Preparation and application of ovarian cancer cell membrane bionic nano sound-sensitive agent

Abstract

The invention discloses preparation and application of an ovarian cancer cell membrane bionic nano sound-sensitive agent, and belongs to the field of pharmacy. The technical problem to be solved by the invention is to provide the preparation method of the ovarian cancer cell membrane bionic nano sound-sensitive agent. The method is based on the homologous targeting property and immune escape property of an ovarian cancer cell membrane to ovarian tumor cells, the SKOV-3 source ovarian cancer cell membrane (CCM) is innovatively coated on the surfaces of mesoporous silica-titanium nanoparticles (MSTN) encapsulated by manganese dioxide (BSA-MnO2), and a water-soluble sound-sensitive agent azoic compound 2, 2 ', 2', 2 '-tetramethyldisiloxane is added into the mesoporous silica-titanium nanoparticles (MSTN) to prepare the SKOV-3 source ovarian cancer cell membrane. And encapsulating 2, 2 '-azobis [2-(2-imidazoline-2-yl) propane] dihydrochloride (AIPH) into mesopores of the MSTN to prepare the ovarian cancer cell membrane bionic nano sound-sensitive agent. The ovarian cancer cell membrane bionic nano sound-sensitive agent prepared by the invention is high in biocompatibility, strong in targeting specificity and good in stability, and can effectively escape from immune system recognition and elimination. On the basis, the ovarian cancer cell membrane bionic nano sound-sensitive agent can effectively realize targeted ultrasound and magnetic resonance (MRI) bimodal imaging of the sound-sensitive agent to ovarian cancer cells, effectively realize fixed-point and quantitative release of drugs, achieve adjustable diagnosis and treatment of ovarian cancer, and is expected to provide a new thought for clinical early diagnosis and precise treatment of ovarian cancer.

Description

Technical Field

[0001] The invention belongs to the field of pharmacy, and in particular relates to the preparation and application of an ovarian cancer cell membrane bionic nano sonosensitizer. Background Art

[0002] Ovarian cancer is one of the common malignant tumors of the female reproductive system, and its incidence rate ranks third among the most common tumors, second only to cervical cancer and uterine body cancer. At present, surgery combined with chemotherapy is the main method for treating ovarian malignant tumors, but there are still many problems such as high recurrence rate and easy drug resistance, and the overall clinical prognosis is poor. Therefore, studying new treatment measures has become the focus of current research.

[0003] As a promising noninvasive treatment for malignant tumors, sonodynamic therapy (SDT) has attracted increasing attention in recent years. SDT treats cancer by inducing the activation of sonosensitizers by low-intensity ultrasound, which can be activated by low-intensity ultrasound to produce lethal reactive oxygen species (ROS) for tumor treatment. Due to these recognized therapeutic advantages, a large number of studies on SDT have been reported. However, the poor ROS generation ability and lack of targeting of existing sonosensitizers limit the efficacy of SDT. Therefore, it is necessary to optimize the properties of sonosensitizers according to the basic mechanism of sonodynamic therapy. In recent years, advances in nanotechnology have promoted the development of nanoplatforms in this emerging field with enhanced therapeutic effects, including their potential to increase sonosensitizer delivery, enhance ultrasonic cavitation, amplify ROS generation, and mediate combined treatment.

[0004] Titanium dioxide (TiO 2 ) is a widely used inorganic sonosensitizer with high biocompatibility, stability and acoustic sensitivity. As a typical semiconductor material, TiO 2 When triggered by US with energy greater than the band gap, its special band structure causes electrons to transfer from the valence band to the conduction band, thereby forming electron vacancies in the valence band. The resulting highly active holes and electrons react with water and oxygen molecules to produce ROS that lead to cell death. The ideal sonosensitizer should not only have a strong ROS generation ability, but also have good stability, biocompatibility and tumor targeting. Therefore, TiO-based 2 New sonosensitizers must be designed to overcome their inherent shortcomings such as oxygen dependence and non-targeting.

