Hybrid cell membrane nanodelivery vehicles that can target intracranial inflammatory lesions, drugs and methods of making and use thereof

Abstract

The present application relates to a hybrid cell membrane nano delivery carrier which can target intracranial inflammatory lesions, which is a spherical vesicle coated with a hybrid cell membrane coating, wherein the hybrid cell membrane coating is composed of platelet cell membrane proteins and HEK293t cell membrane proteins overexpressing chemokine receptor CCR2 genes at a weight ratio of 1:2-2:1. The present application also provides a nano drug which can target intracranial inflammatory lesions, which is composed of drug-loaded liposomes coated with the hybrid cell membrane coating. The present application also provides a preparation method of the nano delivery carrier and the nano drug and applications thereof. The nano delivery carrier of the present application simultaneously expresses platelet membrane marker proteins and chemokine receptor CCR2 genes on the surface, and the nano drug formed by loading specific small molecule drugs in the nano delivery carrier can significantly improve the cognitive function of Alzheimer's disease model mice and reduce the deposition of amyloid plaques in the brain.

Description

Technical Field

[0001] This invention relates to a cell membrane-coated nanodelivery carrier that targets intracranial inflammatory lesions across the blood-brain barrier, its preparation, and its application in the treatment of central nervous system diseases. Background Technology

[0002] The protective effect of the blood-brain barrier (BBB) ​​on the central nervous system (CNS) hinders drug entry and therapeutic effects, making targeted drug delivery across the BBB a major challenge. Under normal physiological conditions, the tight junctions and active efflux of the BBB prevent the entry of 98% of small molecule drugs and all large molecules. Although in some neurological diseases (such as Alzheimer's disease), disruption of the tight junctions and increased permeability of the BBB may facilitate the entry of small molecule drugs into the CNS, studies have shown that even in severe Alzheimer's disease, only molecules with a particle size smaller than 20 nm can cross the BBB via this pathway. Many small molecule drugs (such as rapamycin and curcumin) are hampered in their application in CNS diseases due to low BBB permeability and bioavailability.

[0003] Nanomaterial carriers are a series of nanoscale particles capable of encapsulating functional substances such as small-molecule drugs, nucleotides, and proteins. They can overcome the body's barriers that limit drug action and have been reported as delivery systems for the treatment of central nervous system diseases. The earliest nanocarriers primarily improved blood-brain barrier penetration by reducing nanoparticle size. Currently, a novel nanocarrier with carbon dots of only 1-10 nm has been verified to have good blood-brain barrier penetration. However, this carrier requires high molecular weight functional substances and lacks specific central targeting. During blood-brain barrier penetration, in addition to passive diffusion through intermolecular spaces such as tight junctions, numerous receptors and carriers participate in the transport of macromolecules to the central nervous system. Therefore, chemical target modification of nanocarriers can improve their transport efficiency; however, biocompatibility and safety remain the biggest limitations to their clinical translation.

[0004] Disruption of the blood-brain barrier can induce peripheral cell infiltration, offering new opportunities for cell membrane-coated nanomaterials in the treatment of central nervous system diseases. This biomimetic strategy demonstrates good biocompatibility and a longer metabolic cycle. Numerous biomembrane-coated nanosystems derived from erythrocytes, leukocytes, and tumor cells have been developed and proven effective in treating central nervous system diseases such as Alzheimer's and cerebrovascular diseases. Platelet membranes offer significant advantages as coatings for drug delivery in the central nervous system. Firstly, platelet membranes express various membrane proteins. The presence of CD47 on the platelet membrane surface allows it to interact with signal regulatory proteins on immune cells and inhibit immune cell-mediated clearance of nanoparticles, thereby prolonging the circulation cycle of nanomedicines in vivo. Platelet membranes also express unique surface ligands for targeting tissues or cells, especially damaged vascular systems. Furthermore, disruption of the blood-brain barrier is essentially a form of microvascular injury, suggesting that platelet membranes can cross the blood-brain barrier.

[0005] Beyond single-cell membrane coating technology, hybrid cell membrane technology derived from multiple cell types can provide nanomaterials with diverse biological functions or properties for complex physiological environments. However, research on hybrid cell membrane-coated nanoparticles for the treatment of nervous system diseases is still in its early stages. To date, hybridization of blood cells and tumor cells has been applied to the treatment of brain tumors, but there is no research on the application of hybrid cell membrane nanocarriers to other nervous system diseases. Furthermore, continuous advancements in cell separation technology and engineering have facilitated the development of more complex cell membrane-coated nanomaterials, such as adding additional functions (specific ligands, antibodies, or imaging agents) to the surface of nanoparticles through biological modification. This further improves their targeting precision, therapeutic efficacy, and diagnostic capabilities. Neuroinflammation, as a common pathological feature of central nervous system diseases, has been reported to target inflammatory lesions using biomembranes derived from microglia (BV2) or macrophages; however, the existence of subpopulations with different polarization states in these cells may limit their application.

[0006] In summary, biologically modified hybrid cell membrane nanocarriers possess multifunctionality, high biocompatibility, and a relatively long biological circulation period. When used for drug delivery in central nervous system diseases, they can cross the blood-brain barrier and accumulate in the lesion area. However, hybrid cell membrane nanocarriers capable of efficiently delivering drugs for treating intracranial inflammation (especially Alzheimer's disease) still require further development. Summary of the Invention

[0007] The primary objective of this invention is to construct a hybrid cell membrane nanocarrier that can target intracranial inflammatory lesions. This carrier can not only cross the blood-brain barrier, but also has good targeting ability to intracranial inflammatory lesions, making it particularly suitable for delivering drugs for the treatment of Alzheimer's disease.

[0008] Another objective of this invention is to construct nanomedicines coated with hybrid cell membranes that can target intracranial inflammatory lesions. These nanomedicines can not only cross the blood-brain barrier but also have good targeting properties for intracranial inflammatory lesions, making them particularly suitable for the treatment of Alzheimer's disease.

[0009] Another object of the present invention is to provide the carrier and a method for preparing the nanomedicine.

[0010] Another object of the present invention is to provide the application of the said carrier and the said nanomedicine in the treatment of central nervous system diseases.

[0011] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0012] In a first aspect, the present invention provides a hybrid cell membrane nanodelivery carrier that can target intracranial inflammatory lesions. It is a spherical vesicle coated with a hybrid cell membrane, wherein the hybrid cell membrane coating is composed of platelet cell membrane proteins and HEK293t cell membrane proteins that overexpress the chemokine receptor CCR2 gene in a weight ratio of 1:2 to 2:1.

[0013] The spherical vesicles of the preferred nanodelivery carrier of the present invention have a diameter of 25-60 nm.

