AMYLOID B SHORT PEPTIDE MEDIATED BRAIN TARGETED DELIVERY SYSTEM, PREPARATION METHOD THEREFOR AND USE THEREOF

Abstract

A polypeptide modified complex in the pharmaceutical field that can specifically adsorb apolipoproteins in plasma and can mediate a drug across the blood-brain barrier, a target delivery system, and use thereof in preparation of a formulation for diagnosing and treating brain tumors and other brain diseases. The polypeptide fragment (SP) of the amyloid β (relating to one type) is modified shown that the modified delivery system increases uptake the of the amyloid β by vascular endothelial cells after the modified delivery system forms a protein crown with plasma proteins. The modified liposome delivery system delivers a drug to the lesion site more effectively, significantly improving the therapeutic effect of the drug. After the SP adsorbs plasma proteins, a drug may be mediated across the blood-brain barrier and/or targeted to tumor neovascular and tumor cells, and the modified drug and delivery system thereof obtain a better therapeutic effect when treating brain tumors and other diseases in the brain.

Claims

1.-15. (canceled)

16. A method for mediating targeted delivery of a composition to a brain of a subject in need thereof, wherein the composition comprises an amyloid β short peptide (SP) combined with a drug molecule, a diagnostic molecule, a delivery system, or combinations thereof, wherein the SP mediates targeted delivery of the composition to the brain and can adsorb apolipoprotein in plasma, wherein the method comprises administering the composition to the subject in need thereof.

17. The method according to claim 16, wherein the amyloid β short peptide including the sequence of GSNKGAIIGLM; preferably, wherein the method further comprises diagnosing a brain tumor or a brain disease in the subject, wherein the composition comprises an imaging substance X, wherein the amyloid β short peptide (SP) is linked to the imaging substance X by covalent bond to prepare SP-X, wherein SP-X to image a brain tumor or a diseased brain tissue or cell in the subject.

18. The method according to claim 17, wherein X is a fluorescent molecule selected from a Fluorescein, or a near-infrared dye molecule selected from Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, or DiI.

19. The method according to claim 16, wherein the amyloid β short peptide including the sequence of GSNKGAIIGLM; preferably, wherein the method further comprises targeted intervention of a brain tumor or a brain disease in the subject, wherein the amyloid β short peptide (SP) is linked to an antitumor drug Y by covalent bond to prepare SP-Y, wherein SP-Y targets the brain tumor or the brain disease.

20. The method according to claim 19, wherein Y comprises gefitinib, icotinib, anlotinib, crizotinib, erlotinib, osimertinib, alectinib, paclitaxel, docetaxel, cabazitaxel, adriamycin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocellulose camptothecine or vincristine small molecule antitumor drugs, p53 activating peptide, polypeptide toxin, or polypeptide antitumor drug.

21. The method according to claim 16, wherein the amyloid β short peptide including the sequence of GSNKGAIIGLM; preferably, wherein the amyloid β short peptide (SP) is linked to polyethylene glycol-Z by covalent bond to prepare SP-polyethylene glycol-Z, wherein SP-polyethylene glycol-Z is used to prepare a nanometer delivery system.

22. The method according to claim 21, wherein (1) Z comprises a phospholipid, a polylactic acid, a lactic-co-glycolic acid or a polycaprolactone; (2) wherein the nanometer delivery system comprises a liposome delivery system, a micelle delivery system, a nano-disc delivery system, a polymer nanoparticle delivery system, or combinations thereof; or both (1) and (2).

23. The method according to claim 22, (a) wherein the SP-polyethylene glycol-phospholipid is used for preparing the liposome delivery system, the micelle delivery system or the nano-disc delivery system; (b) wherein the SP-polyethylene glycol-polylactic acid, the SP-polyethylene glycol-lactic-co-glycolic acid, or the SP-polyethylene glycol-polycaprolactone is used to prepare the micelle delivery system and the polymer nanoparticle delivery system; or both (a) and (b).

24. The method according to claim 23, wherein the method further comprises diagnosing a brain tumor or a brain disease, and wherein the liposome delivery system, the micellar delivery system, the nano-disc delivery system or the polymer nano-particle delivery system is used to encapsulate a diagnostic molecule to trace the brain tumor or the diseased brain tissue or cell.

25. The method according to claim 24, wherein the diagnostic molecule encapsulated in the delivery system comprises 5-carboxyfluorescein 5-FAM, near-infrared dye selected from Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, or DiI.

