Elastin-like polypeptide-based composite material and cell membrane bionic elastin-like polypeptide-based nano-drug

Abstract

The invention relates to an elastin-like polypeptide-based composite material and a cell membrane bionic elastin-like polypeptide-based nano-drug. After the nano-drug is injected into the body of a mouse through caudal vein, the nano-drug can be promoted to cross a blood brain barrier to enter in-situ brain glioma, meanwhile, MR imaging, chemical kinetic treatment and sonodynamic treatment of tumors are achieved, and by activating a cGAS-STING pathway, immunogenic cell death is caused to activate anti-tumor immune response, so that the anti-tumor effect is achieved. The obvious anti-tumor effect and the potential clinical application value are realized.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and particularly relates to a composite material based on elastin-like polypeptide and a cell membrane bionic nanomedicine based on elastin-like polypeptide. Background Art

[0002] Gliomas are the most invasive and lethal primary brain tumors of the central nervous system, with poor prognosis, easy recurrence, and a five-year survival rate of less than 10%. The current treatment of gliomas is mainly surgical resection, supplemented by radiotherapy and chemotherapy, but due to its high invasiveness, genetic heterogeneity and protection of the blood-brain barrier (BBB), it has brought great resistance to the treatment of gliomas (Wu et al.Pharmacol.Res.2021,171,105780). With the continuous development of biology and the deepening of tumor research, targeted therapy and immunotherapy applied to gliomas have made progress, greatly improving the prognosis of glioma patients, but facing the inevitable drug resistance of gliomas and other problems is still unsolved. Therefore, it is urgent to prepare a suitable treatment system in combination with existing technical means to achieve efficient diagnosis and treatment of gliomas.

[0003] Sonodynamic therapy is a new type of tumor treatment with the advantages of being non-invasive, non-invasive, and having a high tissue penetration depth. It kills tumor cells by delivering small molecule sonosensitizers to the tumor site and inducing tumor cells to produce singlet oxygen through ultrasonic stimulation, thereby destroying tumor cell membrane components and nucleic acids (Zhang et al. Adv. Sci. 2023, 10(6), 2206009).

[0004] However, small molecule drug ingredients, including sonosensitizers that can be used for sonodynamic therapy, are easily metabolized during in vivo delivery and often lack selectivity for lesion sites. Therefore, they are often accompanied by serious side effects and low therapeutic efficiency during use. With the continuous development of nanomedicine, various nanosystems that can be designed on demand provide feasible strategies for solving these problems and provide new opportunities for achieving effective cancer diagnosis and treatment. Among the many organic / inorganic components used to construct nanodelivery platforms, biomacromolecules have attracted more and more attention due to their versatility and natural biocompatibility (Cheng et al. Polym. Int. 2022, 71 (4), 371-378). Elastin-like polypeptide (ELP) is an artificial polypeptide with elastin characteristics designed and synthesized based on the amino acid sequence of natural elastin. Its amino acid sequence is mainly composed of pentapeptide (Val-Pro-Gly-Xaa-Gly, VPGXG) repeating units. These artificial peptides exhibit temperature-dependent phase transition behavior and can be used to prepare stimulus-responsive biomaterials. By changing their sequence composition or modifying specific amino acid residues, their response to stimuli such as temperature, pH and light can be adjusted (Ibrahimova et al. Bioconjugate Chem. 2021, 32 (8), 1719-1728). At present, there are many literature reports that hydrophobic small molecule drugs such as DOX, PTX, and TT1 are connected to ELP, and the resulting complexes self-assemble to form nanomicelle structures, which enhance the water solubility of hydrophobic drugs and improve drug delivery efficiency for tumor treatment (Andrew MacKay et al. Nat. Mater. 2009, 8 (12), 993-999; Bhattacharyya et al. Nat. Commun. 2015, 6, 7939; Ibrahimova et al. Bioconjugate Chem. 2021, 32 (8), 1719-1728). A large number of studies have verified that ELP can be used as a non-immunogenic and precisely controllable nanocarrier building block.

[0005] Tannic acid (TA) and manganese ion (Mn 2+ ) can form a metal polyphenol network through coordination for tumor diagnosis and treatment. 3+ , Mn 2+ It can not only inhibit tumor cells through chemodynamic therapy, but also has the potential to activate immunity. The research of Jiang Zhengfan's team at Peking University found that Mn 2+It can strongly activate the intracellular cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, release a large amount of type I interferon, and activate the natural immune response of macrophages and dendritic cells (DCs) (Zhao et al. Cell Rep. 2020, 32 (7), 108053). 2+ The "manganese-free therapy" combined with PD-1 antibodies significantly enhanced the therapeutic effect of tumors by activating cellular immunity (Lv et al. Cell Res. 2020, 30(11), 966-979). The Dai Yunlu team of the University of Macau combined the phenolic hydroxyl groups of chemically synthesized polyphenol derivatives with Mn 2+ A new metal-polyphenol network was constructed by coordination, using Mn 2+ Activate cGAS and increase its responsiveness to intracellular dsDNA, activate the STING pathway, thereby maturing DCs and activating anti-tumor immune responses, reshaping the tumor immune microenvironment (Yanet.al.Adv.Mater.,2022,34(10),2105783). The nanomedicine prepared in this design showed good anti-tumor effects, but active targeting was not involved in the design.

[0006] Although the nano-drug delivery system can achieve drug loading, exogenous substances are easily captured by the mononuclear phagocytic system and quickly cleared from the body. In addition, the existence of the BBB greatly limits the accumulation of drugs in situ brain gliomas, which greatly reduces the diagnosis and treatment efficiency of brain gliomas.

[0007] After searching relevant domestic and foreign literature and patents, no relevant reports have been found on the construction of new nanomedicines using ELP nanomicelles and metal polyphenol networks as components for combined dynamic therapy and immunotherapy of in situ brain gliomas. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a composite material based on elastin-like polypeptides, a cell membrane biomimetic nanomedicine based on elastin-like polypeptides, and the preparation and application of nanomedicines that have good biocompatibility and can cross the BBB to achieve efficient treatment of brain gliomas.

[0009] The invention provides a composite material based on elastin-like polypeptide. The composite material comprises a metal polyphenol network and nano micelles. The nano micelles are elastin-like polypeptide ELP coupled sonosensitizer nano micelles, and the metal polyphenol network is coated on the surface of the nano micelles.

[0010] Preferably, the sonosensitizer comprises dihydrochlorin e6; the metal polyphenol network comprises drugs and Mn 2+ ; The drug includes tannic acid.

[0011] Furthermore, the metal polyphenol network is tannic acid / manganese (TA / Mn).

[0012] Furthermore, the molecular weight of the elastin-like polypeptide ELP is 33735Da, and the protein sequence is MW[VPGVG-VPGMG-(VPGVG) 2 ] 20 .

[0013] Furthermore, the composite material is ELP loaded with sonosensitizer Ce6 via amide bonds, and nano-micelles are prepared by using the ELP, and coated with tannic acid and Mn 2+ Metal polyphenol network.

