 2-3 fold increase in lipid peroxidation products; Figure C: ROS probe showed a 3-5 fold increase in reactive oxygen species levels; Figure D: qPCR detection of ferroptosis-related genes (PTGS2 upregulated, GPX4 downregulated); Figures E and F: TEM observation of mitochondrial damage showed cristae breakage and membrane integrity disruption.

[0025] Figure 5 This study illustrates ultrasound-sensitized OMV-Ce6@Fe NPs-mediated ferroptosis and mitochondrial damage in renal cell carcinomas. Figure A shows that the ferroptosis inhibitor Fer-1 reverses the cytotoxic effect of nanoparticles; Figure B shows that Fer-1 reduces MDA levels, confirming the involvement of lipid peroxidation; and Figure C shows that Fer-1 inhibits ROS production, enhancing the specificity of the ferroptosis pathway.

[0026] Figure 6 Iron-based nanoparticles coated with engineered probiotic exosomes exhibit good targeting activity against solid tumors in vivo. Specifically, A:DIR-labeled nanoparticles show fluorescence enrichment at tumor sites (red area). Figure B: Quantitative fluorescence analysis of major organs; tumor enrichment is 3-5 times that of normal tissue.

[0027] Figure 7 Iron-based nanoparticles coated with engineered probiotic exosomes enhance the internal immunotherapy effect of PD1 monoclonal antibody. Figure A: Tumor volume growth curve, with the combined group showing an inhibition rate of over 80%; Figure B: Tumor weight statistics, showing a significant reduction; Figure C: Survival curve, with the combined group prolonging the survival of mice by over 50%.

[0028] Figure 8Iron-based nanoparticles coated with engineered probiotic exosomes enhance the in vivo tumor-suppressing effect of ultrasound. Figure A shows the experimental design (ultrasound irradiation after intravenous injection); Figure B shows tumor volume changes, with the combined group almost completely inhibiting growth; Figure C shows tumor weight comparison, with the combined group showing a 70% reduction; and Figure D shows prolonged survival, demonstrating the durability of the treatment. Detailed Implementation

[0029] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0030] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0031] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0032] The overall concept of this invention is as follows: The iron-based nanoparticles in this patent application are defined by their structural composition characteristics and have the following innovative features: 1. Core structural features The nanoparticle core consists of Ce6 molecules and Fe. 3+ Ions are formed through intermolecular coordination and self-assembly. This specific assembly method gives the nanoparticles a regular spherical or near-spherical structure with a uniform particle size distribution. In the core structure, Ce6 molecules are arranged in an ordered manner through π-π stacking interactions, while Fe³⁺… + The ions form stable coordination bonds with the carboxyl and amino groups of the Ce6 molecule. The core nanoparticle structure is unique: the core layer consists of Ce6 and Fe. 3+ The carrier-free nanoparticles formed through coordination self-assembly possess a specific molar ratio (~1:2) and size (~100 nm). This structure not only achieves the co-delivery of Ce6 and iron ions but also ensures their stability and activity under ultrasound. This is not a conventional drug loading method.

[0033] 2. Surface modification characteristics When encapsulated with a cell membrane nanovesicle layer, the vesicles retain the complete membrane protein composition and biological functions of the source bacteria, particularly the activity of catalase. This structural design allows the nanoparticles to possess both the acoustic sensitivity and ferroptosis-inducing ability of an iron-based core, as well as the tumor-targeting and microenvironment-regulating functions of bacterial membrane vesicles. 3. Functional characteristics The nanoparticles, under ultrasonic excitation, can generate a large amount of reactive oxygen species through Ce6 molecules, and simultaneously through Fe³⁺. + / Fe² + The transformation promotes lipid peroxidation and synergistically induces ferroptosis. This multi-mechanism effect enables the nanoparticles to exhibit a significant sensitizing effect in the sonodynamic therapy of solid tumors.