26. The method according to claim 23, wherein the method further comprises targeting a brain tumor or a diseased brain tissue or cell, wherein the nanoparticle delivery system, the liposome delivery system, the micelle delivery system, the polymer nanoparticle delivery system or the nano-disc delivery system is used for encapsulating an antitumor drug.

27. The method according to claim 26, wherein the antitumor drug encapsulated in the delivery system comprises gefitinib, icotinib, anlotinib, crizotinib, erlotinib, osimertinib, alectinib, paclitaxel, docetaxel, cabazitaxel, adriamycin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocellulose camptothecine, vincristine, p53 activating peptide, or polypeptide toxin.

28. The method of claim 16, wherein the subject suffers from a brain metastatic tumor, a primary brain tumor, or both.

29. A nano delivery system comprising an amyloid β short peptide (SP), polyethylene glycol, and a Z molecule, wherein the nano delivery system is prepared by linking the amyloid short peptide (SP) with polyethylene glycol-Z to form SP-polyethylene glycol-Z.

30. The nano delivery system according to claim 29, wherein Z comprises a phospholipid, a polylactic acid, a lactic-co-glycolic acid or a polycaprolactone.

31. The nano delivery system according to claim 30, wherein the nano delivery system comprises one or more selected from the group consisting of a liposome delivery system, a micelle delivery system, a nano-disc delivery system, and a polymer nanoparticle delivery system.

32. The nano delivery system according to claim 31, wherein (1) the SP-polyethylene glycol-Z is SP-polyethylene glycol-phospholipid, and the nano delivery system is selected from the group consisting of the liposome delivery system, the micelle delivery system, the nano-disc delivery system, and a combination thereof; or (2) the SP-polyethylene glycol-Z is SP-polyethylene glycol-polylactic acid, SP-polyethylene glycol-lactic-co-glycolic acid or SP-polyethylene glycol-polycaprolactone, and the nano delivery system is selected from the group consisting of the micelle delivery system, the polymer nanoparticle delivery system, and a combination thereof.

33. The nano delivery system according to claim 32, wherein the liposome delivery system, the micelle delivery system, the nano-disc delivery system or the polymer nanoparticle delivery system encapsulates a diagnostic molecule or an antitumor drug.

34. The nano delivery system according to claim 33, (I) wherein the diagnostic molecule comprises 5-carboxyfluorescein 5-FAM, near-infrared dye Cy5, Cy5.5, Cy7, IR820, ICG, DiR, DiD, or DiI; (II) wherein the antitumor drug comprises gefitinib, icotinib, anlotinib, crizotinib, erlotinib, osimertinib, alectinib, paclitaxel, docetaxel, cabazitaxel, adriamycin, epirubicin, camptothecin, hydroxycamptothecin, 9-nitrocellulose camptothecine, vincristine, p53 activating peptide, or polypeptide toxin, or both (I) and (II).

35. A method for the preparation of the nano delivery system according to claim 17, comprising: (A) combining a natural phospholipid, cholesterol, methoxy-PEG-DSPE, and the SP-polyethylene glycol-Z with the diagnostic molecule or the antitumor drug to form a mixture, dissolving the mixture in a solvent, forming a film from the dissolved mixture, hydrating the film, passing the film through a membrane, and removing the free diagnostic molecules or antitumor drugs by column chromatography to make the nano delivery system comprising the diagnostic molecule or the antitumor drug; or (B) dissolving a natural phospholipid, cholesterol, methoxy-PEG-DSPE, and SP-polyethylene glycol-Z in a solvent to form a mixture, forming a film from the mixture, hydrating the film, passing the film through the membrane to form a nano delivery system, encapsulating the diagnostic molecules or antitumor drugs, and removing the free diagnostic molecule or the antitumor drug by column chromatography to make the nano delivery system comprising the diagnostic molecule or the antitumor drug.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG. 1 is the in vitro binding identification of SP modified liposomes and plasma proteins.

[0084] SP was modified on the surface of liposome by chemical coupling to obtain SP-sLip, which was mixed with mouse plasma and incubated at 37° C. for 1 h, and then centrifuged at high speed to obtain liposome precipitate 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 FIG. 1A). Compared with methoxy polyethylene glycol modified long circulating liposome (sLip), the bands of plasma protein adsorbed by SP-sLip at 45 KDa (band 1), 38 KDa (band 2) and 25 KDa (band 3) increased significantly. The results of LC-MS/MS and western blot (as shown in FIG. 1B) were analyzed on PAGE glue corresponding to the same position in three places, and the three proteins were identified as ApoJ, ApoE and ApoA1 from mice. Western blot semi-quantitative method also confirmed that SP-sLip adsorbed three apolipoproteins significantly more than sLip (as shown in FIG. 1C-E).

