Multi-mode brain-targeted intelligent nano-drug system aiming at brain glioma and preparation method of multi-mode brain-targeted intelligent nano-drug system

Abstract

The invention discloses a multi-mode brain-targeted intelligent nano-drug system aiming at brain glioma and a preparation method of the multi-mode brain-targeted intelligent nano-drug system. According to the system, a dual enhancer MnO2 nanoparticle capable of combining a chemodynamic therapy and an immunotherapy is loaded with a traditional chemotherapeutic drug doxorubicin hydrochloride to serve as an inner core, and the outer layer is coated with a bionic lipid membrane with vitamin D3 and a neutrophile granulocyte membrane. According to the system, the inflammation chemotaxis and adhesion capacity of bionic neutrophil are utilized, specific delivery of glioma is achieved, manganese dioxide is further reduced into divalent manganese ions by responding to glutathione (GSH) in the tumor microenvironment in the glioma microenvironment, doxorubicin hydrochloride and vitamin D3 are released, finally cGAS-STING immune response is synergistically activated, and the glioma is specifically delivered. The compound can promote tumor cell apoptosis and reverse glioma immunosuppression microenvironment, and is especially used for multi-mode combined treatment of brain glioma.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a multimodal brain-targeted intelligent nanomedicine system for brain glioma and a preparation method thereof. Background Art

[0002] Gliomas are malignant tumors originating from glial cells, accounting for about 80% of all malignant tumors of the central nervous system. At present, the clinical treatment principle for gliomas is to remove glioma tissue as much as possible through surgery, and then combine temozolomide (TMZ) for radiotherapy and chemotherapy. However, since gliomas usually invade deep into the brain and functional areas of the brain, the range of surgical resection is limited, and there are characteristics of infiltrative growth, which makes it difficult to completely remove tumor cells during surgery. Due to the existence of the blood-brain barrier, many small molecule chemotherapy drugs are difficult to reach the central nervous system. Temozolomide, which is currently used as a first-line drug, has the disadvantage of easy drug resistance. Radiotherapy is intolerant and has short-term and long-term toxicity, which may cause side effects such as radiation brain damage and hearing loss. Therefore, the limitations of clinical treatment options have led to an extremely high recurrence rate in patients with gliomas receiving treatment. Even with the most aggressive treatment measures, the median survival of patients with malignant gliomas is still less than 15 months. However, the emerging immuno-oncology therapies are limited by high costs and low response rates, resulting in only 20% of patients benefiting from them. Most importantly, immune T lymphocytes are difficult to infiltrate into the glioma tumor microenvironment, making gliomas, a "cold tumor", naturally low in response to immune checkpoint inhibitor therapy. The response and maintenance of T cell immune responses require the activation of antigen presenting cells and innate immunity. Stimulating the innate immune system to increase the activation, expansion and infiltration of cytotoxic T lymphocytes in the glioma microenvironment may be a breakthrough in improving the current brain glioma immunotherapy.

[0003] The cGAS-STING signaling pathway is part of the innate immune system and plays an important role in signaling in sensing cytoplasmic DNA and immune defense. Its activation is not limited to a specific tumor type, but has a broad-spectrum immune activation effect on a variety of tumors, and can trigger systemic specific immune responses, including stimulating the activity of immune cells such as T cells and dendritic cells, thereby enhancing the body's resistance to tumors, which makes it one of the current hot research directions. The pattern recognition receptor cGAS (Circular GMP-AMP synthetase) is a nuclease that recognizes double-stranded DNA in the cytoplasm and catalyzes the production of endogenous cyclic dinucleotides (CDN): 2′-3′ cyclic GMP-AMP (cGAMP), thereby activating the interferon activator protein (Stimulator of interferon genes, STING). STING is a transmembrane protein located on the endoplasmic reticulum. After being activated, it regulates cells to produce type I interferon and pro-inflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor (TNF-α), which can then activate human immune cells (such as dendritic cells and natural killer cells), thereby increasing the infiltration of cytotoxic T lymphocytes and the effect of natural killer cells killing tumors. At the same time, it also inhibits myeloid-derived suppressor cells to achieve the purpose of inhibiting tumor growth. However, because the second messenger cGAMP synthesized by the cGAS receptor is a hydrophilic and negatively charged molecule, it is difficult to transport between cells to activate other immune cells. This situation makes it difficult to amplify the immune response mediated by the cGAS-STING pathway, which in turn affects its ability to achieve the desired therapeutic effect.

