Ultrasonic response type drug-loaded nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and relates to ultrasonic response type drug-loaded nanoparticles as well as a preparation method and application thereof. The ultrasonic response type drug-loaded nanoparticle comprises a matrix, wherein an ultrasonic response type nitric oxide donor and an immunosuppressive agent are dispersed in the matrix; wherein the ultrasonic response type nitric oxide donor releases nitric oxide under ultrasonic triggering, and diffusion of the nitric oxide induces the matrix to form a pore structure for releasing the immunosuppressive agent. The ultrasonic response type drug-loading nanoparticle can be targeted to an immunological rejection part. Before ultrasonic triggering, the nanoparticles show a good slow release characteristic, and systemic burst release of drugs can be effectively avoided; after ultrasonic triggering, the drug release rate is remarkably increased, switching from passive slow release to active induced release is achieved, and the problem that the concentration of drugs at the focus position is insufficient is solved.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an ultrasound-responsive drug-loaded nanoparticle, its preparation method, and its application. Background Technology

[0002] Heart transplantation is an important treatment for end-stage heart failure, but acute rejection after the procedure still significantly affects the long-term survival of transplanted hearts. Immunosuppressants, such as rapamycin (RAPA), are commonly used in clinical practice. They can inhibit T cell proliferation and regulate the immune response, but long-term systemic use can easily cause adverse reactions such as liver and kidney toxicity, infection, and tumor risk, and lacks precise regulation of the local inflammatory microenvironment of the transplanted heart.

[0003] Current nanomedicine delivery technologies, when addressing the complex need for immune regulation in organ transplantation, often rely on traditional delivery systems, such as polylactic-co-glycolic acid (PLGA) nanoparticles, which typically exhibit passive and continuous release. This makes it difficult to regulate the delivery based on the dynamic process of transplant rejection, resulting in insufficient concentrations at the lesion site. Summary of the Invention

[0004] This invention provides an ultrasound-responsive drug-loaded nanoparticle, its preparation method, and its application, to solve the problem in related technologies where passive drug release leads to insufficient drug concentration at the lesion site, making it impossible to administer drugs as needed based on the course of the transplant response.

[0005] Based on the above technical problems, the present invention provides the following technical solution: In a first aspect, the present invention provides an ultrasound-responsive drug-loaded nanoparticle, the ultrasound-responsive drug-loaded nanoparticle comprising a matrix in which an ultrasound-responsive nitric oxide donor and an immunosuppressant are dispersed; wherein the ultrasound-responsive nitric oxide donor releases nitric oxide upon ultrasound triggering, and the diffusion of the nitric oxide induces the matrix to form a porous structure for releasing the immunosuppressant.

[0006] In some specific embodiments of the present invention, particles with a diameter in the range of 100-400 nanometers account for 85%-90% of the total number of ultrasonically responsive drug-loaded nanoparticles.

[0007] In some specific embodiments of the present invention, the immunosuppressant is rapamycin.

[0008] In some specific embodiments of the present invention, the ultrasonically responsive nitric oxide donor is BNN6.

[0009] In some specific embodiments of the present invention, the matrix is ​​a polylactic acid-hydroxyacetic acid copolymer.

[0010] Secondly, the present invention provides a method for preparing ultrasound-responsive drug-loaded nanoparticles, comprising the following steps: The matrix, an ultrasound-responsive nitric oxide donor, and an immunosuppressant are dissolved together in an organic solvent to form an organic phase; The surfactant is dissolved in water to form an aqueous phase; The organic phase is added to the aqueous phase and emulsified to form an emulsion; The organic solvent in the emulsion is evaporated and solidified to form coarse nanoparticles. The crude nanoparticles were collected, washed, and freeze-dried to obtain the ultrasonically responsive drug-loaded nanoparticles.

[0011] In some specific embodiments of the present invention, the mass ratio of matrix, ultrasonic responsive nitric oxide donor, and immunosuppressant is (4-6):(0.8-1.2):(1.5-2.5), and the volume ratio of organic phase to aqueous phase is 1:(4-6).

[0012] In some specific embodiments of the present invention, ultrasound is used during emulsification, with a power of 280-310W and a duration of 3-10 minutes.

[0013] Thirdly, the present invention provides the application of ultrasound-responsive drug-loaded nanoparticles in the preparation of drugs for treating acute rejection reactions after organ transplantation.

[0014] In some specific embodiments of the present invention, the organ transplant is a heart transplant.

[0015] The beneficial effects of the present invention include at least the following: The ultrasound-responsive drug-loaded nanoparticles of this invention can target sites of immune rejection. Before ultrasound triggering, the nanoparticles exhibit good sustained-release characteristics, effectively avoiding systemic burst release of the drug; after ultrasound triggering, the drug release rate is significantly increased, realizing a switch from passive sustained release to active induced release, solving the problem of insufficient drug concentration at the lesion site.

[0016] This invention co-loads an ultrasound-responsive nitric oxide donor with rapamycin, allowing them to synergistically exert therapeutic effects under ultrasound. Experimental results show that these ultrasound-responsive drug-loaded nanoparticles effectively inhibit the secretion of pro-inflammatory factors such as TNF-α, IL-6, and IL-1β, while increasing the levels of anti-inflammatory factors such as IL-10 and TGF-β, significantly promoting the transformation of macrophages from pro-M1 inflammatory to M2 anti-inflammatory. In a heart transplantation model, this synergistic effect significantly alleviated acute rejection and reduced inflammatory cell infiltration, thereby effectively prolonging the survival time of the transplanted heart. Attached Figure Description

[0017] 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 only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the preparation process of P-BNN6 / RAPA nanoparticles obtained in Example 1: a diagram of the preparation of PLGA nanoparticles by the single emulsion-solvent evaporation method.

[0019] Figure 2 The morphology and physicochemical properties of P-RAPA, P-BNN6, and P-BNN6 / RAPA nanoparticles prepared in Example 1 are characterized. SEM / TEM images show the spherical morphology and structure of the nanoparticles. (A–C) Scanning electron microscopy (SEM) was used to observe the morphology of P-RAPA, P-BNN6, and the P-BNN6 / RAPA nanoparticles prepared in Example 1. Scale bar: 100 μm; (D–F) Transmission electron microscopy (TEM) was used to observe the morphology and particle size distribution of the above nanoparticles. Scale bar: 500 μm. (G) Average hydrated particle size was measured by dynamic light scattering (DLS).

[0020] (H) Zeta potential measurement results.

[0021] (I) Histogram of P-BNN6 / RAPA nanoparticle size distribution obtained by DLS particle size analysis in Example 1. Data are expressed as mean ± SD (n=3).

[0022] Figure 3 The chemical structure and composition characterization of the P-BNN6 / RAPA nanoparticles prepared in Example 1 are described. (A) High-resolution X-ray photoelectron spectroscopy (XPS) analysis: The N 1s spectra (top row) of BNN6, PLGA, and P-BNN6 / RAPA nanoparticles prepared in Example 1 are shown, as well as the C 1s spectra (bottom row) of RAPA, PLGA, and P-BNN6 / RAPA nanoparticles prepared in Example 1. Peak fitting analysis shows that the P-BNN6 / RAPA nanoparticles prepared in Example 1 simultaneously contain nitrogen-containing bond characteristic peaks associated with BNN6 and carbon bond and carbonyl characteristic peaks associated with RAPA / PLGA, indicating that both drugs were successfully loaded into the PLGA matrix. (B) Comparative analysis of Raman and FT-IR spectra: The spectral signals of PLGA, RAPA, BNN6, and different nanoparticle formulations (P-RAPA, P-BNN6, and P-BNN6 / RAPA nanoparticles prepared in Example 1) are presented respectively. The P-BNN6 / RAPA nanoparticles prepared in Example 1 simultaneously exhibit characteristic vibrational peaks such as C=O and N=O, further verifying the successful construction of dual-drug co-loaded nanoparticles.

[0023] Figure 4 The encapsulation efficiency of P-BNN6 / RAPA nanoparticles prepared in Example 1 is shown. (A) The linear regression equation for the absorbance-concentration standard curve of BNN6: Y = 0.0217X + 0.0732, R 2 =0.9995, used for calculating the BNN6 content in subsequent samples; (B) The linear regression equation for the absorbance-concentration standard curve of RAPA: Y = 0.0258X + 0.1901, R 2 =0.9996, used for calculating the RAPA content in subsequent samples; (C) Linear regression equation for the NaNO2 absorbance-concentration standard curve: Y = 0.0098X + 0.0371, R 2 =0.9959, used for subsequent calculation of NO content in samples; (D) Encapsulation efficiency and drug loading of P-BNN6, P-RAPA and P-BNN6 / RAPA nanoparticle formulations prepared in Example 1.

[0024] Figure 5 It is a BNN6 ultrasound responsiveness assessment and drug release kinetics assay: (A) Power intensity-concentration curve of NO generated by ultrasonic irradiation of BNN6; (B) Duty cycle-concentration curve of NO generated by ultrasonic irradiation of BNN6; (C) Changes in NO release concentration of different formulations (BNN6, BNN6+US, P-BNN6+US, P-BNN6 / RAPA+US) under different ultrasonic irradiation times; (D) Cumulative release curves of RAPA at different time points for different formulations (RAPA, P-RAPA, P-RAPA+US, P-BNN6 / RAPA, P-BNN6 / RAPA+US).

