WO2021147917A1 - 一种全过程靶向分子及其药物复合物在构建递药系统中的应用 - Google Patents

一种全过程靶向分子及其药物复合物在构建递药系统中的应用 Download PDF

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WO2021147917A1
WO2021147917A1 PCT/CN2021/072910 CN2021072910W WO2021147917A1 WO 2021147917 A1 WO2021147917 A1 WO 2021147917A1 CN 2021072910 W CN2021072910 W CN 2021072910W WO 2021147917 A1 WO2021147917 A1 WO 2021147917A1
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drug
molecule
tumor
drugs
following
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French (fr)
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陆伟跃
周建芬
柴芝兰
吴荪奕
谢操
王浩
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复旦大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present invention belongs to the field of pharmacy, and relates to the whole process of targeting brain capillary endothelial cells (crossing the blood-brain barrier), tumor neovascular endothelial cells (crossing the blood-tumor barrier), tumor mimicking blood vessels, tumor cells and tumor stem cells
  • the application of molecules, modified drug complexes and drug-carrying systems in tumor diagnosis and targeted therapy specifically relates to whole-process targeting molecules and modified imaging molecules and therapeutic drug complexes, modified targeting functional carrier materials and the liposomes, polymer micelles, polymer discs, nanoparticles, Nano drug delivery systems such as biofilm-coated nano preparations, and their applications in the diagnosis and targeted therapy of brain tumors or peripheral tumors with brain metastasis characteristics.
  • Tumor is a serious threat to human life and health, and the mortality rate is the highest among all diseases.
  • the morbidity and mortality of primary brain tumors are among the top 10 in my country’s tumor rankings, while gliomas account for about 45% of primary brain tumors.
  • the median survival time of its patients is less than 16 months, which is harmful Great sex.
  • Drug chemotherapy is still the main method for the treatment of brain tumors, but it has disadvantages such as poor selectivity to tumor tissue, high toxicity, narrow therapeutic window, easy to produce multi-drug resistance, and chemotherapy drugs are also extremely limited. In recent years, active targeted drug delivery has become an important strategy to improve the targeting efficiency of brain tumor tissues.
  • Active targeting strategies mainly target highly expressed receptors or transporters in brain tumor tissues, and use corresponding ligands that specifically recognize and bind to the receptors or transporters to deliver drugs or drug delivery systems to brain tumor tissues or In the cell. Most of the current ligands only target a certain receptor or transporter and a certain cell. However, brain tumor tissues are not only tumor cells, but also tumor stem cells, tumor mimics, brain capillaries and their blood-brain barrier. (BBB), tumor neovascularization and its blood-tumor barrier (BTB), etc.
  • the BBB In the early stage of brain tumors, the BBB remains intact and restricts the entry of drugs into the brain, so that about 98% of small-molecule chemotherapeutics and almost 100% of large-molecule drugs such as proteins cannot enter the brain through the BBB, resulting in almost ineffective drug treatment; With the occurrence and development of tumors, tumor angiogenesis is generated, but the new blood vessels of brain tumors are relatively dense and less permeable than peripheral tumors. The resulting BTB has become a major obstacle to drug delivery, and BBB still exists in the glioma infiltration area. Obstruct drug delivery; at the same time, brain tumor stem cells have the characteristics of self-renewal, proliferation and high tumorigenicity.
  • the inventor of the present application has further modified the existing targeting molecules, using the principle of molecular fusion, to covalently link the brain targeting molecule and the tumor targeting molecule into a whole process targeting molecule, so that it has a brain Some tumors or peripheral tumors with the characteristics of brain metastasis are targeted for the whole process of growth and development.
  • the purpose of the present invention is to provide a whole process targeting molecule that targets brain capillary endothelial cells (trans BBB), tumor neovascular endothelial cells (trans BTB), tumor mimic blood vessels, tumor cells, and tumor stem cells.
  • the targeting molecule and the tumor targeting molecule are covalently linked to form.
  • Use whole-process targeting molecules to modify imaging molecules, therapeutic drugs, and polymer carrier materials, to construct targeted molecule-drug complexes, and targeted molecule-modified nano-drug delivery systems to improve the effects of drugs on brain, brain tumors or brain metastases Features targeted diagnosis and treatment of peripheral tumors.
  • the first aspect of the present invention provides a whole-process targeting molecule, which is formed by covalently connecting two parts of a brain-targeting molecule and a tumor-targeting molecule, which can mediate imaging molecules, therapeutic drugs, and Nano drug delivery system achieves targeted delivery;
  • the whole process targeting molecule is composed of a combination of small molecules, polypeptide molecules or protein molecules that have cross-blood-brain barrier and cross-blood-tumor barrier and target tumor mimic blood vessels, tumor cells and their stem cells, and other targeting molecules;
  • the whole process targeting molecule can 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 mimic blood vessels, tumor cells and tumors stem cell;
  • the whole-process targeting molecule can mediate imaging molecules, therapeutic drugs and nano-drug carrier systems to cross the blood-brain barrier and blood-tumor barrier, and target tumor mimic blood vessels, tumor cells and/or tumor stem cells to achieve Targeted delivery of imaging molecules, therapeutic drugs and nano-drug delivery systems to brain tumors and peripheral tumors with brain metastasis characteristics.
  • the targeting molecules such as small molecules, polypeptide molecules or protein molecules that cross the blood-brain barrier are selected from: p-hydroxybenzoic acid (pHA) and its derivatives , Fatty acids such as myristic acid (MC) and its derivatives, D8 polypeptide, WSW polypeptide, D WSW polypeptide, TGN polypeptide, D TGN polypeptide, CDX polypeptide, D CDX polypeptide, T7 polypeptide, and D T7 polypeptide and its derivatives.
  • pHA p-hydroxybenzoic acid
  • MC myristic acid
  • D8 polypeptide WSW polypeptide, D WSW polypeptide, TGN polypeptide, D TGN polypeptide, CDX polypeptide, D CDX polypeptide, T7 polypeptide, and D T7 polypeptide and its derivatives.
  • the targeting molecules such as polypeptide molecules or protein molecules that cross the blood-tumor barrier are selected from: VAP polypeptide, cVAP polypeptide, S VAP polypeptide, D VAP polypeptide, A7R polypeptide, cA7R polypeptide, D A7R polypeptide, RGD polypeptide, Stapled- RGD polypeptide, RW polypeptide, mn polypeptide, RAP12 polypeptide, D RAP12 polypeptide and derivatives thereof.
  • the whole process targeting molecule according to the first aspect of the present invention wherein active functional groups can be introduced into the whole process targeting molecule to construct its modified imaging molecule complex, therapeutic drug complex, targeting functional carrier material and the like Nano drug delivery system.
  • the imaging molecule X is introduced into the whole process of targeting molecules to prepare the whole process of targeting molecule-X complex
  • the therapeutic drug molecule Y is introduced into the whole process of targeting molecules to make the whole process of targeting molecule-Y complex
  • Drug molecules or hydrophilic ligand molecules are further prepared into a nano drug-carrying system with targeted molecules modified on the surface during the whole process.
  • the second aspect of the present invention provides a drug complex, the drug complex is composed of the whole-process targeting molecule described in the first aspect, an imaging molecule, and a therapeutic drug;
  • active functional groups are introduced into the whole process targeting molecule to construct its modified imaging molecule complex and therapeutic drug complex; wherein, the imaging molecule X is introduced into the whole process targeting molecule to produce the whole process targeting molecule -X complex; the therapeutic drug molecule Y is introduced on the whole process targeting molecule to prepare the whole process targeting molecule-Y complex.
  • X is selected from one or more of the following: optical imaging molecule , Magnetic resonance imaging agent, radionuclide imaging agent, used for imaging diagnosis and tracing of brain tumors or peripheral tumors;
  • the optical imaging molecule is selected from one or more of the following: fluorescent probe molecules FITC, FAM, 6-TET, 5-TAMRA, HEX, 6-JOE, near infrared dye molecules Cy3, Cy3.5, Cy5 , Cy5.5, Cy7, IR783, IR820, DiR, DiD, BIDIPY630/650-X, BIDIPY650/665-X, BIDIPY665/676, TO-PRO-3, TO-PRO-5, chemiluminescent substance molecules luminol, isoluminol , AMPPD, CSPD, CDP-star, lucigenin, Raman probe molecules;
  • the magnetic resonance imaging agent is a chelate of a Gd magnetic resonance substance.
  • the radionuclide imaging agent is selected from one or more of the following: 18 F, 32 P, 35 S, 64 Cu, 67/68 Ga, 75 Se, 89 Zr, 86 Y, 99m Tc, 111/111m In , 123/125 I, 177 Lu, 149/161 Tb radionuclide chelate for imaging;
  • the magnetic resonance imaging agent and the radionuclide imaging agent are composed of a bifunctional chelating agent and a substance for magnetic resonance imaging or a radionuclide for imaging.
  • the bifunctional chelating agent is selected from one or more of the following: DOTA, DOTAGA, NOTA, NOTAGA, NODA, DTPA, TETA, CB-TE2A, Cyclam, DFO, MAG3, EC, EDTA, DADT, HYNIC, CE -DTS, NS3, used to chelate magnetic resonance imaging substances or radioactive imaging nuclides.
  • Y is selected from one or more of the following: tumor chemotherapeutics Anthracycline drugs, taxane drugs, camptothecin drugs, vinblastine drugs, proteasome inhibitor drugs, anti-tumor stem cell drugs, molecular targeted drugs, peptide drugs, antibody drugs , Therapeutic radionuclide chelate;
  • the anthracyclines in the tumor chemotherapy drugs are selected from one or more of the following: doxorubicin and epirubicin;
  • the taxane drug is selected from one or more of the following: paclitaxel, docetaxel, cabazitaxel;
  • the camptothecin drug is selected from one or more of the following: camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan;
  • the vinblastine drug is selected from one or more of the following: vinblastine and vincristine;
  • the proteasome inhibitor drug is bortezomib
  • the anti-tumor stem cell drug is selected from parthenolide and its derivatives;
  • the molecular targeted drugs are selected from one or more of the following: trametinib, imatinib, nilotinib, dasatinib, everolimus, erlotinib, sulatinib Nitinib, sorafenib, ibrutinib, regorafenib, verofenib, olaparib; and/or
  • the polypeptide drug is selected from one or more of the following: p53 activating peptide, melittin, scorpion venom peptide, antibacterial peptide;
  • the antibody drug is selected from one or more of the following: rituximab, bevacizumab, trastuzumab, cetuximab, pertuzumab, ipilimumab Anti-, nivolumab, PD-L1 monoclonal antibodies, and combinations of genetically engineered antibody fragments include Fab fragments, single domain antibodies, Fv fragments, single chain antibodies, bivalent small molecule antibodies, micro antibodies, and nano antibodies; and / or
  • the therapeutic radionuclide chelate is selected from one or more of the following: 90 Y, 131 I, 152/155 Tb, 153 Sm, 177 Lu, 186/188 Re, 211 At, 212/213 Chelate of Bi, 212 Pb, 225 Ac, 227 Th therapeutic radionuclides;
  • the therapeutic radionuclide chelate is composed of a bifunctional chelating agent and a therapeutic radionuclide, wherein the bifunctional chelating agent is selected from DOTA, DOTAGA, NOTA, NOTAGA, NODA, DTPA, TETA, CB -TE2A, Cyclam, DFO, MAG3, EC, EDTA, DADT, HYNIC, CE-DTS, NS3.
  • the bifunctional chelating agent is selected from DOTA, DOTAGA, NOTA, NOTAGA, NODA, DTPA, TETA, CB -TE2A, Cyclam, DFO, MAG3, EC, EDTA, DADT, HYNIC, CE-DTS, NS3.
  • the third aspect of the present invention provides a targeted functional carrier material, the targeted functional carrier material is composed of the whole process targeting molecule described in the first aspect and a polymer carrier material, and can be used to prepare the whole process targeting molecule Nano drug delivery system modified on the surface;
  • active functional groups are introduced into the whole process targeting molecule to construct a modified polymer carrier material complex; wherein, polyethylene glycol-Z complex molecules are introduced into the whole process targeting molecule to prepare the target molecule.
  • the whole process of the functional carrier material targets the molecule-polyethylene glycol-Z complex, where Z is a lipophilic material molecule or a lipophilic drug molecule or a hydrophilic ligand molecule.
  • the lipophilic material molecule Z is selected from one or more of the following: phospholipids, polylactic acid (PLA), Copolymer of lactic acid and glycolic acid (PLGA), polycaprolactone (PCL);
  • the lipophilic drug molecule Z is selected from one or more of the following: anthracycline drugs, taxane drugs, camptothecin drugs, vinblastine drugs, proteasome inhibitor drugs, lactones anti-tumor Drugs; and/or
  • the hydrophilic ligand molecule Z is biotin.
  • the targeted functional carrier material can be further prepared into a nano drug-carrying system with targeted molecules modified on the surface during the whole process.
  • the fourth aspect of the present invention provides the application of the whole process targeting molecule of the first aspect or the targeted functional carrier material of the third aspect in the preparation of a nano-drug carrier system, wherein when the whole process targeting molecule is introduced
  • Z in the polyethylene glycol-Z complex is a lipophilic material molecular phospholipid
  • the targeted functional carrier material is used to prepare a liposome drug delivery system, a micellar drug delivery system or a disc drug delivery system;
  • the targeting molecule is introduced into the polyethylene glycol-Z complex, when Z is a lipophilic material molecule polylactic acid complex, lactic acid glycolic acid copolymer and/or polycaprolactone, the targeting The functional carrier material is used to prepare micellar drug delivery system or nanoparticle drug delivery system; and/or
  • the targeting functional carrier material can be used to prepare a biofilm-coated nano drug delivery system .
  • the liposome drug-carrying system, micellar drug-carrying system, disc drug-carrying system, nano-particle drug-carrying system, and biofilm-coated nano-drug loading system contain diagnostic drugs , Used for imaging diagnosis and tracing of brain tumors or peripheral tumors;
  • the diagnostic drug is selected from one or more of the following: optical imaging materials, magnetic resonance imaging agents, and radionuclide imaging agents;
  • the optical imaging substance is selected from one or more of the following: fluorescent probe coumarin 6, FITC, FAM, DiI, Rhodamine B, Rhodamine 6G, 5-TAMRA, 6-TET, HEX, 6- JOE, near infrared dyes Cy3, Cy3.5, Cy5, Cy5.5, Cy7, IR783, IR820, DiR, DiD, Alexa Fluor 680, BIDIPY630/650-X, BIDIPY650/665-X, BIDIPY665/676, TO-PRO -3, TO-PRO-5, chemiluminescent substances luminol, isoluminol, AMPPD, CSPD, CDP-star, lucigenin, Raman probe;
  • the magnetic resonance imaging agent is a chelate of a magnetic resonance imaging substance such as Gd; and/or
  • the radionuclide imaging agent is selected from one or more of the following: 18 F, 32 P, 35 S, 64 Cu, 67/68 Ga, 75 Se, 89 Zr, 86 Y, 99m Tc, 111 /111m In, 123/125 I, 177 Lu, 149/161 Tb chelate of radionuclides for imaging.
  • the liposome drug delivery system, micellar drug delivery system, disc drug delivery system, nanoparticle drug delivery system and biofilm coated nano drug delivery system are used for encapsulation Tumor therapy drugs for targeted therapy of brain tumors or peripheral tumors with brain metastasis characteristics;
  • the tumor treatment drugs are selected from one or more of the following: anthracyclines, taxanes, camptothecins, vinblastines, platinum drugs, proteasome inhibitors in chemotherapy Class drugs, anti-tumor stem cell drugs, molecular targeted drugs, peptide drugs, antibody drugs, therapeutic radionuclide chelates;
  • the anthracycline in the chemotherapy is selected from one or more of the following: adriamycin and epirubicin;
  • the taxane drug is selected from one or more of the following: paclitaxel, docetaxel, cabazitaxel;
  • the camptothecin drug is selected from one or more of the following: camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan;
  • the vinblastine drug is selected from one or more of the following: vinblastine and vincristine;
  • the platinum-based drug is selected from one or more of the following: cisplatin, carboplatin, oxaliplatin, and miplatin;
  • the proteasome inhibitor drug is selected from one or more of the following: bortezomib and carfilzomib;
  • the anti-tumor stem cell drug is selected from one or more of the following: parthenolide and its derivatives;
  • the molecular targeted drug is selected from one or more of the following: trametinib, imatinib, nilotinib, dasatinib, everolimus, erlotinib, sunitin Ni, Sorafenib, Ibrutinib, Regorafenib, Verofenib, Olaparib;
  • the polypeptide drug is selected from one or more of the following: p53 activating peptide, melittin, scorpion venom peptide and antibacterial peptide;
  • the antibody drug is selected from one or more of the following: rituximab, bevacizumab, trastuzumab, cetuximab, pertuzumab, ipilimumab , Nivolumab, PD-L1 monoclonal antibodies, and combinations of antibody fragments modified by genetic engineering methods, including Fab fragments, single domain antibodies, Fv fragments, single chain antibodies, bivalent small molecule antibodies, micro antibodies, and nano antibodies; and / or
  • the therapeutic radionuclide chelate is selected from one or more of the following: 90 Y, 131 I, 152/155 Tb, 153 Sm, 177 Lu, 186/188 Re, 211 At, 212/213 Bi , 212 Pb, 225 Ac, 227 Th chelate of therapeutic radionuclides.
  • the fifth aspect of the present invention provides a nano drug delivery system.
  • the nano drug delivery system includes the whole process targeting molecule of the first aspect or the targeted functional carrier material of the third aspect, and the nano drug delivery system is It is constructed by introducing polyethylene glycol-Z complex to the targeting molecule in the whole process, where Z is a lipophilic material molecule or a lipophilic drug molecule or a hydrophilic ligand molecule;
  • the lipophilic material molecule Z is selected from one or more of the following: phospholipid, polylactic acid (PLA), lactic acid glycolic acid copolymer (PLGA), polycaprolactone (PCL);
  • the lipophilic drug molecule Z is selected from one or more of the following: anthracycline drugs, taxane drugs, camptothecin drugs, vinblastine drugs, proteasome inhibitor drugs, lactones anti-tumor Drugs; and/or
  • the hydrophilic ligand molecule Z is biotin.