[0005] Ultrasound (US) irradiation decomposes the water-soluble azo compound 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH), causing it to produce nitrogen and oxygen-independent free radicals. US can also penetrate deep tissues and control the generation of alkyl groups by synergistic power size and irradiation time. To date, nitrogen has been a special treatment method for cancer treatment and is often used as a US contrast agent due to its nonlinear oscillation in the US field. Based on this, AIPH can be used for synergistic SDT treatment of hypoxic tumor microenvironments. It is worth noting that the bubbles generated by US can also be used as ultrasound contrast agents to enhance ultrasound imaging to guide tumor treatment. It can be seen that the AIPH-based nanoplatform is expected to solve the problem of tumor penetration depth and break through the limitations of hypoxic tumor treatment strategies.

[0006] In recent years, cell membrane mimicry technology has gradually attracted great interest from researchers due to its good biocompatibility, stability and tumor targeting, and has become a promising targeting strategy. It is reported that cell membrane-coated nanoparticles can completely retain the cell membrane protein components, allowing them to disguise themselves as source cells in vivo without complex modifications. At present, commonly used cell membranes mainly include macrophage membranes, erythrocyte membranes, bacterial membranes and cancer cell membranes (CCM). Among them, CCM has become a common method for modifying sonosensitizers due to its homologous targeting and immune escape capabilities, in order to prepare more promising biomimetic nanosonosensitizers. Summary of the invention:

[0007] The technical problem to be solved by the present invention is to provide a bionic nanosonosensitizer with high biocompatibility, strong targeting specificity, low toxicity and side effects, good stability and the ability to effectively evade recognition and clearance by the immune system, so as to achieve sonodynamic enhancement therapy for ovarian cancer under the guidance of dual-modality imaging of ultrasound and MRI.

[0008] A method for preparing an ovarian cancer biomimetic sonosensitizer AMBC comprises the following steps:

[0009] a. Synthesis of mesoporous silica (MSN) by microemulsion method, refer to the literature [Lee J, Kim JH, You DG, et al. "Cavitation-Inducible Mesoporous Silica-Titania Nanoparticles for Cancer Sonotheranostics". Advanced Healthcare Materials, 2020, 9(19): 2000877.].

[0010] b. The preparation of mesoporous silica-titanium nanoparticles (MSTN) refers to the literature [Lee J, Kim JH, You DG, et al. "Cavitation-Inducible Mesoporous Silica-Titania Nanoparticles for Cancer Sonotheranostics". Advanced Healthcare Materials, 2020, 9(19): 2000877.].

[0011] c. Thiolation of MSTN and its loading 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH): 0.5 g of MTSNs was dispersed in anhydrous toluene (80 mL) containing 0.74 mL of 3-mercaptopropyltrimethoxysilane (MPTMS), and the mixed solution was refluxed at 60° C. overnight. After the reaction was complete, the prepared thiolated MSTN (MSTN-SH) was washed with ethanol and dried in a vacuum oven at 35° C. Next, 40 mg of AIPH and 20 mg of MSTN-SH were co-dispersed in 10 mL of deionized water and stirred at room temperature for 12 hours. After high-speed centrifugation 3 times, MSTN-SH loaded with AIPH (AIPH-MSTN) was obtained.

[0012] d. Biomineralization strategy to prepare protein-linked manganese dioxide (BSA-MnO 2 ) References [Fang J, Wang Q, Yang G, et al. “Albumin-MnO 2 gated hollow mesoporous silica nanosystem formodulating tumor hypoxia and synergetic therapy of cervical carcinoma". Colloids and Surfaces B: Biointerfaces, 2019, 179: 250-259.].

[0013] e. AIPH-MSTN surface connected with BSA-MnO 2 (AIPH-MSTN@BSA-MnO 2 ): After dispersing 50 mg of AIPH-MSTN in 50 mL of PBS solution, 4 mL of BSA-MnO was slowly added to the solution. 2 After stirring at room temperature for 24 h, the prepared nanoparticles were separated by high-speed centrifugation and washed with deionized water, thereby successfully preparing BSA-MnO2 AIPH-MSTN(AIPH-MSTN@BSA-MnO 2 ).

[0014] f. Extraction of SKOV-3 derived ovarian cancer cell membrane (CCM) refers to [Li Yuzhen. Research on bionic drug delivery system based on hemoglobin combined with sound therapy and chemotherapy. Zhengzhou University, 2019.].