[0014] The performance of the nanodelivery carrier described in this invention is significantly affected by the ratio of platelet cell membrane protein to HEK293t cell membrane protein overexpressing the CCR2 chemokine receptor gene. When the proportion of platelet cell membrane protein is too high and the proportion of HEK293t cell membrane protein overexpressing the CCR2 chemokine receptor gene is too low, the nanocarrier's ability to accumulate in intracranial inflammatory lesions is weakened. Conversely, when the proportion of platelet membrane protein is too low and the proportion of HEK293t cell membrane protein overexpressing the CCR2 chemokine receptor gene is too high, the blood-brain barrier penetration rate of the nanocarrier is reduced. Therefore, the hybrid cell membrane described in this invention is composed of platelet cell membrane protein and HEK293t cell membrane protein overexpressing the CCR2 chemokine receptor gene in a ratio of 1:2 to 2:1. Within this ratio range, the hybrid cell membrane can balance blood-brain barrier penetration rate and targeting of inflammatory lesions; a more preferred ratio is 2:1.

[0015] In the nanodelivery carrier of this invention, the hybrid cell membrane is obtained by fusing a platelet cell membrane and a HEK293t cell membrane overexpressing the chemokine receptor CCR2 gene in the specified ratio. The fusion method is not particularly limited; existing fusion methods capable of cell membrane hybridization, such as co-extrusion, can be used to prepare the hybrid cell membrane.

[0016] Furthermore, in the nanodelivery carrier of the present invention, the platelet cell membrane protein can be obtained by various existing methods, such as by gradient centrifugation and repeated freeze-thaw cycles of blood cells from an organism.

[0017] Furthermore, in the nanodelivery carrier of the present invention, the HEK293t cell membrane protein overexpressing the chemokine receptor CCR2 gene can be extracted from HEK293t stable transgenic cells overexpressing the CCR2 gene by repeated freeze-thaw cycles.

[0018] The HEK293t stable cell line overexpressing the CCR2 gene is preferably obtained by the following method: a plasmid overexpressing the CCR2 gene is constructed using the PGMLV-CMV-MCS-mScarlet-PGK-Puro vector, which is then used to infect the HEK293t cell line, and finally, the HEK293t stable cell line overexpressing the CCR2 gene is obtained by screening with puromycin.

[0019] In a preferred embodiment of the present invention, the spherical vesicles are composed of liposomes coated with a hybrid cell membrane; the liposomes account for 40%-60% of the total weight of the nanodelivery carrier. In a further preferred embodiment, the liposomes contain natural phospholipids and synthetic phospholipids; the natural phospholipids may be selected from soybean lecithin or egg yolk lecithin, preferably soybean lecithin; the synthetic phospholipids may be selected from DSPE-PEG, DSPC, or DOPC, preferably DSPE-PEG.

[0020] Secondly, the present invention also provides a nanomedicine that can target intracranial inflammatory lesions, wherein the nanomedicine is composed of a hybrid cell membrane coating coated with a drug-loaded liposome; the hybrid cell membrane coating is composed of platelet cell membrane protein and HEK293t cell membrane protein overexpressing the chemokine receptor CCR2 gene in a weight ratio of 1:2 to 2:1; the drug-loaded liposome is a complex phospholipid internally loaded with a therapeutic drug, wherein the therapeutic drug includes one or more small molecule drugs that have therapeutic effects on Alzheimer's disease.

[0021] In the nanomedicine of the present invention, the preferred hybrid cell membrane coating is composed of platelet cell membrane protein and HEK293t cell membrane protein overexpressing the chemokine receptor CCR2 gene in a weight ratio of 2:1.

[0022] In the nanomedicine of the present invention, the preferred weight ratio of the hybrid cell membrane coating to the composite phospholipid is 0.5 to 1.5:1; more preferably 1:1.

[0023] In the nanomedicine of the present invention, the composite phospholipid is a mixture of natural phospholipid and synthetic phospholipid; the natural phospholipid can be selected from soybean lecithin or egg yolk lecithin, preferably soybean lecithin; the synthetic phospholipid can be selected from DSPE-PEG, DSPC or DOPC, preferably DSPE-PEG.

[0024] In the nanomedicines of the present invention, the preferred small molecule drugs that have therapeutic effects on Alzheimer's disease are selected from one or more of rapamycin, TPPU, curcumin, and resveratrol; more preferably, a combination of rapamycin and TPPU.

[0025] In the nanomedicine of this invention, the hybrid cell membrane coating is obtained by fusing a platelet cell membrane and a HEK293t cell membrane overexpressing the chemokine receptor CCR2 gene in the specified ratio. The fusion method is not particularly limited; existing fusion methods capable of cell membrane hybridization, such as co-extrusion, can be used to prepare the hybrid cell membrane.

[0026] Furthermore, in the nanomedicine of the present invention, the platelet cell membrane protein can be obtained by various existing methods, such as by gradient centrifugation and repeated freeze-thaw cycles of blood cells from an organism.

[0027] Furthermore, in the nanomedicine of the present invention, the HEK293t cell membrane protein overexpressing the chemokine receptor CCR2 gene can be extracted from HEK293t stable cell line overexpressing the CCR2 gene by repeated freeze-thaw cycles.

[0028] Furthermore, the HEK293t stable cell line overexpressing the CCR2 gene is preferably obtained by the following method: constructing a plasmid overexpressing the CCR2 gene using the PGMLV-CMV-MCS-mScarlet-PGK-Puro vector, then infecting the HEK293t cell line, and finally selecting the HEK293t stable cell line overexpressing the CCR2 gene by puromycin.

[0029] The nanomedicine described in this invention is in the form of nanoparticles with a particle size between 35 nm and 70 nm, which is very conducive to penetrating the blood-brain barrier.

[0030] Thirdly, the present invention also provides a method for preparing the nanodelivery carrier described in the first aspect of the present invention and a method for preparing the nanomedicine described in the second aspect of the present invention.

[0031] The method for preparing the nanodelivery carrier according to the first aspect of the present invention includes:

[0032] 1) Platelet cell membranes were obtained by gradient centrifugation and repeated freeze-thaw cycles;

[0033] 2) A plasmid overexpressing the CCR2 gene was constructed based on the PGMLV-CMV-MCS-mScarlet-PGK-Puro vector. The plasmid was then used to infect the HEK293t cell line. Stable HEK293t cells overexpressing the CCR2 gene were screened from the infected HEK293t cell line. The cell membrane of the stable HEK293t cells overexpressing the CCR2 gene was then extracted by repeated freeze-thaw cycles.

