Bionic nano-drug delivery system for targeted regulation of immune function of microglial cells

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and relates to a bionic nano-drug delivery system for targeted regulation of microglial cell immune function, in particular to a bionic nano-drug delivery system for targeted regulation of microglial cell immune function and having the effect of an immune checkpoint inhibitor and application of the bionic nano-drug delivery system in brain glioma immunotherapy. The drug delivery system is a bionic nano drug delivery system prepared from an oleanolic acid nanocrystal core (OA-core), an M0 or M2 type macrophage membrane and an acid-sensitive lipid material connected with immune checkpoint antagonistic polypeptide. The bionic nano-drug delivery system constructed by the invention can realize co-loading of small molecule drugs and polypeptide drugs and precise delivery and graded release of focus parts, regulates and controls the brain glioma immune process in a multi-target manner, synergistically enhances the anti-glioma immune effect, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and relates to a bionic nano drug delivery system for targeted regulation of microglial immune function, and specifically to a bionic nano drug delivery system for targeted regulation of microglial immune function and having the effect of immune checkpoint inhibitor, and its application in brain glioma immunotherapy. Background Art

[0002] According to literature, glioblastoma (abbreviated as brain glioma) is the most malignant intracranial tumor. Currently, the clinical practice mainly adopts a combination of surgical resection combined with radiotherapy and chemotherapy, which has a poor prognosis and a high tumor recurrence rate. The 5-year survival rate of patients is only 5.8%. The emerging immunotherapy can inhibit tumor growth by mobilizing the body's own immune function and can produce a long-term immune memory effect to inhibit tumor recurrence, bringing hope to the treatment of brain glioma. However, commonly used immunotherapy strategies, such as immune checkpoint blockade (ICB), tumor vaccines, chimeric antigen receptor T cell (CAR-T) and oncolytic viruses, have not shown good anti-glioma effects. The core reasons are mainly two: (1) The existence of barrier systems such as the blood-brain barrier (BBB) ​​makes it difficult for large-molecule antibody drugs such as ICB to enter the brain; (2) Current immunotherapy strategies mainly target peripheral immune cells such as T cells and dendritic cells, but gliomas are immune "cold" tumors and lack infiltration of peripheral immune cells. Therefore, promoting the entry of immunotherapy-related drugs into the brain and improving the function of innate immune cells in the brain are new development directions for improving the effect of immunotherapy for gliomas.

[0003] Microglia are innate immune cells in the central nervous system. Studies have confirmed that microglia are also the largest proportion of immune cells in the microenvironment of gliomas. When gliomas occur, microglia infiltrate into the tumor site and promote T cell-mediated anti-tumor immune responses. However, due to the large presence of immunosuppressive cytokines such as interleukin-6 (IL-6) in the tumor microenvironment, the immunosuppressive pathway STAT-3 (Signal Transducer and Activator of Transcription 3) in microglia is overactivated, resulting in a significant weakening of immune functions such as microglial recruitment, activation of T cells, and antigen presentation, ultimately leading to the progression of gliomas. Therefore, inhibiting the abnormal activation of the STAT-3 pathway in microglia is expected to improve their immune function and promote the recruitment and activation of effector T cells in gliomas. However, considering that the highly expressed immune checkpoint proteins in brain gliomas, such as PD-L1 (Programmed cell deathligand-1), can inhibit the recognition and killing function of T cells on tumor cells, the combination of regulating microglia function and ICB strategy is expected to achieve multi-target immunotherapy for brain gliomas.

[0004] Oleanolic acid (OA) can reduce the expression of phosphorylated STAT-3 (p-STAT-3) protein and inhibit the activation of the STAT-3 pathway, so it can be used as a microglial immunopotentiator. Immune checkpoint antagonist peptides are a new type of peptide ICBs, which have been shown to have strong affinity with PD-L1 / PD-1 and can promote the killing function of T cells. In addition, compared with traditional macromolecular antibody ICBs, peptide ICBs have the advantages of simple preparation, low production cost, and easy functionalization, so they are suitable for the treatment of brain gliomas. However, the two drugs have different physicochemical properties (OA: hydrophobic; immune checkpoint antagonist peptides: hydrophilic), different target cells (OA: microglia; immune checkpoint antagonist peptides: PD-L1 on the surface of tumor cells), and the two drugs also have problems such as poor stability in the blood and poor BBB permeability. It is difficult to fully exert the synergistic effect by direct combination. Therefore, it is necessary to construct a suitable nanosystem to co-deliver OA and immune checkpoint antagonist peptides and achieve the concentration of the two drugs at their respective target sites.

[0005] Cell membrane bionic nanosystems have been widely used in cancer treatment research due to their good biological properties. Cell membranes can inherit the biological functions of specific source cells (such as red blood cells, monocytes, tumor cells and platelets), which not only helps the nanosystem escape immune clearance and prolong blood circulation time, but also can use the inflammatory tendency of cell membrane proteins to target the nanosystem to the tumor lesion site. The occurrence and development of brain gliomas are accompanied by a large number of macrophage infiltrations. Studies have found that integrin α4 / β1 (Integrin α4 / β1) and lymphocyte function-associated antigen-1 (Lymphocyte Function-associated Antigen-1, LFA-1) are overexpressed on the surface of macrophage membranes, which can specifically bind to intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the surface of inflammatory BBB. Therefore, some researchers have used this characteristic to design macrophage membrane bionic nanosystems to promote drug entry into the brain. Our previous work confirmed that ICAM-1 and VCAM-1 are also highly expressed on the surface of microglia, so the biomimetic strategy of macrophage membrane is also expected to achieve microglia targeting, which has not been reported in the literature. In addition, the phenotype of macrophages in patients and lesions (mainly divided into M0, M1 and M2) will change with the progression of tumors and other diseases. It is not clear which phenotype of macrophage membrane has better tropism for brain glioma, which needs to be studied in depth.

[0006] At present, the literature studies the use of polymer nanoparticles prepared from polymer materials such as polylactic acid and polylactic-co-glycolic acid as the core of the cell membrane. Such polymer nanoparticles have strong rigidity and good biosafety, but the introduction of polymer materials will greatly reduce the drug loading of the nanosystem and increase the preparation cost. Observing the molecular structure of OA, the molecule contains exposed hydroxyl / carboxyl groups (-OH / -COOH) and pentacyclic triterpenoid structures. The intermolecular hydroxyl / carboxyl groups can form hydrophilic hydrogen bonds, while the pentacyclic triterpenoid skeleton structure in the molecule can serve as a hydrophobic group. Therefore, it is expected that the hydrophilic and hydrophobic interactions between OA molecules can be used to prepare nanocores through self-assembly to increase the drug loading of the nanosystem and simplify the preparation process.

[0007] Clinical practice studies have shown that the nanosystem must quickly release immune checkpoint antagonist peptides after reaching the brain glioma microenvironment to facilitate its interaction with the PD-L1 receptor on the surface of tumor cells.

