Bionic anti-inflammatory nano ultrasonic contrast agent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of ultrasonic molecular imaging, and relates to a bionic anti-inflammatory nano ultrasonic contrast agent as well as a preparation method and application thereof. The bionic anti-inflammatory nano ultrasonic contrast agent takes an erythrocyte membrane loaded with rapamycin as a shell membrane material, and tiny mesoporous silica loaded with TAT peptide and siRNA and perfluoropentane or perfluorohexane are wrapped in the bionic anti-inflammatory nano ultrasonic contrast agent. The prepared bionic anti-inflammatory nano ultrasonic contrast agent is high in biological safety, can efficiently transfer siRNA and rapamycin in vivo, and realizes non-invasive targeted anti-inflammatory treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of ultrasonic molecular imaging, and more specifically, to a nano-scale bionic ultrasonic contrast agent that can be used for anti-inflammatory treatment, and a preparation method and application thereof. Background Art

[0002] With the rapid development of ultrasonic molecular imaging technology and bio-nanotechnology, nano-scale ultrasound contrast agents have shown great potential in the treatment of tumors, thrombotic plaques and other medical fields. The advantage of in vivo nano-targeted therapy is that it concentrates drugs in the targeted area in the body, which significantly enhances the local drug concentration and therapeutic effect on the one hand, and greatly reduces the accumulation of drugs in non-lesional areas and the side effects caused by them on the other hand. Nano-ultrasound contrast agents can not only achieve the role of targeted transport of drugs and other small molecule preparations in the body, but also can observe this process in real time and accurately through in vitro ultrasound imaging, so as to adjust the treatment plan at any time.

[0003] Gene therapy based on siRNA has the advantages of simple design and can be used to treat different pathogenic targets. However, unmodified siRNA is easily degraded in the body, has a short half-life, and cannot maintain local effective drug concentrations. In addition, unmodified siRNA also has the risk of off-target and toxic side effects in the body. Therefore, how to establish a safe and efficient in vivo delivery system to enable siRNA to continue to play a role in the lesion site is still one of the focuses of current clinical research. Currently available targeted drugs such as Patisiran and Givosiran are mostly delivered by LNP or GalNAc. Their targeted delivery systems contain lipids or glycoproteins to varying degrees, which have the risk of causing severe allergic reactions, bone marrow suppression, and cardiotoxicity in clinical applications, greatly reducing their biosafety.

[0004] Rapamycin has multiple anti-inflammatory effects. However, the clinical risks of systemic use of rapamycin mainly include metabolic defects, peripheral edema, pancytopenia and proteinuria. Chinese patent document CN119055615A produced multifunctional biomimetic nanoparticles that can load rapamycin. The nanoparticles use the organic compound dopamine hydrochloride as a carrier for targeted delivery and the chemical reagent dimethyl sulfoxide as a solvent, which cannot guarantee the biosafety of its clinical application. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a bionic anti-inflammatory nano-ultrasound contrast agent and a preparation method and application thereof. The prepared nano-ultrasound contrast agent has high biological safety, a high siRNA loading rate and a rapamycin loading amount, and can be used for in vivo targeted anti-inflammatory treatment.

[0006] In a first aspect, the present invention provides a bionic anti-inflammatory nano-ultrasound contrast agent, which uses an erythrocyte membrane loaded with rapamycin as a shell membrane material, and the interior of the shell membrane is wrapped with micro-mesoporous silica loaded with TAT peptide and siRNA and perfluoropentane or perfluorohexane.

[0007] Preferably, the average particle size of the nano-scale ultrasound contrast agent is 250-255 nm.

[0008] Preferably, the particle size of the nano-scale ultrasound contrast agent is in the range of 30-900 nm.

[0009] Preferably, the mass ratio of micro-mesoporous silica to TAT transmembrane protein is (15-20): (1-3).

[0010] Preferably, the diameter of the micro-mesoporous silica is 50-80 nm.

[0011] In a second aspect, the present invention provides a method for preparing a biomimetic nanoscale ultrasound contrast agent, comprising the following steps:

[0012] (1) mesoporous silica nanoparticles, (3-aminopropyl) triethoxysilane and glacial acetic acid are mixed, stirred at 65-78° C. for 4-5 hours, the mixture is centrifuged at 4000-5000 rpm for 5-10 minutes, the supernatant is discarded, and the mixture is redispersed in phosphate buffer, CIITA-siRNA is added, and stirred at 4° C. for 2 hours to prepare micro-mesoporous silica containing siRNA;

[0013] (2) weighing TAT peptide and dissolving it in MES buffer with a pH value of 5.5-6.5, then adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in sequence and stirring at room temperature for 1 hour to prepare a TAT peptide solution, adding the TAT peptide solution to the siRNA-micro-mesoporous silica prepared in step (1), stirring at 37° C. for 5-6 hours, and preparing a mixed suspension;

[0014] (3) repeatedly freezing and thawing mouse red blood cells between -80°C and 37°C to obtain red blood cell fragments, centrifuging the obtained red blood cell fragments at 4-6°C at a speed of 10000-14000 rpm for 20-30 min, removing the upper layer of liquid, filtering the red blood cell membrane fragments at the lower layer through a filter membrane, and resuspending them in a phosphate buffer, mixing them with perfluoropentane or perfluorohexane and the mixed suspension obtained in step (2), and stirring them at high speed using an ultrasonic disruptor for 5-10 min to obtain nanoparticles;

[0015] (4) Dissolving rapamycin in N,N-dimethylformamide in advance and conjugating it with the carboxyl group of phospholipid polyethylene glycol, then mixing and stirring it with the nanoparticles prepared in step (3), and obtaining a biomimetic anti-inflammatory nano-ultrasound contrast agent by ultrafiltration.

[0016] Preferably, in step (1), the mass ratio of (3-aminopropyl)triethoxysilane and glacial acetic acid is 20-25:0.1; the concentration of siRNA in the added CIITA-siRNA is 20uM, and the diameter of the mesoporous silica is 50-80nm.

[0017] Preferably, in step (2), the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to TAT peptide is 2-3:1; the mass ratio of N-hydroxysuccinimide to TAT peptide is 3.5-4:1.

[0018] Preferably, in step (3), the pore size of the filter membrane is 0.22 μm, the conditions for ultrasonic high-speed stirring are an ice bath, a power of 650-700 W, a frequency of 20-25 kHz, and a time of 5-10 min.

[0019] Preferably, in step (4), the mass ratio of rapamycin to phospholipid polyethylene glycol carboxyl group is 1-2:9, and the molecular weight cut-off of the dialysis bag used for filtration is 3500D.

