AUTAC bionic nano-particle, preparation method and application of AUTAC bionic nano-particle

Abstract

The invention provides an AUTAC bionic nano-particle as well as a preparation method and application of the AUTAC bionic nano-particle. The nanoparticle has a core-shell structure, and sequentially comprises a core, a functional peptide layer and a cell membrane shell from inside to outside, the core comprises a PLGA-PEG-Mal copolymer carrier, and entrapped temozolomide and cationic lipid DOTAP (Dioctyl Terephthalate); the functional peptide layer is formed by covalently connecting a multifunctional peptide with a maleimide group through sulfydryl; the cell membrane shell is a microcolloid cell membrane; the nanoparticles can efficiently pass through a blood brain barrier, are enriched at a brain tumor part in a targeted manner, reduce MGMT expression by degrading beta-catenin, reverse TMZ drug resistance and enhance a chemotherapy effect, and are suitable for treating temozolomide drug-resistant glioblastoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an AUTAC biomimetic nanoparticle, a preparation method and application thereof. BACKGROUND

[0002] Glioblastoma (GBM) is the most common and most invasive primary malignant brain tumor in the central nervous system, with high recurrence rate, high mortality and poor prognosis. The current standard treatment regimen is mainly surgical resection, postoperative adjuvant radiotherapy combined with temozolomide (TMZ) chemotherapy. However, the efficacy of TMZ is often limited by the presence of DNA repair mechanisms in tumor cells, and the high expression of O6-methylguanine-DNA methyltransferase (MGMT) is a key factor leading to TMZ resistance. Clinical studies have confirmed that the expression level of MGMT is significantly negatively correlated with the efficacy of TMZ.

[0003] Studies have shown that the Wnt / β-catenin signaling pathway is involved in the regulation of MGMT transcription and is an important mechanism for mediating TMZ resistance. Therefore, inhibiting this signaling pathway has become a potential strategy to enhance TMZ sensitivity. However, the current drug development targeting this pathway faces the following bottlenecks: the side effects of upstream target inhibition are large, and existing studies have focused on targeting Tankyrase, CK1 epsilon, GSK-3 beta and other upstream proteins, but these targets are involved in multiple physiological processes, and their inhibition is prone to cause non-specific toxicity; β-catenin itself is "undruggable", as a key downstream effector molecule of the pathway, β-catenin is structurally disordered and lacks typical drug binding sites, making it difficult to be effectively targeted by traditional small molecule inhibitors; the blood-brain barrier limits drug delivery, even if an effective compound is developed, the blood-brain barrier (BBB) will severely limit its brain entry efficiency, and most drugs lack tumor targeting, which is prone to cause damage to normal brain tissue.

[0004] In recent years, targeted protein degradation technology, especially the AUTAC (Autophagy-Targeting Chimera) strategy based on the autophagy-lysosome pathway, has provided a new way for the degradation of "undruggable" targets. AUTAC guides target proteins into the autophagy degradation pathway through a bifunctional molecule, which is theoretically applicable to proteins such as β-catenin that are difficult to target. However, how to efficiently and specifically deliver AUTAC molecules to intracranial tumor regions and overcome the BBB barrier remains a key challenge for the application of this technology in GBM treatment.

[0005] Therefore, the present application proposes a new type of nanosystem that combines the biomimetic camouflage of microglial cells with the autophagy-mediated protein degradation, aiming to synergistically solve the two core problems of target undruggability and low brain delivery efficiency of drugs, and provide a new solution for reversing TMZ resistance. SUMMARY

[0006] To solve the above problems, the application provides an AUTAC biomimetic nanoparticle, a preparation method and an application thereof. Through microglial cell membrane camouflage, the natural BBB penetration ability and tumor chemotaxis are utilized to realize the precise enrichment of the nanoparticle at the brain tumor site. At the same time, with the help of the specific capture of β-catenin by the surface modified multifunctional peptide, the autophagy activity induced by DOTAP in the core is utilized to guide it to the lysosomal degradation pathway, thereby down-regulating the expression of MGMT from the source. At this time, the expression of MGMT has been inhibited, and the encapsulated TMZ is released in response to the tumor microenvironment, and the DNA damage effect of TMZ can be fully played, thereby realizing the dual synergy of degrading drug-resistant proteins and enhancing the effect of chemotherapy.

