WO2018103660A1 - Vap多肽及其在制备靶向诊疗肿瘤药物中的应用 - Google Patents

Vap多肽及其在制备靶向诊疗肿瘤药物中的应用 Download PDF

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WO2018103660A1
WO2018103660A1 PCT/CN2017/114796 CN2017114796W WO2018103660A1 WO 2018103660 A1 WO2018103660 A1 WO 2018103660A1 CN 2017114796 W CN2017114796 W CN 2017114796W WO 2018103660 A1 WO2018103660 A1 WO 2018103660A1
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vap
polypeptide
drug
tumor
polypeptide complex
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PCT/CN2017/114796
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English (en)
French (fr)
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陆伟跃
冉丹妮
毛佳妮
谢操
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复旦大学
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Priority to AU2017372268A priority Critical patent/AU2017372268B2/en
Priority to JP2019528582A priority patent/JP7035050B2/ja
Priority to CA3045367A priority patent/CA3045367A1/en
Priority to US16/467,149 priority patent/US11622990B2/en
Priority to KR1020197019624A priority patent/KR102631204B1/ko
Priority to EP17878861.8A priority patent/EP3553074A4/en
Priority to CN201780069749.3A priority patent/CN110114367B/zh
Publication of WO2018103660A1 publication Critical patent/WO2018103660A1/zh

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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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Definitions

  • the present invention belongs to the field of pharmacy, and relates to a VAP polypeptide and its use in pharmacy, in particular to a multifunctional D-configuration polypeptide which is highly stable and can target the glucose-regulating protein GRP78, and an L-configuration polypeptide and a stable D-configuration polypeptide.
  • Drug complexes and modified nano-delivery systems especially involving D-configuration peptide D VAP (D configuration amino acid sequence D P D A D V D R D T D N D S) and S VAP (D configuration amino acid sequence D S D N D T D R D V D A D P), and L-configuration polypeptide L VAP (amino acid sequence SNTRVAP) and stable D-configuration polypeptide diagnostic and therapeutic drug complexes, modified polymeric carrier materials and Nano-delivery systems such as liposomes, polymer micelles, polymer disks, nanoparticles, and the like, and applications in the preparation of tumor diagnostic and targeted therapeutic drugs.
  • D VAP D configuration amino acid sequence D P D A D V D R D T D N D S
  • S VAP D configuration amino acid sequence D S D N D T D R D V D A D P
  • L-configuration polypeptide L VAP amino acid sequence SNTRVAP
  • ligands commonly used in cells include monoclonal antibodies, polypeptides, aptamers, small molecule compounds, etc.; ligand-modified drugs or nano-delivery systems can be specific to cell surface receptors or transporters and ligands. Sexual recognition, binding, internalization, delivery of drugs into tumor tissues and cells, thereby achieving the active targeting of drugs to tumors.
  • Glucose regulatory protein GRP78 also known as immunoglobulin heavy chain binding protein (Bip) is one of the major molecular chaperones of the endoplasmic reticulum and plays an important role in protein folding and endoplasmic reticulum stress response. Studies have shown that the expression of GRP78 is significantly increased in several tumor cell lines, solid tumors and human cancer tissue biopsy samples. Studies have shown that GRP78 protein is highly expressed in various tumors such as breast cancer, liver cancer, colon cancer and gastric cancer, and The occurrence, progression, prognosis and drug resistance of these tumors are closely related.
  • GRP78 can be transferred to the surface of cell membranes in tumor cells, but no such metastasis is observed on the membrane surface of normal cells; studies have also shown that GRP78 plays a role in the regulation of cancer stem cells.
  • the role of the Cripto/GRP78 signaling pathway regulates the function of adult stem cells and cancer stem cells to maintain stem cell dryness. Therefore, specific delivery of drugs or nano-drug systems to tumor tissues via GRP78 receptor-mediated pathways The diagnosis and treatment of tumors will be of great value.
  • L VAP (L-configuration amino acid sequence SNTRVAP) is a heptapeptide selected by phage display technology. It has been shown to have high affinity activity against GRP78, but no research report on tumor targeted diagnosis has been reported so far.
  • the inventors of the present application intend to provide the application of VAP polypeptide in the targeted diagnosis and treatment of tumors and further optimize the stability of existing polypeptides to achieve better tumor targeting effects in vivo.
