Brain-targeted delivery system mediated by amyloid beta short peptides, its preparation method and use

Abstract

The present invention relates to a polypeptide-modified conjugate capable of specifically adsorbing apolipoproteins in plasma and mediating drug delivery across the blood-brain barrier, as well as its use in the preparation of formulations for diagnosing and treating brain tumors and other brain diseases. A polypeptide fragment (SP) of amyloid beta (type 1 related) is modified, and the modified delivery system has been shown to increase the uptake of amyloid beta by vascular endothelial cells after forming a protein crown with plasma proteins. The modified liposome delivery system more effectively delivers drugs to lesion sites, significantly improving the therapeutic efficacy of the drug. After SP adsorbs plasma proteins, the drug can be mediated across the blood-brain barrier and / or target tumor neovasculature and tumor cells. The modified drug and delivery system can achieve better therapeutic effects in treating brain tumors and other brain diseases.

Description

[Technical field]

[0001] This application claims the benefit of Chinese Patent No. CN201810626652.4, filed on June 19, 2018, which is incorporated herein by reference in its entirety.

[0002] The present invention belongs to the field of pharmacology and relates to a brain-targeting delivery system mediated by amyloid-β short peptide (SP), in particular to a polypeptide-modified compound and a targeted delivery system that can specifically adsorb apolipoproteins in plasma and mediate the passage of drugs through the blood-brain barrier, and its application in the preparation of preparations for diagnosing and treating brain tumors (including brain metastases and primary brain tumors) and other brain diseases. [Background technology]

[0003] Although diseases of the central nervous system have become diseases that seriously endanger human health, the development progress of research on related new drugs is still very slow, as disclosed in the prior art. One of the main reasons is that most drug molecules in drugs have difficulty passing through the blood-brain barrier, and designing drugs or drug delivery systems that can pass through the blood-brain barrier is one of the key and difficult points in the field of current drug research and development. Brain-targeting drug delivery systems mainly use receptor-mediated methods to pass through the blood-brain barrier to reach the brain, but current drug design mainly focuses on the binding efficiency of brain-targeting molecules and associated receptors on the blood-brain barrier, and research has revealed that the influence of body fluid components is often ignored in the process of in vivo delivery of drug delivery systems. For example, combining plasma proteins to form a protein corona can have a significant impact on the targeting and in vivo circulation time of drug delivery systems, which results in a decrease in the targeting efficiency in vivo of in vitro designed brain-targeting drug delivery systems. How to design a brain-targeting drug delivery system from a brand new perspective and precisely regulate its interaction with related proteins or cells during the process of in vivo delivery, including the adsorption and separation of plasma proteins, phagocytosis of other cells, protein degradation, immune response, etc., to achieve more effective targeting function, is the hottest research topic in the current industry. Summary of the Invention [Problem to be solved by the invention]

[0004] Based on the current state of the prior art, the inventors of the present application intend to provide a brain-targeting delivery system mediated by amyloid beta short peptide (hereinafter referred to as SP), particularly to a polypeptide-modified compound and targeted delivery system that can specifically adsorb apolipoproteins in plasma and mediate drugs to pass through the blood-brain barrier, and its application in preparing preparations for diagnosing and treating brain tumors (including brain metastasis and primary brain tumors) and other brain diseases. The polypeptide-modified targeted delivery system overcomes the defects of traditional targeted nano-drugs by adjusting the components and bioactivity of nano-drugs that form protein coronas in plasma, constructing SP-drug complexes and SP-modified nano-drug delivery systems that can pass through the blood-brain barrier and target vascular endothelial cells and tumor cells simultaneously, thereby realizing the targeted diagnosis and treatment of brain tumors and drug delivery of other brain diseases.

[0005] The purpose of the present invention is to provide a brain-targeting delivery system mediated by amyloid beta short peptide (SP), in particular, a polypeptide-modified compound and a targeted delivery system that can specifically adsorb apolipoproteins in plasma and mediate drugs to pass through the blood-brain barrier, and its application in the preparation of preparations for diagnosing and treating peripheral tumors, brain tumors and other brain diseases. The polypeptide-modified targeted delivery system overcomes the defects of traditional targeted nano-drugs by adjusting the components and bioactivity of nano-drugs that form protein coronas in plasma, constructing SP-drug complexes and SP-modified nano-drug delivery systems that can pass through the blood-brain barrier and target vascular endothelial cells and tumor cells simultaneously, thereby realizing the targeted diagnosis and treatment of brain tumors (including brain metastases and primary brain tumors) and drug delivery of other brain diseases.

[0006] Before describing the present invention, the terms used herein are defined as follows: The term "SP" refers to amyloid beta short peptide. The term "PLA" means polylactic acid. The term "PLGA" means poly(lactic-co-glycolic acid). The term "PCL" means polycaprolactone. The sequence (N to C) of the SP polypeptide of the present invention is NH2-GSNKGAIIGLM-CONH2. [Means for solving the problem]

[0007] A first aspect of the present invention provides the use of an amyloid β short peptide in the preparation of a preparation for mediating a drug molecule, a fluorescent probe or a delivery system for targeting lesions of brain tumors and / or other brain diseases, wherein the amyloid β short peptide is an amyloid β short peptide capable of specifically adsorbing apolipoproteins in plasma; preferably, the brain tumor is a brain metastatic tumor and / or a primary brain tumor.

[0008] A short amyloid beta peptide is covalently linked to an imaging agent X to prepare SP-X, which is used to track lesions of brain tumors and other brain diseases; Preferably, in the SP-X, X is a fluorescent molecule fluorescein or a near-infrared dye molecule Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, or DiI; More preferably, the use according to the first aspect of the invention, wherein said brain tumour is a brain metastatic tumour and / or a primary brain tumour.

[0009] A short amyloid beta peptide is covalently linked to an antitumor drug Y to prepare SP-Y, which is used for targeted intervention of the lesions of brain tumors and other brain diseases; Preferably, in said SP-Y, Y is gefitinib, icotinib, anlotinib, crizotinib, erlotinib, osimertinib, alectinib, paclitaxel, docetaxel, cabazitaxel, adriamycin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocellulose camptothecin or vincristine small molecule antitumor drug; and / or In the SP-Y, Y is a p53 activating peptide or a polypeptide poison polypeptide antitumor drug; More preferably, the use according to the first aspect of the invention, wherein said brain tumour is a brain metastatic tumour and / or a primary brain tumour.