[0034] 3) The platelet cell membrane obtained in 1) and the HEK293t stable cell membrane overexpressing the CCR2 gene obtained in 2) are fused at a protein weight ratio of 1:2 to 2:1 (preferably 2:1), preferably by co-extrusion, to obtain the hybrid cell membrane nanodelivery carrier that can target intracranial inflammatory lesions as described in the first aspect of the present invention.

[0035] The method for preparing the preferred nanodelivery carrier of the first aspect of the present invention includes:

[0036] 1) Platelet cell membranes were obtained by gradient centrifugation and repeated freeze-thaw cycles;

[0037] 2) A plasmid overexpressing the CCR2 gene was constructed based on the PGMLV-CMV-MCS-mScarlet-PGK-Puro vector. The plasmid was then used to infect the HEK293t cell line. Stable HEK293t cells overexpressing the CCR2 gene were screened from the infected HEK293t cell line. The cell membrane of the stable HEK293t cells overexpressing the CCR2 gene was then extracted by repeated freeze-thaw cycles.

[0038] 3) The platelet cell membrane obtained in 1), the HEK293t stable cell membrane overexpressing the CCR2 gene obtained in 2), and the mixed phospholipid solution are mixed, and the concentration ratio of membrane protein to phospholipid is controlled to be 1:2 to 2:1, preferably 1:1. At the same time, the protein concentration ratio of the platelet cell membrane to the HEK293t stable cell membrane overexpressing the CCR2 gene is controlled to be 1:2 to 2:1, preferably 2:1. Then, the mixture is fused, preferably by co-extrusion, to obtain the nano-delivery carrier composed of hybrid cell membrane coating coated with liposomes as described in the first aspect of the present invention.

[0039] The method for preparing the nanomedicine according to the second aspect of the present invention includes:

[0040] I) Platelet cell membranes were obtained by gradient centrifugation and repeated freeze-thaw cycles;

[0041] II) A plasmid overexpressing the CCR2 gene was constructed based on the PGMLV-CMV-MCS-mScarlet-PGK-Puro vector. The plasmid was then used to infect the HEK293t cell line. Stable HEK293t cells overexpressing the CCR2 gene were screened from the infected HEK293t cell line. The cell membrane of the stable HEK293t cells overexpressing the CCR2 gene was then extracted by repeated freeze-thaw cycles.

[0042] III) Add one or more drugs that have therapeutic effects on Alzheimer's disease to a mixed phospholipid solution, and use rotary evaporation to make the phospholipids self-assemble and encapsulate the drugs to obtain drug-loaded liposomes.

[0043] IV) The platelet cell membrane obtained in I), the HEK293t stable cell membrane overexpressing the CCR2 gene obtained in II), and the drug-loaded liposome obtained in III) are mixed, and the concentration ratio of membrane protein to phospholipid is controlled at 1:2 to 2:1, preferably 1:1. At the same time, the protein concentration ratio of the platelet cell membrane to the HEK293t stable cell membrane overexpressing the CCR2 gene is controlled at 1:2 to 2:1, preferably 2:1. Then, the mixture is fused, preferably by co-extrusion, to obtain the nanomedicine that can target intracranial inflammatory lesions as described in the second aspect of the present invention.

[0044] Furthermore, the mixed phospholipid solution described above preferably contains natural phospholipids and synthetic phospholipids; the natural phospholipids can be selected from soybean lecithin or egg yolk lecithin, preferably soybean lecithin; the synthetic phospholipids can be selected from DSPE-PEG, DSPC, or DOPC, preferably DSPE-PEG. In the most preferred embodiment, the mixed phospholipid solution is composed of 35 mg of soybean lecithin, 5 mg of cholesterol, and 10 mg of DSPE-PEG dissolved in 10 mL of chloroform.

[0045] Fourthly, the present invention also provides the use of the nanomedicine described in the second aspect of the present invention in the preparation of medicaments for treating diseases of the central nervous system in humans or mammals.

[0046] Furthermore, the central nervous system disease is preferably Alzheimer's disease, multiple sclerosis, or Parkinson's disease; Alzheimer's disease is the most preferred.

[0047] Furthermore, the preferred mammal is the mouse.

[0048] Furthermore, the nanomedicine is prepared into an injectable formulation.

[0049] Fifthly, the present invention also provides the application of the nanomedicine described in the second aspect of the present invention in the treatment of diseases of the central nervous system in humans or mammals.

[0050] Furthermore, the central nervous system diseases mentioned are preferably Alzheimer's disease, multiple sclerosis, and Parkinson's disease; Alzheimer's disease is the most preferred.

[0051] Furthermore, the preferred mammal is the mouse.

[0052] Furthermore, the nanomedicine is preferably administered via injection.

[0053] Furthermore, the injection administration regimen for mice was as follows: based on 1 mg / ml of hybrid cell membrane, the injection dose was 100 μl / 20 g body weight, administered 3 times a week for a total of 6 weeks.

[0054] This invention provides a hybrid cell membrane nanodelivery carrier that can target intracranial inflammatory lesions. By fusing platelet cell membranes and cell membranes overexpressing the chemokine receptor CCR2 gene in a specific ratio, a specific biomimetic vesicle morphology with a hybrid cell membrane coating is formed. Its surface can simultaneously express platelet membrane marker proteins and the chemokine receptor CCR2, exhibiting a stable surface potential under normal physiological conditions and allowing for prolonged circulation in the bloodstream. This hybrid cell membrane nanodelivery carrier can cross the blood-brain barrier to enter the central nervous system and accumulate in intracranial inflammatory lesion areas, particularly around microglia and astrocytes. The nanodelivery carrier of this invention, after encapsulating specific small molecule drugs, forms nanoparticles. Experiments have demonstrated that the nanoparticles of this invention can be used to treat central nervous system diseases in mice, especially when the nanodelivery carrier encapsulates rapamycin and TPPU in combination, effectively improving cognitive function and reducing amyloid plaque deposition in Alzheimer's disease model mice. Attached Figure Description

[0055] Figure 1 Transmission electron microscopy image of the hybrid cell membrane nanodelivery carrier prepared in Example 4 of the present invention.

[0056] Figure 2 This demonstrates the expression of surface receptor proteins (A) and fusion localization (B) of the hybrid cell membrane nanodelivery carrier in Experiment Example 1.

[0057] Figure 3 This demonstrates the particle size (A) and potential (B) of different cell membranes, nanodelivery carriers, and nanomedicines in Experiment 1 and Experiment 2.

[0058] Figure 4 This demonstrates the blood-brain barrier crossing (A) and tissue distribution (B) of hybrid cell membrane nanodelivery carriers with different fusion ratios in Experiment Example 1.

[0059] Figure 5The results demonstrate the intracranial inflammatory lesion targeting function of the two hybrid cell membrane nanodelivery carriers in Experiment Example 1, showing the enrichment around microglia (A) and astrocytes (B).