[0004] This paper explores a new idea for the combined treatment of brain glioma based on chemotherapy, chemodynamic therapy and immunotherapy. For the first time, MnO, a dual enhancer that can combine chemodynamic therapy and immunotherapy, is used to treat glioma. 2 The combination of nanoparticles and doxorubicin hydrochloride for the treatment of brain gliomas has complementary advantages and provides a highly potential clinical treatment strategy. Summary of the invention

[0005] In view of the limitations of current clinical treatment methods for gliomas and the low targeting of brain tumor drug delivery systems, the purpose of the present invention is to provide a multimodal brain-targeted intelligent nanodrug system for gliomas. The nanodrug delivery system has excellent biocompatibility and can actively target the glioma site by crossing the blood-brain barrier.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned cell membrane bionic co-delivery system.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] A multimodal brain-targeted smart nanodrug system for glioma, consisting of an inner core and an outer layer; the inner core is manganese dioxide nanoparticles loaded with doxorubicin hydrochloride; the outer layer is vitamin D 3 The inner core and the outer layer are wrapped and fused by ultrasound, and the weight ratio of the inner core to the outer layer is 1:5-10. The doxorubicin hydrochloride is a small molecule topoisomerase II inhibitor.

[0009] Specifically, the above is a dual enhancer MnO combined with chemodynamic therapy and immunotherapy. 2 Nanoparticles are used as carriers, with traditional chemotherapy drugs such as doxorubicin hydrochloride as the core of the drug-loaded nanoparticles, and the outer layer is wrapped with VD 3 The bionic lipid membrane with neutrophil membrane components; the inner core drug-loaded nanoparticles and the outer bionic lipid membrane are wrapped by ultrasound and co-extrusion.

[0010] The dual reinforcing agent MnO 2 The nanoparticles are prepared from potassium permanganate and oleic acid as raw materials, and are used to carry the chemotherapy drug doxorubicin hydrochloride. The nanoparticles are sea urchin-like in appearance, with an average particle size of about 160nm, and the particle size increases slightly after drug loading.

[0011] MnO 2 On the one hand, nanoparticles enhance the efficacy of chemodynamic therapy by depleting glutathione and increasing hydroxyl radicals in the tumor area, and on the other hand, they participate in tumor immunotherapy by activating the cGAS-STING pathway. 3 It is effectively released in the glioma area, synergistically activates and amplifies the cGAS-STING pathway, maximizes the efficacy and reduces toxic side effects.

[0012] Preferably, the MnO 2 The preparation method of nanoparticles comprises the following steps: dissolving potassium permanganate in ultrapure water, adding oleic acid dropwise to make the molar mass ratio of oleic acid to potassium permanganate be 1:5, stirring the solution at 200 rpm until it turns brown-black, centrifuging at 16000 r / min for 30 min, dispersing the brown precipitate in water, and obtaining MnO 2 Nanoparticles.

[0013] The manganese dioxide nanoparticles loaded with doxorubicin hydrochloride include the following steps: preparing MnO 2Nanoparticles and doxorubicin hydrochloride were dissolved in 5 mL of ultrapure water at a mass ratio of 1:1, stirred at 200 rpm for 2 h at room temperature and protected from light, and then centrifuged at 16000 r / min for 30 min. The red supernatant was the free doxorubicin hydrochloride solution without loading, and the brown precipitate was the manganese dioxide nanoparticles (MnO 2 -DOX nanoparticles).