[0010] Amyloid β short peptide is covalently linked to polyethylene glycol-Z to prepare SP-polyethylene glycol-Z, which is used to prepare the nanometer delivery system; Preferably, in said SP-polyethylene glycol-Z, Z is a phospholipid, polylactic acid, lactic acid-co-glycolic acid or polycaprolactone; and / or Preferably, the nano-delivery system is selected from one or more of a liposome delivery system, a micelle delivery system, a nanodisc delivery system, and a polymeric nanoparticle delivery system; More preferably, SP-polyethylene glycol-phospholipid is used to prepare a liposome delivery system, a micellar delivery system or a nanodisc delivery system; and / or SP-polyethylene glycol-polylactic acid, SP-polyethylene glycol-lactic acid-co-glycolic acid, SP-polyethylene glycol-polycaprolactone are used to prepare a micellar delivery system and a polymeric nanoparticle delivery system; More preferably, a liposomal, micelle, nanodisc or polymeric nanoparticle delivery system is used to encapsulate a diagnostic molecule for tracking the lesions of brain tumors or other brain diseases; preferably, the diagnostic molecule encapsulated in the delivery system is 5-carboxyfluorescein 5-FAM or the near infrared dyes Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, DiI; and / or Nanoparticle delivery systems, liposomal delivery systems, micelle delivery systems, polymeric nanoparticle delivery systems or nanodisc delivery systems are used to encapsulate antitumor drugs for targeted intervention of lesions of brain tumors and other brain diseases; preferably, the drug encapsulated in the delivery system is gefitinib, icotinib, anlotinib, crizotinib, erlotinib, osimertinib, alectinib, paclitaxel, docetaxel, cabazitaxel, adriamycin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocellulose camptothecin, vincristine, p53 activating peptide or polypeptide toxin; Even more preferably, the use according to the first aspect of the invention, wherein said brain tumour is a brain metastatic tumour and / or a primary brain tumour.

[0011] A second aspect of the present invention provides an amyloid β short peptide for preparing a preparation for mediating a drug molecule, a fluorescent probe or a delivery system for targeting lesions of brain tumors and / or other brain diseases, the amyloid β short peptide being an amyloid β short peptide capable of specifically adsorbing apolipoproteins in plasma; preferably, the brain tumor is a brain metastatic tumor and / or a primary brain tumor.

[0012] A third aspect of the present invention is a tracer molecule for brain tumors and other brain disease lesions, the tracer molecule being SP-X prepared by covalently linking the amyloid beta short peptide of claim 5 to an imaging substance X; Preferably, in the SP-X, X is a fluorescent molecule fluorescein or a near-infrared dye molecule Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, or DiI; More preferably, the tracer molecule is provided wherein said brain tumor is a brain metastatic tumor and / or a primary brain tumor.

[0013] A fourth aspect of the present invention is a drug for targeted intervention of lesions of brain tumors and other brain diseases, said drug being SP-Y prepared by covalently linking the amyloid beta short peptide according to claim 5 to an antitumor drug Y; Preferably, in said SP-Y, Y is gefitinib, icotinib, anlotinib, crizotinib, erlotinib, osimertinib, alectinib, paclitaxel, docetaxel, cabazitaxel, adriamycin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocellulose camptothecin or vincristine small molecule antitumor drug; and / or In the SP-Y, Y is a p53 activating peptide or a polypeptide poison polypeptide antitumor drug; More preferably, the brain tumor is a brain metastatic tumor and / or a primary brain tumor.

[0014] A fifth aspect of the present invention is a nano-delivery system comprising SP-polyethylene glycol-Z prepared by linking the amyloid beta short peptide of the second aspect with polyethylene glycol-Z; Preferably, in the SP-polyethylene glycol-Z, Z is a phospholipid, polylactic acid, lactic acid-co-glycolic acid, or polycaprolactone; More preferably, the nano-delivery system is selected from one or more of a liposome delivery system, a micelle delivery system, a nanodisc delivery system, and a polymeric nanoparticle delivery system; More preferably, when SP-polyethylene glycol-Z is SP-polyethylene glycol-phospholipid, said nano-delivery system is selected from one or more of a liposome delivery system, a micelle delivery system, a nanodisc delivery system; when SP-polyethylene glycol-Z is SP-polyethylene glycol-polylactic acid, SP-polyethylene glycol-lactic acid-co-glycolic acid or SP-polyethylene glycol-polycaprolactone, said nano-delivery system is a micelle delivery system and / or a polymeric nanoparticle delivery system; More preferably, said liposomal, micelle, nanodisc or polymeric nanoparticle delivery system is used to encapsulate a diagnostic molecule for tracking the lesions of brain tumors or other brain diseases; preferably, the diagnostic molecule encapsulated in the delivery system is 5-carboxyfluorescein 5-FAM or the near infrared dyes Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, DiI; and / or More preferably, a nanoparticle delivery system, a liposomal delivery system, a micelle delivery system, a polymeric nanoparticle delivery system or a nanodisc delivery system is used to encapsulate an antitumor drug for targeted intervention of lesions of brain tumors or other brain diseases; preferably, the drug encapsulated in the delivery system is gefitinib, icotinib, anlotinib, crizotinib, erlotinib, osimertinib, alectinib, paclitaxel, docetaxel, cabazitaxel, adriamycin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocellulose camptothecin, vincristine, p53 activating peptide or polypeptide toxin; Even more preferably, the nanodelivery system is provided wherein said brain tumor is a brain metastatic tumor and / or a primary brain tumor.

[0015] A sixth aspect of the present invention provides a method for preparing a nanodelivery system of the fifth aspect, comprising the steps of: Weighing out the natural phospholipids, cholesterol, methoxy-PEG-DSPE, and SP-polyethylene glycol-Z, model drug or probe molecules, dissolving them in a solvent, forming a film, hydrating, passing them through a membrane, and removing the free probe molecules or drugs by column chromatography to create a nano-delivery system; and / or Measure out the natural phospholipid, cholesterol, methoxy-PEG-DSPE, and SP-polyethylene glycol-Z, dissolve them in a solvent, form a film, hydrate, pass them through the membrane, actively encapsulate the probe molecule or drug, and remove the free probe molecule or drug by column chromatography to create the nano-delivery system. The present invention provides a method comprising:

[0016] A seventh aspect of the present invention provides a pharmaceutical composition comprising: 1) a short amyloid β peptide according to the second aspect, a tracer molecule according to the third aspect, a drug according to the fourth aspect and / or a nanodelivery system according to the fifth aspect; and 2) a pharma- ceutically acceptable carrier.