[0025] Figure 6 The effect of P-BNN6 / RAPA nanoparticles prepared in Example 1 on LPS-induced polarization and secretion of inflammatory factors in NR8383 macrophages; (A) Representative scatter plot and gating diagram of CD86 (M1 marker) expression detected by flow cytometry; (B)CD86 + Statistical analysis of the proportion of (M1) macrophages; (C) Representative scatter plot and gating diagram of CD163 (M2 marker) expression detected by flow cytometry; (D)CD163 + Statistical analysis of the proportion of (M2) macrophages.

[0026] (E–I) Cell supernatant ELISA detection of inflammatory factor levels: IL-6, IL-1β, TNF-α (pro-inflammatory factor) and IL-10, TGF-β (anti-inflammatory factor).

[0027] Figure 7 The cell compatibility of the P-BNN6 / RAPA nanoparticles prepared in Example 1 and their effect on the migration ability of NR8383 are discussed. (A) CCK-8 assay was used to detect the cell viability of NR8383 cells after treatment with different concentrations of P-BNN6 / RAPA nanoparticles (25, 50, 100, 200, 400 μg / mL) for 24 h and 48 h. (B) CCK-8 assay was used to detect the cell viability of H9c2 cells after treatment with different concentrations of P-BR for 24 h and 48 h. (C) Representative images of Transwell migration assay: migration of NR8383 cells under Blank, P-BNN6 / RAPA nanoparticle and P-BNN6 / RAPA nanoparticle + US treatment conditions (crystal violet staining); (D) Statistics on the number of Transwell-migrated cells.

[0028] Figure 8 This is an in vivo biosafety evaluation of the P-BNN6 / RAPA nanoparticles prepared in Example 1 (serum biochemical indicators and HE staining of major organs); (A–D) Serum biochemical parameters: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) were measured in each group of rats to assess liver and kidney function. There were no statistically significant differences among the groups (ns). (E) Histological analysis of major organs: Heart, liver, spleen, lung and kidney were stained with HE. Representative sections showed no obvious differences in tissue damage or inflammatory infiltration among the groups.

[0029] Figure 9 The transplantation model was established: a rat peritoneal heterotopic heart transplantation model was created (the orthotopic heart of a BN rat was transplanted into the peritoneal cavity of a Lewis rat).

[0030] Figure 10 It is used for fluorescence tracing to assess the in vivo distribution of nanoparticles and ultrasound-enhanced local drug release in transplanted hearts; (A) Schematic diagram of in vitro IVIS fluorescence imaging of transplanted heart after administration of fluorescently labeled ultrasound-responsive drug-loaded nanoparticles (PLGA-BNN6 / RAPA-DiD) and free DiD, showing that nanoparticle delivery can improve fluorescence enrichment of target tissue; (B) Quantitative fluorescence signal analysis of the transplanted heart region (ROI) in (A) (photons / sec / cm) 2 / sr); (C) Quantitative detection of RAPA concentration in transplanted heart tissue using liquid chromatography-tandem mass spectrometry.

[0031] Figure 11 The P-BNN6 / RAPA nanoparticles prepared in Example 1, which were triggered by ultrasound, alleviated acute rejection and remodeled the immune microenvironment in a heart transplantation model. (A) Experimental procedure diagram: After establishing a rat heart transplantation model, the rat was drugged and subjected to ultrasound treatment (lasting for 3 days). Samples were taken on the 7th day after the operation for histological examination, and the survival time of the transplanted heart was followed up. (B) Representative HE-stained images of transplanted hearts (PBS, RAPA, P-RAPA, P-BNN6 / RAPA nanoparticles prepared in Example 1 + US), showing the effects of different treatments on tissue damage and inflammatory infiltration (bottom row shows magnified images). Scale bar: 50 μm; (C) Statistical results of pathological grading of transplant heart rejection (OR–3R); (D) Immunofluorescence staining of transplanted hearts to detect macrophage polarization-related markers: CD86 (red, M1) and CD163 (green, M2), with DAPI counterstaining of cell nuclei (blue). Scale bar: 50 μm; (E–F) Quantification of mean fluorescence intensity (MFI) of CD86 and CD163 fluorescence signals in (D). (G) Kaplan–Meier curves show the differences in transplanted heart survival time among the groups; ELISA detection of inflammatory factors in (H–L) transplanted heart homogenate: IL-6, IL-1β, TNF-α (pro-inflammatory factor) and IL-10, TGF-β (anti-inflammatory factor).

[0032] Figure 12 This study investigated the effects of ultrasound-responsive drug-loaded nanoparticle therapy on the expression of inflammatory factors and immune cell infiltration in transplanted hearts. The transplanted heart drug administration groups were further divided into PBS, free RAPA, P-RAPA, and P-BNN6 / RAPA nanoparticle + US groups. (A) Immunofluorescence staining of transplanted hearts to detect pro-inflammatory factors IL-2, IL-6, and IFN-γ (red); DAPI counterstaining of cell nuclei (blue). Scale bar: 50 μm; (B–D) Quantitative analysis of the average fluorescence intensity (MFI, AU) of the fluorescence signals of IL-2, IL-6 and IFN-γ in (A); (E) Immunohistochemical (IHC) staining of transplanted heart to detect CD3 + Representative image of T cell infiltration (DAB staining, brown), scale bar: 50 μm; (F) Quantitative statistics of CD3 positive signal (proportion of positive cells); (G) Immunohistochemical staining of transplanted heart to detect CD68 + Representative image of macrophage infiltration, scale bar: 50 μm; Quantitative statistics on the proportion of (H)CD68 positive cells.

[0033] Figure 13 This is a flow cytometry analysis of the proportion of T cell subsets in the spleen and inguinal lymph nodes on postoperative day 7. Single-cell suspensions were prepared from inguinal lymph nodes (LN) and spleen (SP) of recipient rats on postoperative day 7. After surface staining with CD3, CD4, and CD8, flow cytometry analysis was performed, and the proportions of CD3+ T cells were analyzed. + CD4 count within T cell gate + With CD8 + Subgroup proportions; (A) CD4 in inguinal lymph nodes + T cells (CD4) + of CD3 + Representative scatter plot and gating diagram; (B) CD4 in inguinal lymph nodes + Statistical analysis of the proportion of T cells; (C) CD8 in inguinal lymph nodes + T cells (CD8) + of CD3 + Representative scatter plot and gating diagram; (D) CD8 in inguinal lymph nodes + Statistical analysis of the proportion of T cells; (E) CD4 in the spleen + Representative scatter plot of T cells and gating diagram; (F) CD4 in the spleen + Statistical analysis of the proportion of T cells; (G) CD8 in the spleen + Representative scatter plot of T cells and gating diagram; (H) CD8 in the spleen+ Statistical analysis of the proportion of T cells. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] The abbreviations involved in this invention have the following meanings: PLGA is polylactic acid-glycolic acid copolymer, which is the matrix of this invention; PVA is polyvinyl alcohol; BNN6 is N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine, which is an ultrasound-responsive nitric oxide donor; RAPA is rapamycin; P-BNN6 is a single-loaded ultrasound-responsive nitric oxide donor nanoparticle, P-RAPA is a single-loaded rapamycin nanoparticle, P-BNN6 / RAPA nanoparticles are ultrasound-responsive drug-loaded nanoparticles, and US stands for ultrasound. In the BNN6+US group, P-BNN6+US group, and P-BNN6 / RAPA nanoparticle+US group, US refers to ultrasound irradiation applied to the transplanted heart region after drug administration, with ultrasound parameters of ultrasound power intensity of 1 W / cm². 2 Duty cycle 50%, frequency 1 MHz, duration 5 minutes.

[0036] In existing technologies, immunosuppressants or gaseous donors are mostly delivered via systemic administration or passively targeted nanocarriers. Their release behavior mainly depends on material degradation and diffusion, and usually presents as a continuous passive release mode. It is difficult to achieve on-demand administration according to the course of transplant rejection, and it is difficult to form and maintain an effective drug concentration in the transplanted heart. At the same time, it is easy to increase the exposure of non-target organs and cause toxic side effects.

[0037] To overcome the above shortcomings, this invention provides an ultrasound-responsive drug-loaded nanoparticle that enables the controlled release of drugs and gaseous signaling molecules under ultrasound, and increases local drug exposure in the transplanted heart when needed. This allows for dynamic regulation of the transplanted inflammatory microenvironment and immune response, thereby improving therapeutic efficacy and reducing systemic adverse reactions.

[0038] Ultrasonic-responsive drug-loaded nanoparticles The ultrasound-responsive drug-loaded nanoparticles include a matrix in which an ultrasound-responsive nitric oxide donor and an immunosuppressant are dispersed; wherein the ultrasound-responsive nitric oxide donor releases nitric oxide upon ultrasound triggering, and the diffusion of the nitric oxide induces the matrix to form a porous structure for releasing the immunosuppressant.

[0039] This invention significantly increases the local concentration of rapamycin in graft tissue through the passive targeting effect of ultrasound-responsive drug-loaded nanoparticles and ultrasound-triggered release. Compared to systemic administration, this local delivery strategy effectively reduces the toxic side effects of immunosuppressants on non-target organs while maintaining efficacy, demonstrating good biocompatibility.

[0040] In some specific embodiments of the present invention, particles with a diameter in the range of 100-400 nanometers account for 85%-90% of the total number of ultrasonically responsive drug-loaded nanoparticles.