[0008] Based on the foundation and current status of the prior art, the inventors of the present application took into account that the pH of the slightly acidic environment of the tumor is 6.5-6.8, and therefore connected the immune checkpoint antagonist polypeptide to the end of the lipid material distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) through a pH-sensitive bond, and then inserted it into the cell membrane of the bionic nano-preparation, in order to provide a bionic nano-drug delivery system for targeted regulation of the immune function of microglia. The bionic nano-drug delivery system of the present invention can ensure that the pH-sensitive bond is located on the outside of the nano-system, which is beneficial to its hydrolysis and cleavage in the weak acid environment of the glioma to release the immune checkpoint antagonist polypeptide, while not affecting the remaining nano-system to deliver OA into the microglia. Summary of the invention

[0009] The purpose of the present invention is to overcome the defects of existing brain glioma immunotherapy by constructing a bionic nano drug delivery system to efficiently regulate the immune function of microglia and deliver immune checkpoint inhibitors into the brain, thereby synergistically enhancing the immunotherapy effect of brain glioma based on the foundation and status of the prior art. The nano system is simple to prepare, has the advantages of strong targeting, good safety, and high drug loading, and can achieve the co-loading of drugs with different physical and chemical properties, as well as the graded release of drugs in different target sites (microglia and tumor cell extracellular microenvironment), thereby effectively inhibiting the progression and recurrence of brain glioma.

[0010] The bionic nano drug delivery system of the present invention has a core-shell structure. Its core is composed of oleanolic acid nanocrystals; the shell is an M0 or M2 macrophage membrane, which, by coating the core, gives the delivery system long circulation characteristics, inflammatory BBB and microglia cascade targeting; the acid-sensitive lipid material connected with the immune checkpoint antagonist polypeptide is inserted into the macrophage membrane, and when the bionic nano system reaches the tumor microenvironment, the acid-sensitive bond can be hydrolyzed and broken under pH 6.5-6.8 to release the immune checkpoint antagonist polypeptide.

[0011] The oleanolic acid nanocrystal core described in the present invention is prepared by a solvent exchange method. Specifically, OA powder is dissolved in an appropriate amount of organic solvent, sucked with a syringe, and dripped into stirred (500 rpm to 2000 rpm) water at a speed of 0.1 mL / min, and after continuing to stir for 1 to 2 hours, the probe is ultrasonically treated (temperature: 0°C to 5°C, power: 60W to 220W, time: 30s to 120s), and the organic solvent is removed by rotary evaporation to obtain the oleanolic acid nanocrystal core. The organic solvent is selected from one of methanol, ethanol, and acetone, and the ratio of the organic phase to the aqueous phase is 1:10 to 1:30 (v / v). In order to reduce the aggregation tendency of nanocrystals, a stabilizer is added in the preparation, and the stabilizer is selected from one of lecithin, sodium cholate, vitamin E polyethylene glycol succinate (TPGS), poloxamer 188 (F-68), hydroxypropyl methyl cellulose (HPMC), sodium dodecyl sulfate (SDS), and Tween-80. The stabilizer is pre-added to an organic solvent or water according to its properties, and the concentration range of the stabilizer is 0.1% to 2.0%.

[0012] The macrophage membrane described in the present invention is extracted from M0 or M2 macrophages. The extraction of cell membrane is mainly divided into the following two steps: (1) Cell culture and polarization: The mouse mononuclear macrophage leukemia cells RAW264.7 with good growth status are inoculated into a cell culture dish, and serum-free culture medium and culture medium containing IL-4 (10-100 ng / mL) are added to culture the cells for 24-48 hours. The cells cultured in serum-free culture medium are M0 macrophages. The cells cultured in IL-4 culture medium are taken out and blown away, and CD206 are obtained by flow cytometry sorting. + Cell group, inoculate the group of cells into serum-containing culture medium, and continue to culture for 48 hours to obtain M2 macrophages. (2) Cell membrane extraction: Wash the cells with PBS (5mL), add 0.25×PBS hypotonic buffer 10mL, let stand at room temperature for 10min, scrape the cells in the culture bottle with a cell scraper, pipette the cell suspension into a centrifuge tube, let stand for 12h, freeze at -80℃ for 30min, take out and place in a constant temperature water bath (37℃) to thaw, repeat the freezing-thawing process 3 times. The treated suspension is centrifuged at low speed (1200rpm, 10min), the cell content precipitate is discarded, the supernatant is centrifuged at high speed (14500rpm, 2h, 4℃), the supernatant is discarded, and the precipitate is collected as M0 or M2 macrophage membrane.

[0013] The M0 and M2 macrophage membranes described in the present invention have significantly higher expression levels of targeting molecules such as Integrins α4 and LFA-1 on their surfaces than those of the M1 macrophage membranes; the targeting properties of the two macrophage membrane-coated bionic nanopreparations to brain capillary endothelial cells (representing the inflammatory BBB) and microglia pretreated with tumor conditioned medium are also significantly better than those of the M1 macrophage membrane-coated bionic nanopreparations.

[0014] In the present invention, the macrophage membrane is coated with oleanolic acid nanocrystals by ultrasonic extrusion. Specifically, (1) the macrophage membrane and oleanolic acid nanocrystals are fully mixed in water at a mass ratio of 1:1 to 8:1, and probe ultrasound is applied (temperature: 0°C to 5°C, power: 60W to 120W, time: 10s to 30s); (2) the system after ultrasound treatment is absorbed by a liposome extruder, and extruded through 400nm and 200nm polycarbonate porous membranes in turn, and a total of 3 to 5 cycles are performed to obtain macrophage membrane-coated nanoparticles (M@OA).

[0015] The immune checkpoint antagonist polypeptide of the present invention is selected from D One of PPA-1 peptide (d-NYSKPTDRQYHF), PD-L1Pep1 peptide (CLQKTPKQC), PD-L1Pep2 peptide (CVRARTR), SPAM peptide (MPIFLDHILNKFWILHYA), DT-300 peptide (SNGLSQPV), TPP-1 peptide (SGQYASYHCWCWRDPGRSGGSK). These peptides have been shown to have high binding affinity with PD-L1 and have the effect of blocking PD-L1.

[0016] The acid-sensitive lipid material described in the present invention is synthesized from DSPE-PEG-OH lipid raw material with a molecular weight of 2000-8000Da; the acid-sensitive bond connecting the immune checkpoint antagonist polypeptide and DSPE-PEG is selected from a maleic acid amide bond and a cis-aconitic acid amide bond.

[0017] The acid-sensitive lipid material described in the present invention is inserted into the macrophage membrane by co-incubation. Specifically, the acid-sensitive lipid material connected to the immune checkpoint antagonist polypeptide is mixed with M@OA and stirred at room temperature (speed: 500 rpm ~ 2000 rpm, time: 1 ~ 4h) to obtain a bionic nano drug delivery system. In order to ensure the efficacy of the immune checkpoint antagonist polypeptide without affecting the targeting effect of the macrophage membrane, the modification ratio of the acid-sensitive lipid material connected to the immune checkpoint antagonist polypeptide on the nanosystem is controlled at 5% ~ 30% (w / w).

[0018] The particle size of the biomimetic nano drug delivery system prepared by the present invention is 100 to 160 nm, the Zeta potential is -16 to -27 mV, and the drug loading of OA is 12% to 25%. The biomimetic nano drug delivery system shows the same protein bands as the macrophage membrane and the expression of key targeting ligands (Integrin α4 / β1 and LFA-1), which provides a molecular basis for its targeting. The biomimetic nano drug delivery system can be stable for at least 4 days in the environment of PBS or 10% fetal bovine serum, and it has good acid-sensitive response drug release characteristics, and is almost non-cytotoxic (cell viability>80%) at the tested concentration.