[0020] The present invention uses erythrocyte membrane as the shell membrane material, and uses micro-mesoporous silica loaded with TAT peptide and siRNA and perfluorohexane or perfluoropentane core to obtain a nano-scale ultrasound contrast agent with small and uniform particle size through high-speed emulsification. The erythrocyte membrane has good biocompatibility and is not easy to cause allergic reactions in the body.

[0021] When the bionic anti-inflammatory nano-ultrasound contrast agent reaches the thyroid gland and enters the glandular tissue through ultrasonic fragmentation, the mesoporous silica protects the siRNA from being degraded in the local body fluid environment, and its coated TAT peptide assists the mesoporous silica loaded with siRNA to enter the thyroid cells. The carboxyl group of phospholipid polyethylene glycol can be inserted into the phospholipid bilayer of the cell membrane, and on the basis of not changing the activity of rapamycin and the siRNA encapsulated in the contrast agent, it assists rapamycin to be loaded on the surface of the nano-ultrasound contrast agent, and brought into the thyroid tissue to exert its effect.

[0022] In a third aspect, the present invention provides an application of a bionic anti-inflammatory nano-ultrasound contrast agent, and the application of the bionic anti-inflammatory nano-ultrasound contrast agent in a drug for treating Hashimoto's thyroiditis.

[0023] In summary, the present invention has the following beneficial effects:

[0024] 1. The bionic anti-inflammatory nano-ultrasound contrast agent prepared by the present invention has a red blood cell membrane as a shell membrane, and mesoporous silica loaded with TAT protein-siRNA and perfluorohexane or perfluoropentane as the inside of the shell membrane. The particle size of the bionic anti-inflammatory nano-ultrasound contrast agent is 30 to 900 nm, and the average particle size is 255 nm. It is in the nanoscale range, has tissue targeting under ultrasound guidance, and is highly effective in the treatment of Hashimoto's thyroiditis.

[0025] 2. The bionic anti-inflammatory nano-ultrasound contrast agent prepared by the present invention takes perfluorohexane or perfluoropentane liquid as the core. Perfluorohexane or perfluoropentane can significantly improve the output efficiency of the contrast agent, has a high bubbling rate, and makes the structure of the ultrasound contrast agent more stable.

[0026] 3. The bionic anti-inflammatory nano-ultrasound contrast agent prepared by the present invention has high in vivo biocompatibility and high biosafety, and has no obvious toxicity when injected into the tail vein of mice.

[0027] 4. The bionic anti-inflammatory nano-ultrasound contrast agent prepared by the present invention has a strong in vivo enhanced imaging capability, and has a high drug encapsulation rate and drug loading capacity.

[0028] 5. The present invention utilizes the prepared bionic anti-inflammatory nano-ultrasound contrast agent to treat Hashimoto's thyroiditis by ultrasonic irradiation. On the one hand, the ultrasound-assisted directional rupture of the contrast agent can promote specific siRNA and the anti-inflammatory drug rapamycin to enter the thyroid cells, exerting a significant anti-inflammatory effect. On the other hand, it can enhance the local development of the nano-scale ultrasound contrast agent to achieve integrated diagnosis and treatment.

[0029] 6. The present invention applies targeted siRNA to thyroid tissue to block HLA-DR expression on the surface of thyroid cells and the lymphocyte activation induced by it, and combines it with rapamycin for targeted anti-inflammatory treatment to fundamentally treat Hashimoto's thyroiditis, so as to avoid adverse reactions such as arrhythmias and progressive bone loss that may be caused by lifelong thyroid hormone replacement therapy, and reduce the potential risk of thyroid cancer caused by inflammation.

[0030] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of protection of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a synthetic process flow chart of the bionic anti-inflammatory nano ultrasonic contrast agent prepared by the present invention;

[0032] Figure 2 This is a graph showing the differences in HLA-DR and CIITA expression levels between patients with Hashimoto's thyroiditis and the control group;

[0033] Figure 3 This is an effect analysis diagram of the bionic anti-inflammatory nano-ultrasound contrast agent prepared by the present invention;

[0034] Figure 4 It is an analysis diagram of the in vivo and in vitro ultrasound imaging ability of the bionic anti-inflammatory nano-ultrasound contrast agent prepared by the present invention and a diagram showing the effect of the contrast agent in inhibiting cellular oxidative stress;

[0035] Figure 5 This is a diagram showing the effect of the bionic anti-inflammatory nano-ultrasound contrast agent prepared by the present invention on inhibiting cell apoptosis and promoting autophagy;

[0036] Figure 6 This is a diagram showing the effect of the bionic anti-inflammatory nano-ultrasound contrast agent prepared by the present invention on inhibiting the expression of CIITA and HLA-DR in thyroid cells;

[0037] Figure 7 This is a diagram showing the targeting and sustained release effects of the bionic anti-inflammatory nano-ultrasound contrast agent prepared by the present invention in vivo;

[0038] Figure 8 This is a diagram showing the therapeutic effect of the bionic anti-inflammatory nano-ultrasound contrast agent prepared by the present invention on Hashimoto's thyroiditis in vivo;

[0039] Fig. 9 This is a diagram showing the biosafety test results of the biomimetic anti-inflammatory nano-ultrasound contrast agent prepared in the present invention. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with the examples. It is particularly noted that if no specific conditions are specified in the following examples, the reactions are carried out according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from common commercial sources.

[0041] In this embodiment, mesoporous silica nanoparticles (MSNPs) with a diameter of 50 nm were purchased from Ruixi Biotechnology Co., Ltd.

[0042] CIITA-siRNA was synthesized by GenePharma (Shanghai, China).

[0043] Rapamycin was purchased from Sigma, Germany.

[0044] TAT protein was purchased from Nanjing Peptide Biotechnology Co., Ltd.

[0045] Interleukin-23 was purchased from Abcam.

[0046] Recombinant human interferon-γ was purchased from PrimeGene.

[0047] 1. Human thyroid tissue RNA extraction and transcriptome sequencing:

[0048] (1) To confirm whether the expression of CIITA and human leukocyte antigen-DR (HLA-DR) in thyroid tissue is different between patients with Hashimoto's thyroiditis and the general population, we recruited 10 patients who were hospitalized for thyroid cancer surgery at Qilu Hospital of Shandong University. These patients required total thyroidectomy due to suspected contralateral nodules or other personal reasons, and some of them also had Hashimoto's thyroiditis. All patients signed informed consent.