[0007] In a first aspect, the application provides an AUTAC biomimetic nanoparticle, characterized in that the nanoparticle has a core-shell structure, and comprises, from inside to outside, a core, a functional peptide layer and a cell membrane shell. The core comprises a PLGA-PEG-Mal copolymer as a carrier, and temozolomide and a cationic lipid DOTAP encapsulated in the carrier. The functional peptide layer is composed of a multifunctional peptide, and the multifunctional peptide is covalently connected to the maleimide group at the end of the PLGA-PEG-Mal copolymer through a thioether bond formed by the thiol group at the end of the multifunctional peptide. The cell membrane shell is a microglial cell membrane wrapped outside the functional peptide layer.

[0008] As a preferred, the multifunctional peptide comprises, in sequence and end-to-end, a β-catenin binding peptide, a cleavable linker peptide for caspase B, and a phosphatidylserine targeting peptide; and the phosphatidylserine targeting peptide is the terminal module of the multifunctional peptide.

[0009] As a preferred, the amino acid sequence of the multifunctional peptide is shown in SEQ ID NO: 1.

[0010] As a preferred, the microglial cell membrane is derived from BV2 cells.

[0011] In a second aspect, the application provides a preparation method of an AUTAC biomimetic nanoparticle, characterized by comprising the following steps: S1: dissolving a PLGA-PEG-Mal copolymer, temozolomide and a cationic lipid DOTAP in an organic solvent to obtain a nanoparticle precursor; S2: coupling the multifunctional peptide with the nanoparticle precursor of S1 through a thiol-maleimide reaction to obtain a peptide-modified nanoparticle; S3: preparing a cell membrane vesicle; S4: co-incubating the peptide-modified nanoparticle of S2 and the cell membrane vesicle of S3 to form the biomimetic nanoparticle with core-shell structure by specific interaction self-assembly.

[0012] Preferably, the mass ratio of the PLGA-PEG-Mal copolymer, temozolomide and cationic lipid DOTAP in S1 is 5:1:1.

[0013] Preferably, the mass ratio of the multifunctional peptide and the nanoparticle precursor in S2 is 1:2-5.

[0014] Preferably, the mass ratio of the peptide-modified nanoparticle and the cell membrane vesicle in S4 is 1:1.

[0015] In a third aspect, the present application provides a use of the AUTAC biomimetic nanoparticle in the preparation of a drug for treating temozolomide-resistant glioblastoma.

[0016] In summary, the present application has at least one of the following beneficial technical effects: (1) The nanoparticle-loaded TMZ provided by the present application can directly exert DNA damage effect, and the beta-catenin degradation mediated by DOTAP and functional peptides can down-regulate MGMT expression from the source and weaken the DNA repair ability. The two synergistic effects fundamentally reverse the TMZ resistance and exhibit a significantly better antitumor effect than the traditional single therapy.

[0017] (2) The nanoparticle provided by the present application breaks through the double barrier of brain drug delivery through its microglial cell membrane shell and surface functional peptides. The membrane camouflage endows the nanoparticle with excellent blood-brain barrier penetration ability, and the targeting module in the functional peptide ensures the precise binding of the particle with tumor cells, so that the nanoparticle can be efficiently enriched in brain tumor lesions.