[0041] In some specific embodiments of this invention, the immunosuppressant is rapamycin. Rapamycin is a novel macrolide immunosuppressant. Rapamycin exerts its immunosuppressive effect by blocking signal transduction through different cytokine receptors, thereby blocking the progression of T lymphocytes and other cells from the G1 phase to the S phase. In practical applications, this invention utilizes the universal encapsulation properties of the PLGA matrix to achieve sustained drug release. Experimental data show that free rapamycin is almost completely released within 24 hours, while the nanoparticles prepared in this invention exhibit good sustained-release characteristics even without ultrasound, with a release rate of only about 35%-40% after 24 hours. Crucially, this invention significantly accelerates rapamycin release by triggering the release of NO from BNN6 through ultrasound. Under ultrasound, the P-BNN6 / RAPA group achieved a release rate of approximately 70% after 12 hours and increased to approximately 80% after 24 hours, significantly better than the group without added NO donor (approximately 45% after 24 hours). This drug delivery system can rapidly reach therapeutic concentrations at the lesion site, thereby reducing systemic toxicity and significantly improving biosafety and efficacy.

[0042] In some specific embodiments of the present invention, the ultrasound-responsive nitric oxide donor is BNN6. BNN6 is stable in the physiological environment and its NN=O bond can be broken by ultrasound, with each molecule releasing two molecules of nitric oxide quantitatively.

[0043] In some specific embodiments of the present invention, the matrix is ​​polylactic acid-glycolic acid copolymer (PLGA). PLGA is a biodegradable functional polymeric organic compound formed by the random polymerization of lactic acid and glycolic acid. This material is biocompatible, non-toxic, and its degradation cycle can be controlled by adjusting the monomer ratio. It is readily soluble in DMSO and chloroform, but sparingly soluble in water.

[0044] Preparation method of ultrasound-responsive drug-loaded nanoparticles Includes the following steps: S1. The matrix, the ultrasound-responsive nitric oxide donor, and the immunosuppressant are dissolved together in an organic solvent to form an organic phase; S2. Dissolve the surfactant in water to form an aqueous phase; S3. The organic phase is added to the aqueous phase for emulsification to form an emulsion; S4. The organic solvent in the emulsion is evaporated and solidified to form a crude ultrasonically responsive drug-loaded nanoparticle; S5. Collect, wash, and freeze-dry the crude ultrasonic-responsive drug-loaded nanoparticles to obtain the ultrasonic-responsive drug-loaded nanoparticles.

[0045] In some specific embodiments of the present invention, the mass ratio of matrix, ultrasonic responsive nitric oxide donor, and immunosuppressant is (4-6):(0.8-1.2):(1.5-2.5), and the volume ratio of organic phase to aqueous phase is 1:(4-6).

[0046] Furthermore, the mass ratio of matrix, ultrasound-responsive nitric oxide donor, and immunosuppressant is (4.5-5.5):(0.9-1.1):(1.8-2.1), and the volume ratio of organic phase to aqueous phase is 1:(4.5-5.5).

[0047] Preferably, the mass ratio of the matrix, the ultrasound-responsive nitric oxide donor, and the immunosuppressant is 5:1:2, and the volume ratio of the organic phase to the aqueous phase is 1:5.

[0048] In some specific embodiments of the present invention, ultrasound is used during emulsification, with a power of 280-310W and a duration of 3-10 minutes.

[0049] The emulsification-solvent evaporation method of this invention is simple and reproducible. The resulting ultrasonically responsive nanoparticles have uniform particle size, with particles in the 100-400 nm range accounting for 85%-90% of the total particles. They exhibit good dispersibility and high drug loading and encapsulation efficiency, with BNN6 encapsulation efficiency >80% and RAPA encapsulation efficiency >70%, making them suitable for further industrial production and clinical application.

[0050] Applications of ultrasound-responsive drug-loaded nanoparticles Application of ultrasound-responsive drug-loaded nanoparticles in the preparation of drugs for treating acute rejection after organ transplantation.

[0051] In some specific embodiments of the present invention, the organ transplant is a heart transplant.

[0052] The PLGA involved in this embodiment of the invention was purchased from Merck China Shanghai Co., Ltd., the PVA was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and both BNN6 and RAPA were purchased from MCE Corporation of the United States.

[0053] Example 1: See Figure 1A method for preparing ultrasound-responsive drug-loaded nanoparticles includes the following steps: S1. Weigh out 10 mg of PLGA and dissolve it in 500 μL of dichloromethane, 2 mg of BNN6 and 4 mg of RAPA and dissolve them in 200 μL of dichloromethane respectively, and mix the above solutions to form a homogeneous organic phase.

[0054] S2. Based on the volume ratio of aqueous phase to organic phase of 5:1, add 4.5 mL of 0.5% PVA aqueous solution to the organic phase. Use a cell disruptor to process the mixture at 300 W for 5 min to form a stable emulsion. To avoid local overheating, place the emulsion in an ice-water bath and perform probe ultrasonic emulsification in a low-temperature environment to ensure that the emulsion is uniformly mixed during the disruption process. S3. Quickly add the above emulsion to 10 mL of ultrapure water and stir in a fume hood for 3 h to evaporate the dichloromethane; S4. Collect P-BNN6 / RAPA nanoparticles by centrifuging at 20000 g for 20 min using a high-speed centrifuge; S5. Wash the nanoparticles three times with ultrapure water, centrifuging after each wash to remove impurities. After the final centrifugation, discard the supernatant and resuspend the nanoparticles in 2 mL of ultrapure water. Pre-freeze the concentrated nanoparticle suspension in an ultra-low temperature freezer at -80℃ for 12 hours to ensure complete freezing. Then, transfer the sample to a vacuum freeze dryer and freeze-dry for 48 hours at -50℃ and a vacuum of <10 Pa. Once the sample is completely dry and appears as a white, loose powder, remove it to obtain P-BNN6 / RAPA nanoparticles. Seal and store in a freezer at -20℃ for later use.

[0055] Comparative Example 1 Preparation method of single-loaded ultrasound-responsive nitric oxide donor (P-BNN6) nanoparticles: S1. Weigh 10 mg of PLGA and dissolve it in 500 μL of dichloromethane, and dissolve 2 mg of BNN6 in 200 μL of dichloromethane. Mix the above solutions to form a homogeneous organic phase.

[0056] S2. Based on the volume ratio of aqueous phase to organic phase of 5:1, add 3.5 mL of 0.5% PVA aqueous solution to the organic phase. Use a cell disruptor to process the mixture at 300 W for 5 min to form a stable emulsion. To avoid local overheating, place the emulsion in an ice-water bath and perform probe ultrasonic emulsification in a low-temperature environment to ensure that the emulsion is uniformly mixed during the disruption process. S3. Quickly add the above emulsion to 10 mL of ultrapure water and stir in a fume hood for 3 h to evaporate the dichloromethane; S4. P-BNN6 nanoparticles were collected after centrifugation at 20000 g for 20 min using a high-speed centrifuge; S5. Wash the nanoparticles three times with ultrapure water, centrifuging after each wash to remove impurities. After the final centrifugation, discard the supernatant and resuspend the nanoparticles in 2 mL of ultrapure water. Pre-freeze the concentrated nanoparticle suspension in an ultra-low temperature freezer at -80℃ for 12 hours to ensure complete freezing. Then, transfer the sample to a vacuum freeze dryer and freeze-dry for 48 hours at -50℃ and a vacuum of <10 Pa. Once the sample is completely dry and appears as a white, loose powder, remove it to obtain P-BNN6 nanoparticles. Seal and store in a freezer at -20℃ for later use.

[0057] Comparative Example 2 Preparation method of single-loaded rapamycin (P-RAPA) nanoparticles: S1. Weigh 10 mg of PLGA and dissolve it in 500 μL of dichloromethane, and dissolve 4 mg of P-RAPA in 200 μL of dichloromethane. Mix the above solutions to form a homogeneous organic phase.

[0058] S2. Based on the volume ratio of aqueous phase to organic phase of 5:1, add 3.5 mL of 0.5% PVA aqueous solution to the organic phase. Use a cell disruptor to process the mixture at 300 W for 5 min to form a stable emulsion. To avoid local overheating, place the emulsion in an ice-water bath and perform probe ultrasonic emulsification under low temperature conditions to ensure that the emulsion is uniformly mixed during the disruption process. S3. Quickly add the above emulsion to 10 mL of ultrapure water and stir in a fume hood for 3 h to evaporate the dichloromethane; S4. Collect P-RAPA nanoparticles by centrifuging at 20000 g for 20 min using a high-speed centrifuge; S5. Wash the nanoparticles three times with ultrapure water, centrifuging after each wash to remove impurities. After the final centrifugation, discard the supernatant and resuspend the nanoparticles in 2 mL of ultrapure water. Pre-freeze the concentrated nanoparticle suspension in an ultra-low temperature freezer at -80℃ for 12 hours to ensure complete freezing. Then, transfer the sample to a vacuum freeze dryer and freeze-dry for 48 hours at -50℃ and a vacuum of <10 Pa. Once the sample is completely dry and appears as a white, loose powder, remove it to obtain P-RAPA nanoparticles. Seal and store in a freezer at -20℃ for later use.

[0059] The preparation methods of the P-BNN6 nanoparticles, P-RAPA nanoparticles, and P-BNN6 / RAPA nanoparticles used in the various performance tests and effect verifications below have been described in Comparative Example 1, Comparative Example 2, and Example 1, respectively.