[0049] (2) Based on the postoperative pathological results, 4 patients with contralateral thyroid cancer TC or lymph node metastasis were excluded. The remaining six thyroid tissue samples were divided into HT group (3 cases) and control group (3 cases) for specific polymerase chain reaction (PCR) and high-throughput transcriptome sequencing. All experimental procedures were carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) and approved by the Research Ethics Committee of Qilu Hospital.

[0050] The quantitative RT-PCR and transcriptome sequencing techniques were used to analyze the genes of thyroid tissues of patients with Hashimoto's thyroiditis and controls. Figure 2 As shown, (A) is the result of PCR analysis, and the expression of CIITA and HLA-DR in patients with Hashimoto's thyroiditis was significantly increased (P < 0.01). (B, C) are the results of transcriptome sequencing analysis. Compared with the control group, the up-regulation frequency of CIITA, HLA-DRA, HLA-DRB1-5, HLA-DQA1 and HLA-DPB1 in patients with Hashimoto's thyroiditis was higher. (D) is the gene ontology (GO) and molecular function (MF) analysis performed simultaneously with transcriptome sequencing, showing that these up-regulated genes are mainly involved in peptide antigen binding in vivo, MHC class II receptor activity, immune receptor activity and transmembrane signaling receptor activity. (E) is the KEGG enrichment analysis, indicating that the above genes play an important role in antigen processing and presentation signaling pathways and pathways related to influenza A, asthma and type 1 diabetes.

[0051] like Figure 2 As shown, the results suggest that CIITA and HLA-DR are potential targets for the treatment of Hashimoto's thyroiditis.

[0052] Example

[0053] Example 1

[0054] A method for preparing a bionic anti-inflammatory nano-ultrasound contrast agent, comprising the following steps:

[0055] (1) Mix mesoporous silica nanoparticles with excess (3-aminopropyl) triethoxysilane and an appropriate amount of glacial acetic acid and stir at 78°C for 5 hours. Centrifuge the suspension at 5000 rpm for 5 minutes and redisperse it in phosphate buffered saline (PBS). Then add an appropriate amount of CIITA-siRNA and incubate overnight at 4°C to prepare micro-mesoporous silica of siRNA. Among them, the mass ratio of (3-aminopropyl) triethoxysilane to glacial acetic acid is 20:0.1; the concentration of siRNA in CIITA-siRNA is 20uM, and the diameter of mesoporous silica is 50nm.

[0056] (2) Dissolve the accurately weighed TAT peptide in MES buffer at pH 6.5, and then add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in sequence. Add the prepared TAT peptide solution to the micro-mesoporous silica of siRNA in step (1), stir at 37°C for 6 hours to obtain a mixed suspension. The mass ratio of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to TAT peptide is 2:1; the mass ratio of N-hydroxysuccinimide to TAT peptide is 3.5:1.

[0057] (3) Mouse red blood cells were repeatedly frozen and thawed between -80°C and 37°C to obtain broken red blood cell membranes. Then, the mixture was centrifuged at 14,000 rpm for 20 minutes at 4°C. The upper liquid was discarded, and the red blood cell membrane fragments in the lower layer were filtered through a 0.22 μm filter membrane and resuspended in phosphate buffered saline (PBS). The mixture was mixed with perfluorohexane or perfluoropentane and the mixed suspension obtained in step (2). The suspension was stirred at high speed in an ice bath using an ultrasonicator (650 W, 20-25 kHz) for 5 minutes to obtain nanoparticles.

[0058] (4) Rapamycin is pre-dissolved in N,N-dimethylformamide and conjugated with phospholipid polyethylene glycol carboxyl. Then, it is mixed with the nanoparticles prepared in step (3) and ultrafiltration is performed to obtain a biomimetic anti-inflammatory nano-ultrasound contrast agent. The mass ratio of rapamycin to phospholipid polyethylene glycol carboxyl is 1:9, and the molecular weight cut-off of the dialysis bag used for filtration is 3500D.

[0059] Example 2

[0060] A method for preparing a bionic anti-inflammatory nano-ultrasound contrast agent, comprising the following steps:

[0061] (1) Mix mesoporous silica nanoparticles with excess (3-aminopropyl) triethoxysilane and an appropriate amount of glacial acetic acid and stir at 78°C for 5 hours. Centrifuge the suspension at 5000 rpm for 5 minutes and redisperse it in phosphate buffered saline (PBS). Then add an appropriate amount of CIITA-siRNA and incubate overnight at 4°C to prepare micro-mesoporous silica of siRNA. Among them, the ratio of (3-aminopropyl) triethoxysilane to glacial acetic acid is 22:0.1; the concentration of siRNA in CIITA-siRNA is 20uM, and the diameter of mesoporous silica is 60nm.

[0062] (2) Dissolve the accurately weighed TAT peptide in MES buffer at pH 6.5, and then add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in sequence. Add the prepared TAT peptide solution to the micro-mesoporous silica of siRNA in step (1), stir at 37°C for 6 hours to obtain a mixed suspension. The mass ratio of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to TAT peptide is 3:1; the mass ratio of N-hydroxysuccinimide to TAT peptide is 3.5:1.

[0063] (3) Mouse red blood cells were repeatedly frozen and thawed between -80°C and 37°C to obtain broken red blood cell membranes. Then, the mixture was centrifuged at 14,000 rpm for 20 minutes at 4°C. The upper liquid was discarded, and the red blood cell membrane fragments in the lower layer were filtered through a 0.22 μm filter membrane and resuspended in phosphate buffered saline (PBS). The mixture was mixed with perfluorohexane or perfluoropentane and the mixed suspension obtained in step (2). The suspension was stirred at high speed in an ice bath using an ultrasonicator (650 W, 20-25 kHz) for 5 minutes to obtain nanoparticles.

[0064] (4) Rapamycin is pre-dissolved in N,N-dimethylformamide and conjugated with phospholipid polyethylene glycol carboxyl. Then, it is mixed with the nanoparticles prepared in step (3) and ultrafiltration is performed to obtain a biomimetic anti-inflammatory nano-ultrasound contrast agent. The mass ratio of rapamycin to phospholipid polyethylene glycol carboxyl is 1:9, and the molecular weight cut-off of the dialysis bag used for filtration is 3500D.