  • the nano drug delivery system is a liposome drug delivery system, a micellar drug delivery system or a disc drug delivery system system;
  • the nano drug delivery system is a micellar drug delivery system or a nanoparticle drug delivery system;
  • the nano drug delivery system is a biofilm coated nano drug delivery system.
  • nano drug delivery system according to the fifth aspect of the present invention, wherein the liposome drug delivery system, micellar drug delivery system, disc drug delivery system, nanoparticle drug delivery system and biofilm coated nano drug delivery system Packed with diagnostic drugs, used for imaging diagnosis and tracing of brain tumors or peripheral tumors;
  • the diagnostic drug is selected from one or more of the following: optical imaging materials, magnetic resonance imaging agents, and radionuclide imaging agents;
  • the optical imaging substance is selected from one or more of the following: fluorescent probe coumarin 6, FITC, FAM, DiI, Rhodamine B, Rhodamine 6G, 5-TAMRA, 6-TET, HEX, 6- JOE, near infrared dyes Cy3, Cy3.5, Cy5, Cy5.5, Cy7, IR783, IR820, DiR, DiD, Alexa Fluor 680, BIDIPY630/650-X, BIDIPY650/665-X, BIDIPY665/676, TO-PRO -3, TO-PRO-5, chemiluminescent substances luminol, isoluminol, AMPPD, CSPD, CDP-star, lucigenin, Raman probe;
  • the magnetic resonance imaging agent is a chelate of a magnetic resonance imaging substance such as Gd; and/or
  • the radionuclide imaging agent is selected from one or more of the following: 18 F, 32 P, 35 S, 64 Cu, 67/68 Ga, 75 Se, 89 Zr, 86 Y, 99m Tc, 111 /111m In, 123/125 I, 177 Lu, 149/161 Tb chelate of radionuclides for imaging.
  • nano drug delivery system according to the fifth aspect of the present invention, wherein the liposome drug delivery system, micellar drug delivery system, disc drug delivery system, nanoparticle drug delivery system and biofilm coated nano drug delivery system Encapsulated tumor therapy drugs for targeted therapy of brain tumors or peripheral tumors with brain metastasis characteristics;
  • the tumor treatment drugs are selected from one or more of the following: anthracyclines, taxanes, camptothecins, vinblastines, platinum drugs, proteasome inhibitors in chemotherapy Class drugs, anti-tumor stem cell drugs, molecular targeted drugs, peptide drugs, antibody drugs, therapeutic radionuclide chelates;
  • the anthracycline in the chemotherapy is selected from one or more of the following: adriamycin and epirubicin;
  • the taxane drug is selected from one or more of the following: paclitaxel, docetaxel, cabazitaxel;
  • the camptothecin drug is selected from one or more of the following: camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan;
  • the vinblastine drug is selected from one or more of the following: vinblastine and vincristine;
  • the platinum-based drug is selected from one or more of the following: cisplatin, carboplatin, oxaliplatin, and miplatin;
  • the proteasome inhibitor drug is selected from one or more of the following: bortezomib and carfilzomib;
  • the anti-tumor stem cell drug is selected from one or more of the following: parthenolide and its derivatives;
  • the molecularly targeted drug is selected from one or more of the following: trametinib, imatinib, nilotinib, dasatinib, everolimus, erlotinib, sunitin Ni, Sorafenib, Ibrutinib, Regorafenib, Verofenib, Olaparib;
  • the polypeptide drug is selected from one or more of the following: p53 activating peptide, melittin, scorpion venom peptide and antibacterial peptide;
  • the antibody drug is selected from one or more of the following: rituximab, bevacizumab, trastuzumab, cetuximab, pertuzumab, ipilimumab , Nivolumab, PD-L1 monoclonal antibodies, and combinations of antibody fragments modified by genetic engineering methods, including Fab fragments, single domain antibodies, Fv fragments, single chain antibodies, bivalent small molecule antibodies, micro antibodies, and nano antibodies; and / or
  • the therapeutic radionuclide chelate is selected from one or more of the following: 90 Y, 131 I, 152/155 Tb, 153 Sm, 177 Lu, 186/188 Re, 211 At, 212/213 Bi , 212 Pb, 225 Ac, 227 Th chelate of therapeutic radionuclides.
  • the sixth aspect of the present invention provides a method for the diagnosis and/or treatment of brain tumors or peripheral tumors with brain metastasis characteristics, the method comprising: administering the whole process targeting molecules of the first aspect to subjects in need , The drug complex of the second aspect or the targeted functional carrier material of the third aspect, or the nano drug delivery system of the fifth aspect;
  • the method is to target brain capillary endothelial cells and cross the blood-brain barrier to target tumor neovascular endothelium through the whole process targeting molecules, drug complexes, targeted functional carrier materials or nano drug delivery systems.
  • Cells cross the blood-tumor barrier and simultaneously target tumor mimic blood vessels, tumor cells and tumor stem cells;
  • the method is to cross the blood-brain barrier and blood-tumor barrier through the whole process of targeting molecules, drug complexes, targeted functional carrier materials or nano-drug carrier systems to target tumor mimic blood vessels and tumor cells. And/or tumor stem cells to achieve targeted delivery of imaging molecules, therapeutic drugs and nano-drug delivery systems to brain tumors and peripheral tumors with brain metastasis characteristics.
  • the present invention uses the principle of molecular fusion to covalently connect brain targeting molecules and tumor targeting molecules to prepare a whole process targeting molecule, so that it has the targeting ability of both molecules at the same time, and can target brain capillaries.
  • Endothelial cells (trans-BBB), tumor neovascular endothelial cells (trans-BTB), tumor mimicking blood vessels, tumor cells and tumor stem cells have a targeting effect on the whole process of brain tumor growth and development.
  • Targeting molecules such as small molecules, polypeptide molecules or protein molecules that cross the blood-brain barrier involved in the present invention include: p-hydroxybenzoic acid (pHA) and its derivatives, fatty acids, especially myristic acid (MC) and its derivatives , D8 polypeptide, WSW polypeptide, D WSW polypeptide, TGN polypeptide, D TGN polypeptide, CDX polypeptide, D CDX polypeptide, T7 polypeptide and D T7 polypeptide and other polypeptides and their derivatives, transferrin, lactoferrin and other proteins and their derivatives Things.
  • pHA p-hydroxybenzoic acid
  • MC myristic acid
  • Targeting molecules such as polypeptide molecules or protein molecules that cross the blood-tumor barrier involved in the present invention include: VAP polypeptide, cVAP polypeptide, S VAP polypeptide, D VAP polypeptide, A7R polypeptide, cA7R polypeptide, D A7R polypeptide, RGD polypeptide, Stapled -RGD polypeptide, RW polypeptide, mn polypeptide, RAP12 polypeptide, D RAP12 polypeptide and other polypeptides and their derivatives.
  • Each polypeptide sequence is shown in Table 1 of the specification (Table 1-Polypeptide amino acid sequence list).
  • the whole process targeting molecule designed in the present invention can construct its modified imaging molecular complex, therapeutic drug complex, and targeting functional carrier material by introducing active functional groups into the molecule.
  • the sulfhydryl group and maleimide in the molecule are used to functionalize optical imaging molecules (such as fluorescent probe molecules FITC, FAM, 6-TET, 5-TAMRA).
  • near infrared dyes such as Cy3, Cy3.5, Cy5, Cy5.5, Cy7, IR783, IR820, DiR, DiD, BIDIPY630/650-X, BIDIPY650/665-X, BIDIPY665/676, TO-PRO-3, TO-PRO-5, etc., chemiluminescent substance molecules luminol, isoluminol, AMPPD, CSPD, CDP-star, lucigenin, etc., Raman probe molecules, etc.) react to form a complex.
  • near infrared dyes such as Cy3, Cy3.5, Cy5, Cy5.5, Cy7, IR783, IR820, DiR, DiD, BIDIPY630/650-X, BIDIPY650/665-X, BIDIPY665/676, TO-PRO-3, TO-PRO-5, etc.
  • chemiluminescent substance molecules luminol, isoluminol, AMPPD, CSPD, CDP-star, lucigenin
  • the whole process targeting molecule designed in the present invention is combined with magnetic resonance imaging agents (such as Gd and other magnetic resonance imaging materials), or with radionuclide imaging agents (such as 18 F, 32 P, 35 S, 64 Cu, 67/68 Ga, 75 Se, 89 Zr, 86 Y, 99m Tc, 111/111m In, 123/125 I, 177 Lu, 149/161 Tb and other imaging radionuclides), or with Therapeutic radionuclide chelates (such as 90 Y, 131 I, 152/155 Tb, 153 Sm, 177 Lu, 186/188 Re, 211 At, 212/213 Bi, 212 Pb, 225 Ac, 227 Th, etc.
  • magnetic resonance imaging agents such as Gd and other magnetic resonance imaging materials
  • radionuclide imaging agents such as 18 F, 32 P, 35 S, 64 Cu, 67/68 Ga, 75 Se, 89 Zr, 86 Y, 99m Tc, 111/111m In, 123/125 I, 177
  • a complex is formed by reacting with a chelate of a radionuclide, wherein the chelate is composed of a bifunctional chelating agent and a radionuclide for magnetic resonance imaging, or a bifunctional chelating agent and a radionuclide for imaging, or a bifunctional chelating agent And composed of therapeutic radionuclides.
  • the bifunctional chelating agent in the chelate includes DOTA, DOTAGA, NOTA, NOTAGA, NODA, DTPA, TETA, CB-TE2A, Cyclam, DFO, MAG3, EC, EDTA, DADT, HYNIC, CE-DTS, NS3, etc.
  • the whole process targeted molecular modification drugs designed by the present invention include the formation of pH-sensitive hydrazone bonds through the reaction of maleimidohexylhydrazine derivatives (involving drugs containing ketone or aldehyde groups such as doxorubicin and epirubicin), Or through the reaction of 3-(2-pyridinedimercapto) propionic acid derivatives to form a disulfide bond (involving paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, Iraqi Rinotecan, vincristine, and vinorelbine and other drugs containing hydroxyl or amino groups), or through the reaction of dopamine with the boric acid group in the drug to form pH-sensitive borates (involving drugs containing boric acid groups such as bortezomib), or through Solid-phase synthesis directly forms amide bonds (involving peptide drugs such as p53 activating peptide, melitt
  • PEG-DSPE polyethylene glycol-distearoyl phosphatidylethanolamine
  • PEG-DSPE polyethylene glycol containing maleimide functional groups
  • -Polylactic acid (PEG-PLA) polyethylene glycol-lactic acid glycolic acid copolymer (PEG-PLGA), polyethylene glycol-polycaprolactone (PEG-PCL) and other polymer carrier materials, used in the whole process Construction of nano drug delivery systems such as liposomes, micelles, discs, and nanoparticles modified by targeting molecules.
  • cysteine is introduced into the whole process targeting molecule designed by the present invention, it is modified on targeting materials such as polyethylene glycol-biotin (PEG-Biotin) containing maleimide functional groups for use in the whole process Construction of a biofilm-coated nano-drug carrier system modified by targeting molecules.
  • PEG-Biotin polyethylene glycol-biotin
  • the nano drug-carrying system designed by the present invention for the whole process of targeted molecular modification is used for loading anthracycline drugs such as doxorubicin and epirubicin, and taxanes such as paclitaxel, docetaxel and cabazitaxel.
  • anthracycline drugs such as doxorubicin and epirubicin
  • taxanes such as paclitaxel, docetaxel and cabazitaxel.
  • Drugs including camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan and other camptothecin drugs, including vincristine, vinorelbine, and other vinblastine drugs, including cis Platinum, carboplatin, oxaliplatin, miplatin and other platinum drugs, including proteasome inhibitors such as bortezomib and carfilzomib, including parthenolide and other lactone drugs, including trameti Ni, imatinib, nilotinib, dasatinib, everolimus, erlotinib, sunitinib, sorafenib, ibrutinib, regorafenib, verofinil Molecular targeted drugs such as nigra, olaparib, etc., containing peptide drugs such as p53 activating peptide, melittin, scorpion venom peptide, and antimicrobial peptide, and
  • optical imaging molecules such as fluorescent probe molecules FITC, FAM, 6-TET, 5-TAMRA, HEX, 6-JOE, etc.
  • the whole process targeting molecule designed in the present invention is used to mediate drugs or nano drug delivery systems across BBB and BTB, target tumor neovascularization, tumor mimicry blood vessels, tumor cells and tumor stem cells, and be used for brain, brain tumor or Targeted diagnosis and treatment of peripheral tumors with brain metastasis characteristics.
  • PHA-VAP was prepared by solid-phase synthesis; pHA-VAP-Cy7 was synthesized by Michael addition reaction of maleimide group and sulfhydryl group; its structure was characterized by HPLC and MS.
  • pHA-VAP-DTPA or pHA-VAP-DOTA was synthesized by the Michael addition reaction of the maleimide group and the sulfhydryl group, and pHA-VAP-DTPA-Gd or pHA-VAP-DTPA- was obtained by chelating Gd or 99m Tc. 99m Tc, or pHA-VAP-DOTA-Gd.
  • pHA-VAP-Cy7 brain capillary endothelial cells (BCEC), umbilical vein endothelial cells (HUVEC) and model tumor cells (such as glioma cells U87).
  • BCEC brain capillary endothelial cells
  • U87 umbilical vein endothelial cells
  • model tumor cells such as glioma cells U87.
  • mice and nude mice bearing U87 subcutaneous tumors and U87 intracranial orthotopic tumor models were injected with pHA-VAP-Cy7 through the tail vein to investigate its distribution in animals at various time points.
  • pHA-VAP reacts with the maleimide hydrazine derivative on the drug to form a polypeptide-drug complex containing a pH-sensitive hydrazone bond.
  • the drugs involved include doxorubicin, epirubicin, etc. Drugs containing ketone or aldehyde groups.
  • pHA-VAP After introducing cysteine, pHA-VAP reacts with 3-(2-pyridinedimercapto)propionic acid derivatives on the drug to form a disulfide bond-containing polypeptide-drug complex.
  • the drugs involved include paclitaxel and docetaxel , Cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine, vinorelbine and other drugs containing hydroxyl or amino groups.
  • pHA-VAP modifies the dopamine and reacts with the boronic acid group on the drug to form a pH-sensitive boronic acid-containing polypeptide-drug complex.
  • the drugs involved include borate-containing drugs such as bortezomib.
  • pHA-VAP is directly condensed with polypeptide drugs through solid-phase synthesis.
  • the drugs involved include p53 activating peptides, antibacterial peptides, polypeptide toxins and other polypeptide drugs.
  • pHA-VAP is targeted by random site modification (the free amino group in the antibody is activated and then covalently linked to pHA-VAP) or site-specific modification (the targeting molecule is non-covalently linked to the antibody through affinity coupling)
  • Functional molecule modified antibody complex the drugs involved include Rituximab, Bevacizumab, Trastuzumab, Cetuximab, Pertuzumab, Ipilimumab, Nivolu Antibody drugs such as monoclonal antibodies and PD-L1 monoclonal antibodies and combinations of antibody fragments modified by genetic engineering methods (including Fab fragments, single domain antibodies, Fv fragments, single chain antibodies, bivalent small molecule antibodies, micro antibodies, nano antibodies) Wait).
  • pHA-VAP-doxorubicin complex obtained by condensing the pHA-VAP with cysteine and the maleimidohexyl hydrazine derivative (MAL-DOX) on doxorubicin (pHA-VAP-DOX), with MTT
  • MAL-DOX maleimidohexyl hydrazine derivative
  • pHA-VAP modified polymer materials pHA-VAP-PEG-DSPE, pHA-VAP-PEG-PLA, pHA-VAP-PEG-PLGA, pHA-VAP-PEG-PCL, pHA-VAP-PEG-Biotin, etc. .
  • the imide reaction realizes the preparation of the above-mentioned targeted polymer materials, namely: dissolve Mal-PEG-DSPE, Mal-PEG-PLA, Mal-PEG-PLGA, Mal-PEG-PCL, Mal-PEG-biotin, etc.
  • pHA-VAP modified nano drug delivery system was constructed.
  • the pHA-VAP modified nano drug delivery system containing tumor therapeutic drugs was used to investigate its performance at various time points. Distribution within the tumor.
  • the MTT method was used to investigate the in vitro growth inhibitory effects of the pHA-VAP modified nano drug delivery system containing tumor therapeutic drugs on tumor cells (U87 cells, 4T1 cells) and HUVEC cells; Nude mice or bearing U87 intracranial orthotopic tumor models were used 4T1 breast cancer orthotopic model balb/c mice were injected with a pHA-VAP modified nano-drug carrier system containing tumor therapeutic drugs into the tail vein for survival time, tumor suppression curve, tumor tissue cell apoptosis, neovascularization and stem cell number, etc. As an index to evaluate its anti-tumor effect in vivo.
  • the method provided by the present invention is used to design and prepare the whole process targeting molecule pHA-VAP and its modified drug complex and nano drug-carrying system.
  • the test results of the present invention show that pHA-VAP also has the ability of pHA to target brain capillaries and cross the BBB, VAP targets tumor neovascular endothelial cells and cross BTB, target tumor mimic blood vessels, tumor cells and tumor stem cells.
  • Animals have good brain and tumor tissue targeting capabilities and show better brain tumor targeting effects; pHA-VAP modified drug complexes and nano drug delivery systems show good tumor targeting performance and better The diagnosis and treatment of brain tumors.
  • Figure 1 shows the HPLC and ESI-MS spectra of pHA-VAP-Cys
  • Chromatographic method chromatographic column (YMC, C18): 150 ⁇ 4.6mm; mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid); elution program: 0- 45min 5%B-65%B; flow rate: 0.7mL/min; column temperature: 40°C; detection: UV 214nm, retention time: 16min.
  • ESI-MS 1080.4, which is consistent with the theoretical molecular weight.