[0034] 4. Functional synergy, not simple addition: The role of OMVs (cell membrane nanovesicles): They are not only targeting carriers, but their surface catalase can also improve hypoxia and facilitate ferroptosis (Fe2+). 2+ The Fenton reaction and sonodynamic effects (Ce6 producing singlet oxygen) provide a favorable oxidizing microenvironment. The role of the Ce6@Fe core: not only provides the acoustic sensor Ce6, but also releases Fe³⁺. + / Fe² + Simultaneously, the ferroptosis pathway was activated. Ultrasound-induced ROS production from Ce6 further exacerbated ferroptosis-related lipid peroxidation. Synergistic evidence: Example data show that the tumor-suppressing effect (~80% inhibition rate) produced by OMV-Ce6@Fe combined with ultrasound is far superior to the simple sum of the components, achieving a synergistic effect of "1+1 is far greater than 2" (see Figure 2 B, Figure 8 B). This powerful synergy is an outcome that those skilled in the art could not have foreseen when combining existing technologies.

[0035] The present application will now be described in detail with reference to embodiments and experimental data.

[0036] Example 1: Synthesis of iron-based nanoparticles encapsulated in engineered bacterial exosomes and verification of their inhibitory and killing effects on solid tumor growth mediated by sensitized ultrasound.

[0037] 1. Synthesis of iron-based nanoparticles (OMV-Ce6@Fe) encapsulated by bacterial exosomes (1) Preparation of iron-based nanoparticles (Ce6@Fe) Ce6@Fe NPs nanoparticles were synthesized using a self-assembly strategy and intermolecular π-π stacking attraction. 100 mg of FeCl3 was dissolved in 5 mL of ultrapure water to obtain 5 mL of 20 mg / mL FeCl3 solution, which was then stirred uniformly at room temperature using a magnetic stirrer. 20 mg of Ce6 was dissolved in 1 mL of dimethyl sulfoxide (DMSO) and slowly added dropwise to the FeCl3 solution under rotating stirring. The mixture was stirred for another 24 h at room temperature. After centrifugation at 12000 rpm for 40 min at 4°C, the supernatant was slowly discarded. The precipitate was repeatedly resuspended in 10 mL of ddH2O, rinsed, and centrifuged three times to obtain self-assembled Ce6@Fe nanoparticles (Ce6@FeNPs). Since FeCl3 was added in excess during synthesis, theoretically all Ce6 molecules were reacted and assembled into nanoparticles. The drug concentration of the nanoparticles was calculated based on a mass of 20 mg of Ce6 molecules.

[0038] The morphology and size of the nanoparticles were detected using transmission electron microscopy (TEM; JM-3010, 300 kV, dot resolution: 0.17 nm). Dynamic light scattering (Zetasizer Pro) was used to measure the particle size and potential of Ce6@Fe NPs. After centrifugation of a certain concentration of Ce6@Fe NPs and discarding the supernatant, the nanoparticles were dried in a 37°C oven and analyzed under infrared spectroscopy along with pure Ce6 drug to detect the Ce6 drug and Fe content within the nanoparticles. 3+ The self-assembly ratio.

[0039] 2. Preparation of cell membrane nanovesicles; Escherichia coli Nissle 1917 strain was purchased from Sangon Biotech Co., Ltd. (catalog number A330171-0001) and cultured in sterilized LB broth for 12 hours (37°C, 220 rpm). Hydrogen peroxide (50 μM) was then added to the medium, and the culture was continued for another 24 hours to induce catalase expression in the cell membrane of the 1917 probiotic under hydrogen peroxide pressure. The bacterial solution was collected during the culture process. The bacterial solution was centrifuged at 5000 rpm for 30 minutes at 4°C, and the supernatant was filtered through a 0.45 μm filter. The supernatant was then centrifuged at 150,000 rpm for 2 hours at 4°C to obtain outer membrane vesicles (OMVs). The membrane protein concentration of the OMVs was determined using a Thermo Coomassie (Bradford) protein assay kit (Lot: 23200). Catalase activity was analyzed using a catalase assay kit (Bejomt, S0051).

[0040] 3. Bacterial exosomes encapsulating iron-based nanoparticles (OMV-Ce6@Fe) The number of bacterial exosomes OMV of catalase and iron-based nanoparticles (Ce@Fe) was measured using a nanoparticle tracking analyzer (NTA). The OMV nanofilm and Ce@Fe nanoparticle NTA were mixed at a ratio of 2:1. The mixed nanoparticle solution was then repeatedly extruded more than 20 times under a 400 nm polycarbonate porous membrane on a liposome extruder to obtain OMVs-Ce@Fe nanomaterials.