[0019] The bionic nano drug delivery system prepared by the present invention has a pharmacokinetic study in rats, which shows that its half-life in blood (t 1 / 2 ) was significantly prolonged and the clearance rate (CL) was significantly reduced compared with that of OA nanocrystals alone, indicating that macrophage membrane coating prolonged the drug circulation time in the blood. After intravenous injection of the bionic nanodrug delivery system, its distribution in the brain of tumor-bearing mice increased significantly, and it was mainly concentrated in the microglia in the glioma area.

[0020] The bionic nano drug delivery system prepared by the present invention can effectively inhibit the over-activation of the STAT-3 pathway in brain gliomas, significantly increase the expression of T cell recruiting cytokines (CXCL-9), immune co-stimulatory molecules (CD80 & CD86) and histocompatibility complex (MHC II) on microglia, and reduce the differentiation of M2 (tumor-promoting) microglia, thereby effectively improving the immune function of microglia in the tumor microenvironment.

[0021] The bionic nano drug delivery system prepared by the present invention can significantly increase the two effector T cells in the glioma site - cytotoxic T cells (CTL, CD3 + CD8 + ) and helper T cells (Th, CD3 + CD4+)--, reducing the proportion of regulatory T cells (Treg, CD3 + CD4 + Foxp3 + ), and promoted the production of granzyme B in the tumor site, increasing the apoptosis rate of tumor cells, confirming that the bionic nanodrug delivery system played a synergistic role between the two drugs and effectively enhanced the killing effect of effector T cells on tumor cells.

[0022] The bionic nano drug delivery system prepared by the present invention can significantly inhibit the growth of brain gliomas and prolong the median survival of tumor-bearing mice when used alone or in combination with temozolomide. After the bionic nano drug delivery system is administered to recurrent model mice stimulated by tumor cell fragments, the growth of recurrent tumors is significantly slowed down, the immune memory response of the body is stimulated, and the recurrence of brain gliomas is effectively inhibited.

[0023] The outstanding advantages of the present invention are:

[0024] (1) By utilizing the excellent targeting properties of M0 or M2 macrophage membranes, the drug's brain penetration efficiency and ability to target microglia were greatly improved, providing an experimental basis for the expansion of macrophage membrane bionic strategies.

[0025] (2) Hydrophobic drugs and hydrophilic drugs are co-loaded into the same drug delivery system, thus avoiding the difference in in vivo pharmacokinetic between the two drugs due to their different physicochemical properties. The two drugs can be delivered to the site of brain glioma at a preset therapeutic ratio, thus fully exerting their synergistic effect.

[0026] (3) The immune checkpoint antagonist peptide is connected to the outside of the nanoformulation through an acid-sensitive bond, so that it can be released at a specific point in the tumor microenvironment and better exert the PD-L1 blocking effect.

[0027] (4) It provides a therapeutic approach to improve the immunosuppressive microenvironment in brain gliomas by enhancing the immune function of microglia inherent to the central nervous system. This approach overcomes the difficulty of existing immunotherapy strategies in enhancing the function of peripheral immune lymphocytes to inhibit the progression of gliomas, and provides a new idea for improving the immunotherapy effect of brain gliomas. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : DSPE-PEG-Ama-Peptide H NMR spectrum.

[0029] Figure 2 : DSPE-PEG-UAslink-Peptide H NMR spectrum.

[0030] Figure 3 : DSPE-PEG-Ama- D PPA-1, DSPE-PEG-Aac- D PPA-1 and DSPE-PEG-UAslink- D Degradation of PPA-1 in buffers with different pH values ​​(pH=6.6 or 7.4).

[0031] Figure 4 :(A) Expression of ICAM-1 and VCAM-1 on bEnd.3 cells under tumor inflammation state (B) Expression of ICAM-1 and VCAM-1 on BV-2 cells.

[0032] Figure 5 :The expression of Integrinα4, Integrinβ1 and LFA-1 on the membrane of M0, M1 and M2 macrophages.

[0033] Figure 6 : Fluorescence images of M0@OA, M1@OA and M2@OA taken up by bEnd.3 cells and BV2 cells.

[0034] Figure 7 : Effects of different peptide modification ratios (5% to 30%) on the targeting effect of PSM@OA.

[0035] Figure 8 : Transmission electron microscopy images of OA-core, PSR@OA and PSM@OA.

[0036] Fig. 9 :Surface protein characterization of biomimetic nanodrug delivery system: SDS-PAGE and Western Blot images.

[0037] Fig.10 : Characterization of release behavior of bionic nanodrug delivery system at different pH: (A) TPP-1 peptide release curve; (B) OA release curve.

[0038] Fig.11 :Blood drug concentration-time curve of bionic nano drug delivery system.

[0039] Fig.12 : Evaluation of brain targeting of biomimetic nano-drug delivery system.

[0040] Fig.13 :Evaluation of microglia targeting of biomimetic nanodrug delivery system: (A) Fluorescence observation of BV2 cell uptake; (B) Flow cytometry of BV2 cell uptake; (C) Distribution of the preparation in the brain glioma area. Fig.14 :Bionic nanodrug delivery system inhibits overexpression of STAT-3 pathway in microglia.

[0041] Fig.15 :The bionic nano drug delivery system promotes microglia (A) to secrete CXCL-9 (B) to express CD80 / CD86 (C) to express MHC II.

[0042] Fig.16 :Bionic nanodrug delivery system reduces the differentiation of microglia into M2 type (tumor-promoting type).

[0043] Fig.17 :Bionic nano-drug delivery system enhances T cell-mediated anti-tumor immune effect: (A) Th (CD45 + CD3 + CD4 + ) cells (B) CTL (CD45 + CD3 + CD8 +) cells and (C) Treg (CD45 + CD3 + CD4 + Foxp3 + ) Changes in cell ratio; (D) Positive signals of granzyme B in brain glioma; (E) TUNEL staining results.

[0044] Fig.18 :Bionic nanodrug delivery system combined with temozolomide inhibits the growth of brain glioma: (A) Representative bioluminescence images of tumor-bearing mice; (B) Tumor growth curves of each group; (C) Survival curves of tumor-bearing mice.

[0045] Fig.19 :Bionic nano-drug delivery system inhibits the recurrence of brain glioma: (A) Representative bioluminescence images of relapsed mice; (B) Growth curve of relapsed tumors; (C) H&E staining images of the brains of tumor-bearing mice; (D) Survival curve of relapsed mice; (E) PSM@OA promotes the differentiation of immune memory T cells. DETAILED DESCRIPTION

[0046] The present invention is illustrated by the following description and examples, which are non-limiting and do not limit the scope of the claims of the present invention.

[0047] Example 1: Synthesis of Acid-Sensitive Lipid Materials Connected by Maleic Acid Amide Bonds

[0048]

[0049] 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid (CDM) (0.5mmol) was dissolved in 3mL dry dichloromethane, oxalyl chloride (4mmol) was added, and the mixture was fully mixed under nitrogen protection, and the mixture was stirred at 0℃ for 3h, and then stirred at room temperature for 12h. The unreacted oxalyl chloride was removed by rotary evaporation, and the product was dissolved by adding dry dichloromethane (3mL). Under nitrogen protection, pyridine (10μL) and DSPE-PEG-OH (0.1mmol) were added and reacted at room temperature for 48h. The reaction was terminated by adding an excess of saturated ammonium chloride solution, and the organic phase was collected. Cold ether (-20℃, 10mL) was added and the precipitation was repeated 3 times, and the solid was collected and vacuum dried for 24h to obtain the intermediate product DSPE-PEG-S1 with a yield of 70%.