[0085] FIG. 2 is the identification of the adsorption of plasma functional apolipoprotein by fluorescent labeled sLip and SP-sLip in vivo.

[0086] In order to verify that liposomes modified by target small peptides can still adsorb related target proteins after systemic administration in animals, we injected the same amount of DiI fluorescence labeled liposomes into mice via tail vein, and then took the blood 1 h later to separate liposomes containing protein corona in plasma. As shown in FIG. 2A, western blot showed that there were significant differences in the contents of three apolipoproteins (ApoJ, ApoE and ApoA1) adsorbed by liposomes with the same content in mice. This result proves that SP modified liposome (SP-sLip) can quickly and specifically adsorb apolipoproteins in plasma proteins in mice compared with ordinary liposomes. (as shown the semi-quantitative analysis in FIG. 2B-D)

[0087] FIG. 3 is the evaluation of binding activity of sLip and SP-sLip to LRP-1 receptor after adsorption of plasma protein.

[0088] SP modified liposome (SP-sLip) itself can specifically adsorb recombinant protein LRP-1 in solution (as shown in FIG. 3A). We confirmed by western blot that SP-sLip still retains the ability to bind LRP-1 after preincubation with ApoE (as shown in FIG. 3B-C). FIG. 3DE proved by ELISA that both free SP and modified liposome (SP-sLip) can compete for the interaction between SP and antibody fixed on 96-well plate, while SP-sLip losed the ability to compete for antibody binding after plasma incubation, which indicated that the functional domain of SP on the surface of liposome was blocked after binding with plasma proteins such as ApoE. Apolipoprotein, such as ApoE, which can specifically binds to the surface of SP-sLip, has the activity of binding to BBB receptor LRP-1, which has become an effective way to mediate SP-sLip to enter the brain across BBB.

[0089] FIG. 4 is the uptake of liposomes by vascular endothelial cells before and after plasma incubation.

[0090] After SP modified liposome (SP-sLip) was incubated with plasma to form protein corona, the uptake of SP-sLip by endothelial cells increased (as shown in FIG. 4A-B).

[0091] FIG. 5 is the pharmacokinetic parameters and immunogenicity evaluation of SP modified liposome in mice.

[0092] SP was modified on the surface of liposome (SP-sLip), which did not affect the pharmacokinetic parameters of liposome in vivo (as shown in FIG. 5A), and did not increase the immunogenicity of common long-circulating liposomes (taking IgG and IgM in blood as evaluation indexes, as shown in FIG. 5B-D).

[0093] FIG. 6 is the evaluation of brain entry efficiency of SP modified liposomes in mice.

[0094] Through in vivo detection of fluorescein in mouse brain, it was found that the amount of cross BBB in SP-sLip group was significantly higher than that in sLip group, which indicated that SP could mediate the delivery system across blood-brain barrier.

[0095] FIG. 7 is the anti-glioma efficacy of adriamycin encapsulated liposomes in vivo.

[0096] The median survival time of mice in saline group, DOX group, sLip/DOX group and SP-sLip/DOX group were 27 days, 31 days, 33 days and 50 days, respectively. SP modified liposome could significantly prolong the median survival time of brain orthotopic tumor model mice.

[0097] FIG. 8 is the effect of adriamycin encapsulated liposomes on neovascularization in tumor tissue.

[0098] The vascular density in tumor tissue of SP modified liposome encapsulating adramycin (SP-sLip/DOX) group was significantly lower than that of unmodified group (sLip/DOX), which indicated that SP could mediate the delivery system to target tumor neovascularization.

[0099] FIG. 9 is the effect of adriamycin loaded liposomes on apoptosis of glioma cells.

[0100] The number of apoptosis in tumor tissue of SP modified liposome encapsulating adramycin (SP-sLip/DOX) group was significantly higher than that of unmodified group, which indicated that SP could mediate drug delivery system to target tumor cells.

[0101] FIG. 10 is the safety evaluation.