[0015] g. CCM coated AIPH-MSTN@BSA-MnO 2 : 1 mg / mL AIPH-MSTN@BSA-MnO 2 After the PBS solution and 2 mg / mL CCM were uniformly mixed, the mixed solution was passed through a 200 nm polycarbonate membrane using an extruder and extruded 21 times to obtain the CCM-coated biomimetic drug delivery system AIPH-MSTN@BSA-MnO 2 @CCM (i.e. AMBC).

[0016] Furthermore, the ovarian cancer cell membrane biomimetic sonosensitizer AMBC is in the form of a uniformly dispersed sphere with a particle size of 80 nm to 300 nm.

[0017] Furthermore, the surface charge of the ovarian cancer cell membrane biomimetic sonosensitizer is substantially the same as the charge of the ovarian cancer cell membrane, which is -8Mv to -11Mv.

[0018] Furthermore, the pore size of the MSTN is 1 nm to 100 nm, and the pore volume is 0.1 m 3 / g~0.5m 3 / g. Furthermore, the drug loading of the AIPH is 20% to 80%, and the encapsulation efficiency is 45% to 85%.

[0019] The invention discloses an application of an ovarian cancer cell membrane biomimetic nanosonosensitizer to realize the sonodynamic enhancement therapy of ovarian cancer under the dual-modality guidance of ultrasound and MRI.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) AMBC has good biocompatibility, biosafety, and immune evasion. The ROS it produces not only contributes to the sonodynamic therapy of ovarian cancer, but also enables MR and ultrasound imaging, thus achieving sonodynamic therapy of ovarian cancer under the guidance of ultrasound / MRI dual-modality imaging.

[0022] (2) MnO 2 O produced by nanoparticles 2 and Mn 2+ Ions can both improve the hypoxic environment of tumors and enable magnetic resonance imaging.

[0023] (3) The new water-soluble azo compound AIPH can generate alkyl free radicals and nitrogen under the action of ultrasound, which can effectively enhance the reactive oxygen in the tumor microenvironment and avoid competing with titanium dioxide for the already scarce oxygen in tumor cells. It can also perform ultrasound imaging and realize real-time dynamic tracking of tumors.

[0024] (4) The ovarian cancer cell membrane itself has the ability to target ovarian cancer cells and escape immune evasion, which solves the problem of targeting of inorganic sonosensitizers. This enables AMBC modified with ovarian cancer cell membranes to be accurately delivered to ovarian cancer, increasing the enrichment of sonosensitizers in tumor cells and providing favorable conditions for effective treatment.

[0025] (5) The preparation method of AMBC is stable, reliable and reproducible. The prepared ovarian cancer biomimetic sonosensitizer not only has strong homologous targeting and immune escape capabilities, but also can effectively achieve targeted ultrasound and MRI dual-modality imaging of sonosensitizers to ovarian cancer cells, achieving effective sonodynamic therapy for ovarian cancer, which is expected to provide new ideas for the clinical early diagnosis and precision treatment of ovarian cancer. Description of the drawings:

[0026] Figure 1 A is the transmission electron microscope and elemental analysis diagram of AIPH-MSTN of the present invention;

[0027] Figure 1 B is BSA-MnO of the present invention 2 Transmission electron microscopy images of

[0028] Figure 1 C is the albumin nanoparticles BSA-MnO loaded on AIPH-MSTN of the present invention 2 AIPH-MSTN@BSA-MnO 2 (AMB) transmission electron microscopy image;

[0029] Figure 1 D is a transmission electron microscopic image of the SKOV-3 derived ovarian cancer cell membrane (CCM) of the present invention;

[0030] Figure 1 E is the CCM coated with AIPH-MSTN@BSA-MnO 2 (AMB) AIPH-MSTN@BSA-MnO 2 @ Transmission electron microscopy image of CCM (AMBC);

[0031] Figure 2 A is the hydrated particle size diagram of AMBC of the present invention;

[0032] Figure 2 B is a physical picture of AMBC of the present invention at different time points;

[0033] Figure 3 A is a Zeta potential diagram of different nanoparticles of the present invention;

[0034] Figure 3 B is the SDS-PAGE protein analysis diagram of different nanoparticles of the present invention;

[0035] Figure 4 It is the standard curve of AIPH concentration and ultraviolet absorption peak value of the present invention;