[0060] I. Characterization of ultrasound-responsive drug-loaded P-BNN6 / RAPA nanoparticles: ① Particle size potential of P-BNN6 / RAPA nanoparticles was measured using a particle size potential analyzer. P-BNN6, P-RAPA, and P-BNN6 / RAPA nanoparticle powders were prepared into a 1 mg / mL stock solution using ultrapure water. The stock solution was then diluted 20-fold, and 1 mL of each solution was added to a quartz cuvette and measured using a particle size analyzer. This process was repeated three times. The stability of each sample was assessed by measuring it three times at different time points: 24 h, 48 h, and 72 h after preparation. All prepared samples were stored at 4 ℃, and the measurement temperature was 25 ℃.

[0061] ②Scanning electron microscopy (SEM) observation of the morphology of P-BNN6 / RAPA nanoparticles The P-BNN6 / RAPA nanoparticle powder was prepared into a stock solution of 1 mg / mL using ultrapure water. The stock solution was then diluted 20 times and dropped onto a silicon wafer. After the sample dropped onto the silicon wafer was allowed to air dry naturally, the silicon wafer was attached to the conductive adhesive. To enhance the conductivity of the P-BNN6 / RAPA nanoparticles prepared in Example 1, a layer of gold was plated on them. Finally, the morphology of the P-BNN6 / RAPA nanoparticles was observed using a scanning electron microscope.

[0062] ③ Observation of the morphology of P-BNN6 / RAPA nanoparticles by transmission electron microscopy (TEM) P-BNN6 / RAPA nanoparticle powder was prepared into a 1 mg / mL stock solution using ultrapure water. The stock solution was then dropped onto a copper mesh at a 20-fold dilution and allowed to air dry. 10 μL of 1% phosphotungstic acid was then added as a contrast agent. After about 1 minute, the excess liquid was blotted off with filter paper and allowed to air dry naturally. The morphological characteristics were then observed using a transmission electron microscope (TEM).

[0063] Depend on Figure 2 The morphology and physicochemical properties of P-RAPA, P-BNN6, and P-BNN6 / RAPA nanoparticles were characterized. Scanning electron microscopy and transmission electron microscopy showed that the P-BNN6 / RAPA nanoparticles were generally regular spherical with a relatively uniform particle size distribution and a smooth surface, without obvious collapse or severe aggregation. The morphology was consistent with that of the single-drug nanoparticles, suggesting that the co-loading of the two drugs did not destroy the particle formation and structural stability of PLGA.

[0064] ④ Fourier transform infrared spectroscopy (FT-IR) was used to observe the absorption peaks of specific functional groups in P-BNN6 / RAPA nanoparticles. Small amounts of P-BNN6, P-RAPA, and P-BNN6 / RAPA nanoparticle powders were taken respectively, and the scanning range was set to 400~4000 cm⁻¹. 1The recorded infrared spectra were analyzed, with particular attention paid to the absorption peaks of specific functional groups in each sample, to verify whether the ultrasonically responsive drug-loaded nanoparticles were successfully loaded with drugs.

[0065] ⑤ Raman spectroscopy observation of the changes in characteristic vibrational peaks of P-BNN6 / RAPA nanoparticles Raman spectroscopy analysis was performed on small amounts of P-BNN6, P-RAPA, and P-BNN6 / RAPA nanoparticle powders to compare the changes in characteristic vibrational peaks of different samples, further verifying the presence of drug molecule characteristic structures in P-BNN6 / RAPA nanoparticles and their interaction with the carrier, such as changes in peak position / peak intensity.

[0066] ⑥ X-ray photoelectron spectroscopy (XPS) was used to detect and observe the elemental composition of P-BNN6 / RAPA nanoparticles. Small amounts of P-BNN6, P-RAPA, and P-BNN6 / RAPA nanoparticle powders were taken respectively, and full-spectrum surveys were performed on the samples to obtain elemental composition information. High-resolution scans of C 1s, N 1s, and O 1s were performed to analyze the surface chemical bonding state.

[0067] Depend on Figure 3 As can be seen, comparative analysis of Fourier transform infrared (FT-IR) and Raman spectroscopy yielded the spectral signals of PLGA, RAPA, BNN6, and P-RAPA, P-BNN6, and P-BNN6 / RAPA nanoparticles, respectively. The P-BNN6 / RAPA nanoparticles simultaneously exhibited characteristic vibrational peaks such as C=O and N=O, further verifying the successful construction of the dual-drug co-loaded nanoparticles. Peak fitting in X-ray photoelectron spectroscopy (XPS) showed that the P-BNN6 / RAPA nanoparticles simultaneously contained nitrogen-containing bond characteristic peaks associated with BNN6 and carbon bond and carbonyl group characteristic peaks associated with PLGA / RAPA, indicating that both drugs were successfully loaded into the PLGA matrix.

[0068] II. Microplate reader assay for drug loading and encapsulation of P-BNN6 / RAPA nanoparticles In step S4 of the P-BNN6 / RAPA nanoparticle preparation process, the supernatant was collected and its absorbance was measured using a microplate reader. The concentrations of BNN6 and RAPA in the supernatant were calculated based on the standard curve, and then the specific contents of BNN6 and RAPA were calculated based on the solution volume. The drug loading of BNN6 and RAPA in the ultrasound-responsive drug-loaded nanoparticles was calculated using an indirect method, with the following formula: Encapsulation efficiency (%) = "Drug loading" / "Drug dosage" × 100% Drug loading rate (%) = "Drug loading amount" / "Total carrier mass" × 100% Drug loading refers to the total amount of BNN6 or RAPA actually contained in P-BNN6 / RAPA nanoparticles; carrier mass refers to the total amount of PLGA used in the preparation of P-BNN6 / RAPA nanoparticles; and drug loading refers to the total amount of BNN6 or RAPA added during the preparation of P-BNN6 / RAPA nanoparticles. We used an indirect method with a microplate reader to determine the encapsulation efficiency and drug loading rate of BNN6 and RAPA. Figure 4 According to the drug standard curves and calculation formulas, the encapsulation efficiency of BNN6 in the single-drug nanoparticle P-BNN6 was 91.45±0.10%, and the drug loading rate was 26.78±0.02%, indicating that PLGA has a high encapsulation capacity for BNN6. In the P-BNN6 / RAPA nanoparticles, the encapsulation efficiency of BNN6 was 82.78±0.16%, and the drug loading rate was 22.32±0.03%, which was slightly lower than that of the single-drug group, but still remained at a high level, indicating that the co-loading of the ultrasound-responsive nitric oxide donor and the immunosuppressant in this invention did not significantly weaken the loading efficiency of BNN6.

[0069] The encapsulation efficiency of RAPA in the single-drug nanoparticle P-RAPA was 73.67±0.40%, and the drug loading rate was 12.84±0.06%. In the P-BNN6 / RAPA nanoparticles, the encapsulation efficiency of RAPA was 76.06±0.25%, and the drug loading rate was 10.26±0.03%. Compared with the single-drug nanoparticles, the encapsulation efficiency of RAPA was slightly improved while the drug loading rate was reduced after co-loading with the ultrasound-responsive nitric oxide donor and the immunosuppressant. This suggests that co-loading with two drugs may affect the drug distribution ratio and carrier mass composition in the nanoparticles, but overall, stable and effective co-loading with two drugs can still be achieved, providing a foundation for subsequent studies on synergistic delivery and ultrasound-triggered release.

[0070] III. Stability of P-BNN6 / RAPA Nanoparticles, In Vitro Ultrasonic Response, and Synergistic Drug Release ①BNN6 working solution preparation Weigh 2.79 mg of BNN6 powder and add DMSO to 1 mL to prepare a 10 mM BNN6 stock solution. Before the experiment, take an appropriate amount of the stock solution and dilute it stepwise with ultrapure water to obtain a BNN6 aqueous solution with a final concentration of 100 μM for later use.

[0071] ② Effects of different power intensities and frequencies on nitric oxide release from BNN6 At room temperature, 100 μM BNN6 solution was added to a 12-well plate. The ultrasound gene transfection instrument probe was placed under the plate, and coupling agent was applied to ensure acoustic energy transmission. With a fixed duty cycle of 50% and irradiation time of 5 min, different power intensities (0, 0.2, 0.5, 1, 1.5, 2, 2.5 W / cm²) were set. 2The sample was irradiated at two frequencies: 1 MHz and 3 MHz. After irradiation, 50 μL of sample was taken from each well, and Griess reagent was added sequentially. After incubation in the dark for 10 min, the absorbance (OD) was measured.

[0072] ③ The effect of different duty cycles and frequencies on nitric oxide release from BNN6 Fixed power intensity 1 W / cm 2 Irradiation time was 5 min, with duty cycles set at 10%, 20%, 30%, 40%, 50%, and 75%, and irradiation was performed at 1 MHz and 3 MHz respectively. After irradiation, 50 μL samples (n=3) were taken, and Griess reagent was added. The reaction was carried out in the dark for 10 min, and the OD value was measured.

[0073] ④ Determination of the ultrasonic responsiveness of P-BNN6 and P-BNN6 / RAPA nanoparticles At a power intensity of 1 W / cm 2 Under conditions of 50% duty cycle and 1 MHz frequency, 1 mL of P-BNN6 or P-BNN6 / RAPA nanoparticle suspension with a final concentration of 100 μM (based on BNN6) was subjected to ultrasonic irradiation for different durations. The ultrasonic operation was paused at cumulative irradiation times of 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min (time points and...). Figure 5 (The x-axis corresponds completely). The control group without ultrasound was placed under the same conditions. The NO concentration in each group was determined by the Griess method.