  • Figure 1A shows the ESI-MS spectrum
  • Figure 1B shows the HPLC spectrum.
  • Figure 2 shows the HPLC and ESI-MS spectra of pHA-VAP-Cy7
  • Figure 3 shows the HPLC and ESI-MS spectra of pHA-VAP-DOTA-Gd
  • FIG. 4 shows the uptake of Cy7-labeled pHA-VAP by BCEC of primary brain capillary endothelial cells
  • the picture shows the quantitative ( Figure 4A) and qualitative (Figure 4B) results of flow cytometric fluorescence detection after Cy7-labeled VAP and pHA-VAP were incubated with BCEC cells for 4 hours. It can be seen that the uptake of pHA-VAP by BCEC cells is significantly higher than that of VAP and free fluorescein.
  • FIG. 5 shows the uptake of Cy7-labeled pHA-VAP by HUVEC in umbilical vein endothelial cells
  • the picture shows the quantitative ( Figure 5A) and qualitative (Figure 5B) results of flow cytometry fluorescence detection after Cy7-labeled VAP and pHA-VAP were incubated with HUVEC cells for 4 hours. It can be seen that the uptake of pHA-VAP by U87 cells is significantly higher than that of VAP and free fluorescein.
  • Figure 6 shows the uptake of Cy7-labeled pHA-VAP by glioma cells U87
  • the picture shows the quantitative ( Figure 6A) and qualitative ( Figure 6B) results of flow cytometry fluorescence detection after Cy7-labeled VAP and pHA-VAP were incubated with U87 cells for 4 hours. It can be seen that the uptake of pHA-VAP by U87 cells is significantly higher than that of VAP and free fluorescein.
  • Figure 7 shows the uptake of pHA-VAP-DOTA-Gd by tumor cells HCC1806 or A549
  • the picture shows the intracellular Gd uptake measured by ICP-MS after pHA-VAP-DOTA-Gd and DOTA-Gd were incubated with HCC1806 or A549 cells for 4 hours. It can be seen that the uptake of pHA-VAP-DOTA-Gd by HCC1806 and A549 cells was significantly higher than that of the unmodified group.
  • FIG. 8 shows the tissue distribution map of Cy7-labeled pHA-VAP in mice bearing U87 subcutaneous transplantation tumor model
  • FIG. 9 shows the tissue distribution map of Cy7-labeled pHA-VAP in mice bearing U87 brain tumor in situ
  • Figure 10 shows the HPLC and ESI-MS spectra of pHA-VAP-DOX
  • Figure 11 shows the in vitro anti-U87 cell activity curve of pHA-VAP-DOX
  • Figure 12 shows the in vitro anti-HUVEC cell activity curve of pHA-VAP-DOX
  • Figure 13 shows the survival curve of pHA-VAP-DOX against U87 in situ glioma
  • the picture shows the survival curve of nude mice with U87 orthotopic glioma model.
  • the median survival time of model animals was used as an indicator, compared with normal saline (median survival time of 26 days), DOX (10 mg, 20 mg, 40 mg median survival time of 27, 17, and 13 days, respectively, medium and high dose doxorubicin toxicity Large leads to shorter survival period of model nude mice), pHA-VAP (median survival period of 28 days), pHA-VAP-DOX (median survival period of 10mg, 20mg, 40mg respectively 29, 32, 40 days) prolonged model animals Survival time, and dose-dependent.
  • Figure 14 shows the electron micrograph of the nano-drug delivery system of pHA-VAP modified lipid membrane coated with cabazitaxel nanocrystals
  • the cabazitaxel nanocrystals are spherical with a particle size of about 80nm;
  • the pHA-VAP modified lipid membrane coated cabazitaxel nanocrystals are spherical with obvious cores.
  • -Membrane structure, particle size is about 100nm.
  • Figure 15 shows the particle size characterization of the nano-drug delivery system in which pHA-VAP modified red blood cell membranes are coated with docetaxel/parthenolide nano-cocrystals
  • the particle size of the docetaxel/parthenolide nano-eutectic is about 130nm and the potential is -20mV; the size of the erythrocyte membrane is about 140nm and the potential is -25mV; the pHA-VAP modification pair
  • the particle size of the constructed nano-drug delivery system has no obvious effect, but the potential of the modified nano-drug delivery system rises to -15mV because the molecule itself is positively charged.
  • Figure 16 shows the uptake of pHA-VAP modified lipid membrane-coated cabazitaxel nanocrystals by umbilical vein endothelial cells HUVEC and breast cancer cell 4T1
  • HUVEC cells Figure 16A
  • 4T1 cells Figure 16B
  • uptake of pHA-VAP modified lipid membrane-coated cabazitaxel nanocrystals was significantly higher than that of the free drug group, nanocrystal group and no target lipid
  • the film is coated with the cabazitaxel nanocrystal group.
  • Figure 17 shows the uptake of primary brain capillary endothelial cells BCEC, umbilical vein endothelial cells HUVEC and glioma cells U87 on pHA-VAP modified red blood cell membrane-coated docetaxel/parthenolide nano co-crystals
  • BCEC cells Figure 17A, Figure 17B
  • HUVEC cells Figure 17C, Figure 17D
  • U87 cells Figure 17E, Figure 17F
  • pHA-VAP modified red blood cell membrane-coated drug nano-cocrystal Docetal The ingestion of ceramide and parthenolide was significantly higher than that of the nano-eutectic group and the non-target erythrocyte membrane-coated drug nano-eutectic group, which was comparable to the free drug group.
  • Figure 18 shows the tissue distribution map of pHA-VAP modified lipid membrane coated cabazitaxel nanocrystals in mice bearing 4T1 breast cancer in situ tumor model
  • pHA-VAP modification can significantly increase the accumulation of lipid membrane-coated cabazitaxel nanocrystals at the 4T1 tumor site at different time points, and better target the tumor site.
  • Figure 19 shows the tissue distribution of pHA-VAP modified erythrocyte membrane-coated docetaxel/parthenolide nano-cocrystal in mice bearing U87 in situ glioma model
  • pHA-VAP modification can significantly increase the accumulation of erythrocyte membrane-coated drug nano-cocrystals (Docetaxel ( Figure 19A) and Parthenolide (Figure 19B)) at different time points in the glioma site , To better target the tumor site.
  • Docetaxel Figure 19A
  • Parthenolide Figure 19B
  • Figure 20 shows the in vitro anti-HUVEC and 4T1 cell activity curve of pHA-VAP modified lipid membrane coated cabazitaxel nanocrystals
  • Figure 21 shows the in vitro anti-HUVEC and U87 cell activity curve of pHA-VAP modified erythrocyte membrane coated with docetaxel/parthenolide nano-cocrystal
  • Figure 22 shows the volume change curve of anti-4T1 breast cancer in situ tumors with pHA-VAP modified lipid membrane coated with cabazitaxel nanocrystals
  • the figure shows the curve of tumor volume in each group of balb/c mice over time.
  • each administration group had an inhibitory effect on tumor growth.
  • the pHA-VAP modified lipid membrane coated cabazitaxel nanocrystals has the best in vivo efficacy .
  • Figure 23 shows the comparison of tumor weights of anti-4T1 breast cancer in situ tumors with pHA-VAP modified lipid membrane coated with cabazitaxel nanocrystals
  • the balb/c mice were sacrificed and the tumor tissues were taken out and weighed and statistically analyzed.
  • Figure 24 shows the anti-U87 in situ glioma survival curve of pHA-VAP modified red blood cell membrane coated with docetaxel/parthenolide nano co-crystal
  • Co-crystal with PBS (median survival time 38 days), free docetaxel/parthenolide (median survival time 40.5 days), red blood cell membrane-coated docetaxel/parthenolide nano co-crystal (median survival time Compared with pHA-VAP modified erythrocyte membrane-coated docetaxel/parthenolide nano-cocrystal mice, the survival time (median survival period of 77 days) was significantly prolonged (***p ⁇ 0.001) .
  • Figure 25 shows the inhibitory effect of pHA-VAP modified lipid membrane coated cabazitaxel nanocrystals on tumor cell apoptosis and angiogenesis in 4T1 breast cancer in situ
  • the picture shows two kinds of cells with free cabazitaxel, cabazitaxel nanocrystals, lipid membrane-coated cabazitaxel nanocrystals, and pHA-VAP modified lipid membrane-coated cabazitaxel nanocrystals to inhibit 4T1 in situ tumor growth
  • Blood vessels (CD31 staining) are brownish red or tan, and apoptotic cells (TUNEL staining) are green.
  • Figure 26 shows the effect of pHA-VAP modified red blood cell membrane-coated docetaxel/parthenolide nano-cocrystal on tumor cell apoptosis, angiogenesis inhibition and tumor stem cell killing in U87 in situ glioma
  • the picture shows the TUNEL staining (green), CD31 staining (red) of neovascularization and CD133 staining (red) of apoptotic tumor cells at the site of U87 tumor in situ.
  • the blue is the nuclear DAPI staining.
  • the specific technology or condition is not indicated in the embodiment, it shall be carried out according to the technology or condition described in the literature in the field, or according to the product specification.
  • the reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased through formal channels.
  • pHA-VAP polypeptide (amino acid sequence of p-hydroxybenzoic acid-Ahx-Cys-pavrtns; uppercase letters indicate L-configuration amino acids, lowercase letters indicate D-configuration amino acids).
  • N-hydroxysuccinimide NHS
  • graft the VAP peptide synthesized by Boc-protected solid-phase peptide synthesis to cys(trt)-acp-4-tert-butyl benzoic acid 95% PHA-VAP-Cys is obtained by TFA deprotection, and the crude polypeptide is separated and purified with an acetonitrile/water (containing 0.1% TFA) system.
  • HPLC and ESI-MS characterize the purity and molecular weight (Mw) of pHA-VAP-Cys. See attached Figure 1 for the HPLC spectrum and mass spectrum of pHA-VAP-Cys.
  • the pHA-VAP-DOTA-Gd was prepared as an example of the pHA-VAP-imaging agent. 0.1 mmol of pHA-VAP was dissolved in pH 7 phosphate buffer, 0.1 mmol of MAL-DOTA was added and stirred for 1 h, and the reaction solution was purified by liquid phase preparation and freeze-dried to obtain pHA-VAP-DOTA. The lyophilized pHA-VAP-DOTA was dissolved in an aqueous ammonium acetate solution, then GdCl 3 ⁇ 6H 2 O was added, and the mixture was stirred at room temperature for 1 h. The reaction solution was purified by preparative chromatography and lyophilized to obtain pHA-VAP-DOTA-Gd. The purity and molecular weight (Mw) of pHA-VAP-DOTA-Gd were characterized by HPLC and ESI-MS.
  • the brains of 4-week-old SD rats were decapitated, and the cerebral cortex was quickly separated in pre-cooled D-Hanks solution. After rolling off the meninges and large blood vessels in the brain, they were cut into pieces, and collagenase and DNase were added and digested at 37°C for 90 minutes Centrifuge at 1000 rpm for 8 minutes, discard the supernatant, and transfer to 20% BSA in DMEM solution, centrifuge at 1000 g/min at 4°C for 20 minutes, discard the upper and middle layer of liquid, and transfer the bottom capillaries to DMEM culture medium.
  • VEC cells umbilical vein endothelial cells
  • DMEM culture medium containing 10% fetal bovine serum.
  • 1 ⁇ 10 5 cells per well were inoculated in a 12-well culture plate with a volume of 1 mL per well. The culture plate was moved into a carbon dioxide incubator and incubated at 37° C., 5% CO 2 and saturated humidity for 24 hours. The same experiment was followed. See Figure 5 for the results of flow cytometry analysis.
  • U87 cells in the logarithmic growth phase were resuspended in an appropriate amount of PBS solution at a cell concentration of 1.3 ⁇ 10 8 /mL.
  • Nude mice were anesthetized by intraperitoneal injection of 8% chloral hydrate solution, fixed on the brain stereotaxic device, and 5 ⁇ L of U87 cell suspension was injected into the brain striatum to construct a U87 in situ glioma model.
  • the same dose of fluorescein-labeled pHA-VAP polypeptide was injected into the tail vein, and the mice were sacrificed at 30 min, 1, 4, and 24 h after injection. Blood, heart, liver, spleen, lung, kidney, brain, and tumor were taken and weighed. , Add 1mL of distilled water, homogenize the tissue, measure by a microplate reader, and quantify the fluorescence. The results are shown in Figure 9.
  • the pHA-VAP-doxorubicin complex was prepared as an example of the targeting molecule to connect drugs containing ketone or aldehyde groups. 9.4 mg of thiolated pHA-VAP was dissolved in 3 mL of phosphate buffer (0.1 mM, pH 7.4), and an equimolar amount of doxorubicin 6-maleimide hexylhydrazine derivative was added, and reacted for 1 h at room temperature in the dark. The reaction solution is purified by preparative liquid phase and freeze-dried to obtain the pHA-VAP-doxorubicin complex. See Figure 10 for the HPLC spectrum and mass spectrum.
  • the pHA-VAP-paclitaxel complex is used as an example in which the targeting molecule is connected to a drug containing a hydroxyl group or an amino group via a disulfide bond.
  • Paclitaxel 3-(2-pyridinedimercapto)propionic acid derivative was dissolved in 5mL DMF, 1.5 times the molar amount of pHA-VAP-Cys was dissolved in PBS/DMF, and the pH of the solution was maintained at 4 ⁇ 5.
  • Paclitaxel 3-(2 -Pyridinedimercapto)propionic acid derivative is added dropwise to the sulfhydryl polypeptide solution, reacted at room temperature for 6 hours, and purified by preparation liquid phase and freeze-dried to obtain a polypeptide-paclitaxel complex.
  • the pHA-VAP-bortezomib complex is used as an example of the targeting molecule to connect drugs containing boronic acid groups.
  • amino acids are sequentially connected to the resin. After all the amino acid residues of the polypeptide are connected, trifluoroacetic acid removes the Boc protection of the nitrogen terminal.
  • DMF solution containing 3 times the molar amount of succinic anhydride and DIEA, and react at room temperature for 30 minutes. After washing the resin, add 5 times the molar amount of trimethylchlorosilane to protect the dopamine, and use HBTU/DIEA as the condensing agent, and react at room temperature for 1 hour.
  • the resin was cut with HF and purified by preparative HPLC to obtain polypeptide-dopamine derivatives.
  • the pHA-VAP-dopamine derivative and bortezomib are mixed at a molar ratio of 1:1 to obtain a pHA-VAP-bortezomib complex.
  • the pHA-VAP-PMI fusion polypeptide is used as an example of targeting molecules to connect polypeptide drugs.
  • the specific method is: after determining the pHA-VAP-PMI polypeptide sequence, the amino acids are sequentially connected according to the same method as the preparation of pHA-VAP, and the pHA-VAP-PMI fusion is obtained after HF cutting and purification. Peptides.
  • Example 6 In vitro pharmacodynamic test of targeted molecular modification nano drug delivery system
  • U87 cells in the logarithmic growth phase were digested with 0.25% trypsin and pipetted into single cells.
  • the cells were suspended in DMEM medium containing 10% FBS and seeded on a 96-well cell culture plate at a density of 3000 cells per well.
  • the volume of each well is 0.2 mL, and three wells are left as blank holes with cell-free culture medium, and cultured in a carbon dioxide incubator for 24 hours.
  • mice On the 7th day after the establishment of the U87 tumor in situ animal model, the mice were randomly divided into 8 groups with 10 rats in each group. DOX, pHA-VAP-DOX, pHA-VAP, physiological saline and doxorubicin were injected into the tail vein every two days. The total dose is 10mg/kg, 20mg/kg, 40mg/kg, and the peptide is converted into the amount of peptide in the pHA-VAP-DOX 40mg/kg complex. The survival time of each group of nude mice was recorded, and the survival curve was drawn (Figure 13).
  • pHA-VAP-PEG-DSPE is synthesized by the reaction between the free sulfhydryl group of pHA-VAP-Cys and the maleimide contained in Mal-PEG-DSPE.
  • cabazitaxel and an appropriate amount of surfactant TPGS into a 25 ml eggplant-shaped bottle, add an appropriate amount of dichloromethane to dissolve and then form a film for hydration to prepare cabazitaxel nanocrystals with good dispersibility.
  • the membrane protein concentration was measured with BCA kit; 4mg docetaxel, 1.2mg parthenolide and appropriate amount Surfactant F127 was placed in a 25mL eggplant-shaped bottle, and an appropriate amount of methanol was added to dissolve it to form a film for hydration, and a well-dispersed docetaxel/parthenolide nano co-crystal was prepared; 40 ⁇ L of streptavidin-PEG 3400- The PBS solution of DSPE (5 mg/mL) and the erythrocyte membrane vesicles obtained from 100 ⁇ L of whole blood were incubated in a 37°C water bath for 30 minutes to obtain streptavidin-erythrocyte membrane vesicles.
  • streptavidin-erythrocyte membrane vesicles are mixed with docetaxel/parthenolide nano-cocrystals and then sonicated to obtain a nano-drug delivery system with surface-modified streptavidin-coated erythrocyte membranes nanocrystals, Then add 100 ⁇ L of biotin-PEG 2000- VAP-pHA in PBS solution (0.1mg/mL), and incubate in a water bath at 37°C for 10 minutes to obtain pHA-VAP modified red blood cell membrane coated with docetaxel/parthenolide nano co Crystal's nano drug delivery system. See Figure 15 for the characterization of particle size and potential.
  • HUVEC and 4T1 cell plating methods are as above.
  • Two kinds of cells with corresponding concentrations are prepared with 10% FBS-containing DMEM medium and respectively combined with free cabazitaxel, cabazitaxel nanocrystals, lipid membrane-coated cabazitaxel nanocrystals, pHA-
  • the VAP modified lipid membrane is coated with cabazitaxel nanocrystals, the DMEM culture solution in the 12-well plate is aspirated, the drug solution is added, and the solution is incubated at 37°C for 4 hours, and the drug solution is discarded.