[0018] (3) The nanoparticle provided by the present application realizes efficient loading and controlled release of drugs, and at the same time realizes precise drug release at the lesion site and activation of the synergistic mechanism through the response of the surface functional peptide to the tumor microenvironment, thereby significantly reducing the systemic toxic side effects while enhancing the therapeutic effect. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the description of the embodiments of the present application will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0020] Figure 1A schematic diagram of synthesis of BM@pPPD / TMZ is shown in the embodiments provided in the present application. Figure 2 A diagram showing in vivo targeted delivery and brain tumor enrichment evaluation of BM@pPPD biomimetic nanoparticles provided in the embodiments of the present application is shown; wherein Figure 2 a is a diagram of in vivo blood circulation curves of different nanoparticles; Figure 2 b is a live fluorescence imaging diagram of tumor-bearing mice; Figure 2 c is an ex vivo brain tissue fluorescence imaging diagram; Figure 2 d is a tumor region fluorescence quantitative analysis diagram; Figure 2 e is a brain tissue fluorescence quantitative analysis diagram; Figure 2 f is a quantitative analysis diagram of nanoparticles in brain tumor tissue; Figure 2 g is a CLSM image diagram of brain tumor frozen sections; Figure 3 A diagram showing mechanism research of BM@pPPD nanoparticles degrading β-catenin through autophagy-lysosome pathway provided in the embodiments of the present application is shown; wherein Figure 3 a is a diagram of β-catenin targeted degradation mechanism; Figure 3 b is a diagram of intracellular co-localization analysis of nanoparticles and β-catenin; Figure 3 c is a diagram of influence of different concentrations of BM@pPPD on expression of autophagy marker protein LC3-II; Figure 3 d is a diagram of influence of BM@pPPD treatment time on expression of autophagy marker protein LC3-II; Figure 3 e is a diagram of flow cytometry detection of autophagosome formation induced by nanoparticles; Figure 3 f is a diagram of complete autophagy flow analysis based on mRFP-GFP-LC3 reporter system; Figure 3 g is a diagram of influence of different concentrations of BM@pPPD on expression of β-catenin and MGMT; Figure 3 h is a diagram of influence of different concentrations of BM@pPPD on expression of β-catenin and MGMT; Figure 3 i is a diagram of influence of autophagy inhibitor 3-MA on BM@pPPD-mediated protein degradation; Figure 3 j is a diagram of influence of lysosome inhibitor CQ on BM@pPPD-mediated protein degradation; Figure 4 A diagram showing in vitro BBB penetration, tumor cell uptake and CTSB-responsive release evaluation of BM@pPPD nanoparticles provided in the embodiments of the present application is shown; wherein Figure 4 a is a schematic diagram of in vitro BBB model construction; Figure 4 b is a diagram of quantitative analysis of BBB penetration efficiency of different nanoparticles; diagram c is a schematic diagram of tumor cell uptake experiment model; Figure 4 d is a CLSM image of GL261 / TR cells uptaking nanoparticles;Figure 4 e is a flow cytometry analysis chart of intracellular nanoparticle content; Figure 4 f is a quantitative statistical chart of cell uptake efficiency; Figure 4 g is a CLSM verification chart of CTSB-responsive connecting peptide cleavage; Figure 5 The in vivo anti-tumor efficacy evaluation chart of BM@pPPD / TMZ in a TMZ-resistant GBM orthotopic model is shown. Wherein Figure 5 a is a schematic diagram of the experimental treatment scheme; Figure 5 b is a live imaging monitoring chart of tumor growth in different treatment groups; Figure 5 c is a quantitative analysis chart; Figure 5 d is a curve chart of the change of mouse body weight during treatment; Figure 5 e is a survival curve chart of tumor-bearing mice; Figure 5 f is a H&E staining and immunohistochemical analysis chart of TUNEL, Ki67, β-catenin, MGMT, and γ-H2A.X of tumor tissue sections; Figure 6 The in vitro anti-tumor activity evaluation chart of BM@pPPD / TMZ on TMZ-resistant GL261 / TR cells is shown. Wherein Figure 6 a is a schematic diagram of synergistic anti-tumor mechanism; Figure 6 b is a CLSM image chart of β-catenin expression after treatment with different preparations; Figure 6 c is a Western blot analysis chart of key proteins LC3, β-catenin, and MGMT; Figure 6 d is a chart of long-term cell proliferation ability detected by colony formation experiment; Figure 6 e is a cell migration ability detection chart; Figure 6 f is a cell invasion ability detection chart; Figure 6 g is a flow cytometry detection chart of cell apoptosis; Figure 6 h is a Western blot analysis chart of DNA damage marker γ-H2A.X; and chart i is a CLSM image chart of γ-H2A.X focus formation. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present application will be described clearly and completely in the present application with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way limiting on the application or its applications or uses. Based on the embodiments in the present application, any person skilled in the art obtains any product same or similar to the present application under the inspiration of the present application or combines the present application with other prior art features, which falls within the protection scope of the present application. In addition, all other embodiments obtained by the person skilled in the art without carrying out creative labor fall within the protection scope of the present application.