[0060] Figure 6 The figure shows the drug release curves of the dual-drug nanomedicine prepared in Example 7, including the release curve of TPPU (A) and the release curve of rapamycin (B).

[0061] Figure 7 This demonstrates the improvement in cognitive function achieved by injecting various drug-loaded hybrid cell membrane nanomedicines into Alzheimer's disease mice, as shown in Experiment Example 3.

[0062] Figure 8 This demonstrates the pathological improvement results of various drug-loaded hybrid cell membrane nanomedicines injected into Alzheimer's disease mice in Experiment Example 3. Detailed Implementation

[0063] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. Those skilled in the art can implement the present invention using various other specific embodiments based on the content disclosed herein, or any simple changes or modifications made to the design structure and concept of the present invention fall within the scope of protection of the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0064] This invention provides a hybrid cell membrane nanodelivery carrier that can cross the blood-brain barrier to target intracranial inflammatory lesions, and a nanomedicine formed by encapsulating a therapeutic drug on the nanodelivery carrier.

[0065] The hybrid cell membrane nanodelivery carrier of this invention is a spherical vesicle coated with a hybrid cell membrane. The hybrid cell membrane comprises a platelet cell membrane and a HEK293t cell membrane overexpressing the chemokine receptor CCR2 gene, with a weight ratio of 1:2 to 2:1 (e.g., 1:2, 1:1, or 2:1, with 2:1 being the most preferred). The nanodelivery carrier has a diameter of 25-60 nm.

[0066] In a preferred nanodelivery carrier of the present invention, the surface of the spherical vesicles is the hybrid cell membrane coating, and the interior is coated with liposomes; the liposomes are a mixture of natural phospholipids and synthetic phospholipids; the natural phospholipids can be selected from soybean lecithin or egg yolk lecithin, preferably soybean lecithin; the synthetic phospholipids can be selected from DSPE-PEG, DSPC or DOPC, preferably DSPE-PEG; the liposomes account for 40% to 60% of the total weight of the nanodelivery carrier.

[0067] The preferred hybrid cell membrane nanodelivery carrier of the present invention is prepared by the following method:

[0068] 1. Platelet cell membranes are obtained using gradient centrifugation and repeated freeze-thaw cycles; this may include, for example, the following specific steps:

[0069] 100g of mouse whole blood was centrifuged for 20 minutes to collect serum and the intermediate white blood cell layer. The mixture was then centrifuged at 800g for 20 minutes, the supernatant was discarded, and an appropriate amount of erythrocyte lysis buffer was added. Lysis was performed on ice for 5 minutes, and repeated until no obvious red blood cells were observed. The platelet pellet was then collected. The pellet was dissolved in a pre-added protease inhibitor solution, flash-frozen in liquid nitrogen, thawed at 37°C, and subjected to three freeze-thaw cycles. The pellet was then sonicated at 10% working intensity for 5 minutes, centrifuged at 12000 rpm at 4°C for 30 minutes, the supernatant was discarded, and the pellet was the platelet cell membrane. After dissolving in an appropriate amount of Milli-Q containing 10% glycerol, BCA protein was quantified and stored at -80°C.

[0070] 2. Construct a plasmid overexpressing the CCR2 gene based on the PGMLV-CMV-MCS-mScarlet-PGK-Puro vector, then infect the HEK293t cell line with the plasmid, screen stable HEK293t cells overexpressing the CCR2 gene from the infected HEK293t cell line, and then extract the cell membrane of HEK293t cells overexpressing the CCR2 gene using a repeated freeze-thaw method; for example, the following specific steps may be included:

[0071] A plasmid overexpressing the CCR2 gene was constructed using the PGMLV-CMV-MCS-mScarlet-PGK-Puro vector (from Jimon Biotechnology Co., Ltd.). The plasmid was used to infect the HEK293t cell line, and stable transgenic HEK293t cells overexpressing the CCR2 gene were obtained through puromycin selection. Stable transgenic HEK293t cells were collected by trypsin digestion, washed three times with pre-chilled PBS, and an appropriate amount of cell membrane protein extraction buffer (with protease inhibitors added beforehand) was added according to the cell volume. The cells were incubated on ice for 15 minutes, then flash-frozen in liquid nitrogen and thawed at 37°C, repeating the freeze-thaw cycle six times. The cells were centrifuged at 1000g at 4°C for 10 minutes, and the gelatinous substance was discarded. The cells were then centrifuged at 14000g at 4°C for 30 minutes, and the supernatant was discarded. The precipitate was the HEK293t cell membrane overexpressing the CCR2 gene. After dissolving the membrane in an appropriate amount of milliQ containing 10% glycerol, BCA protein was quantified, and the membrane was stored at -80°C.

[0072] 3. Preparation of mixed phospholipid solution

[0073] Dissolve 35 mg of soybean lecithin, 5 mg of cholesterol and 10 mg of DSPE-PEG in 10 mL of chloroform to obtain a mixed phospholipid solution of 5 mg / mL.

[0074] 4. Preparation of hybrid cell membrane nanodelivery carriers

[0075] The platelet cell membrane obtained in step 1 and the HEK293t cell membrane overexpressing the CCR2 gene obtained in step 2) were dispersed together in a buffer solution to form a mixed membrane solution with a concentration of 1 mg / ml. The mixed phospholipid solution obtained in step 3 was diluted to a concentration of 1 mg / ml. Then, the 1 mg / ml mixed phospholipid solution and the 1 mg / ml mixed membrane solution were mixed in equal volumes (i.e., the phospholipid mg:membrane protein mg after mixing = 1:1), and the particles were collected after extrusion using a liposome extruder, thus obtaining spherical vesicles coated with hybrid cell membrane, which is the hybrid cell membrane nanodelivery carrier described in this invention.

[0076] The nanomedicine formed by encapsulating therapeutic drugs in the nanodelivery carrier of the present invention is preferably formed by encapsulating small molecule drugs (TPPU and rapamycin) in combination within the above-mentioned hybrid cell membrane nanodelivery carrier, and can be used for the treatment of specific neurological diseases (Alzheimer's disease).

[0077] The preferred nanomedicines of this invention are prepared by the following methods:

[0078] I. Obtaining platelet cell membranes via gradient centrifugation and repeated freeze-thaw cycles; this may include, for example, the following specific steps:

[0079] 100g of mouse whole blood was centrifuged for 20 minutes to collect serum and the intermediate white blood cell layer. The mixture was then centrifuged at 800g for 20 minutes, the supernatant was discarded, and an appropriate amount of erythrocyte lysis buffer was added. Lysis was performed on ice for 5 minutes, and repeated until no obvious red blood cells were observed. The platelet pellet was then collected. The pellet was dissolved in a pre-added protease inhibitor solution, flash-frozen in liquid nitrogen, thawed at 37°C, and subjected to three freeze-thaw cycles. The pellet was then sonicated at 10% working intensity for 5 minutes, centrifuged at 12000 rpm at 4°C for 30 minutes, the supernatant was discarded, and the pellet was the platelet cell membrane. After dissolving in an appropriate amount of Milli-Q containing 10% glycerol, BCA protein was quantified and stored at -80°C.