[0017] An eighth aspect of the present invention provides a diagnostic preparation comprising a short amyloid beta peptide according to the second aspect, a tracer molecule according to the third aspect, a drug according to the fourth aspect and / or a nanodelivery system according to the fifth aspect.

[0018] A ninth aspect of the present invention relates to a method for treating a brain disease, comprising administering to a subject in need thereof an amyloid beta short peptide according to the second aspect, a tracer molecule according to the third aspect, a drug according to the fourth aspect and / or a nanodelivery system according to the fifth aspect, Preferably, the brain disease is a brain tumor or other brain disease; More preferably, the method is provided wherein said brain tumor is a brain metastatic tumor and / or a primary brain tumor.

[0019] A tenth aspect of the present invention provides a method for diagnosing brain tumours and other brain disease lesions, comprising administering to a subject in need thereof an amyloid beta short peptide as described in the second aspect, a tracer molecule as described in the third aspect, a drug as described in the fourth aspect and / or a nanodelivery system as described in the fifth aspect, preferably wherein the brain tumour is a brain metastatic tumour and / or a primary brain tumour.

[0020] An eleventh aspect of the present invention provides a method for targeted intervention of brain tumours and other brain disease lesions, comprising the step of administering to a subject in need thereof a short amyloid beta peptide as described in the second aspect, a tracer molecule as described in the third aspect, a drug as described in the fourth aspect and / or a nanodelivery system as described in the fifth aspect, preferably wherein the brain tumour is a brain metastatic tumour and / or a primary brain tumour.

[0021] A twelfth aspect of the present invention provides a drug delivery method comprising the step of administering to a subject in need thereof an amyloid β short peptide as described in the second aspect, a tracer molecule as described in the third aspect, a drug as described in the fourth aspect and / or a nanodelivery system as described in the fifth aspect.

[0022] According to the present invention, the clearance mechanism of amyloid beta in the brain is simulated by using apolipoprotein as a molecular partner and receptor-mediated transport to the periphery, and amyloid beta is used as a template to design a short peptide (SP) that can specifically bind to apolipoprotein lipid-binding domain. By modifying the short peptide on the surface of liposome, apolipoprotein in plasma can be specifically adsorbed, and its biological activity can be maintained during the process of blood circulation in vivo, and then apolipoprotein can combine with various receptors on the blood-brain barrier [including low-density lipoprotein-related protein 1 (LRP-1), scavenger receptor class b member 1 (srb1) and low-density liposome-related protein 2 (LRP-2)] to mediate liposome transport to the brain. The polypeptide-modified liposomes overcome the deficiencies of traditional targeted nano-drugs by adjusting the components and biological activity of nano-drugs that form a protein corona in plasma, and construct SP-drug complexes and SP-modified nano-drug delivery systems that can cross the blood-brain barrier and simultaneously target vascular endothelial cells and tumor cells, thereby realizing the targeted diagnosis and treatment of brain tumors and drug delivery for other brain diseases.

[0023] In the present invention, SP is covalently modified with polyethylene glycol-distearoylphosphoethanolamine (PEG-DSPE), polyethylene glycol-polylactic acid (PEG-PLA), polyethylene glycol-lactic acid-co-glycolic acid (PEG-PLGA), polyethylene glycol-polycaprolactone (PEG-PCL) and other substances to construct delivery systems such as liposomes, micelles, nanodiscs, nanoparticles, etc. modified with SP short peptides.

[0024] The SP-modified nano-delivery system can encapsulate antitumor drugs such as gefitinib, icotinib, anlotinib, crizotinib, erlotinib, osimertinib, alectinib, paclitaxel, docetaxel, cabazitaxel, adriamycin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocellulose camptothecin, vincristine, p53 activating peptide, bee venom peptide, scorpion venom peptide, etc. It can also encapsulate fluorescent substances such as FAM, near-infrared dyes Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, DiI, etc.

[0025] In the present invention, the SP modified drug or probe comprises a pH-sensitive hydrazone bond formed by the reaction of a maleimide hydrazide derivative with a drug, such as adriamycin, epirubicin, p53 activating peptide, and polypeptide toxin; or comprises a disulfide bond formed by the reaction of a 3-(2-pyridinedimercapto)propionic acid derivative with a drug, such as gefitinib, icotinib, anlotinib, crizotinib, erlotinib, osimertinib, alectinib, paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocellulose camptothecin, vincristine, p53 activating peptide, and polypeptide toxin; or comprises a fluorescent probe connected via a stable chemical bond, such as fluorescein, Cy5, Cy5.5, Cy7, ICG, and IR820.

[0026] Furthermore, the present invention provides an application of amyloid beta short peptides in the preparation of a preparation for mediating a drug molecule, a fluorescent probe or a delivery system for targeting a brain tumor (including brain metastasis and primary brain tumor) or a lesion of another brain disease, wherein the amyloid beta short peptide is an amyloid beta short peptide capable of specifically adsorbing apolipoproteins in plasma.

[0027] A short amyloid-β peptide (SP) is covalently linked to an imaging agent X to prepare SP-X, which is used to track lesions of brain tumors (including brain metastases and primary brain tumors) and other brain diseases; in the SP-X, X is the fluorescent molecule fluorescein or the near-infrared dye molecules Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, DiI.

[0028] Amyloid-β short peptides (SP) are covalently linked to an antitumor drug Y to prepare SP-Y for targeted intervention of brain tumors (including brain metastases and primary brain tumors) and other brain disease lesions; in the SP-Y, Y is gefitinib, icotinib, anlotinib, crizotinib, erlotinib, osimertinib, alectinib, paclitaxel, docetaxel, cabazitaxel, adriamycin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocellulose camptothecin, or vincristine small molecule antitumor drug; in the SP-Y, Y is a p53 activating peptide or a polypeptide poison polypeptide antitumor drug.