[0022] If the specific experimental steps or conditions are not indicated in the embodiments, the operation or conditions can be carried out according to the conventional experimental steps described in the prior art in the field. If the reagents and other instruments are not indicated by the manufacturers, they are all conventional reagent products that can be obtained by purchase. In addition, the drawings are only schematic illustrations of the embodiments of the present application, and are not necessarily drawn to scale. The same reference numerals in the drawings represent the same or similar parts, and thus repeated description thereof will be omitted. Some block diagrams shown in the drawings are functional entities, and do not necessarily correspond to physically or logically independent entities.

[0023] The technologies, methods and devices known to the person skilled in the relevant field can not be discussed in detail, but under appropriate circumstances, the technologies, methods and devices should be regarded as part of the present application.

[0024] In the description of the present application, it should be understood that the use of the words "first", "second" and the like to qualify elements is only for the convenience of distinguishing the corresponding elements, and the above words have no special meaning unless otherwise stated, and therefore cannot be understood as limiting the protection scope of the present application.

[0025] In addition, the technical features involved in different embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0026] In order for the person skilled in the art to more clearly understand the present application, the present application provides an AUTAC biomimetic nanoparticle, a preparation method and an application thereof, which are described in detail through the following embodiments.

[0027] Embodiments Embodiment 1 Preparation of nanoparticles (BM@pPPD / TMZ) S1: weigh PLGA 5k -PEG 5k-Mal copolymer 10 mg, cationic lipid DOTAP 2 mg, TMZ 2 mg, dissolved in 1 mL of dimethyl sulfoxide (DMSO) together, ultrasonic treatment to form a uniform solution. Under the condition of magnetic stirring, the above solution is slowly added to 10 mL of ultrapure water, and stirred for 1 hour, and the nanoparticle precursor is self-assembled by nanoprecipitation method.

[0028] S2: Then, 3 mg of multifunctional peptide is added to the system, and the reaction is stirred at room temperature for 12 hours to complete the covalent coupling of the thiol group at the end of the peptide segment and the maleimide group at the end of PLGA-PEG-Mal through click chemistry. After the reaction is completed, the reaction solution is transferred to a dialysis bag with a molecular weight cut-off of 10,000 Da, and dialyzed in ultrapure water for 12 hours to completely remove uncoupled peptide segments, free TMZ and other small molecular impurities, and finally obtain a purified peptide-modified drug-loaded nanoparticle pPPD / TMZ aqueous dispersion.

[0029] S3: Take the BV2 microglial cells in good growth state, wash with pre-cooled phosphate buffered saline (PBS) for three times, add appropriate amount of cell lysis buffer, and perform ultrasonic crushing under ice bath condition to make the cells fully lysed. The cell lysate is first centrifuged at 800g, 4°C for 10 minutes to remove unbroken cells, nuclei and large cell fragments. The supernatant is carefully collected, and then centrifuged at 12,000g, 4°C for 30 minutes, and the obtained precipitate is the preliminarily purified microglial cell membrane. The membrane precipitate is resuspended with appropriate PBS, and ultrasonic treatment is performed to form a uniform cell membrane vesicle suspension.

[0030] S4: The pPPD / TMZ nanoparticles prepared in S1 are mixed with the microglial cell membrane vesicles prepared in S3 according to a mass ratio of 1:1, and incubated at 37°C for 30 minutes. In this process, the phosphatidylserine (PS) on the inner side of the cell membrane vesicles specifically interacts with the PS targeting peptide at the end of the multifunctional peptide on the surface of the pPPD / TMZ nanoparticles, driving the cell membrane to spontaneously and directionally wrap on the surface of the nanoparticles, and finally forming an AUTAC biomimetic nanoparticle BM@pPPD / TMZ with complete core-shell structure (as shown in Figure 1