[0041] 4. Characterization of iron-based nanoparticles (OMV-Ce6@Fe) encapsulated by bacterial exosomes The morphology of Ce6@Fe nanoparticles was characterized by transmission electron microscopy. The results showed that the self-assembled Ce6@Fe nanoparticles had a regular morphology and were approximately 100 nm in size, resembling spherical particles. Figure 2 A); Dynamic light scattering (DLS) analysis showed that the hydrodynamic size of Ce6@Fe nanoparticles was approximately 95 nm. Figure 2 B). Energy-dispersive X-ray spectroscopy (EDX) scans by scanning electron microscopy also confirmed that Ce6@Fe NPs nanoparticles contain significant amounts of Fe, with the Fe content exceeding 26%. Figure 2 C); Fourier transform infrared spectroscopy analysis of Ce6 and Ce6@Fe nanoparticles showed that the spectra of NH and OH peaks changed significantly after Ce6 and Fe particles were assembled into nanoparticles, and the molar ratio of Ce6 to Fe in the nanoparticles was calculated to be approximately 1:2. Figure 2 D). Furthermore, the exosomes OMVs of engineered probiotic 1917 are lipid cell membrane nanoparticles with a particle size of approximately 120 nm. Figure 2 E); The exosome-encapsulated nanoparticles OMVs-Ce@Fe are composite nanoparticles encapsulated in membrane vesicles, with a particle size of approximately 130 nm. Figure 2 F); In addition, scanning electron microscopy mapping scans revealed the distribution of Fe and O elements in the OMVs-Ce@Fe nanoparticles (F). Figure 2 G); DLS analysis of Ce6@Fe and OMV-Ce6@Fe nanoparticles revealed that the particle size of Ce6@Fe nanoparticles tended to increase after OMVs encapsulated Ce6@Fe nanoparticles. Figure 2 H), but the Zate potential did not change significantly with the combination of composite nanoparticle sizes ( Figure 2 I), DLS analysis of the overall particle size of OMV-Ce6@Fe nanoparticles after standing for 1 to 7 days showed that the particle size of OMV-Ce6@Fe was stable under static conditions, with no degradation or significant agglomeration. Figure 2 J).

[0042] Electron spin resonance (ESR) was used to detect the ability of different nanoparticles to generate ·OH under hydrogen peroxide conditions. The results showed that Ce6@Fe nanoparticles could generate ·OH under hydrogen peroxide conditions. After OMV encapsulation, the new composite nanoparticles OMV-Ce6@Fe had a stronger ability to generate ·OH under hydrogen peroxide conditions. Figure 2 K).

[0043] Furthermore, to demonstrate that OMV derived from probiotic exosomes retains bacterial cell membrane proteins, we performed protein extraction on OMV and OMV-encapsulated nanoparticles, followed by SDS-PAGE electrophoresis verification. The results showed that both OMV and OMV-Ce6@Fe nanoparticles retained a large number of functional membrane proteins. Figure 2 This study lays the foundation for the biological effects of Ce6@Fe NPs on OMV. All the above results demonstrate that Ce6@Fe NPs nanoparticles were successfully synthesized and can be used in subsequent in vitro and in vivo experiments.

[0044] Example 2: Iron-based nanoparticles encapsulated in bacterial exosomes can enhance ultrasound-mediated killing of 786-0 renal cell carcinoma cells, promote apoptosis of renal cell carcinoma cells, and inhibit the migration and invasion of renal cell carcinoma cells. 1. Select 786-0 cells in the logarithmic growth phase, and use 3 × 10⁻⁶ cells. 3 Cells were seeded at a density of cells / well in 96-well cell culture plates. After 8 h of culture, Ce6, Ce6@Fe, and OMV-Ce6@Fe nanoparticles at gradient concentrations (0, 10, 20, 40, 50, 80, 100, 200 μg / mL) were added to each well, and the cells were cultured for another 24 h. Then, 10 µL of CCK-8 reagent was added to each group, and the cells were incubated at 37°C for 2 h. The absorbance at 450 nm was measured using a microplate reader, and the proliferation capacity of each group was calculated based on the absorbance.