[0036] Figure 5 The encapsulation efficiency and drug loading rate of MSTN and AIPH at different mass ratios of the present invention;

[0037] Figure 6 A is the DPBF ultraviolet absorption spectrum under different ultrasonic durations of the present invention;

[0038] Figure 6 B is the DPBF detection of active oxygen generation performance of each nanoparticle in the present invention;

[0039] Figure 7 A is the T1-mapping diagram of AMBC with or without GSH in the present invention;

[0040] Figure 7 B is the ultrasonic relaxation rate of the AMBC of the present invention;

[0041] Figure 7 C shows the bubble generation of AMBC under the microscope before and after ultrasound of the present invention;

[0042] Figure 7 D is the ultrasonic imaging diagram of AMBC with different ultrasonic durations of the present invention;

[0043] Fig. 8A The CLSM observation of ROS produced in SKOV-3 by different control groups in the present invention;

[0044] Figure 8B The flow cytometer of the present invention is used to detect the ROS produced by SKOV-3;

[0045] Fig. 9 is the percentage of ROS in SKOV-3 cells measured in the present invention;

[0046] Fig. 10A The T1-weighted magnetic resonance imaging images at different time points after the injection of AMB and AMBC according to the present invention;

[0047] Fig. 10B These are two-dimensional ultrasound images of mouse ovarian cancer of AMB and AMBC before and after ultrasound excitation according to the present invention. Specific implementation method:

[0048] The technical solutions in the embodiments of the present invention will be further described below in conjunction with the drawings in the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] (1) Synthesis of mesoporous silica (MSN) by microemulsion method: 0.1 g CTAB was dissolved in 30 mL deionized water, and octane, styrene monomer, L-lysine, tetraethyl orthosilicate (TEOS) and 2,2-azobis(2-methylpropylimidamide) dihydrochloride (AIBA) were added. The total mass ratio of water, octane, styrene monomer, TEOS, L-lysine and CTAB was 310:100:10:10:0.22:1, and the concentration of AIBA was 0.84 mg / mL. Under nitrogen protection, the mixed solution was reacted at 60°C for 3 hours. After the reaction was completed, the suspension was decanted and cooled to room temperature overnight. The mixture was collected by centrifugation with ethanol three times, and the obtained mixture was vacuum dried. Finally, the mixture was calcined at 500°C in a tube furnace for 5 hours to remove the template residue to obtain mesoporous silica (MSN).

[0050] (2) Preparation of mesoporous silica-titanium nanoparticles (MSTN): 0.03 g of MSNs was dissolved in 30 mL of isopropanol to prepare solution A. 0.1 mL of tetraisopropyl titanate (TTIP) was slowly added dropwise to 0.3 mL of isopropanol to prepare solution B. After solution B was slowly added dropwise to solution A, the mixture was stirred at 600 rpm for 1 h at 20 °C. Then, 1 mL of deionized water was slowly added to the mixture and stirred at room temperature overnight. The resulting mixture was purified by centrifugation with ethanol and heat treated in a tube furnace at 550 °C for 5 h to prepare anatase TiO 2 (MSTN).

[0051] (3) Thiolation of MSTN and its loading with 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH): 0.5 g of MTSNs was dispersed in anhydrous toluene (80 mL) containing 0.74 mL of 3-mercaptopropyltrimethoxysilane (MPTMS), and the mixed solution was refluxed at 60°C overnight. After the reaction was complete, the prepared thiolated MSTN (MSTN-SH) was washed with ethanol and dried in a vacuum oven at 35°C. Next, 40 mg of AIPH and 20 mg of MSTN-SH were co-dispersed in 10 mL of deionized water and stirred at room temperature for 12 h. After high-speed centrifugation 3 times, MSTN-SH loaded with AIPH (AIPH-MSTN) was obtained. Figure 1As shown in A, AIPH-MSTN is a uniformly dispersed sphere with a particle size of about 50nm. Elemental analysis shows that Si (red), O (blue), Ti (green) and C (yellow) are uniformly distributed in the sphere, which indicates that TiO 2 and AIPH were successfully loaded onto the mesoporous silica surface.