  • BCEC cell extraction method and HUVEC, U87 cell plating method are as above, use 10% FBS-containing DMEM medium to prepare corresponding concentrations of free docetaxel/parthenolide, docetaxel/parthenolide nano co-crystals, Red blood cell membrane coated with docetaxel/parthenolide nano co-crystal, pHA-VAP modified red blood cell membrane coated with docetaxel/parthenolide nano co-crystal, aspirate the DMEM medium in the 12-well plate, and add Incubate the drug solution at 37°C for 1 h, and discard the drug solution.
  • Example 10 In vivo targeting verification of targeted molecule modified nano drug delivery system
  • Example 11 In vitro pharmacodynamic test of targeted molecule modified nano drug delivery system
  • Example 12 In vivo pharmacodynamic test of targeted molecular modification nano drug delivery system
  • the constructed 4T1 orthotopic breast cancer animal model When the tumor size was 100mm 3 , the group was divided into groups, and PBS, free cabazitaxel, cabazitaxel nanocrystals, lipid membrane coated cabazitaxel nanocrystals, and pHA-VAP modified lipid membrane coated cabazitaxel nanocrystals were injected into the tail vein. crystal.
  • the total dose of cabazitaxel in the administration group was 16 mg/kg, divided into 4 times, with an interval of two days between each administration. On the next day, the long diameter (a) and short diameter (b) of the tumor were measured with vernier calipers. Calculate the tumor volume of each group of balb/c mice according to the formula, draw the curve of tumor volume change over time, and calculate the statistical difference of each group. Calculate the tumor volume and draw the growth inhibition curve according to the following formula ( Figure 22):
  • V tumor volume 0.5 (a ⁇ b 2 )
  • Tumor-bearing balb/c mice were sacrificed on the 2nd day after the completion of the administration, the tumor tissues were taken out, fixed, paraffin sections or frozen sections were made, and the inhibition of neovascularization was detected by CD31 staining, or the promotion of apoptosis was detected by TUNEL staining .
  • the results are shown in Figure 25.
  • Tumor-bearing nude mice were sacrificed on the 10th day after the completion of the administration, the tumor tissues were taken out, fixed, and frozen sections were made to detect the promotion of apoptosis, the inhibition of neovascularization and the killing of tumor stem cells by TUNEL staining, CD31 and CD133 antibody staining. The results are shown in Figure 26.

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Abstract

本发明属于药学领域，披露了一种具有靶向脑毛细血管内皮细胞(跨血-脑屏障)、肿瘤新生血管内皮细胞(跨血-肿瘤屏障)、肿瘤拟态血管、肿瘤细胞和肿瘤干细胞的全过程靶向分子，以及其修饰的药物复合物、纳米载药系统在肿瘤诊断和靶向治疗中的用途。本发明的全过程靶向分子、其药物复合物及其构建的纳米载药系统，可将所携带的影像分子、治疗药物、纳米载药系统靶向递送至脑内肿瘤组织，或靶向递送至具备脑转移特征的外周肿瘤组织。

Description

一种全过程靶向分子及其药物复合物在构建递药系统中的应用
相关申请的交叉引用
本申请要求2020年01月23日提交的第CN202010078153.3号中国发明专利申请的优先权，所述申请以引用的方式整体并入本文。
技术领域
本发明属药学领域，涉及具有靶向脑毛细血管内皮细胞(跨血-脑屏障)、肿瘤新生血管内皮细胞(跨血-肿瘤屏障)、肿瘤拟态血管、肿瘤细胞和肿瘤干细胞的全过程靶向分子，以及其修饰的药物复合物、载药系统在肿瘤诊断和靶向治疗中的用途。本发明具体涉及全过程靶向分子及其修饰的影像分子和治疗药物复合物、修饰的靶向功能载体材料及其所构建的脂质体、聚合物胶束、聚合物圆盘、纳米粒、生物膜包被纳米制剂等纳米递药系统，及其在脑部肿瘤或具有脑转移特征的外周肿瘤诊断和靶向治疗中的应用。
背景技术
肿瘤是严重威胁人类生命和健康的疾病，死亡率高居所有疾病死亡率前位。其中脑原发瘤发病率和死亡率均处于我国肿瘤排行榜的前10位，而脑胶质瘤占了脑原发瘤的45％左右，其患者的中位生存期小于16个月，危害性极大。药物化疗仍然是脑肿瘤治疗的主要手段，但存在对肿瘤组织选择性差、毒性大、治疗窗窄、易产生多药耐药等缺陷，且化疗药物也极为有限。近年来，主动靶向递药成为了提高脑肿瘤组织靶向效率的重要策略。主动靶向策略主要针对脑肿瘤组织中高表达的受体或转运体，利用与该受体或转运体具有特异性识别、结合能力的对应配体，将药物或载药系统递送至脑肿瘤组织或细胞中。目前的配体大多数只是针对某一受体或转运体和某一细胞的靶向，然而脑肿瘤组织不只有肿瘤细胞，还有肿瘤干细胞、肿瘤拟态血管、脑毛细血管及其血-脑屏障(BBB)、肿瘤新生血管及其血-肿瘤屏障(BTB)等。在脑肿瘤发生早期，BBB仍然保持完整并限制着药物进入脑部，使约98％小分子化疗药物和几乎100％蛋白类等大分子药物无法透过BBB进入脑内，导致药物治疗几乎无效；随着肿瘤的发生发展，肿瘤新生血管生成，但脑肿瘤新生血管较外周肿瘤相对致密且通透性差，所形成的BTB成为药物递送的主要障碍，且在脑胶质瘤浸润区依然存在BBB也阻碍药物转运；同时，脑肿瘤干细胞具有自我更新、增殖和高致瘤性的特点，尽管其在肿瘤组织中数量极少，但对药物治疗表现出高度的耐受性，易导致脑胶质瘤复发。鉴于此，选择一种对BBB及BTB具有更好跨越能力、对脑肿瘤具有更好亲和能力的靶向分子至关重要。
基于现有技术现状，本申请发明人进一步将已有靶向分子进行了改造，利用分子融合原理，将脑靶向分子与肿瘤靶向分子共价连接成全过程靶向分子，使其拥有对脑部肿瘤或具有脑转移特征外周肿瘤生长发展全过程靶向功能。同时，构建全过程靶向分子修饰的影像分子和治疗药物复合物、修饰的靶向功能载体材料及其所构建的载药系统，从而更有效地发挥对脑部肿瘤或具有脑转移特征外周肿瘤的影像诊断和靶向治疗作用。
发明内容
本发明目的是，提供一种具有靶向脑毛细血管内皮细胞(跨BBB)、肿瘤新生血管内皮细胞(跨BTB)、肿瘤拟态血管、肿瘤细胞和肿瘤干细胞的全过程靶向分子，其由脑靶向分子与肿瘤靶向分子共价连接形成。用全过程靶向分子修饰影像分子、治疗药物、高分子载体材料，构建靶向分子-药物复合物、靶向分子修饰的纳米载药系统，以提高药物对脑、脑部肿瘤或具有脑转移特征外周肿瘤的靶向诊疗效果。
本发明的第一方面提供了一种全过程靶向分子，所述全过程靶向分子由脑靶向分子与肿瘤靶向分子两部分分子共价连接形成，可介导影像分子、治疗药物和纳米载药系统实现靶向递送；
所述全过程靶向分子由具有跨血-脑屏障与跨血-肿瘤屏障并靶向肿瘤拟态血管和肿瘤细胞及其干细胞的小分子、多肽分子或蛋白分子等靶向分子组合而成；
优选地，所述全过程靶向分子可靶向脑毛细血管内皮细胞并跨血-脑屏障、靶向肿瘤新生血管内皮细胞并跨血-肿瘤屏障，同时靶向肿瘤拟态血管、肿瘤细胞和肿瘤干细胞；
更优选地，所述全过程靶向分子可介导影像分子、治疗药物和纳米载药系统跨血-脑屏障和血-肿瘤屏障，靶向肿瘤拟态血管、肿瘤细胞和/或肿瘤干细胞，实现影像分子、治疗药物和纳米载药系统对脑部肿瘤和具备脑部转移特征外周肿瘤的靶向递送。
根据本发明第一方面的全过程靶向分子，其中，所述的跨血-脑屏障的小分子、多肽分子或蛋白分子等靶向分子选自：对羟基苯甲酸(pHA)及其衍生物，脂肪酸如肉豆蔻酸(MC)及其衍生物，D8多肽、WSW多肽、 DWSW多肽、TGN多肽、 DTGN多肽、CDX多肽、 DCDX多肽、T7多肽及 DT7多肽及其衍生物，转铁蛋白、乳铁蛋白及其衍生物；和/或
所述的跨血-肿瘤屏障的多肽分子或蛋白分子等靶向分子选自：VAP多肽、cVAP多肽、 SVAP多肽、 DVAP多肽、A7R多肽、cA7R多肽、 DA7R多肽、RGD多肽、Stapled-RGD多肽、RW多肽、mn多肽、RAP12多肽及 DRAP12多肽及其衍生物。
根据本发明第一方面的全过程靶向分子，其中，在所述的全过程靶向分子中可引入活性官能团构建其修饰的影像分子复合物、治疗药物复合物和靶向功能载体材料及其纳米载药系统。其中，在全过程靶向分子上引入影像分子X，制得全过程靶向分子-X复合物；在全过程靶向分子上引入治疗药物分子Y，制得全过程靶向分子-Y复合物；在全过程靶 向分子上引入聚乙二醇-Z复合物，制得靶向功能载体材料全过程靶向分子-聚乙二醇-Z复合物，其中Z是亲脂性材料分子或亲脂性药物分子或亲水性配体分子，并进一步制备成全过程靶向分子修饰在表面的纳米载药系统。
本发明的第二方面提供了一种药物复合物，所述药物复合物由第一方面所述的全过程靶向分子与影像分子、治疗药物组成；
优选地，在所述全过程靶向分子中引入活性官能团构建其修饰的影像分子复合物、治疗药物复合物；其中，在全过程靶向分子上引入影像分子X，制得全过程靶向分子-X复合物；在全过程靶向分子上引入治疗药物分子Y，制得全过程靶向分子-Y复合物。
根据本发明第一方面的全过程靶向分子或第二方面的药物复合物，其中，所述的全过程靶向分子-X复合物中，X选自以下一种或多种：光学影像分子、磁共振影像剂、放射性核素影像剂，用于脑部肿瘤或外周肿瘤的影像诊断和示踪；
优选地，所述光学影像分子选自以下一种或多种：荧光探针分子FITC、FAM、6-TET、5-TAMRA、HEX、6-JOE，近红外染料分子Cy3、Cy3.5、Cy5、Cy5.5、Cy7、IR783、IR820、DiR、DiD、BIDIPY630/650-X、BIDIPY650/665-X、BIDIPY665/676、TO-PRO-3、TO-PRO-5，化学发光物质分子luminol、isoluminol、AMPPD、CSPD、CDP-star、光泽精，拉曼探针分子；
优选地，所述磁共振影像剂为Gd磁共振物质的螯合物；和/或
优选地，放射性核素影像剂选自以下一种或多种： 18F、 32P、 35S、 64Cu、 67/68Ga、 75Se、 89Zr、 86Y、 99mTc、 111/111mIn、 123/125I、 177Lu、 149/161Tb影像用放射性核素的螯合物；
更优选地，所述的磁共振影像剂和放射性核素影像剂，由双功能螯合剂和磁共振影像用物质或影像用放射性核素组成。其中，所述双功能螯合剂选自以下一种或多种：DOTA、DOTAGA、NOTA、NOTAGA、NODA、DTPA、TETA、CB-TE2A、Cyclam、DFO、MAG3、EC、EDTA、DADT、HYNIC、CE-DTS、NS3，用于螯合磁共振影像用物质或放射性影像用核素。
根据本发明第一方面的全过程靶向分子或第二方面的药物复合物，其中，所述的全过程靶向分子-Y复合物中，Y选自以下一种或多种：肿瘤化疗药物中的蒽环类药物、紫杉烷类药物、喜树碱类药物、长春碱类药物、蛋白酶体抑制剂类药物、抗肿瘤干细胞类药物、分子靶向类药物、多肽类药物、抗体类药物、治疗用放射性核素螯合物；
优选地，所述肿瘤化疗药物中的蒽环类药物选自以下一种或多种：阿霉素、表阿霉素；
优选地，所述紫衫烷类药物选自以下一种或多种：紫杉醇、多西他赛、卡巴他赛；
优选地，所述喜树碱类药物选自以下一种或多种：喜树碱、羟基喜树碱、9-硝基喜树碱、伊立替康；
优选地，所述长春碱类药物选自以下一种或多种：长春碱、长春新碱；
优选地，所述蛋白酶体抑制剂类药物为硼替佐米；
优选地，所述抗肿瘤干细胞类药物选自小白菊内酯及其衍生物；
优选地，所述分子靶向类药物选自以下一种或多种：曲美替尼、伊马替尼、尼罗替尼、达沙替尼、依维莫司、厄洛替尼、舒尼替尼、索拉非尼、依鲁替尼、瑞戈非尼、威罗非尼、奥拉帕尼；和/或
优选地，所述多肽类药物选自以下一种或多种：p53激活肽、蜂毒肽、蝎毒肽、抗菌肽；
优选地，所述抗体类药物选自以下一种或多种：利妥昔单抗、贝伐珠单抗、曲妥珠单抗、西妥昔单抗、帕妥珠单抗、伊匹单抗、纳武单抗、PD-L1单抗，及其通过基因工程改造的抗体片段组合包括Fab片段、单域抗体、Fv片段、单链抗体、双价小分子抗体、微抗体、纳米抗体；和/或
优选地，所述治疗用放射性核素螯合物选自以下一种或多种： 90Y、 131I、 152/155Tb、 153Sm、 177Lu、 186/188Re、 211At、 212/213Bi、 212Pb、 225Ac、 227Th治疗用放射性核素的螯合物；
更优选地，所述的治疗用放射性核素螯合物由双功能螯合剂和治疗用放射性核素组成，其中双功能螯合剂选自DOTA、DOTAGA、NOTA、NOTAGA、NODA、DTPA、TETA、CB-TE2A、Cyclam、DFO、MAG3、EC、EDTA、DADT、HYNIC、CE-DTS、NS3。
本发明的第三方面提供了一种靶向功能载体材料，所述靶向功能载体材料由第一方面所述的全过程靶向分子与高分子载体材料组成，可用于制备全过程靶向分子修饰在表面的纳米载药系统；
优选地，在所述全过程靶向分子中引入活性官能团构建其修饰的高分子载体材料复合物；其中，在全过程靶向分子上引入聚乙二醇-Z复合物分子，制得靶向功能载体材料全过程靶向分子-聚乙二醇-Z复合物，其中Z是亲脂性材料分子或亲脂性药物分子或亲水性配体分子。
根据本发明第一方面的全过程靶向分子或第三方面的靶向功能载体材料，其中，所述的亲脂性材料分子Z选自以下一种或多种：磷脂、聚乳酸(PLA)、乳酸羟基乙酸共聚物(PLGA)、聚己内酯(PCL)；
