Whole-process targeted polypeptide and its application in constructing tumor-targeting diagnosis and treatment drug delivery system

Abstract

The application belongs to the field of pharmacy, and particularly relates to a whole-process targeting polypeptide molecule with the functions of targeting brain capillary endothelial cells, tumor neovascular endothelial cells, tumor pseudo vascular, tumor cells and tumor stem cells, a stable and optimized polypeptide molecule, and a modified complex and drug delivery system for tumor diagnosis and treatment. The application prepares a whole-process targeting polypeptide WVAP and a WVAP modified drug and high polymer carrier material, and applies the WVAP to the construction of a drug delivery system for tumor imaging and targeted therapy. The results show that the WVAP can cross the blood-brain barrier, target tumor neovascular and cross the blood-tumor barrier, target tumor pseudo vascular, tumor cells and tumor stem cells; and the nano drug delivery system constructed by the WVAP modified high polymer carrier material can effectively deliver the loaded drug to the brain, target tumor tissues, and significantly improve the anti-tumor efficacy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmacy, and relates to a whole-process targeting polypeptide and use thereof, in particular to a whole-process targeting polypeptide molecule having the functions of crossing blood-brain barrier and blood-tumor barrier, targeting tumor neovasculature, tumor vasculogenic mimicry, tumor cells and tumor stem cells, and a pharmaceutical complex and a modified nanodelivery system thereof. BACKGROUND

[0002] The existing technology discloses that tumor mortality is ranked first among all disease mortalities, and has become a serious threat to human life and health. In clinical treatment, traditional chemotherapy as a main means of tumor drug treatment has defects such as poor selectivity for tumor tissues, great toxicity, narrow treatment window, and easy production of multidrug resistance. Therefore, in order to overcome the limitations of traditional treatment means, in recent years, active targeting has become an important strategy for improving tumor tissue targeting efficiency. The active targeting strategy mainly targets receptors or transporters highly expressed in tumor tissues, and uses corresponding ligands having recognition and binding capacity with specific receptors or transporters to deliver drugs or nanodelivery systems to tumor tissues or cells. Research shows that drugs or nanodelivery systems modified by ligands can deliver drugs to tumor tissues and cells through specific recognition, binding and internalization of cell surface receptors or transporters and ligands, so as to realize active targeting of tumors. According to data, most of the current ligands are only targeted to a certain receptor or a certain cell. However, tumor tissues have tumor cells, tumor stem cells, tumor vasculogenic mimicry, tumor neovasculature and blood-tumor barrier (BTB), and brain tumors also have blood-brain barrier (BBB), which makes about 98% of small molecule chemotherapy drugs and almost 100% of protein and other macromolecular drugs unable to penetrate BBB into the brain, resulting in almost ineffective drug treatment. Therefore, it is of great practical significance to develop ligands having the function of targeting the whole process of tumor growth and development, especially brain tumors.

[0003] VAP( L VAP1 (L-type amino acid sequence SNTRVAP) and its stable optimized polypeptide D VAP (D-type amino acid sequence D P D A D V D R D T D N D S)、 L VAP2 (L-type amino acid sequence PAVR TNS) and its stable optimized polypeptide S VAP (D-type amino acid sequence D S D N D T D R D V D AD P)) is a 7-peptide with high binding activity to glucose-regulated protein GRP78 verified in the early stage of the present application, which can target tumor neovascular endothelial cells and cross BTB, target tumor cells and tumor stem cells, and has good targeting ability in vivo, but cannot cross BBB.

[0004] WSW( L WSW(L type amino acid sequence is SYPGWSW) and its stable optimized polypeptide D WSW(D type amino acid sequence D W D S D W D G D P D Y D S)) is also a 7-peptide with high binding activity to quorum sensing receptors verified in the early stage of the present application, which can target brain capillary endothelial cells and penetrate BBB, target tumor neovascular endothelial cells and penetrate BTB, and target tumor cells, but has no tumor stem cell targeting function.

[0005] Based on the current situation and foundation of the prior art, the inventors of the present application intend to provide a whole-process targeting polypeptide and its application in constructing a tumor-targeting diagnosis and treatment drug delivery system, so as to more effectively exert the targeting diagnosis and treatment effect on brain tumors or peripheral tumors with brain metastasis characteristics, and to exert a synergistic anti-tumor effect by combining the drug delivery system with a tumor treatment drug

[0006] Further modification of the existing targeting polypeptide molecules, using fusion protein principle, VAP and WSW are spliced into WVAP( L WVAP: PAVRTNS-linker-SYPGWSW( L WVAP1), SNTRVAP-linker-SYPGWSW( L WVAP2), SYPGWSW-linker-PAVRTNS( L WVAP3) and SYPGWSW-linker-SNTRVAP( L WVAP4); D WVAP: D S D N D T D R D V D A D P-linker- D W D S D W D G D PD Y D S( D WVAP1), D P D A D V D R D T D N D S-linker- D W D S D W D G D P D Y D S( D WVAP2), D W D S D W D G D P D Y D S-linker- D S D N D T D R D V D ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​WVAP2), D W D S D W D G D P D Y D S-linker-PAVRTNS( LD WVAP3), D W D S D W D G D P D Y D S-linker-SNTRVAP( LD WVAP4), D S D N D T D R D V D A D P-linker-SYPGWSW( LD WVAP5), D P D A D V D R D T D N D S-linker-SYPGWSW( LD WVAP6), SYPGWSW-linker- D S D N D T D R D V D A D P( LD WVAP7) and SYPGWSW-linker- D P D A D V D R D T D N D S( LD WVAP8), SUMMARY

[0007] The present application is based on the current situation and foundation of the prior art, and aims to provide a whole-process targeted polypeptide and its application in constructing a tumor-targeting diagnosis and treatment drug delivery system, in particular to a whole-process targeted polypeptide molecule with the functions of crossing blood-brain barrier and blood-tumor barrier, targeting tumor neovasculature, tumor vasculature, tumor cells and tumor stem cells, and a drug complex and a modified nano drug delivery system thereof.

[0008] The present application provides a whole-process targeted polypeptide WVAP and a modified diagnosis and treatment drug complex thereof, a modified high molecular carrier material, and a nano drug delivery system such as a liposome, a polymer micelle, a polymer disc, a nanoparticle, etc. constructed by the same, and the application of the same in the diagnosis and targeted treatment of brain tumors or peripheral tumors with brain metastasis characteristics; and the application of a tumor treatment drug loaded in a WVAP modified drug delivery system in synergistic anti-tumor combination therapy.

[0009] Specifically, the whole-process targeted polypeptide molecule WVAP with the functions of crossing blood-brain barrier and blood-tumor barrier, and targeting tumor neovasculature, tumor vasculature, tumor cells and tumor stem cells is covalently connected by a linker to form WSW and VAP L WVAP: PAVRTNS-linker-SYPGWSW L WVAP1), SNTRVAP-linker-SYPGWSW L WVAP2), SYPGWSW-linker-PAVRTNS L WVAP3), and SYPGWSW-linker-SNTRVAP L WVAP4); D WVAP: D S D N D T D R D V D A D P-linker- D W D S D W D G D P D Y D S D WVAP1), D P D A D V D R D T D N D S-linker- D W D SD W D G D P D Y D S( D WVAP2), D W D S D W D G D P D Y D S-linker- D S D N D T D R D V D A D P( D WVAP3) and D W D S D W D G D P D Y D S-linker- D P D A D V D R D T D N D S( D WVAP4); LD WVAP: PAVRTNS-linker- D W D S D W D G D P D Y D S( LD WVAP1), SNTRVAP-linker- D W D S D W D G D P D Y D S( LD WVAP2), D W D S D W D G D P D Y D S-linker-PAVRTNS( LD WVAP3), D W D S D WD G D P D Y D S-linker-SNTRVAP ( LD WVAP4) D S D N D T D R D V D A D P-linker-SYPGWSW( LD WVAP5) D P D A D V D R D T D N D S-linker-SYPGWSW( LD WVAP6), SYPGWSW-linker- D S D N D T D R D V D A D P( LD WVAP7) and SYPGWSW-linker- D P D A D V D R D T D N D S( LD WVAP8));

[0010] In this invention, WVAP is used to modify drug molecules and polymeric carrier materials to construct WVAP-drug complexes and WVAP-modified nanodelivery systems, which can improve the targeted therapeutic effects of drugs on the brain, brain tumors, or peripheral tumors with brain metastasis characteristics. Furthermore, the WVAP delivery system encapsulating tumor therapeutic drugs can be used in combination with other drugs to achieve a synergistic anti-tumor effect.