[0102] SP did not show cytotoxicity on the neural cell line PC12 cultured in vitro (as shown in FIG. 10A), and there was no obvious abnormality in the tissue sections of mice organs in SP modified liposome encapsulating adramycin (SP-sLip/DOX) group (as shown in FIG. 10B), which indicated that the SP modification did not cause toxic and side effects on the liposome surface.

DETAILED DESCRIPTION OF EMBODIMENTS

[0103] The present invention is further illustrated by specific examples below, but it should be understood that these examples are only for more detailed and specific explanation, and should not be understood as limiting the present invention in any form.

[0104] This section gives a general description of the materials and test methods used in the test of this invention. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is described in as much detail as possible here. It is clear to the person skilled in the art that in the context, unless otherwise specified, the materials and operation methods used in the present invention are well known in the art.

Example 1 Preparation of Liposome

[0105] Preparation of Liposome sLip/DiI and SP-sLip/DiI:

[0106] Preparation of Liposome sLip/DiI: Weigh the membrane material for preparing liposome: 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 CHCl.sub.3, suspend in 40° C. water bath to form a film, dry in vacuum to remove organic solvent, dissolve the film in 1 mL double distilled water, shake and hydrate in 60° C. water bath, and extrude in liposome extruder at 400 nm, 200 nm and 100 nm aperture to obtain sLip/DiI.

[0107] Preparation of liposome SP-sLip/DiI: Weigh 50 mg Mal-PEG-DSPE, dissolve it in 5 mL CHCl.sub.3 at 37° C. to form a film, dry in vacuum for half an hour, dissolve it in 4 mL double distilled water, hydrate at 37° C., and remove large particles by ultrasound. 26 mg SP-Cys protein is weighed and dissolved in 2 mL of double distilled water and mixed with the solution of above membrane material. After washing the container with 1 mL of double distilled water, the mixture was mixed, 40 μL EDTA solution (500 mM, pH 8.0) and 3 mL PB solution (0.1 M, pH 7.4) are added, and stirred at room temperature to react for 6 h, so that there is no flocculent precipitation. Dialysis in distilled water for 48 hours to 72 hours by using dialysis membrane with aperture of 8000-10000 Da, and the freeze-dried product of solution obtained is SP-PEG-DSPE.

[0108] Weigh the membrane material for preparing liposome: HSPC: 7.85 mg; cholesterol: 3.35 mg; mPEG-2000-DSPE: 1.67 mg; SP-PEG-DSPE: 1.82 mg; DiI:0.4 mg, dissolve them in 10 mL CHCl.sub.3, suspend in 40° C. water bath to form a film, dry in vacuum to remove organic solvent, dissolve the film in 1 mL double distilled water, shake and hydrate in 60° C. water bath, and extrude in liposome extruder at 400 nm, 200 nm and 100 nm aperture to obtain SP-SLIP/DII.

[0109] Preparation of Liposome sLip/DOX and SP-sLip/DOX:

[0110] Preparation of liposome sLip/DOX: Weigh the membrane material for preparing liposome: HSPC: 7.85 mg; cholesterol: 3.35 mg; MPEG-2000-DSPE: 2.78 mg, dissolve them in 10 mL CHCl.sub.3, suspend in 40° C. water bath to form a film, dry in vacuum to remove organic solvent, dissolved in 1 mL ammonium sulfate solution (0.32 M), shake and hydrate in 60° C. water bath, and extruded in liposome extruder with apertures of 400 nm, 200 nm and 100 nm to obtain sLip hydrated by ammonium sulfate solution. After being replaced by physiological saline solvent by G50 elution column, adriamycin aqueous solution is added and mixed (drug-lipid ratio was 1:10), and the uncoated adriamycin is removed by G50 chromatography column to obtain sLip/DOX.

[0111] Preparation of liposome SP-sLip/DOX: Weigh the membrane material for preparing liposome: 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), so as to obtain SP-sLip/DOX.