[0014] The multimodal brain-targeted intelligent nanomedicine system is a system that contains vitamin D 3 Lipid membrane (VD@MnO 2 -DOX nanoparticles) and neutrophil membranes by ultrasonic fusion. Specifically, the following steps are included: first, the neutrophil membrane is ultrasonicated in an ice bath by an ultrasonic cell disruptor, the power is set to 500W, each ultrasonication is 10s, repeated 5 times, the power is set to 500W, each ultrasonication is 5s, repeated 5 times, and the nano-scale neutrophil membrane is obtained; then, the vitamin D-containing 3 Lipid membrane (VD@MnO 2 -DOX nanoparticles) were mixed with neutrophil cell membranes and ultrasonicated in an ice bath at a power of 500 W for 10 s each time, repeated 5 times. VD was contained during the ultrasonication process. 3 The phospholipid bilayer of the lipid membrane was fused with the neutrophil membrane. After ultrasound, the liquid was squeezed out through a 200 nm polycarbonate membrane and centrifuged at 16000 r / min for 30 min to collect the precipitate, which was the multimodal brain-targeted intelligent nanodrug system (NED@MnO 2 -DOX).

[0015] Vitamin D 3 The lipid film preparation includes the following steps: hydrogenated soybean lecithin, cholesterol, vitamin D 3 The mixture was dissolved in anhydrous ethanol at a mass ratio of 4:1:1 and completely dissolved by water bath ultrasound as the oil phase. The manganese dioxide nanoparticles (MnO 2 -DOX) PBS buffer was used as the aqueous phase. The oil phase containing phospholipid components was slowly dripped into the aqueous phase by ethanol injection method, stirred at 200 rpm for 2 h, and then extruded through a 200 nm polycarbonate membrane to obtain uniformly dispersed nanoparticles VD@MnO 2 -DOX.

[0016] The neutrophil membrane can be a neutrophil membrane conventionally extracted or preserved in the prior art, or a uniform neutrophil membrane obtained by breaking the membrane of mouse neutrophils through repeated freezing and thawing extraction, centrifugation, and ultrasonic treatment.

[0017] The neutrophils can be obtained by conventional methods or specifically include the following steps: intraperitoneal injection of 100 μL lipopolysaccharide (administered dose 1 mg LPS kg -1) to activate neutrophils in mice. After 12 hours, mice were killed by cervical dislocation and immersed in 75% ethanol solution for 5-10 minutes. The femurs of the lower limbs of mice were separated on a clean bench and the joints at both ends of the femurs were cut off. The bone marrow in the femurs was completely flushed out with pre-cooled PBS, filtered with a 70μm cell strainer and transferred to a 15mL centrifuge tube. Cells were centrifuged at 1000rpm for 3min to obtain cell pellets. Red blood cell lysis buffer (3-5mL) was added to the cell pellets, and the cells were resuspended by blowing thoroughly. After standing at room temperature for 4-5min, they were centrifuged at 1000g for 3min. The above operation was repeated 2-3 times until the cell pellets had no obvious red color. Prepare percoll separation solution containing 3mL81%, 2mL62%, 2mL55% and 2mL50%, resuspend the cell pellet with RPMI 1640 medium (1mL), draw it with a 1mL syringe and carefully add it to the top layer of Percoll separation solution, centrifuge at 3000g low brake for 30min, and the neutrophils will gather at the liquid level between 81% and 62%. Carefully draw out the neutrophil suspension with a syringe, wash it 3 times with 10mL PBS to remove the percoll solution, and resuspend it with 1mL pre-cooled PBS to obtain freshly extracted neutrophils.

[0018] The extraction of the neutrophil membrane specifically includes the following steps: adding 1 mL of PBS to suspend and disperse the neutrophils, and performing 3 freeze-thaw cycles: first freezing at -80°C for 30 min, then thawing at room temperature for 30 min, and repeating this three times. Collect the above-mentioned cell membrane suspension after disruption, place it on ice and ultrasonicate the probe 5 times, the power is set to 500 W, each ultrasonication is 5s, and repeated 3 times to fully lyse the cells and remove the nuclei, centrifuge at 4°C, 1000g for 5 min, discard the cell nuclei under the precipitate, and collect the supernatant in a new centrifuge tube. Centrifuge at 4°C, 20000g for 30 min, discard the supernatant, and the resulting precipitate is the neutrophil membrane (NEM).