[0029] Amyloid beta short peptides (SP) are covalently linked to polyethylene glycol-Z to prepare SP-polyethylene glycol-Z, which is used to prepare nanometer delivery systems; in the SP-polyethylene glycol-Z, Z is phospholipid, polylactic acid (PLA), lactic-co-glycolic acid (PLGA) or polycaprolactone (PCL).

[0030] The SP-polyethylene glycol-phospholipids of the present invention are used to prepare liposomal, micelle or nanodisc delivery systems.

[0031] The SP-polyethylene glycol-polylactic acid, SP-polyethylene glycol-lactic acid-co-glycolic acid, SP-polyethylene glycol-polycaprolactone of the present invention are used to prepare micellar delivery systems and polymeric nanoparticle delivery systems.

[0032] The liposomal, micelle, nanodisc or polymeric nanoparticle delivery system of the present invention is used to encapsulate diagnostic molecules to track the lesions of brain tumors (including brain metastases and primary brain tumors) or other brain diseases; the diagnostic molecules encapsulated in the delivery system are 5-carboxyfluorescein 5-FAM or near infrared dyes Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, DiI.

[0033] The nanoparticle delivery system, liposomal delivery system, micelle delivery system, polymeric nanoparticle delivery system or nanodisc delivery system of the present invention is used to encapsulate anti-tumor drugs for targeted intervention of lesions of brain tumors (including brain metastases and primary brain tumors) or other brain diseases; the encapsulated drug is gefitinib, icotinib, anlotinib, crizotinib, erlotinib, osimertinib, alectinib, paclitaxel, docetaxel, cabazitaxel, adriamycin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocellulose camptothecin, vincristine, p53 activating peptide or polypeptide toxin.

[0034] More specifically, the present invention is realized by the following technical solutions: 1. Preparation of SP-modified Liposome Delivery System The SP-containing cysteine ​​sulfhydryl and maleimide-PEG-DSPE are reacted in phosphate buffer at pH 7.4. After purification, the product is lyophilized to prepare SP-PEG-DSPE for later use.

[0035] A certain amount of natural phospholipid, cholesterol, methoxy-PEG-DSPE (mPEG-DSPE) is weighed together with SP-PEG-DSPE, model drug or probe molecule, dissolved in a solvent, formed into a film, hydrated, passed through a membrane by a liposome extruder, and free probe molecule or drug is removed by column chromatography to prepare SP polypeptide-modified liposomes. The particle size and surface potential are characterized by a dynamic light scattering particle size analyzer. Alternatively, a certain amount of natural phospholipid, cholesterol, methoxy-PEG-DSPE (mPEG-DSPE) and SP-PEG-DSPE is weighed, dissolved in a solvent, formed into a film, hydrated, passed through a membrane by a liposome extruder, actively encapsulated the probe molecule or drug, and free probe molecule or drug is removed by column chromatography to prepare SP polypeptide-modified liposomes. The particle size and surface potential are characterized by a dynamic light scattering particle size analyzer.

[0036] 2. Experiments on the effect of SP-modified delivery systems on the binding activity after incubation with plasma to form a protein corona After the SP-modified delivery system is incubated with fresh plasma for a while, its binding activity to LRP-1 receptor is detected by Western blot and radiolabeling method. The SP-modified delivery system without serum incubation and the unmodified SP-delivery system are used as negative controls.

[0037] 3. Experiment of the effect of SP modification on the uptake of the delivery system by vascular endothelial cell lines The uptake of SP-modified and unmodified delivery systems by vascular endothelial cells (such as bEND.3 cells) is compared before and after serum incubation.

[0038] 4. Assessment of the Ability of the SP-Modified Delivery System to Cross the BBB in Normal Mice Normal mice (Kunmin, C57BL / 6, etc.) are injected with the fluorescein-loaded delivery system via the tail vein, and the accumulation of the SP-modified and unmodified delivery systems in the mouse brain at different time points is compared.

[0039] 5. Pharmacokinetic evaluation of the SP-modified delivery system in normal rats Normal SD rats are injected with the fluorescein DiI-labeled polypeptide modified and unmodified delivery systems through the tail vein. Blood is collected at different time points, plasma is separated, and the content of DiI in the plasma is quantitatively detected by fluorescence to evaluate the pharmacokinetics of the SP-modified delivery system in rats.

[0040] 6. Evaluation of Immunogenicity of the SP-modified Delivery System in Normal Mice The polypeptide-modified and unmodified delivery systems containing lipid A are prepared and injected intraperitoneally into Balb / c mice once every 7 days for 4 weeks. Blood is collected from the orbit on the 7th day after each injection, and plasma is collected and frozen for later use. ELISA is used to detect the contents of IgG and IgM against PEG and SP in mice at different time points to evaluate immunogenicity.

[0041] 7. Evaluation of the Antitumor Efficacy of the SP-modified Delivery System In Vivo The SP-modified delivery system, unmodified polypeptide delivery system, free drug and normal saline were injected into nude mice bearing orthotopic U87 glioma model via tail vein to evaluate the antitumor effect of the SP-modified delivery system in vivo using the median survival time, apoptosis and angiogenesis density of tumor tissue as indicators.

[0042] The study shows that the SP-mediated drug or drug delivery system has the ability to target cells and tissues with high LRP-1 expression and cross biological membrane barriers, especially the blood-brain barrier (BBB), and can be used for targeted diagnosis and treatment of brain tumors (including brain metastases and primary brain tumors) and other diseases.