[0045] 2. The Ce6@Fe nanoparticle concentration, which inhibited the proliferation of renal cell carcinoma 786-0 cells by 30%, was used in subsequent experiments combining ultrasound sensitization. 786-0 cells were again seeded in 96-well plates and divided into 8 groups: Control, Ce6, US, Ce6@Fe, OMV-Ce6@Fe, US+Ce6@Fe, and OMV-Ce6@Fe+US. Eight hours after seeding, the two groups were treated with the determined Ce6@Fe nanoparticle concentrations, while the control group received an equal volume of PBS. After 12 hours of incubation, some experimental groups were treated with 1 MHz, 1 W / cm²... 2 The renal cell carcinoma cells were treated with ultrasound for 3 min, and then cultured for 12 h. After adding CCK8 reagent and culturing for 2 h, the cell viability was detected by enzyme-linked immunosorbent assay (ELISA).

[0046] 3. CCK8 cell proliferation experiments revealed that Ce6, Ce6@Fe, and OMV-Ce6@Fe all exhibited inhibitory effects on tumor growth in 786-0 cells in a concentration-dependent manner. At the same Ce6 concentration, OMV-Ce6@Fe showed a stronger inhibitory effect on the growth of 786-0 cells. Figure 3 A); Ultrasound significantly enhanced the inhibitory effect of low concentration OMV-Ce6@Fe NPs on the growth of 786-0 cells ( Figure 3 B). Live / dead staining results showed that ultrasound significantly enhanced the killing effect of low-concentration iron-containing nanoparticles on 786-0 cells, and the sensitizing effect of ultrasound was even stronger on OMV-Ce6@Fe nanoparticles encapsulated in OMV. Figure 3 C). Transwell cell migration assays showed that Ce6@Fe and OMV-Ce6@Fe nanoparticles significantly inhibited the migration and invasion of 786-0 cells. Figure 3 D and Figure 3 F), the OMV-Ce6@Fe nanoparticle effect is stronger, and ultrasound significantly enhances the OMV-Ce6@Fe NPs-mediated inhibition of 786-0 cell migration and invasion (F). Figure 3 (D and 3E), the two exert a synergistic tumor-suppressing effect.

[0047] Furthermore, apoptosis assays (apoptosis detection kit, batch number: C1086) showed that Ce6@Fe NPs and OMV-Ce@Fe nanoparticles significantly induced apoptosis in 786-0 cells. Figure 3 (F) Ultrasound significantly promoted apoptosis in Ce6@Fe NPs and OMV-Ce@Fe-mediated 786-0 cells, with OMV-encapsulated OMV-Ce@Fe exhibiting a stronger ultrasound-sensitizing effect.

[0048] Example 3: OMV-Ce6@Fe induces kidney cancer cell death mediated by ferroptosis and sensitized ultrasound.

[0049] 1. To further evaluate the therapeutic effects of Ce6@Fe and OMV-Ce6@Fe nanoparticles on sensitized ultrasound and their mechanism of inhibiting the proliferation and metastasis of renal cell carcinoma, we detected lipid oxidation and ferroptosis-related indicators in 786-0 cells treated with OMV-Ce6@Fe and Ce6@Fe NPs. MDA (batch number: S0131S), GSH / GSSG (batch number: S0056), and the reactive oxygen species detection kit (S0033S) were all purchased from Beyotime Biotechnology Co., Ltd., and the experimental methods were performed according to the kit instructions.

[0050] 2. Experimental results show that treatment with OMV-Ce6@Fe and Ce6@Fe nanoparticles reduces the level of reduced GSH in renal cell carcinoma cells. Figure 4 A), increased the MDA content in renal cell carcinoma cells ( Figure 4 B), promotes the production of ROS in renal cell carcinoma cells ( Figure 4 (C) Furthermore, ultrasound enhanced the reduction of GSH in renal cell carcinoma cells mediated by OMV-Ce6@Fe nanoparticles, as well as the increase of MDA and ROS, indicating a stronger effect of OMV-Ce6@Fe nanoparticles. In addition, OMV-Ce6@Fe and Ce6@Fe NPs significantly increased ferroptosis-related genes in renal cell carcinoma cells. PTGS2 and FTH1 mRNA expression, inhibited GPX4 mRNA expression was reduced, and ultrasound enhanced this effect. Figure 4 D), OMV-Ce6@Fe nanoparticles have a stronger ultrasound-enhancing effect.