[0014] The present invention provides a method for preparing a composite material based on elastin-like polypeptide, comprising:

[0015] (1) activating the sonosensitizer to obtain the activated sonosensitizer; mixing the activated sonosensitizer and ELP for reaction, and post-treating to obtain a sonosensitizer / ELP conjugate;

[0016] (2) adding the sonosensitizer / ELP solution into stirred water and centrifuging to obtain sonosensitizer / ELP nanomicelles;

[0017] (3) Mixing the sonosensitizer / ELP nanomicelles, metal salt, and drug, stirring for reaction, and centrifuging to obtain a composite material based on elastin-like polypeptide.

[0018] Preferably, the sonosensitizer activation in step (1) comprises: adding DCC and NHS to the sonosensitizer solution for activation; wherein the molar ratio of DCC, NHS and sonosensitizer is 90-110:90-110:9-11; the activation reaction time is 2-4 hours; the sonosensitizer comprises dihydrochlorin e6 (Ce6);

[0019] In the step (1), the activated sonosensitizer and ELP mixed reaction is performed by dropping the activated sonosensitizer solution into the stirred ELP solution and stirring for reaction.

[0020] Preferably, in step (1), the molar ratio of sonosensitizer to ELP is 28-32:0.9-1.1; the reaction is stirred at room temperature for 2-3 days;

[0021] The post-treatment in step (1) is ether precipitation, vacuum drying, dialysis, and freeze drying; wherein the ratio of the volume of ether to the volume of the reaction solution during ether precipitation is 9-11:0.9-1.1.

[0022] Preferably, the solvent of the sonosensitizer / ELP solution in step (2) is dimethyl sulfoxide (DMSO); the water is ultrapure water, wherein the ratio of DMSO to ultrapure water is 0.8-1.2:38-40; the centrifugation is ultrafiltration centrifugation, wherein the molecular weight cutoff of the ultrafiltration centrifuge tube is 50,000 Da, and the centrifugation conditions are 4,000-6,000 rpm, 4-6 min, and centrifugation 1-3 times.

[0023] Furthermore, the sonosensitizer / ELP in step (2) is dissolved in a trace amount of solvent, slowly added dropwise into ultrapure water that is vigorously stirred, and ultrafiltered and centrifuged.

[0024] Preferably, in step (3), the sonosensitizer / ELP nano-micelles, metal salt (MnCl 2 The mass ratio of the metal salt to the drug is 3-7:1-5:0.8-1.2; the metal salt includes a manganese salt; the drug includes tannic acid; the manganese salt is MnCl 2 ·4H 2 O.

[0025] Preferably, the stirring reaction in step (3) is performed for 8-12 hours; the centrifugation is ultrafiltration centrifugation, wherein the molecular weight cut-off of the ultrafiltration centrifuge tube is 5000Da, wherein the centrifugation conditions are 4000-6000rpm, 4-6min, and centrifugation 1-3 times;

[0026] In the step (3), the sonosensitizer / ELP nanomicelles, metal salt and drug are mixed to form a mixture of sonosensitizer / ELP nanomicelles nanomicelle solution, metal salt solution and drug solution; wherein the solvents of the metal salt solution and the drug solution are both water.

[0027] The invention provides a cell membrane bionic nanomedicine, which comprises a cell membrane and the composite material based on elastin-like polypeptide, wherein the cell membrane covers the composite material based on elastin-like polypeptide.

[0028] Furthermore, the cell membrane biomimetic nanomedicine comprises preparing an amphiphilic copolymer by loading an ELP with a sonosensitizer, and using the amphiphilic copolymer to prepare nanomicelles, coating a nanomice composed of tannic acid and Mn 2+ A metal polyphenol network is composed, and finally a nanomedicine is obtained by cell membrane biomimetic; wherein the amphiphilic copolymer is prepared by ELP loading the sonosensitizer Ce6 with amide bonds.

[0029] The cell membrane is a macrophage membrane.

[0030] The present invention provides a method for preparing a cell membrane bionic nanomedicine, comprising:

[0031] The composite material based on elastin-like polypeptide and cell membrane are mixed, extruded, and centrifuged to remove the supernatant to obtain the cell membrane bionic nanomedicine.

[0032] The cell membrane includes a macrophage membrane MM; the extrusion is repeated 8 to 11 times using an Avanti micro extruder with a filter membrane pore size of 300 to 500 nm; the centrifugal conditions are 6000 to 8000 rpm and 5 to 15 min.

[0033] The cell membrane is a cell membrane suspension, which includes: adding a cell lysate to a macrophage precipitate, repeatedly freezing and thawing the cells, extracting MM (MM, macrophage membrane) by gradient centrifugation, and resuspending in a PBS solution to obtain an MM suspension;

[0034] The ratio of macrophages to cell lysate was 1×10 7 The cell lysis buffer contained 1% phenylmethylsulfonyl chloride (PMSF). The freeze-thaw process parameters were as follows: freezing in liquid nitrogen, thawing at 37°C, and repeated 3 times. The gradient centrifugation was as follows: the temperature was 4°C, first centrifuged at 700g for 10min, the precipitate was removed, then centrifuged at 14000g for 30min, the supernatant was removed, and the precipitate was resuspended in 0.3mL PBS solution.

[0035] The present invention provides an application of the composite material based on elastin-like polypeptide or the cell membrane bionic nanomedicine in the preparation of MR imaging medicine.

[0036] The present invention provides an application of the composite material based on elastin-like polypeptide or the cell membrane bionic nanomedicine in combination with sonodynamic therapy and / or chemodynamic therapy and / or immunotherapy in the preparation of anti-tumor drugs.

[0037] The present invention includes ELP coupling dihydrochlorin e6 (Ce6) to obtain amphiphilic macromolecules and prepare nano micelles, coating tannic acid (TA) / manganese metal polyphenol network structure, and MM biomimetic. In the present invention, after the nano drug is injected into the mouse through the tail vein, it can promote the nano drug to cross the blood-brain barrier and enter the in situ brain glioma, while realizing MR imaging, chemodynamic therapy and sonodynamic therapy of the tumor, and can activate anti-tumor immune response by activating the cGAS-STING pathway and causing immunogenic cell death, and has significant anti-tumor effect and potential clinical application value.

[0038] The present invention uses biological macromolecular protein substances as components to construct nanomedicines, and uses MM biomimetic to achieve brain delivery of nanomedicines across the BBB, and has good biodegradability. At the same time, the nanomedicine is mainly based on dynamic therapy, has good biocompatibility, can achieve chemodynamic therapy after reaching the tumor site, and achieve sonodynamic therapy of the tumor after ultrasound irradiation and induce immunogenic cell death (ICD) to activate anti-tumor immunity, which is expected to achieve efficient treatment of tumors.