[0052] (4) Preparation of BSA-MnO by biomineralization 2 : Dissolve 0.2 g BSA in 10 mL deionized water and add 1 mL MnCl dropwise under vigorous stirring. 2 Aqueous solution (10 mM). After stirring at room temperature for 30 min, the pH of the system was adjusted to 12 with 2 M NaOH solution. After the mixed solution was vigorously stirred at 37 ° C for 2 h, the mixture was further dialyzed in deionized water for 24 h to remove excess impurities. Finally, the mixture was freeze-dried to obtain BSA-MnO 2 Powder. Figure 1 As shown in B, BSA-MnO 2 It is a spherical particle with a particle size of about 7nm, which is evenly distributed in the aqueous solution.

[0053] (5) AIPH-MSTN surface connected with BSA-MnO 2 (AIPH-MSTN@BSA-MnO 2 ): After dispersing 50 mg of AIPH-MSTN in 50 mL of PBS solution, 4 mL of BSA-MnO was slowly added to the solution. 2 After stirring at room temperature for 24 h, the prepared nanoparticles were separated by high-speed centrifugation and washed with deionized water, thereby successfully preparing BSA-MnO 2 AIPH-MSTN(AIPH-MSTN@BSA-MnO 2 ).like Figure 1 As shown in C, AIPH-MSTN@BSA-MnO 2 The surface of (AMB) is granular, indicating that BSA-MnO 2 The nanoparticles were successfully attached to the MSTN surface.

[0054] (6) Extraction of SKOV-3 derived human ovarian cancer cell membrane (CCM): 3 × 10 7Add 1 mL of hypotonic lysis buffer to each SKOV-3 human ovarian cancer cell, and let the mixture stand at 4°C overnight. After complete lysis, centrifuge at 3200 r / min for 5 min at 4°C. Collect the supernatant, and resuspend the obtained cell pellet in hypotonic lysis buffer. Homogenize the buffer 20 times with a glass homogenizer, centrifuge at 3200 r / min for 5 min at 4°C, and collect the supernatant. Repeat this process 6 times, and centrifuge the supernatant obtained from 8 centrifugations at 12000 r / min for 30 min at 4°C. After removing the supernatant, resuspend the obtained cell membrane pellet in PBS, and use an extruder to extrude it 21 times through a 400nm polycarbonate membrane to obtain a SKOV-3-derived cancer cell membrane (CCM) solution. Figure 1 As shown in D, CCM shows the structure of the cell membrane under transmission electron microscopy.

[0055] (7) CCM coated AIPH-MSTN@BSA-MnO 2 (AMBC): 1 mg / mL AIPH-MSTN@BSA-MnO 2 After the PBS solution and 2 mg / mL CCM were uniformly mixed, the mixed solution was passed through a 200 nm polycarbonate membrane using an extruder and extruded 21 times to obtain the CCM-coated biomimetic drug delivery system AIPH-MSTN@BSA-MnO 2 @CCM(AMBC). Figure 1 As shown in Figure E, the surface of AMBC obtained after co-extrusion with AMB is smoother than that of AMB and presents a spherical "core-shell" structure, which indicates that CCM is successfully coated on the surface of the nanocarrier.

[0056] The hydrated particle size and Zeta potential of the nanoprobe were tested using a Malvern particle size tester. Figure 2 As shown in A, the average hydrated particle size of AMBC is 225.9±1.7nm, and the PDI is 0.1833±0.01. The narrow particle size distribution and small PDI indicate that the hydrated particle size of the ovarian cancer biomimetic sonosensitizer AMBC is relatively uniform and has good dispersibility. Under 4°C storage conditions, the stability of the nanoparticles was evaluated by monitoring the particle size changes of AMBC in PBS for 7 days. The results are shown in Figure 2 As shown in B. With the extension of time, the average water content and particle size of AMBC remained basically unchanged (P>0.05), indicating that it has good dispersibility and stability. The surface potential of the nanoprobe is shown in Figure 3As shown in A: The potentials of CCM, MSTN, MSTN-SH, AIPH-MSTN, AMB and AMBC are -8.887±0.69mV, -22.37±0.22mV, -20.03±0.23mV, -16.10±0.44mV, -28.13±0.41mV, -10.90±0.06mV respectively. After comparison, it was found that the Zeta potential of AMBC was similar to that of CCM, indicating that CCM was successfully coated on the surface of nanoparticles. The SDS-PAGE protein analysis results of different samples showed ( Figure 3 B), AMBC, CCM, and ovarian cancer cell (SKOV-3) lysates have almost identical protein profiles (samples stained with Coomassie blue), further demonstrating the successful coating of CCM on the nanoprobe surface.