[0051] 3. TEM electron microscopy was used to observe the mitochondria of 786-0 renal cell carcinoma cells treated with nanoparticles and ultrasound, either alone or in combination. The results showed that OMV-Ce6@Fe and Ce6@Fe nanoparticles significantly disrupted the integrity of mitochondria in renal cell carcinoma cells, and ultrasound enhanced this effect. The ultrasound-sensitizing effect of OMV-Ce6@Fe nanoparticles was stronger. Figure 4 E and Figure 4 F).

[0052] Example 4: OMV-Ce6@Fe-sensitized ultrasound-mediated renal cell carcinoma death is achieved through ferroptosis. To further evaluate whether OMV-Ce6@Fe-sensitized ultrasound-mediated renal cell carcinoma death is achieved through ferroptosis, we simultaneously treated 786-0 cells treated with OMV-Ce6@Fe and Ce6@Fe NPs with the ferroptosis inhibitor Fer-1 (MCE, 347174-05-4). The viability of 786-0 cells was detected by CCK8 assay, and the levels of lipid oxidation and ferroptosis-related indicators in 786-0 cells were detected by MDA probe and DCFH-DA probe (Beyotime: S0034S).

[0053] The results showed that treatment with the ferroptosis-specific inhibitor Fer-1 significantly reduced the killing activity of OMV-Ce6@Fe and Ce6@Fe nanoparticles against renal cell carcinoma cells. Figure 5 A), reduced the level of intracellular lipid oxidation MDA induced by OMV-Ce6@Fe and Ce6@Fe nanoparticles in 786-0 cells ( Figure 5 B), reducing ROS levels induced by iron-based nanoparticles ( Figure 5 C). This indicates that OMV-Ce6@Fe-sensitized ultrasound-mediated renal cell carcinoma death is achieved through the induction of ferroptosis.

[0054] Example 5: Engineered probiotic exosomes coated with iron-based nanoparticles exhibit good targeting activity against solid tumors in vivo. To confirm whether engineered probiotic exosome coating modification endows iron-based nanoparticles with targeting effects on solid tumors in vivo, we constructed subcutaneous solid tumors in BALB nude mice using 786-0 human renal cell carcinoma cells and intravenously administered iron-based nanoparticles coated with DIR-modified OMV and DIR controls. The enrichment of fluorescence signals in the tumor was monitored in real time using a small animal in vivo imaging system.

[0055] Experimental results showed that, compared with DIR dye alone, cell membrane coating modification significantly enhanced the targeting effect of iron-based nanoparticles on in vivo solid tumor sites. Figure 6 A); Statistical analysis of fluorescence distribution in major organs clearly shows that cell membrane-coated nanoparticles account for approximately 12.4% of the major organs ( Figure 6 B), while the enrichment rate of pure DIR fluorescent dye in tumors was only 1 / 4 that of OMV-coated iron-based nanoparticles, indicating that engineered OMV coating modification enhances the targeting effect of iron-based nanoparticles on solid tumors in vivo.

[0056] Example 6: Engineered probiotic exosome-coated iron-based nanoparticles exhibit good ultrasound-enhancing and solid tumor growth-inhibiting effects in vivo. We constructed a solid tumor renal cell carcinoma model in C57 mice using Renca mouse renal cell carcinoma cells. The mice were intravenously administered OMV-Ce6@Fe nanoparticles (150 μg / mouse), mouse-derived PD-1 mAb (200 μg), and a combination of mouse-derived PD-1 mAb and OMV-Ce6@Fe. The control group received an equal volume of PBS intravenously. The treatment was administered every other day, and tumor growth and mouse survival were monitored. When the tumor size in the control group approached 1200 mm... 3 The experiment was terminated and the mice were recorded as dead.

[0057] Experimental results showed that OMV-Ce6@Fe nanoparticles significantly enhanced the in vivo immunotherapeutic effect of PD1 monoclonal antibodies. Figure 7 A), inhibiting tumor growth ( Figure 7 B) significantly prolonged the survival of mice bearing renal cell carcinoma. Figure 7 C).