[0043] The experiment shows that SP can be modified on the surface of the drug delivery system, combine with apolipoproteins in plasma to form a protein corona, and increase the uptake of vascular endothelial cells; SP can mediate drugs across the blood-brain barrier and / or target tumor angiogenesis and tumor cells at the same time. The modified drugs and drug delivery systems can achieve better therapeutic effects in the treatment of brain tumors (including brain metastases and primary brain tumors) and other diseases of the brain. [Brief description of the drawings]

[0044] [Figure 1] Figure 1 shows the binding identification of SP-modified liposomes and plasma proteins in vitro. SP was modified on the surface of liposomes by chemical binding to obtain SP-sLip, which was mixed with mouse plasma, incubated at 37°C for 1 h, and then centrifuged at high speed to obtain liposome precipitates containing protein corona. After Biorad 4%-20% gradient SDS-PAGE separation, the plasma protein components adsorbed by liposomes were analyzed by rapid silver staining (as shown in Figure 1A). Compared with methoxypolyethylene glycol-modified long circulating liposomes (sLip), the bands of plasma proteins adsorbed by SP-sLip at 45 KDa (band 1), 38 KDa (band 2), and 25 KDa (band 3) were significantly increased. The LC-MS / MS and Western blot results (as shown in Figure 1B) were analyzed on PAGE adhesive corresponding to the same positions at three places, and the three proteins were identified as ApoJ, ApoE, and ApoA1 from mice. Western blot semiquantitative methods also confirmed that SP-sLip adsorbed significantly more of the three apolipoproteins than sLip (as shown in Fig. 1C–E ). [Diagram 2]Figure 2A shows the identification of plasma functional apolipoprotein adsorption by fluorescently labeled sLip and SP-sLip in vivo. To demonstrate that liposomes modified by targeted small peptides can adsorb relevant target proteins even after systemic administration to animals, we injected the same amount of DiI fluorescently labeled liposomes into mice via the tail vein, then collected blood 1 h later to isolate liposomes containing protein corona in plasma. As shown in Figure 2A, Western blot showed that there was a significant difference in the content of three apolipoproteins (ApoJ, ApoE, and ApoA1) adsorbed by the same content of liposomes in mice. This result demonstrates that SP-modified liposomes (SP-sLip) can rapidly and specifically adsorb apolipoproteins in mouse plasma proteins compared with normal liposomes. (As shown in the semi-quantitative analysis in Figure 2B-D) [Diagram 3] Figure 3 shows the evaluation of the binding activity of sLip and SP-sLip to the LRP-1 receptor after adsorption of plasma proteins. The SP-modified liposome (SP-sLip) itself can specifically adsorb the recombinant protein LRP-1 in solution (as shown in Figure 3A). We confirmed by Western blot that SP-sLip retained the ability to bind to LRP-1 even after preincubation with ApoE (as shown in Figures 3B-C). Figure 3D shows that ELISA demonstrated that both free SP and modified liposome (SP-sLip) could compete for the interaction of SP with antibodies immobilized on a 96-well plate, but SP-sLip lost the ability to compete for antibody binding after plasma incubation, which indicated that the functional domain of SP on the surface of the liposome was blocked after binding to plasma proteins such as ApoE. Apolipoproteins such as ApoE, which can specifically bind to the surface of SP-sLip, have the activity of binding to the BBB receptor LRP-1, making this an effective way to mediate SP-sLip to cross the BBB and enter the brain. [Figure 4]Figure 4 shows the uptake of liposomes by vascular endothelial cells before and after plasma incubation. After SP-modified liposomes (SP-sLip) were incubated with plasma to form a protein corona, the uptake of SP-sLip by endothelial cells was increased (as shown in Figure 4A-B). [Diagram 5] Figure 5 shows the pharmacokinetic parameters and immunogenicity evaluation of SP-modified liposomes in mice. SP was modified on the surface of liposomes (SP-sLip), which did not affect the pharmacokinetic parameters of liposomes in vivo (as shown in Figure 5A), and did not increase the immunogenicity of general long-circulating liposomes (IgG and IgM in blood were used as evaluation indexes, as shown in Figures 5B-D). [Figure 6] Figure 1 shows the evaluation of the brain penetration efficiency of SP-modified liposomes in mice.Through the in vivo detection of fluorescein in mouse brain, it was found that the amount of SP-sLip group passing through the BBB was significantly higher than that of the sLip group, indicating that SP can mediate the delivery system across the blood-brain barrier. [Figure 7] Figure 1 shows the anti-glioma effect of adriamycin-encapsulated liposomes in vivo. The median survival times of mice in the saline, DOX, sLip / DOX and SP-sLip / DOX groups were 27, 31, 33 and 50 days, respectively. SP-modified liposomes can significantly extend the median survival time of brain orthotopic tumor model mice. [Figure 8] Figure 1 shows the effect of adriamycin-encapsulated liposomes on the angiogenesis of tumor tissues. The blood vessel density of tumor tissues in the SP-modified liposomes encapsulating adriamycin (SP-sLip / DOX) group was significantly lower than that in the unmodified group (sLip / DOX), indicating that SP could mediate the delivery system to target tumor angiogenesis. [Figure 9]Figure 1 shows the effect of adriamycin-loaded liposomes on the apoptosis of glioma cells. The number of apoptosis in tumor tissues of the adriamycin-encapsulated SP-modified liposome (SP-sLip / DOX) group was significantly higher than that of the unmodified group, indicating that SP can mediate a drug delivery system targeting tumor cells. [Figure 10] Figure 10 shows safety evaluation. SP showed no cytotoxicity to the in vitro cultured neuronal cell line PC12 (as shown in Figure 10A), and there was no obvious abnormality in the tissue sections of mouse organs in the group of SP-modified liposomes encapsulating adriamycin (SP-sLip / DOX) (as shown in Figure 10B), which indicated that SP modification on the liposome surface did not cause toxicity and side effects. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The present invention will be further described by the following specific examples, which should be understood as being for the purpose of more detailed and specific illustration only, and should not be understood as limiting the present invention in any manner.

[0046] This section provides a general description of the materials and test methods used in the testing of the present invention. Although many of the materials and methods of operation used to achieve the objectives of the present invention are well known in the art, the present invention is described herein in as much detail as possible. It will be apparent to those skilled in the art that, unless otherwise specified in the context, the materials and methods of operation used in the present invention are well known in the art. EXAMPLES

[0047] Preparation of liposomes Preparation of liposomal sLip / DiI and SP-sLip / DiI: Preparation of liposome sLip / DiI: Weigh the membrane materials for preparing liposomes: natural phospholipid (HSPC): 7.85 mg; cholesterol: 3.35 mg; mPEG-2000-DSPE: 2.78 mg; DiI: 0.4 mg, dissolve them in 10 mL of CHCl3, suspend them in a 40 °C water bath to form a film, vacuum dry to remove the organic solvent, dissolve the film in 1 mL of double distilled water, shake and hydrate in a 60 °C water bath, and extrude with a liposome extruder with pores of 400 nm, 200 nm, and 100 nm to obtain sLip / DiI.