[0080] II. Construct a plasmid overexpressing the CCR2 gene based on the PGMLV-CMV-MCS-mScarlet-PGK-Puro vector, then infect the HEK293t cell line with the plasmid, screen stable HEK293t cells overexpressing the CCR2 gene from the infected HEK293t cell line, and then extract the HEK293t cell membrane overexpressing the CCR2 gene using a repeated freeze-thaw method; for example, it may include the following specific steps:

[0081] A plasmid overexpressing the CCR2 gene was constructed using the PGMLV-CMV-MCS-mScarlet-PGK-Puro vector. HEK293t cell lines were infected with the plasmid, and stable transgenic HEK293t cells overexpressing the CCR2 gene were obtained through puromycin selection. Stable transgenic HEK293t cells overexpressing the CCR2 gene were collected by trypsin digestion, washed three times with pre-chilled PBS, and an appropriate amount of cell membrane protein extraction buffer (with protease inhibitors added beforehand) was added according to the cell volume. The cells were incubated on ice for 15 minutes, then flash-frozen in liquid nitrogen and thawed at 37°C, repeating the freeze-thaw cycle six times. The cells were centrifuged at 1000g at 4°C for 10 minutes, and the gelatinous substance was discarded. The cells were then centrifuged at 14000g at 4°C for 30 minutes, and the supernatant was discarded. The precipitate was the HEK293t cell membrane overexpressing the CCR2 gene. After dissolving the membrane in an appropriate amount of Milli-Q containing 10% glycerol, BCA protein was quantified, and the membrane was stored at -80°C.

[0082] III. Preparation of mixed phospholipid solution and drug stock solution; for example, may include the following specific steps:

[0083] 35 mg of soybean lecithin, 5 mg of cholesterol, and 10 mg of DSPE-PEG were dissolved in 10 ml of CHCl3 to obtain a 5 mg / ml mixed phospholipid solution. TPPU and rapamycin were separately prepared into 1 mg / ml stock solutions with ethanol, which were then aliquoted and stored.

[0084] IV. Utilizing phospholipid self-assembly to prepare drug-loaded liposomes (or drug-loaded phospholipid particles); for example, this may include the following specific steps:

[0085] Take 0.2 ml of the 5 mg / ml mixed phospholipid solution obtained in step III above, add 5 ml of CHCl3, then add 100 μl of the 1 mg / ml TPPU stock solution prepared in step III and 64 μl of the 1 mg / ml rapamycin stock solution prepared in step III. Remove the solvent by rotary evaporation, then add 1 ml of PBS pre-filtered with a filter head. The resulting solution system has a TPPU concentration of 0.1 mg / mL, a rapamycin concentration of 0.064 mg / mL, and a phospholipid concentration of 1 mg / mL. Sonicate the solution in an ice-water bath for 20 min to allow the phospholipids to self-assemble, yielding drug-loaded liposomes (or drug-loaded phospholipid particles). Remove the free drug by ultrafiltration twice (collecting particles from the filter membrane by blowing), and redisperse the precipitate in 1 ml of PBS.

[0086] V. Prepare nanomedicines by co-extrusion of drug-loaded phospholipid particles and hybrid cell membranes; for example, this may include the following specific steps:

[0087] Take a solution containing 1 mg / ml of drug-loaded phospholipid particles prepared in step IV and mix it with an equal volume of a mixed membrane solution containing 1 mg / ml of cell membrane prepared in steps I and II. The phospholipids and membrane proteins are fused in equal proportions, i.e., phospholipid mg: membrane protein mg = 1:1. Then, the particles are collected after being extruded using a liposome extruder, thus obtaining a nanomedicine with a hybrid cell membrane coating on the surface and TPPU and rapamycin loaded inside.

[0088] The present invention provides the following specific embodiments based on the above-described specific implementation methods:

[0089] Example 1. Preparation of hybrid cell membrane nanocarriers

[0090] According to the above-described embodiments, platelet cell membranes were obtained by gradient centrifugation and repeated freeze-thaw cycles. The membranes of the HEK293t stable cell line overexpressing the CCR2 gene were extracted by repeated freeze-thaw cycles. Cell membrane protein concentrations were determined using the BCA method. The platelet cell membranes and the HEK293t stable cell line overexpressing the CCR2 gene were dispersed together in a buffer solution at a protein concentration ratio of 2:1 to form a mixed membrane solution with a concentration of 1 mg / ml. The mixed membrane solution was extruded using a liposome extruder, and the particles were collected to obtain the hybrid cell membrane nanodelivery carrier-I.

[0091] Example 2. Preparation of hybrid cell membrane nanodelivery carriers

[0092] According to the above implementation method, platelet cell membranes were obtained by gradient centrifugation and repeated freeze-thaw cycles. The cell membranes of the HEK293t stable cell line overexpressing the CCR2 gene were extracted by repeated freeze-thaw cycles. The cell membrane protein concentration was determined by the BCA method. The platelet cell membranes and the cell membranes of the HEK293t stable cell line overexpressing the CCR2 gene were dispersed together in a buffer solution at a protein concentration ratio of 1:1 to form a mixed membrane solution with a concentration of 1 mg / ml. The mixed membrane solution was extruded using a liposome extruder, and the particles were collected to obtain the hybrid cell membrane nanodelivery carrier-II.

[0093] Example 3. Preparation of hybrid cell membrane nanocarriers

[0094] According to the above implementation method, platelet cell membranes were obtained by gradient centrifugation and repeated freeze-thaw cycles. The cell membranes of the HEK293t stable cell line overexpressing the CCR2 gene were extracted by repeated freeze-thaw cycles. The cell membrane protein concentration was determined by the BCA method. The platelet cell membranes and the cell membranes of the HEK293t stable cell line overexpressing the CCR2 gene were dispersed together in a buffer solution at a protein concentration ratio of 1:2 to form a mixed membrane solution with a concentration of 1 mg / ml. The mixed membrane solution was extruded using a liposome extruder, and the particles were collected to obtain the hybrid cell membrane nanodelivery carrier-III.