[0011] More specifically, this invention utilizes the principle of fusion proteins to design and prepare the polypeptide molecule WVAP, which simultaneously possesses the targeting capabilities of WSW and VAP. It can exert a targeting effect across the blood-brain barrier and the blood-tumor barrier, and target the entire process of tumor growth and development, especially brain tumors, including tumor angiogenesis, tumor-mimicking vessels, tumor cells, and tumor stem cells.

[0012] In the present application, the splicing of WSW and VAP is covalently connected through a suitable bridging structure, and the bridging structure used is amino aliphatic acid and its derivatives, such as cysteine, aminocaproic acid, etc.; amino aromatic acid and its derivatives, such as p-aminobenzoic acid, etc.; or oligopeptide, such as GG, GSSG, etc.

[0013] The WVAP designed in the present application can further introduce active functional groups in the molecule to construct modified drug complexes and polymer carrier materials, and the site for introducing the functional groups is the nitrogen end, the carbon end or the bridging structure of the WVAP, preferably the bridging structure, so that the molecule forms a Y-shaped structure, which is beneficial to the exertion of the targeting function.

[0014] After introducing cysteine, the WVAP designed in the present application can form a complex by reacting the thiol group in the molecule with a maleimide functionalized imaging material (such as Fluorescein, near-infrared dye Cy5.5, IR820, DiR, magnetic resonance imaging agent Gd-DTPA, radio imaging agent Tc-DTPA, etc.). 99m

[0015] The WVAP modified drug designed in the present application includes a WVAP-drug complex formed by the reaction of a maleimide hexahydrate derivative to form a pH-sensitive hydrazone bond (involving drugs containing ketone or aldehyde groups such as doxorubicin and epirubicin), or by the reaction of a 3-(2-pyridine dimercapto) propionic acid derivative to form a disulfide bond (involving drugs containing hydroxyl or amino groups such as paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine and vinorelbine), or by the reaction of dopamine with a boronic acid group in the drug to form a pH-sensitive boronic acid ester (involving drugs containing a boronic acid group such as bortezomib), or by direct formation of an amide bond through solid-phase synthesis (involving polypeptide drugs such as p53 activating peptide).

[0016] After introducing cysteine, the WVAP designed in the present application can be modified on polyethylene glycol-distearylphosphatidylethanolamine (PEG-DSPE), polyethylene glycol-polylactic acid (PEG-PLA), polyethylene glycol-poly(lactic-co-glycolic acid) (PEG-PLGA), polyethylene glycol-polycaprolactone (PEG-PCL) and other high molecular weight carrier materials containing maleimide functional groups, and can be used for the construction of WVAP modified liposomes, polymer micelles, polymer discs, nanoparticles and other nanomedicine systems.

[0017] ​The WVAP modified nanodelivery system designed in the application can encapsulate anthracycline drugs such as doxorubicin and epirubicin, taxol and docetaxel, and taxane drugs such as cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine and vinorelbine, lactone drugs such as parthenolide, polypeptide toxins such as p53 activating peptide, melittin and scorpion peptide, and antitumor drugs such as antibacterial peptide; and can also encapsulate imaging substances such as coumarin 6, FAM, near-infrared dye Cy5.5, IR820, DiR, DiD, and magnetic resonance imaging agent Gd-DTPA.

[0018] The WVAP designed in the application can mediate drugs or nanodelivery systems to cross the blood-brain barrier and blood-tumor barrier, target tumor neovasculature, tumor vasculature, tumor cells and tumor stem cells, and be used for targeted diagnosis and treatment of brain, brain tumors or peripheral tumors with brain metastasis characteristics.

[0019] The WVAP modified nanodelivery system designed in the application encapsulates tumor treatment drugs (such as doxorubicin, epirubicin, taxol, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine, vinorelbine, bortezomib, carfilzomib, parthenolide, p53 activating peptide, melittin and scorpion peptide, and antibacterial peptide), and is combined with other tumor treatment drugs (such as temozolomide, cyclophosphamide, etoposide, mercaptopurine, gemcitabine, cytarabine, 5-fluorouracil, teniposide, epothilone, dactinomycin, mitoxantrone, mitomycin, bleomycin, teni drugs, platinum drugs, bevacizumab, trastuzumab, etc.) to achieve a synergistic antitumor effect.

[0020] The WVAP modified nanodelivery system designed in the application encapsulates different tumor treatment drugs for combination therapy, such as a nanodelivery system encapsulating doxorubicin, epirubicin, taxol, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine, vinorelbine, etc., which inhibit tumor cell growth, combined with a nanodelivery system encapsulating bortezomib, carfilzomib, parthenolide, etc., which have an antitumor stem cell effect, or combined with a nanodelivery system encapsulating polypeptide toxins such as p53 activating peptide, melittin and scorpion peptide, and antibacterial peptide, which have an antitumor effect, to achieve a synergistic antitumor effect.

[0021] In the application, the following experiments were conducted:

[0022] 1) Synthesis of WVAP, WVAP-Cys and their fluorescent markers (WVAP-Fluorescein, WVAP-Cy7),

[0023] WVAP and WVAP-Cys were prepared by solid phase synthesis; WVAP-Fluorescein or WVAP-Cy7 were synthesized by Michael addition reaction between maleimide group and thiol group; the structure was characterized by MS.

[0024] 2) Synthesis of WVAP-DTPA-Gd and WVAP-DTPA- 99m Synthesis of Tc

[0025] WVAP-DTPA was synthesized by Michael addition reaction between maleimide group and thiol group, chelated with Gd or 99m Tc to form WVAP-DTPA-Gd or WVAP-DTPA- 99m Tc.

[0026] 3) Evaluation of the affinity of WVAP to GRP78

[0027] The binding constant K D value of WVAP peptide to GRP78 receptor protein was determined by surface plasmon resonance method (SPR) to evaluate the affinity.

[0028] 4) Evaluation of the stability of WVAP

[0029] The stability of WVAP in serum was investigated; WVAP was incubated with mouse serum at 37℃, and the concentration of polypeptide was detected at different time points to evaluate the stability.

[0030] 5) Evaluation of the in vitro targeting ability of WVAP

[0031] The in vitro targeting ability of WVAP-Fluorescein to brain capillary endothelial cells (BCEC), umbilical vein endothelial cells (HUVEC) and model tumor cells (such as brain glioma cell U87) was investigated; the uptake ability of 3D tumor sphere model to WVAP-Fluorescein in vitro was investigated.

[0032] 6) Preparation of WVAP-drug complex

[0033] WVAP with introduced cysteine was reacted with maleimide hydrazine derivative of drugs to form polypeptide-drug complex containing pH-sensitive hydrazone bond, wherein the involved drugs include doxorubicin, epirubicin and other drugs containing ketone or aldehyde group;

[0034] WVAP reacts with 3-(2-pyridyl disulfide) propionic acid derivatives of drugs to form disulfide-containing polypeptide-drug complexes, wherein the drugs involved include taxol, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine, vinorelbine and other drugs containing hydroxyl or amino groups;

[0035] WVAP reacts with the boronic acid group of drugs to form pH-sensitive boronic acid-containing polypeptide-drug complexes, wherein the drugs involved include bortezomib and other drugs containing boronic acid groups;

[0036] WVAP is directly condensed with polypeptide drugs through solid-phase synthesis, wherein the drugs involved include p53 activating peptide and other polypeptide drugs.