[0019] The preparation method of the novel multimodal brain-targeted intelligent nanomedicine system for glioma is as follows: 2 Nanoparticles were used as carriers to prepare drug-loaded nanoparticles with doxorubicin hydrochloride, and then the drug-loaded nanoparticles were used as the core to achieve the outer layer wrapping fusion containing vitamin D through ultrasound. 3 and a bionic lipid membrane of a neutrophil membrane; the weight ratio of the drug-loaded nanoparticles to the bionic lipid membrane is 1:5-10.

[0020] Bionic coated nanoparticles are a new type of nanotechnology that can achieve precise identification of specific areas such as tumor cells and inflammatory sites by coating the cell membrane on the surface. This bionic targeting strategy helps to increase the accumulation of drugs at the lesion site, reduce the impact on normal tissues, improve the biocompatibility of the carrier and improve the therapeutic effect. Neutrophils are the most numerous white blood cells in the body and play a key role in the regulation of the immune system. Neutrophils are naturally capable of crossing the blood-brain barrier and can be used as drug carriers that efficiently target gliomas. Under the induction of chemokines, they can quickly aggregate and attach to the site where gliomas occur.

[0021] Based on these characteristics, the present invention intends to use nano-bionic technology to connect neutrophil membranes and VD 3 The lipid membrane is coated on the surface of honeycomb manganese dioxide nanoparticles by ultrasound and co-extrusion. First, the biomimetic coating of neutrophil membrane can give manganese dioxide nanoparticles the ability to cross the blood-brain barrier and blood-tumor barrier; secondly, the biomimetic lipid membrane fused with VD 3 , so that it can synergistically activate and amplify the cGAS-STING pathway with manganese ions and doxorubicin hydrochloride, and the lipid membrane coating can enhance the stability of manganese dioxide nanoparticles and help to better disperse; furthermore, after the tumor tissue activates divalent manganese ions, the proinflammatory cytokines and chemokines released by the tumor cells help to continuously chemotacticize the neutrophil membrane-coated manganese dioxide nanoparticles, thereby achieving good targeting performance.

[0022] Compared with the prior art, the present invention has the following beneficial effects: the present invention combines the dual enhancer MnO2, which can be used to enhance chemodynamic therapy and immunotherapy. 2 Nanoparticles were used as carriers, and small molecule topoisomerase II inhibitor doxorubicin hydrochloride was loaded to prepare drug-loaded nanoparticles. The drug-loaded nanoparticles were then used as the core, and the outer layer was wrapped and fused with neutrophils and VD by ultrasound. 3 biomimetic lipid membrane.

[0023] The novel multimodal brain-targeted intelligent nano-drug system for glioma constructed by the present invention has good biocompatibility and the characteristics of active targeting of glioma due to the biomimetic lipid membrane wrapped in the outer layer. At the same time, the nanoparticles in the inner layer have excellent adsorption capacity and can effectively carry chemotherapy drugs. The co-delivery system has excellent biocompatibility and can actively target the brain glioma area. On the one hand, the intelligent nano-drug system can directly kill tumors by producing hydroxyl free radicals through the Fenton-like reaction, and on the other hand, it can release doxorubicin hydrochloride and vitamin D in the tumor area. 3 , inducing the immunogenic cell death of the tumor while maximally activating the cGAS-STING pathway, achieving the goal of combined chemotherapy-chemodynamic therapy-immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The multimodal brain-targeted intelligent nanomedicine system NED@MnO 2 -DOX particle size distribution diagram.

[0025] Figure 2 The manganese dioxide nanoparticles and multimodal brain-targeted intelligent nanomedicine system NED@MnO 2 -DOX transmission electron microscopy image (where A is MnO 2 ,B is NED@MnO 2 -DOX, scale bar: 200nm)

[0026] Figure 3 This is the in vitro release diagram of the multimodal brain-targeted intelligent nanodrug system of the present invention.