  • the object of the present invention is to provide a VAP polypeptide and a tumor-targeted diagnosis and treatment thereof according to the defects of the prior art, and further optimize the stability of the existing polypeptide to achieve better tumor targeting effect in vivo.
  • a first aspect of the invention provides a D-configuration polypeptide, wherein the D-configuration polypeptide is D VAP and/or S VAP, and the amino acid sequence of the D VAP is D P D A D V D R D T D N D S, the amino acid sequence of the S VAP D S D N D T D R D V D A D P.
  • a second aspect of the invention provides a D VAP and/or S VAP polypeptide complex, the D VAP and/or S VAP polypeptide complex of claim 1 comprising the D VAP and/or S VAP polypeptide modification
  • An imaging substance of a maleimide group wherein the structure of the D VAP and/or S VAP polypeptide complex is D VAP-X and/or S VAP-X, and X is the image substance; preferably, The X is selected from one or more of a fluorescent substance, a near-infrared dye, a magnetic resonance imaging agent, and a radiographic agent;
  • the fluorescent substance is Fluorescein
  • the near-infrared dye is Cy7, IR820, DiR
  • the magnetic resonance imaging agent is Gd-DTPA
  • the radioactive agent 99m Tc-DTPA is 99m Tc-DTPA.
  • a third aspect of the present invention provides an L-shaped configuration of the VAP L polypeptide complex, said polypeptide complex the VAP-modified L image maleimide group-containing substance is the VAP L polypeptide, wherein said the VAP L
  • the amino acid sequence of the polypeptide is SNTRVAP, and the structure of the L VAP polypeptide complex is L VAP-X, and X is the image substance;
  • the X is selected from one or more of fluorescein, a near-infrared dye, a magnetic resonance imaging agent, and a radiographic agent;
  • the fluorescein is Fluorescein
  • the near-infrared dye is Cy7, IR820, DiR
  • the magnetic resonance imaging agent is Gd-DTPA
  • the radioactive agent is 99m Tc-DTPA.
  • a fourth aspect of the invention provides a D VAP and/or S VAP polypeptide complex, wherein the D VAP and/or S VAP polypeptide complex is the D VAP and/or S VAP polypeptide modification antibody of claim 1 a tumor drug, wherein the structure of the D VAP and/or S VAP polypeptide complex is D VAP-Y and/or S VAP-Y, and Y is the antitumor drug;
  • the anti-tumor drug is selected from the group consisting of anthracyclines such as doxorubicin and epirubicin, taxanes such as paclitaxel and docetaxel and cabazitaxel, camptothecin and hydroxycamptothecin and y Camptothecins such as rituximab, vinca alkaloids such as vincristine and vinorelbine, proteasome inhibitors such as bortezomib and carfilzomib, lactones such as parthenolide, p53 activating peptide And one or more of polypeptide drugs such as melittin, muscarinic peptides and antimicrobial peptides;
  • anthracyclines such as doxorubicin and epirubicin
  • taxanes such as paclitaxel and docetaxel and cabazitaxel
  • the antitumor drug is selected from the group consisting of ketone or aldehyde-based doxorubicin or epirubicin, hydroxyl or amino-containing paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, 9-nitro Camptothecin, vincristine, etoposide, gemcitabine, cytarabine, 5-fluorouracil, teniposide, moritinib, epothilone, vinorelbine, actinomycin D, mitoxantrone Anthraquinone, mitomycin, bleomycin, irinotecan, boric acid-containing bortezomib or carfilzomib, and/or one of the polypeptide drugs p53 activating peptide, melittin and scorpion venom Or a variety.
  • a fifth aspect of the present invention provides an L-shaped configuration of the VAP L polypeptide complex, said polypeptide complex L the VAP-modified antitumor agent to the VAP L polypeptide, wherein the amino acid sequence of the polypeptide is L the VAP SNTRVAP, and The structure of the L VAP polypeptide complex is L VAP-Y, and Y is the antitumor drug;
  • the anti-tumor drug is selected from the group consisting of anthracyclines such as doxorubicin and epirubicin, taxanes such as paclitaxel and docetaxel and cabazitaxel, camptothecin and hydroxycamptothecin and y Camptothecins such as rituximab, vinca alkaloids such as vincristine and vinorelbine, proteasome inhibitors such as bortezomib and carfilzomib, lactones such as parthenolide, p53 activating peptide And one or more of polypeptide drugs such as melittin, muscarinic peptides and antimicrobial peptides;
  • anthracyclines such as doxorubicin and epirubicin
  • taxanes such as paclitaxel and docetaxel and cabazitaxel
  • the antitumor drug is selected from the group consisting of ketone or aldehyde-based doxorubicin or epirubicin, hydroxyl or amino-containing paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, 9-nitro Camptothecin, vincristine, etoposide, gemcitabine, cytarabine, 5-fluorouracil, teniposide, moritinib, epothilone, vinorelbine, actinomycin D, mitoxantrone Anthraquinone, mitomycin, bleomycin, irinotecan, boric acid-containing bortezomib or carfilzomib, and/or one of the polypeptide drugs p53 activating peptide, melittin and scorpion venom Or a variety.