[0074] ⑤ Calculation and data processing of nitric oxide concentration Bilinitrite (NO2) was determined by the Griess method. - At the specified levels, precisely pipette 50 μL of reaction solution from each sample group into a 96-well plate. Under strict light-protected conditions, add 50 μL of Griess Reagent I (1% sulfonamide solution) and 50 μL of Griess Reagent II (0.1% N-1-naphthylethylenediamine dihydrochloride solution) sequentially to each sample group. After mixing, incubate at room temperature in the dark for 10 min, observing the solution color change from colorless to pink / purple-red. Measure the absorbance (OD value) of each well at 540 nm using a microplate reader. Utilize a pre-constructed sodium nitrite (NaNO2) standard curve (see [link to standard curve]). Figure 4 C) Convert the measured OD value to NO (in NO2). - The molar concentration (μM) of NO was calculated. NO release kinetics curves were plotted with cumulative ultrasonic irradiation time on the x-axis and NO concentration on the y-axis to evaluate the ultrasonic response performance of the nanoparticles.

[0075] ⑥ Assess cumulative RAPA release using in vitro dialysis Samples were prepared by dispensing free rapamycin (RAPA), P-RAPA nanoparticles, and P-BNN6 / RAPA nanoparticles at equal RAPA dosages (1 mg). RAPA, P-RAPA, P-RAPA+US, P-BNN6 / RAPA nanoparticle, and P-BNN6 / RAPA nanoparticle+US groups were established to verify whether BNN6 promotes RAPA release under ultrasound. The samples were placed in dialysis bags (Mw=8000Da, Beijing Solarbio Co., Ltd.), sealed, and placed in a container containing the release medium. The release medium was PBS, pH=7.4, with Tween-80 added to maintain sedimentation conditions at 0.5% w / v. The total external phase volume was kept constant at 50 mL, and the in vitro release experiment was conducted under constant temperature shaking at 37℃ and 150 rpm.

[0076] To simulate the ultrasonic-triggered controlled release process, before sampling and measurement at each preset sampling time point (2, 4, 6, 8, 10, 12, 24, 48, 72, 96, 120, 144, 168 h), a power intensity of 1 W / cm² was used. 2 Under conditions of 50% duty cycle and 1 MHz frequency, the dialysis system was subjected to ultrasonic irradiation, followed immediately by a 1.0 mL sample taken from the external phase release medium. An equal volume of fresh release medium, preheated to 37°C, was then added back to maintain a constant external phase volume. The sample was centrifuged, filtered through a 0.22 μm filter, and the RAPA concentration was determined using an ELISA reader. The release amount at each time point was calculated based on the standard curve.

[0077] Depend on Figure 5 It can be seen that, regarding the effect of different duty cycles and frequencies on the release of nitric oxide from BNN6, as the ultrasonic power intensity gradually increases from low to high (see...), the effect is positive. Figure 5 A) The concentration of nitric oxide in the system generally showed an increasing trend, reaching approximately 2.0 W / cm². 2 It reaches a relatively high level nearby; when the power is further increased to 2.5 W / cm 2 At this point, the nitric oxide concentration decreased, suggesting that excessive sound energy may be accompanied by a more pronounced thermal effect, thus affecting the stable existence of nitric oxide. Further adjustments to the duty cycle under constant power conditions (see...) Figure 5 B) It can be seen that nitric oxide release gradually increases with increasing duty cycle, suggesting that a higher duty cycle and a longer effective irradiation time are conducive to triggering BNN6 breakage and generating nitric oxide.

[0078] In the ultrasonic responsiveness determination of P-BNN6 and P-BNN6 / RAPA nanoparticles, groups were set up as BNN6 group, BNN6+US group, P-BNN6+US group, and P-BNN6 / RAPA nanoparticle+US group. In the BNN6 group without ultrasound, the NO concentration remained close to the background level within 5–60 min, indicating that the release of nitric oxide from BNN6 is significantly ultrasound-dependent. After ultrasound application, the NO concentrations in the BNN6+US group, P-BNN6+US group, and P-BNN6 / RAPA nanoparticle+US group all showed an increasing trend with increasing cumulative irradiation time, reaching a plateau around 30 min. Taking a representative time point as an example, at 20 min, the NO concentration in the BNN6+US group was approximately 44 μM, while that in the nanoparticle group was approximately 32–34 μM; the plateau period was from 30–60 min, with all three groups stabilizing in the range of approximately 51–54 μM, indicating that good ultrasonic responsiveness was maintained after encapsulation, but early release was slightly delayed (see [link to relevant documentation]). Figure 5 C).

[0079] In vitro dialysis was used to detect the release of RAPA. Free RAPA showed the fastest release rate, approximately 94% at 12 h and nearly 100% at 24 h. In contrast, the P-RAPA group showed approximately 38% release at 24 h, reaching 50% release in about two days. The P-RAPA+US group increased to approximately 46% at 24 h. The P-BNN6 / RAPA nanoparticle group maintained its sustained-release characteristics at approximately 41% at 24 h, but the P-BNN6 / RAPA nanoparticle+US group showed approximately 71% at 12 h and approximately 82% at 24 h, suggesting that ultrasound triggering had a more significant effect on promoting the release of P-BNN6 / RAPA nanoparticles (see...). Figure 5 D).

[0080] IV. Verification of Immune Regulation (1) Effects of P-BNN6 / RAPA nanoparticles on LPS-induced polarization and secretion of inflammatory factors in NR8383 macrophages ① NR8383 macrophage M1 phenotype induction and material processing Rat alveolar macrophages NR8383 were cultured in medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2. Cells were seeded into 6-well plates for flow cytometry analysis. After cell adhesion and reaching a suitable density, further treatment was performed. Cell experiments included a control group (LPS model group, i.e., M1 group), a RAPA single-drug group, a P-RAPA group, and a P-BNN6 / RAPA nanoparticle + US group. To establish the pro-inflammatory M1 model, each group was stimulated with lipopolysaccharide (LPS) (final concentration 1 μg / mL) for 24 h. After induction, the culture medium was discarded, and the cells were washed once with PBS. Then, different drugs (RAPA single-drug, P-RAPA nanoparticles, and P-BNN6 / RAPA nanoparticles + US) were added and incubated for another 24 h. Nanoparticles were prepared at an equivalent RAPA concentration of 500 ng / mL (even if the drug loading of different ultrasound-responsive drug-loaded nanoparticles was different, the final RAPA concentration in the culture system was consistent). After treatment, flow cytometry was performed and the supernatant was collected for ELISA to assess macrophage polarization.

[0081] ② Flow cytometry detection of CD86(M1) / CD163(M2) and ELISA detection of cytokines After 24 h of treatment, the cell culture supernatant from each group was collected for cytokine detection; the supernatant was centrifuged at 1000 g for 10 min to remove cell debris and then aliquoted. Store at 80℃ for later use. Then, perform flow cytometry on the cells: discard residual liquid in the wells, wash once with PBS, gently scrape adherent cells with a cell scraper and collect them into centrifuge tubes, centrifuge at 300g for 5 min, discard the supernatant, resuspend in PBS containing 2% BSA and block, incubate at 4℃ for 15 min. Add fluorescently labeled antibody and incubate in the dark at 4℃ for 30 min. Detect CD86 (M1 phenotypic marker) and CD163 (M2 phenotypic marker) separately, wash twice with PBS, resuspend and analyze. FlowJo software was used to analyze CD86. + CD163 + Cell proportions and all data were plotted and statistically analyzed using GraphPad Prism 9.0 software. All measurement data are expressed as mean ± standard deviation (Mean ± SD). Independent samples t-tests were used for individual comparisons between two groups, and one-way ANOVA was used for comparisons between multiple groups. P < 0.05 was considered statistically significant.

[0082] Cytokine profiles were determined using ELISA kits following the manufacturer's instructions, detecting pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, and anti-inflammatory cytokines IL-10 and TGF-β. Concentrations were calculated based on standard curves, and statistical analysis and graphical representation were performed. All experiments were repeated at least three times. Results are expressed as mean ± SD. Differences between groups were analyzed using t-tests or one-way ANOVA, with P < 0.05 considered statistically significant.

[0083] Depend on Figure 6 It can be seen that, compared with other control groups, M1 group (i.e., LPS group), RAPA group and P-RAPA group, P-BNN6 / RAPA nanoparticles in the US group can reduce pro-inflammatory factors and increase anti-inflammatory factor levels. At the same time, the transformation of macrophage phenotype from M1 to M2 and the improvement of inflammation spectrum are more significant.