[0037] 7) Construction and characterization of WVAP-modified nanodrug delivery system

[0038] First, synthesize WVAP-modified polymer materials WVAP-PEG-DSPE, WVAP-PEG-PLA, WVAP-PEG-PLGA, WVAP-PEG-PCL, etc. Synthesis of materials is achieved by reaction of free thiol of WVAP with maleimide contained in Mal-PEG-DSPE, Mal-PEG-PLA, Mal-PEG-PLGA, Mal-PEG-PCL, etc. through introduction of cysteine, i.e. dissolve Mal-PEG-DSPE, Mal-PEG-PLA, Mal-PEG-PLGA, Mal-PEG-PCL, etc. in acetonitrile, rotary evaporation, film formation, add thiol-containing WVAP in PBS (pH 8.0) to prepare D WVAP-modified polymer materials;

[0039] Then prepare WVAP-modified nanodrug delivery system. A certain amount of WVAP-PEG-DSPE and mPEG-DSPE and phospholipids and cholesterol, or WVAP-PEG-DSPE and mPEG-DSPE, or WVAP-PEG-PLA and mPEG-PLA, or WVAP-PEG-PLGA and mPEG-PLGA, or WVAP-PEG-PCL and mPEG-PCL, and a certain amount of the above drugs, are used to prepare corresponding D WVAP-modified liposomes, polymer micelles, polymer discs, polymer nanoparticles and other nanodrug delivery systems; laser scattering particle size instrument is used to characterize the particle size and particle size distribution of the nanodrug delivery system.

[0040] 8) Evaluation of in vitro and in vivo tumor targeting of WVAP-modified nanodrug delivery system

[0041] To investigate the uptake of the WVAP modified nanodelivery system loaded with fluorescent substance by U87 cells, HUVEC cells and U87 tumor spheroid model;

[0042] To investigate the intratumoral distribution of the WVAP modified nanodelivery system loaded with fluorescent substance at each time point by tail vein injection of the system to the U87 subcutaneously transplanted tumor model nude mice.

[0043] 9) In vitro and in vivo anti-tumor effect evaluation of the WVAP modified nanodelivery system

[0044] To investigate the in vitro growth inhibition effect of the WVAP modified nanodelivery system loaded with tumor therapeutic drugs on U87 cells and HUVEC cells by MTT method; to evaluate the in vivo anti-tumor effect by tail vein injection of the WVAP modified nanodelivery system loaded with tumor therapeutic drugs to the U87 orthotopic tumor model nude mice, and to take survival time, tumor tissue cell apoptosis, neovascularization, vasculogenic mimicry and stem cell number as indexes.

[0045] 10) Combination drug experiment

[0046] To evaluate the anti-tumor effect by tail vein injection of the WVAP modified nanodelivery system loaded with tumor therapeutic drugs to the U87 orthotopic tumor model nude mice in combination with temozolomide and other above-mentioned clinically commonly used tumor therapeutic drugs; to evaluate the anti-tumor effect by combination of the WVAP modified nanodelivery system loaded with tumor therapeutic drugs and the nanodelivery system loaded with tumor therapeutic drugs such as bortezomib and small white chrysanthemum lactone which have anti-tumor stem cell effect; to evaluate the anti-tumor effect by combination of the WVAP modified nanodelivery system loaded with tumor therapeutic drugs and polypeptide toxins such as p53 activating peptide, melittin and scorpion venom peptide and polypeptide drugs such as antimicrobial peptide, and to take median survival time as an index.

[0047] The present application provides the material basis of the WVAP preparation and property investigation and the above-mentioned modified drug compound and nanodelivery system for tumor diagnosis and treatment, and the results show that the WVAP has the targeting ability of WSW and VAP at the same time, has good brain and tumor tissue targeting ability and imaging effect in model animals, and shows better brain tumor targeting ability; the WVAP modified nanodelivery system shows good tumor targeting performance and stronger anti-brain tumor effect; the WVAP modified nanodelivery system loaded with tumor therapeutic drugs has obvious synergistic anti-tumor effect in combination with other tumor therapeutic drugs or in combination of the nanodelivery system loaded with different tumor therapeutic drugs. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 、 D ESI-MS spectrum of WVAP1-Cys,

[0049] The results show that the ESI-MS value is 1822.4, which is consistent with the theoretical molecular weight.

[0050] Figure 2 , D ESI-MS spectrum of WVAP1-Fluorescein

[0051] The results show that the ESI-MS value is 2250.4, which is consistent with the theoretical molecular weight.

[0052] Figure 3 , D ESI-MS spectrum of WVAP1-Cy7

[0053] The results show that the ESI-MS value is 2517.8, which is consistent with the theoretical molecular weight.

[0054] Appendix Figure 4 , D WVAP1-PEG-PLA 1 H-NMR spectrum,

[0055] Among them, the NMR spectrum of Mal-PEG-PLA showed a maleimide peak at 7.0 ppm, while D The disappearance of this peak in the NMR spectrum of WVAP1-PEG-PLA indicates that the maleimide group in Mal-PEG-PLA has reacted completely.

[0056] Figure 5 , D WVAP1 binding activity to GRP78 protein,

[0057] It shows D WVAP1 has high binding activity with GRP78, and D Similar to VAP, but D WSW does not bind to the GRP78 protein. D WVAP1's K D The value is 0.686 μM, and D VAP equivalent (0.102 μM).

[0058] Figure 6 , D Serum stability of WVAP1

[0059] The vertical axis of the graph represents the percentage of intact polypeptide remaining. It can be seen that the polypeptide has high stability in 50% mouse serum and hardly degrades after 24 hours of incubation.

[0060] Figure 7The uptake of fluorescein-labeled polypeptides by rat primary brain capillary endothelial cells (BCEC), human glioma cells (U87) and human umbilical vein endothelial cells (HUVEC),

[0061] Figure A is a laser confocal image of fluorescein-labeled D WVAP1, D WSW, D VAP and free fluorescein after 12 h of interaction with BCEC cells, 4 h of interaction with U87 cells and 4 h of interaction with HUVEC cells; Figure B, Figure C and Figure D are the results of flow cytometry for the uptake of the above samples by BCEC cells, U87 cells and HUVEC cells, respectively, which shows that the uptake of D WVAP1 and D WSW by BCEC cells is significantly higher than that of free fluorescein and D VAP, and there is no significant difference in the uptake of D WVAP1 and D WSW, and U87 cells and HUVEC cells have good uptake of the three polypeptides.

[0062] Figure 8 D WVAP1-Cy7 and DiR-loaded D WVAP modified polymer micelles in situ tumor distribution and normal mouse brain distribution,

[0063] Figure A is a fluorescence distribution image of Cy7-labeled polypeptides 1 h after tail vein injection of U87-bearing nude mice, Figure B is the fluorescence semi-quantitative results of the brain at each time point after administration, which shows that the accumulation of Cy7-labeled D WVAP1, D WSW and D VAP in the tumor is significantly higher than that of free fluorescein Cy7 (***p<0.001), and the tumor targeting effect of D WVAP1-Cy7> D WSW-Cy7> D VAP-Cy7; Figure C is a fluorescence distribution image of DiR-loaded polymer micelles 24 h after tail vein injection of U87-bearing nude mice, Figure D is the fluorescence semi-quantitative results of the brain tumor ex vivo 24 h after administration, and Figure E is the distribution of four different polymer micelles in the brain of normal mice, which shows that the brain targeting effect of D WVAP1 modified micelles is not inferior to that of D WSW modified polymer micelles; Figure F is the fluorescence quantitative results of the brain tumor 2 h and 24 h after administration, which shows that D WVAP1 modified polymer micelles can better target the tumor site. ​