  • a sixth aspect of the invention provides a D VAP and/or S VAP polypeptide complex, wherein the D VAP and/or S VAP polypeptide complex is modified by the D VAP and/or S VAP polypeptide of claim 1 support material molecule, wherein said D VAP and / or structure S VAP polypeptide complex is -Z D VAP- polyethylene glycol and / or polyethylene glycol -Z S VAP-, the Z is a macromolecular carrier material;
  • the polymeric carrier material is selected from one or more of phospholipids, polylactic acid, lactic acid glycolic acid copolymers, and polycaprolactones.
  • a seventh aspect of the present invention provides an L-shaped configuration of the VAP L polypeptide complex, said polypeptide complex L the VAP-modified polymeric carrier material is the VAP L polypeptide, wherein the amino acid sequence of the polypeptide of the VAP L is SNTRVAP, L VAP and the structure of the polypeptide complex is L VAP- polyethylene glycol -Z, Z is a polymer support material;
  • the polymeric carrier material is selected from one or more of phospholipids, polylactic acid, lactic acid glycolic acid copolymers, and polycaprolactones.
  • An eighth aspect of the invention provides a delivery system comprising the complex of the sixth aspect or the seventh aspect; preferably, the delivery system is a liposome delivery system, polymerization Micelle delivery system, polymer disc delivery system or nanoparticle delivery system.
  • the delivery system further comprises (1) a diagnostic drug and/or (2) an antitumor other than the D VAP, S VAP and/or L VAP polypeptide complex.
  • Drug preferably:
  • the (1) diagnostic drug is selected from one or more of a fluorescent substance, a near-infrared dye, and a magnetic resonance imaging agent. More preferably, the fluorescent substance is Fluorescein, and the near-infrared dye is selected from the group consisting of Cy7 and IR820. DiR, and/or the magnetic resonance imaging agent is Gd-DTPA, and/or
  • the (2) antitumor drug is selected from the group consisting of anthracyclines such as doxorubicin and epirubicin, taxanes such as paclitaxel and docetaxel and cabazitaxel, camptothecin and hydroxycamptothecin, and Inhibitors such as irinotecan and other camptothecin drugs, vincristine and vinorelbine, proteasome inhibitors such as bortezomib and carfilzomib, lactones such as parthenolide, and p53 activation
  • anthracyclines such as doxorubicin and epirubicin
  • taxanes such as paclitaxel and docetaxel and cabazitaxel
  • camptothecin and hydroxycamptothecin and Inhibitors
  • irinotecan and other camptothecin drugs vincristine and vinorelbine
  • proteasome inhibitors such as bortezomib and
  • the ninth aspect of the present invention provides a D VAP the first aspect and / or S VAP polypeptide, the second to seventh aspects of the D VAP, S VAP and / or L VAP polypeptide complex, an eighth aspect
  • the use of the delivery system described herein for the preparation of a medicament or medical product for the diagnosis, tracing and/or treatment of a tumor preferably:
  • the tumor is a high expression of the glucose regulatory protein GRP78 tumor.
  • a tenth aspect of the invention provides a method for tumor diagnosis and/or targeted therapy, administered to a subject in need thereof:
  • Polypeptide complex and/or
  • a stabilized D-configuration VAP polypeptide of the invention said D-configuration VAP polypeptide is D VAP and/or S VAP, and the amino acid sequence of said D VAP is D P D A D V D R D T D N D S The amino acid sequence of the S VAP is D S D N D T D R D V D A D P.
  • the present invention designs and prepares the D-configuration polypeptide D VAP and/or S VAP, both of which have high stability to serum and high affinity to GRP78.