[0058] Example 7: Engineered probiotic exosomes coated with iron-based nanoparticles enhance ultrasound-mediated tumor suppression in vivo. We constructed a subcutaneous solid tumor model in Balb / c immunodeficient mice using 786-0 human renal cell carcinoma cells and administered Ce6@Fe nanoparticles and OMV-Ce6@Fe nanoparticles intravenously. Twelve hours after administration, some mice were subjected to ultrasound irradiation of the tumor (1 MHz, 1 W / cm²). 2(5 min), the control group was intravenously injected with an equal volume of PBS, and the drugs were administered or irradiated with ultrasound every other day for a total of 3 administrations and irradiations. The growth of the tumor and the survival of the mice were monitored. When the tumor in the control group approached 1200 mm 3 The experiment was terminated and mouse deaths were recorded. Figure 8 A).

[0059] Experimental results showed that engineered probiotic exosomes coated with iron-based nanoparticles significantly enhanced ultrasound-mediated inhibition of solid tumor growth. Figure 8 B), the combination therapy group almost completely suppressed the growth of solid tumors ( Figure 8 B), reduced the tumor weight of solid tumors ( Figure 8 C) significantly prolonged the survival of tumor-bearing mice ( Figure 8 D).

[0060] Finally, it should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention. If such modifications and variations of the invention fall within the scope of the claims and their equivalents, the invention also intends to include these modifications and variations.

Claims

1. An iron-based nanoparticle for ultrasound sensitization, characterized in that, The iron-based nanoparticles used for ultrasound sensitization have a core-shell structure and include: Core layer: composed of Ce6 molecules and Fe 3+ Nanoparticle cores formed by ion self-assembly through coordination; Shell: A cell membrane nanovesicle that encloses the core of the nanoparticle, wherein... Cell membrane nanovesicles are derived from engineered probiotics, and their surface expresses catalase.

2. The iron-based nanoparticles for ultrasound sensitization according to claim 1, characterized in that, Ce6 molecules and Fe in the nanoparticle core 3+ The molar ratio of ions is 1:1 to 1:

3.

3. The iron-based nanoparticles for ultrasound sensitization according to claim 1, characterized in that, The engineered probiotic is Escherichia coli Nissle 1917, and the cell membrane nanovesicles are obtained by adding 50-100 μM H2O2 during the culture process to induce catalase overexpression.

4. The iron-based nanoparticles for ultrasound sensitization according to claim 1, characterized in that, The iron-based nanoparticles used for ultrasound sensitization have a particle size of 120-150 nm, wherein the core layer has a particle size of 90-110 nm and the shell layer has a thickness of 15-25 nm.

5. A method for preparing iron-based nanoparticles for ultrasound sensitization as described in any one of claims 1-4, characterized in that, The method includes: Step S1: Mix the iron salt solution with the Ce6 solution to form nanoparticle cores through intermolecular coordination self-assembly and π-π stacking; then obtain purified Ce6@Fe nanoparticles by centrifugation and washing. Step S2: During the culture of probiotics, H2O2 is added to induce catalase overexpression, and then cell membrane nanovesicles are isolated from the bacterial culture supernatant. Step S3: Mix the Ce6@Fe nanoparticles and the cell membrane nanovesicles at a particle number ratio of 2-3:1, and fuse them by extrusion to obtain iron-based nanoparticles for ultrasound sensitization.

6. The preparation method according to claim 5, characterized in that, In step S1, the iron salt solution is a FeCl3 solution with a concentration of 15-25 mg / mL, and the Ce6 solution is a solution of dihydroporphyrin e6 molecules dissolved in dimethyl sulfoxide.

7. The preparation method according to claim 5, characterized in that, In step S2, the concentration of H2O2 added is 45-55 μM, and the culture conditions are 180-300 rpm for 12-48 h.

8. The use of the iron-based nanoparticles according to any one of claims 1-4 in the preparation of an ultrasound-sensitized sonodynamic therapy drug for solid tumors.

9. The application according to claim 8, characterized in that, The solid tumor includes at least one of renal cell carcinoma, melanoma, breast cancer, and colorectal cancer.

10. The use of the iron-based nanoparticles according to any one of claims 1-4 in combination with PD-1 monoclonal antibody in the preparation of a drug for treating solid tumors.

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