[0064] Figure 9 、 D In vitro blood-brain barrier, blood-tumor barrier transport and tumor spheroid uptake of WVAP1-modified polymeric micelles, wherein,

[0065] Figures A and B are the particle size of coumarin 6-loaded D WVAP1-modified polymeric micelles D WVAP1-Micelle / C6) and control samples D WSW-modified polymeric micelles D WSW-Micelle / C6), D VAP-modified polymeric micelles D VAP-Micelle / C6) and polymeric micelles (Micelle / C6) at different time points, showing that at 30 min, 1, 2 and 4 h, D WVAP1-Micelle / C6 and D WSW-Micelle / C6 transported through the in vitro BBB model were significantly higher than the non-target Micelle / C6 and D VAP-Micelle / C6, and there was no significant difference in the transport percentage of the two; at 30 min, 1, 2 and 4 h, D WVAP1-Micelle / C6, D WSW-Micelle / C6 and D VAP-Micelle / C6 transported through the in vitro BTB model were significantly higher than the non-target Micelle / C6, and there was no significant difference in the transport percentage of the three; Figure C is the three-dimensional tumor spheroid uptake after crossing the BBB or BTB, showing that D WVAP1-Micelle / C6 and D WSW-Micelle / C6 can effectively cross the BBB or BTB and be taken up by the lower chamber U87 three-dimensional tumor spheroid, D VAP-Micelle / C6 can cross the BTB and be taken up by the lower chamber U87 three-dimensional tumor spheroid, but lacks the ability to cross the BBB.

[0066] Figure 10 , particle size of paclitaxel and small white chrysanthemum lactone-loaded polymeric micelles,

[0067] Among them, respectively, paclitaxel-loaded D WVAP-modified polymeric micelles D WVAP1-Micelle / PTX) and small white chrysanthemum lactone-loaded D WVAP1-modified polymeric micellesD The particle size pictures of WVAP1-Micelle / PTX show that, D WVAP1-Micelle / PTX and D The particle sizes of WVAP1-Micelle / PTX are 45nm and 30nm respectively, and the particle size distribution is narrow.

[0068] Figure 11 The growth curves of U87 cells and HUVEC cells inhibited by paclitaxel and small white chrysanthemum lactone polymer micelles in vitro, wherein,

[0069] Fig. A and Fig. B are Taxol (Taxol), Micelle / PTX, D The growth curves of U87 cells and HUVEC cells inhibited by WVAP1-Micelle / PTX show that, Fig. A shows that the IC 50 of U87 cells is 2.87, 1.09 and 0.342μM respectively after 4h of administration and 72h of culture, D The in-vitro activity of WVAP1-Micelle / PTX is 3.18 times that of Micelle / PTX; Fig. B shows that the IC 50 of HUVEC cells is 1.173, 0.749 and 0.267μM respectively after 4h of administration and 72h of culture, D The in-vitro activity of WVAP1-Micelle / PTX is 2.81 times that of Micelle / PTX; Fig. C and Fig. D are PTL (free small white chrysanthemum lactone), Micelle / PTL, D The growth curves of U87 cells and HUVEC cells inhibited by WVAP1-Micelle / PTL show that, Fig. C shows that the IC 50 of U87 cells is 19.13, 15.08 and 7.59μM respectively after 4h of administration and 72h of culture, D The in-vitro activity of WVAP1-Micelle / PTL is 1.99 times that of Micelle / PTL; Fig. D shows that the IC 50 of HUVEC cells is 5.670, 3.828 and 2.766μM respectively after 4h of administration and 72h of culture, D The in-vitro activity of WVAP1-Micelle / PTL is 1.38 times that of Micelle / PTL.

[0070] Figure 12 The growth curves of U87 cells and HUVEC cells inhibited by paclitaxel and small white chrysanthemum lactone polymer micelles in vitro, wherein,

[0071] Fig. A and Fig. B are Taxol (Taxol), Micelle / PTX, D WVAP1-Micelle / PTX andD Inhibition photos of WVAP1-Micelle / PTL on the in vitro models of neovascularization (A) and vasculogenic mimicry (B) compared with free PTL and Taxol, D WVAP1-Micelle / PTX, D WVAP1-Micelle / PTL inhibited the formation of neovascularization and vasculogenic mimicry more significantly. Figure 13 Survival curves of the model nude mice against brain glioma by polymeric micelles loaded with paclitaxel,

[0072] Among them, the average survival time of the saline group, the Taxol group, the Micelle / PTX group, D the VAP-Micelle / PTX group, D the WSW-Micelle / PTX group, D The average survival time of the WVAP1-Micelle / PTX group was 19, 20, 21, 23.5, 27.5 and 29.5 days, respectively, and the results showed that compared with the other groups, D WVAP1-Micelle / PTX can significantly prolong the survival time of the model nude mice against brain glioma (n = 8).

[0073] Figure 14 Survival curves of the model nude mice against brain glioma by polymeric micelles loaded with parthenolide,

[0074] Among them, the average survival time of the saline group, the PTL group, the Micelle / PTL group, D The average survival time of the WVAP1-Micelle / PTL group was 29.5, 32, 32 and 37 days, respectively, and the results showed that compared with the other groups, D WVAP1-Micelle / PTL can significantly prolong the survival time of the model nude mice against brain glioma (n = 8).

[0075] Figure 15 Survival curves of the model nude mice against brain glioma by polymeric micelles loaded with parthenolide and paclitaxel,

[0076] Among them, the average survival time of the saline group, D the WVAP1-Micelle / PTL group, D the WVAP1-Micelle / PTX group, D WVAP1-Micelle / PTL and D The average survival time of the WVAP1-Micelle / PTX combined administration group was 33.5, 47.5, 51.5 and 74 days, respectively, and the results showed that compared with the other groups, D WVAP1-Micelle / PTL andD WVAP1-Micelle / PTX combination therapy can significantly prolong the survival time of nude mice with brain glioma model (n=8).

[0077] Figure 16 Figure 6 shows the survival curves of nude mice with brain glioma model treated with artemin micelles and temozolomide combination therapy,

[0078] Among them, the saline group, D WVAP1-Micelle / PTL group, TMZ (temozolomide) group, D The average survival time of WVAP1-Micelle / PTL and TMZ combination therapy group was 29.5, 37, 39 and 59 days, respectively, and the results showed that compared with the other groups, the combination therapy can significantly prolong the survival time of nude mice with brain glioma model (n=8). Figure 17 Figure 7 shows the TUNEL staining results of brain glioma model,

[0079] Among them, Figure A is the saline group, TMZ group, D WVAP1-Micelle / PTX group, D WVAP1-Micelle / PTL group, D WVAP1-Micelle / PTL and TMZ combination therapy group, D WVAP1-Micelle / PTL and D Figure B is the quantitative results of WVAP1-Micelle / PTX combination therapy group leading to brain glioma apoptosis TUNEL staining photos (bar=50μm), in which the positive nuclei of apoptosis are brownish yellow or brown, and Figure B is the quantitative results.

[0080] Figure 18 Figure 8 shows the CD31 / PAS double staining results of brain glioma model,

[0081] Among them, Figure A is the saline group, TMZ group, D WVAP1-Micelle / PTX group, D WVAP1-Micelle / PTL group, D WVAP1-Micelle / PTL and TMZ combination therapy group, D WVAP1-Micelle / PTL and D Figure B is the quantitative results of WVAP1-Micelle / PTX combination therapy group inhibiting the formation of new blood vessels CD31 / PAS double staining photos (bar=100μm), in which the nuclei of new blood vessels are brownish yellow or brown, and Figure B is the quantitative results.

[0082] Figure 19 Figure 9 shows the CD133 staining results of brain glioma model,

[0083] Figure A is the physiological saline group, TMZ group, D WVAP1-Micelle / PTX group, D WVAP1-Micelle / PTL group, D WVAP1-Micelle / PTL combined with TMZ group, D WVAP1-Micelle / PTL combined with TMZ group, D WVAP1-Micelle / PTX combined group killing tumor stem cell CD133 staining photographs (bar = 100 μm), wherein the tumor stem cells are brownish yellow or brown, and Figure B is the quantitative results. DETAILED DESCRIPTION

[0084] The present application will be further understood by the following examples, but the present application is not limited to the following description.