所述亲脂性药物分子Z选自以下一种或多种：蒽环类药物、紫衫烷类药物、喜树碱类药物、长春碱类药物、蛋白酶体抑制剂类药物、内酯类抗肿瘤药物；和/或
所述亲水性配体分子Z是生物素。
所述靶向功能载体材料可进一步制备成全过程靶向分子修饰在表面的纳米载药系统。
本发明的第四方面提供了第一方面的全过程靶向分子或第三方面的靶向功能载体材料在制备纳米载药系统中的应用，其中，当所述的全过程靶向分子上引入聚乙二醇-Z复合物中的Z为亲脂性材料分子磷脂时，所述靶向功能载体材料用于制备脂质体载药系统、胶束载药系统或圆盘载药系统；
当所述的全过程靶向分子上引入聚乙二醇-Z复合物中的Z为亲脂性材料分子聚乳酸复合物、乳酸羟基乙酸共聚物和/或聚己内酯时，所述靶向功能载体材料用于制备胶束载药系统或纳米粒载药系统；和/或
当所述的全过程靶向分子上引入聚乙二醇-Z复合物中的Z为亲水性材料分子生物素时，所述靶向功能载体材料可用于制备生物膜包被纳米载药系统。
根据本发明第四方面的应用，其中，所述脂质体载药系统、胶束载药系统、圆盘载药系统、纳米粒载药系统和生物膜包被纳米载药系统包载诊断药物，用于脑部肿瘤或外周肿瘤的影像诊断和示踪；
优选地，所述诊断药物选自以下一种或多种：光学影像物质、磁共振影像剂、放射性核素影像剂；
更优选地，所述光学影像物质选自以下一种或多种：荧光探针香豆素6、FITC、FAM、DiI、Rhodamine B、Rhodamine 6G、5-TAMRA、6-TET、HEX、6-JOE，近红外染料Cy3、Cy3.5、Cy5、Cy5.5、Cy7、IR783、IR820、DiR、DiD、Alexa Fluor 680、BIDIPY630/650-X、BIDIPY650/665-X、BIDIPY665/676、TO-PRO-3、TO-PRO-5，化学发光物质luminol、isoluminol、AMPPD、CSPD、CDP-star、光泽精，拉曼探针；
更优选地，所述磁共振影像剂为Gd等磁共振影像物质的螯合物；和/或
更优选地，所述放射性核素影像剂选自以下一种或多种： 18F、 32P、 35S、 64Cu、 67/68Ga、 75Se、 89Zr、 86Y、 99mTc、 111/111mIn、 123/125I、 177Lu、 149/161Tb影像用放射性核素的螯合物。
根据本发明第四方面的应用，其中，所述脂质体载药系统、胶束载药系统、圆盘载药系统、纳米粒载药系统和生物膜包被纳米载药系统用于包载肿瘤治疗药物，用于脑部肿瘤或具有脑转移特征外周肿瘤的靶向治疗；
优选地，所述肿瘤治疗药物选自以下一种或多种：化疗中的蒽环类药物、紫杉烷类药物、喜树碱类药物、长春碱类药物、铂类药物、蛋白酶体抑制剂类药物、抗肿瘤干细胞类药物、分子靶向药物、多肽类药物、抗体类药物、治疗用放射性核素螯合物；
更优选地，所述化疗中的蒽环类药物选自以下一种或多种：阿霉素、表阿霉素；
更优选地，紫杉烷类药物选自以下一种或多种：紫杉醇、多西他赛、卡巴他赛；
更优选地，喜树碱类药物选自以下一种或多种：喜树碱、羟基喜树碱、9-硝基喜树碱、伊立替康；
更优选地，长春碱类药物选自以下一种或多种：长春碱、长春新碱；
更优选地，铂类药物选自以下一种或多种：顺铂、卡铂、奥沙利铂、米铂；
更优选地，蛋白酶体抑制剂类药物选自以下一种或多种：硼替佐米、卡非佐米；
更优选地，抗肿瘤干细胞类药物选自以下一种或多种：小白菊内酯及其衍生物；
更优选地，分子靶向药物选自以下一种或多种：曲美替尼、伊马替尼、尼罗替尼、达沙替尼、依维莫司、厄洛替尼、舒尼替尼、索拉非尼、依鲁替尼、瑞戈非尼、威罗非尼、奥拉帕尼；
更优选地，多肽类药物选自以下一种或多种：p53激活肽、蜂毒肽、蝎毒肽和抗菌肽；
更优选地，抗体类药物选自以下一种或多种：利妥昔单抗、贝伐珠单抗、曲妥珠单抗、西妥昔单抗、帕妥珠单抗、伊匹单抗、纳武单抗、PD-L1单抗，及其通过基因工程手段改造的抗体片段组合包括Fab片段、单域抗体、Fv片段、单链抗体、双价小分子抗体、微抗体、纳米抗体；和/或
更优选地，治疗用放射性核素螯合物选自以下一种或多种： 90Y、 131I、 152/155Tb、 153Sm、 177Lu、 186/188Re、 211At、 212/213Bi、 212Pb、 225Ac、 227Th治疗用放射性核素的螯合物。
本发明的第五方面提供了一种纳米载药系统，所述纳米载药系统包括第一方面的全过程靶向分子或第三方面的靶向功能载体材料，且所述纳米载药系统是通过全过程靶向分子上引入聚乙二醇-Z复合物构建而成，其中Z是亲脂性材料分子或亲脂性药物分子或亲水性配体分子；
所述的亲脂性材料分子Z选自以下一种或多种：磷脂、聚乳酸(PLA)、乳酸羟基乙酸共聚物(PLGA)、聚己内酯(PCL)；
所述亲脂性药物分子Z选自以下一种或多种：蒽环类药物、紫衫烷类药物、喜树碱类药物、长春碱类药物、蛋白酶体抑制剂类药物、内酯类抗肿瘤药物；和/或
所述亲水性配体分子Z是生物素。
根据本发明第五方面的纳米载药系统，其中，当所述亲脂性材料分子Z为磷脂时，所述纳米载药系统为脂质体载药系统、胶束载药系统或圆盘载药系统；
当所述亲脂性材料分子Z为聚乳酸、乳酸羟基乙酸共聚物和/或聚己内酯时，所述纳米载药系统为胶束载药系统或纳米粒载药系统；和/或
当所述亲水性配体分子Z为生物素时，所述纳米载药系统为生物膜包被纳米载药系统。
根据本发明第五方面的纳米载药系统，其中，所述的脂质体载药系统、胶束载药系统、圆盘载药系统、纳米粒载药系统和生物膜包被纳米载药系统包载诊断药物，用于脑部肿瘤或外周肿瘤的影像诊断和示踪；
优选地，所述诊断药物选自以下一种或多种：光学影像物质、磁共振影像剂、放射性核素影像剂；
更优选地，所述光学影像物质选自以下一种或多种：荧光探针香豆素6、FITC、FAM、DiI、Rhodamine B、Rhodamine 6G、5-TAMRA、6-TET、HEX、6-JOE，近红外染料Cy3、Cy3.5、Cy5、Cy5.5、Cy7、IR783、IR820、DiR、DiD、Alexa Fluor 680、BIDIPY630/650-X、BIDIPY650/665-X、BIDIPY665/676、TO-PRO-3、TO-PRO-5，化学发光物质luminol、isoluminol、AMPPD、CSPD、CDP-star、光泽精、拉曼探针；
更优选地，所述磁共振影像剂为Gd等磁共振影像物质的螯合物；和/或
更优选地，所述放射性核素影像剂选自以下一种或多种： 18F、 32P、 35S、 64Cu、 67/68Ga、 75Se、 89Zr、 86Y、 99mTc、 111/111mIn、 123/125I、 177Lu、 149/161Tb影像用放射性核素的螯合物。
根据本发明第五方面的纳米载药系统，其中，所述的脂质体载药系统、胶束载药系统、圆盘载药系统、纳米粒载药系统和生物膜包被纳米载药系统包载肿瘤治疗药物，用于脑部肿瘤或具有脑转移特征外周肿瘤的靶向治疗；
优选地，所述肿瘤治疗药物选自以下一种或多种：化疗中的蒽环类药物、紫杉烷类药物、喜树碱类药物、长春碱类药物、铂类药物、蛋白酶体抑制剂类药物、抗肿瘤干细胞类药物、分子靶向药物、多肽类药物、抗体类药物、治疗用放射性核素螯合物；
更优选地，所述化疗中的蒽环类药物选自以下一种或多种：阿霉素、表阿霉素；
更优选地，紫杉烷类药物选自以下一种或多种：紫杉醇、多西他赛、卡巴他赛；
更优选地，喜树碱类药物选自以下一种或多种：喜树碱、羟基喜树碱、9-硝基喜树碱、伊立替康；
更优选地，长春碱类药物选自以下一种或多种：长春碱、长春新碱；
更优选地，铂类药物选自以下一种或多种：顺铂、卡铂、奥沙利铂、米铂；
更优选地，蛋白酶体抑制剂类药物选自以下一种或多种：硼替佐米、卡非佐米；
更优选地，抗肿瘤干细胞类药物选自以下一种或多种：小白菊内酯及其衍生物；
更优选地，分子靶向药物选自以下一种或多种：曲美替尼、伊马替尼、尼罗替尼、达沙替尼、依维莫司、厄洛替尼、舒尼替尼、索拉非尼、依鲁替尼、瑞戈非尼、威罗非尼、奥拉帕尼；
更优选地，多肽类药物选自以下一种或多种：p53激活肽、蜂毒肽、蝎毒肽和抗菌肽；
更优选地，抗体类药物选自以下一种或多种：利妥昔单抗、贝伐珠单抗、曲妥珠单抗、西妥昔单抗、帕妥珠单抗、伊匹单抗、纳武单抗、PD-L1单抗，及其通过基因工程手段改造的抗体片段组合包括Fab片段、单域抗体、Fv片段、单链抗体、双价小分子抗体、微抗体、纳米抗体；和/或
更优选地，治疗用放射性核素螯合物选自以下一种或多种： 90Y、 131I、 152/155Tb、 153Sm、 177Lu、 186/188Re、 211At、 212/213Bi、 212Pb、 225Ac、 227Th治疗用放射性核素的螯合物。
本发明的第六方面提供了一种脑部肿瘤或具有脑转移特征外周肿瘤的诊断和/或治疗方法，所述方法包括：对有需要的受试者给予第一方面的全过程靶向分子、第二方面的药物复合物或第三方面的靶向功能载体材料，或第五方面的纳米载药系统；
优选地，所述方法是通过所述全过程靶向分子、药物复合物、靶向功能载体材料或纳米载药系统靶向脑毛细血管内皮细胞并跨血-脑屏障、靶向肿瘤新生血管内皮细胞并跨血-肿瘤屏障，同时靶向肿瘤拟态血管、肿瘤细胞和肿瘤干细胞实现的；
更优选地，所述方法是通过所述全过程靶向分子、药物复合物、靶向功能载体材料或纳米载药系统跨血-脑屏障和血-肿瘤屏障，靶向肿瘤拟态血管、肿瘤细胞和/或肿瘤干细胞， 实现影像分子、治疗药物和纳米载药系统对脑部肿瘤和具备脑转移特征外周肿瘤的靶向递送。
具体的，本发明利用分子融合原理，将脑靶向分子与肿瘤靶向分子通过共价连接制备了全过程靶向分子，使其同时具有两种分子的靶向能力，可靶向脑毛细血管内皮细胞(跨BBB)、肿瘤新生血管内皮细胞(跨BTB)、肿瘤拟态血管、肿瘤细胞和肿瘤干细胞，具有对脑部肿瘤生长发展全过程的靶向作用。
本发明所涉及的跨血-脑屏障的小分子、多肽分子或蛋白分子等靶向分子包括：对羟基苯甲酸(pHA)及其衍生物，脂肪酸特别是肉豆蔻酸(MC)及其衍生物，D8多肽、WSW多肽、 DWSW多肽、TGN多肽、 DTGN多肽、CDX多肽、 DCDX多肽、T7多肽及 DT7多肽等多肽及其衍生物，转铁蛋白、乳铁蛋白等蛋白及其衍生物。各多肽序列见说明书表1(表1-多肽氨基酸序列表)。
本发明所涉及的跨血-肿瘤屏障的多肽分子或蛋白分子等靶向分子包括：VAP多肽、cVAP多肽、 SVAP多肽、 DVAP多肽、A7R多肽、cA7R多肽、 DA7R多肽、RGD多肽、Stapled-RGD多肽、RW多肽、mn多肽、RAP12多肽及 DRAP12多肽等多肽及其衍生物。各多肽序列见说明书表1(表1-多肽氨基酸序列表)。
表1-多肽靶向分子的氨基酸序列表
Figure PCTCN2021072910-appb-000001
Figure PCTCN2021072910-appb-000002
本发明所设计的全过程靶向分子可通过在分子中引入活性官能团，构建其修饰的影像分子复合物、治疗药物复合物、靶向功能载体材料。
本发明所设计的全过程靶向分子引入半胱氨酸后，利用其分子中巯基与马来酰亚胺功能化光学影像分子(如荧光探针分子FITC、FAM、6-TET、5-TAMRA、HEX、6-JOE等，近红外染料如Cy3、Cy3.5、Cy5、Cy5.5、Cy7、IR783、IR820、DiR、DiD、BIDIPY630/650-X、BIDIPY650/665-X、BIDIPY665/676、TO-PRO-3、TO-PRO-5等，化学发光物质分子luminol、isoluminol、AMPPD、CSPD、CDP-star、光泽精等，拉曼探针分子等)反应而形成复合物。
本发明所设计的全过程靶向分子，与磁共振影像剂(如Gd等磁共振影像用物质的螯合物)、或与放射性核素影像剂(如 18F、 32P、 35S、 64Cu、 67/68Ga、 75Se、 89Zr、 86Y、 99mTc、 111/111mIn、 123/125I、 177Lu、 149/161Tb等影像用放射性核素的螯合物)、或与治疗用放射性核素螯合物(如 90Y、 131I、 152/155Tb、 153Sm、 177Lu、 186/188Re、 211At、 212/213Bi、 212Pb、 225Ac、 227Th等治疗用放射性核素的螯合物)反应而形成复合物，其中的螯合物由双功能螯合剂和磁共振影像用核素、或双功能螯合剂和影像用放射性核素、或双功能螯合剂和治疗用放射性核素所组成。螯合物中的双功能螯合剂包括DOTA、DOTAGA、NOTA、NOTAGA、NODA、DTPA、TETA、CB-TE2A、Cyclam、DFO、MAG3、EC、EDTA、DADT、HYNIC、CE-DTS、NS3等。
本发明所设计的全过程靶向分子修饰药物，包括通过马来酰亚胺己肼衍生物反应形成pH敏感腙键(涉及阿霉素、表阿霉素等含酮或醛基的药物)、或通过3-(2-吡啶二巯基)丙酸衍生物反应形成二硫键(涉及紫杉醇、多烯紫杉醇、卡巴他赛、喜树碱、羟基喜树碱、9-硝基喜树碱、伊立替康、长春新碱和长春瑞滨等含羟基或氨基的药物)、或通过多巴胺与药物中硼酸基团反应形成pH敏感硼酸脂(涉及硼替佐米等含硼酸基团的药物)、或通过固相合成直接形成酰胺键(涉及p53激活肽、蜂毒肽、蝎毒肽和抗菌肽等等多肽药物)、或通过共价或非共价连接(涉及利妥昔单抗、贝伐珠单抗、曲妥珠单抗、西妥昔单抗、帕 妥珠单抗、伊匹单抗、纳武单抗、PD-L1单抗等抗体类药物及其通过基因工程手段改造的抗体片段组合，包括Fab片段、单域抗体、Fv片段、单链抗体、双价小分子抗体、微抗体、纳米抗体等)合成的全过程靶向分子-药物复合物。
本发明所设计的全过程靶向分子引入半胱氨酸后，修饰在含马来酰亚胺功能基的聚乙二醇-二硬脂酰基磷脂酰乙醇胺(PEG-DSPE)、聚乙二醇-聚乳酸(PEG-PLA)、聚乙二醇-乳酸羟基乙酸共聚物(PEG-PLGA)、聚乙二醇-聚己内酯(PEG-PCL)等高分子载体材料上，用于全过程靶向分子修饰的脂质体、胶束、圆盘、纳米粒等纳米载药系统的构建。
本发明所设计的全过程靶向分子引入半胱氨酸后，修饰在含马来酰亚胺功能基的聚乙二醇-生物素(PEG-Biotin)等靶向材料上，用于全过程靶向分子修饰的生物膜包被纳米载药系统的构建。
本发明所设计的全过程靶向分子修饰的纳米载药系统，用于包载阿霉素、表阿霉素等蒽环类药物，包载紫杉醇、多烯紫杉醇、卡巴他赛等紫杉烷类药物，包载喜树碱、羟基喜树碱、9-硝基喜树碱、伊立替康等喜树碱类药物，包载长春新碱、长春瑞滨等长春碱类药物，包载顺铂、卡铂、奥沙利铂、米铂等铂类药物，包载硼替佐米和卡非佐米等蛋白酶体抑制剂，包载小白菊内酯等内酯类药物，包载曲美替尼、伊马替尼、尼罗替尼、达沙替尼、依维莫司、厄洛替尼、舒尼替尼、索拉非尼、依鲁替尼、瑞戈非尼、威罗非尼、奥拉帕尼等分子靶向药物，包载p53激活肽、蜂毒肽、蝎毒肽、抗菌肽等多肽药物，包载利妥昔单抗、贝伐珠单抗、曲妥珠单抗、西妥昔单抗、帕妥珠单抗、伊匹单抗、纳武单抗、PD-L1单抗等抗体类药物及其通过基因工程手段改造的抗体片段组合(包括Fab片段、单域抗体、Fv片段、单链抗体、双价小分子抗体、微抗体和纳米抗体等)，包载 90Y、 131I、 152/155Tb、 153Sm、 177Lu、 186/188Re、 211At、 212/213Bi、 212Pb、 225Ac、 227Th等治疗用放射性核素的螯合物。
本发明所设计的全过程靶向分子修饰的纳米载药系统，用于包载光学影像分子(如荧光探针分子FITC、FAM、6-TET、5-TAMRA、HEX、6-JOE等，近红外染料Cy3、Cy3.5、Cy5、Cy5.5、Cy7、IR783、IR820、DiR、DiD、BIDIPY630/650-X、BIDIPY650/665-X、BIDIPY665/676、TO-PRO-3、TO-PRO-5等，化学发光物质luminol、isoluminol、AMPPD、CSPD、CDP-star、光泽精等，拉曼探针分子)，包载磁共振影像剂(如Gd等磁共振物质的螯合物)，包载放射性核素影像剂(如 18F、 32P、 35S、 64Cu、 67/68Ga、 75Se、 89Zr、 86Y、 99mTc、 111/111mIn、 123/125I、 177Lu、 149/161Tb等影像用放射性核素的螯合物)。
本发明所设计的全过程靶向分子，用于介导药物或纳米载药系统跨越BBB和BTB，靶向肿瘤新生血管、肿瘤拟态血管、肿瘤细胞和肿瘤干细胞，用于脑、脑部肿瘤或具有脑转移特征外周肿瘤的靶向诊断和治疗。
1、pHA-VAP及其荧光标记物(pHA-VAP-Cy7)的制备
采用固相合成方法制备pHA-VAP；通过马来酰亚胺基团与巯基的Michael加成反应合成pHA-VAP-Cy7；HPLC、MS表征其结构。
2、pHA-VAP-影像剂的制备