[0057] The linear regression equation of AIPH concentration and its absorbance was calculated as y = 0.070x-0.0030 (R 2 =0.9998) Figure 4 ), by adjusting the mass ratio of MSTN to AIPH, the drug loading rate and encapsulation rate of the nanoprobe for AIPH were optimized. Figure 5 When the mass ratio of MSTN to AIPH is 1:2, the drug loading rate is as high as 50.70%. We will use this as a guide to prepare drug-loaded nanoprobes and conduct subsequent experiments.

[0058] As a fluorescent probe, DPBF is often used to detect the generation of ROS due to its high specificity for ROS. Therefore, DPBF was used to evaluate the ability of AMBC, a biomimetic sonosensitizer for ovarian cancer, to generate ROS. Figure 6 As shown in A. As the time of ultrasound acting on AMBC increases, the UV absorption peak intensity of DPBF gradually decreases. After testing the generation of ROS by different nanoprobes, the results are as follows Figure 6 B. Compared with the blank control group, MSTN@CCM(MC) and pure BSA-MnO 2 After ultrasound, both produced significant reactive oxygen species. The increase in reactive oxygen species produced by AMC+US compared with MC+US can be attributed to the decomposition of AIPH under ultrasound to produce alkyl free radicals. In the presence of hydrogen peroxide, MnO 2 The generated oxygen increases the efficiency of TiO2 in generating ROS under ultrasound, so that AMBC+US generates more ROS than AMC. By testing the MRI performance and ultrasound performance of AMBC in the presence or absence of glutathione (GSH), it can be seen that AMBC has a higher ROS production rate in the presence of glutathione. 1 The signal intensity gradually increases with the increase of its concentration, while in the absence of GSH, the magnetic resonance T 1 The signal is not obvious ( Figure 7A) Through calculation, it can be seen that GSH increases the r1 value of the AMBC group from 0.346mM -1 s -1 Increased to 8.112mM -1 s -1 ( Figure 7 B) Figure 7 As shown in C, the generation of bubbles in AMBC can be observed under a microscope after ultrasound. The images of AMBC before ultrasound and after 60s, 120s, 180s, and 5min of ultrasound were observed using an ultrasound imager. The results are shown in Figure 7 D. After 120s of ultrasound, the ultrasonic signal generated by AMBC is the strongest.

[0059] DCF is a green fluorescent compound that can be detected by fluorimeter, flow cytometer and fluorescence microscope. Its maximum excitation spectrum and emission spectrum are 495nm and 529nm respectively. Therefore, the content of ROS in SKOV-3 cells can be detected by the intracellular green fluorescence intensity. Fig. 8A As shown in the figure, the blank control group, the pure ultrasound group and the pure AMBC group had almost no green fluorescence, while the green fluorescence of AMC was stronger than that of MC. This is because azo compounds produce cytotoxic alkyl free radicals (R·) and N· ... 2 No oxygen supply is required, and N2 enhances the penetration of nanoparticles through ultrasonic cavitation effect under ultrasound. In addition, it was found that the green color of AMBC+US group was the strongest, which can be attributed to BSA-MnO 2 Decomposition within cells produces oxygen, enhancing the sonosensitizer TiO 2 The ability to produce ROS. At the same time, this is also the result of CCM's homologous targeting effect. Quantitative determination of ROS content in different groups by flow cytometry showed that ( Figure 8B and Fig. 9 ), the ROS production of AMBC+US group was nearly 4.8 times higher than that of MSTN@CCM(MC)+US group, which may be due to the decomposition of AIPH to produce alkyl radicals and BSA-MnO 2 Provide oxygen to jointly enhance TiO 2 The result of acoustic dynamics.