[0085] Example 1

[0086] D WVAP1-Cys, D WVAP1-Fluorescein, D WVAP1-Cy7, D WVAP1-drug, D Synthesis and characterization of WVAP1-PEG-PLA

[0087] 1) D Synthesis and characterization of WVAP1-Cys

[0088] WVAP1-Cys was synthesized by solid-phase polypeptide synthesis D WVAP1-Cys (the sequence is D S D N D T D R D V D A D P D C-amino hexanoic acid- D W D S D W D G D P D Y DS), Specific method: Amino acids were sequentially added to Boc-Cys PAM resin, using HBTU / DIEA as the condensing agent and TFA as the deprotecting agent. After the reaction, the resin was cleaved with hydrogen fluoride containing P-cresol, and the reaction was stirred in an ice bath for 1 hour. After the reaction, the hydrogen fluoride in the tube was removed under reduced pressure, the precipitate was precipitated with ice-cold ether and washed three times, the precipitate was redissolved in 20% acetonitrile, the filtrate was collected and rotary evaporated to obtain a crude peptide solution, and the crude peptide was separated and purified using an acetonitrile / water (containing 0.1% TFA) system. The molecular weight (Mw) was characterized by ESI-MS, and the mass spectrum is shown below. Figure 1 As shown;

[0089] 2) D WVAP1-Fluorescein and D Synthesis and Characterization of WVAP1-Cy7

[0090] The results obtained from the above steps D WVAP1-Cys was dissolved in 0.1M PBS solution (pH 7.2). Fluorescein-5-maleimide or Cy7-maleimide was dissolved in DMF. The mixture was then magnetically stirred, and the reaction was monitored by HPLC. D After the WVAP-Cys reaction was completed, the reaction was stopped. Liquid chromatography was then performed for purification, followed by separation and purification using an acetonitrile / water system (containing 0.1% TFA). The purified product was then freeze-dried to obtain... D WVAP-Fluorescein or D Pure WVAP1-Cy7, mass spectrum as shown below. Figure 2 , 3 As shown;

[0091] 3) Preparation D WVAP1-DTPA-Gd and D WVAP1-DTPA- 99m Tc

[0092] Will D WVAP1-Cys was dissolved in 0.1M PBS (pH 7.2), and maleimide-DTPA was dissolved in DMF. The mixture was magnetically stirred, and the reaction was monitored by HPLC. After the reaction was complete, the solution was purified by preparative HPLC using an acetonitrile / water (containing 0.1% TFA) system, and then freeze-dried to obtain the final product. D WVAP1-DTPA pure product, chelated with Gd or 99m Tc is obtained D WVAP1-DTPA-Gd or D WVAP1-DTPA- 99m Tc;

[0093] 4. Preparation DWVAP1-drug complex

[0094] WVAP1-doxorubicin complex was prepared as D WVAP1-doxorubicin complex was prepared as D WVAP1-doxorubicin complex was prepared as D WVAP1-Cys polypeptide was dissolved in phosphate buffer 3 mL (0.1 mM, pH 7.0), 10 times molar amount of tris(2-carboxyethyl)phosphine (TCEP) was added, stirred at 4°C for 20 min, then 4 times molar amount of doxorubicin-6-maleimide hexahydrate derivative was added, reacted at room temperature for 1 h in dark, the reaction solution was purified by preparative liquid phase, freeze-dried to obtain D WVAP1-doxorubicin complex was prepared as

[0095] WVAP1-doxorubicin complex was prepared as D WVAP1-doxorubicin complex was prepared as D WVAP1-doxorubicin complex was prepared as D WVAP1-doxorubicin complex was prepared as D WVAP1-doxorubicin complex was prepared as

[0096] WVAP1-doxorubicin complex was prepared as D WVAP1-doxorubicin complex was prepared as D WVAP1-doxorubicin complex was prepared as D WVAP1-doxorubicin complex was prepared as D WVAP1-doxorubicin complex was prepared as D WVAP1-doxorubicin complex was prepared asD WVAP1 -borzoxil complex;

[0097] By D WVAP1 -PMI (a p53 activation peptide) as D WVAP1 -PMI polypeptide drug, directly prepared by solid-phase polypeptide synthesis method, including: determine D WVAP1 -PMI polypeptide sequence, according to the same method as the preparation D WVAP1, sequentially access amino acids, after HF cutting and purification D WVAP1 -PMI complex;

[0098] 5) D Synthesis and characterization of WVAP1 -high molecular material complex

[0099] By D WVAP1 upstream free thiol and maleimide contained in Mal-PEG-PLA as preparation D WVAP1 -high molecular material complex, 40 mg Mal-PEG-PLA was dissolved in 5 mL acetonitrile, rotary evaporation, film, 3 mL PBS (pH 8.0, 0.2M) was added to hydrate the micelles at 37℃, 9.6 mg D WVAP1 -Cys was added within 8 h, the reaction was detected by HPLC, and the excess D WVAP1 -Cys was removed by dialysis and freeze-dried; 1 H-NMR characterization (as shown in Figure 4 ).

[0100] Example 2 D WVAP1 and glucose-regulated protein GRP78 binding activity experiment

[0101] Pre-binding analysis was carried out by biacore system, and pH 5.0 was selected as the best pH for GRP78 and CM5 chip binding, and the RU value reached the target value, and D WVAP1 was configured into different concentration sample solutions, and was injected from low to high, and the Biacore T200 Evaluation software was used for analysis D WVAP1 and protein binding activity, respectively calculate its K D value, and compared with D VAP and D WSW (as shown in Figure 5 ).

[0102] Example 3 D Serum stability test of WVAP1

[0103] Will D WVAP1 was prepared as a 1 mg / mL aqueous solution. 0.1 mL of this solution was added to 0.9 mL of 25% mouse serum and incubated at 37°C. At 0 and 15 min, 0.5, 1, 2, and 4 h, 100 μL of the reaction solution was collected, and 20 μL of trichloroacetic acid (TCA) was added to precipitate serum proteins. The mixture was allowed to stand at 4°C for 20 min, then centrifuged at 12000 rpm for 10 min. 20 μL of the supernatant was collected for HPLC analysis. Serum stability results (e.g., ...) were obtained. Figure 6 As shown in the figure, D WVAP1 exhibits good serum stability.

[0104] Example 4 D In vitro cell targeting validation experiment of WVAP1

[0105] 1) D In vitro targeting of brain capillary endothelial cells by WVAP1

[0106] Brains were harvested from decapitated 4-week-old SD rats. The cerebral cortex was rapidly isolated in ice-cold D-Hanks solution. After removing the meninges and major cerebral blood vessels, the cortex was minced, and collagenase and DNase were added for digestion at 37°C for 90 min. The cortex was then centrifuged at 1000 rpm for 8 min, the supernatant was discarded, and the contents were transferred to 20% BSA. The cortex was centrifuged at 1000 g / min at 4°C for 20 min, the middle and upper layers were discarded, and the basal microvessels were transferred to culture medium and centrifuged at 1000 rpm for 5 min. Primary brain capillary endothelial cells were obtained by preparing microvascular segment suspensions using DMEM culture medium containing 20% ​​fetal bovine serum and seeding them in confocal dishes or 12-well plates. The suspensions were cultured at 37°C, 5% CO2, and saturated humidity for 24 hours. The culture medium was then replaced with endothelial-specific culture medium containing puromycin for another 72 hours, followed by 72 hours of culture in endothelial-specific culture medium containing cell growth factors. A 5 μM solution of FAM was prepared using DMEM culture medium containing 10% fetal bovine serum. D WVAP1-Fluorescein, D VAP-Fluorescein and D WSW-Fluorescein solution. Aspirate the culture medium from the culture plate and add the above solution. Incubate at 37°C for 12 hours, then discard the supernatant. Wash three times with PBS solution, fix cells with formaldehyde fixative, stain nuclearly with DAPI, and observe using laser confocal microscopy. Cell internalization images are shown below. Figure 7 As shown in Figure A, after washing three times with PBS, flow cytometry analysis was performed, and the results are as follows. Figure 7 As shown in B;