[0048] Preparation of liposome SP-sLip / DiI: Weigh 50 mg of Mal-PEG-DSPE, dissolve it in 35 mL of CHCl at 37 °C to form a film, vacuum dry it for 30 minutes, dissolve it in 4 mL of double distilled water, hydrate it at 37 °C, and remove large particles by ultrasonication. Weigh 26 mg of SP-Cys protein, dissolve it in 2 mL of double distilled water, and mix it with the solution of the membrane material above. After washing the container with 1 mL of double distilled water, mix the mixture, add 40 μL of EDTA solution (500 mM, pH 8.0) and 3 mL of PB solution (0.1 M, pH 7.4), and react for 6 hours with stirring at room temperature to ensure that there is no flocculent precipitation. Dialysis in distilled water is performed for 48 hours to 72 hours using a dialysis membrane with pores of 8000 to 10000 Da, and the lyophilized product of the resulting solution is SP-PEG-DSPE.

[0049] Membrane materials for preparing liposomes: HSPC: 7.85 mg; cholesterol: 3.35 mg; mPEG-2000-DSPE: 1.67 mg; SP-PEG-DSPE: 1.82 mg; DiI: 0.4 mg were weighed, dissolved in 10 mL of CHCl3, suspended in a 40°C water bath to form a film, vacuum dried to remove organic solvent, and the film was dissolved in 1 mL of double distilled water, shaken and hydrated in a 60°C water bath, and extruded by a liposome extruder with pores of 400 nm, 200 nm, and 100 nm to obtain SP-SLIP / DII.

[0050] Preparation of liposomal sLip / DOX and SP-sLip / DOX: Preparation of liposome sLip / DOX: Membrane materials for preparing liposomes: HSPC: 7.85mg; Cholesterol: 3.35mg; MPEG-2000-DSPE: 2.78mg are weighed, dissolved in 10mL of CHCl3, suspended in a 40℃ water bath to form a film, vacuum dried to remove organic solvent, dissolved in 1mL of ammonium sulfate solution (0.32M), shaken and hydrated in a 60℃ water bath, extruded with a liposome extruder with pores of 400nm, 200nm and 100nm to obtain sLip hydrated by ammonium sulfate solution. After replacing with saline solvent by G50 elution column, add adriamycin aqueous solution, mix (drug to lipid ratio is 1:10), and remove uncoated adriamycin by G50 chromatography column to obtain sLip / DOX.

[0051] Preparation of liposome SP-sLip / DOX: Weigh out the membrane materials to prepare liposomes: HSPC: 7.85 mg; cholesterol: 3.35 mg; mPEG-2000-DSPE: 1.67 mg; SP-PEG-DSPE: 1.82 mg, other steps are the same as above (Preparation of liposome sLip / DOX) to obtain SP-sLip / DOX. EXAMPLES

[0052] Effects of protein corona formation in serum on LRP-1 receptor binding activity and uptake by vascular endothelial cells Characteristics of the protein corona formed by liposomes in serum: Serum from C57BL / 6 mice (containing protease inhibitors and EDTA as anticoagulants) was mixed with liposomes in a 1:1 ratio, incubated at 37°C for 1 h, centrifuged at 15000 rpm for 30 min, washed twice with cold PBS, and dissolved in 30 μL of PBS. Serum was used as a positive control and PBS as a negative control. 6 μL of SDS-PAGE loading buffer and 3 μL of β-mercaptoethanol were added and boiled for 10 min to denature the proteins. Proteins with different molecular weights were separated using a 4-20% polyacrylamide gel and stained with a rapid silver staining kit. Bands with obvious differences at the same position of the PAGE adhesive and control (as indicated by red arrows) were excised, digested with trypsin, and resuspended in 0.1% formic acid solution. The protein components of each band were analyzed by LC-MS / MS, and the experimental results are shown in Figure 1.

[0053] In vivo experiments were performed as follows: modified and unmodified liposomes labeled with fluorescent DiI were injected into C57BL / 6 mice via the tail vein, blood was collected 1 h later, and plasma was centrifuged at low speed. The isolation method of the protein corona was as described in the in vitro experiments above, and the protein corona components adsorbed in vivo were identified by SDS-PAGE and Western blot as shown in Figure 2.

[0054] Binding activity of SP-sLip to LRP-1 receptor before and after serum incubation: The ELISA method was used to detect the binding activity between SP-sLip and SP antibody before and after serum incubation, to judge the change of the active binding domain between SP and receptor after plasma protein interaction, and to detect the activity of apolipoprotein adsorbed on it. The specific operations were as follows: 0.1 μg of SP-PEG-DSPE was added to each well of the ELISA plate, left at room temperature overnight, washed three times with PBS, sealed with 3% BSA for 1 h, and aspirated the BSA solution. SP antibody was added, incubated at 37 ° C for 1 h, washed three times with PBS, liposomes previously incubated with serum or PBS and diluted in gradient were added, incubated at 37 ° C for 1 h, washed three times with PBS, and the corresponding horseradish peroxidase-labeled secondary antibody was added, reacted with TMB color-developing solution for 3 to 15 min after 1 h, the reaction was stopped with 2M H2SO4, and its absorbance value was measured at 450 nm wavelength. The experimental results are shown in Figure 3.

[0055] Effect of SP-sLip on uptake by endothelial cells after forming a protein corona in serum: Resuscitation of vascular endothelial cells bEND3: Frozen bEND3 cells were thawed quickly, transferred to a centrifuge tube filled with culture medium, centrifuged at 1000r / min for 3 minutes, the supernatant was discarded, and DMEM culture medium containing 10% FBS was added, gently blown evenly, and then dropped into a culture dish and mixed evenly. The morphology and proliferation of the cells were observed under a microscope, and the cells were cultured in an incubator at 37℃, 5%CO2 and 95% relative humidity.

[0056] Culture of vascular endothelial cells bEND3: The proliferation of bEND3 cells, i.e., the number, shape and adhesion of the cells, was observed. The old culture medium was drained, and fresh DMEM culture medium containing 10% FBS was added, mixed evenly, and the culture was continued in an incubator at 37°C, 5% CO2 and saturated humidity. The proliferation of the cells was observed every day, and the cells were subcultured every 2 to 3 days. The cells entered the logarithmic growth phase for about 10 days, and they could be used for in vitro cell experiments.