[0031] Example 2 Evaluation of the distribution and blood-brain barrier penetration ability of BM@pPPD in vivo ​The embodiment evaluates and verifies the in vivo long-circulating characteristics of the biomimetic nanoparticles BM@pPPD and its ability to cross the blood-brain barrier and target enrichment in glioblastoma by small animal live imaging technology. To perform in vivo tracing, three kinds of fluorescently labeled nanoparticles, pPP / DiD, pPPD / DiD and BM@pPPD / DiD, were prepared. The preparation method is exactly the same as the preparation process of BM@pPPD / TMZ in Example 1, only the loaded drug TMZ is replaced with an equal amount of near-infrared fluorescent dye DiD, so as to obtain nanoparticles with fluorescent signal for subsequent in vivo imaging studies. A C57BL / 6 mouse orthotopic GL261 / TR temozolomide-resistant glioblastoma model was established. After the tumor grew to an appropriate size, the tumor-bearing mice were randomly divided into groups, and an equal dose of pPP / DiD, pPPD / DiD or BM@pPPD / DiD nanoparticles was injected into the tail vein of each group.

[0032] The in vivo fluorescence tracking results show that Figure 2 a), the blood circulation half-life of pPP / DiD and pPPD / DiD nanoparticles without cell membrane coating is relatively short, about 1.7 hours and 1.5 hours respectively. In contrast, BM@pPPD / DiD coated with microglial cell membrane shows significant long-circulating characteristics, with a half-life of about 3.4 hours, which is consistent with the report that cell membrane camouflage technology can effectively avoid rapid clearance by the immune system.

[0033] In the orthotopic GBM model, BM@pPPD / DiD showed rapid accumulation at the brain tumor site, reaching a peak at 6 hours after injection and maintaining until 24 hours after injection Figure 2 b, 2d). The fluorescence signal intensity of pPP / DiD and pPPD / DiD in the brain was always at a low level. Ex vivo brain tissue fluorescence imaging further confirmed that the brain tissue fluorescence intensity of the BM@pPPD / DiD group was significantly higher than that of the other two groups Figure 2 c, 2e).

[0034] The CLSM observation results of brain tissue sections Figure 2 f, 2g) clearly show that BM@pPPD / DiD has the most extensive distribution and the strongest signal in glioblastoma tissue, which is significantly higher than that of pPP / DiD and pPPD / DiD, indicating that it can effectively cross the blood-brain barrier and achieve efficient enrichment in the core area of the tumor.

[0035] Example 3 Mechanism of BM@pPPD degrading β-catenin through autophagy-lysosome pathway The present embodiment verifies the molecular mechanism of BM@pPPD nanoparticles degrading β-catenin through the autophagy-lysosome pathway by various cell molecular biology techniques. Figure 3 b), pPP, pPPD and BM@pPPD can all produce high co-localization with β-catenin, proving that the β-catenin binding peptide in the functional peptide can effectively mediate the specific recognition and binding of the nanoparticles to the target protein. Western blot analysis shows that after BM@pPPD containing DOTAP treatment, the expression level of LC3-II significantly increases in a dose-dependent and time-dependent manner ( Figure 3 c, 3d). MDC staining flow results further confirm that the number of autophagosomes in the pPPD and BM@pPPD treatment groups is significantly higher than that in the pPP group ( Figure 3 e). The mRFP-GFP-LC3 reporter system shows that after pPPD / DiD and BM@pPPD / DiD treatment, the GFP green fluorescence is significantly quenched, and the red fluorescence signal is enhanced, and is significantly co-localized with the nanoparticle signal (DiD) ( Figure 3 f), indicating that the nanoparticles can induce a complete autophagy flow and promote the effective fusion of autophagosomes and lysosomes. Western blot results show that BM@pPPD can promote β-catenin degradation in a dose and time-dependent manner ( Figure 3 g, 3h). At the same time, the expression level of the downstream DNA repair protein MGMT is significantly decreased, indicating that the Wnt / β-catenin signaling pathway is effectively inhibited. After treatment with autophagy inhibitor 3-MA or lysosome inhibitor CQ, the BM@pPPD-mediated β-catenin degradation is significantly inhibited ( Figure 3 i, 3j), proving that the degradation process completely depends on the autophagy-lysosome pathway.