[0050] Under nitrogen protection, the immune checkpoint antagonist peptide and DSPE-PEG-S1 were dissolved in dry DMSO (7 mL) at a molar ratio of 1:1, stirred at room temperature for 0.5 h, and triethylamine (10 μL) was added dropwise. The mixture was reacted at 45 ° C for 48 h, cooled to room temperature, dialyzed in a Na2CO3 aqueous solution (pH = 8.0) for 48 h, and freeze-dried to obtain the acid-sensitive lipid material DSPE-PEG-Ama-Peptide connected by maleic acid amide bonds, with a yield of 53%. Figure 1 As shown, it shows the NMR spectrum of DSPE-PEG-Ama-Peptide: 1 H NMR (600MHz, DMSO-d6): -CH3(inDSPE): δ=0.85ppm, -CH2-CH2-: δ=1.34–1.14ppm, -CH3(inmaleic acid amide bond): δ=2.26ppm, Pyrrolidine-H: 2.61ppm, -OCH2-CH2-O-: δ=3.51ppm, -N-CH2-CH2-CH2-N-: δ=1.55–1.39ppm, -N-CH2-CH2-CH2-N-: δ=1.61ppm, -COO-CH2-CH2-: δ=1.72ppm, Ph-H: 7.29–6.59ppm, Imidazole-H: 8.28–7.58ppm.

[0051] Example 2: Synthesis of acid-sensitive lipid materials linked by cis-aconitic acid amide bonds

[0052]

[0053] Dissolve cis-3-carboxyglutamate anhydride (DAA, 0.5mmol) in 5mL dry dichloromethane, add oxalyl chloride (4mmol), mix thoroughly under nitrogen protection, stir at room temperature for 4h, add oxalyl chloride (4mmol) in an ice-water bath, and continue to react for 8h. Remove unreacted oxalyl chloride by rotary evaporation, add 5mL dry dichloromethane to dissolve the product, add pyridine (10μL) and DSPE-PEG-OH (0.1mmol), and react at room temperature for 48h under nitrogen. Add saturated ammonium chloride solution to terminate the reaction, collect the organic phase, repeat precipitation with cold ether (-20℃, 10mL) 3 times, filter, collect the solid and vacuum dry for 24h to obtain the intermediate product DSPE-PEG-S2, with a yield of 55% to 65%.

[0054] Under nitrogen protection, the immune checkpoint antagonist peptide and DSPE-PEG-S2 were dissolved in dry DMSO (10 mL) at a molar ratio of 1:1.5, stirred at room temperature for 0.5 h, and triethylamine (20 μL) was added dropwise. The reaction was carried out at room temperature for 72 h. The product was dialyzed in a Na2CO3 aqueous solution (pH = 7.6) for 48 h and freeze-dried to obtain the acid-sensitive lipid material DSPE-PEG-Aac-Peptide connected by cis-aconitic acid amide bonds, with a yield of 60% to 65%.

[0055] Example 3: Synthesis of acid-insensitive lipid materials

[0056]

[0057] Distearoylphosphatidylethanolamine-polyethylene glycol-succinimide ester (DSPE-PEG-NHS) and immune checkpoint antagonist peptide were dissolved in dry DMF (5 mL) at a molar ratio of 1:1, and stirred at room temperature for 48 hours under nitrogen protection. The product was dialyzed in pure water for 48 hours and freeze-dried to obtain the acid-insensitive lipid material DSPE-PEG-UAslink-Peptide with a yield of 76%.

[0058] Attached Figure 2 This is the NMR spectrum of DSPE-PEG-UAslink-peptide: 1 HNMR(600MHz,DMSO-d6):-CH3(in DSPE): δ=0.85ppm, -CH2-CH2-: δ=1.23ppm, Pyrrolidine-H: 2.61ppm, -OCH2-CH2-O-: δ=3.51ppm, -N-CH2-CH2-CH2-N-: δ=1.5 9–1.43ppm, -N-CH2-CH2-CH2-N-: δ=1.61ppm, -COO-CH2-CH2-: δ=1.72ppm, Ph-H: 7.34–6.53ppm, Imidazole-H: 8.16–7.54ppm.

[0059] Example 4: Investigation of pH sensitivity of acid-sensitive lipid materials

[0060] Weigh two peptides connected with immune checkpoint antagonists D Acid-sensitive lipid material of PPA-1 (DSPE-PEG-Ama- D PPA-1, DSPE-PEG-Aac- D PPA-1 and acid-insensitive lipid materials (DSPE-PEG-UAslink- DPPA-1) 5 mg each was dissolved in 1 mL of phosphate buffer with different pH values ​​(pH 6.6 and pH 7.4), placed in a 37°C thermostat for shaking, and samples were taken at 0, 1, 2, and 4 h, respectively, and analyzed by HPLC to see if there was D The signal peak of PPA-1 peptide was generated.

[0061] Figure 3 The three lipid materials were shown to break and release under different pH conditions. D In the case of PPA-1 peptide, the two acid-sensitive lipid materials have good stability under normal physiological conditions (pH = 7.4), but are degraded and released under the slightly acidic environment of the tumor (pH = 6.6). D The rate of PPA-1 peptide was significantly accelerated, and DSPE-PEG-Ama- D PPA-1, DSPE-PEG-Aac- D PPA-1 was degraded by about 76% and 65% in 4 hours, respectively, which confirmed that the two materials had good acid sensitivity and could release peptides in the tumor microenvironment. D PPA-1 was not degraded and released under both pH conditions D The PPA-1 peptide was shown to be pH-insensitive and was suitable as a control for comparative studies.

[0062] Example 5: Preparation of Oleanolic Acid Nanocrystal Core

[0063] Weigh 3 mg of OA accurately, dissolve in 1 mL of methanol, ethanol and acetone respectively, and sonicate in a water bath until completely dissolved. Draw with a syringe and drip into purified water containing different stabilizers at a rate of 100 μL / min, with the volume ratio of organic phase to aqueous phase being 1:10 to 1:30. After the addition is completed, stir vigorously for 1 hour, use probe sonication, and remove the organic solvent by rotary evaporation to obtain OA nanocrystals. The selected stabilizers are as follows: TPGS (0.5%), lecithin (0.5%, 0.2%) are added to the organic solvent to dissolve; F-68 (0.5%), HPMC (2%, 0.5%, 0.1%), sodium cholate (0.5%), SDS (0.5%), and Tween-80 (1%, 0.5%) are added to the water to dissolve.

[0064] Table 1 shows that the particle size of OA nanocrystals prepared by three organic solvents is <200nm. However, if no stabilizer is added during preparation, the OA nanocrystals tend to aggregate with the prolonged storage time, and the particle size and PDI increase significantly. After adding several stabilizers, the aggregation trend of OA nanocrystals can be inhibited, and the particle size distribution range is narrow (as shown in Table 2). Further, the particle size and PDI of OA nanocrystals can be reduced by probe ultrasound, indicating that their distribution is more uniform (as shown in Table 3).