通过马来酰亚胺基团与巯基的Michael加成反应合成了pHA-VAP-DTPA或pHA-VAP-DOTA，螯合Gd或 99mTc得pHA-VAP-DTPA-Gd或pHA-VAP-DTPA- 99mTc，或pHA-VAP-DOTA-Gd。
3、pHA-VAP体内外靶向能力的评价
考察pHA-VAP-Cy7对脑毛细血管内皮细胞(BCEC)、脐静脉内皮细胞(HUVEC)和模型肿瘤细胞(如：脑胶质瘤细胞U87)的体外亲和性。考察了pHA-VAP-DOTA-Gd被模型肿瘤细胞(HCC1806和A549细胞)的摄取。
通过正常小鼠和荷U87皮下移植瘤、U87颅内原位瘤模型裸鼠的尾静脉注射pHA-VAP-Cy7，考察其在各时间点的动物体内分布。
4、pHA-VAP-药物复合物的制备
引入半胱氨酸后的pHA-VAP与药物上马来酰亚胺己肼衍生物反应，形成含pH敏感腙键的多肽-药物复合物，其中所涉及药物包括阿霉素、表阿霉素等含酮或醛基的药物。
引入半胱氨酸后的pHA-VAP与药物上3-(2-吡啶二巯基)丙酸衍生物反应，形成含二硫键的多肽-药物复合物，其中所涉及药物包括紫杉醇、多烯紫杉醇、卡巴他赛、喜树碱、羟基喜树碱、9-硝基喜树碱、伊立替康、长春新碱、长春瑞滨等含羟基或氨基的药物。
pHA-VAP通过修饰上多巴胺进而与药物上硼酸基团反应，形成含pH敏感硼酸脂的多肽-药物复合物，其中所涉及药物包括硼替佐米等含硼酸基团的药物。
pHA-VAP通过固相合成直接与多肽药物缩合，其中所涉及药物包括p53激活肽、抗菌肽、多肽毒素等多肽药物。
pHA-VAP通过随机位点修饰(将抗体中游离氨基活化后与pHA-VAP共价连接)或定点位点修饰(通过亲和偶联作用将靶向分子与抗体非共价连接)得到靶向功能分子修饰的抗体复合物，其中所涉及药物包括利妥昔单抗、贝伐珠单抗、曲妥珠单抗、西妥昔单抗、帕妥珠单抗、伊匹单抗、纳武单抗、PD-L1单抗等抗体类药物及其通过基因工程手段改造的抗体片段组合(包括Fab片段、单域抗体、Fv片段、单链抗体、双价小分子抗体、微抗体、纳米抗体等)。
5、pHA-VAP-阿霉素复合物体内外抗肿瘤效果的评价
连接半胱氨酸后的pHA-VAP与阿霉素上马来酰亚胺己肼衍生物(MAL-DOX)缩合得的pHA-VAP-阿霉素复合物(pHA-VAP-DOX)，以MTT法考察的pHA-VAP-DOX对U87细胞和HUVEC细胞的体外生长抑制效果；通过荷U87颅内原位瘤模型裸鼠的尾静脉给药，以中位生存时间为指标评价其体内抗肿瘤效果。
6、pHA-VAP修饰纳米载药系统的构建与表征
首先制备pHA-VAP修饰高分子材料pHA-VAP-PEG-DSPE、pHA-VAP-PEG-PLA、pHA-VAP-PEG-PLGA、pHA-VAP-PEG-PCL、pHA-VAP-PEG-生物素等。通过pHA-VAP上引入半胱氨酸，其游离巯基与Mal-PEG-DSPE、Mal-PEG-PLA、Mal-PEG-PLGA、 Mal-PEG-PCL、Mal-PEG-生物素等所含马来酰亚胺反应而实现上述靶向高分子材料制备，即：将Mal-PEG-DSPE、Mal-PEG-PLA、Mal-PEG-PLGA、Mal-PEG-PCL、Mal-PEG-生物素等分别溶解在乙腈中，旋转蒸发，成膜，加入含巯基pHA-VAP的PBS(pH 8.0)反应制备得到pHA-VAP修饰的高分子材料。
然后构建pHA-VAP修饰的纳米载药系统。一定量的pHA-VAP-PEG-DSPE、mPEG-DSPE、磷脂和胆固醇，或pHA-VAP-PEG-DSPE和mPEG-DSPE，或pHA-VAP-PEG-PLA和mPEG-PLA，或pHA-VAP-PEG-PLGA和mPEG-PLGA，或pHA-VAP-PEG-PCL和mPEG-PCL，以及一定量上述药物，采用成膜水化等方法，分别构建相应的pHA-VAP修饰脂质体、胶束、圆盘、纳米粒等纳米载药系统；一定量的pHA-VAP-PEG-生物素和预先修饰亲和素的生物膜孵育后，包被载有上述药物的聚合物纳米粒、硅纳米粒、纳米凝胶、纳米晶等纳米载药系统，构建生物膜包被纳米载药系统。激光散射粒度仪表征纳米载药系统粒径及电势，透射电镜表征其形貌特征。
7、pHA-VAP修饰纳米载药系统体内外靶向能力的评价
考察BCEC细胞、肿瘤细胞(U87细胞、4T1细胞)、HUVEC细胞对包载肿瘤治疗药物的pHA-VAP修饰纳米载药系统的摄取情况。
通过荷U87颅内原位瘤模型裸鼠或荷4T1乳腺癌原位模型balb/c小鼠的尾静脉注射包载肿瘤治疗药物的pHA-VAP修饰纳米载药系统，考察其在各时间点的肿瘤内分布。
8、pHA-VAP修饰纳米载药系统体内外抗肿瘤效果的评价
以MTT法考察包载肿瘤治疗药物的pHA-VAP修饰纳米载药系统对肿瘤细胞(U87细胞、4T1细胞)和HUVEC细胞的体外生长抑制效果；通过荷U87颅内原位瘤模型裸鼠或荷4T1乳腺癌原位模型balb/c小鼠的尾静脉注射包载肿瘤治疗药物的pHA-VAP修饰纳米载药系统，以生存时间、肿瘤抑制曲线、肿瘤组织细胞凋亡、新生血管和干细胞数量等为指标评价其体内抗肿瘤效果。
利用本发明提供的方法设计制备了全过程靶向分子pHA-VAP及其修饰的药物复合物和纳米载药系统。本发明的试验结果表明：pHA-VAP同时具备pHA靶向脑毛细血管而跨BBB能力，VAP靶向肿瘤新生血管内皮细胞而跨BTB、靶向肿瘤拟态血管、肿瘤细胞和肿瘤干细胞能力，在模型动物体内具有良好的脑和肿瘤组织靶向能力并表现出更优脑部肿瘤靶向效果；pHA-VAP修饰的药物复合物及纳米载药系统显示出了良好的肿瘤靶向性能和更优的脑部肿瘤诊断和治疗效果。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1示出了pHA-VAP-Cys的HPLC和ESI-MS图谱
色谱方法：色谱柱(YMC，C18)：150×4.6mm；流动相A：水(含0.1％三氟乙酸)，流动相B：乙腈(含0.1％三氟乙酸)；洗脱程序：0-45min 5％B-65％B；流速：0.7mL/min； 柱温：40℃；检测：UV 214nm，保留时间：16min。ESI-MS：1080.4，与理论分子量相符合。其中图1A示出了ESI-MS图谱，图1B示出了HPLC图谱。
图2示出了pHA-VAP-Cy7的HPLC和ESI-MS图谱
色谱方法同上，保留时间：25min。ESI-MS：1750.6，与理论分子量相符合。其中图2A示出了ESI-MS图谱，图2B示出了HPLC图谱。
图3示出了pHA-VAP-DOTA-Gd的HPLC和ESI-MS图谱
除流动相中不含三氟乙酸外，色谱方法同上，保留时间：21min。ESI-MS：1831.8，与理论分子量相符合。
图4示出了原代脑毛细血管内皮细胞BCEC对Cy7标记pHA-VAP的摄取
图为Cy7标记的VAP和pHA-VAP与BCEC细胞孵育4h后流式细胞荧光检测定量(图4A)和定性(图4B)结果。可见BCEC细胞对pHA-VAP的摄取明显高于VAP和游离荧光素。
图5示出了脐静脉内皮细胞HUVEC对Cy7标记pHA-VAP的摄取
图为Cy7标记的VAP和pHA-VAP与HUVEC细胞孵育4h后流式细胞荧光检测定量(图5A)和定性(图5B)结果。可见U87细胞对pHA-VAP的摄取明显高于VAP和游离荧光素。
图6示出了脑胶质瘤细胞U87对Cy7标记pHA-VAP的摄取
图为Cy7标记的VAP和pHA-VAP与U87细胞孵育4h后流式细胞荧光检测定量(图6A)和定性(图6B)结果。可见U87细胞对pHA-VAP的摄取明显高于VAP和游离荧光素。
图7示出了肿瘤细胞HCC1806或A549对pHA-VAP-DOTA-Gd的摄取
图为pHA-VAP-DOTA-Gd和DOTA-Gd与HCC1806或A549细胞孵育4h后，ICP-MS测定的细胞内Gd的摄取量。可见HCC1806和A549细胞对pHA-VAP-DOTA-Gd的摄取量均明显高于未修饰组。
图8示出了Cy7标记pHA-VAP在荷U87皮下移植瘤模型小鼠体内组织分布图
由图可见，与游离荧光素和VAP-Cy7相比，4h内pHA-VAP-Cy7在皮下肿瘤中分布提高，24h后蓄积量降低。
图9示出了Cy7标记pHA-VAP在荷U87脑内原位瘤模型小鼠体内组织分布图
由图可见，与游离荧光素和VAP-Cy7相比，24h内pHA-VAP-Cy7在原位肿瘤中分布提高，说明靶向分子能跨越血-脑屏障及血-脑肿瘤屏障并显著增加药物在脑肿瘤部位的蓄积量。
图10示出了pHA-VAP-DOX的HPLC和ESI-MS图谱
色谱方法：色谱柱(YMC，C18)：150×4.6mm；流动相A：水(含0.01％甲酸)，流动相B：纯乙腈；洗脱程序：0-45min 5％B-65％B；流速：0.7mL/min；柱温：40℃；检测：UV 214nm，保留时间：17min。ESI-MS：1832.2，与理论分子量相符合。其中图 10A示出了ESI-MS图谱，图10B示出了HPLC图谱。
图11示出了pHA-VAP-DOX体外抗U87细胞活性曲线
由图可见，U87细胞分别与DOX、MAL-DOX、VAP-DOX或pHA-VAP-DOX孵育72h后，其IC 50分别为0.06、1.45、1.57和0.38μM。结果说明，pHA-VAP修饰的阿霉素体外抗肿瘤活性优于马来酰胺阿霉素和VAP修饰阿霉素。
图12示出了pHA-VAP-DOX体外抗HUVEC细胞活性曲线
由图可见，HUVEC细胞分别与DOX、MAL-DOX、VAP-DOX或pHA-VAP-DOX孵育72h后，其IC 50分别为0.20、1.50、0.70和0.30μM。结果说明，pHA-VAP修饰的阿霉素体外抗肿瘤活性优于马来酰胺阿霉素和VAP修饰阿霉素。
图13示出了pHA-VAP-DOX抗U87原位脑胶质瘤生存曲线
图为U87原位脑胶质瘤模型裸鼠的生存曲线。以模型动物中位生存期为指标，与生理盐水(中位生存期26天)、DOX(10mg、20mg、40mg中位生存期分别27、17、13天，中剂量和高剂量阿霉素毒性大而导致模型裸鼠生存期更短)、pHA-VAP(中位生存期28天)，pHA-VAP-DOX(10mg、20mg、40mg中位生存期分别29、32、40天)延长模型动物的生存时间，且剂量存在依赖性。
图14示出了pHA-VAP修饰脂质膜包被卡巴他赛纳米晶的纳米递药系统电镜照片
由图可见，卡巴他赛纳米晶(图14A)呈球形，粒径在80nm左右；pHA-VAP修饰脂质膜包被卡巴他赛纳米晶(图14B、图14C)呈球形，具有明显的核-膜结构，粒径在100nm左右。
图15示出了pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶的纳米递药系统粒径表征
由图可见，多西他赛/小白菊内酯纳米共晶粒径在130nm左右，电势为-20mV；经红细胞膜包被后粒径为140nm左右，电势为-25mV；pHA-VAP修饰对所构建的纳米递药系统粒径无明显影响，但因分子本身带正电荷，所修饰纳米递药系统电势升至-15mV。
图16示出了脐静脉内皮细胞HUVEC、乳腺癌细胞4T1对pHA-VAP修饰脂质膜包被卡巴他赛纳米晶的摄取
由图可见，HUVEC细胞(图16A)和4T1细胞(图16B)对pHA-VAP修饰脂质膜包被卡巴他赛纳米晶的摄取量明显高于游离药组、纳米晶组及无靶脂质膜包被卡巴他赛纳米晶组。
图17示出了原代脑毛细血管内皮细胞BCEC、脐静脉内皮细胞HUVEC和脑胶质瘤细胞U87对pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶的摄取
由图可见，BCEC细胞(图17A，图17B)、HUVEC细胞(图17C，图17D)和U87细胞(图17E，图17F)对pHA-VAP修饰红细胞膜包被药物纳米共晶(多西他赛和小白菊内酯)的摄取量明显高于纳米共晶组及无靶红细胞膜包被药物纳米共晶组，与游离药组相当。
图18示出了pHA-VAP修饰脂质膜包被卡巴他赛纳米晶在荷4T1乳腺癌原位瘤模型小鼠体内组织分布图
由图可知，pHA-VAP修饰可显著提升不同时间点脂质膜包被卡巴他赛纳米晶在4T1肿瘤部位的蓄积量，更好地靶向至肿瘤部位。
图19示出了pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶在荷U87原位脑胶质瘤模型小鼠体内组织分布图
由图可知，pHA-VAP修饰可显著提升不同时间点红细胞膜包被药物纳米共晶(多西他赛(图19A)和小白菊内酯(图19B))在脑胶质瘤部位的蓄积量，更好地靶向至肿瘤部位。
图20示出了pHA-VAP修饰脂质膜包被卡巴他赛纳米晶的体外抗HUVEC、4T1细胞活性曲线
由图可见，两种细胞分别与游离卡巴他赛、卡巴他赛纳米晶、脂质膜包被卡巴他赛纳米晶、pHA-VAP修饰脂质膜包被卡巴他赛纳米晶孵育48h后，显示其对4T1的IC 50分别为0.88、8.51、2.93和0.06nM(图20A)，对HUVEC细胞的IC 50分别为30.3、1.86、1.61和1.49nM(图20B)。结果表明，pHA-VAP修饰的脂质膜包被卡巴他赛纳米晶的体外抗肿瘤效果优于所有组。
图21示出了pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶的体外抗HUVEC、U87细胞活性曲线
由图可见，两种细胞分别与游离多西他赛/小白菊内酯、多西他赛/小白菊内酯纳米共晶、红细胞膜包被多西他赛/小白菊内酯纳米共晶、pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶孵育48h后，显示其对HUVEC细胞的IC 50分别为23.7、44.6、67.0和7.0nM(图21A)，对U87的IC 50分别为45.6、79.9、90.3和3.2nM(图21B)。结果说明，pHA-VAP修饰的红细胞膜包被多西他赛/小白菊内酯纳米共晶的体外抗肿瘤效果优于所有组。
图22示出了pHA-VAP修饰脂质膜包被卡巴他赛纳米晶的抗4T1乳腺癌原位瘤体积变化曲线
图为各组balb/c小鼠肿瘤体积随时间变化的曲线。与PBS组相比，各给药组对肿瘤生长均有抑制作用。无靶脂质膜包被卡巴他赛纳米晶和pHA-VAP修饰后相比具有显著性差异(n＝6)，pHA-VAP修饰脂质膜包被卡巴他赛纳米晶的体内药效最佳。
图23示出了pHA-VAP修饰脂质膜包被卡巴他赛纳米晶的抗4T1乳腺癌原位瘤瘤重比较图
将balb/c小鼠处死取出肿瘤组织后称重并进行统计分析，pHA-VAP修饰脂质膜包被卡巴他赛纳米晶组瘤重显著低于其余各组(n＝6)。
图24示出了pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶的抗U87原位脑胶质瘤生存曲线
图为各组U87原位脑胶质瘤裸鼠的生存曲线。采用单次尾静脉给药，以模型动物生存期为评价指标(n＝10)。与PBS(中位生存期38天)、游离多西他赛/小白菊内酯(中位生存期40.5天)、红细胞膜包被多西他赛/小白菊内酯纳米共晶(中位生存期41.5天)相比，pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶小鼠生存时间(中位生存期77天)极显著延长(***p<0.001)。
图25示出了pHA-VAP修饰脂质膜包被卡巴他赛纳米晶对4T1乳腺癌原位瘤内肿瘤细胞凋亡及新生血管抑制的效果
图为两种细胞分别与游离卡巴他赛、卡巴他赛纳米晶、脂质膜包被卡巴他赛纳米晶、pHA-VAP修饰脂质膜包被卡巴他赛纳米晶的抑制4T1原位瘤新生血管(上图)和促进肿瘤细胞凋亡(下图)的染色照片，其中血管(CD31染色)呈棕红色或棕褐色、凋亡细胞(TUNEL染色)呈绿色。
图26示出了pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶对U87原位脑胶质瘤内肿瘤细胞凋亡、新生血管抑制及肿瘤干细胞杀伤的效果
图为U87原位瘤部位凋亡肿瘤细胞的TUNEL染色(绿)、新生血管的CD31染色(红)及肿瘤干细胞的CD133染色(红)照片，其中蓝色为细胞核DAPI染色。由图可见，相比于其余三组，pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶可显著促进肿瘤凋亡、抑制新生血管生成并杀伤肿瘤干细胞。
实施发明的最佳方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1 靶向分子、靶向分子-Cys、靶向分子-Cy7的合成与表征
1、靶向分子和靶向分子-Cys的合成与表征
采用固相多肽合成法，设计并合成pHA-VAP多肽(氨基酸序列对羟基苯甲酸-Ahx-Cys-pavrtns；大写字母表示L构型氨基酸，小写字母表示D构型氨基酸)。