  • the image material is modified, wherein the structure of the D VAP, S VAP, L VAP polypeptide complex is D VAP-X, S VAP-X and/or L VAP-X, and X is the image substance.
  • the X is selected from one or more of fluorescein, a near-infrared dye, a magnetic resonance imaging agent, and a radiographic agent, and more preferably, the fluorescein is Fluorescein, and the near-infrared dye is selected from the group consisting of cy7 One or more of IR820 and DiR, the magnetic resonance imaging agent is Gd-DTPA, and the radioactive agent is 99m Tc-DTPA.
  • the D VAP, S VAP of the present invention or the L VAP thiolated by the literature can utilize the thiol and maleimide functionalized fluorescent substances in the molecule (such as Fluorescein, near-infrared dye Cy7, IR820, DiR, etc.).
  • the magnetic resonance imaging agent Gd-DTPA and the radioimaging agent 99m Tc-DTPA react to form a complex.
  • the D VAP, S VAP of the present invention or the L VAP complex reported in the literature the D VAP, S VAP, L VAP polypeptide complex is a D VAP, S VAP, L VAP modified antitumor drug, wherein the D VAP
  • the structure of the S VAP, L VAP polypeptide complex is D VAP-Y, S VAP-Y and/or L VAP-Y, and Y is the antitumor drug.
  • the anti-tumor drug is selected from the group consisting of anthracyclines such as doxorubicin and epirubicin, taxanes such as paclitaxel and docetaxel and cabazitaxel, camptothecin and hydroxycamptothecin and y Camptothecins such as rituximab, vinca alkaloids such as vincristine and vinorelbine, proteasome inhibitors such as bortezomib and carfilzomib, lactones such as parthenolide, p53 activating peptide And one or more of polypeptide drugs such as melittin, muscarinic peptides and antimicrobial peptides.
  • anthracyclines such as doxorubicin and epirubicin
  • taxanes such as paclitaxel and docetaxel and cabazitaxel
  • the antitumor drug is selected from the group consisting of ketone or aldehyde-based doxorubicin or epirubicin, hydroxyl or amino-containing paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, 9- Nitrocamptothecin, vincristine, etoposide, gemcitabine, cytarabine, 5-fluorouracil, teniposide, moritinib, epothilone, vinorelbine, actinomycin D, rice Toxic, mitomycin, bleomycin, irinotecan, boric acid-containing bortezomib or carfilzomib, and/or peptide drug p53 activating peptide, melittin and muscarinic peptide One or more.
  • the D VAP, S VAP of the present invention or the L VAP modified drug reported in the literature, including the reaction of a maleimide hexanthene derivative to form a pH-sensitive hydrazone bond (involving ketones such as doxorubicin and epirubicin) Or an aldehyde-based drug, or a 3-(2-pyridyldithio)propionic acid derivative to form a disulfide bond (involving paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, 9-nitrocime Alkali, vincristine, etoposide, gemcitabine, cytarabine, 5-fluorouracil, teniposide, moritinib, epothilone, vinorelbine, actinomycin D, mitoxantrone, a drug containing hydroxyl or amino groups such as mitomycin, bleomycin, irinotecan, or by reacting dopamine with a male
  • the D VAP, S VAP or the reported L VAP complex of the present invention is a D VAP, S VAP, L VAP modified polymer carrier material, wherein the D The structure of the VAP, S VAP, L VAP polypeptide complex is D VAP-polyethylene glycol-Z, S VAP-polyethylene glycol-Z and/or L VAP-polyethylene glycol-Z, Z is the high Molecular carrier material.
  • the polymeric carrier material is selected from one or more of phospholipids, polylactic acid, lactic acid glycolic acid copolymers, and polycaprolactones.
  • the D VAP, S VAP of the present invention or L VAP thiolated in the literature can be modified to contain a maleimide functional group-containing polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE).
  • PEG-DSPE polyethylene glycol-distearoylphosphatidylethanolamine
  • PEG-PLA polyethylene glycol-polylactic acid
  • PEG-PLGA polyethylene glycol-lactic acid glycol copolymer
  • PEG-PCL polyethylene glycol-polycaprolactone
  • nano drug delivery systems such as D VAP, S VAP, L VAP modified liposomes, polymer micelles, polymer disks, and nanoparticles.
  • the delivery system comprises the aforementioned D VAP, S VAP, L VAP polypeptide complex.