[0027] Figure 4 The results of in vitro cell uptake of the multimodal brain-targeted intelligent nanomedicine system of the present invention. (Scale bar: 100μm)

[0028] Figure 5 This is the mouse brain tissue distribution map of the multimodal brain-targeted intelligent nanomedicine system of the present invention. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0030] The methods are conventional methods unless otherwise specified, and the raw materials are available from public commercial channels unless otherwise specified.

[0031] Example 1

[0032] MnO 2 Preparation and characterization of nanoparticles

[0033] Potassium permanganate was dissolved in ultrapure water and stirred for 30 minutes to obtain a clear and transparent potassium permanganate solution. Oleic acid solution was added dropwise at a mixing ratio of 8 mL / g, so that the molar mass ratio of oleic acid to potassium permanganate was 1:5. The solution was stirred at 200 rpm until it turned brown-black, centrifuged at 16000 r / min for 30 minutes, and the brown precipitate was dispersed in water to obtain MnO 2 Nanoparticles.

[0034] The MnO was characterized by laser granulometer (DLS) and high-resolution transmission electron microscopy (TEM). 2 Nanoparticle morphology, the results show that MnO 2The nanoparticles are uniform in size, with a hydrated particle size of about 155 nm (see Figure 1 Further transmission electron microscopy results showed that the morphology of manganese dioxide nanoparticles was similar to that of sea urchins (see Figure 2 (shown in A).

[0035] Example 2

[0036] Neutrophil extraction

[0037] The mice were intraperitoneally injected with 100 μL of lipopolysaccharide (administered dose 1 mg LPS kg -1 ) to activate neutrophils in mice. After 12 hours, mice were killed by cervical dislocation, and the femurs of the lower limbs of mice were separated and the two ends of the femurs were carefully cut off. The bone marrow in the femurs was completely flushed out with pre-cooled PBS, filtered with a 70μm cell strainer and transferred to a 15mL centrifuge tube. The cells were centrifuged at 1000rpm for 3min to obtain a cell pellet. Red blood cell lysis buffer (3-5mL) was added to the cell pellet, and the cells were fully blown to resuspend them. After standing at room temperature for 4-5min, they were centrifuged at a speed of 1000g for 3min. The above operation was repeated 2-3 times until the cell pellet had no obvious red color. Percoll separation solution containing 3mL81%, 2mL62%, 2mL55% and 2mL50% was prepared, and the cell pellet was resuspended with RPMI 1640 culture medium (1mL), and carefully added to the top layer of Percoll separation solution after being drawn with a 1mL syringe. The neutrophils were gathered at the liquid level between 81% and 62% after centrifugation at 3000g with low brake for 30min. The neutrophil suspension was carefully aspirated with a syringe, washed three times with 10 mL of PBS to remove the Percoll solution, and resuspended with 1 mL of pre-cooled PBS to obtain freshly extracted neutrophils. That is, after lysing the red blood cells, the bone marrow cell suspension was centrifuged by Percoll density gradient method to obtain purified neutrophils.

[0038] Add 1 mL of pre-cooled PBS to resuspend and disperse the neutrophils, and perform 3 freeze-thaw cycles: first freeze at -80°C for 30 minutes, then thaw at room temperature for another 30 minutes, and repeat this three times. Collect the above-mentioned cell membrane suspension after disruption, place it on ice and ultrasonicate it with a probe for 5 times, with a power setting of 500W, and ultrasonicate for 5 seconds each time, repeat 3 times, so that the cells are fully lysed and nucleated, centrifuge at 4°C, 1000g for 5 minutes, discard the cell nuclei under the precipitate, and collect the supernatant in a new centrifuge tube. Centrifuge at 4°C, 20,000g for 30 minutes, discard the supernatant, and the precipitate is the neutrophil membrane (NEM).

[0039] Example 3

[0040] Multimodal brain-targeted intelligent nanodrug system (NED@MnO 2 -DOX) preparation and characterization

[0041] The MnO prepared in Example 1 2 Nanoparticles and doxorubicin hydrochloride were dissolved in 5 mL of ultrapure water, stirred at 200 rpm for 2 h at room temperature and protected from light, and then centrifuged at 16000 r / min for 30 min. The red supernatant was the free doxorubicin hydrochloride solution, and the brown precipitate was the manganese dioxide nanoparticles loaded with doxorubicin hydrochloride (MnO 2 -DOX) nanoparticles.