具体方法：首先以Fmoc固相多肽合成法合成Cys(Trt)-Acp-4-叔丁基苯甲酸，在PAM-Fmoc树脂上按序列依次接入氨基酸，以HBTU/DIEA为缩合剂、TFA为脱保护剂进行反应。然后用N-羟基琥珀酰亚胺(NHS)激活侧链羧基，将Boc保护固相肽合成法合成的VAP肽接枝到cys(trt)-acp-4-叔丁基苯甲酸上，95％TFA脱保护得到pHA-VAP-Cys，多肽粗品用乙腈/水(含0.1％TFA)体系分离纯化。HPLC和ESI-MS表征pHA-VAP-Cys的纯度和分子量(Mw)。pHA-VAP-Cys的HPLC图谱、质谱图见附图1。
2、靶向分子-Cy7的合成与表征
将上述步骤得到的pHA-VAP-Cys溶于0.1M的PBS溶液中(pH7.2)，取Cy7-maleimide溶于DMF，两者混合后磁力搅拌反应，HPLC监测，待pHA-VAP-Cys反应完全后停止反应，制备液相纯化，用乙腈/水(含0.1％TFA)体系分离纯化。冷冻干燥得pHA-VAP-Cy7纯品。HPLC图谱、质谱图见附图2。
实施例2 pHA-VAP-影像剂的制备
以pHA-VAP-DOTA-Gd制备作为pHA-VAP-影像剂的实施例。0.1mmol的pHA-VAP溶于pH 7的磷酸缓冲液中，加入0.1mmol的MAL-DOTA搅拌反应1h，反应液经制备液相纯化并冷冻干燥得pHA-VAP-DOTA。冻干的pHA-VAP-DOTA溶于醋酸铵水溶液中，然后加入GdCl 3·6H 2O，室温搅拌1h，反应溶液经制备色谱纯化，冻干即得到pHA-VAP-DOTA-Gd。HPLC和ESI-MS表征pHA-VAP-DOTA-Gd的纯度和分子量(Mw)，图谱见附图3。
实施例3 靶向分子的体外细胞靶向性验证
1、靶向分子对原代脑毛细血管内皮细胞BCEC的体外靶向性
4周龄SD大鼠断头后取脑，于预冷D-Hanks溶液中迅速分离得到大脑皮层，滚去脑膜和脑部大血管后剪碎，加入胶原酶和DNA酶后37℃消化90分钟，1000转/分钟离心8分钟，弃去上清，转移至20％BSA的DMEM溶液中，1000g/分钟4℃离心20分钟，弃去中上层液体，将底部微血管转移至DMEM培养液中，1000转/分钟离心5分钟，用含20％胎牛血清的DMEM培养液重悬微血管段，接种于12孔板中，37℃，5％CO 2及饱和湿度条件下培养24h，换含有嘌呤霉素的内皮专用培养液继续培养72h后，再换含有细胞生长因子的内皮专用培养液培养72h，得原代脑毛细血管内皮细胞。
用含10％FBS的DMEM培养液配制荧光浓度为5μM的荧光标记多肽溶液，将12孔板中的DMEM培养液吸出，加入药液，37℃孵育4h，弃去荧光素溶液。用PBS洗板两次，加入胰蛋白酶消化细胞，用DMEM培养液分散细胞后离心，弃上清，PBS洗涤两次，最后将每孔细胞分散于200μL PBS，流式细胞仪测定。结果见附图4。
2、靶向分子对脐静脉内皮细胞HUVEC的体外靶向性
取对数生长期的单层培养的脐静脉内皮细胞细胞(HUVEC细胞)，用0.25％胰蛋白酶消化单层培养细胞，用含10％胎牛血清的DMEM培养液配成单细胞悬液，以每孔1×10 5个细胞接种于12孔培养板中，每孔体积1mL，将培养板移入二氧化碳培养箱中，37℃、5％CO 2及饱和湿度条件下培养24h后，同上实验。流式细胞仪分析结果见附图5。
3、靶向分子对胶质瘤细胞U87的体外靶向性
取对数生长期的单层培养的胶质瘤细胞(U87细胞)，同上实验。流式细胞仪分析结果见附图6。
4、肿瘤细胞HCC1806或A549对pHA-VAP-DOTA-Gd的摄取
取对数生长期的单层培养的HCC1806或A549细胞，用0.25％胰蛋白酶消化单层培养细胞，用含10％胎牛血清的RPMI 1640培养液配成单细胞悬液，以每孔1×10 5个细胞接种于6孔培养板中，每孔体积2mL，将培养板移入二氧化碳培养箱中，37℃、5％CO2及饱和湿度条件下培养24h后，分别加入100μl浓度为2.5μmol/mL的pHA-VAP-DOTA-Gd或DOTA-Gd溶液，孵育4h，然后弃去溶液，用PBS洗板两次。每孔加入0.5mL浓硝酸，硝解20min，加入4.5mL纯化水，混合均匀后，IC-PMS测定每孔细胞中Gd的含量，结果见附图7。
实施例4 靶向分子的体内靶向性验证
1、pHA-VAP-Cys在荷U87皮下移植瘤模型裸鼠体内组织分布检测
构建U87皮下移植瘤模型。分别尾静脉注射同等剂量的荧光素标记的pHA-VAP多肽，分别在注射后30min和1、4、24h时处死老鼠，取血、心、肝、脾、肺、肾、脑和肿瘤，称重，加入1mL蒸馏水，组织匀浆，经酶标仪测量，荧光定量。结果见附图8。
2、pHA-VAP-Cys在荷U87原位脑胶质瘤模型裸鼠体内组织分布检测
将对数生长期的U87细胞重悬于适量的PBS溶液中，细胞浓度为1.3×10 8/mL。用8％水合氯醛溶液腹腔注射麻醉裸鼠，固定于脑立体定位仪上，于脑纹状体区域注射5μL的U87细胞悬液，构建成U87原位脑胶质瘤模型。分别尾静脉注射同等剂量的荧光素标记的pHA-VAP多肽，分别在注射后30min和1、4、24h时处死老鼠，取血、心、肝、脾、肺、肾、脑和肿瘤，称重，加入1mL蒸馏水，组织匀浆，经酶标仪测量，荧光定量。结果见附图9。
实施例5 靶向分子-药物复合物的制备
以pHA-VAP-阿霉素复合物制备作为靶向分子连接含酮或醛基药物的实施例。9.4mg巯基化pHA-VAP溶于磷酸盐3mL缓冲液(0.1mM，pH 7.4)，加入等摩尔量的阿霉素6-马来酰亚胺己肼衍生物，于室温避光反应1h。反应液用制备液相纯化，冷冻干燥得pHA-VAP-阿霉素复合物。HPLC图谱、质谱图见附图10。
以pHA-VAP-紫杉醇复合物作为靶向分子以二硫键连接含羟基或氨基药物的实施例。200mg紫杉醇溶于10mL氯仿中，冷却至0-5℃，先后加入39.99mg DCC及60.4mg 3-(2-吡啶二巯基)丙酸，加料完毕后，升至室温反应过夜。反应液过滤，经柱层析纯化(CHCl3/MeOH＝50:1-15:1，V/V洗脱)得紫杉醇3-(2-吡啶二巯基)丙酸衍生物。紫杉醇3-(2-吡啶二巯基)丙酸衍生物溶解在5mL DMF中，1.5倍摩尔量的pHA-VAP-Cys溶解在PBS/DMF中，溶液pH值保持4～5将紫杉醇3-(2-吡啶二巯基)丙酸衍生物滴加至巯基多肽溶液中，于室温反应6h，经制备液相纯化冻干得多肽-紫杉醇复合物。
以pHA-VAP-硼替佐米复合物作为靶向分子连接含硼酸基团药物的实施例。依照 pHA-VAP的合成在树脂上依次接入氨基酸，待多肽的所有氨基酸残基接入完毕，三氟乙酸脱去氮端的Boc保护。加入含3倍摩尔量的丁二酸酐与DIEA的DMF溶液，于室温反应30min。洗涤树脂后，加入5倍摩尔量的三甲基氯硅烷保护多巴胺，并以HBTU/DIEA为缩合剂，于室温反应1h。树脂用HF切割，并经制备型HPLC纯化得多肽-多巴胺衍生物。在pH 7.4的缓冲液中，pHA-VAP-多巴胺衍生物与硼替佐米以摩尔比1:1混合即得pHA-VAP-硼替佐咪复合物。
以pHA-VAP-PMI融合多肽作为靶向分子连接多肽药物的实施例。直接通过固相多肽合成法制得，具体方法为：确定pHA-VAP-PMI多肽序列后，按与制备pHA-VAP相同的方法依次接入氨基酸，经HF切割并纯化后得pHA-VAP-PMI融合多肽。
实施例6 靶向分子修饰纳米递药系统的体外药效学试验
1、pHA-VAP-DOX对胶质瘤细胞U87的体外药效试验
取对数生长期的U87细胞，用0.25％胰蛋白酶消化并吹打成单个细胞，细胞悬浮于含10％FBS的DMEM培养液中，以每孔3000个细胞的密度接种于96孔细胞培养板中，每孔体积0.2mL，留出三孔加不含细胞的培养液作为空白孔，二氧化碳培养箱内培养24h。用细胞培养液将各组药物依次六倍稀释。吸去96孔板内细胞培液，各孔加入200μL系列浓度的药液。每个浓度均设三复孔，留出三个仅加入培养液的孔作为对照孔。培养72h后在实验孔、对照孔和空白孔中加入MTT试剂(5mg/mL)20μL孵育4h，弃去孔内培养液，每孔加入二甲亚砜150μL，振荡使生成的蓝紫色结晶充分溶解后，用酶标仪测定各孔在490nm处的吸光度(A)，按照以下公式计算细胞存活率：
存活率＝(A 490实验孔-A 490空白孔)/(A 490对照孔-A 490空白孔)×100％
用GraphPad Prism软件将存活率对药物浓度对数值做图(附图11)，计算半数抑制浓度(IC 50)。
2、pHA-VAP-DOX对脐静脉内皮细胞HUVEC的体外药效试验
取对数生长期的HUVEC细胞，同上试验。结果见附图12。
实施例7 靶向分子修饰药物复合物的体内药效学试验
构建U87原位瘤动物模型后第7天，将鼠随机分为8组，每组10只，每隔两天尾静脉注射DOX、pHA-VAP-DOX、pHA-VAP和生理盐水，阿霉素总剂量分别为10mg/kg、20mg/kg、40mg/kg，多肽换算成pHA-VAP-DOX 40mg/kg复合物中多肽的量。记录各组裸鼠生存时间，绘制生存曲线(附图13)。
实施例8 靶向分子修饰纳米递药系统的制备及表征
1、pHA-VAP修饰脂质膜包被卡巴他赛纳米晶体的制备
pHA-VAP-PEG-DSPE通过pHA-VAP-Cys的游离巯基与Mal-PEG-DSPE所含马来酰 亚胺的反应合成。称取4mg的卡巴他赛及适量的表面活性剂TPGS于25ml茄形瓶中，加入适量二氯甲烷溶解后成膜水化，制备得到分散性良好的卡巴他赛纳米晶。将适量脂质体膜材(摩尔量比：HSPC:Chol:DSPE-PEG2000＝50:45:5)旋干，加入纳米晶溶液于65℃条件下水化磷脂膜，探头超声10min(120W)。醋酸铀负染色电镜法观察形态，结果见附图14。
2、pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶的制备
取雄性ICR小鼠全血，1000g/分钟4℃离心5分钟，弃去上层血清与白细胞层，用1×PBS洗涤下层红细胞后在0.25×PBS中4℃重悬30分钟，15000g/分钟4℃离心7分钟去除血红蛋白，所得到的浅红色红细胞膜重悬并保存于双蒸水中，以BCA试剂盒检测其膜蛋白浓度；称取4mg的多西他赛、1.2mg小白菊内酯及适量的表面活性剂F127于25mL茄型瓶中，加入适量甲醇溶解成膜水化，制备得到分散性良好的多西他赛/小白菊内酯纳米共晶；将40μL链霉亲和素-PEG 3400-DSPE的PBS溶液(5mg/mL)与从100μL全血中取得的红细胞膜囊泡在37℃水浴中孵育30分钟，得到链霉亲和素-红细胞膜囊泡。将所得链霉亲和素-红细胞膜囊泡与多西他赛/小白菊内酯纳米共晶混合后超声，得到表面修饰链霉亲和素的红细胞膜包被纳米晶的纳米递药系统，然后加入100μL生物素-PEG 2000-VAP-pHA的PBS溶液(0.1mg/mL)，37℃水浴中孵育10分钟，得到pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶的纳米递药系统。粒径及电势表征见附图15。
实施例9 靶向分子修饰纳米递药系统的体外靶向性
1、脐静脉内皮细胞HUVEC、乳腺癌细胞4T1对pHA-VAP修饰脂质膜包被卡巴他赛纳米晶体的摄取试验
HUVEC、4T1细胞铺板方法如上，用含10％FBS的DMEM培养液配制相应浓度的两种细胞分别与游离卡巴他赛、卡巴他赛纳米晶、脂质膜包被卡巴他赛纳米晶、pHA-VAP修饰脂质膜包被卡巴他赛纳米晶，将12孔板中的DMEM培养液吸出，加入药液，37℃孵育4h，弃去药液。PBS溶液洗板两次，加入胰蛋白酶消化细胞，用DMEM培养液分散细胞后计数，离心，弃上清，PBS洗涤两次，最后将每孔细胞分散于100μL PBS，超声破碎细胞后加入3倍体积甲醇沉淀蛋白，10000rpm离心10min后取上清，HPLC测定药物含量。结果见附图16。
2、原代脑毛细血管内皮细胞BCEC、脐静脉内皮细胞HUVEC、胶质瘤细胞U87对pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶的摄取试验
BCEC细胞提取方法及HUVEC、U87细胞铺板方法如上，用含10％FBS的DMEM培养液配制相应浓度的游离多西他赛/小白菊内酯、多西他赛/小白菊内酯纳米共晶、红细胞膜包被多西他赛/小白菊内酯纳米共晶、pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶，将12孔板中的DMEM培养液吸出，加入药液，37℃孵育1h，弃去药液。PBS溶液洗板两次，加入胰蛋白酶消化细胞，用DMEM培养液分散细胞后计数，离心， 弃上清，PBS洗涤两次，最后将每孔细胞分散于100μL PBS，超声破碎细胞后加入3倍体积甲醇沉淀蛋白，10000rpm离心10min后取上清，HPLC测定药物含量。结果见附图17。
实施例10 靶向分子修饰纳米递药系统的体内靶向性验证
1、pHA-VAP修饰脂质膜包被卡巴他赛纳米晶体在荷4T1原位乳腺癌模型裸鼠体内组织分布检测
构建4T1原位乳腺癌模型。分别尾静脉注射同等剂量的游离卡巴他赛、卡巴他赛纳米晶、脂质膜包被卡巴他赛纳米晶、pHA-VAP修饰脂质膜包被卡巴他赛纳米晶，分别在注射后2、12及24h时处死老鼠，取血、心、肝、脾、肺、肾、脑和肿瘤，称重，加入1mL蒸馏水，组织匀浆，萃取，HPLC定量(附图18)。
2、pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶在荷U87原位脑胶质瘤模型裸鼠体内组织分布检测
构建U87原位脑胶质瘤模型。分别尾静脉注射同等剂量的度的游离多西他赛/小白菊内酯、红细胞膜包被多西他赛/小白菊内酯纳米共晶、pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶，分别在注射后2及12h时处死老鼠，取血、心、肝、脾、肺、肾、脑及脑肿瘤，称重，加入1mL蒸馏水，组织匀浆，以甲基叔丁基醚萃取两次，挥干，甲醇复溶后进行HPLC定量(附图19)。
实施例11 靶向分子修饰纳米递药系统的体外药效学试验
1、pHA-VAP修饰脂质膜包被卡巴他赛纳米晶体的体外药效试验
取对数生长期的HUVEC细胞或4T1细胞，同上实验。结果如附图20。
2、pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶的体外药效试验
取对数生长期的HUVEC细胞或U87细胞，同上实验。结果如附图21。
实施例12 靶向分子修饰纳米递药系统的体内药效学试验
1、pHA-VAP修饰脂质膜包被卡巴他赛纳米晶体的体内药效学试验
构建的4T1原位乳腺癌动物模型。待肿瘤大小为100mm 3时分组，尾静脉分别注射PBS、游离卡巴他赛、卡巴他赛纳米晶、脂质膜包被卡巴他赛纳米晶、pHA-VAP修饰脂质膜包被卡巴他赛纳米晶。给药组的卡巴他赛总给药剂量为16mg/kg，分为4次，每次给药间隔为两天。隔天以游标卡尺测量肿瘤的长径(a)及短径(b)。根据公式计算各组balb/c小鼠肿瘤体积，绘制肿瘤体积随时间的变化曲线，计算各组统计学差异。按照以下公式计算肿瘤体积并绘制生长抑制曲线(附图22)：
V 瘤体积＝0.5(a×b 2)
给药20天后，断颈处死所有balb/c小鼠，取下皮下肿瘤称重，并计算各组统计学差异(附图23)。
2、pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶的体内药效学试验
构建U87原位脑胶质瘤动物模型。种瘤10天后分别尾静脉注射PBS(pH 7.4)、度的游离多西他赛/小白菊内酯、红细胞膜包被多西他赛/小白菊内酯纳米共晶、pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶。多西他赛总给药剂量为25mg/kg，小白菊内酯给药总剂量为6mg/kg，单次给药。记录裸鼠生存时间(附图24)。
3、pHA-VAP修饰脂质膜包被卡巴他赛纳米晶体的促凋亡和新生血管抑制检测
荷瘤balb/c小鼠在给药完成后的第2天，处死取出瘤组织进行固定，作石蜡切片或作冰冻切片，通过CD31染色检测新生血管抑制情况，或通过TUNEL染色检测凋亡促进情况。结果见附图25。
4、pHA-VAP修饰红细胞膜包被多西他赛/小白菊内酯纳米共晶的促凋亡、新生血管抑制和肿瘤干细胞杀伤检测
荷瘤裸鼠在给药完成后的第10天，处死取出瘤组织进行固定，作冰冻切片，通过TUNEL染色、CD31及CD133抗体染色检测凋亡促进、新生血管抑制及肿瘤干细胞杀伤情况。结果见附图26。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (16)

  1. 一种全过程靶向分子，其特征在于，所述全过程靶向分子由脑靶向分子与肿瘤靶向分子两部分分子共价连接形成，可介导影像分子、治疗药物和纳米载药系统的靶向递送；