[0084] (2) Cell compatibility of P-BNN6 / RAPA nanoparticles and their effect on the migration ability of NR8383 ① Evaluation of the effect of P-BNN6 / RAPA nanoparticles on the viability of NR8383 and H9c2 cells using the CCK-8 assay NR8383 and H9c2 cells were cultured in media containing 10% FBS and 1% penicillin-streptomycin, respectively, and maintained at 37°C and 5% CO2. After cell counting, the cells were seeded into 96-well plates (1 x 102 cells per well). 5 Cells were cultured overnight at varying densities. The culture medium was then replaced with fresh medium, and different concentrations of P-BNN6 / RAPA nanoparticles were added to each well, with final concentrations of 25, 50, 100, 200, and 400 μg / mL. After incubation for 24 h and 48 h, CCK-8 reagent (generally 10% of the culture medium volume, according to the kit instructions) was added to each well. The cells were incubated at 37°C in the dark for an appropriate time of 2 h. The absorbance (OD) at 450 nm was then measured using a microplate reader. 450 Relative cell viability was calculated using the control group as a normalized benchmark: Cell viability (%) = (OD of treatment group) / (OD of treatment group) Blank hole OD) / (Control group OD) The blank well OD) × 100%. Each group was set with ≥3 replicates, and the experiment was independently repeated ≥3 times (the treatment group was the P-BNN6 / RAPA nanoparticle group, the blank group was the culture medium group only, and the control group was the culture medium group with cells added).

[0085] ② Transwell assay to evaluate the effect of P-BNN6 / RAPA nanoparticles on the migration function of NR8383 macrophages. Cell migration assays were performed using Transwell chambers (8.0 μm pore size). NR8383 cells were prepared as single-cell suspensions. A certain amount of cell suspension (serum-free medium) was added to the upper chamber, and three groups were established: Blank group, P-BNN6 / RAPA nanoparticle group (50 μg / mL), and P-BNN6 / RAPA nanoparticle + US (50 μg / mL). Chemotactic agents were added to the lower chamber, with complete medium containing 10% FBS as the chemotactic source. After incubation at 37℃ and 5% CO2 for 24 h, unmigrated cells from the upper chamber were discarded, and the upper membrane surface was gently wiped with a cotton swab. The cells were then fixed with 4% paraformaldehyde for 15 min and stained with crystal violet for 10 min. After washing with PBS, multiple fields of view were randomly selected under a microscope for photographing, and the number of migrating cells on the lower membrane surface was counted. The number of migrating cells was statistically analyzed, and inter-group comparisons were performed. The P-BNN6 / RAPA nanoparticles + US group was subjected to ultrasonic irradiation (ultrasonic power 1 W / cm²) after sample addition, according to the set parameters. 2 The control group (duty cycle 50%, sonication time 5 min) was placed under the same conditions but without sonication. The experiment was independently repeated ≥3 times, and the results are expressed as mean ± SD.

[0086] Depend on Figure 7 The CCK-8 assay results showed that when the concentration of P-BNN6 / RAPA nanoparticles was in the range of 25–50 μg / mL, the cell viability remained above 85% after co-culturing with both cell types for 24 h and 48 h, demonstrating good cell safety. As the material concentration increased to 100–400 μg / mL, the cell viability decreased in a concentration-dependent manner. In summary, P-BNN6 / RAPA nanoparticles exhibit good biocompatibility at low concentrations ≤50 μg / mL, meeting the safety requirements for subsequent drug delivery studies.

[0087] Transwell assays confirmed that P-BNN6 / RAPA nanoparticles did not affect the normal migration ability of macrophages, and macrophage recruitment and migration are fundamental to their immune surveillance and regulatory functions. To further evaluate the effects of P-BNN6 / RAPA nanoparticles on macrophage physiological function, we used Transwell assays to examine the effects of P-BNN6 / RAPA nanoparticles and their combined sonication treatment on the vertical migration ability of NR8383 cells. Figure 7As shown in C and 7D, compared with the Blank group, the number of migrating cells in the P-BNN6 / RAPA nanoparticle group and the P-BNN6 / RAPA nanoparticle + US group was slightly reduced after 24 h, but statistical analysis showed no significant difference between the groups. This result indicates that at the selected safe therapeutic concentration, P-BNN6 / RAPA nanoparticles do not significantly impair or inhibit the normal motility and migration ability of macrophages during the ultrasound response, further confirming the excellent in vitro biocompatibility of this delivery system.

[0088] V. In vivo biosafety verification Serum biochemical indicators and HE staining of major organs were used for evaluation. Healthy rats were randomly divided into PBS group, BNN6 group, RAPA group, PLGA group, and P-BNN6 / RAPA nanoparticle group. All treatment groups were administered drugs via tail vein injection, with the dosage converted to 1 mg / kg (based on the drug) according to the clinical equivalent dose; the PBS group was administered an equal volume of PBS via tail vein injection as a control.

[0089] On day 7 after drug administration, rats were anesthetized and peripheral blood was collected. After coagulation at room temperature, the serum was separated by centrifugation for the detection of serum biochemical indicators. A fully automated biochemical analyzer was used to measure bile and liver function related indicators: alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were used to assess liver function, and blood urea nitrogen (BUN) and creatinine (CREA) were used to assess kidney function.

[0090] Animals were subsequently euthanized, and major organs, including the heart, liver, spleen, lungs, and kidneys, were rapidly harvested. After rinsing with PBS to remove blood, the organs were fixed in 4% paraformaldehyde. Following fixation, the organs were routinely dehydrated, cleared, embedded in paraffin, and sectioned to a thickness of approximately 5 μm for hematoxylin-eosin (HE) staining. Morphological changes in each organ tissue, such as inflammatory cell infiltration, necrosis, hemorrhage, and edema, were observed using an optical microscope, and representative images were captured for intergroup comparisons. Serum biochemical data are expressed as mean ± SD and statistically analyzed. The in vivo biocompatibility of the ultrasound-responsive drug-loaded nanoparticles was comprehensively assessed in conjunction with the histological results.

[0091] Depend on Figure 8It was found that the levels of liver function indicators (ALT, AST) and kidney function indicators (BUN, CREA) in rats from each group were comparable (P>0.05), with no abnormal increases observed. These results indicate that continuous intravenous injection of P-BNN6 / RAPA nanoparticles did not cause significant systemic toxicity or liver and kidney damage in rats. Further pathological observation of the major organs of the rats was performed using H&E staining. The results showed that after 7 days of continuous administration, the histomorphology of the heart, liver, spleen, lungs, and kidneys in the P-BNN6 / RAPA nanoparticle group was consistent with that of the PBS control group, with no significant pathological changes observed. Figure 8 As shown in Figure E, the myocardial fibers were neatly arranged without breakage; the liver lobule structure was clear, and the hepatic cords were arranged regularly; the alveolar structure was intact, without significant thickening or inflammatory infiltration; the glomeruli and renal tubules were normal in morphology, and no cell necrosis or inflammatory cell infiltration was observed. These histopathological results were consistent with the blood biochemical analysis results, indicating that P-BNN6 / RAPA nanoparticles have good in vivo biosafety, laying the foundation for their subsequent in vivo application.

[0092] VI. In vivo targeting and efficacy verification (1) Establishing a rat model of heterotopic peritoneal heart transplantation ① Laboratory animals and anesthesia Male BN rats weighing 250 g were selected as donors, and male Lewis rats weighing 250 g were selected as recipients. Surgery was performed under isoflurane inhalation anesthesia: anesthesia was induced with 4% isoflurane in an induction chamber, followed by maintenance anesthesia with 2% isoflurane via face mask inhalation. After the animals were fixed on the operating table, the depth of anesthesia was assessed by stimulating the toes; the surgical procedure began after confirming no significant response.

[0093] ② Donor heart retrieval and perfusion After routine disinfection of the donor's abdominal skin, a 5 cm incision was made in the midline of the abdomen to enter the abdominal cavity. The intestines were gently pushed to one side to expose the inferior vena cava. 2 mL of pre-cooled heparinized saline solution (500 U / mL heparin) was injected through the inferior vena cava, and allowed to stand for approximately 2 minutes to complete systemic heparinization. The inferior vena cava and abdominal aorta were then cut to release blood. The ribs were cut along both sides of the abdominal incision to enter the thoracic cavity. The heart was exposed and covered with a small amount of crushed ice to induce rapid cardiac arrest, reducing heat ischemia-reperfusion injury. 20 mL of pre-cooled heparinized saline solution was slowly infused into the heart through the inferior vena cava below the diaphragm. Perfusion was complete when the lung tissue gradually turned white, the superficial blood vessels became clear, and the outflowing fluid gradually became clear. The thymus and surrounding tissues were then separated, the pericardium was opened, and the ascending aorta was separated and neatly cut before its bifurcation. The superior vena cava, inferior vena cava, and pulmonary veins were then sequentially separated, ligated, and cut. The adipose tissue surrounding the main pulmonary artery was removed using blunt dissection. The pulmonary artery was then cut before its bifurcation, leaving an appropriate length. Except for the aorta, pulmonary artery, and inferior vena cava, all other vessels and tissues were ligated before the donor heart was removed and stored in 4°C saline for later use.

[0094] ③ Receptor preparation Recipient rats were fasted for 24 hours and deprived of water for 6 hours prior to surgery. After anesthesia using the method described above, they were fixed on the operating table, their abdominal hair was shaved, and the surgical area was disinfected. A midline abdominal incision was made, extending from below the xiphoid process to approximately 1 cm above the external urethral orifice. The intestines were covered and protected with saline-moistened gauze, and pushed towards the left upper abdomen to fully expose the abdominal aorta and inferior vena cava. Blunt dissection was performed using cotton swabs, carefully freeing the tissues surrounding the abdominal aorta and inferior vena cava; larger branches were ligated and severed, and slipknots were tied at the left and right branches. During the procedure, care was taken to avoid damaging nerves and the ureter.