[0057] Passage of vascular endothelial cells bEND3: Discard the culture medium, wash twice with PBS, add a small amount of 0.25% trypsin, place in an incubator for 1 minute, then add 2-3 mL of culture medium to stop digestion, transfer the cell suspension to several centrifuge tubes in equal amounts, centrifuge, discard the supernatant, add new culture medium, transfer it to a culture dish, and place in an incubator to culture.

[0058] Freezing of vascular endothelial cells bEND3: After cell experiments, the cells were frozen for the next use, and the freezing solution (containing 10% DMSO and 90% fetal bovine serum) was prepared in advance and pre-cooled at 4°C. The cells were digested with trypsin, and the pre-cooled freezing solution was added, gently blown with a dropper, and mixed. 1 mL of cell liquid was added to each freezing tube, and after sealing, the name and date of freezing were written down, and left at -80°C overnight, which were then stored in a liquid nitrogen tank and registered in the records.

[0059] Appropriate densities of BEND3 were seeded in 6-well plates, placed in a cell incubator, and cultured overnight. Fluorescein DiI-labeled liposomes (serum-incubated and serum-unincubated) were diluted 50-fold in serum-free DMEM medium, 1 mL was added to each well, and incubated with the cells for 4 h. The culture medium was drained and rapidly washed twice with PBS, and the cells were digested with trypsin, the digestion was stopped with serum-containing medium, centrifuged, and washed again with PBS. Finally, the cells were dispersed in 300 μL of PBS, and the percentage of positive cells and the fluorescence intensity of DiI-loaded liposomes (excitation wavelength 555 nm, emission wavelength 570 nm) were measured by flow cytometry. The experimental results are shown in Figure 4.

[0060] Effect of SP modification on pharmacokinetic parameters and immunogenicity of liposomes in vivo: Three SD rats in each group were injected with sLip / DiI and SP-sLip / DiI via the tail vein. Blood was collected from the tail (EDTA anticoagulation) before injection, 5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after injection, respectively. The supernatant was separated from the blood samples, and the DiI in the plasma was serially diluted with normal rat serum. A fluorescence spectrophotometer was used for quantification (excitation wavelength 555 nm, emission wavelength 570 nm), and the experimental results are shown in Figure 5a. Lipid A-loaded sLip and SP-sLip were intraperitoneally injected into Balb / c mice once every 7 days, and blood was collected 7 days after each injection for later use. ELISA was used to detect the contents of IgG and IgM against PEG and SP produced in mice at different time points, and the experimental results are shown in Figure 5b-c. EXAMPLES

[0061] SP-modified liposomes can cross the BBB In vivo experiments confirmed that fluorescein DiI-labeled SP-modified liposomes (SP-sLip / DiI) could cross the blood-brain barrier: Fluorescently labeled SP-sLip / DiI (phospholipid content 10mg / mL, DiI content 0.4mg / mL) was injected into C57BL / 6 mice via the tail vein (10μL / g). After 1h, the mice were anesthetized with ether, and the brain tissue was taken out, which was fixed in 4% paraformaldehyde for 24h, dehydrated in 30% sucrose, embedded in OCT, frozen, and sectioned. Nuclei were stained by DAPI, blood vessels were labeled by CD31 antibody, which were observed and photographed under a microscope, and fluorescein DiI-labeled methoxyliposomes were treated with the same procedure as a control, and the experimental results are shown in Figure 6. EXAMPLES

[0062] In vivo pharmacodynamics of adriamycin-encapsulated SP-sLip Median survival time in orthotopic tumor model mice: Establishment of glioma model in nude mice: Take U87 cells in logarithmic growth phase, digest and count the cells, suspend them in an appropriate amount of PBS buffer, and add 6 × 105 Each nude mouse was seeded with 100 cells (dispersed in 5 μL of PBS buffer). Before the experiment, the nude mice were anesthetized with 7% chloral hydrate, fixed in a stereotaxic apparatus, and the suspended cells were seeded in the striatum (i.e., 0.6 mm anterior and 1.8 mm right of the anterior fontanelle, at a depth of 3 mm). The condition of the nude mice was regularly observed after surgery.

[0063] Nude mice of orthotopic brain tumor model were randomly divided into 4 groups (n=13), and their body weights were recorded on days 7, 9, 11, 13, and 15, respectively. 200 μL of saline, adriamycin, sLip / DOX, and SP-sLip / DOX were injected into the tail vein, the single injection dose of adriamycin was 2 mg / kg, and the survival time of the model nude mice was recorded. The experimental results are shown in Figure 7.

[0064] Inhibitory effects of adriamycin-encapsulated SP-sLip on angiogenesis and apoptosis of tumor cells: On the 18th day after administration, the orthotopic glioma model mice were anesthetized with chloral hydrate, and the brain tissues were isolated, fixed with paraformaldehyde, dehydrated, and embedded in paraffin. The inhibitory effect on angiogenesis was observed and detected using immunofluorescence staining of CD31 antibody. The apoptosis degree of tumor cells was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and observed and photographed using a confocal fluorescence microscope. The experimental results are shown in Figures 8 and 9.

[0065] Safety evaluation: SP was co-cultured with PC12 neuronal cell line at gradient concentrations to evaluate its cytotoxicity in vitro. At the same time, the heart, liver, spleen, lung and kidney tissues of mice in the orthotopic tumor pharmacodynamic test were dissected, fixed with 4% paraformaldehyde in PBS, paraffin-embedded sections were prepared, stained with HE, and observed and photographed under a microscope. The experimental results are shown in Figure 10.

[0066] The invention has been described to some extent, but it will be apparent that various conditions can be suitably modified without departing from the spirit and scope of the invention. It is to be understood that the invention is not limited to the described embodiments, but falls within the scope of the appended claims, which include equivalent substitutions of each described element.

Claims

1. Use of an amyloid beta short peptide in the preparation of a preparation for mediating a drug molecule, a fluorescent probe or a delivery system for targeting a brain tumor by binding to a receptor on the blood-brain barrier and passing through the blood-brain barrier, wherein the amyloid beta short peptide is an amyloid beta short peptide capable of specifically adsorbing apolipoproteins in plasma; The sequence of the amyloid β short peptide is NH ２ -GSNKGAIIIGLM-CONH ２ and the receptor is selected from one or more of low density lipoprotein-related protein 1 (LRP-1), scavenger receptor class b member 1 (srb1) and low density liposome-related protein 2 (LRP-2); The preparation is a nanometer delivery system; The amyloid β short peptide is covalently linked to polyethylene glycol-Z to prepare amyloid β short peptide-polyethylene glycol-Z; The above use, wherein in the amyloid β short peptide-polyethylene glycol-Z, Z is a phospholipid, polylactic acid, lactic acid-co-glycolic acid, or polycaprolactone.