[0065] Table 1 Oleanolic acid nanocrystals prepared by three organic solvents (n=3)

[0066]

[0067] Table 2 Oleanolic acid nanocrystals prepared with different stabilizers (n=3)

[0068]

[0069] Table 3 Oleanolic acid nanocrystals prepared under different ultrasonic conditions (n=3)

[0070]

[0071]

[0072] Example 6: Preparation of cell membrane-coated nanoparticles

[0073] Macrophage membranes and erythrocyte membranes were collected by the cell membrane extraction method described in the aforementioned invention content. The cell membrane was coated on OA nanocrystals using a method combining probe ultrasound and extruder extrusion. Macrophage membranes were mixed with OA nanocrystals at a mass ratio of 1:1, 2:1, 4:1 and 8:1, and after probe ultrasound (120w, 30s, ultrasound 1s interval 1s), the system was sequentially extruded through 400nm and 200nm polycarbonate porous membranes using an extruder to obtain macrophage membrane-coated nanoparticles (M@OA) of different membrane thicknesses. Table 4 shows that with the increase in the proportion of cell membranes, the particle size of the nanoparticles increased slightly and the Zeta potential decreased slightly. The same method was used to prepare erythrocyte membrane-coated nanoparticles (R@OA) as a non-targeted control.

[0074] Table 4 Biomimetic nanoformulations with different OA / macrophage membrane ratios (n=3)

[0075]

[0076] Example 7: Targeting receptor expression on inflammatory endothelial cells and microglia

[0077] Co-incubation of cells in the tumor microenvironment with tumor culture medium is a relatively mature method to simulate the tumor microenvironment. Mouse glioma cells GL-261 were cultured until the cell density was greater than 90%, the culture medium was discarded, PBS was washed three times, DMEM basal culture medium was added and cultured for 12 hours, the culture medium was collected, centrifuged at 1000rpm for 4 minutes, and the upper liquid was taken to obtain the tumor conditioned medium.

[0078] Take brain capillary endothelial cells bEnd.3 or microglial cells BV-2 in good growth state and plant them in 6-well plates. Add 2 mL of serum-free, anti-antibody-free DMEM basal medium or 2 mL of the above-collected tumor conditioned medium to each well. After culturing for 24 hours, add FITC-labeled ICAM-1 flow cytometry antibody (1:100) or PE-labeled VCAM-1 flow cytometry antibody (1:100) for staining, and detect the fluorescence intensity by flow cytometry. Figure 4 As shown in the figure, after stimulation with tumor conditioned medium, the positive rate of ICAM-1 on bEnd.3 cells was increased by about 13.7%, and the positive rate of VCAM-1 was increased by about 44.9%; there was no obvious change in the expression of ICAM-1 on BV2 cells (the positive rate itself was 85.7%), while the positive rate of VCAM-1 was increased by about 18.7%. These results indicate that under the influence of the tumor microenvironment, the BBB exhibits an inflammatory state, and the high expression of ICAM-1 and VCAM-1 on the two cells provides a molecular basis for the bionic nanodrug delivery system to penetrate the BBB and target microglia.

[0079] Example 8: Extraction and targeting ability comparison of macrophage membranes of different phenotypes

[0080] RAW264.7 cells in good growth state were inoculated in a cell culture dish, and serum-free medium, medium containing lipopolysaccharide (100 ng / mL), and medium containing IL-4 (100 ng / mL) were added to culture the cells for 48 hours. Cells cultured in serum-free medium are M0 macrophages. Cells cultured in lipopolysaccharide or IL-4 medium were taken out and blown away, and stained with two flow cytometry antibodies: APC anti-mouse CD86 and PE anti-mouse CD80, respectively, and sorted by flow cytometry to obtain CD86 + and CD206 + Cell populations. The two cell populations were inoculated into serum-containing culture medium and cultured for 48 hours to obtain M1 and M2 macrophages.

[0081] According to the cell membrane extraction method described in the above invention, macrophage membranes of three phenotypes were collected, and appropriate amounts were added to the cell membrane protein extraction kit, lysed on ice, and centrifuged to obtain cell membrane protein solutions. The expression of three key target proteins (Integrinα4, Integrinβ1, and LFA-1) was detected by Western blot. Figure 5It can be seen that the expression levels of Integrinβ1 on the membranes of the three phenotypes of macrophages are basically the same, but the expression levels of Integrinα4 and LFA-1 on the membranes of M0 and M2 macrophages are significantly higher than those on the membranes of M1 macrophages. To further investigate the effect of different phenotypes of macrophage membranes on the targeting of nanoparticles, three macrophage membrane-coated nanoparticles were prepared according to Example 6, and the preparations were labeled with the fluorescent dye DiI, which were recorded as M0@OA, M1@OA and M2@OA, respectively. The nanoparticles were co-incubated with bEnd.3 cells or BV2 cells pretreated with tumor conditioned medium for 2 hours. The cell uptake results showed that the uptake of M0@OA and M2@OA by both cells was significantly higher than that of M1@OA (see Appendix). Figure 6 ), indicating that these two phenotypes of macrophage membranes have better targeting. Therefore, M0 and M2 macrophage membranes were selected for the construction of bionic nano-drug delivery system.

[0082] Example 9: Preparation of biomimetic nano drug delivery system

[0083] The acid-sensitive lipid material with immune checkpoint antagonist peptides was inserted into the macrophage membrane by co-incubation method to obtain a biomimetic nano drug delivery system (PSM@OA). 1 mg DSPE-PEG-Ama-Peptide or DSPE-PEG-Aac-Peptide was added to 2.5 mg M@OA, stirred at low speed at 37 ° C, and samples were taken at 0.5, 1, 2, 4 and 6 h to determine the co-incubation time. According to the experimental results in Table 5, after incubation for 1 h, the particle size of the obtained PSM@OA gradually stabilized, the PDI change decreased, and the final incubation time was 1 to 4 h.

[0084] According to the optimized incubation time, PSM@OA with immune checkpoint antagonist peptide modification ratio of 5% to 30% (w / w) was prepared. In addition, DSPE-PEG-Ama-Peptide or DSPE-PEG-Aac-Peptide was co-incubated with red blood cell membrane-coated nanoparticles (R@OA) to prepare a non-targeted biomimetic nanodrug delivery system (PSR@OA); DSPE-PEG-UAslink-Peptide was co-incubated with M@OA to prepare a biomimetic nanodrug delivery system without pH response characteristics (PNM@OA).

[0085] Table 5 Bionic nano drug delivery system obtained at different co-incubation times (n=6)

[0086]

[0087] Example 10: Effects of different peptide modification ratios on the targeting of biomimetic nano-drug delivery systems

[0088] According to the optimization method of Example 9, PSM@OA with immune checkpoint antagonist peptide modification ratios of 5%, 15% and 30% (w / w) were prepared respectively. Figure 7 The results showed that PSM@OA with three modification ratios could be taken up in large quantities by bEnd.3 cells and BV2 cells, and the cell fluorescence intensity was close to that of the M@OA group. There was no significant difference between the different polypeptide modification ratios, indicating that this modification range (5% to 30%) had no significant effect on the targeting of the delivery system.