[0095] ④ Heart transplantation and vascular anastomosis The recipient's abdominal aorta and inferior vena cava are clamped at their proximal and distal ends using vascular clips, with the proximal end clamped first followed by the distal end to maintain vascular refill. Anastomoses are created in the recipient's abdominal aorta and inferior vena cava according to the donor's vessel diameter. After anastomosis, the inferior vena cava is flushed with saline to reduce residual blood and the risk of thrombosis. The donor heart is placed in the recipient's right abdominal cavity and covered with moist gauze for moisture retention. The donor aorta and recipient's abdominal aorta are anastomosed end-to-side using a continuous suture technique; the donor pulmonary artery and recipient's inferior vena cava are anastomosed end-to-side using a unilateral seton technique, ensuring vertical needle insertion, uniform needle spacing, and appropriate suture tension. After anastomosis, the clamped vessels are released sequentially, and hemostasis is achieved: first, open the branch vessels, then release the distal clamps first, followed by the proximal clamps, and apply pressure with cotton swabs for hemostasis, supplemented with gelatin sponge if necessary. After blood flow is restored, refilling of the donor's coronary arteries is visible, and the donor heart resumes pulsation and gradually becomes regular within approximately 1 minute.

[0096] ⑤ Abdominal closure and postoperative management After clearing peritoneal effusion and blood clots, and observing for 3 minutes to confirm no significant active bleeding, the intestines were gently returned to the abdominal cavity, and the abdominal wall was closed in layers using 4-0 sutures. Postoperatively, 4 mL of normal saline was injected subcutaneously for rehydration. Patients were kept NPO (no food, but water) for 6 hours postoperatively, and resumed normal feeding 24 hours later.

[0097] Depend on Figure 9 It can be seen that the rat peritoneal heterotopic heart transplantation model has been successfully established, and all in vivo experimental results are based on this model.

[0098] (2) Fluorescence tracing assessment of in vivo distribution of ultrasound-responsive drug-loaded nanoparticles and ultrasound-enhanced local drug release in transplanted hearts: ①Preparation and drug delivery of DiD / DiR labeled P-BNN6 / RAPA nanoparticles To evaluate the in vivo distribution of P-BNN6 / RAPA nanoparticles, the near-infrared fluorescent dye DiD was used for tracer labeling. DiD and PLGA were co-dissolved in an organic phase, allowing the dye to be embedded within the polymer matrix during the preparation of the P-BNN6 / RAPA nanoparticles, resulting in fluorescently labeled, ultrasound-responsive drug-loaded nanoparticles (PLGA-BNN6 / RAPA-DiD). After preparation, free dye was removed by centrifugation and washing until the fluorescence signal of the supernatant was close to the background. The PLGA-BNN6 / RAPA-DiD nanoparticles were then resuspended in PBS for later use.

[0099] After establishing a rat heart transplantation model, fluorescently labeled P-BNN6 / RAPA nanoparticles were injected via tail vein at set time points (dose was based on clinical dosage, with RAPA at 1 mg / kg). A free dye group (DiD) was set up as a control to distinguish the difference between "free dye distribution" and "ultrasound-responsive drug-loaded nanoparticle delivery distribution".

[0100] ②IVIS ex vivo imaging assessment of the distribution of the transplanted heart and major organs Animals were subjected to ex vivo imaging at preset time points after drug administration. Animals were euthanized after anesthesia, and the transplanted heart was quickly removed for ex vivo imaging. Using Living Image software, fluorescence intensity was recorded and quantified in the defined region of interest (ROI) of the transplanted heart. The enrichment differences between the free dye group (DiD) and the PLGA-BNN6 / RAPA-DiD group in the transplanted heart were compared, and statistical graphs were plotted.

[0101] ③ LC-MS / MS quantification of RAPA content in transplanted hearts and verification of ultrasound-enhanced drug release To verify the effect of ultrasound on promoting local drug release in transplanted hearts, transplanted hearts were harvested at preset time points after drug administration. Surface liquid was aspirated, the hearts were weighed, and pre-cooled. 200 μL of pre-cooled methanol solution containing the internal standard (Ascomycin, 50 ng / mL) was added to the tubes. The tissue was thoroughly homogenized using a high-throughput tissue homogenizer (60 Hz, 2 min) to precipitate proteins and extract the drug. The homogenate was centrifuged at 4 ℃ and 12,000 rpm for 10 min, and 100 μL of the supernatant was precisely pipetted into a clean glass tube. The solvent was dried under a nitrogen stream at 40 ℃. The residue was reconstituted with 100 μL of the initial mobile phase (acetonitrile:water = 80:20), vortexed for 1 min, centrifuged again, and the supernatant was used for analysis.

[0102] A RAPA standard curve was established using LC-MS / MS, and MRM quantification was performed to calculate the RAPA content in the samples, which was expressed as ng / g after tissue weight normalization. P-RAPA and P-BNN6 / RAPA nanoparticle + US groups were set up. The "+US" group underwent ultrasound irradiation (ultrasound power intensity 1 W / cm²) on the transplanted heart region after drug administration. 2 The study used an ultrasound-enhanced local drug release and improved drug exposure in transplanted hearts (with a duty cycle of 50%, a frequency of 1 MHz, and a duration of 5 min) to compare the differences in RAPA concentration in different groups of transplanted hearts, in order to verify the effect of ultrasound-enhanced local drug release and improved drug exposure in transplanted hearts.

[0103] Depend on Figure 10 As shown in A and 10B, 24 hours after tail vein injection, fluorescence imaging of ex vivo organs revealed a diffuse distribution of fluorescence signals in the free DiD group, with weaker signals in the transplanted heart. In contrast, the PLGA-BNN6 / RAPA-DiD group exhibited significantly enhanced fluorescence intensity (red / yellow area) in the transplanted heart, suggesting that the nanoparticles can effectively accumulate in the transplanted heart site where rejection occurs through phagocytosis by inflammatory cells (macrophages).

[0104] Based on the confirmation that P-BNN6 / RAPA nanoparticles can target and reach the transplanted heart, LC-MS / MS technology was further used to quantitatively detect the RAPA concentration in the tissue to verify the promoting effect of ultrasound irradiation on drug release. Figure 10 As shown in C, although the P-RAPA group achieved a drug concentration of approximately 280 ng / g in the transplanted heart due to passive targeting, the tissue drug concentration of the P-BNN6 / RAPA nanoparticle + US group was significantly higher, reaching approximately 450 ng / g.

[0105] (3) Verification of P-BNN6 / RAPA nanoparticles regulating macrophage polarization in vivo ① Animal grouping, drug administration, and ultrasound treatment A rat model of heterotopic peritoneal heart transplantation was established. Post-operatively, rats were randomly divided into four groups: PBS group, free RAPA group, P-RAPA group, and P-BNN6 / RAPA nanoparticle + US group. All groups were administered an equivalent dose of RAPA to ensure consistent RAPA exposure across different formulations (dose was based on clinical dosage, with RAPA calculated as 1 mg / kg), via tail vein injection. In the US group, ultrasound irradiation was applied to the transplanted heart region after administration, with the same ultrasound parameters as above (ultrasound power intensity 1 W / cm²). 2 (Duty cycle 50%, frequency 1MHz, duration 5min), the other groups were treated under the same conditions but without ultrasound.

[0106] ② Immunofluorescence staining of transplanted heart to assess macrophage polarization (CD86 / CD163) Seven days after the predetermined time point, some animals were euthanized and their transplanted hearts were harvested. After rinsing with PBS to remove blood, the hearts were fixed in 4% paraformaldehyde, routinely dehydrated, embedded, and sectioned. After dewaxing and rehydration, antigen retrieval was performed, followed by blocking with 5% BSA blocking solution. Primary antibodies CD86 (M1 marker) and CD163 (M2 marker) were then added and incubated overnight at 4 °C. The next day, the corresponding fluorescent secondary antibodies (the secondary antibody being the agent that develops the primary antibody) were added and incubated in the dark. Cell nuclei were counterstained with DAPI, and the sections were mounted. Images were acquired using a fluorescence microscope, and multiple fields of view were randomly selected for semi-quantitative analysis using ImageJ software to compare the differences in the expression of M1 / M2 related markers in the transplanted hearts of different groups.

[0107] ③ELISA detection of inflammatory factor spectrum in transplanted heart Seven days after the preset time point, animal transplanted hearts were harvested and homogenized to extract total protein. The animal transplanted heart samples were weighed, homogenized in pre-cooled PBS (containing protease inhibitors), centrifuged, and the supernatant was collected. Total protein was determined using the BCA method and normalized to protein content. ELISA kits were used to detect pro-inflammatory factors IL-6, IL-1β, and TNF-α, and anti-inflammatory factors IL-10 and TGF-β according to the manufacturer's instructions, and their concentrations were calculated based on the standard curve. Results are expressed as mean ± SD, and statistical comparisons were performed between groups to evaluate the material's regulatory effect on the transplanted inflammatory microenvironment.

[0108] ④ Monitoring of transplanted heart survival time and Kaplan-Meier analysis Postoperatively, the recipient's abdomen was palpated daily to assess and record the strength of the transplanted heart's pulsation. The absence or significant failure of the pulsation was used as the criterion for transplanted heart failure. Transplanted heart survival time was recorded, and Kaplan-Meier survival curves were plotted. Differences between groups were evaluated using the log-rank test.