[0039] The present invention first couples ELP and Ce6 to prepare an amphiphilic copolymer, and prepares nano micelles by dialysis. Subsequently, a tannic acid / manganese metal polyphenol network structure is coated on the surface of the micelles. MM is then extracted and coated on the surface of the nano drug by co-extrusion to obtain the nano drug Ce6-ELP@TM / MM.

[0040] The present invention prepares an amphiphilic substance by coupling Ce6 with ELP, prepares nano-micelles, and then coats the metal polyphenol network and prepares nano-drugs by MM biomimetic. The nano-drugs prepared by the present invention have high biosafety, are easy to degrade in vivo, and have good dispersibility. The coated MM can promote the nano-drugs to cross the BBB to achieve in situ brain glioma enhanced tumor treatment effect. The nano-drugs can kill tumor cells by generating reactive oxygen species (ROS) under the action of tumor microenvironment and ultrasound, and destroy DNA while killing tumor cells. Combined with Mn 2+ Co-activation of the cGAS-STING pathway to achieve tumor immunotherapy. After the nanodrug is injected into the mouse through the tail vein, it can not only achieve MR imaging of the tumor, but also achieve sonodynamic therapy and chemodynamic therapy, and can activate the cGAS-STING pathway and cause ICD to activate anti-tumor immune response, which has significant anti-tumor effects and potential clinical application value.

[0041] The present invention uses Zeta potential and dynamic light scattering analysis (DLS), nuclear magnetic resonance hydrogen spectrum ( 1H NMR), fluorescence spectroscopy, inductively coupled plasma atomic emission spectroscopy (ICP-OES), nuclear magnetic resonance imaging analyzer, SDS-PAGE and other methods were used to characterize the physical and chemical properties of the prepared nanodrugs. The cytotoxicity of the nanodrugs was evaluated by CCK-8 analysis; the uptake of the nanodrugs by cells, and the effects of the nanodrugs on the intracellular ROS level, cell apoptosis level and dendritic cell maturation were detected by flow cytometry; the release of HMGB-1 in cancer cells treated with the materials was evaluated by ELISA; the effects of the nanodrugs on the expression of cGAS-STING pathway-related proteins p-STING, p-IRF3 and p-TBK1 were evaluated by Western blot. Finally, an orthotopic brain glioma mouse model was established to evaluate the anti-tumor performance in vivo.

[0042] The present invention provides a MM bionic ELP-based nanomedicine, which is mainly composed of macromolecular substances of biological origin, has good biocompatibility and biodegradability, and the nanomedicine is mainly used to achieve dynamic therapy, reducing its own toxicity. 2+ Not only can chemodynamic therapy be achieved, but also the cGAS-STING pathway in tumor cells can be activated, and the ICD of tumor cells induced by combined chemodynamic therapy and sonodynamic therapy can be further promoted to activate anti-tumor immunity. 2+ It also has excellent MR imaging performance, and the sonosensitizer Ce6 itself has near-infrared fluorescence imaging properties, which can realize MR / fluorescence dual-modality imaging visualization of nanomedicines in the body, improving the efficiency of tumor diagnosis and treatment.

[0043] The present invention provides a preparation method and application of MM bionic ELP-based nanomedicine, which has good biocompatibility and realizes efficient tumor combined treatment through sonodynamic therapy, chemodynamic therapy, cGAS-STING pathway immune activation and tumor cell ICD. At the same time, the platform uses MM bionics to realize drug delivery across the BBB to the brain to enhance the therapeutic effect of in situ brain glioma.

[0044] Beneficial Effects

[0045] (1) The process of the present invention is simple, the cost is low, the operation and separation are easy, and it has a good development prospect.

[0046] (2) The nanomedicine prepared by the present invention has good dispersibility, water solubility and biocompatibility, providing a new idea for constructing safe and efficient nanomedicine.

[0047] (3) The nanomedicine prepared by the present invention can be complexed with Mn 2+It produces a "Fenton-like reaction" to achieve chemodynamic therapy, and can also activate the cGAS-STING pathway in tumor cells to induce anti-tumor immune response, thereby combining with the ICD caused by sonodynamic therapy to achieve efficient combined treatment of tumors.

[0048] (4) After the nanomedicine prepared by the present invention is injected into the mouse body through the tail vein, it can cross the BBB through MM bionics, showing a significant combined sonodynamic / chemodynamic / immunological therapeutic effect on in situ brain glioma, and has potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the synthesis and application of Ce6-ELP@TM / MM prepared in the present invention.

[0050] Figure 2 The Ce6 and ELP used in the present invention are 1 H NMR spectra (ab) and (c) are of the prepared Ce6-ELP copolymers. 1 H NMR spectrum;

[0051] Figure 3 The results of the critical micelle concentration of the Ce6-ELP copolymer prepared by the present invention were determined using the fluorescent probe pyrene;

[0052] Figure 4 The standard curve (a) of the emission spectrum of Ce6 at 669 nm under 405 nm excitation light used in the present invention and the emission spectrum (b) of ELP and Ce6-ELP copolymer prepared in the present invention under 405 nm excitation;

[0053] Figure 5 SDS-PAGE results of Ce6-ELP@TM, Ce6-ELP@TM / MM and MM prepared in the present invention;

[0054] Figure 6 The T of Ce6-ELP@TM / MM prepared in this invention is 1 Linear relationship between the reciprocal of relaxation time and Mn concentration;

[0055] Figure 7 The flow cytometric analysis results after the Ce6-ELP@TM / MM prepared by the present invention was co-incubated with C6 cells for different time periods;

[0056] Figure 8 (a) to (d) are cell viability graphs after Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM and Ce6-ELP@TM / MM prepared by the present invention were co-incubated with C6 cells for 24 hours;

[0057] Fig. 9 (a) to (d) are cell viability diagrams after Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM and Ce6-ELP@TM / MM prepared by the present invention were co-incubated with C6 cells and then treated with ultrasound;

[0058] Fig.10 The flow cytometric analysis results of the changes in intracellular ROS levels after Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM and Ce6-ELP@TM / MM prepared by the present invention were co-incubated with C6 cells in the absence (ab) or presence (cd) of ultrasound;

[0059] Fig.11 Flow cytometry detection results (a) and quantitative analysis diagrams (bc) of cell apoptosis after Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM and Ce6-ELP@TM / MM prepared by the present invention were co-incubated with C6 cells in the presence of ultrasound for 16 hours;

[0060] Fig.12 The results of detecting the ICD-related factors ATP (a) and HMGB-1 (b) secreted by the cells after Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM and Ce6-ELP@TM / MM prepared by the present invention were co-incubated with C6 cells in the presence or absence of ultrasound for 24 hours;

[0061] Fig.13 Figure 2 is the flow cytometric analysis (a) and related quantitative analysis (b) of the effects of Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM and Ce6-ELP@TM / MM on DCs maturation after 24 h of co-incubation with C6 cells in the presence or absence of ultrasound by Transwell analysis;