[0089] Example 11: Characterization of biomimetic nano drug delivery system

[0090] The PSM@OA prepared in Example 9 was characterized, and the results showed that its average particle size was 100-160 nm, the Zeta potential was -16-27 mV, the PDI was <0.25, the drug loading of OA was 12%-25%, and the modification ratio of the immune checkpoint antagonist peptide on the nanoparticles was 5%-30%. The transmission electron microscopy results showed that compared with OA-core, PSM@OA had a distinct cell membrane layer structure (attached Figure 8 ). By comparing the total protein staining results of PSM@OA and macrophage membrane, it was found that the protein bands of the two were basically the same, and it successfully retained several key ligands such as Integrinα4 / β1 and LFA-1 on the cell membrane surface, laying the foundation for its in vivo targeting (Appendix Fig. 9 OA nanocrystals alone were stable for 2 days under storage conditions (PBS) and physiological conditions (10% FBS). On the third day, the particle size increased significantly and even precipitated, indicating poor stability. However, the particle size of PSM@OA did not change after 4 days, indicating that macrophage membrane coating significantly improved the stability of the nanoparticles.

[0091] Example 12: Acid-sensitive responsive drug release of biomimetic nano drug delivery system

[0092] The acid-sensitive drug release characteristics of PSM@OA prepared in Example 9 were investigated, and the drug release of PNM@OA without pH responsiveness was used as a control. PBS with different pH values ​​was used to simulate different release environments after the preparation entered the body: normal physiological environment (pH 7.4), weakly acidic tumor microenvironment (pH 6.6), and degradation environment of intracellular acidic organelles (late endosomes / lysosomes) (pH 5.5). Fig.10A shows that PSM@OA can quickly release the immune checkpoint antagonist peptide TPP-1 at pH 6.6, with a cumulative release of about 69% in 24 hours, while at pH 7.4, the cumulative release of TPP-1 peptide is less than 10%, indicating that the prepared PSM@OA has good stability under normal physiological conditions, and once it reaches the tumor microenvironment, it can quickly release the peptide, which is beneficial for it to play a role in blocking the highly expressed PD-L1 on tumor cells. PNM@OA, which is not pH sensitive, almost does not release peptides under two pH conditions.

[0093] The cumulative release of OA in 24h of PSM@OA under normal physiological conditions and in the micro-acid environment of tumors was 26.9% and 23.9%, respectively, while the cumulative release in the intracellular degradation environment reached 78.5%, confirming that the nanoparticles had less OA leakage due to the coating of macrophage membranes before entering the target cells, and after entering the cells, the degradation of cell membranes by lysosomes would accelerate the release of OA molecules (attached Fig.10 B).

[0094] Example 13: Cytotoxicity Evaluation of Biomimetic Nano-drug Delivery System

[0095] The CCK-8 method was used to investigate the toxicity of PSM@OA to two target cells. bEnd.3 cells and BV2 cells were cultured at 5×10 4 / hole and 1×10 4 / well were inoculated in a 96-well culture plate. After 24h of cell culture, free OA and PSM@OA were added, respectively, with OA concentrations ranging from 0 to 100μmol / L, and incubated at 37℃ for 24h. The drug-containing culture medium was discarded, and 100μL of diluted CCK8 solution was added to each well and incubated for 1h. The absorbance value was measured and recorded using a multifunctional microplate reader, and the cell survival rate (Cell Survial Rate, CSR) was calculated. Results Within the safe concentration range (0.5-10μmol / L) of OA to inhibit STAT-3 overactivation, the CSR of both cells was greater than 80%, proving that PSM@OA had no obvious growth inhibitory effect on both cells and had good safety.

[0096] Example 14: Pharmacokinetic evaluation of biomimetic nano drug delivery system

[0097] Six healthy SD rats were randomly divided into two groups: OA-core group and PSM@OA group. The rats were injected with tail vein at a dose of 10 mg OA / kg. Blood (500 μL) was collected from the rats' orbits at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h after administration and placed in sodium heparin anticoagulation tubes. The rats were centrifuged at 4°C and 5000 rpm for 15 min, and the upper plasma was collected. The OA concentration in the blood at each time point was determined by LC-MS / MS, and the blood drug concentration-time curve was drawn, and the pharmacokinetic parameters were calculated (as shown in Table 6).

[0098] Table 6 Main pharmacokinetic parameters (n=3)

[0099]

[0100] Depend on Fig.11 The experimental results shown in Table 6 show that after intravenous injection of OA-core, its blood concentration decreased rapidly and was lower than the quantitative limit after 4 hours, indicating that the OA nanocrystal core was easily cleared. The blood concentration of PSM@OA decreased significantly, and its half-life (t 1 / 2 ) was significantly prolonged and the clearance rate (CL) was significantly reduced, indicating that PSM@OA can significantly prolong the circulation time of OA in the blood and increase its chance of entering the brain.

[0101] Example 15: Evaluation of brain targeting of biomimetic nano-drug delivery system

[0102] Orthotopic tumor-bearing mice were used to investigate the brain targeting of PSM@OA. Five days after tumor inoculation, mice were injected with free dye DiR, PSR@OA / DiR or PSM@OA / DiR (1 mg DiR / kg) via tail vein. 24 hours after administration, the mice were anesthetized and cardiac perfused with saline. The heart, liver, spleen, lung, kidney and brain were removed and observed and photographed using a small animal in vivo imaging device to record the fluorescence value. Fig.12 The results of in vitro imaging showed that the accumulation of PSM@OA in the brain was significantly higher than that of the red blood cell membrane-coated nanoparticle (PSR@OA) and the free fluorescent probe DiR, and its fluorescence signal intensity was 3.49 times that of the PSR@OA group and 4.03 times that of the free DiR group. In addition, brain fluorescence was mainly distributed in the glioma site, confirming that macrophage membrane coating effectively promoted the nanoparticles to pass through the BBB and reach the brain tumor area.

[0103] Example 16: Evaluation of microglial targeting of biomimetic nano-drug delivery system

[0104] PSM@OA and PSR@OA were labeled with fluorescent dye DiI, and the two nanoparticles were incubated with microglial cells BV2 at 37°C for 2 h. Laser confocal microscopy was used to observe and photograph, and flow cytometry was used for quantitative analysis. Fig.13AB shows that the uptake of free DiI, PSR@OA and PSM@OA by BV2 cells increased successively, among which the uptake of PSM@OA group increased by 2.89 times compared with that of PSR@OA group.

[0105] Tumor-bearing mice were injected with free DiI and DiI-labeled PSM@OA and PSR@OA (1 mg DiI / kg) via the tail vein. 24 hours after administration, the mice were anesthetized and cardiac perfused, brain tumor tissues were removed, frozen sections were prepared, microglia were immunofluorescently stained (using TMEM-119 primary antibody), and laser confocal microscopy was used to observe and photograph. The results are shown in the attached figure. Fig.13 As shown in Figure C, microglia mainly accumulate in the tumor edge area, free DiI and PSR@OA hardly co-localize with microglia, while PSM@OA can clearly co-localize with microglia. These results fully confirm that nanoparticles coated with M0 or M2 macrophage membranes can effectively target microglia in the microenvironment of brain glioma.

[0106] Example 17: Biomimetic nano-drug delivery system inhibits excessive activation of microglial STAT-3 pathway

[0107] BV2 cells were inoculated in 12-well plates and cultured for 24 h. DMEM culture medium containing PBS, IL-6, IL-6+OA, IL-6+PSR@OA or IL-6+PSM@OA was added to the wells, respectively. The concentration of inflammatory cytokine IL-6 was 100 ng / mL and the concentration of OA was 5 nmol / mL. The cells were incubated at 37°C for 24 h. The expression of STAT-3 and p-STAT-3 (phospho Y705) in microglia was detected by Western blot. The results are shown in the attached figure. Fig.14 As shown in the figure, IL-6 can significantly activate the STAT-3 pathway in BV2 cells by promoting the phosphorylation of STAT-3 protein (p-STAT-3). OA, PSR@OA and PSM@OA can significantly downregulate the expression of p-STAT-3 after administration, among which PSM@OA has the strongest inhibitory effect, and the expression of p-STAT-3 is reduced by 1.82 times and 1.37 times compared with the OA group and PSR@OA group, respectively.