[0107] 2) DIn vitro targeting experiment of WVAP1 to human glioma cell U87

[0108] The monolayer culture of brain glioma cells (U87 cells) in logarithmic growth phase were digested with 0.25% trypsin, and the single cell suspension was prepared with DMEM medium containing 10% fetal bovine serum at 1x10 5 cells per well in a 12-well culture plate with 1 mL volume. The culture plate was moved into a carbon dioxide incubator and cultured at 37°C, 5% CO2 and saturated humidity for 24 h. The FAM, D WVAP1-Fluorescein, D VAP-Fluorescein and D WSW-Fluorescein solution was prepared with DMEM medium containing 10% fetal bovine serum at a concentration of 5 μM. The culture medium in the culture plate was aspirated, and the above solution was added, respectively, and incubated at 37°C for 4 h. The supernatant was aspirated and discarded. The cells were washed with PBS solution for three times, fixed with formaldehyde solution, and stained with DAPI. The laser confocal observation was performed, and the intracellular photos are shown in Figure 7 A. The cells were washed with PBS solution for three times, and flow cytometry analysis was performed. The results are shown in Figure 7 C.

[0109] 3) D In vitro targeting experiment of WVAP1 to human umbilical vein endothelial cell HUVEC

[0110] The monolayer culture of human umbilical vein endothelial cells (HUVEC cells) in logarithmic growth phase were digested with 0.25% trypsin, and the single cell suspension was prepared with DMEM medium containing 10% fetal bovine serum at 1x10 Figure 7 cells per well in a 12-well culture plate with 1 mL volume. The culture plate was moved into a carbon dioxide incubator and cultured at 37°C, 5% CO2 and saturated humidity for 24 h. The FAM, Figure 7 WVAP1-Fluorescein, VAP-Fluorescein and

[0111] WSW-Fluorescein solution was prepared with DMEM medium containing 10% fetal bovine serum at a concentration of 5 μM. The culture medium in the culture plate was aspirated, and the above solution was added, respectively, and incubated at 37°C for 4 h. The supernatant was aspirated and discarded. The cells were washed with PBS solution for three times, fixed with formaldehyde solution, and stained with DAPI. The laser confocal observation was performed, and the intracellular photos are shown in A. The cells were washed with PBS solution for three times, and flow cytometry analysis was performed. The results are shown in

[0112] Example 5 D In vivo tumor targeting verification of WVAP1 polypeptide

[0112] The in situ glioma model mice were established. The U87 cells in logarithmic growth phase were taken, and 5x10 5 cells (dispersed in 5 μL PBS buffer) were inoculated into each nude mouse. After the nude mice were anesthetized, the cells were inoculated into the right part of the striatum (0.6 mm in front of the bregma, 1.8 mm in side, and 3 mm in depth) using a brain stereotaxic instrument. The state of the nude mice was observed regularly. The Cy7, D WVAP1-Cy7, D WSW-Cy7 and DVAP-Cy7 solution was injected into tumor-bearing nude mice via the tail vein. In vivo fluorescence distribution was recorded using an in vivo imaging system at 15, 30, 45, and 120 minutes post-injection. Two hours later, the mice were routinely treated, and brain tumors were harvested. The fluorescence distribution of the tumors was then detected using an in vivo imaging system (e.g., ...). Figure 8 As shown in A), and perform semi-quantitative fluorescence calculations (such as...). Figure 8 (as shown in B).

[0113] Example 6 D In vitro targeting validation of WVAP1-modified polymer micelles

[0114] 1) Preparation of coumarin-6 loaded polymer micelles

[0115] Weigh 1mg D WVAP1-PEG-PLA, 9 mg mPEG-PLA, and 5 μg C6 were dissolved in 2 mL of acetonitrile and rotary evaporated under reduced pressure (~0.085 MPa) in a water bath at 37 °C to form a film. The film was then dried under vacuum at room temperature overnight, hydrated with 2 mL of physiological saline, and the free C6 was removed by CL-4B column chromatography to obtain the desired film. D WVAP1-Micelle / C6, store at 4℃ protected from light, for later use;

[0116] 2) D WVAP1-Micelle's ability to cross the extracorporeal blood-brain barrier

[0117] Brains were harvested from decapitated 4-week-old SD rats. The cerebral cortex was rapidly isolated in ice-cold D-Hanks solution. After removing the meninges and major cerebral blood vessels, the cortex was minced and digested with collagenase and DNase at 37°C for 90 min. The brain was then centrifuged at 1000 rpm for 8 min, the supernatant was discarded, and the fragments were transferred to 20% BSA. The fragments were centrifuged at 1000 g / min at 4°C for 20 min, the middle and upper layers were discarded, and the basal microvessels were transferred to culture medium. The fragments were centrifuged at 1000 rpm for 5 min, and a microvessel suspension was prepared using DMEM medium containing 20% ​​fetal bovine serum. This suspension was then seeded into 24-well transwells pre-coated with rat tail collagen. The transwells were placed in a CO2 incubator and cultured at 37°C, 5% CO2, and saturated humidity for 24 h. The culture medium was then replaced with puromycin-containing endothelial culture medium for 72 h, followed by another 72 h culture with cell growth factors. The electrical resistance was measured to be over 200 Ω·cm. 2 This means that the in vitro BBB model was successfully established.

[0118] A C6 concentration of 50 ng / mL was prepared using DMEM culture medium containing 10% FBS. D WVAP1-Micelle / C6 D WSW-Micelle / C6D VAP-Micelle / C6 and Micelle / C6 solution, the culture solution in the upper chamber of transwell was sucked out, and the above solution was added, respectively, and the culture solution in the lower chamber was taken out at 30 min, 1, 2 and 4 h to measure the fluorescence concentration, and the BBB transport results of each polymer micelle were as shown in Table B. Figure 9 A;

[0119] 3) D WVAP1-Micelle trans-blood-tumor barrier ability in vitro

[0120] The umbilical vein endothelial cells HUVEC and U87 were respectively plated in the upper and lower chambers of transwell at a ratio of 1:5, and cultured for 72 h, and the fluorescence concentration of 50 ng / mL was prepared by using DMEM culture solution containing 10% FBS. D WVAP1-Micelle / C6, D WSW-Micelle / C6, D VAP-Micelle / C6 and Micelle / C6 solution, the culture solution in the upper chamber of transwell was sucked out, and the above solution was added, respectively, and the culture solution in the lower chamber was taken out at 30 min, 1, 2 and 4 h to measure the fluorescence concentration, and the BBB transport results of each polymer micelle were as shown in Table B. Figure 9 B;

[0121] 4、 D WVAP1-Micelle in vitro trans-blood-brain barrier or blood-tumor barrier targeting brain tumor verification

[0122] 2% low molecular weight agarose solution was added into a 48-well plate while hot, 150 μL per well, and after cooling and solidification at room temperature, 400 μL of U87 cell suspension was inoculated in each well, with a cell density of 2 x 10 3 4 per well, and placed in a carbon dioxide incubator at 37°C, 5% CO2 and saturated humidity for 7 days to form tumor spheres, and the U87 tumor spheres were transferred to the lower chamber of the BBB model transwell for culture to obtain a BBB / U87 tumor sphere co-culture model, and the U87 tumor spheres were transferred to the lower chamber of the BTB model transwell for culture to obtain a BTB / U87 tumor sphere co-culture model.