[0042] Hydrogenated soy lecithin, cholesterol, vitamin D 3 Dissolved in anhydrous ethanol at a mass ratio of 4:1:1, and completely dissolved in water bath ultrasound as the oil phase. 2 -DOX PBS buffer was used as the aqueous phase. The oil phase containing phospholipid components was slowly dripped into the aqueous phase using the ethanol injection method, stirred for 2 hours, and then extruded through a 200nm polycarbonate membrane to obtain uniformly dispersed nanoparticles containing vitamin D 3 Lipid membrane (VD@MnO 2 -DOX).

[0043] The obtained neutrophil membrane (NEM) was resuspended in PBS and VD@MnO 2 -DOX mixed, ultrasonic cell crusher for 3 minutes, during the ultrasonic process containing VD 3 The phospholipid bilayer of the lipid membrane was fused with the neutrophil membrane, and the ultrasonic liquid was squeezed out through a 200 nm polycarbonate membrane. The precipitate was collected by centrifugation at 16000 r / min for 30 min to obtain a multimodal brain-targeted intelligent nanodrug system (NED@MnO 2 -DOX).

[0044] Its morphology was characterized by laser particle size analyzer (DLS) and high-resolution transmission electron microscopy (TEM). 2 -DOX is uniform in size and has a 2 In comparison, its hydrated particle size increases to 160nm ( Figure 1 Further transmission electron microscopy results show that NED@MnO 2 -DOX has a dense lipid membrane on its surface ( Figure 2 (shown in B).

[0045] Example 4

[0046] NED@MnO 2 -DOX release in vitro

[0047] This experiment used the dialysis method to investigate the in vitro release behavior. The glutathione (GSH) solution selected in this experiment was used to simulate the high-concentration glutathione microenvironment of the tumor. Among them, the 10μM GSH solution simulates the normal physiological conditions in the body, and the 10mM GSH solution is used to simulate the tumor microenvironment. Constant temperature oscillation (37°C, 100rpm), sampling at different time points, aspirating 1mL of release medium, and supplementing with isothermal equal volume blank release medium. The medium taken out was filtered through a 0.22μm microporous filter membrane and diluted to an appropriate multiple. The content of doxorubicin hydrochloride in the sample was determined using a full-wavelength fluorescence microplate reader. The samples were operated in parallel for 3 times. At the same time, a PBS solution with a pH of 7.4 was prepared to simulate the normal physiological conditions in the body, and a PBS solution with a pH of 6.8 was used to simulate the acidic microenvironment of the tumor. Using the dialysis method, samples were taken at different time points, and the vitamin D in the released samples was determined by high-performance liquid chromatography. 3 The results of the in vitro release of doxorubicin hydrochloride are shown in Figure 3 As shown in Figure A, NED@MnO 2 -DOX has glutathione responsiveness to the release of doxorubicin hydrochloride, and the cumulative release rate in 24 hours reaches 73.27%. 3 The release results are as follows Figure 3 As shown in Figure B, compared with the neutral condition, NED@MnO 2 -DOX can release VD better under acidic conditions 3 The cumulative release rate under acidic conditions for 24 hours was 13.74% higher than that under neutral conditions. This may be because the acidic components destroyed the phospholipid components of the lipid membrane, promoting the release of VD 3 The above release results show that NED@MnO 2 -DOX does not produce a burst release phenomenon under physiological conditions and is not easily degraded or inactivated during the circulation process in the body, which helps to prolong the drug's action time in the body and improve the therapeutic effect. 2 -DOX can effectively release doxorubicin hydrochloride and vitamin D 3 This selective drug release behavior can reduce the adverse effects of drugs on normal tissues, while increasing the drug concentration in the tumor area to enhance the therapeutic effect of the drug.