  • the delivery system is a liposome delivery system, a polymeric micellar delivery system, a polymer disc delivery system, or a nanoparticle delivery system.
  • the present invention also provides the aforementioned delivery system comprising (1) a diagnostic drug and/or (2) an antitumor drug other than the D VAP, S VAP, L VAP polypeptide complex.
  • the (1) diagnostic drug is selected from one or more of a fluorescent substance, a near-infrared dye, and a magnetic resonance imaging agent. More preferably, the fluorescent substance is Fluorescein, the near-infrared dye is selected from one or more of Cy7, IR820, DiR, and/or the magnetic resonance imaging agent is Gd-DTPA.
  • the (2) antitumor drug is selected from the group consisting of anthracyclines such as doxorubicin and epirubicin, taxanes such as paclitaxel and docetaxel and cabazitaxel, camptothecin and hydroxy camptothecin Camptothecins such as alkali and irinotecan, vinca alkaloids such as vincristine and vinorelbine, proteasome inhibitors such as bortezomib and carfilzomib, lactones such as parthenolide,
  • the p53 activates one or more of a peptide and a polypeptide such as melittin, muscarinic peptide and antimicrobial peptide.
  • the D VAP, S VAP, L VAP modified nano-delivery system designed by the present invention may contain anthracyclines such as doxorubicin and epirubicin, paclitaxel, docetaxel and cabazitaxel.
  • anthracyclines such as doxorubicin and epirubicin, paclitaxel, docetaxel and cabazitaxel.
  • Protease inhibition of cedar, camptothecin and hydroxycamptothecin and irinotecan and other camptothecin drugs such as vincristine, vinorelbine and vinorelbine, bortezomib and carfilzomib Agents, such as lactones such as lactone lactones, p53 activating peptides and melittin, muscarinic peptides and antimicrobial peptides; and fluorescent substances, near-infrared dyes and magnetic resonance imaging agents such as Fluorescein , Cy7, IR820, DiR, Gd-DTPA, etc.
  • the present invention also provides the aforementioned D VAP, S VAP polypeptide, the aforementioned D VAP, S VAP, L VAP polypeptide complex, the aforementioned delivery system for preparing a drug or medical treatment for diagnosis, tracing and/or treatment of a tumor. Application in the product.
  • the tumor is a high expression GRP78 tumor.
  • the D VAP, S VAP of the first aspect of the invention or the L VAP reported in the literature can mediate a drug or nano-delivery system targeting cells and tissues thereof with high expression of GRP78 for targeted diagnosis and treatment of tumors.
  • the invention also provides a combination product for diagnosing, tracing and/or treating a tumor, the combination product comprising one or more components selected from the group consisting of the aforementioned D VAP, S VAP, L VAP polypeptide complex And the aforementioned delivery system.
  • the combination product is a kit, and/or
  • the tumor is a high expression GRP78 tumor.
  • a method of diagnosing, tracing, and/or treating a tumor of the present invention comprising administering an effective dose of one or the following to an orally or non-oral route to a patient having or having the tumor
  • an effective dose of one or the following to an orally or non-oral route to a patient having or having the tumor
  • materials the aforementioned D VAP, S VAP, L VAP polypeptide complexes, the aforementioned delivery systems, and combinations of the foregoing.
  • the tumor is a high expression GRP78 tumor.
  • the oral or parenteral route can be delivered to the patient by oral, injection, patch, spray, and other known one or more.
  • the effective amount can include an amount effective to treat, reduce, alleviate, alleviate, eliminate, or condition one or more symptoms, the condition seeking to be treated, or alternatively, the condition seeking to be avoided, or otherwise A clinically identifiable favorable change is produced in the condition or its effect.
  • the present invention provides the use of the aforementioned D VAP, S VAP, L VAP polypeptides in the preparation of tumor targeting products.
  • the tumor targeting product is for targeting a tumor with high expression of GRP78; and/or the tumor targeting product is a drug, experimental reagent and/or medical product for diagnosing, tracing and/or treating a tumor .
  • the invention provides a drug complex of L VAP polypeptide (SNTRVAP) modification and a modified nano drug delivery system; and the problem that the L-configuration polypeptide has poor stability in vivo and is easily degraded in blood may cause a decrease in tumor targeting ability.