[0123] D WVAP1-Micelle / C6, D WSW-Micelle / C6, D VAP-Micelle / C6 and Micelle / C6 solution, and 50% serum was incubated for 4 h, and the micelles before and after incubation were prepared into 50 ng / mL of DWVAP1-Micelle / C6, D WSW-Micelle / C6, D VAP-Micelle / C6 and Micelle / C6 solution, the culture solution in the upper chamber of the transwell was sucked out, and the above solution was added, respectively, and the tumor spheres in the lower chamber were taken out after 4 h of continuous culture and observed under a fluorescence microscope. The results are shown in Figure 9 C.

[0124] Example 7 D In vivo targeting verification of WVAP1-modified polymer micelles

[0125] 1) Preparation of Micelle / DiR and Micelle / DiD loaded

[0126] The preparation method of Micelle / DiR and Micelle / DiD loaded is the same as that of Micelle / C6 loaded;

[0127] 2) D Qualitative experiment of in vivo targeting verification of WVAP1-Micelle / DiR

[0128] U87 orthotopic glioma model mice were injected with 100 μL of Micelle / DiR, D WVAP1-Micelle / DiR, D WSW-Micelle / DiR, and D VAP-Micelle / DiR, respectively, by tail vein injection. After 24 h of injection, the mice were anesthetized, and the distribution of DiR fluorescence in the mice was recorded by a live imaging instrument and fluorescence semi-quantitative calculation was performed. Then, the tumor was dissected and photographed, and fluorescence semi-quantitative calculation was performed (as shown in Figure 8 C and 8D);

[0129] 3) D Distribution of WVAP1-Micelle / DiD in normal mouse brain

[0130] ICR mice were injected with 100 μL of Micelle / DiD, D WVAP1-Micelle / DiD, D WSW-Micelle / DiD, and D VAP-Micelle / DiD, respectively, by tail vein injection. The mice were sacrificed at 2 h and 24 h after injection, respectively, and then the brain tissue was homogenized and the fluorescence was measured (as shown in Figure 8 E);

[0131] 4) D Quantitative experiment of distribution of WVAP1-Micelle / DiD in tumor-bearing mouse brain

[0132] U87 orthotopic glioma model mice were injected with 100 μL of Micelle / DiD, D WVAP1-Micelle / DiD, D WSW-Micelle / DiD and D VAP-Micelle / DiD, respectively, and the nude mice were sacrificed at 2 h and 24 h after injection, respectively, and then the brain tissues containing tumors were dissected out, homogenized and the fluorescence was measured (as shown in Figure 8 F).

[0133] Example 8 In vitro pharmacodynamics test of polymeric micelles loaded with paclitaxel or parthenolide

[0134] 1) Preparation and characterization of polymeric micelles loaded with paclitaxel or parthenolide

[0135] 1 mg D WVAP1-PEG-PLA, 9 mg mPEG-PLA and 2 mg PTX (paclitaxel) were weighed and dissolved in 2 mL acetonitrile, and then evaporated to dryness under reduced pressure (~0.085 MPa) in a water bath at 37°C to form a film, which was dried in a vacuum overnight at room temperature, and then hydrated with 2 mL of normal saline, and the free drug was removed by CL-4B column chromatography to obtain D WVAP1-modified polymeric micelles loaded with paclitaxel D (WVAP1-Micelle / PTX). The preparation method of D WVAP1-modified polymeric micelles loaded with parthenolide (PTL) D (WVAP1-Micelle / PTL) was the same as above; the particle size and PDI were measured by a laser particle size analyzer (as shown in Figure 10 F); 2、 D WVAP1-Micelle / PTX and D WVAP1-Micelle / PTL in vitro pharmacodynamics test

[0136] U87 cells were seeded in a 96-well plate at a density of 4.0 x 10 3 U87 cells were seeded in a 96-well plate at a density of 4.0 x 10 D WVAP1-Micelle / PTX, D WSW-Micelle / PTX, D VAP-Micelle / PTX, Micelle / PTX and Taxol (paclitaxel), and then MTT solution was added and incubated for another 4 h, and then the culture solution was discarded, 150 μL of DMSO was added, and the plate was shaken until the purple particles were dissolved, and then the absorbance value was measured at 590 nm by an enzyme-labeled instrument, the cell survival rate was determined by MTT method, and the cell survival rate and median lethal dose were calculated,D WVAPl-Micelle / PTL in vitro efficacy test was the same as above, and the results were shown in Figure 11

[0137] 3) Inhibition test of neovascularization

[0138] A 24-well culture plate was taken, 50 μL Matrigel was added to each well, and was spread in the 24-well plate. After incubation in a 37°C incubator for 30 min, the Matrigel was solidified. U87 cells were trypsinized, and a single cell suspension was prepared in DMEM medium containing 10% FBS, 1% penicillin-streptomycin, and 2 mM L-glutamine, at a concentration of 1 x 10 D WVAPl-Micelle / PTX, D WVAPl-Micelle / PTL, PTL and Taxol, at a concentration of 1 x 10 5 cells per well. After incubation in a 37°C, 5% CO2 and saturated humidity incubator for 12 h, the formation of vascular-like structures was observed (as shown in Figure 12 A).

[0139] 4) Inhibition test of vasculogenic mimicry

[0140] A 24-well culture plate was taken, 50 μL Matrigel was added to each well, and was spread in the 24-well plate. After incubation in a 37°C incubator for 30 min, the Matrigel was solidified. U87 cells were trypsinized, and a single cell suspension was prepared in DMEM medium containing 10% FBS, 1% penicillin-streptomycin, and 2 mM L-glutamine, at a concentration of 1 x 10 D WVAPl-Micelle / PTX, D WVAPl-Micelle / PTL, PTL and Taxol, at a concentration of 1 x 10 5 cells per well. After incubation in a 37°C, 5% CO2 and saturated humidity incubator for 12 h, the formation of vascular-like structures was observed (as shown in Figure 12 B).

[0141] Example 9 In vivo pharmacodynamics test of polymeric micelles loaded with paclitaxel or parthenolide

[0142] 1) D WVAPl-Micelle / PTX in vivo pharmacodynamics test

[0143] The nude mice bearing orthotopic glioma model were injected with normal saline, Taxol, Micelle / PTX, D WVAPl-Micelle / PTX, D WSW-Micelle / PTX and D VAP-Micelle / PTX, respectively, through the tail vein. The total dose of PTX was 24 mg / kg, and the administration was performed on the 6th, 9th, 12th and 15th day after tumor implantation. The survival time of the nude mice was recorded, Figure 13 ​The survival time curve of nude mice is shown, compared with other groups, D WVAP1-Micelle / PTX significantly prolongs the survival time of nude mice with orthotopic glioma model;

[0144] 2) D In vivo pharmacodynamic test of WVAP1-Micelle / PTL

[0145] Nude mice with orthotopic glioma model were injected with normal saline, PTL, Micelle / PTL and D WVAP1-Micelle / PTL, respectively, through the tail vein. The total dose of PTL was 25 mg / kg, and it was administered on the 6th, 9th, 12th, 15th and 17th day after tumor implantation. The survival time of nude mice was recorded, and the survival time curve of nude mice is shown in Figure 14 Compared with other groups, D WVAP1-Micelle / PTL significantly prolongs the survival time of nude mice with orthotopic glioma model.