Example 2 Binding Activity of Liposome to LRP-1 Receptor after Forming Protein Corona in Serum and the Effect on Uptake by Vascular Endothelial Cells

[0112] Characteristics of Protein Corona Formed by Liposomes in Serum:

[0113] Serum of C57BL/6 mice (containing protease inhibitor and EDTA as anticoagulant) was mixed with liposome at a ratio of 1:1, incubated at 37° C. for 1 h, centrifuged at 15000 rpm for 30 min, washed twice with cold PBS, and dissolved in 30 μL PBS. With serum as positive control and PBS as negative control, 6 μL SDS-PAGE loading buffer and 3 μL β-mercaptoethanol were added, boiled for 10 minutes to denature the protein, separated the proteins with different molecular weights with 4-20% polyacrylamide gel, and color development with rapid silver staining kit. Cut off the obvious difference band (as shown by red arrow) in the PAGE glue and the same position of the control, digested them with trypsin, and resuspended them in 0.1% formic acid solution. Analyzed the protein components of each band by LC-MS/MS, and the experimental results are shown in FIG. 1.

[0114] In vivo experiments were carried out as follows: the modified and unmodified liposomes labeled with fluorescent DiI were injected into C57BL/6 mice through tail vein, and the blood was collected after 1 hour and plasma was centrifuged at low speed. The separation method of protein corona was as described in the above in vitro experiment, and the protein corona components adsorbed in vivo were identified by SDS-PAGE and western blot, as shown in FIG. 2.

[0115] Binding Activity of SP-sLip and LRP-1 Receptor Before and after Serum Incubation:

[0116] ELISA method was used to detect the binding activity between SP-sLip and SP antibody before and after serum incubation, so as to judge the change of active binding domain between SP and receptor after plasma protein interaction, and to detect the activity of apolipoprotein adsorbed by it. The specific operation was as follows: 0.1 μg SP-PEG-DSPE was added to each well of ELISA plate, left overnight at room temperature, washed with PBS for 3 times, sealed with 3% BSA for 1 h, and the BSA solution was sucked off. Added SP antibody, incubated at 37° C. for 1 h, washed with PBS for three times, added liposome incubated with serum or PBS in advance and diluted in gradient, incubated at 37° C. for 1 h, washed with PBS for three times, added corresponding horseradish peroxidase labeled secondary antibody, reacted with TMB chromogenic solution for 3-15 min after 1 h, stopped the reaction with 2 M H2504, and measured its absorbance value at 450 nm wavelength. The experimental results are shown in FIG. 3.

[0117] Effect of SP-sLip on Uptake by Vascular Endothelial Cells after Forming Protein Corona in Serum:

[0118] Resuscitation of vascular endothelial cell bEND3: Thawed freezing bEND3 cells quickly and transferred them to a centrifuge tube pre-filled with culture solution, centrifuged at 1000 r/min for 3 min, discarded the supernatant, added DMEM culture solution containing 10% FBS, gently blew evenly and dropped them into a culture dish, and mixed evenly. The morphology and growth of cells were observed under a microscope and cultured in an incubator at 37° C., 5% CO.sub.2 and 95% relative humidity.

[0119] Culture of vascular endothelial cells bEND3: Observed the growth of bEND3 cells, i.e. the number, shape and adherence of cells. Drained the old culture solution, add fresh DMEM culture solution containing 10% FBS and mixed evenly, continued to culture in an incubator with 37° C., 5% CO.sub.2 and saturated humidity, observed the growth of cells every day, and passaged every 2-3 days, and the cells were in logarithmic growth phase for about 10 days, which can be used for in vitro cell experiments.

[0120] Passage of vascular endothelial cells bEND3: Drained the culture solution, washed twice with PBS, added a little 0.25% trypsin, put it in an incubator for 1 min, then added 2-3 mL culture solution to stop digestion, put the cell suspension into several centrifugal tubes in equal parts, centrifuged and discarded the supernatant, added new culture solution and transferred it to a culture dish and put it in an incubator for culture.

[0121] Freeze vascular endothelial cells bEND3: After the cell experiment, the cells were frozen for the next use, and the frozen solution (containing 10% DMSO and 90% fetal bovine serum) was prepared in advance and precooled at 4° C. Digested cells with trypsin, added precooled frozen solution, blew gently with dropper and mixed. Added 1 mL cell fluid into each freezing tube, marked the name and date of freezing after sealing, left it at −80° C. overnight, stored it in liquid nitrogen tank and registered for the record.

[0122] BEND3 with proper density was inoculated in a 6-well plate and placed in a cell incubator for overnight culture. Fluorescein DiI-labeled liposomes (serum-incubated and serum-non-incubated) were diluted 50 times with DMEM medium without serum, and 1 mL was added to each well to incubate with the cells for 4 h. The culture solution was drained, washed twice with PBS quickly, 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 PBS, and the percentage of positive cells and fluorescence intensity (excitation wavelength 555 nm, emission wavelength 570 nm) of liposome loaded with DiI were measured by flow cytometry. The experimental results are shown in FIG. 4.