[0048] Example 5

[0049] NED@MnO 2 -DOX in vitro cellular uptake results

[0050] In order to study the tumor cell uptake and targeting ability of nanoparticles, GL261 was co-incubated with DIO fluorescently labeled nanoparticles. GL261 cells were first cultured to the logarithmic growth phase. When the density reached 80%-90%, the cells were digested with trypsin. After the digestion was terminated with complete medium, the cell suspension was collected and centrifuged to obtain the cell pellet. The concentration of GL261 cell suspension was adjusted to 5×10 4 The cells were inoculated into a 24-well plate at a density of 100 cells / well, gently shaken, and cultured overnight. When the cells were completely attached to the wall, the drug treatment was started when the appropriate density was reached. Different concentrations of DIO fluorescently labeled VD@MnO 2 -DOX group and NED@MnO 2 -DOX group, after the incubation, the medium in the wells was aspirated and discarded, and the wells were washed with PBS three times. DAPI staining solution was added to mark the GL261 cell nuclei, and the cell uptake was observed using a fluorescence microscope. Figure 4 As shown in the figure, doxorubicin hydrochloride represented by red fluorescence co-localizes with the cell nucleus and shows concentration dependence like the fluorescence signal intensity of the biomimetic lipid membrane represented by green fluorescence. 2 -DOX group fluorescence was higher than VD@MnO 2 -DOX group was significantly stronger. This result showed that: (1) NED@MnO 2 -DOX-loaded doxorubicin hydrochloride can be effectively released inside tumor cells and exert a chemotherapeutic effect in the cell nucleus, thereby producing a cytotoxic effect on tumor cells. (2) After coextrusion and ultrasound, the biomimetic lipid membrane can effectively encapsulate manganese dioxide nanoparticles and remain stable during cell uptake. (3) Neutrophil membrane encapsulation can make NED@MnO 2 -DOX can more effectively attach to the surface of tumor cell membranes and promote the endocytosis of nanoparticles. This adhesion-uptake ability helps nanoparticles deliver drugs more effectively into tumor cells, thereby enhancing the therapeutic effect.

[0051] Example 6

[0052] NED@MnO 2 -DOX distribution in mouse brain tissue

[0053] In order to investigate the ability of the carrier to cross the blood-brain barrier and target glioma sites, VD@MnO 2 In the process, the near-infrared dye DIR was encapsulated in the bionic lipid membrane of the carrier to prepare DIR fluorescently labeled non-targeted carriers (VD@MnO 2 ) and targeting carrier (NED@MnO 2 ). At the same time, the mice were divided into the Sham group and the VD@MnO2 Group and (NED@MnO 2 In the second group, after the tail vein injection of dye-labeled nanoparticles, the fluorescence signal intensity in the brain of mice was detected by small animal in vivo imaging at 2h and 5h, and the mice were killed at 5h. The heart, liver, spleen, lung, kidney, and brain were separated, and the distribution of fluorescence in major organs was observed in vitro to evaluate the targeting of nanoparticles in brain glioma tissues and the distribution of nanoparticles in other organs in vivo. Figure 5 , NED@MnO 2 The fluorescence signal in the brain of mice in the group was relative to that of VD@MnO 2 group and sham operation group (mice were not modeled and NED@MnO was injected into the tail vein 2 ) increased significantly. This result confirms that in mice, NED@MnO 2 The inherited inflammatory chemotactic properties of -DOX enable it to efficiently cross the blood-brain barrier along the vascular system and accumulate in tumor areas.

Claims

1. A multimodal brain-targeted intelligent nanomedicine system for glioma, characterized in that: It consists of an inner core and an outer layer; the inner core is manganese dioxide nanoparticles loaded with doxorubicin hydrochloride, and the outer layer is a bionic lipid membrane containing vitamin D3 and neutrophil membrane; the inner core and the outer layer are wrapped and fused by ultrasound, and the weight ratio of the inner core to the outer layer is 1:5-10.