[0062] Fig.14 Figure 1 is a Western blot study result of the expression of cGAS-STING pathway-related proteins p-STING, p-IRF3 and p-TBK1 in cells after Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM and Ce6-ELP@TM / MM were co-incubated with C6 cells in the presence or absence of ultrasound for 24 hours, wherein (a) is a protein band diagram of Western blot, and (bd) are grayscale relative quantitative diagrams of protein bands;

[0063] Fig.15MR imaging results of tumor volume changes observed during ultrasound treatment of tumor-bearing mice injected with Ce6, Ce6-ELP@TM and Ce6-ELP@TM / MM via tail vein (a), as well as the relative tumor volume change curve (b) and body weight change curve (c) within 10 days of treatment;

[0064] Fig.16 The results of flow cytometry detection and corresponding quantitative analysis (cd) of the changes in CD4+T cells (a) and CD8+T cells (b) in the tumor sites of tumor-bearing mice after treatment in different groups. DETAILED DESCRIPTION

[0065] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0066] Unless otherwise specified, all chemical reagents were commercially available and used without further purification. DCC, NHS, TA, and MnCl 2 Purchased from Bailingwei Technology Co., Ltd. (Shanghai). The elastin-like polypeptide ELP has a molecular weight of 33735 Da and a protein sequence of MW [VPGVG-VPGMG-(VPGVG) 2 ] 20 , prepared by the team of Professor Sébastien Lecommandoux of the University of Bordeaux, France. Ce6 was purchased from Shanghai Hongye Biotechnology Co., Ltd. (Shanghai). C6 cells (rat glioma cells) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. DCs (immature dendritic cell line derived from mouse bone marrow) were obtained from the Affiliated Cancer Hospital of Fudan University. DMEM culture medium, penicillin-streptomycin double antibody, and trypsin were purchased from Hangzhou Gino Biomedical Technology Co., Ltd. (Hangzhou). Fetal bovine serum was purchased from Gibco Life Technologies Co., Ltd. (Carlsbad, CA). Cell Counting Kit-8 (CCK-8) was purchased from Shanghai Qihai Biotechnology Co., Ltd. ROS detection kit and ATP detection kit were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Cell apoptosis detection kit was purchased from Jiangsu Keygen Biotechnology Co., Ltd. HMGB-1 detection kit, Anti-CD80-PE, Anti-CD86-FITC, Anti-CD3-PE, Anti-CD8-FITC, and Anti-CD4-FITC were purchased from Thermo Fisher Scientific (Waltham, MA). ICR mice were purchased from Shanghai Slake Experimental Animal Center

[0067] Example 1

[0068] (1) Weigh 21 mg of Ce6 and dissolve it in 2 mL of DMSO; weigh 72 mg of DCC and 40 mg of NHS and dissolve them in 1 mL of DMSO respectively. First, add the DCC solution dropwise to the Ce6 solution and stir at room temperature for 30 min, then add the NHS solution dropwise to the reaction solution and continue stirring for 3 h to activate the carboxyl groups in Ce6. Then, weigh 40 mg of ELP and dissolve it in 2 mL of DMSO. Add the activated Ce6 solution dropwise to the vigorously stirred ELP solution and continue stirring at room temperature for 3 days. After the reaction is completed, transfer the reaction solution to a centrifuge tube and add ten times the volume of anhydrous ether to precipitate the prepared Ce6-ELP conjugate. Let it stand overnight, remove the upper layer of anhydrous ether, and vacuum dry the obtained Ce6-ELP precipitate. The dried Ce6-ELP was resuspended in 5 mL of DMSO and transferred to a dialysis bag with a molecular weight of 3500 Da. It was dialyzed against PBS buffer for one day to further remove impurities, and then dialyzed against water for 2 days to obtain the purified Ce6-ELP conjugate, which was freeze-dried and sealed and stored at -20°C.

[0069] (2) Weigh 20 mg of Ce6-ELP and dissolve it in 0.25 mL of DMSO. Add the Ce6-ELP solution dropwise into 9.75 mL of vigorously stirred ultrapure water and stir overnight. Transfer the solution to an ultrafiltration centrifuge tube with a molecular weight of 50,000 Da and centrifuge three times (5,000 rpm, 5 min) to obtain a Ce6-ELP nanomicelle suspension.

[0070] (3) Weigh MnCl 2 ·4H 2 Dissolve 20 mg of O in 1 mL of ultrapure water, weigh 20 mg of tannic acid and dissolve it in 1 mL of ultrapure water, take 1 mL of 1 mg / mL Ce6-ELP nanomicelle dispersion, and add 30 μL of MnCl while stirring vigorously. 2 ·4H 2 O and 10 μL of tannic acid solution, stirred for 4 h, transferred to an ultrafiltration centrifuge tube with a molecular weight of 5000 Da, and centrifuged three times (5000 rpm, 5 min) to obtain a Ce6-ELP@TM dispersion.

[0071] (4) adding the cell lysate to the macrophage pellet, repeatedly freezing and thawing, extracting MMs by gradient centrifugation, and resuspending in PBS solution to obtain MM suspension;

[0072] (5) The Ce6-ELP@TM in step (3) is mixed with the MM suspension in step (4), and the mixture is repeatedly squeezed, centrifuged at 8000 rpm for 15 min, and the supernatant is removed to obtain the MM bionic nanomedicine Ce6-ELP@TM / MM, wherein the mass ratio of the cell membrane suspension to the Ce6-ELP@TM is 1:2-4.

[0073] Example 2

[0074] 1 mg of Ce6-ELP nanomicelles, Ce6-ELP@TM and Ce6-ELP@TM / MM synthesized in Example 1 were dissolved in 1 mL of ultrapure water to measure the surface potential, hydrodynamic diameter and polymerization dispersion coefficient. The results are shown in Table 1. The potential of Ce6-ELP nanomicelles is -8.3 mV, and the hydration kinetic size is about 141.9 nm. 2+ The surface potential of the nanodrug Ce6-ELP@TM decreased slightly after the metal polyphenol network was formed, and the particle size increased to 155.2nm. After coating with MM, the surface potential of the nanodrug further decreased to -15.2mV, and the particle size increased to 172.6nm.

[0075] Table 1 Surface potential, hydrodynamic diameter and polymerization dispersion index of each material

[0076] Sample ζ-potential(mV) Size(nm) PDI Ce6-ELP nanomicelles -8.3±0.3 141.9±6.7 0.4±0.1 Ce6-ELP@TM -11.3±0.8 155.2±1.4 0.2±0.02 Ce6-ELP@TM / MM -15.2±0.4 172.6±1.5 0.4±0.1

[0077] Example 3

[0078] 7 mg of Ce6-ELP, Ce6 and ELP prepared in Example 1 were taken and dissolved in 600 μL of deuterated DMSO, and then the nuclear magnetic resonance hydrogen spectrum of the material was measured using a nuclear magnetic resonance spectrometer. The nuclear magnetic resonance results are as follows: Figure 2 c, compared with the simple Ce6 ( Figure 2 a) and ELP( Figure 2 b), in the NMR results of Ce6-ELP, the proton peak at the chemical shift of 6-6.5 ppm represents the methylene proton peak of Ce6, proving that Ce6 was successfully coupled with ELP to obtain the Ce6-ELP conjugate.