2. 2. The use according to claim 1, characterized in that the brain tumor is a brain metastatic tumor and / or a primary brain tumor.

3. 3. The use according to claim 1 or 2, characterized in that the nanometric delivery system is selected from one or more of a liposomal delivery system, a micelle delivery system, a nanodisc delivery system and a polymeric nanoparticle delivery system.

4. Amyloid β short peptide-polyethylene glycol-phospholipid is used to prepare a liposomal, micelle or nanodisc delivery system; and / or The use according to claim 3, characterized in that amyloid beta short peptide-polyethylene glycol-polylactic acid, amyloid beta short peptide-polyethylene glycol-lactic acid-co-glycolic acid, amyloid beta short peptide-polyethylene glycol-polycaprolactone are used to prepare micellar delivery systems and polymeric nanoparticle delivery systems.

5. 5. The use according to claim 4, characterized in that a liposomal, micelle, nanodisc or polymeric nanoparticle delivery system is used to encapsulate diagnostic molecules for tracking brain tumors.

6. Use according to claim 5, characterised in that the diagnostic molecule encapsulated in the delivery system is 5-carboxyfluorescein 5-FAM or the near infrared dyes Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, DiI.

7. The use described in claim 4, characterized in that a liposomal delivery system, a micellar delivery system, a nanodisc delivery system or a polymeric nanoparticle delivery system is used to encapsulate an antitumor drug for targeted intervention of brain tumors.

8. 8. The use according to claim 7, characterized in that the drug incorporated in the delivery system is gefitinib, icotinib, anlotinib, crizotinib, erlotinib, osimertinib, alectinib, paclitaxel, docetaxel, cabazitaxel, adriamycin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocellulose camptothecin, vincristine, a p53 activating peptide or a polypeptide toxin.

9. A nanometer delivery system, comprising amyloid beta short peptide-polyethylene glycol-Z, which is prepared by linking amyloid beta short peptide with polyethylene glycol-Z; In the amyloid β short peptide-polyethylene glycol-Z, Z is a phospholipid, polylactic acid, lactic acid-co-glycolic acid, or polycaprolactone; The amyloid beta short peptide is for preparing a preparation for mediating a drug molecule, a fluorescent probe, or a delivery system for targeting a brain tumor; the amyloid β short peptide is an amyloid β short peptide capable of specifically adsorbing apolipoprotein in plasma, The sequence of the amyloid β short peptide is NH ２ -GSNKGAIIIGLM-CONH ２ The nanometer delivery system,

10. The nanometric delivery system according to claim 9, characterized in that the brain tumor is a brain metastatic tumor and / or a primary brain tumor.

11. 11. The nanometric delivery system according to claim 9 or 10, characterized in that it is selected from one or more of a liposomal delivery system, a micelle delivery system, a nanodisc delivery system and a polymeric nanoparticle delivery system.

12. The nanometer delivery system according to claim 11, characterized in that when the amyloid beta short peptide-polyethylene glycol-Z is amyloid beta short peptide-polyethylene glycol-phospholipid, the nanometer delivery system is selected from one or more of a liposome delivery system, a micelle delivery system, and a nanodisc delivery system; and when the amyloid beta short peptide-polyethylene glycol-Z is amyloid beta short peptide-polyethylene glycol-polylactic acid, amyloid beta short peptide-polyethylene glycol-lactic acid-co-glycolic acid, or amyloid beta short peptide-polyethylene glycol-polycaprolactone, the nanometer delivery system is a micelle delivery system and / or a polymer nanoparticle delivery system.

13. The nanometric delivery system according to claim 12, characterized in that a liposomal delivery system, a micelle delivery system, a nanodisc delivery system or a polymeric nanoparticle delivery system is used to encapsulate diagnostic molecules for tracking brain tumors.

14. The nanometric delivery system according to claim 13, characterized in that the diagnostic molecule encapsulated in the delivery system is 5-carboxyfluorescein 5-FAM or near infrared dyes Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, DiI.

15. The nanometric delivery system described in claim 12, characterized in that a liposomal delivery system, a micellar delivery system, a nanodisc delivery system or a polymeric nanoparticle delivery system is used to encapsulate an antitumor drug for targeted intervention of brain tumors.

16. The nanometric delivery system according to claim 15, characterized in that the drug entrapped in the delivery system is gefitinib, icotinib, anlotinib, crizotinib, erlotinib, osimertinib, alectinib, paclitaxel, docetaxel, cabazitaxel, adriamycin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocellulose camptothecin or vincristine p53 activating peptide or polypeptide toxin.

17. The nanometric delivery system according to claim 16, characterized in that the brain tumor is a brain metastatic tumor and / or a primary brain tumor.

18. A method for preparing a nanometric delivery system according to any one of claims 9 to 17, comprising the steps of: preparing said nanometer delivery system by weighing out natural phospholipids, cholesterol, methoxy-PEG-DSPE, and amyloid beta short peptide-polyethylene glycol-Z, model drug or probe molecules, dissolving them in a solvent, forming a film, hydrating, passing them through a membrane, and removing free probe molecules or drugs by column chromatography; The method comprising:

19. A method for preparing a nanometric delivery system according to any one of claims 9 to 17, comprising the steps of: Weighing out natural phospholipids, cholesterol, methoxy-PEG-DSPE, and amyloid beta short peptide-polyethylene glycol-Z, dissolving them in a solvent, forming a film, hydrating them, passing them through a membrane, actively encapsulating the probe molecules or drugs, and removing the free probe molecules or drugs by column chromatography to prepare the nanometer delivery system. The method comprising:

20. A pharmaceutical composition comprising: 1) a nanometric delivery system according to any one of claims 9 to 17; and 2) a pharma- ceutically acceptable carrier.

21. A diagnostic preparation, characterized in that it comprises a nanometric delivery system according to any one of claims 9 to 17.

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