[0060] Next, we studied the T function of AMB and AMBC in animals. 1 Weighted magnetic resonance imaging and ultrasound imaging, the results are shown in Figure 10. After intravenous injection of AMBC, it was found that the magnetic resonance image in the tumor area showed a clear and strong brightening effect ( Fig. 10A ). Moreover, T 1The magnetic resonance signal increased rapidly with time and reached the maximum intensity at 5 hours, which also confirmed the targeting of ovarian cancer cell membrane to tumor. Two-dimensional ultrasound and contrast-enhanced ultrasound (CEUS) were performed on mouse ovarian cancer tumors and found that ( Fig. 10B ), the injected AMBC showed a heterogeneous enhancement effect in the mouse tumor, and the ultrasound signal was significantly better than that of the mice injected with AMB, further indicating that AMBC has the potential to be used as an effective dual-mode nanoprobe for in vivo magnetic resonance and ultrasound imaging.

Claims

1. A biomimetic nanosonosensitizer for ovarian cancer cell membrane for sonodynamic diagnosis and treatment, characterized in that The SKOV-3 derived ovarian cancer cell membrane (CCM) was innovatively coated on the surface of mesoporous silica-titanium nanoparticles (MSTN) encapsulated by manganese dioxide (BSA-MnO2), and the water-soluble sonosensitizer azo compound 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) was encapsulated into the mesopores of MSTN to prepare an ovarian cancer cell membrane bionic nanosonosensitizer.

2. The surface charge of the ovarian cancer cell membrane biomimetic nanosonosensitizer as claimed in claim 1 is substantially the same as the charge of the ovarian cancer cell membrane, which is -8Mv to -11Mv.

3. A method for preparing an ovarian cancer cell membrane biomimetic nanosonosensitizer, characterized in that The method comprises the following steps: (1) Synthesis of mesoporous silica (MSN) by microemulsion method: 0.1g-1.0g CTAB was dissolved in 10mL-100mL deionized water, and octane, styrene monomer, L-lysine, tetraethyl orthosilicate (TEOS) and 2,2-azobis(2-methylpropylimidazole) dihydrochloride (AIBA) were added. The total mass ratio of water, octane, styrene monomer, TEOS, L-lysine and CTAB was 200-500: 50-200: 5-20: 5-20: 0.1-0.5: 0.5-5, and the concentration of AIBA was 0.5mg / mL-1.5mg / mL. Under nitrogen protection, the mixed solution was reacted at 40℃-100℃ for 1h-10h. After the reaction was completed, the suspension was decanted and cooled to room temperature overnight. The mixture was collected by centrifugation with ethanol three times, and the obtained mixture was vacuum dried. Finally, the mixture was calcined in a tube furnace at 300°C to 800°C for 3h to 8h to remove the template residues to obtain mesoporous silica (MSN); (2) Preparation of mesoporous silica-titanium nanoparticles (MSTN): 0.01g~0.09g MSNs were dissolved in 10mL~50mL isopropanol to prepare solution A. 0.05mL~0.5mL tetraisopropyl titanate (TTIP) was slowly added dropwise to 0.1mL~0.6mL isopropanol to prepare solution B. After solution B was slowly added dropwise to solution A, the mixed solution was stirred at 5℃-50℃ at a speed of 10000rpm~15000rpm for 0.5h~5h. Then, 0.5mL~5mL deionized water was slowly added to the mixed solution and stirred at room temperature overnight. The obtained mixture was purified by centrifugation with ethanol and heat treated in a tube furnace at 250℃~750℃ for 3h~8h to prepare anatase TiO2 (MSTN); (3) Thiolation of MSTN and its loading with 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH): 0.3 g to 1.0 g of MTSNs were dispersed in 50 mL to 100 mL of anhydrous toluene containing 0.4 mL to 1.4 mL of 3-mercaptopropyltrimethoxysilane (MPTMS), and the mixed solution was refluxed at 30°C to 100°C overnight. After the reaction was complete, the prepared thiolated MSTN (MSTN-SH) was washed with ethanol and dried in a vacuum oven at 15°C to 55°C. Then, 20 mg to 70 mg of AIPH and 20 mg to 70 mg of MSTN-SH were co-dispersed in 5 mL to 50 mL of deionized water and stirred at room temperature for 5 h to 15 h. After high-speed centrifugation 3 times, MSTN-SH loaded with AIPH (AIPH-MSTN) was obtained; (4) Preparation of protein-linked manganese dioxide (BSA-MnO2) by biomineralization strategy: Dissolve 0.1g~0.5g bovine serum albumin (BSA) in 5mL~40mL deionized water, and add 0.5mL~5mL manganese chloride (MnCl2) aqueous solution (5mM~50mM) dropwise under vigorous stirring. After stirring at room temperature for 0.1h~1.5h, adjust the pH of the system to 7~12 with 1.0M~5.0M sodium hydroxide (NaOH) solution. After the mixed solution is vigorously stirred at 20℃~50℃ for 1h~5h, the mixture is further dialyzed in deionized water for 12h~72h to remove excess impurities. Finally, the mixture is freeze-dried to obtain BSA-MnO2 powder; (5) AIPH-MSTN surface connected with BSA-MnO2 (AIPH-MSTN@BSA-MnO2): 30mg~100mg AIPH-MSTN was dispersed in 30mL~100mL PBS solution, and 2mL~9mL BSA-MnO2 solution (1mg / mL~5mg / mL) was slowly added to the solution. After stirring at room temperature for 12h~72h, the prepared nanoparticles were separated by high-speed centrifugation and washed with deionized water, and AIPH-MSTN connected with BSA-MnO2 (AIPH-MSTN@BSA-MnO2) was successfully prepared. (6) Extraction of SKOV-3 derived ovarian cancer cell membrane (CCM): 1×10 7 ~9×10 7 Add 0.5mL to 5mL of hypotonic lysis buffer to each SKOV-3 human ovarian cancer cell, and let the mixture stand overnight at 4°C. After complete lysis, centrifuge at 8000r / min to 15000r / min at 4°C for 3min to 15min. Collect the supernatant, and resuspend the obtained cell pellet in hypotonic lysis buffer. Homogenize the buffer 20 times with a glass homogenizer, and centrifuge at 8000r / min to 15000r / min at 4°C for 3min to 15min, and collect the supernatant. Repeat this process 3 to 9 times, and centrifuge the supernatant obtained from 5 to 15 centrifuges at 8000r / min to 15000r / min at 4°C for 0.1h to 1h. After removing the supernatant, resuspend the obtained cell membrane pellet in PBS, and squeeze it through a 200nm to 800nm ​​polycarbonate membrane with an extruder for 15 to 35 times to obtain a SKOV-3 derived cancer cell membrane (CCM) solution. (7) CCM-coated AIPH-MSTN@BSA-MnO2: After uniformly mixing 0.5 mg / mL to 5 mg / mL AIPH-MSTN@BSA-MnO2 PBS solution with 1 mg / mL to 5 mg / mL CCM, the mixed solution was passed through a 100 nm to 500 nm polycarbonate membrane using an extruder and extruded 15 to 35 times to obtain the CCM-coated bionic drug delivery system AIPH-MSTN@BSA-MnO2@CCM (AMBC).