2. The multimodal brain-targeted intelligent nanomedicine system for glioma according to claim 1 is characterized in that The preparation steps of the doxorubicin hydrochloride-loaded manganese dioxide nanoparticles are as follows: dissolve manganese dioxide nanoparticles and doxorubicin hydrochloride in 5 mL of ultrapure water at a mass ratio of 1:1, stir at 200 rpm for 2 hours at room temperature in the dark, and centrifuge at 16000 r / min for 30 minutes to obtain brown precipitates as manganese dioxide nanoparticles loaded with doxorubicin hydrochloride.

3. The multimodal brain-targeted intelligent nanomedicine system for glioma according to claim 2, characterized in that: The manganese dioxide nanoparticles are nanoparticles prepared by taking potassium permanganate and oleic acid as raw materials.

4. The multimodal brain-targeted intelligent nanomedicine system for glioma according to claim 2, characterized in that: The preparation method of the manganese dioxide nanoparticles comprises the following steps: dissolving potassium permanganate in ultrapure water, adding oleic acid dropwise to make the molar mass ratio of oleic acid to potassium permanganate be 1:5, stirring the solution at 200 rpm until it turns brown-black, centrifuging at 16000 r / min for 30 min, dispersing the brown precipitate in water, and obtaining manganese dioxide nanoparticles.

5. The novel multimodal brain-targeted intelligent nanomedicine system for glioma according to claim 1 is characterized in that The preparation method of the multimodal brain-targeted intelligent nano-drug system comprises the following steps: firstly, subjecting the neutrophil membrane to ice-bath ultrasonication with an ultrasonic cell disruptor, with the power set to 500 W, each ultrasonication for 10 seconds, repeated 5 times, and the power set to 500 W, each ultrasonication for 5 seconds, repeated 5 times, to obtain a nano-scale neutrophil membrane; mixing the vitamin D3-containing lipid membrane with the neutrophil membrane, subjecting them to ice-bath ultrasonication with the power set to 500 W, each ultrasonication for 10 seconds, repeated 5 times, and after ultrasonication, extruding the liquid through a 200 nm polycarbonate membrane, and collecting the precipitate by centrifugation at 16000 r / min for 30 minutes.

6. The novel multimodal brain-targeted intelligent nanomedicine system for glioma according to claim 5 is characterized in that The preparation of the vitamin D3-containing lipid film comprises the following steps: dissolving hydrogenated soybean lecithin, cholesterol and vitamin D3 in anhydrous ethanol at a mass ratio of 4:1:1, completely dissolving them by water bath ultrasound as an oil phase, using a PBS buffer solution containing manganese dioxide nanoparticles loaded with doxorubicin hydrochloride as an aqueous phase, slowly dripping the oil phase containing phospholipid components into the aqueous phase by an ethanol injection method, stirring at 200 rpm for 2 hours and extruding through a 200 nm polycarbonate membrane to obtain uniformly dispersed nanoparticles.

7. The novel multimodal brain-targeted intelligent nanomedicine system for glioma according to claim 5 is characterized by: The extraction of the neutrophil membrane specifically includes the following steps: adding 1 mL of PBS to suspend and disperse the neutrophils, and performing 3 freeze-thaw cycles: first freezing at -80°C for 30 min, then thawing at room temperature for 30 min, and repeating this three times; collecting the above-mentioned cell membrane suspension after disruption, placing it on ice and ultrasonicating the probe 5 times, setting the power to 500 W, ultrasonicating for 5 s each time, repeating 3 times, so that the cells are fully lysed and nucleated, centrifuging at 4°C, 1000g for 5 min, discarding the cell nuclei under the precipitate, and collecting the supernatant in a new centrifuge tube. Centrifuging at 4°C, 20000g for 30 min, discarding the supernatant, the resulting precipitate is the neutrophil membrane.

8. A method for preparing the novel multimodal brain-targeted intelligent nanomedicine system for glioma according to claim 1, characterized in that: MnO2 nanoparticles are used as carriers and loaded with doxorubicin hydrochloride to prepare drug-loaded nanoparticles, which are then used as the core and ultrasound is used to wrap and fuse the outer layer with a bionic lipid membrane containing vitamin D3 and a neutrophil membrane; the weight ratio of the drug-loaded nanoparticles to the bionic lipid membrane is 1:5-10.

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