[0079] Example 4

[0080] In order to determine the critical micelle concentration (CMC) of the copolymer Ce6-ELP in Example 1, the CMC of Ce6-ELP was detected by using the fluorescent probe pyrene. Ce6-ELP solutions of different concentrations (0.001-2 mg / mL) were prepared, and the Ce6-ELP solution was mixed with the fluorescent probe pyrene to make the pyrene concentration be 4.0×10 7M, ultrasonic for 30 min, and stored at room temperature overnight. The emission spectrum (350-435 nm) of the solution at an excitation wavelength of 333 nm was measured by a fluorescence spectrophotometer. The Lg value of Ce6-ELP concentration was compared with I 373 / I 393 The fluorescence intensity ratio was used as the horizontal and vertical coordinates for analysis. Figure 3 As shown in Figure 2, as the concentration of Ce6-ELP increases, I 373 / I 393 The fluorescence intensity of the samples increased first and reached the position I where the concentration was about 0.36 mg / mL. 373 / I 393 It began to decrease, indicating that Ce6-ELP copolymer can form nanomicelles, and its CMC is 0.36 mg / mL.

[0081] Example 5

[0082] To determine the loading of Ce6 in the prepared Ce6-ELP, an appropriate amount of Ce6 was weighed and dissolved in DMSO, and gradient solutions (0.05, 0.1, 0.2, 0.5 and 1 μg / mL) were prepared to measure the fluorescence intensity at 669 nm under 405 nm excitation light, as shown in Fig. Figure 4 (a) As shown. At the same time, the Ce6-ELP prepared in Example 1 was taken for fluorescence spectrum detection, and the results were as follows Figure 4 (b) As shown. Compared with ELP (curve 1), Ce6-ELP (curve 2) showed an obvious emission spectrum after 405nm light excitation, which can prove the successful coupling of Ce6 and ELP. Subsequently, the molar ratio of Ce6 to ELP in the prepared Ce6-ELP was calculated to be 8.7:1 based on the standard curve.

[0083] Example 6

[0084] To prove the successful coating of MM, Ce6-ELP@TM, Ce6-ELP@TM / MM and extracted MM prepared in Example 1 were subjected to SDS-PAGE characterization. The protein content in Ce6-ELP@TM, Ce6-ELP@TM / MM and extracted MM was determined by BCA protein quantification kit, and the protein content in each sample was adjusted to 0.5 mg / mL with PBS solution. 5 μL of protein Marker was added to the first protein lane, and then 20 μL of Ce6-ELP@TM, Ce6-ELP@TM / MM and MM were added to the lanes respectively. The voltage was set to 150 V and the time was set to 30 min. After the electrophoresis, it was stained with Coomassie Brilliant Blue and decolorized. The results are as follows: Figure 5As shown, a protein band appeared at about 37 kDa in the Ce6-ELP@TM group, which belonged to Ce6-ELP, while a band similar to MM appeared in the Ce6-ELP@TM / MM group. This result proved the successful coating of MM on the surface of Ce6-ELP@TM.

[0085] Example 7

[0086] 5 mg of Ce6-ELP@TM / MM was weighed and digested with 1 mL of aqua regia for 3 h, and ultrapure water was added to terminate the digestion. The content of Mn in Ce6-ELP@TM / MM was determined by ICP-OES. It was calculated that the mass fraction of Mn in Ce6-ELP@TM / MM was 4.6%.

[0087] In order to test the magnetic resonance imaging performance of the prepared Ce6-ELP@TM / MM, Ce6-ELP@TM / MM solutions with different Mn concentrations were prepared (the Mn concentrations were 0.019, 0.038, 0.076, 0.19, 0.38 and 0.76 mM, respectively). The T values ​​of the material at different Mn concentrations were measured by magnetic resonance imaging analyzer. 1 Relaxation time, the reciprocal of the relaxation time is linearly fitted with the Mn concentration, and its slope is the relaxation rate r 1 .like Figure 6 As shown, the r 1 3.663mM -1 s -1 , indicating that Ce6-ELP@TM / MM has good T 1 MR imaging effects.

[0088] Example 8

[0089] C6 cells were used as model cells to study the phagocytosis of Ce6-ELP@TM / MM prepared in Example 1. C6 cells in the logarithmic growth phase were collected and cultured at 1×10 5 The cells were seeded at a density of 100 cells per well in a 12-well plate. The culture medium used was DMEM complete culture medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. The plate was placed in a 5% CO 2 , and incubate at 37°C for 24 h. Discard the original culture medium, add culture medium containing Ce6-ELP@TM / MM (Ce6 concentration of 10 μg / mL) and incubate the cells at 5% CO 2 The cells were then digested, centrifuged, and collected at 37°C. The fluorescence intensity in the cells was detected by flow cytometry. Figure 7As shown in the figure, as the incubation time increases, the phagocytic amount of Ce6-ELP@TM / MM by C6 cells gradually increases and reaches the highest phagocytic amount at 4 hours. Therefore, in subsequent experiments, Ce6-ELP@TM / MM was first incubated with cells for 4 hours, and then the cells were ultrasonically treated to achieve sonodynamic therapy of tumors.

[0090] Example 9

[0091] C6 cells were used as model cells to study the in vitro antitumor activity of Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM and Ce6-ELP@TM / MM prepared in Example 1. C6 cells in the logarithmic growth phase were collected and cultured at 1×10 4 The cells were seeded in 6 96-well plates at a density of 100 cells per well. The culture medium used was DMEM complete culture medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin and 10% FBS. The plates were placed in a 5% CO 2 , incubate at 37°C for 24h. Discard the original culture medium, and add complete culture medium containing different concentrations of Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM and Ce6-ELP@TM / MM (the concentrations of the materials used are the corresponding concentrations when the Ce6 concentration is 0.2, 0.5, 1, 2, 5, 10, 20, 50 μg / mL) to each well plate. Then place the well plate in 5% CO 2 , incubate at 37°C for 24h, discard the original culture medium, add DMEM medium containing 10% (v / v) CCK-8, and continue to culture for 2-4h. Finally, use a multifunctional microplate reader to test the absorbance value of each well at a wavelength of 450nm. The cells treated with PBS are used as blank controls, and the cell viability is recorded as 100%. The results are as follows Figure 8 As shown in the figure, within the experimental concentration range, the cell viability of Ce6 and Ce6-ELP nanoemulsions did not decrease significantly, indicating that the nanoemulsions have good cell compatibility. 2+ After the addition of Mn 2+ It has a chemodynamic therapeutic effect, which gives the nanomedicine certain cancer cell killing properties. In contrast, the cell viability of the Ce6-ELP@TM / MM group was lower than that of the Ce6-ELP@TM group, which may be due to the fact that after MM bionics, the nanomedicine enhanced the uptake of nanomedicine by cancer cells through homologous targeting.