  • D VAP D configuration amino acid sequence D P D A D V D R D T D N D S
  • S VAP D configuration amino acid
  • the present invention produces a D-configuration polypeptide D VAP having a high stability (D configuration amino acid sequence D P D A D V D R D T D N D S) and S VAP (D configuration amino acid sequence D S D N D T D R D V D A D P), and modify the drug molecule and polymer carrier material with L VAP (L configuration amino acid sequence SNTRVAP), D VAP and S VAP to construct VAP drug complex, VAP modified Nano drug delivery system.
  • D VAP high stability
  • S VAP D configuration amino acid sequence D S D N D T D R D V D A D P
  • a D-configuration polypeptide targeting molecule D VAP (D configuration amino acid sequence D P D A D V D R D T D N D S) and S VAP (D) are designed and prepared by solid phase polypeptide synthesis technology.
  • the configuration amino acid sequence D S D N D T D R D V D A D P), both polypeptides have high stability to serum and high affinity with GRP78.
  • the sulfhydryl group and the maleimide functionalized image substance react to form a complex.
  • L VAP and the designed D VAP, S VAP modified drug including the reaction of maleimide hexanthene derivative to form a pH sensitive oxime bond (involving ketone or aldehyde containing doxorubicin, epirubicin, etc.) a disulfide bond formed by the reaction of a 3-(2-pyridinyl)propionic acid derivative (involving paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, a drug containing a hydroxyl group or an amino group such as vincristine, or a reaction of a dopamine with a boric acid group in a drug to form a pH-sensitive boric acid ester (a drug containing a boric acid group such as bortezomib), or an amide bond directly formed by solid phase synthesis
  • a polypeptide-drug complex involving a polypeptide drug such as a p53 activating peptide
  • the polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) having a maleimide functional group can be modified.
  • PEG-PLA polyethylene glycol-polylactic acid
  • PEG-PLGA polyethylene glycol-lactic acid glycol copolymer
  • PEG-PCL polyethylene glycol-polycaprolactone
  • the designed L VAP, D VAP and S VAP modified nano drug delivery systems contain anthracyclines such as doxorubicin and epirubicin, paclitaxel and docetaxel, and taxanes such as cabazitaxel.
  • camptothecin and hydroxycamptothecin and irinotecan and other camptothecins such as vincristine, vincristine and vinorelbine, proteasome inhibitors such as bortezomib and carfilzomib, and small white chrysanthemum Antitumor drugs such as esters and other lactones, p53 activating peptides and melittin, muscarinic peptides and antibacterial peptides; or imaging materials such as Fluorescein, near-infrared dye Cy7, IR820, DiR, magnetic resonance imaging agents Gd-DTPA, etc.
  • the D VAP, S VAP and L VAP of the present invention can mediate drugs or nano drug delivery systems targeting cells and tissues thereof with high expression of the glucose regulatory protein GRP78 for targeted diagnosis and treatment of tumors.
  • the present invention provides D VAP, S VAP preparation and property investigation, and the above-mentioned L VAP, D VAP and S VAP modified drug complexes and nano drug delivery systems for the preparation of the material basis of tumor diagnosis and treatment; and L VAP, D VAP and S VAP-mediated in vivo active targeting assays, including,
  • VAP-Fluorescein, VAP-Cy7 Synthesis of VAP, VAP-Cys and its fluorescent markers
  • L VAP, L VAP-Cys, D VAP, D VAP-Cys, S VAP, S VAP-Cys were prepared by solid phase synthesis.
  • L VAP-Fluorescein, D VAP-Fluorescein, S VAP-Fluorescein, L VAP-Cy7, D VAP-Cy7, S VAP-Cy7 were synthesized by Michael addition reaction of maleimide group with thiol group. The structure was characterized by HPLC and MS.
  • D VAP and S VAP were examined from three aspects: serum stability, ability to bind to glucose-regulated protein GRP78, and cellular uptake ability to express this protein.
  • D VAP, S VAP and L VAP were incubated with mouse serum at 37 ° C, respectively, and the concentration of the polypeptide was measured at different time points for stability comparison.