[0095] Comparative Example 1. Preparation of control hybrid cell membrane nanodelivery carrier:

[0096] Following the method described in Example 1, platelet cell membranes were obtained using gradient centrifugation and repeated freeze-thaw cycles. Conventional HEK293t cell membranes were extracted. Cell membrane protein concentrations were determined using the BCA method. Platelet cell membranes and conventional HEK293t cell membranes were co-dispersed in a buffer solution at a protein concentration ratio of 2:1 to form a mixed membrane solution with a concentration of 1 mg / ml. The mixed membrane solution was extruded using a liposome extruder, and the particles were collected to obtain the control hybrid cell membrane nanocarrier.

[0097] Example 4. Preparation of a nanocarrier for liposomes coated with a hybrid cell membrane:

[0098] A 1 mg / ml mixed membrane solution was prepared according to the method in Example 1. 35 mg of soybean phospholipids, 5 mg of cholesterol, and 10 mg of DSPE-PEG were dissolved in 10 mL of chloroform to obtain a 5 mg / mL mixed phospholipid solution, which was then diluted to obtain a 1 mg / ml mixed phospholipid solution. 0.5 mL of the 1 mg / ml mixed phospholipid solution was mixed with 0.5 mL of the 1 mg / ml mixed membrane solution. The phospholipids and membrane proteins were fused in an equal ratio (phospholipid mg:membrane protein mg = 1:1). The mixture was extruded using a liposome extruder, and the particles were collected to obtain spherical vesicles formed by liposomes coated with a hybrid cell membrane coating, designated as hybrid cell membrane nanodelivery carrier-IV. Its transmission electron microscopy image is shown below. Figure 1 .

[0099] Example 5. Preparation of a nanocarrier for liposomes coated with a hybrid cell membrane:

[0100] A 1 mg / ml mixed membrane solution was prepared according to the method in Example 2. 35 mg of soybean phospholipids, 5 mg of cholesterol, and 10 mg of DSPE-PEG were dissolved in 10 mL of chloroform to obtain a 5 mg / mL mixed phospholipid solution, which was then diluted to obtain a 1 mg / ml mixed phospholipid solution. 0.5 mL of the 1 mg / ml mixed phospholipid solution was mixed with 0.5 mL of the 1 mg / ml mixed membrane solution. The phospholipids and membrane proteins were fused in equal proportions, i.e., phospholipid mg: membrane protein mg = 1:1. The particles were extruded using a liposome extruder and collected to obtain spherical vesicles formed by liposomes coated with a hybrid cell membrane coating, denoted as hybrid cell membrane nanodelivery carrier-V.

[0101] Example 6. Preparation of a nanocarrier for liposomes coated with a hybrid cell membrane:

[0102] A 1 mg / ml mixed membrane solution was prepared according to the method in Example 3. 35 mg of soybean phospholipids, 5 mg of cholesterol, and 10 mg of DSPE-PEG were dissolved in 10 mL of chloroform to obtain a 5 mg / mL mixed phospholipid solution, which was then diluted to obtain a 1 mg / ml mixed phospholipid solution. 0.5 mL of the 1 mg / ml mixed phospholipid solution was mixed with 0.5 mL of the 1 mg / ml mixed membrane solution. The phospholipids and membrane proteins were fused in equal proportions, i.e., phospholipid mg:membrane protein mg = 1:1. The particles were extruded using a liposome extruder and collected to obtain spherical vesicles formed by liposomes coated with a hybrid cell membrane, denoted as hybrid cell membrane nanodelivery carrier-VI.

[0103] Experimental Example 1. Characterization and Performance Testing of Hybrid Cell Membrane Nanodelivery Carriers

[0104] 1) Take 20 μg each of the platelet cell membrane extracted according to the method described in Example 1, the HEK293t stable cell membrane overexpressing the CCR2 gene, and the hybrid cell membrane co-extruded and fused in Example 1 (all at a concentration of 1 mg / ml in buffer), add protein loading buffer, incubate at 95°C for 5 minutes, and perform Western blotting to detect the expression of receptor proteins on the surface of the hybrid cell membrane. The results are shown in [reference needed]. Figure 2 ,like Figure 2 Part A shows that the fused hybrid cell membrane simultaneously expresses platelet surface receptors CD41, CD61P, CD62 and stable transgene overexpressing receptor CCR2.

[0105] 2) Platelet cell membranes (PLT) and stable HK293t cell membranes overexpressing the CCR2 gene (293T) obtained according to the method in Example 1 were respectively labeled with PKH67 lipid fluorescent markers. The stable HK293t cell membranes expressed red fluorescent protein. The labeled platelet membranes and labeled HK293t cell membranes overexpressing the CCR2 gene were prepared into three mixed membrane solutions with a total membrane protein concentration of 1 mg / ml, respectively, according to the methods in Examples 1, 2, and 3. Specifically, the protein weight ratio (PLT:293T) of the platelet cell membrane to the HK293t cell membrane overexpressing the CCR2 gene in the mixed membrane solutions was 2:1, 1:1, and 1:2, respectively. The three mixed membrane solutions were extruded using a liposome extruder, and the particles were collected to obtain the labeled hybrid cell membrane nanodelivery carriers. The co-localization of the two cell membranes in the three labeled hybrid cell membrane nanodelivery carriers was observed using a fluorescence microscope. The results are shown in [link to relevant documentation]. Figure 2 .like Figure 2 Part B shows that the hybrid cell membranes of the three fusion ratios all exhibited approximately 80% colocalization.

[0106] 3) The particle size of the hybrid cell membrane nanodelivery carrier obtained in Example 1 was measured, and the results are as follows: Figure 3 As shown in section A, the CPL group has a hybrid cell membrane (CPL) particle size of approximately 35 nm. Surface potential was measured using CPL, platelet cell membrane (PLTCM) extracted according to the method in Example 1, and the cell membrane of the HEK293t stable transgenic cell line overexpressing the CCR2 gene (CCR2-RFPCM). The results are as follows... Figure 3 As shown in Part B, there was no significant difference in surface potential between the single cell membrane (PLT CM group and CCR2-RFPCM group) and the hybrid cell membrane (CPL group), indicating that the surface molecules did not change.