[0065] Example 3

[0066] A method for preparing a bionic anti-inflammatory nano-ultrasound contrast agent, comprising the following steps:

[0067] (1) Mix mesoporous silica nanoparticles with excess (3-aminopropyl) triethoxysilane and an appropriate amount of glacial acetic acid and stir at 78°C for 5 hours. Centrifuge the suspension at 5000 rpm for 5 minutes and redisperse it in phosphate buffered saline (PBS). Then add an appropriate amount of CIITA-siRNA and incubate overnight at 4°C to prepare micro-mesoporous silica of siRNA. Among them, the ratio of (3-aminopropyl) triethoxysilane to glacial acetic acid is 25:0.1; the concentration of siRNA in CIITA-siRNA is 20uM, and the diameter of mesoporous silica is 80nm.

[0068] (2) Dissolve the accurately weighed TAT peptide in MES buffer at pH 6.5, and then add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in sequence. Add the prepared TAT peptide solution to the micro-mesoporous silica of siRNA in step (1), stir at 37°C for 6 hours to obtain a mixed suspension. The mass ratio of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to TAT peptide is 3:1; the mass ratio of N-hydroxysuccinimide to TAT peptide is 4:1.

[0069] (3) Mouse red blood cells were repeatedly frozen and thawed between -80°C and 37°C to obtain broken red blood cell membranes. Then centrifuged at 14,000 rpm for 20 minutes at 4°C. The upper liquid was discarded, and the red blood cell membrane fragments in the lower layer were filtered through a 0.22 μm filter membrane and resuspended in phosphate buffered saline (PBS). It was mixed with perfluorohexane or perfluoropentane and the mixed suspension prepared in step (2). The suspension was stirred at high speed in an ice bath using an ultrasonicator (650 W, 20-25 kHz) for 5 minutes to obtain nanoparticles.

[0070] (4) Rapamycin is pre-dissolved in N,N-dimethylformamide and conjugated with phospholipid polyethylene glycol carboxyl. Then, it is mixed with the nanoparticles prepared in step (3) and ultrafiltration is performed to obtain a biomimetic anti-inflammatory nano-ultrasound contrast agent. The mass ratio of rapamycin to phospholipid polyethylene glycol carboxyl is 2:9, and the molecular weight cut-off of the dialysis bag used for filtration is 3500D.

[0071] like Figure 1 As shown, the synthesis process flow chart of the bionic anti-inflammatory nano ultrasonic contrast agent prepared by the present invention, wherein (A) is a schematic diagram of the synthesis of the TAT-siRNA-MSNPs core structure, and (B) is a schematic diagram of the synthesis of the bionic anti-inflammatory nano ultrasonic contrast agent shell structure.

[0072] The biomimetic anti-inflammatory nano-ultrasound contrast agent prepared in Example 1-3 was diluted and dropped onto a glass slide. The surface morphology of the contrast agent was observed under an optical microscope. The results were as follows: Figure 3As shown, (A) is a light microscopic image of the prepared biomimetic anti-inflammatory nano-ultrasound contrast agent, (B) is a transmission electron microscopy (TEM) image of the prepared biomimetic anti-inflammatory nano-ultrasound contrast agent, (C) is the size distribution of the prepared biomimetic anti-inflammatory nano-ultrasound contrast agent, (D) is a fluorescence microscopic image of the prepared biomimetic anti-inflammatory nano-ultrasound contrast agent encapsulating FITC-TAT-siRNA-MSNPs, (E) is a fluorescence microscopic image of the prepared biomimetic anti-inflammatory nano-ultrasound contrast agent encapsulating TAT-CY3-siRNA-MSNPs, and (F) is the loading efficiency of siRNA in different dose groups (added siRNA The dosages were 50 μL, 70 μL and 90 μL respectively), (G) is the fluorescence microscopy image of the bionic anti-inflammatory nano-ultrasound contrast agent prepared in Nthy-ori3-1 cells, (HK) is the chromatographic overlap of the bionic anti-inflammatory nano-ultrasound contrast agent (rapamycin) prepared by four different methods (chromatographic column: 5 μm, Agilent Z type: 5 μm, Agilent ZORBAXS-C18250×4.6 mm; temperature 40°C; UV detection wavelength 277 nm), (L) Rapamycin encapsulation efficiency of four different preparation methods, data are expressed as mean ± SD, ***P < 0.001.

[0073] like Figure 3 As can be seen from pictures (A) and (B), under an optical microscope, the contrast agents are spherical, with uniform particle size and are evenly dispersed without aggregation.

[0074] Take the nano-scale ultrasound contrast agent prepared in Example, dilute it, and use a nano-laser particle size and Zeta potential analyzer to detect the particle size and potential of the ultrasound contrast agent. The results are as follows: Figure 2 As shown, the average particle size of the contrast agent is 255 nm, and PI: 0.23.

[0075] Performance Testing:

[0076] 1. Detection of the siRNA and rapamycin loading capacity of the bionic anti-inflammatory nano-ultrasound contrast agent prepared in the example:

[0077] (1) To test the loading efficiency of mesoporous silica (MSNPs) in the biomimetic anti-inflammatory nano-ultrasound contrast agent for siRNA, MSNPs were mixed and incubated with fluorescent CIITA-siRNA (20 μM) at volume ratios of 50 μl:1 ml, 70 μl:1 ml and 90 μl:1 ml, respectively, according to the method of Example 2. After centrifugation, the fluorescence intensity of the supernatant was measured using a microplate spectrophotometer to calculate the loading amount and loading efficiency of siRNA.

[0078] (2) To test the loading efficiency of the bionic anti-inflammatory nano-ultrasonic contrast agent for rapamycin, the bionic anti-inflammatory nano-ultrasonic contrast agent prepared in Example 2 was taken, and an appropriate proportion of acetonitrile was added to the bionic anti-inflammatory nano-ultrasonic contrast agent suspension. The suspension was injected into an Agilent ZORBAXSB-C18 chromatographic column (5 μm, 4.6×250 mm) using high performance liquid chromatography (HPLC; Agilent 1260 Infinity II; Agilent Technologies, Inc.), and detected by refractive index (RI) and ultraviolet (UV) absorbance at a wavelength of 280 nm.

[0079] Figure (3E) shows that the biomimetic anti-inflammatory nano-ultrasound contrast agent encapsulating siRNA-MSNPs appears as red fluorescent particles under an optical microscope. Figure (3F) shows that after different doses of CIITA-siRNA (50, 70 and 90 μl) were mixed and stirred with MSNPs for 6 hours, more than 90% of the siRNA was successfully loaded onto the MSNPs, as determined by microplate spectrophotometry. The DOX-BCNDs involved in the following examples were all prepared by adding 90 μl CIITA-siRNA.