[0092] To evaluate the effect of sonodynamic therapy, C6 cells in the logarithmic growth phase were collected and cultured at 1×10 4The cells were seeded in 6 96-well plates at a density of 100 cells per well. The culture medium used was DMEM complete culture medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin and 10% FBS. The plates were placed in a 5% CO 2 , incubate at 37°C for 24h. Discard the original culture medium, and add complete culture medium containing different concentrations of Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM and Ce6-ELP@TM / MM (the concentrations of the materials used are the corresponding concentrations when the Ce6 concentration is 0.2, 0.5, 1, 2, 5, 10, 20, 50 μg / mL) to each well plate. Then place the well plate in 5% CO 2 , incubated at 37°C for 4 h, at 1 MHz, 1 W cm -2 , 40s of conditioned cells were then incubated for 8h, the original culture medium was discarded, and DMEM culture medium containing 10% (v / v) CCK-8 was added and cultured for 2-4h. Finally, the absorbance of each well was tested at a wavelength of 450nm using a multifunctional microplate reader. Cells treated with PBS were used as blank controls, and cell viability was recorded as 100%. The results are shown in Fig. 9 As shown in Figure 2, after ultrasound irradiation, the cell viability level gradually decreased with the increase of Ce6 concentration. Figure 8 Compared with the group without ultrasound, the cell viability level after ultrasound treatment was significantly reduced, which indicates that sonodynamic therapy plays a stronger role in the anti-tumor activity mediated by the nanodrug than chemodynamic therapy and can more effectively inhibit the activity of cancer cells. After MM biomimetic, the uptake of nanodrugs by cancer cells was enhanced, further improving its effect. According to the experimental results, the half-maximal inhibition concentration of different nanodrugs under ultrasound assistance can be calculated, which are 5.14μg / mL (Ce6), 6.38μg / mL (Ce6-ELP nanomicelles), 4.63μg / mL (Ce6-ELP@TM) and 4.02μg / mL (Ce6-ELP@TM / MM).

[0093] Example 10

[0094] C6 cells were used as a cell model to evaluate the effects of different materials on intracellular ROS levels. C6 cells in the logarithmic growth phase were collected and counted at 1×10 5 The cells were inoculated in a 12-well plate at a density of 100 cells / mL and cultured for 24 h. The original culture medium was discarded and culture medium containing PBS or culture medium containing Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM and Ce6-ELP@TM / MM (the concentrations of the materials used were the corresponding concentrations when the concentration of Ce6 was 4 μg / mL) was added to each well and incubated with the cells under 5% CO. 2 The cells and materials were incubated at 37°C for 6 h. In the ultrasound treatment group, cells and materials were incubated for 4 h at 1 MHz and 1 W cm-2 The cells were treated with the conditions of 100 μL PBS for 30 s and then incubated for 2 h. The original culture medium was discarded, the cells were washed with PBS three times, and the cells were collected by trypsin digestion. They were incubated with DCFH-DA probe diluted with serum-free culture medium for 30 min in the dark. After washing with PBS three times, the cells were resuspended in 300 μL PBS and the fluorescence intensity was detected by flow cytometry. Fig.10 (ab) As shown, without ultrasound, the Mn 2+ Ce6-ELP@TM and Ce6-ELP@TM / MM can significantly increase the level of intracellular ROS, which is due to the fact that Mn 2+ It can catalyze the production of hydroxyl radicals through a Fenton-like reaction, thus achieving chemodynamic therapy for cancer cells. After the cells were co-incubated with the material and further irradiated with ultrasound, the ROS levels in each group increased significantly (cd), which was due to the generation of singlet oxygen by the sonosensitizer Ce6 under ultrasound irradiation. These results indicate that the prepared Ce6-ELP@TM / MM can utilize Mn 2+ Hydroxyl free radicals are produced, which can further produce singlet oxygen under the action of ultrasound, thereby increasing the ROS level in cancer cells through chemodynamic / sonodynamic therapy.

[0095] Embodiment 11

[0096] C6 cells were used as a cell model to evaluate the effects of different materials prepared in Example 1 on inducing apoptosis of cancer cells. C6 cells in the logarithmic growth phase were collected and plated at 2×10 5 The cells were inoculated at a density of 100 cells / mL in a 6-well plate. The culture medium used was DMEM complete culture medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. The plate was placed in a 5% CO 2 The cells were incubated at 37°C for 24 h. The original culture medium was discarded, and the culture medium containing Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM, and Ce6-ELP@TM / MM (the concentrations of the materials used were the corresponding concentrations when the concentration of Ce6 was 4 μg / mL) was added to each well and incubated at 5% CO. 2 The cells were incubated at 37°C for 16 h. The ultrasound treatment group was incubated with the cells and materials for 4 h at 1 MHz, 1 W cm -2 , 30s of treatment, and then continued to incubate for 12h. Then, all the cells in the wells were digested, centrifuged, collected, and incubated with cell apoptosis detection reagent Annexin V-FITC and PI diluted 100 times with PBS for 10min, and cell apoptosis and necrosis were detected by flow cytometry. The results are shown in Fig.11 As shown in Figure 2, without ultrasound irradiation, cells treated with Ce6 and Ce6-ELP nanomicelles did not show significant cell killing, but after integrating Mn2+ After that, the level of cell death increased, which was due to the Mn 2+ After the cells were treated with the same material, the level of cell apoptosis increased significantly compared with that before ultrasound, which once again proved that Ce6-ELP@TM / MM-mediated sonodynamic therapy played a major role, and combined with chemodynamic therapy and MM bionics, it could exert a stronger therapeutic effect.

[0097] Example 12

[0098] In order to verify the ICD effect caused by the nanomaterials prepared in Example 1 in treating cancer cells, C6 cells were used as a cell model to evaluate the release of ICD markers high mobility group protein (HMGB-1) and adenosine triphosphate (ATP) after treatment with Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM and Ce6-ELP@TM / MM (the concentrations of the materials used were the corresponding concentrations when the concentration of Ce6 was 4 μg / mL) and ultrasonic irradiation. Cancer cells that were not treated with ultrasonic waves were used as controls.