[0107] 4) The hybrid cell membrane nanodelivery carrier-IV from Example 4, the hybrid cell membrane nanodelivery carrier-V from Example 5, and the hybrid cell membrane nanodelivery carrier-VI from Example 6 were injected into randomly grouped Alzheimer's disease model mice, respectively. The nanocarrier content in the mouse brain and other major organs was measured 12 hours later. Furthermore, the enrichment of the optimal hybrid cell membrane nanocarrier in different organs within 0-24 hours was also measured. See the results below. Figure 4 Part A shows that all three fusion ratios of hybrid cell membrane nanocarriers can cross the blood-brain barrier, and that increased platelet count improves the blood-brain barrier crossing rate. The group with a weight ratio of platelet cell membrane (PLT CM) to HEK293t stable cell membrane overexpressing the CCR2 gene (239TCM) of 2:1 has the highest blood-brain barrier crossing rate. Figure 4 In Part A, the three curves G3, G1, and G2 correspond to the hybrid cell membrane nanodelivery carrier-IV of Example 4, the hybrid cell membrane nanodelivery carrier-V of Example 5, and the hybrid cell membrane nanodelivery carrier-V of Example 6, respectively; Part B shows that the hybrid cell membrane nanodelivery carrier-IV can also be enriched in the liver, spleen, and kidney.

[0108] 5) The hybrid cell membrane nano-delivery carriers obtained in Example 1 and Comparative Example 1 were injected into randomly grouped Alzheimer's disease model mice, and the enrichment of nanomaterials in the inflammatory lesion areas of the brain was detected 12 hours later. See the results below. Figure 5 The results showed that the hybrid cell membrane nanocarrier overexpressing CCR2 (CPL group) could specifically target microglia and astrocytes compared with the control hybrid cell membrane nanocarrier (PL group), indicating that it can achieve the aggregation of inflammatory lesions.

[0109] Example 7. Preparation of nanomedicines encapsulating two combined small molecule drugs:

[0110] 35 mg of soybean lecithin, 5 mg of cholesterol, and 10 mg of DSPE-PEG were dissolved in 10 ml of CHCl3 to obtain a mixed phospholipid solution with a concentration of 5 mg / ml. TPPU and rapamycin were separately prepared into 1 mg / ml stock solutions with ethanol and aliquoted for storage. 0.2 ml of the 5 mg / ml mixed phospholipid solution was taken, and 5 ml of CHCl3 was added, followed by 100 μl of 1 mg / ml TPPU stock solution and 64 μl of 1 mg / ml rapamycin stock solution. The solvent was removed by rotary evaporation, and 1 ml of pre-filtered PBS was added. The resulting drug-loaded solution had a TPPU concentration of 0.1 mg / mL, a rapamycin concentration of 0.064 mg / mL, and a phospholipid concentration of 1 mg / ml. The solution was ultrasonically dispersed in an ice-water bath for 20 min to obtain drug-loaded liposomes (or drug-loaded phospholipid particles). Free drug was removed by ultrafiltration twice (particles on the filter membrane were collected by blowing away impurities), and the precipitate was redispersed in 1 ml of PBS. Take 0.5 ml of a solution containing 1 mg / ml drug-loaded phospholipid particles and mix it with 0.5 ml of a mixed membrane solution with a concentration of 1 mg / ml prepared in Example 1. The phospholipids and membrane proteins are fused in equal proportions, i.e., phospholipid mg: membrane protein mg = 1:1. Then, the particles are collected after being extruded using a liposome extruder, thus obtaining a nanomedicine with a hybrid cell membrane coating on the surface and TPPU and rapamycin loaded inside.

[0111] Comparative Example 2. Preparation of nanomedicines encapsulating single small molecule drugs:

[0112] The preparation method of the nanomedicine in this comparative example is largely the same as that in Example 7, except that rapamycin stock solution is not added during the preparation of the drug-loaded solution system. That is, the TPPU concentration in the obtained drug-loaded solution system is 0.1 mg / mL, the rapamycin concentration is 0 mg / mL, and the phospholipid concentration is 1 mg / mL. Finally, the particles are collected after extrusion using a liposome extruder, yielding a nanomedicine with a hybrid cell membrane coating on the surface and a single TPPU encapsulation inside.

[0113] Comparative Example 3. Preparation of nanomedicines encapsulating single small molecule drugs:

[0114] The preparation method of the nanomedicine in this comparative example is largely the same as that in Example 7, except that TPPU stock solution is not added during the preparation of the drug-loaded solution system. That is, the TPPU concentration in the resulting drug-loaded solution system is 0 mg / mL, the rapamycin concentration is 0.064 mg / mL, and the phospholipid concentration is 1 mg / mL. The particles are then collected after extrusion using a liposome extruder, yielding a nanomedicine with a hybrid cell membrane coating on the surface and encapsulated with a single rapamycin.

[0115] Comparative Example 4. Preparation of nanomedicines containing two combined small molecule drugs loaded onto the cell membrane of a control hybridization cell:

[0116] The preparation method of the nanomedicine in this comparative example is largely the same as that in Example 7, except that a 1 mg / ml mixed membrane solution prepared in Comparative Example 1 is used instead of the 1 mg / ml mixed membrane solution prepared in Example 1 in Example 7, and mixed with 0.5 ml of a solution containing 1 mg / ml of drug-loaded phospholipid particles. The particles are then extruded using a liposome extruder and collected to obtain a nanomedicine with a control hybridization cell membrane coating on the surface and internally loaded with TPPU and rapamycin.

[0117] Experimental Example 2. Characterization of Nanomedicines:

[0118] The nanomedicines obtained in Example 7, Comparative Example 2, Comparative Example 3, and Comparative Example 4 were subjected to particle size analysis, and the results are as follows: Figure 3 As shown, the nanomedicines loaded with a single drug prepared in Comparative Examples 2 and 3 are shown. Figure 3 The particle size of T@CPL and R@CPL groups in part A is 35-50 nm. The nanomedicines loaded with dual drugs prepared in Example 7 and Comparative Example 4 ( Figure 3 The nanoparticles (TR@CPL and TR@PL group in part A) have a diameter of approximately 70 nm. Comparative Examples 2 and 3 prepared nanomedicines encapsulating single drugs... Figure 3 The T@CPL and R@CPL groups in Part B and the nanomedicines loaded with dual drugs prepared in Example 7 and Comparative Example 4 ( Figure 3 There was no significant difference in surface potential between TR@CPL and TR@PL groups in part B. The drug release curves of TPPU (A) and rapamycin (B) in the dual-drug nanomedicine prepared in Example 7 are shown below. Figure 6 The drug can be released continuously for at least 72 hours.