4. The method for preparing an ovarian cancer cell membrane biomimetic nanosonosensitizer according to claim 3, characterized in that The particle size of the AMBC is 80nm to 300nm.

5. A method for preparing an ovarian cancer cell membrane biomimetic nanosonosensitizer as claimed in claim 3, characterized in that The pore size of the MSTN is 1 nm to 100 nm, and the pore volume is 0.1 m 3 / g~0.5m 3 / g.

6. The method for preparing an ovarian cancer cell membrane biomimetic nanosonosensitizer according to claim 3, characterized in that The drug loading of the AIPH is 20% to 80%, and the encapsulation rate is 45% to 85%.

7. The method for preparing an ovarian cancer cell membrane biomimetic nanosonosensitizer according to claim 3, characterized in that The prepared ovarian cancer cell membrane biomimetic nanosonosensitizer has high biocompatibility, strong targeting specificity, good stability, and can effectively evade recognition and clearance by the immune system.

8. The method for preparing an ovarian cancer cell membrane biomimetic nanosonosensitizer according to claim 3, characterized in that The prepared ovarian cancer cell membrane bionic nanosonosensitizer not only has strong homologous targeting and immune escape capabilities, but can also effectively realize targeted ultrasound and MRI dual-modal imaging of sonosensitizers to ovarian cancer cells, effectively realize targeted and quantitative drug release, and achieve controllable diagnosis and treatment of ovarian cancer, which is expected to provide new ideas for the clinical early diagnosis and precise treatment of ovarian cancer.

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