[0146] In vivo pharmacodynamic test of drug-loaded polymer micelles combined with other drugs

[0147] 1) D In vivo pharmacodynamic test of WVAP1-Micelle / PTL combined with D WVAP1-Micelle / PTX

[0148] Nude mice with orthotopic glioma model were injected with normal saline, D WVAP1-Micelle / PTX, D WVAP1-Micelle / PTL, D WVAP1-Micelle / PTX and D WVAP1-Micelle / PTL combined administration, the total dose of PTX was 30 mg / kg, and the total dose of PTL was 25 mg / kg. They were administered on the 6th, 9th, 12th, 15th and 17th day after tumor implantation. The survival time of nude mice was recorded, and the survival time curve of nude mice is shown in Figure 15 Compared with other groups, combined administration can significantly prolong the survival time of nude mice with orthotopic glioma model;

[0149] 2) D In vivo pharmacodynamic test of WVAP1-Micelle / PTL combined with TMZ

[0150] Nude mice with orthotopic glioma model were injected with normal saline, D WVAP1-Micelle / PTL, oral gavage of TMZ and TMZ combined withD WVAP1-Micelle / PTL combined administration, the total dose of PTL was 25 mg / kg, the total dose of TMZ was 50 mg / kg, and the administration was performed on the 6th, 9th, 12th, 15th and 17th day after tumor implantation, and the survival time of the nude mice was recorded, and the survival time curve of the nude mice is shown in Figure 16 Compared with other groups, the combined administration can significantly prolong the survival time of the nude mice with orthotopic glioma model.

[0151] Example 11 Detection of tumor tissue sections after administration

[0152] 1) Tumor blood vessel inhibition test

[0153] After the administration of the tumor-bearing nude mice was completed, the brain tissue was removed, fixed, paraffin-embedded and sectioned, and CD31 immunohistochemical staining and PAS double staining were performed, and 3 high-power fields were continuously observed under a general optical microscope, and the number of CD31 positive blood vessels was counted, and the results are shown in Figure 17

[0154] 2) Tumor apoptosis test

[0155] After the administration of the tumor-bearing nude mice was completed, the brain tissue was removed, fixed, and frozen sectioned, and the tumor apoptosis was detected by TUNEL method, and the degree of tumor cell apoptosis was detected by Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling (TUNEL), including the steps of: paraffin section was routinely dewaxed to water; PBS was rinsed for 3 times, each for 3 min; 0.3% H2O2 solution was treated at room temperature for 20 min; 20 μg / mL protease K was digested at 37°C for 20 min; PBS was rinsed for 3 times, each for 3 min; 30 μL of TUNEL mixture (TDT and biotin-dNTP) was added to each section and placed in a humidified box for incubation at 37°C for 60 min; the positive result was brown yellow or brown in the cell nucleus, and the brown particles in the cell nucleus were positive, which was determined as apoptotic cells; 5 high-power fields were continuously observed under a general optical microscope, and the number of positive cells was counted, and the percentage of positive cells in the cells in the field was the apoptosis index (as shown in Figure 18

[0156] 3) Tumor stem cell inhibition test

[0157] ​​After the administration was completed, the brain tissues of the tumor-bearing nude mice were routinely treated for fixation, and the number of brain tumor stem cells was detected by CD133 staining method. The positive result was that the cell nucleus was brownish yellow or brown, and the brown particles in the cell nucleus were positive, which was determined as tumor stem cells. Five high-power fields were continuously observed under an ordinary optical microscope, and the number of positive cells was counted, and the results were shown in Table 1. Figure 19 Table 1

Claims

1. A fully targeted polypeptide WVAP, characterized in that, The polypeptide WVAP has the sequence of D S D N D T D R D V D A D P D C-amino hexanoic acid- D W D S D W D G D P D Y D S; wherein the amino acids are in the D configuration, and the whole process targets the polypeptide WVAP to target brain capillary endothelial cells and cross the blood-brain barrier, target tumor neovascular endothelial cells and cross the blood-tumor barrier, and simultaneously target tumor mimetic blood vessels, tumor cells and tumor stem cells.

2. A WVAP-X complex, characterized in that, The WVAP-X complex is obtained by reacting the thiol group in the WVAP molecule of the whole-process targeted peptide described in claim 1 with an imaging substance containing a maleimide group; wherein in the WVAP-X complex, X is a fluorescent substance Fluorescein, a near-infrared dye Cy7, IR820, or DiR, a magnetic resonance imaging agent Gd-DTPA, or a radioactive imaging agent. 99m Tc-DTPA.

3. A WVAP-Y complex, characterized in that, The WVAP-Y complex is prepared by linking the pH-sensitive hydrazone bonds, pH-sensitive borate ester bonds, and disulfide bonds in the whole-process targeted peptide WVAP as described in claim 1 with therapeutic drugs, or by directly condensing with peptide drugs; wherein in the WVAP-Y complex, Y is an antitumor drug such as doxorubicin and epirubicin anthracyclines, paclitaxel and docetaxel and carbataxel taxanes, camptothecin and hydroxycamptothecin and irinotecan camptothecin, vincristine and vinorelbine vinblastine, bortezomib and p53 activating peptides.

4. A WVAP-polyethylene glycol-Z composite, characterized in that, By linking the thiol group in the WVAP molecule of the whole-process targeted polypeptide as described in claim 1 with the maleiminated polyethylene glycol-Z complex, a WVAP-polyethylene glycol-Z complex is obtained; in the WVAP-polyethylene glycol-Z complex, Z is phospholipid, polylactic acid (PLA), lactoglycolic acid copolymer (PLGA), or polycaprolactone (PCL).

5. A delivery system, characterized in that, It is a liposome drug delivery system, polymer micelle drug delivery system or polymer disk drug delivery system prepared from the WVAP-polyethylene glycol-phospholipid complex as described in claim 4.

6. A delivery system, characterized in that, It is a polymer micelle drug delivery system or a nanoparticle drug delivery system prepared from the WVAP-polyethylene glycol-polylactic acid composite, WVAP-polyethylene glycol-lactic acid-glycolic acid copolymer composite or WVAP-polyethylene glycol-polycaprolactone composite as described in claim 4.

7. The delivery system as described in claim 5 or 6, characterized in that, The delivery system is used to encapsulate diagnostic drugs or tumor treatment drugs.

8. The delivery system as claimed in claim 7, characterized in that, The diagnostic drugs are selected from fluorescent substances coumarin 6 and FAM, near-infrared dyes Cy7, IR820, DiR, and DiD, or magnetic resonance imaging agents Gd-DTPA.

9. The delivery system as claimed in claim 7, characterized in that, The tumor treatment drugs mentioned are selected from anthracyclines such as doxorubicin and epirubicin, paclitaxel and docetaxel and carbataxel taxanes, camptothecin and hydroxycamptothecin and irinotecan camptothecin, vincristine or vinorelbine vinca alkaloids, bortezomib or carfilzomib proteasome inhibitors, parthenolide, or p53 activating peptides, melitrizin, scorpion venom peptides, and other antimicrobial peptides.

10. The delivery system as claimed in claim 7, characterized in that, The drug delivery system described herein carries tumor therapeutic drugs in combination with other tumor therapeutic drugs, or carries different tumor therapeutic drugs in combination to prepare synergistic drugs for the treatment of brain tumors or peripheral tumors with brain metastasis characteristics.

11. The delivery system as claimed in claim 10, characterized in that, The tumor therapeutic drugs encapsulated are selected from doxorubicin, epirubicin, paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine, vinorelbine, bortezomib, carfilzomib, parthenolide, p53 activating peptide, melipotassium venom peptide, scorpion venom peptide, and antimicrobial peptide. The other tumor therapeutic drugs used in combination are selected from temozolomide, cyclophosphamide, etoposide, mercaptopurine, gemcitabine, cytarabine, 5-fluorouracil, teniposide, epothilone, actinomycin D, mitoxantrone, mitomycin, bleomycin, tenib drugs, platinum drugs, bevacizumab, or trastuzumab.

12. The delivery system as claimed in claim 10, characterized in that, The aforementioned combination therapy comprises a drug delivery system containing tumor-inhibiting drugs such as doxorubicin, epirubicin, paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine, or vinorelbine, and a drug delivery system containing tumor-inhibiting drugs such as bortezomib, carfilzomib, or parthenolide, or a drug delivery system containing antitumor peptides such as p53-activating peptide, melilotinib, or scorpion venom peptide, or a drug delivery system containing antitumor peptides such as p53-activating peptide, melilotinib, or scorpion venom peptide.

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