[0108] Example 18: Bionic nano drug delivery system improves the immune function of microglia

[0109] BV2 cells were inoculated in 12-well plates and cultured for 24 h. DMEM culture medium containing PBS, IL-6, IL-6+OA, IL-6+PSR@OA or IL-6+PSM@OA was added to the wells, respectively, where the concentration of inflammatory cytokine IL-6 was 100 ng / mL and the concentration of OA was 5 nmol / mL. The cells were incubated at 37°C for 24 h. ELISA was used to detect the secretion of T cell recruitment factor CXCL-9, and flow cytometry was used to determine the expression levels of costimulatory molecules CD80 / CD86 and major histocompatibility complex MHC II on the surface of BV2 cells.

[0110] By the attached Fig.15 A shows that after BV2 cells were stimulated by IL-6, the secretion of CXCL-9 was significantly reduced. The reason is that overactivation of the STAT-3 pathway will reduce the expression of CXCL-9, resulting in a weakened ability of microglia to recruit T cells. PSM@OA can significantly upregulate the expression of CXCL-9, which is 2.32 times that of the IL-6 group and basically the same as the unstimulated group, indicating that PSM@OA is expected to restore the ability of microglia to recruit T cells in the tumor microenvironment. Fig.15 B As can be seen, after administration of PSM@OA, the positive rate of the two co-stimulatory molecules increased significantly compared with the IL-6 group, an increase of about 8.9%, and only about 1.3% lower than that of normal BV2 cells, indicating that PSM@OA has the potential to enhance the expression of CD80 / CD86 on microglia in the brain tumor microenvironment, thereby initiating anti-tumor immune responses in the brain. MHC II molecules are essential for the body's adaptive immune process. After treatment with PSM@OA, the expression of MHC II on BV2 cells increased significantly, increasing by 13.2%, 7.3% and 6.3% compared with the IL-6 group, IL-6+OA group and IL-6+PSR@OA group, respectively (see Appendix). Fig.15 C), indicating that PSM@OA can significantly enhance the expression of MHC II on microglia, thereby promoting the occurrence of antigen presentation process inside brain glioma.

[0111] Example 19: Biomimetic nano-drug delivery system reduces differentiation of M2 microglia in tumors

[0112] Tumor-bearing mice were injected with saline, DT-300 peptide, DT-300+OA, PSR@OA, PNM@OA and PSM@OA through the tail vein. The dose of DT-300 peptide and OA was 10 mg / kg, and the drug was administered once a day for 7 days. After the administration, the mice were anesthetized, and brain tumor tissues were taken. Flow cytometry was used to count the number of M2 microglia (TMEM 119 + CD206 + The results are shown in the attached Fig.16As shown in Figure 2, compared with other groups, PSM@OA could significantly reduce CD206 + The proportion of microglia indicates that PSM@OA can indeed reduce the differentiation of tumor-promoting M2 microglia, which is beneficial to improving the immunosuppressive microenvironment of brain glioma and blocking tumor progression.

[0113] Example 20: Biomimetic nano-drug delivery system enhances T cell-mediated anti-tumor immunity

[0114] Tumor-bearing mice were injected with saline, PD-L1Pep2, PD-L1Pep2+OA, PSR@OA and PSM@OA through the tail vein. The dose of PD-L1Pep2 peptide was 5 mg / kg and the dose of OA was 10 mg / kg. The drugs were administered once a day for 7 days. After the administration, the mice were anesthetized and brain tumor tissues were taken. Flow cytometry was used to count the two main effector T cells that play an anti-tumor role - cytotoxic T cells (CTL, CD3 + CD8 + ) and helper T cells (Th, CD3 + CD4 + ) and the percentage of Treg cells (CD3 + CD4 + Foxp3 + ) percentage. Fig.17 As shown in AC, the number of CTL cells and Th cells in the brain glioma of the Saline group was relatively small, while the number of Treg cells with immunosuppressive effects was large. This result is consistent with the literature report that brain glioma has "cold" tumor characteristics and insufficient effector T cell infiltration. After 7 days of treatment, PSM@OA significantly increased the proportion of Th cells and CTL cells in brain glioma, while greatly reduced the proportion of Treg cells, and the effect was significantly better than other treatment groups, confirming that PSM@OA can promote the infiltration of effector T cells and inhibit the formation of tumor suppressive microenvironment.

[0115] The tumor-killing effect of T cells was further characterized by examining the expression of granzyme B in tumor tissue and the level of TUNEL staining. Fig.17 As shown in D, the PSM@OA group significantly increased the expression level of granzyme B, which was 2.29 times that of the Saline group. Reason: PSM@OA can not only release PD-L1Pep2 peptides in the tumor microenvironment, but also enhance the immune function of microglia and promote the recruitment of effector T cells in brain tumors. The two work together, so the expression of granzyme B in this group is the highest. Due to the enhanced killing ability of T cells, the PSM@OA group has a better effect of promoting tumor cell apoptosis (see Appendix Fig.17E), indicating that the constructed bionic nanodrug delivery system has the effect of promoting T cell-mediated anti-glioma immunity.

[0116] Example 21: Biomimetic Nano-drug Delivery System Enhances Tumor Suppression Effect of Chemotherapeutic Drug Temozolomide

[0117] Temozolomide (TMZ) is a first-line drug for the clinical treatment of brain glioma, but the prognosis of patients using this drug alone is poor. This example investigates whether the bionic nano drug delivery system can enhance the chemotherapy effect of brain glioma from the perspective of evaluating the clinical application potential of PSM@OA.

[0118] Orthotopic tumor-bearing mice were randomly divided into 6 groups: Saline group, TMZ group, PSM@OA group, TMZ+ D In the PPA-1+OA group, TMZ+PSR@OA group, and TMZ+PSM@OA group, TMZ was administered orally at a dose of 50 mg / kg; nanoparticles were administered by tail vein injection. D The dosage of PPA-1 peptide was 5 mg / kg, and the dosage of OA was 10 mg / kg. Each preparation was administered for 7 consecutive days, once a day. The bioluminescence intensity of the brain tumor site of each tumor-bearing mouse was regularly monitored using a small animal in vivo imaging device, and the corresponding tumor growth curve was drawn. At the same time, the survival of each group of mice was observed and recorded, and the median survival time was calculated.

[0119] By the attached Fig.18 AB showed that the use of TMZ or PSM@OA alone had a certain tumor-suppressing effect, and the tumor growth rate was significantly slower than that of the Saline group, but neither of them could effectively inhibit the progression of the tumor. On the 21st day, the tumor signals of the two groups were similar to those of the Saline group. In contrast, the tumor growth curves of all combination groups were flatter than those of the Saline group and the single-use group, indicating that the combination of chemotherapy and immunotherapy improved the therapeutic effect of brain glioma. Among them, the tumor signal of mice in the TMZ+PSM@OA group was still low on the 24th day. The survival curve results were similar. TMZ+PSM@OA treatment was more effective in prolonging the median survival time of tumor-bearing mice than other groups, from 11 days in the Saline group to 41.5 days (such as Fig.18 C). The results indicate that the combination of TMZ and PSM@OA can more effectively inhibit the progression of brain glioma.