[0099] C6 cells in the logarithmic growth phase were collected and counted at 1×10 5 The cells were inoculated at a density of 100 cells / mL in a 12-well plate. The culture medium used was DMEM complete culture medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. The plate was placed in a 5% CO 2 The cells were incubated at 37°C for 24 h. The original culture medium was discarded, and the culture medium containing Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM, and Ce6-ELP@TM / MM (the concentrations of the materials used were the corresponding concentrations when the concentration of Ce6 was 4 μg / mL) was added to each well and incubated at 5% CO. 2 The cells were incubated at 37°C for 24 h. The ultrasound treatment group was incubated with the cells and materials for 4 h at 1 MHz and 1 W cm -2 The cells were treated with the conditions of 10% MgCl2 and 10% MgCl2 for 10 s and 30 s, and then incubated for 20 h. The culture medium was collected and the HMGB-1 ELISA kit and ATP detection kit were used to test the HMGB-1 and ATP content in the cell culture medium, respectively. The results are shown in Fig.12As shown in the figure, similar to the results of apoptosis detection, the HMGB-1 and ATP secreted by cells treated with Ce6-ELP nanomicelles were similar to those in the PBS group without ultrasound irradiation, but after coating with TM, the release levels of HMGB-1 and ATP in the Ce6-ELP@TM and Ce6-ELP@TM / MM groups increased, indicating that chemodynamic therapy has a certain effect in inducing ICD in cancer cells. After further ultrasound irradiation, the release of HMGB-1 and ATP in each treatment group increased significantly. These results indicate that Ce6-ELP@TM / MM can induce ICD in cancer cells under the assistance of ultrasound.

[0100] Embodiment 13

[0101] In order to evaluate the maturation of dendritic cells by the material prepared in Example 1 and ultrasound-induced cancer cell ICD, the maturation level of dendritic cells was verified by Transwell experiment using C6 cells and dendritic cells as cell models. C6 cells in the logarithmic growth phase were collected and 1×10 5 The cells were inoculated into the upper chamber of a 12-well Transwell plate at a density of 100 cells / mL and placed in a new 12-well plate. The culture medium used was DMEM complete culture medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. The plate was placed in a 5% CO 2 At the same time, dendritic cells in the logarithmic growth phase were collected and cultured at 1×10 5 The cells were inoculated into the lower chamber of a 12-well Transwell plate at a density of 100 cells / mL. The culture medium used was DMEM complete culture medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. The plate was placed in a 5% CO 2 After culturing the cancer cells for 24 h, the original culture medium was discarded, and the culture medium containing Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM and Ce6-ELP@TM / MM (the concentrations of the materials used were the corresponding concentrations when the concentration of Ce6 was 4 μg / mL) was added to each well and the cells were cultured under 5% CO 2 The cells were incubated at 37°C for 12 h. The ultrasound treatment group was incubated with the cells and materials for 4 h at 1 MHz and 1 W cm -2 The cells were treated with the conditions of 1% PBS and 30 s, and then incubated for 8 h. Subsequently, the culture medium in the dendritic cells was replaced, and the C6 cells treated with different materials were transferred to the dendritic cell upper chamber for co-culture for 24 h. The lower dendritic cells were collected and co-incubated with dendritic cell maturation marker proteins CD80-FITC and CD86-PE antibodies on ice for 30 min, then washed three times with PBS, and the cells were resuspended in PBS. The maturation of dendritic cells was detected by flow cytometry. The results are shown in Fig.13As shown, without ultrasound, the degree of maturation of dendritic cells in the Ce6-ELP@TM / MM group was slightly higher than that in the PBS group. However, after ultrasound treatment, the maturation level of dendritic cells in each group increased significantly, which was consistent with the release of ICD markers. The above results show that the nanomaterial Ce6-ELP@TM / MM prepared in Example 1 can effectively induce ICD in cancer cells after combining chemodynamic therapy and sonodynamic therapy, promote cell secretion of HMGB-1 and ATP, and induce dendritic cell maturation, and sonodynamic therapy plays a major role in the nanomaterial prepared by the present invention.

[0102] Embodiment 14

[0103] Ce6 cells were used as a cell model to evaluate the effects of different materials on the cGAS-STING pathway in cells. 5 The cells were inoculated at a density of 100 cells / mL in a 6-well plate. The culture medium used was DMEM complete culture medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. The plate was placed in a 5% CO 2 The original culture medium was discarded, and the culture medium containing Ce6, Ce6-ELP nanomicelles, Ce6-ELP@TM and Ce6-ELP@TM / MM (the concentration of the materials used was the corresponding concentration when the concentration of Ce6 was 4 μg / mL) was added to each dish and the cells were incubated at 5% CO. 2 The cells were incubated at 37°C for 24 h. The ultrasound treatment group was incubated with the cells and materials for 4 h at 1 MHz and 1 W cm -2 The cells were treated with the conditions of 10% PBS and 10% PBS for 30 seconds, and then incubated for 20 hours. Then, all the cells in the wells were digested, centrifuged, and collected. The cells were lysed on ice and centrifuged at 4°C and 12,000 rpm for 5 minutes. The supernatant protein solution was collected and the protein concentration was determined. SDS-PAGE electrophoresis, membrane transfer, immunoreaction, and ECL chemical developer fixation experiments were then performed in sequence to study the changes in the content of p-STING, p-IRF3, and p-TBK1 in the cells. The results are shown in Fig.14 As shown in the figure, the expression of STING pathway related proteins changed to varying degrees after treatment with each group of materials, while after treatment with Mn 2+ After treatment with the materials, the levels of various proteins increased significantly, indicating that Mn 2+It can activate the cGAS-STING pathway and activate anti-tumor immunity. After ultrasound irradiation, the upregulation level of STING pathway-related proteins in the Ce6-ELP@TM / MM+US group was more significant, which may be because the singlet oxygen produced by sonodynamic therapy can enhance the DNA damage of cells, thereby promoting the activation of the cGAS-STING pathway. The activation of the cGAS-STING pathway can be combined with ICD caused by sonodynamic / chemodynamic therapy to achieve immunotherapy for tumors.

[0104] Embodiment 15

[0105] All animal experiments were conducted in strict accordance with the standards of the Animal Protection Association. 6-8 week old female ICR mice were purchased from Shanghai Slake Laboratory Animal Center. The orthotopic glioma model was established by the following method: the mice were anesthetized and their heads were fixed using a stereotaxic apparatus. The surgical area was disinfected, the skin was lightly cut with a blade, and a hole was made in the skull. Then 5 μL of 5×10 5 The PBS cell suspension of C6 cells was slowly inoculated into the right striatum of each mouse within 5 minutes (2.0 mm from the dorsal side of the bregma, 2.0 mm from the right lateral side of the sagittal suture, and 3.0 mm in depth). 5-7 days after inoculation, magnetic resonance imaging was used to monitor and confirm whether the orthotopic glioma model was successfully established. The established orthotopic glioma model was used for subsequent in vivo anti-tumor treatment experiments.