[0036] Example 4 Evaluation of BM@pPPD / TMZ in vitro blood-brain barrier penetration ability and tumor cell uptake The BM@pPPD / TMZ constructed in the present application can improve the delivery efficiency of penetrating the BBB in response to the tumor microenvironment (TME). An in vitro BBB model is established to compare the penetration ability of different nanoparticles ( Figure 4 a). The present embodiment systematically evaluates the ability of BM@pPPD / TMZ to penetrate the blood-brain barrier and its uptake efficiency by tumor cells, and verifies its responsive release characteristics in the tumor microenvironment. The results of the BBB penetration experiment show that the BM@pPPD / TMZ nanoparticles have a better penetration ability than the pPPD nanoparticles ( Figure 4b), the fluorescence intensity of the three nanoparticles in the receptor chamber increased with time, and the cumulative penetration of BM@pPPD / DiD was significantly higher than that of pPP / DiD and pPPD / DiD, which proved that the membrane camouflage of microglia cells could effectively enhance the BBB penetration efficiency of nanoparticles.

[0037] In another in vitro BBB model, GL261 / TR cells were seeded in the Transwell receptor chamber to evaluate the uptake of different nanoparticles by GL261 / TR cells Figure 4 c), CLSM observation showed that Figure 4 d), the uptake efficiency of BM@pPPD / DiD by GL261 / TR cells was significantly higher than that of the other two groups. Flow cytometry quantitative analysis further confirmed that Figure 4 e, f), the average fluorescence intensity of the BM@pPPD / DiD group was 4.4 times and 3.7 times that of the pPP / DiD and pPPD / DiD groups, respectively.

[0038] CLSM results showed that Figure 4 g), after co-culturing DiO-BM@pPPD / DiD with GL261 / TR cells, green fluorescence (DiO, labeling cell membrane) was mainly distributed on the cell surface, while red fluorescence (DiD, labeling nanoparticle core) was mainly present in the cytoplasm. This phenomenon indicates that the high expression of caspase B (CTSB) in the tumor microenvironment specifically cleaves the GLFG linker peptide, achieving the detachment of the cell membrane shell and the effective release of the nanoparticle core.

[0039] This example proves that BM@pPPD nanoparticles, with their microglial cell membrane camouflage, can efficiently cross the blood-brain barrier and be taken up by tumor cells. After entering the tumor microenvironment, the GLFG linker peptide on its surface can be specifically cleaved by overexpressed CTSB, achieving targeted release of the nanoparticle core. This feature makes it a promising brain tumor targeted delivery system.

[0040] Example 5 Therapeutic effect of BM@pPPD / TMZ in TMZ-resistant GBM mouse model This example establishes a TMZ-resistant GBM orthotopic mouse model to systematically evaluate the in vivo anti-tumor effect of BM@pPPD / TMZ and its mechanism of action. The treatment scheme is as follows: Figure 6As shown in Figure a. Using C57BL / 6 mice, GL261 / TR TMZ-resistant glioma cells stably expressing luciferase were implanted into the mouse brain via stereotactic injection to construct an orthotopic GBM model. After tumor formation, the tumor-bearing mice were randomly divided into five groups (n=10 per group): control group (saline), free TMZ group, pPP / TMZ group, pPPD / TMZ group, and BM@pPPD / TMZ group.

[0041] In vivo imaging results showed ( Figure 5 (b, c) The bioluminescent signal intensity of the tumors in the BM@pPPD / TMZ treatment group was significantly lower than that in all other control groups, indicating that its tumor-suppressing effect was the most prominent. Survival analysis results ( Figure 5 e) Further, it was shown that the median survival of mice in the BM@pPPD / TMZ group was significantly prolonged.

[0042] Histological analysis confirmed ( Figure 5 f), the BM@pPPD / TMZ group had the smallest tumor area. TUNEL staining showed that this group had the highest proportion of apoptotic cells and the lowest proportion of Ki67-positive cells, indicating that the treatment can effectively induce apoptosis and strongly inhibit tumor cell proliferation.