[0120] Example 22: Bionic nano drug delivery system inhibits the recurrence of brain glioma

[0121] One of the major advantages of immunotherapy is that it can activate the body's own immune system to produce immune memory effects. When antigens, pathogens, or abnormally proliferating cells that have previously invaded the body reappear, the memory cells in the immune system will quickly differentiate into effector cells, produce immune responses, and restore the body's homeostasis in a timely manner. Therefore, this example focuses on whether PSM@OA has the potential to inhibit the recurrence of brain gliomas.

[0122] First, tumor fragments were injected into the mouse brain to simulate the brain microenvironment after radiotherapy and chemotherapy. The mice were randomly divided into 4 groups every other day: Saline group, PD-L1Pep1+OA group, PSR@OA group and PSM@OA group. The drugs were injected into the tail vein. Each group was given the drug once a day for 7 days. The dose of PD-L1Pep1 peptide was 5 mg / kg, and the dose of OA was 10 mg / kg. After the administration, the mice were inoculated with brain glioma cells in situ, and the tumor growth was monitored by bioluminescence. On the ninth day after tumor inoculation, the mice were killed and the brains were removed for HE staining. At the same time, the spleen lymphocytes of the mice were extracted, and the effector memory T cells (CD62L – CD44 + ) and central memory T cells (CD62L + CD44 + ) ratio.

[0123] By the attached Fig.19 AB shows that the PSM@OA group did not show obvious bioluminescent signals in the mouse brain until the 9th day after tumor inoculation, while the other groups showed obvious signals from the 3rd to the 6th day after tumor inoculation; H&E staining results also showed that the tumor volume in the PSM@OA group was the smallest (see Appendix Fig.19 C); in terms of survival time, the median survival time of the Sham group, Saline group, PD-L1Pep1+OA group, PSR@OA group, and PSM@OA group was 8 days, 9 days, 8 days, 12 days, and 25 days, respectively (Appendix Fig.19 D), these results reflect that PSM@OA can effectively inhibit the recurrence of brain glioma and thus prolong the survival time of tumor-bearing mice.

[0124] By the attached Fig.19 E shows that the proportion of effector memory T cells in the spleen of the PSM@OA group reached 19.4%, and the proportion of central memory T cells reached 45.3%, which were significantly higher than those of other control groups. This shows that PSM@OA promotes the differentiation of effector T cells into memory T cells, allowing the body to produce long-term anti-tumor function.

Claims

1. A bionic nano drug delivery system for targeting and regulating the immune function of microglia, characterized in that: The bionic nano drug delivery system is composed of three parts: oleanolic acid nanocrystals as the core, covering the macrophage membrane, and an acid-sensitive lipid material connected to an immune checkpoint antagonist peptide inserted into the membrane: in, The macrophage membrane is extracted from M0 macrophages or M2 macrophages; The modification ratio of the acid-sensitive lipid material connected to the immune checkpoint antagonist polypeptide on the nanosystem is 5% to 30% (w / w).

2. The bionic nano drug delivery system according to claim 1, characterized in that: The preparation method of the oleanolic acid nanocrystal core is a solvent exchange method; the organic solvent is selected from one of methanol, ethanol and acetone; the ratio of the organic phase to the aqueous phase is 1:10-1:30 (v / v).

3. The biomimetic nano drug delivery system according to claim 2, characterized in that: The oleanolic acid nanocrystal core is prepared by adding a stabilizer, which is selected from one of lecithin, sodium cholate, vitamin E polyethylene glycol succinate (TPGS), poloxamer 188 (F-68), hydroxypropyl methyl cellulose (HPMC), sodium dodecyl sulfate (SDS), and Tween-80; the concentration of the stabilizer is 0.1% to 2.0%.

4. The bionic nano drug delivery system according to claim 1, characterized in that: The M0 macrophages are selected from the RAW264.7 cell line; the method for obtaining the M2 macrophages is as follows: the RAW264.7 cell line is stimulated with 10 to 100 ng / mL interleukin-4 (IL-4) for 24 to 48 hours, and then sorted by flow cytometry to obtain the M2 macrophages.

5. The bionic nano drug delivery system according to claim 1, characterized in that: The mass ratio of the macrophage membrane to the oleanolic acid nanocrystal core is 1:1 to 8:

1.

6. The bionic nano drug delivery system according to claim 1, characterized in that: In the acid-sensitive lipid material connected to the immune checkpoint antagonist polypeptide, the immune checkpoint antagonist polypeptide is selected from D One of PPA-1 peptide (d-NYSKPTDRQYHF), PD-L1Pep1 peptide (CLQKTPKQC), PD-L1Pep2 peptide (CVRARTR), SPAM peptide (MPIFLDHILNKFWILHYA), DT-300 peptide (SNGLSQPV), and TPP-1 peptide (SGQYASYHCWCWRDPGRSGGSK).

7. The bionic nano drug delivery system according to claim 1, characterized in that: In the acid-sensitive lipid material connected to the immune checkpoint antagonist polypeptide, the lipid raw material is distearoyl phosphatidylethanolamine-polyethylene glycol-hydroxy (DSPE-PEG-OH) with a molecular weight of 2000 to 8000 Da.

8. The bionic nano drug delivery system according to claim 1, characterized in that: In the acid-sensitive lipid material connected to the immune checkpoint antagonist polypeptide, the acid-sensitive bond connecting the polypeptide can be selected from one of a maleic acid amide bond and a cis-aconitic acid amide bond, which is hydrolyzed and cleaved under acidic conditions of pH 6.5 to 6.

8.

9. The method for preparing the biomimetic nano drug delivery system according to claim 1, characterized in that: It includes: (1) Preparation of oleanolic acid nanocrystals: an organic phase containing oleanolic acid is dripped into water at a rate of 0.1 mL / min, stirred for 1 to 2 h, and then subjected to ultrasonic wave (temperature: 0°C to 5°C, power: 60 W to 220 W, time: 30 s to 120 s) to remove the organic solvent by rotary evaporation; wherein the stabilizer is pre-dissolved in an organic solvent or water according to its properties; (2) extracting M0 or M2 macrophage membranes by hypotonic method; mixing macrophage membranes with oleanolic acid nanocrystals, ultrasonicating with probe (temperature: 0°C to 5°C, power: 60W to 120W, time: 10s to 30s), and then extruding through a liposome extruder (passing through 400nm and 200nm polycarbonate porous membranes in sequence), for 3 to 5 cycles in total, to obtain macrophage membrane-coated nanoparticles (M@OA); (3) Synthesis of an acid-sensitive lipid material connected to an immune checkpoint antagonist peptide: DSPE-PEG-OH is modified to synthesize a lipid material precursor with a pH-sensitive acid anhydride, wherein the solvent and reactants used in the reaction must be anhydrous (water content less than 10 ppm); and then the amino group of the immune checkpoint antagonist peptide undergoes a nucleophilic ring-opening reaction with the lipid material precursor to obtain the acid-sensitive lipid material. (4) The acid-sensitive lipid material connected to the immune checkpoint antagonist peptide was inserted into the M@OA membrane, and the two were incubated at room temperature for 1 to 4 hours.

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