[0106] The ICR mice with successfully constructed C6 orthotopic glioma were randomly divided into 6 groups, with 5 mice in each group: PBS, Ce6-ELP@TM, Ce6-ELP@TM / MM, PBS+US, Ce6-ELP@TM+US, Ce6-ELP@TM / MM+US (Ce6 = 5 mg / kg -1 , 100 μL), the injection method was tail vein injection, and the ultrasound treatment group was treated with ultrasound at 1 MHz, 1.5 W cm 2 h after intravenous injection. -2 , 2 minutes of treatment. The day of treatment was recorded as day 1, and treatment was conducted once every 2 days, for a total of 4 times. At the same time, the magnetic resonance imaging system was used to monitor and record the changes in tumor volume on days 0, 3, 6, and 9, and Magnevist (9.38 mg / mouse) was used as a contrast agent. Tumor volume (V) was calculated according to the following formula: V = a × b 2 / 2, where a represents the measured length of the tumor and b represents the measured width of the tumor. Fig.15(ab), relative to the PBS group, each treatment group showed different degrees of tumor inhibition effect, and the tumor volume growth rate decreased. Compared with the non-ultrasound group, the relative tumor volume of mice after ultrasound treatment was significantly reduced, further proving that sonodynamic therapy still played a major role in Ce6-ELP@TM / MM-mediated in vivo anti-tumor treatment. In addition, Ce6-ELP@TM / MM coated with MM had the best inhibitory effect on tumor growth under ultrasound assistance. This phenomenon is due to the fact that MM can promote the immune escape of nanodrugs and prolong their circulation time in the body on the one hand; on the other hand, it can also help nanodrugs cross the BBB and actively target tumor tissues, thereby increasing drug accumulation at the tumor site. Fig.15 (c) is the curve of mouse body weight changes during treatment. The data show that the body weight of tumor-bearing mice in each group decreased slightly, which may be due to the rapid growth of in situ brain glioma, which has a certain impact on the growth of mice.

[0107] Example 16

[0108] To observe the changes of ICD and Mn caused by different treatments 2+ In order to activate anti-tumor immunity in mice, brain tissues of mice in different groups were collected under sterile conditions, and cells were ground and extracted using a 400-mesh cell sieve, and then filtered through a nylon wool column to obtain T cells. The above-extracted T cell suspension was labeled with Anti-CD3-PE / Anti-CD4-FTIC or Anti-CD3-PE / Anti-CD8-FTIC, and the changes in the levels of CD4+T cells and CD8+T cells in the tumor site were analyzed by flow cytometry. The results are shown in Fig.16 As shown in the figure, after different treatments, the level of T cell infiltration in the mouse tumor site was increased, and the level of tumor-infiltrating T cells in the mouse treated with ultrasound was more significantly increased. The Ce6-ELP@TM / MM plus ultrasound group achieved the best immune activation effect due to enhanced tumor accumulation. The activation of T cells in the tumor site indicates that Ce6-ELP@TM / MM can, on the one hand, induce ICD to activate anti-tumor immune response and recruit T cells into tumor tissue through sonodynamic / chemodynamic therapy, and on the other hand, it can utilize Mn 2+ Activation of the cGAS-STING pathway and the maturation of the generated ICD on DCs are used to promote anti-tumor immune response. In general, the nanomedicine Ce6-ELP@TM / MM prepared by the present invention can achieve immune escape after MM biomimetic, obtain prolonged blood circulation time, and achieve brain delivery of drugs across the BBB, thereby performing chemodynamic / sonic dynamic treatment of in situ brain gliomas, and further achieving immunotherapy of tumors by inducing ICD and activating the cGAS-STING pathway.

Claims

1. A composite material based on elastin-like polypeptide, characterized in that: The composite material comprises a metal polyphenol network and nano micelles; wherein the nano micelles are elastin-like polypeptide ELP-coupled sonosensitizer nano micelles, and the metal polyphenol network is coated on the surface of the nano micelles.

2. The composite material based on elastin-like polypeptide according to claim 1, characterized in that: The sonosensitizer includes dihydrochlorin e6; the metal polyphenol network includes drugs and Mn 2+ ; The drug includes tannic acid; The ELP is MW[VPGVG-VPGMG-(VPGVG)2] 20 .

3. A method for preparing a composite material based on elastin-like polypeptide, comprising: (1) activating the sonosensitizer to obtain the activated sonosensitizer; The activated sonosensitizer and ELP are mixed for reaction and post-processed to obtain a sonosensitizer / ELP conjugate; (2) adding the sonosensitizer / ELP solution into stirred water and centrifuging to obtain sonosensitizer / ELP nanomicelles; (3) Mixing the sonosensitizer / ELP nanomicelles, metal salt, and drug, stirring for reaction, and centrifuging to obtain a composite material based on elastin-like polypeptide.

4. The preparation method according to claim 3, characterized in that: The sonosensitizer activation in step (1) comprises: adding DCC and NHS to the sonosensitizer solution for activation; wherein the molar ratio of DCC, NHS, and sonosensitizer is 90-110:90-110:9-11; the activation reaction time is 2-4 hours; and the sonosensitizer comprises dihydrochlorin e6; The molar ratio of the sonosensitizer to the ELP in the step (1) is 28-32:0.9-1.1; the reaction is stirred at room temperature for 2-3 days; the post-treatment in the step (1) is ether precipitation, vacuum drying, dialysis, and freeze drying; during the ether precipitation, the ratio of the volume of ether to the volume of the reaction solution is 9-11:0.9-1.

1.

5. The preparation method according to claim 3, characterized in that: The solvent of the sonosensitizer / ELP solution in step (2) is dimethyl sulfoxide (DMSO); the water is ultrapure water; The centrifugation in step (2) is ultrafiltration centrifugation, wherein the molecular weight cut-off of the ultrafiltration centrifuge tube is 50,000 Da.

6. The preparation method according to claim 3, characterized in that: In the step (3), the mass ratio of the sonosensitizer / ELP nano-micelles, the metal salt, and the drug is 3-7:1-5:0.8-1.2; the metal salt includes a manganese salt; and the drug includes tannic acid; In the step (3), the reaction is stirred for 8 to 12 hours; the centrifugation is ultrafiltration centrifugation, wherein the molecular weight cut-off of the ultrafiltration centrifuge tube is 5000Da.

7. A cell membrane biomimetic nanomedicine, characterized in that: The cell membrane biomimetic nanomedicine comprises a cell membrane and the composite material based on elastin-like polypeptide according to claim 1, wherein the cell membrane covers the composite material based on elastin-like polypeptide.

8. A method for preparing the cell membrane biomimetic nanomedicine according to claim 7, comprising: The composite material based on elastin-like polypeptide and cell membrane are mixed, extruded and centrifuged to obtain cell membrane biomimetic nanomedicine.

9. The preparation method according to claim 8, characterized in that: The cell membrane includes a macrophage membrane MM; the extrusion is repeated 8 to 11 times using a micro extruder with a filter membrane pore size of 300 to 500 nm.

10. Use of the composite material based on elastin-like polypeptide according to claim 1 or the cell membrane biomimetic nanomedicine according to claim 3 in the preparation of MR imaging drugs.

11. Use of the composite material based on elastin-like polypeptide according to claim 1 or the cell membrane biomimetic nanomedicine according to claim 3 in combination with sonodynamic therapy and / or chemodynamic therapy and / or immunotherapy in the preparation of anti-tumor drugs.

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