[0109] ⑤ Transplant Cardiac Pathological Assessment and Scoring Seven days after the predetermined time point, when the transplanted heart failed, it was harvested, fixed, embedded in paraffin, and sectioned for HE staining. Inflammatory cell infiltration, myocardial edema / necrosis, and perivascular inflammatory pathological changes were assessed under an optical microscope. The transplant rejection pathological scoring was graded according to a pre-established scoring system (performed by a blinded assessor), and intergroup comparisons were conducted to comprehensively evaluate the efficacy.

[0110] Depend on Figure 11 It can be seen that, compared with the PBS group or the free RAPA group, P-BNN6 / RAPA nanoparticles can significantly reduce pro-inflammatory factors and increase anti-inflammatory factor levels under ultrasound triggering, promote the M1 to M2 phenotype conversion and prolong the survival of transplanted hearts.

[0111] (4) P-BNN6 / RAPA nanoparticles improve the levels of inflammatory factors related to transplant immune rejection: A rat model of peritoneal heterotopic heart transplantation was established. After surgery, rats were randomly divided into PBS group (untreated group), free RAPA group, P-RAPA group, and P-BNN6 / RAPA nanoparticle + US group.

[0112] ① Harvesting, fixing, embedding, and preparing transplanted heart sections Seven days after the predetermined time point, recipient rats were sacrificed, the transplanted heart was removed, and tissue blocks from representative sites were harvested after rinsing with PBS to remove blood. The tissues were fixed in 4% paraformaldehyde for 24 h, followed by routine dehydration, clearing, and paraffin embedding. Paraffin sections with a thickness of 5 μm were prepared using a microtome for immunofluorescence detection.

[0113] ② Immunofluorescence detection of inflammation-related biomarkers Sections were rehydrated / permeabilized with 0.1% Triton X-100, followed by blocking with 5% BSA for 60 min. Primary antibodies (anti-IL-2, anti-IL-6, and anti-IFN-γ) were added and incubated overnight at 4°C. The next day, the corresponding fluorescent secondary antibodies were added and incubated at room temperature for 1 h in the dark. Cell nuclei were counterstained with DAPI, and the sections were mounted. Images were acquired using a confocal microscope.

[0114] ③ Immunohistochemical detection of T cell infiltration CD3 and macrophage infiltration CD68 Paraffin sections were dewaxed, rehydrated with graded ethanol, and then subjected to antigen retrieval. After cooling to room temperature, they were treated with 3% H2O2 to block endogenous peroxidase activity, followed by blocking with 5% BSA for 30 min. Primary antibodies (CD3 and CD68 antibodies) were added and incubated overnight at 4°C: anti-CD3 was used to label T cell infiltration, and anti-CD68 was used to label macrophage infiltration. The next day, after thawing, HRP-labeled secondary antibodies were added and incubated (30 min), followed by DAB staining, hematoxylin counterstaining of cell nuclei, graded dehydration and clearing, and mounting. Images were acquired at the same magnification using an optical microscope.

[0115] ④ Image-based quantitative CD3 / CD68 positive ratio and MFI of inflammatory factors and statistical analysis Semi-quantitative analysis of IHC and IF images was performed using ImageJ software. For the IHC portion, multiple fields of view were randomly selected from each slice, and the percentage of CD3 and CD68 positive areas was calculated. For the IF portion, the mean fluorescence intensity (MFI, AU) of IL-2, IL-6, and IFN-γ was calculated under the same ROI and acquisition parameters, and background subtraction was performed. At least ≥3 animals were selected from each group, and ≥3–5 random fields of view were collected from each animal for statistical analysis.

[0116] Depend on Figure 12 It was found that, compared with the PBS group, P-BNN6 / RAPA nanoparticles could reduce the expression of IL-2, IL-6, and IFN-γ in transplanted hearts and decrease CD3 expression. + T cells and CD68 + Macrophage infiltration was observed, with the P-BNN6 / RAPA nanoparticles + US group showing the most significant inhibitory effect.

[0117] (5) Validation of the efficacy of P-BNN6 / RAPA nanoparticles at immunological endpoints: A rat model of peritoneal heterotopic heart transplantation was established. After surgery, rats were randomly divided into PBS group (untreated group), free RAPA group, P-RAPA group, and P-BNN6 / RAPA nanoparticle + US group.

[0118] ①Immune organ sampling and single-cell suspension preparation Recipient rats were sacrificed on day 7 post-heart transplantation. Spleens (SPs) and inguinal lymph nodes (LNs) were aseptically isolated and placed in pre-chilled flow cytometry buffer (PBS + 1% FBS) for later use. The spleen was placed on a 70 μm cell filter, and an appropriate amount of buffer was added. The cells were gently ground using a syringe plunger to obtain a single-cell suspension. The inguinal lymph nodes were ground in the same manner to prepare a single-cell suspension. The cell suspensions were centrifuged at 300 g for 5 min at 4°C, and the supernatant was discarded. Red blood cells were lysed in the spleen sample using erythrocyte lysis buffer (ACK) to remove erythrocytes. The lysis was carried out at room temperature for approximately 5 min, followed by the addition of excess buffer to terminate lysis and centrifugation followed by washing. Lymph node samples typically do not require erythrocyte lysis. Finally, the cells were resuspended in flow cytometry buffer and counted, with the concentration adjusted for subsequent staining.

[0119] ② Flow cytometry surface staining and detection of CD3 / CD4 / CD8 Take 1×10 cells from each sample. 6Each tube was used for Fc receptor blocking at 4°C for 10 min, followed by surface staining with fluorescently labeled antibodies and incubation at 4°C for 30 min in the dark. Antibodies included: anti-CD3 (T cell marker) and anti-CD4 (CD4+). + T cells) and anti-CD8 (CD8) + (T cells). After staining, the cells were washed twice with flow cytometry buffer and resuspended. Dead or live staining dye could be added as needed to remove dead cells, and a single-stain control group was set up to adjust parameter compensation. Data were then collected.

[0120] ③ Gating strategy and T cell subset ratio analysis: LN vs. SP Analysis was performed using software such as FlowJo: lymphocyte populations were selected on FSC / SSC and debris was excluded (dead cells could be further excluded using live / dead dyes), and CD3... + CD4 counts were performed within the T cell population. + With CD8 + Cell percentages. Obtained separately: LN-CD4 + cell of CD3 + (%) and LN-CD8 + cell of CD3 + (%); SP-CD4 + cell of CD3 + (%) and SP-CD8 + cell of CD3 + (%).

[0121] Compare the differences among the PBS group, free RAPA group, P-RAPA group, and P-BNN6 / RAPA nanoparticle + US group and draw statistical graphs.

[0122] Depend on Figure 13 It can be seen that, compared with the PBS group, delivery of free RAPA and P-RAPA can reduce CD4 in LN and SP. + / CD8 + The proportion of T cells was significantly higher in the P-BNN6 / RAPA nanoparticle + US group.

[0123] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features of the invention herein.

Claims

1. An ultrasonically responsive drug-loaded nanoparticle, characterized in that: The ultrasound-responsive drug-loaded nanoparticles include a matrix in which an ultrasound-responsive nitric oxide donor and an immunosuppressant are dispersed; wherein the ultrasound-responsive nitric oxide donor releases nitric oxide upon ultrasound triggering, and the diffusion of the nitric oxide induces the matrix to form a porous structure for releasing the immunosuppressant.

2. The ultrasonically responsive drug-loaded nanoparticles according to claim 1, characterized in that: The ultrasonically responsive drug-loaded nanoparticles have a particle size in the range of 100-400 nanometers, accounting for 85%-90% of the total number of particles.

3. The ultrasonically responsive drug-loaded nanoparticles according to claim 1, characterized in that: The immunosuppressant is rapamycin.

4. The ultrasonically responsive drug-loaded nanoparticles according to claim 1, characterized in that: The ultrasonically responsive nitric oxide donor is BNN6.

5. The ultrasonically responsive drug-loaded nanoparticles according to claim 1, characterized in that: The matrix is ​​a polylactic acid-hydroxyacetic acid copolymer.

6. A method for preparing ultrasonically responsive drug-loaded nanoparticles, characterized in that, Includes the following steps: The matrix, nitric oxide donor, and immunosuppressant are dissolved together in an organic solvent to form an organic phase; The surfactant is dissolved in water to form an aqueous phase; The organic phase is added to the aqueous phase and emulsified to form an emulsion; The organic solvent in the emulsion is evaporated and solidified to form a crude product of ultrasonically responsive drug-loaded nanoparticles. The crude ultrasonic-responsive drug-loaded nanoparticles were collected, washed, and freeze-dried to obtain the ultrasonic-responsive drug-loaded nanoparticles.

7. The method for preparing ultrasonically responsive drug-loaded nanoparticles according to claim 6, characterized in that: The mass ratio of matrix, ultrasound-responsive nitric oxide donor, and immunosuppressant was (4-6):(0.8-1.2):(1.5-2.5), and the volume ratio of organic phase to aqueous phase was 1:(4-6).

8. The method for preparing ultrasonically responsive drug-loaded nanoparticles according to claim 6, characterized in that: During emulsification, use a probe for ultrasound, with a power of 280-310 W and a duration of 3-10 minutes.

9. The use of the ultrasound-responsive drug-loaded nanoparticles of claim 1 in the preparation of drugs for treating acute rejection after organ transplantation.

10. The application according to claim 9, characterized in that: The organ transplant mentioned is a heart transplant.

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