[0080] Rapamycin has anti-inflammatory and immunosuppressive properties. In order to load rapamycin onto the surface of biomimetic anti-inflammatory nano-ultrasound contrast agents assisted by DSPE-PEG2000-COOH, the integrity of the phospholipid bilayer structure of the red blood cell membrane fragment must be maintained as much as possible during the preparation of the biomimetic anti-inflammatory nano-ultrasound contrast agents.

[0081] Figures (3K) and (3L) show that the biomimetic anti-inflammatory nano-ultrasound contrast agent preparation method used in the present invention, that is, the preparation method in Example 2 (the most preferred of the four preparation schemes), can achieve an encapsulation efficiency of rapamycin close to 75%. The following examples are all prepared in this way.

[0082] 2. In vivo and in vitro ultrasound imaging ability testing of the bionic anti-inflammatory nano-ultrasound contrast agent prepared in the example

[0083] (1) For in vitro contrast-enhanced ultrasound imaging, the biomimetic anti-inflammatory nano-ultrasound contrast agent was diluted with PBS and transferred into a 15 ml centrifuge tube, heated to 37°C, and contrast-enhanced ultrasound imaging was performed using an ultrasound diagnostic apparatus.

[0084] (2) For in vivo contrast-enhanced ultrasound imaging, rats anesthetized with sodium pentobarbital were placed on a heating table, and the biomimetic anti-inflammatory nano-ultrasound contrast agent suspension was injected into their tail veins, followed by rinsing with 0.3 ml of PBS, and then contrast imaging was performed using an ultrasound diagnostic apparatus (center frequency, 14 MHz; mechanical index [MI], 0.22; depth, 1.5 cm).

[0085] The ultrasonic wall breaking instrument workstation is used to store image data. Figure 4 As shown, (A) is the ultrasound imaging of the prepared bionic anti-inflammatory nano-ultrasound contrast agent in vitro, (B) is the contrast-enhanced ultrasound imaging of the prepared bionic anti-inflammatory nano-ultrasound contrast agent in vitro, (C) is the ultrasound scan of the rat thyroid gland, (D) is the ultrasound imaging of the thyroid gland, (EI) is the contrast-enhanced ultrasound imaging of the thyroid tissue at different time points after injection of the prepared bionic anti-inflammatory nano-ultrasound contrast agent, (J) is Nthy-ori3-1 cells with or without IL-23 treatment (10 μl), and with or without pretreatment with the bionic anti-inflammatory nano-ultrasound contrast agent; DCFH-DA is used to measure the cellular ROS level, (K) is the detection and analysis of DCF fluorescence using a microplate spectrophotometer, (L) is the detection and analysis of the fluorescence of the MDA content in different groups of cells, *P<0.05, **P<0.01, ***P<0.001.

[0086] In both grayscale imaging and enhanced imaging modes, the bionic anti-inflammatory nano-ultrasound contrast agent can present clear images in vitro. Figure 4 As shown in D, the thyroid gland of a rat injected with a biomimetic anti-inflammatory nano-ultrasound contrast agent was imaged using a high-frequency transducer. The thyroid gland of the rat was clearly visible on the ultrasound grayscale image. Figure 4 EI showed that after the injection of the bionic anti-inflammatory nano-ultrasound contrast agent, the contrast-enhanced imaging of the rat thyroid gland gradually became clear, and then gradually weakened over time. This shows that the contrast agent has a strong ultrasound imaging ability and can obtain ultrasound-enhanced imaging both in vivo and in vitro.

[0087] 3. Detection of the ability of the bionic anti-inflammatory nano-ultrasound contrast agent prepared in the example to inhibit cellular oxidative stress and apoptosis

[0088] (1) Nthy-ori3-1 cells were divided into four groups: (i) control group; (ii) IL-23 group; (iii) biomimetic anti-inflammatory nano-ultrasound contrast agent group; (iv) IL-23+biomimetic anti-inflammatory nano-ultrasound contrast agent group. After the biomimetic anti-inflammatory nano-ultrasound contrast agent suspension was added to the biomimetic anti-inflammatory nano-ultrasound contrast agent group and the IL-23+biomimetic anti-inflammatory nano-ultrasound contrast agent group, the cell culture medium was pretreated with ultrasound (40 seconds; VCX4000).

[0089] (2) Oxidative stress levels, intracellular reactive oxygen species (ROS), and malondialdehyde (MDA) were evaluated using detection kits.

[0090] (3) The levels of Bcl-2, BAX, P62, LC3-II, and LC3-I proteins in cells were determined by Western blotting.

[0091] (4) The apoptotic Nthy-ori3-1 cells were detected using Annexin V-FITC / propidiumiodide (PI) cell apoptosis kit (BD Pharmingen, San Diego, CA, USA).

[0092] Immunofluorescence analysis results Figure 4 J shows that the ROS level in the IL-23 group was significantly increased compared with the control group, indicating that IL-23 induced ROS accumulation. When the biomimetic anti-inflammatory nano-ultrasound contrast agent suspension was used as a pretreatment, the ROS level decreased. The fluorescence intensity of intracellular ROS and MDA was measured using a microplate spectrophotometer. The fluorescence analysis results as shown in Figure (4K, L) show that pretreatment with the biomimetic anti-inflammatory nano-ultrasound contrast agent significantly reduced the accumulation of IL-23-induced ROS and MDA in Nthy-ori3-1 cells.

[0093] Western blot analysis Figure 5 Shown, (AG) are the expression levels and quantitative analysis of Bax, Bcl2, P62, LC3B-I and LC3B-II in Nthy-ori3-1 cells of different groups, (HL) are the analysis of cell apoptosis and quantification after different treatments using flow cytometry (FCM), *p<0.05, **p<0.01.

[0094] Compared with the control group, IL-23 stimulation significantly increased the expression of BAX, LC3-II and P62, while reducing the level of Bcl-2. The biomimetic anti-inflammatory nano-ultrasound contrast agent effectively inhibited the expression of BAX, LC3-II and P62 induced by IL-23, and restored the expression of Bcl-2. The inhibition of BAX expression and the increase of Bcl-2 expression proved that the biomimetic anti-inflammatory nano-ultrasound contrast agent can inhibit IL-23-induced apoptosis of Nthy-ori3-1 cells. The results of flow cytometry are shown in Figure 2. Figure 5 HL showed that apoptotic cells in Nthy-ori3-1 cells in the IL-23 group increased, while pretreatment with biomimetic anti-inflammatory nano-ultrasound contrast agents could partially reverse this phenomenon.