[0123] Effect of SP Modification on Pharmacokinetic Parameters and Immunogenicity of Liposomes In Vivo:

[0124] SD rats, 3 in each group, were injected sLip/DiI and SP-sLip/DiI through tail vein. Blood was taken from tail (EDTA anticoagulation) before the injection, 5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after the injection, respectively. Supernatant was separated from blood samples, and DiI in plasma was gradiently diluted with normal rat serum. The fluorescence spectrophotometer was used for quantification (excitation wavelength 555 nm, emission wavelength 570 nm), and the experimental results are shown in FIG. 5a. The sLip and SP-sLip loaded with Lipid A were injected intraperitoneally into Balb/c mice once every 7 days, and blood was taken for later use on the 7th day after each injection. ELISA was used to detect the content of IgG and IgM against PEG and SP produced in mice at different time points, and the experimental results are shown in FIG. 5b-c.

Example 3 SP Modified Liposomes can Span BBB

[0125] In Vivo Experiments Confirmed that Fluorescein DiI Labeled SP Modified Liposomes (SP-sLip/DiI) can Cross the Blood-Brain Barrier:

[0126] The fluorescently labeled SP-sLip/DiI (phospholipid content is 10 mg/mL, DiI content is 0.4 mg/mL) was injected into C57BL/6 mice by tail vein (10 μL/g). After 1 h, the mice were anesthetized with ether, and the brain tissue was taken out, which was fixed in 4% paraformaldehyde for 24 h, dehydrated by 30% sucrose, embedded by OCT, frozen and sectioned. The nucleus were stained by DAPI and the blood vessel was labeled by CD31 antibody, which were observed and photographed under a microscope, and fluorescein DiI labeled methoxy liposome as control, treated by the same procedure, and the experimental results are shown in FIG. 6.

Example 4 Pharmacodynamic Experiment of Adriamycin Encapsulated SP-sLip In Vivo

[0127] Median Survival Time of Orthotopic Tumor Model Mice:

[0128] Establishment of glioma model in nude mice: Took U87 cells in logarithmic growth phase, digested and counted the cells, suspended them with appropriate amount of PBS buffer, inoculated 6×10.sup.5 cells (dispersed in 5 μL PBS buffer) to each nude mouse. The nude mice was anesthetized with 7% chloral hydrate before the experiment, fixed with a brain stereotactic instrument, and inoculated the suspended cells to the striatum (i.e. 0.6 mm forward, 1.8 mm to the right, and 3 mm deep of the anterior fontanelle). Regularly observed the state of nude mice after operation.

[0129] Nude mice with orthotopic brain tumor model were randomly divided into 4 groups (n=13), and their body weight was recorded on the 7th, 9th, 11th, 13th and 15th day, respectively. 200 μL saline, adriamycin, sLip/DOX and SP-sLip/DOX were injected into tail vein, and 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 FIG. 7.

[0130] Inhibitory Effect of Adriamycin Encapsulated SP-sLip on Angiogenesis and Apoptosis of Tumor Cells:

[0131] On the 18th day after administration, orthotopic glioma model mice was anesthetized by chloral hydrate, and the brain tissue was separated and fixed with paraformaldehyde, dehydrated and embedded in paraffin. Immunofluorescence staining of CD31 antibody was used to observe and detect the inhibitory effect on neovascularization. Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling (TUNEL) was used to detect the apoptosis degree of tumor cells, and confocal fluorescence microscope was used to observe and take pictures. The experimental results are shown in FIGS. 8 and 9.

[0132] Safety evaluation: The gradient concentration of SP was co-cultured with nerve cell line PC12 to evaluate its cytotoxicity in vitro. At the same time, the heart, liver, spleen, lung and kidney tissues of mice in the pharmacodynamic test of orthotopic tumor were dissected and fixed in PBS solution of 4% paraformaldehyde, paraffin-embedded sections were made, stained with HE, observed and photographed under a microscope. the experimental results are shown in FIG. 10.

[0133] Although the present invention has been described to a certain extent, it is obvious that various conditions can be appropriately changed without departing from the spirit and scope of the present invention. It is to be understood that the present invention is not limited to the described embodiments, but belongs to the scope of the claims, which includes equivalent substitutions of each of the described factors.