    所述全过程靶向分子由具有跨血-脑屏障与跨血-肿瘤屏障并靶向肿瘤拟态血管和肿瘤细胞及其干细胞的小分子、多肽分子或蛋白分子靶向分子组合而成；
    优选地，所述全过程靶向分子可靶向脑毛细血管内皮细胞并跨血-脑屏障、靶向肿瘤新生血管内皮细胞并跨血-肿瘤屏障，同时靶向肿瘤拟态血管、肿瘤细胞和肿瘤干细胞；
    更优选地，所述全过程靶向分子可介导影像分子、治疗药物和纳米载药系统跨血-脑屏障和血-肿瘤屏障，靶向肿瘤拟态血管、肿瘤细胞和/或肿瘤干细胞，实现影像分子、治疗药物和纳米载药系统对脑部肿瘤和具备脑转移特征外周肿瘤的靶向递送。
  2. 根据权利要求1所述的全过程靶向分子，其特征在于，所述的跨血-脑屏障的小分子、多肽分子或蛋白分子靶向分子选自：对羟基苯甲酸(pHA)及其衍生物，脂肪酸如肉豆蔻酸(MC)及其衍生物，D8多肽、WSW多肽、 DWSW多肽、TGN多肽、 DTGN多肽、CDX多肽、 DCDX多肽、T7多肽及 DT7多肽及其衍生物，转铁蛋白、乳铁蛋白及其衍生物；和/或
    所述的跨血-肿瘤屏障的多肽分子或蛋白分子靶向分子选自：VAP多肽、cVAP多肽、 SVAP多肽、 DVAP多肽、A7R多肽、cA7R多肽、 DA7R多肽、RGD多肽、Stapled-RGD多肽、RW多肽、mn多肽、RAP12多肽及 DRAP12多肽及其衍生物。
  3. 根据权利要求1或2所述的全过程靶向分子，其特征在于，在所述的全过程靶向分子中引入活性官能团构建其修饰的影像分子复合物、治疗药物复合物和靶向功能载体材料；其中，在全过程靶向分子上引入影像分子X，制得全过程靶向分子-X复合物；
    在全过程靶向分子上引入治疗药物分子Y，制得全过程靶向分子-Y复合物；
    在全过程靶向分子上引入聚乙二醇-Z复合物分子，制得靶向功能载体材料全过程靶向分子-聚乙二醇-Z复合物，其中Z是亲脂性材料分子或亲脂性药物分子或亲水性配体分子。
  4. 一种药物复合物，其特征在于，所述药物复合物由权利要求1或2所述的全过程靶向分子与影像分子、治疗药物组成；
    优选地，在所述全过程靶向分子中引入活性官能团构建其修饰的影像分子复合物、治疗药物复合物；其中，在全过程靶向分子上引入影像分子X，制得全过程靶向分子-X复合物；在全过程靶向分子上引入治疗药物分子Y，制得全过程靶向分子-Y复合物。
  5. 根据权利要求3所述的全过程靶向分子或权利要求4所述的药物复合物，其特征在于，所述的全过程靶向分子-X复合物中，X选自以下一种或多种：光学影像分子、磁共振影像剂、放射性核素影像剂，用于脑部肿瘤或外周肿瘤的影像诊断和示踪；
    优选地，所述光学影像分子选自以下一种或多种：荧光探针分子FITC、FAM、6-TET、5-TAMRA、HEX、6-JOE，近红外染料分子Cy3、Cy3.5、Cy5、Cy5.5、Cy7、IR783、IR820、DiR、DiD、BIDIPY630/650-X、BIDIPY650/665-X、BIDIPY665/676、TO-PRO-3、TO-PRO-5，化学发光物质luminol、isoluminol、AMPPD、CSPD、CDP-star、光泽精，拉曼探针分子；
    优选地，所述磁共振影像剂为Gd磁共振物质的螯合物；和/或
    优选地，放射性核素影像剂选自以下一种或多种： 18F、 32P、 35S、 64Cu、 67/68Ga、 75Se、 89Zr、 86Y、 99mTc、 111/111mIn、 123/125I、 177Lu、 149/161Tb影像用放射性核素的螯合物；
    更优选地，所述的磁共振影像剂和放射性核素影像剂，由双功能螯合剂和磁共振影像用物质或影像用放射性核素组成；其中，所述双功能螯合剂选自以下一种或多种：DOTA、DOTAGA、NOTA、NOTAGA、NODA、DTPA、TETA、CB-TE2A、Cyclam、DFO、MAG3、EC、EDTA、DADT、HYNIC、CE-DTS、NS3，用于螯合磁共振影像用物质或放射性影像用核素。
  6. 根据权利要求3所述的全过程靶向分子或权利要求4所述的药物复合物，其特征在于，所述的全过程靶向分子-Y复合物中，Y选自以下一种或多种：肿瘤化疗药物中的蒽环类药物、紫杉烷类药物、喜树碱类药物、长春碱类药物、蛋白酶体抑制剂类药物、抗肿瘤干细胞类药物、分子靶向类药物、多肽类药物、抗体类药物、治疗用放射性核素螯合物；
    优选地，所述肿瘤化疗药物中的蒽环类药物选自以下一种或多种：阿霉素、表阿霉素；
    优选地，所述紫杉烷类药物选自以下一种或多种：紫杉醇、多西他赛、卡巴他赛；
    优选地，所述喜树碱类药物选自以下一种或多种：喜树碱、羟基喜树碱、9-硝基喜树碱、伊立替康；
    优选地，所述长春碱类药物选自以下一种或多种：长春碱、长春新碱；
    优选地，所述蛋白酶体抑制剂类药物为硼替佐米；
    优选地，所述抗肿瘤干细胞类药物选自小白菊内酯及其衍生物；
    优选地，所述分子靶向类药物选自以下一种或多种：曲美替尼、伊马替尼、尼罗替尼、达沙替尼、依维莫司、厄洛替尼、舒尼替尼、索拉非尼、依鲁替尼、瑞戈非尼、威罗非尼、奥拉帕尼；和/或
    优选地，所述多肽类药物选自以下一种或多种：p53激活肽、蜂毒肽、蝎毒肽、抗菌肽；
    优选地，所述抗体类药物选自以下一种或多种：利妥昔单抗、贝伐珠单抗、曲妥珠单抗、西妥昔单抗、帕妥珠单抗、伊匹单抗、纳武单抗、PD-L1单抗，及其通过基因工程改造的抗体片段组合包括Fab片段、单域抗体、Fv片段、单链抗体、双价小分子抗体、微抗体、纳米抗体；和/或
    优选地，所述治疗用放射性核素螯合物选自以下一种或多种： 90Y、 131I、 152/155Tb、 153Sm、 177Lu、 186/188Re、 211At、 212/213Bi、 212Pb、 225Ac、 227Th治疗用放射性核素的螯合物；
    更优选地，所述的治疗用放射性核素螯合物由双功能螯合剂和治疗用放射性核素组成，其中双功能螯合剂选自DOTA、DOTAGA、NOTA、NOTAGA、NODA、DTPA、TETA、CB-TE2A、Cyclam、DFO、MAG3、EC、EDTA、DADT、HYNIC、CE-DTS、NS3。
  7. 一种靶向功能载体材料，其特征在于，所述靶向功能载体材料由权利要求1或2所述的全过程靶向分子与高分子载体材料组成；
    优选地，在所述全过程靶向分子中引入活性官能团构建其修饰的高分子载体材料复合物；其中，在全过程靶向分子上引入聚乙二醇-Z复合物分子，制得靶向功能载体材料全过程靶向分子-聚乙二醇-Z复合物，其中Z是亲脂性材料分子或亲脂性药物分子或亲水性配体分子。
  8. 根据权利要求3所述的全过程靶向分子或权利要求7所述的靶向功能载体材料，其特征在于，所述的亲脂性材料分子Z选自以下一种或多种：磷脂、聚乳酸(PLA)、乳酸羟基乙酸共聚物(PLGA)、聚己内酯(PCL)；
    所述亲脂性药物分子Z选自以下一种或多种：蒽环类药物、紫衫烷类药物、喜树碱类药物、长春碱类药物、蛋白酶体抑制剂类药物、内酯类抗肿瘤药物；和/或
    所述亲水性配体分子Z是生物素。
  9. 权利要求8所述的全过程靶向分子或靶向功能载体材料在制备纳米载药系统中的应用，其中，当所述亲脂性材料分子Z为磷脂时，所述全过程靶向分子或靶向功能载体材料用于制备脂质体载药系统、胶束载药系统或圆盘载药系统；
    当所述亲脂性材料分子选自聚乳酸复合物、乳酸羟基乙酸共聚物和/或聚己内酯时，所述全过程靶向分子或靶向功能载体材料用于制备胶束载药系统或纳米粒载药系统；和/或
    当所述亲水性配体分子Z为生物素时，所述全过程靶向分子或靶向功能载体材料用于制备生物膜包被纳米载药系统。
  10. 根据权利要求9所述的应用，其特征在于，所述脂质体载药系统、胶束载药系统、圆盘载药系统、纳米粒载药系统和生物膜包被纳米载药系统包载诊断药物，用于脑部肿瘤或外周肿瘤的影像诊断和示踪；
    优选地，所述诊断药物选自以下一种或多种：光学影像物质、磁共振影像剂、放射性核素影像剂；
    更优选地，所述光学影像物质选自以下一种或多种：荧光探针香豆素6、FITC、FAM、DiI、Rhodamine B、Rhodamine 6G、5-TAMRA、6-TET、HEX、6-JOE，近红外染料Cy3、Cy3.5、Cy5、Cy5.5、Cy7、IR783、IR820、DiR、DiD、Alexa Fluor 680、BIDIPY630/650-X、BIDIPY650/665-X、BIDIPY665/676、TO-PRO-3、TO-PRO-5，化学发光物质luminol、isoluminol、AMPPD、CSPD、CDP-star、光泽精、拉曼探针；
    更优选地，所述磁共振影像剂为Gd等磁共振影像物质的螯合物；和/或
    更优选地，所述放射性核素影像剂选自以下一种或多种： 18F、 32P、 35S、 64Cu、 67/68Ga、 75Se、 89Zr、 86Y、 99mTc、 111/111mIn、 123/125I、 177Lu、 149/161Tb影像用放射性核素的螯合物。
  11. 根据权利要求9所述的应用，其特征在于，所述脂质体载药系统、胶束载药系统、圆盘载药系统、纳米粒载药系统和生物膜包被纳米载药系统用于包载肿瘤治疗药物，用于脑部肿瘤或具有脑转移特征外周肿瘤的靶向治疗；
    优选地，所述肿瘤治疗药物选自以下一种或多种：化疗中的蒽环类药物、紫杉烷类药物、喜树碱类药物、长春碱类药物、铂类药物、蛋白酶体抑制剂类药物、抗肿瘤干细胞类药物、分子靶向药物、多肽类药物、抗体类药物、治疗用放射性核素螯合物；
    更优选地，所述化疗中的蒽环类药物选自以下一种或多种：阿霉素、表阿霉素；
    更优选地，紫杉烷类药物选自以下一种或多种：紫杉醇、多西他赛、卡巴他赛；
    更优选地，喜树碱类药物选自以下一种或多种：喜树碱、羟基喜树碱、9-硝基喜树碱、伊立替康；
    更优选地，长春碱类药物选自以下一种或多种：长春碱、长春新碱；
    更优选地，铂类药物选自以下一种或多种：顺铂、卡铂、奥沙利铂、米铂；
    更优选地，蛋白酶体抑制剂类药物选自以下一种或多种：硼替佐米、卡非佐米；
    更优选地，抗肿瘤干细胞类药物选自以下一种或多种：小白菊内酯及其衍生物；
    更优选地，分子靶向药物选自以下一种或多种：曲美替尼、伊马替尼、尼罗替尼、达沙替尼、依维莫司、厄洛替尼、舒尼替尼、索拉非尼、依鲁替尼、瑞戈非尼、威罗非尼、奥拉帕尼；
    更优选地，多肽类药物选自以下一种或多种：p53激活肽、蜂毒肽、蝎毒肽和抗菌肽；
    更优选地，抗体类药物选自以下一种或多种：利妥昔单抗、贝伐珠单抗、曲妥珠单抗、西妥昔单抗、帕妥珠单抗、伊匹单抗、纳武单抗、PD-L1单抗，及其通过基因工程手段改造的抗体片段组合包括Fab片段、单域抗体、Fv片段、单链抗体、双价小分子抗体、微抗体、纳米抗体；
    和/或更优选地，治疗用放射性核素螯合物选自以下一种或多种： 90Y、 131I、 152/155Tb、 153Sm、 177Lu、 186/188Re、 211At、 212/213Bi、 212Pb、 225Ac、 227Th治疗用放射性核素的螯合物。
  12. 一种纳米载药系统，其特征在于，所述纳米载药系统包括权利要求3所述的全过程靶向分子或权利要求7所述的靶向功能载体材料，且所述纳米载药系统是通过全过程靶向分子上引入聚乙二醇-Z复合物构建而成，其中Z是亲脂性材料分子或亲脂性药物分子或亲水性配体分子；
    所述的亲脂性材料分子Z选自以下一种或多种：磷脂、聚乳酸(PLA)、乳酸羟基乙酸共聚物(PLGA)、聚己内酯(PCL)；
    所述亲脂性药物分子Z选自以下一种或多种：蒽环类药物、紫衫烷类药物、喜树碱类药物、长春碱类药物、蛋白酶体抑制剂类药物、内酯类抗肿瘤药物；和/或
    所述亲水性配体分子Z是生物素。
  13. 根据权利要求12所述的纳米载药系统，其特征在于，当所述亲脂性材料分子Z为磷脂时，所述纳米载药系统为脂质体载药系统、胶束载药系统或圆盘载药系统；
    当所述亲脂性材料分子Z为聚乳酸、乳酸羟基乙酸共聚物和/或聚己内酯时，所述纳米载药系统为胶束载药系统或纳米粒载药系统；和/或
    当所述亲水性配体分子Z为生物素时，所述纳米载药系统为生物膜包被纳米载药系统。
  14. 根据权利要求13所述的纳米载药系统，其特征在于，所述的脂质体载药系统、胶束载药系统、圆盘载药系统、纳米粒载药系统和生物膜包被纳米载药系统包载诊断药物，用于脑部肿瘤或外周肿瘤的影像诊断和示踪；
    优选地，所述诊断药物选自以下一种或多种：光学影像物质、磁共振影像剂、放射性核素影像剂；
    更优选地，所述光学影像物质选自以下一种或多种：荧光探针香豆素6、FITC、FAM、DiI、Rhodamine B、Rhodamine 6G、5-TAMRA、6-TET、HEX、6-JOE，近红外染料Cy3、Cy3.5、Cy5、Cy5.5、Cy7、IR783、IR820、DiR、DiD、Alexa Fluor 680、BIDIPY630/650-X、BIDIPY650/665-X、BIDIPY665/676、TO-PRO-3、TO-PRO-5，化学发光物质luminol、isoluminol、AMPPD、CSPD、CDP-star、光泽精、拉曼探针；
    更优选地，所述磁共振影像剂为Gd等磁共振影像物质的螯合物；和/或
    更优选地，所述放射性核素影像剂选自以下一种或多种： 18F、 32P、 35S、 64Cu、 67/68Ga、 75Se、 89Zr、 86Y、 99mTc、 111/111mIn、 123/125I、 177Lu、 149/161Tb影像用放射性核素的螯合物。
  15. 根据权利要求13所述的纳米载药系统，其特征在于，所述的脂质体载药系统、胶束载药系统、圆盘载药系统、纳米粒载药系统和生物膜包被纳米载药系统包载肿瘤治疗药物，用于脑部肿瘤或具有脑转移特征外周肿瘤的靶向治疗；
    优选地，所述肿瘤治疗药物选自以下一种或多种：化疗中的蒽环类药物、紫杉烷类药物、喜树碱类药物、长春碱类药物、铂类药物、蛋白酶体抑制剂类药物、抗肿瘤干细胞类药物、分子靶向药物、多肽类药物、抗体类药物、治疗用放射性核素螯合物；
    更优选地，所述化疗中的蒽环类药物选自以下一种或多种：阿霉素、表阿霉素；
    更优选地，紫杉烷类药物选自以下一种或多种：紫杉醇、多西他赛、卡巴他赛；
    更优选地，喜树碱类药物选自以下一种或多种：喜树碱、羟基喜树碱、9-硝基喜树碱、伊立替康；
    更优选地，长春碱类药物选自以下一种或多种：长春碱、长春新碱；
    更优选地，铂类药物选自以下一种或多种：顺铂、卡铂、奥沙利铂、米铂；
    更优选地，蛋白酶体抑制剂类药物选自以下一种或多种：硼替佐米、卡非佐米；
    更优选地，抗肿瘤干细胞类药物选自以下一种或多种：小白菊内酯及其衍生物；
    更优选地，分子靶向药物选自以下一种或多种：曲美替尼、伊马替尼、尼罗替尼、达沙替尼、依维莫司、厄洛替尼、舒尼替尼、索拉非尼、依鲁替尼、瑞戈非尼、威罗非尼、奥拉帕尼；
    更优选地，多肽类药物选自以下一种或多种：p53激活肽、蜂毒肽、蝎毒肽和抗菌肽；
    更优选地，抗体类药物选自以下一种或多种：利妥昔单抗、贝伐珠单抗、曲妥珠单抗、西妥昔单抗、帕妥珠单抗、伊匹单抗、纳武单抗、PD-L1单抗，及其通过基因工程手段改造的抗体片段组合包括Fab片段、单域抗体、Fv片段、单链抗体、双价小分子抗体、微抗体、纳米抗体；和/或
    更优选地，治疗用放射性核素螯合物选自以下一种或多种： 90Y、 131I、 152/155Tb、 153Sm、 177Lu、 186/188Re、 211At、 212/213Bi、 212Pb、 225Ac、 227Th治疗用放射性核素的螯合物。
  16. 一种脑部肿瘤或具有脑转移特征外周肿瘤的诊断和/或治疗方法，其特征在于，所述方法包括：对有需要的受试者给予权利要求1至8中任一项所述的全过程靶向分子、药物复合物或靶向功能载体材料，或权利要求12至15中任一项所述的纳米载药系统；
    优选地，所述方法是通过所述全过程靶向分子、药物复合物、靶向功能载体材料或纳米载药系统靶向脑毛细血管内皮细胞并跨血-脑屏障、靶向肿瘤新生血管内皮细胞并跨血-肿瘤屏障，同时靶向肿瘤拟态血管、肿瘤细胞和肿瘤干细胞实现的；
    更优选地，所述方法是通过上述述全过程靶向分子、药物复合物、靶向功能载体材料或纳米载药系统跨血-脑屏障和血-肿瘤屏障，靶向肿瘤拟态血管、肿瘤细胞和/或肿瘤干细胞，实现影像分子、治疗药物和纳米载药系统对脑部肿瘤和具备脑转移特征外周肿瘤的靶向递送。
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