[0095] 4. Detection of the bionic anti-inflammatory nano-ultrasound contrast agent prepared in Example 1 inhibiting the expression of CIITA and HLA-DR in cells

[0096] (1) Nthy-ori3-1 cells were divided into four groups: (i) control group; (ii) IFN-γ group; (iii) bionic anti-inflammatory nano-ultrasound contrast agent group; (iv) IFN-γ + bionic anti-inflammatory nano-ultrasound contrast agent group. IFN-γ was used to induce Nthy-ori3-1 cells to express HLA-DR. Nthy-ori3-1 cells in the bionic anti-inflammatory nano-ultrasound contrast agent group and the IFN-γ + bionic anti-inflammatory nano-ultrasound contrast agent group were treated with a bionic anti-inflammatory nano-ultrasound contrast agent suspension and subjected to ultrasound irradiation according to the aforementioned method. After 6 hours, IFN-γ was added to the cell culture of the IFN-γ group and the IFN-γ + bionic anti-inflammatory nano-ultrasound contrast agent group and incubated for 24 hours.

[0097] (2) The messenger RNA (mRNA) levels of CIITA and HLA-DR were measured by reverse transcription-PCR (RT-PCR). The HLA-DR protein expression was quantified by flow cytometry (FCM).

[0098] The results of quantitative RT-PCR analysis were as follows Figure 6 , wherein, (AC) are the changes in the expression levels of CIITA and HLA-DR after Nthy-ori3-1 cells were pretreated with IL-23 and / or the prepared bionic anti-inflammatory nano-ultrasound contrast agent, analyzed by PCR and flow cytometry, (DG) are Group 1: Jurkat cells; Group 2: Group 3: Jurkat cells co-cultured with Nthy-ori3-1 cells stimulated with IFN-γ; Group 4: Jurkat cells co-cultured with Nthy-ori3-1 cells pretreated with IFN-γ and the prepared bionic anti-inflammatory nano-ultrasound contrast agent; flow cytometry was used to analyze the expression of CD25+ and CD69+ on Jurkat cells; (H) is the ELISA detection of IL-17 levels in the cell culture fluid of each group; *p<0.05, **p<0.01, ***p<0.001.

[0099] Compared with the control group, the expression of CIITA, a key transcriptional regulator of HLA class II genes, was significantly increased in the IFN-γ group, and the expression of HLA-DR also showed a similar trend. Pretreatment with biomimetic anti-inflammatory nano-ultrasound contrast agent can prevent the increase in CIITA and HLA-DR expression (p<0.001,). Figure 6 BC, the same trend was observed. These findings suggest that biomimetic anti-inflammatory nano-ultrasound contrast agents can inhibit the expression of CIITA and HLA-DR in Nthy-ori3-1 cells.

[0100] 5. Detection of the bionic anti-inflammatory nano-ultrasound contrast agent obtained in the example to inhibit T cell activation

[0101] (1) Group 1 was a Jurkat cell group without biomimetic anti-inflammatory nano-ultrasound contrast agent or IFN-γ, which served as the control group. Group 2 co-cultured Jurkat cells (target cells) and Nthy-ori3-1 cells in 12-well plates in RPMI1640 containing 5% serum for 48 hours. Nthy-ori3-1 cells in groups 3 and 4 were pretreated with IFN-γ or biomimetic anti-inflammatory nano-ultrasound contrast agent + IFN-γ for 24 hours before co-culture.

[0102] Table 1

[0103]

[0104]

[0105] (2) After an appropriate co-culture period, cells were stained with fluorescent monoclonal antibodies against CD25 or CD69 and then subjected to FCM on a CytoFlexS flow cytometer. Data were acquired and analyzed using FlowJo-V10 software (BD Biosciences, Franklin Lakes, NJ, USA). The concentration of IL-17 in the cell culture medium was detected using an IL-17 enzyme-linked immunosorbent assay kit (Abcam) with a sensitivity of 0.5 pg / mL and a detection range of 1.6-100 pg / mL.

[0106] The results of FCM analysis are as follows Figure 6 DG, co-culture with IFN-γ stimulated Nthy-ori3-1 cells significantly increased the expression of CD25 and CD69 in JurkatT cells. Pretreatment with biomimetic anti-inflammatory nano-ultrasound contrast agents effectively reversed this increase, indicating that biomimetic anti-inflammatory nano-ultrasound contrast agents can inhibit the activation of Jurkat cells induced by IFN-γ stimulated Nthy-ori3-1 cells (p<0.01).

[0107] Jurkat cells normally secrete very little IL-17. Co-culture with IFN-γ-stimulated Nthy-ori3-1 cells resulted in a significant increase in IL-17 secretion. Pretreatment with biomimetic anti-inflammatory nano-ultrasound contrast agents significantly reduced Nthy-ori3-1 cell antigen-induced Jurkat T cell activation and IL-17 secretion ( Figure 6 H). These results suggest that the biomimetic anti-inflammatory nano-ultrasound contrast agent can reduce the antigen presentation response of thyroid cells to IFN-γ, thereby reducing the activation of Jurkat T cells.

[0108] 6. Testing the targeting and sustained release ability of the bionic anti-inflammatory nano-ultrasound contrast agent prepared in the example

[0109] Twenty-four adult mice were randomly divided into two groups: a biomimetic anti-inflammatory nano-ultrasound contrast agent group and a siRNA-MSNPs group, with 12 mice in each group. Each group of mice was injected with a biomimetic anti-inflammatory nano-ultrasound contrast agent (containing CY-3-siRNA) or an equal amount of fluorescent siRNA-MSNPs (containing CY-3-siRNA) via the tail vein. The mice in the biomimetic anti-inflammatory nano-ultrasound contrast agent group received neck ultrasound treatment for 2 minutes (1.25W / cm 2 , 1MHz). Three mice were randomly selected from each group at different time points (0.5, 4, 12 and 24 hours) and their thyroid tissues were removed. Thyroid tissue frozen sections were prepared and their fluorescence intensity was analyzed using a fluorescence microscope.

[0110] Cryosections of thyroid tissue were prepared and observed under a fluorescence microscope. Figure 7 , including: (A) fluorescence imaging of frozen sections of thyroid tissue at different time points (0.5h, 4h, 12h, 24h) after treatment with siRNA-MSNs (B), (C) comparison of the average fluorescence intensity of thyroid tissues in the two groups, and (D) the trend of fluorescence attenuation in thyroid tissues in the two groups.