[0043] Immunohistochemical analysis showed ( Figure 5 f) Treatment with BM@pPPD / TMZ significantly reduced the expression levels of β-catenin and MGMT in tumor tissues, while significantly increasing the positive signal of γ-H2A.X. This result confirms that BM@pPPD / TMZ successfully degraded β-catenin and downregulated its downstream MGMT expression in vivo, thereby weakening DNA repair capacity and enhancing the cytotoxic effect of TMZ.

[0044] This embodiment demonstrates that BM@pPPD / TMZ biomimetic nanoparticles, in the TMZ-resistant GBM in situ model, effectively inhibit tumor growth and significantly prolong the survival of tumor-bearing mice through a synergistic mechanism of actively targeting tumor tissue, degrading β-catenin, downregulating MGMT expression, enhancing DNA damage, and inducing apoptosis.

[0045] Example 6: Study on the in vitro antitumor activity and synergistic mechanism of BM@pPPD / TMZ This embodiment further evaluates its antitumor activity against GL261 / TR cells in vitro. Figure 6 a). Western blot results showed that pPPD / TMZ and BM@pPPD / TMZ treatment significantly increased LC3-II levels and effectively downregulated the expression of β-catenin and MGMT, while other control groups did not show this effect. Figure 6c). Confocal results further confirmed that the expression of β-catenin in the experimental group was significantly reduced Figure 6 b) Clone formation experiments showed that pPPD / TMZ and BM@pPPD / TMZ could significantly inhibit the clone formation ability of GL261 / TR cells, and the inhibition effect was obviously better than that of TMZ and pPP / TMZ groups Figure 6 d) Transwell experiments showed that pPPD / TMZ and BM@pPPD / TMZ treatment could significantly inhibit the migration and invasion ability of GL261 / TR cells, while the inhibition effect of TMZ and pPP / TMZ groups was not obvious Figure 6 e, f) Flow cytometry detection showed that the apoptosis rate of pPPD / TMZ and BM@pPPD / TMZ treatment groups was significantly higher than that of TMZ group and pPP / TMZ group Figure 6 g) Western blot and confocal results showed that pPPD / TMZ and BM@pPPD / TMZ treatment could significantly enhance the expression and focus formation of γ-H2A.X, indicating that it could cause more serious DNA double-strand breaks Figure 6 h, i).

[0046] This example proves that BM@pPPD / TMZ can down-regulate MGMT expression by degrading β-catenin, weaken DNA repair ability; on the other hand, it can enhance the DNA damage induced by TMZ, and together cause serious DNA double-strand breaks and promote tumor cell apoptosis. The synergistic effect ultimately manifests as effective inhibition of tumor cell proliferation, clone formation, migration and invasion ability, providing an in vitro experimental basis for the clinical application of BM@pPPD / TMZ.

[0047] In summary, the present patent relates to a breakthrough AUTAC biomimetic nanoparticle (BM@pPPD / TMZ) that successfully integrates three functional modules of microglial membrane camouflage, targeted protein degradation and chemotherapy drug into one, providing an innovative solution to overcome the treatment challenge of temozolomide (TMZ)-resistant glioblastoma (GBM). The nanoparticle adopts a unique core-shell structure: the core is composed of PLGA-PEG-Mal copolymer, which co-encapsulates TMZ chemotherapy drug and autophagy activator DOTAP; the middle layer is a multifunctional peptide connected by covalent bond, which has the functions of β-catenin specific binding, tumor microenvironment responsive release and membrane targeting; the outer layer is wrapped with microglial cell membrane that retains the natural targeting protein. This ingenious design enables it to efficiently cross the blood-brain barrier, precisely enrich in brain tumor sites, and exert excellent anti-tumor effect through the synergistic mechanism of "degrading β-catenin-down-regulating MGMT expression-enhancing TMZ efficacy". Systematic in vitro and in vivo experiments demonstrate that the nanoparticle not only significantly prolongs the blood circulation time and improves the tumor targeting efficiency, but also effectively degrades β-catenin, inhibits tumor cell proliferation and migration, induces cell apoptosis and DNA damage, and finally exhibits significantly better therapeutic effect than traditional therapy in TMZ-resistant GBM model. The present patent constructs a perfect product, preparation method and medical application right protection system, and provides a new strategy with important transformation value for the clinical treatment of TMZ-resistant glioblastoma.