[0119] Experimental Example 3. Efficacy Experiment of Nanomedicine:

[0120] The nanomedicines containing two combined small molecule drugs obtained in Example 7, TPPU and rapamycin, the nanomedicine containing a single small molecule drug prepared in Comparative Example 2, the nanomedicine containing a single small molecule drug prepared in Comparative Example 3, and the nanomedicine containing two small molecule drugs prepared in Comparative Example 4 were respectively prepared into injections for in vivo efficacy experiments in Alzheimer's disease model mice. This experiment consisted of seven groups: 1) wild-type control mice (WT), 2) Alzheimer's disease model mice injected with a solubilizer (FAD), 3) Alzheimer's disease model mice injected with TPPU + rapamycin (T+R), 4) Alzheimer's disease model mice injected with TPPU monotherapy from CCR2+PLT hybrid cell membranes (T@CPL) as described in Comparative Example 2, 5) Alzheimer's disease model mice injected with rapamycin monotherapy from CCR2+PLT hybrid cell membranes (R@CPL) as described in Comparative Example 3, 6) Alzheimer's disease model mice injected with TPPU and rapamycin monotherapy from CCR2+PLT hybrid cell membranes (TR@CPL) as described in Example 7, and 7) Alzheimer's disease model mice injected with TPPU and rapamycin monotherapy from HEK293t+PLT hybrid cell membranes (TR@PL) as described in Comparative Example 4. The injection method is tail vein injection. The injection dose is 100 μl / 20 g body weight, calculated based on 1 mg / ml of hybrid cell membrane, and is administered 3 times a week.

[0121] Six weeks after drug administration, cognitive function was assessed in mice. Results were as follows: Figure 7 As shown, cognitive function was assessed using a water maze behavioral experiment. The results showed that the cognitive level of mice in group 6) was significantly improved after administration of Example 7. Compared with the drugs injected in other groups, the CCR2-PLT fusion cell membrane nanomedicine prepared in Example 7 with dual drugs exhibited the best therapeutic effect (TR@CPL group).

[0122] For groups 2) to 7) above, amyloid plaque deposition in the brains of Alzheimer's disease model mice was examined 6 weeks after drug administration. Results are shown below. Figure 8 As can be seen, compared with group 2) which received injection of solubilizers and group 3) which received injection of two small molecule drugs, the cortical area of ​​mice in groups 4), 5), 6), and 7) after administration was significantly improved. Figure 8 Part A) and Hippocampus ( Figure 8 In Part B of the study, the deposition of amyloid plaques was significantly reduced. Among them, the CCR2-PLT fusion cell membrane nanomedicine (TR@CPL group) prepared in Example 7, which was injected in Group 6), showed the most significant effect. Compared with the nanomedicines loaded with single drugs injected in Groups 4 and 5 and the nanomedicine containing the control carrier injected in Group 7, the number of amyloid plaques was greatly reduced after administration in Group 6.

[0123] In summary, the delivery vector constructed by fusing platelet cell membranes with HEK293t cell membranes overexpressing the CCR2 gene in this invention significantly improves both blood-brain barrier crossing efficiency and targeting of central nervous system inflammatory lesions. Furthermore, the nanomedicine formed by encapsulating two small molecule drugs, TPPU and rapamycin, on this delivery vector has shown unexpectedly excellent therapeutic effects on Alzheimer's disease, indicating that the two drugs used in combination exert a synergistic therapeutic effect.

Claims

1. A nano-drug capable of targeting intracranial inflammatory lesions, wherein the nano-drug is composed of a hybrid cell membrane coating coated drug-loaded liposome; the hybrid cell membrane coating is composed of platelet cell membranes and HEK293t cell membranes overexpressing CCR2 gene at a weight ratio of 1:2-2:1; the drug-loaded liposome is a complex phospholipid loaded with a therapeutic drug in the interior, wherein the therapeutic drug comprises one or more than two small molecule drugs having therapeutic effects on Alzheimer's disease; and the small molecule drugs having therapeutic effects on Alzheimer's disease are selected from a combination of one or two of rapamycin and TPPU.

2. The nanomedicine of claim 1, wherein: The hybrid cell membrane coating is composed of platelet cell membranes and HEK293t cell membranes overexpressing CCR2 gene at a weight ratio of 2:

1.

3. The nanomedicine as described in claim 1, characterized in that: The weight ratio of the hybrid cell membrane coating to the complex phospholipid is 0.5-1.5:

1.

4. The nanomedicine according to any of claims 1 to 2, wherein: The weight ratio of the hybrid cell membrane coating to the complex phospholipid is 1:

1.

5. Nanomedicine according to any one of claims 1 or 3, characterized in that: The small molecule drugs having therapeutic effects on Alzheimer's disease are a combination of rapamycin and TPPU. 6.A method for preparing the nano-drug of claim 1, comprising: I) obtaining platelet cell membranes by gradient centrifugation and repeated freeze-thaw method; II) constructing a plasmid overexpressing CCR2 gene based on PGMLV-CMV-MCS-mScarlet-PGK-Puro vector, then infecting HEK293t cell line with the plasmid, screening HEK293t stable transfectants overexpressing CCR2 gene from the infected HEK293t cell line, and extracting cell membranes of the HEK293t stable transfectants overexpressing CCR2 gene by repeated freeze-thaw method; III) adding one or more than two drugs having therapeutic effects on Alzheimer's disease to a mixed phospholipid solution, and loading the drugs by phospholipid self-assembly to obtain drug-loaded liposomes; IV) mixing the platelet cell membranes of I), the cell membranes of the HEK293t stable transfectants overexpressing CCR2 gene of II), and the drug-loaded liposomes of III), controlling the concentration ratio of membrane protein to phospholipid to be 1:2-2:1, and controlling the protein concentration ratio of platelet cell membranes to cell membranes of the HEK293t stable transfectants overexpressing CCR2 gene to be 1:2-2:1, and then performing fusion to obtain the nano-drug capable of targeting intracranial inflammatory lesions.

7. The method of claim 6, wherein: In IV), the concentration ratio of membrane protein to phospholipid is controlled to be 1:

1.

8. The method of claim 6, wherein: In IV), the protein concentration ratio of platelet cell membranes to cell membranes of the HEK293t stable transfectants overexpressing CCR2 gene is controlled to be 2:

1.

9. The method of claim 6, wherein: The fusion in IV) is performed by co-extrusion.

10. The method of any one of claims 6-9, characterized in that: The mixed phospholipid solution contains natural phospholipids and synthetic phospholipids; the natural phospholipids are selected from soybean lecithin or egg yolk lecithin; and the synthetic phospholipids are selected from DSPE-PEG, DSPC or DOPC.

11. The method of claim 10, wherein: The natural phospholipid is soybean lecithin.

12. The method of claim 10, wherein: The synthetic phospholipid is DSPE-PEG.

13. The method of claim 10, wherein: The mixed phospholipid solution is composed of 35 mg of soybean lecithin, 5 mg of cholesterol and 10 mg of DSPE-PEG dissolved in 10 mL of chloroform.

14. Use of the nanopharmaceutical of any one of claims 1-5 in the manufacture of a medicament for the treatment of a central nervous system disease in a human or mammal; the central nervous system disease being Alzheimer's disease.

15. The use according to claim 14, wherein: The nanopharmaceutical is prepared into an injection.

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