[0111] like Figure 7 (A, B), Fluorescence intensity analysis as Figure 7 As shown in Figure C, compared with the injection of the same dose of siRNA-MSNPs, the injection of biomimetic anti-inflammatory nano-ultrasound contrast agent followed by ultrasound irradiation can significantly increase the mean fluorescence intensity of rat thyroid tissue. This indicates that the combination of biomimetic anti-inflammatory nano-ultrasound contrast agent and ultrasound enhances the targeted accumulation of CIITA-siRNA in thyroid tissue. After 24 hours of injection, the mean fluorescence intensity of the biomimetic anti-inflammatory nano-ultrasound contrast agent group was still 219.87±18.01IOD.

[0112] Application Example 1

[0113] Effect of the bionic anti-inflammatory nano-ultrasound contrast agent prepared in Example on the treatment of Hashimoto's thyroiditis in vivo

[0114] (1) To establish a mouse model of Hashimoto's thyroiditis, 0.15% sodium iodide (NaI) was added to the drinking water of the mice, and then 200 μg of mouse thyroglobulin (Tg) and complete Freund's adjuvant (Sigma, Germany) were subcutaneously injected. Two weeks later, the same dose of Tg and incomplete Freund's adjuvant were injected again. In order to evaluate the effectiveness of various treatment regimens on thyroid enlargement, 25 NOD.H-2h4 mice were randomly divided into 5 groups (5 mice in each group): (i) normal group; (ii) HT group; (iii) HT (biomimetic anti-inflammatory nano-ultrasound contrast agent prepared in Example 2) group; (iv) HT (biomimetic anti-inflammatory nano-ultrasound contrast agent + US) group; (v) HT (levothyroxine [L-T4]) group. The normal group was injected with normal saline at the same time point.

[0115] Eight weeks after the injection of NaI, mice in the HT (bionic anti-inflammatory nano-ultrasound contrast agent) group and the HT (bionic anti-inflammatory nano-ultrasound contrast agent + US) group were injected with the bionic anti-inflammatory nano-ultrasound contrast agent suspension through the tail vein once every other day, for a total of three injections. After each injection, the thyroid area of ​​the mice in the HT (bionic anti-inflammatory nano-ultrasound contrast agent + US) group was irradiated with ultrasound for 2 minutes (1.25W / cm 2 , 1MHz). The mice in the L-T4 group were intraperitoneally injected with levothyroxine (L-T4; 1.6μg / kg per day).

[0116] (2) Before the end of treatment, serum thyroid peroxidase antibodies (TPOAbs), thyroglobulin antibodies (TgAbs), and T4 levels were measured by ELISA. TPOAb, TgAb, and T4 levels in the supernatant were quantified using quantitative enzyme immunoassay technology using Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. A microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the color intensity of each well. All tests were performed in triplicate.

[0117] (3) 14 days after the start of treatment, mice in each group were anesthetized and euthanized. Thyroid tissues were fixed in formalin and embedded in paraffin for hematoxylin and eosin (H&E) staining. After deparaffinization and rehydration, tissue sections were stained with hematoxylin solution for 3 minutes and then differentiated in acid ethanol for 15 seconds. After rinsing with distilled water, they were stained with eosin solution for 3 minutes, dehydrated in graded alcohols, and cleared in xylene. Sections were observed under an Olympus CX41 microscope (magnification × 200; Olympus, Tokyo, Japan). The expression of CD3 and CD4 in thyroid tissues was evaluated by immunohistochemistry (IHC) analysis.

[0118] The results of anatomical pathological examination of thyroid tissue are as follows Figure 8 , where (A) is hematoxylin and eosin (HE) staining of thyroid tissue after different treatments, (B, C) are CD3 and CD4 immunohistochemical staining of thyroid tissue sections after different treatments, (D, E) are CD3+ and CD4+ cell count analysis in thyroid tissues of different groups, compared with the normal control group, ##P<0.01, ##P<0.001; compared with the HT group, **P<0.01, ***P<0.001, (FH) are the concentrations of TgAb, TPOAb and T4 in the serum of each group detected by ELISA; *P<0.05.

[0119] like Figure 8 As shown in A, under an optical microscope, the thyroid follicles of the normal group were intact and evenly distributed, with minimal lymphocyte infiltration. The HT group showed obvious inflammatory cell infiltration and severe inflammatory response, with obvious atrophy, collapse and uneven distribution of follicles. Severe inflammation leads to the disappearance or fibrosis of thyroid follicles. Similar results were observed in the thyroid tissue of mice in the L-T4 treatment group. Compared with the HT group, the inflammatory cell infiltration and inflammatory response in the bionic anti-inflammatory nano-ultrasound contrast agent group were significantly reduced. Thyroid follicle disorder, destruction and lymphocyte infiltration were reduced. No obvious inflammatory cell infiltration was observed in the bionic anti-inflammatory nano-ultrasound contrast agent + US treatment group.

[0120] Paraffin-embedded thyroid tissue sections were stained with CD3 and CD4 antibodies. Figure 8 As shown in B, CD3+T cells were widely present in the thyroid tissue of the HT group. Figure 8 C shows that the number of CD4+T cells in the HT group was also significantly increased compared with the normal group. There was no significant decrease in the number of CD3+ or CD4+T cells in the thyroid tissue of the HT group treated with L-T4. In contrast, the number of these cells in the thyroid tissue of the biomimetic anti-inflammatory nano-ultrasound contrast agent + US treatment group was significantly reduced. The statistical results are shown in Figure 8 As shown in D and E, unlike L-T4 treatment, the biomimetic anti-inflammatory nano-ultrasound contrast agent combined with ultrasound treatment significantly reduced the infiltration of local lymphocytes in the thyroid tissue of HT mice (p<0.001). This suggests that the combination of biomimetic anti-inflammatory nano-ultrasound contrast agent and ultrasound can reduce the autoimmune response of HT by inhibiting T cell infiltration in thyroid tissue.

[0121] Blood test results such as Figure 8As shown in FG, the levels of TPOAb and TgAb in HT mice were significantly increased compared with normal mice (p<0.05). Treatment with biomimetic anti-inflammatory nano-ultrasound contrast agents reduced the level of TPOAb, and the reduction was more obvious in the biomimetic anti-inflammatory nano-ultrasound contrast agent + US group compared with the HT group (p<0.05). The serum TgAb level of mice treated with biomimetic anti-inflammatory nano-ultrasound contrast agents also decreased, and the decrease in the biomimetic anti-inflammatory nano-ultrasound contrast agent + US group was greater. In contrast, L-T4 treatment did not reduce the serum TPOAb and TgAb levels of HT mice, but instead caused a slight increase in serum T4 levels. These results indicate that biomimetic anti-inflammatory nano-ultrasound contrast agents can significantly reduce the level of thyroid-related autoantibodies in the blood and reduce thyroid autoimmune damage when treated with HT, while L-T4 treatment cannot achieve this therapeutic effect.