[0048] Each embodiment in the specification is described in a progressive manner, and each embodiment focuses on the difference from other embodiments. The same and similar parts between embodiments can be referred to each other.

[0049] Although the preferred embodiments of the present application have been described, those skilled in the art can make additional changes and modifications to the embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the embodiments of the present application.

[0050] Finally, it is to be understood that the phraseology or terminology such as "first" and "second" etc. used herein is merely intended to differentiate one entity or operation from another entity or operation, without necessarily requiring or implying any actual such relationship or order between such entities or operations. Moreover, the terms "comprising", "including", or any other closure, are intended to cover the non-exclusive inclusion such that a process, method, article, or apparatus that comprises a list of elements does not include those elements alone but can include other elements not expressly listed or even include elements inherent in such process, method, article, or apparatus. Without more limitations, the element defined by the statement "comprising a" does not exclude the presence of additional identical elements in the process, method, article, or apparatus that includes the element.

[0051] The above provides a detailed introduction to the AUTAC biomimetic nanoparticles, preparation method and application of the present application. In this paper, specific examples are used to explain the principles and implementation of the present application. The above examples are only used to help understand the method and core idea of the present application; at the same time, for those skilled in the art, according to the idea of the present application, the specific implementation and application range will be changed; in view of the above, the content of the specification should not be understood as a limitation of the present application.

Claims

1. An AUTAC biomimetic nanoparticle, characterized in that, The nanoparticles have a core-shell structure, comprising a core, a functional peptide layer and a cell membrane shell from inside to outside; The core comprises a PLGA-PEG-Mal copolymer as a carrier, and temozolomide and a cationic lipid DOTAP encapsulated in the carrier; The functional peptide layer is composed of a multifunctional peptide covalently connected to the PLGA-PEG-Mal copolymer through a thioether bond formed by the terminal thiol group of the multifunctional peptide and the terminal maleimide group of the PLGA-PEG-Mal copolymer; The cell membrane shell is a microglial cell membrane wrapped outside the functional peptide layer.

2. The AUTAC biomimetic nanoparticle of claim 1, wherein, The multifunctional peptide comprises, in sequence and end-to-end, a β-catenin binding peptide, a cleavable linker peptide for B-type caspase and a phosphatidylserine targeting peptide; and the phosphatidylserine targeting peptide is the terminal module of the multifunctional peptide.

3. The AUTAC biomimetic nanoparticle according to any one of claims 1 or 2, wherein, The amino acid sequence of the multifunctional peptide is shown in SEQ ID NO:

1.

4. The AUTAC biomimetic nanoparticle of claim 1, wherein, The microglial cell membrane is derived from BV2 cells.

5. A method of preparing an AUTAC biomimetic nanoparticle as claimed in any one of claims 1 to 4, characterised in that, The method comprises the following steps: S1: dissolving a PLGA-PEG-Mal copolymer, temozolomide and a cationic lipid DOTAP in an organic solvent to obtain a nanoparticle precursor; S2: coupling a multifunctional peptide with the nanoparticle precursor of S1 through a thiol-maleimide reaction to obtain a peptide-modified nanoparticle; S3: preparing a cell membrane vesicle; S4: co-incubating the peptide-modified nanoparticle of S2 and the cell membrane vesicle of S3 to form the AUTAC biomimetic nanoparticle with a core-shell structure through specific interaction self-assembly.

6. The method for preparing AUTAC biomimetic nanoparticles according to claim 5, characterized in that, The mass ratio of the PLGA-PEG-Mal copolymer, temozolomide and cationic lipid DOTAP in S1 is 5:1:

1.

7. The method for preparing AUTAC biomimetic nanoparticles according to claim 5, characterized in that, The mass ratio of the multifunctional peptide to the nanoparticle precursor in S2 is 1:2-5.

8. The method for preparing AUTAC biomimetic nanoparticles according to claim 5, characterized in that, The mass ratio of the peptide-modified nanoparticle to the cell membrane vesicle in S4 is 1:

1.

9. Use of the AUTAC biomimetic nanoparticle of any one of claims 1-8 in the preparation of a medicament for treating temozolomide-resistant glioblastoma.

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