[0122] In summary, compared with the L-T4 treatment group and the simple bionic anti-inflammatory nano-ultrasound contrast agent treatment group, the bionic anti-inflammatory nano-ultrasound contrast agent combined with ultrasound therapy and endosomal targeted therapy can more effectively treat Hashimoto's thyroiditis.

[0123] Application Example 2

[0124] In vivo and in vitro safety tests of the biomimetic anti-inflammatory nano-ultrasound contrast agent prepared in the embodiment

[0125] (1) Different concentrations of biomimetic anti-inflammatory nano-ultrasound contrast agents were added to the culture medium of Nthyori3-1 cells and incubated for 24 hours, and then CCK-8 test was performed. Fig. 9 As shown, (A) shows the effect of different doses of the prepared bionic anti-inflammatory nano-ultrasound contrast agent on cell survival rate; (B) shows the change in mouse body weight after different treatments; and (C) shows the pathological tissue sections of the heart, liver, spleen, lung and kidney of mice after different treatments.

[0126] Fig. 9 As shown in A, even when the highest concentration of the biomimetic anti-inflammatory nano-ultrasound contrast agent was 600 μg / mL, the cell survival rate remained above 90%.

[0127] (2) The body weight of each group of mice in Example 1 was monitored from the first day of treatment until the seventh day after all treatments were completed, for a total of 14 days. The body weight of each group of mice was statistically analyzed, and the results were as follows: Fig. 9 As shown in B, there was no significant difference in the average body weight of mice in each group after treatment.

[0128] (3) The heart, liver, spleen, lung, and kidney tissues of the mice killed in Application Example 1 were paraffin-embedded and stained with hematoxylin and eosin (H&E). The results of H&E staining were as follows: Fig. 9As shown in C, there were no obvious lesions in the visceral tissues of mice in the bionic anti-inflammatory nano-ultrasound contrast agent group, the bionic anti-inflammatory nano-ultrasound contrast agent + US group, and the L-T4 group.

[0129] These experimental results show that the bionic anti-inflammatory nano-ultrasound contrast agent prepared in the examples has no toxic effect on mice and has high biological safety in vivo.

[0130] The above is only an exemplary embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A bionic anti-inflammatory nano-ultrasound contrast agent, characterized in that: The bionic anti-inflammatory nano-ultrasound contrast agent uses erythrocyte membrane loaded with rapamycin as shell membrane material, and the shell membrane is wrapped with micro-mesoporous silica loaded with TAT peptide and siRNA and perfluoropentane or perfluorohexane.

2. The bionic anti-inflammatory nano-ultrasound contrast agent according to claim 1, characterized in that: The particle size of the bionic anti-inflammatory nano-ultrasound contrast agent is 30 to 900 nm, and the average particle size is 255 nm.

3. The bionic anti-inflammatory nano-ultrasound contrast agent according to claim 1, characterized in that: The mass ratio of micro-mesoporous silica to the TAT peptide loaded thereon is (15-20):(1-3).

4. The bionic anti-inflammatory nano-ultrasound contrast agent according to claim 1, characterized in that: The mass ratio of rapamycin to phospholipid polyethylene glycol carboxyl group is 1:(8-10).

5. The method for preparing the bionic anti-inflammatory nano-ultrasound contrast agent according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) mesoporous silica nanoparticles, (3-aminopropyl) triethoxysilane and glacial acetic acid were mixed, stirred at 65-78° C. for 4-5 hours, the mixture was centrifuged at 4000-5000 rpm for 5-10 minutes, dispersed in phosphate buffer, and CIITA-siRNA was added, and cultured overnight at 4° C. to prepare micro-mesoporous silica loaded with siRNA; (2) weighing TAT peptide and dissolving it in MES buffer with a pH value of 5.5-6.5, then adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in sequence to prepare a TAT peptide solution, adding the TAT peptide solution to the siRNA-micro-mesoporous silica prepared in step (1), stirring at 37° C. for 5-6 hours, and preparing a mixed suspension; (3) repeatedly freezing and thawing mouse red blood cells between -80°C and 37°C to obtain red blood cell fragments, centrifuging the obtained red blood cell fragments at 4-6°C at a speed of 10,000-14,000 rpm for 20-30 min, removing the upper layer of liquid, filtering the red blood cell membrane fragments at the lower layer through a filter membrane, and resuspending them in a phosphate buffer, mixing them with perfluoropentane or perfluorohexane and the mixed suspension obtained in step (2), and stirring them at high speed using an ultrasonic disruptor for 5-10 min to obtain nanoparticles; (4) Dissolving rapamycin in N,N-dimethylformamide in advance and conjugating it with the carboxyl group of phospholipid polyethylene glycol, then mixing and stirring it with the nanoparticles prepared in step (3), and finally obtaining a biomimetic anti-inflammatory nano-ultrasound contrast agent by ultrafiltration.

6. The method for preparing the bionic anti-inflammatory nano-ultrasound contrast agent according to claim 5, characterized in that: In step (1), the mass ratio of (3-aminopropyl)triethoxysilane and glacial acetic acid is 20-25:0.1; the concentration of siRNA in the added CIITA-siRNA is 20uM, and the diameter of the mesoporous silica is 50-80nm.

7. The method for preparing the bionic anti-inflammatory nano-ultrasound contrast agent according to claim 5, characterized in that: In step (2), the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to TAT peptide is 2-3:1; the mass ratio of N-hydroxysuccinimide to TAT peptide is 3.5-4:

1.

8. The method for preparing the bionic anti-inflammatory nano-ultrasound contrast agent according to claim 5, characterized in that: In step (3), the pore size of the filter membrane is 0.22 μm, and ultrasonic high-speed stirring is carried out in an ice bath with a power of 650-700 W, a frequency of 20-25 kHz, and a time of 5-10 min.

9. The method for preparing the bionic anti-inflammatory nano-ultrasound contrast agent according to claim 5, characterized in that: In step (4), the mass ratio of rapamycin to phospholipid polyethylene glycol carboxyl group is 1-2:9, and the molecular weight cut-off of the dialysis bag used for filtration is 3500D.

10. The use of the bionic anti-inflammatory nano-ultrasound contrast agent according to any one of claims 1 to 4, characterized in that: Application of the bionic anti-inflammatory nano-ultrasound contrast agent in medicine for treating Hashimoto's thyroiditis.

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