  • D VAP-Fluorescein, S VAP-Fluorescein and L VAP-Fluorescein eg umbilical vein endothelial cells HUVEC
  • tumor cells eg: U87 glioma cells
  • the L VAP, D VAP and S VAP linked to the cysteine react with the maleimide hexamidine derivative of the drug to form a polypeptide-drug complex containing a pH-sensitive oxime bond, wherein the drug involved includes doxorubicin a drug containing a ketone or an aldehyde group such as epirubicin;
  • L VAP, D VAP and S VAP after attachment of cysteine react with a 3-(2-pyridyldithio)propionic acid derivative of the drug to form a disulfide-containing polypeptide-drug complex, wherein the drug involved includes a drug containing a hydroxyl group or an amino group such as paclitaxel, docetaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, vincristine;
  • L VAP, D VAP and S VAP form a polypeptide-drug complex containing a pH-sensitive borate by modifying dopamine and reacting with a boric acid group of the drug, wherein the drug involved includes a boric acid group-containing drug such as bortezomib;
  • L VAP, D VAP and S VAP are directly condensed with polypeptide drugs by solid phase synthesis to form a fusion polypeptide, and the drugs involved include peptide drugs such as p53 activating peptide, antimicrobial peptide and polypeptide toxin.
  • D VAP after connection cysteine maleimidocaproyl adriamycin hydrazine derivative (MAL-DOX) obtained by condensation of D VAP- doxorubicin complex (D VAP-DOX), by the charge implanted subcutaneously U87
  • MAL-DOX connection cysteine maleimidocaproyl adriamycin hydrazine derivative obtained by condensation of D VAP- doxorubicin complex (D VAP-DOX), by the charge implanted subcutaneously U87
  • the tumor model was administered to the nude mice by tail vein.
  • the antitumor effect was evaluated in vivo by the tumor volume, tumor weight and tumor inhibition rate.
  • the drug was evaluated by the tail vein of the nude mice bearing the U87 tumor model and the median survival time. Its anti-tumor effect in vivo; quantitative administration of pharmacokinetic curves and in vivo distribution by fluorescence injection through mouse tail vein injection.
  • L VAP, D VAP and S VAP modified polymer materials L VAP-PEG-PLA, D VAP-PEG-PLA and S VAP-PEG-PLA were synthesized.
  • the synthesis of the material is achieved by the reaction of the free sulfhydryl group on the cysteine-linked polypeptide with the maleimide contained in the Mal-PEG-PLA.
  • the Mal-PEG-PLA was dissolved in acetonitrile, rotary evaporated, and formed into a membrane, and added with PBS containing peptide (pH 8.0) to prepare L VAP-PEG-PLA, D VAP-PEG-PLA and S VAP-PEG-PLA;
  • L VAP, D VAP, S VAP modified micelles L VAP-Micelle, D VAP-Micelle, S VAP-Micelle
  • a certain amount of VAP-PEG-PLA, mPEG-PLA and drugs were prepared by film formation, and the laser scattering particle size analyzer was used to characterize the micelle size and particle size distribution.
  • L VAP-Micelle/DiR, D VAP-Micelle/DiR, S VAP-Micelle/DiR and mPEG-Micelle/DiR were injected into the tail vein of nude mice bearing the U87 subcutaneous xenograft model, and the tumors of different groups were compared at each time point. distributed.
  • L VAP-Micelle/PTX, S VAP-Micelle/PTX, D VAP-Micelle/PTX and mPEG-Micelle/PTX, clinical preparations, and physiological saline were injected into the tail vein of nude mice bearing the U87 subcutaneous xenograft model. Tumor volume, tumor weight and tumor tissue apoptosis, neovascularization and mimicry of blood vessels were used as indicators to evaluate the in vivo antitumor effects of different paclitaxel delivery systems.
  • the present invention provides a material basis for the preparation and properties of D VAP, S VAP, and the drug complexes and nano drug delivery systems modified by the above L VAP, D VAP and S VAP for preparing a tumor diagnosis and treatment drug; the test results of the present invention indicate : L VAP, D VAP, and S VAP are both mediated in vivo active targeting; compared to L VAP, D VAP and S VAP have better stability in serum, and thus their mediated active targeting in vivo is better.
  • FIG. 1 shows the HPLC and ESI-MS spectra of D VAP:
  • FIG. 2 shows the HPLC and ESI-MS spectra of D VAP-Cys:
  • FIG. 3 shows the HPLC and ESI-MS spectra of S VAP:
  • FIG. 4 shows the HPLC and ESI-MS spectra of S VAP-Cys:
  • FIG. 5 shows the HPLC and ESI-MS spectra of L VAP:
  • Figure 6 shows the HPLC and ESI-MS spectra of L VAP-Cys
  • FIG. 7 shows the HPLC and ESI-MS spectra of D VAP-Fluorescein:
  • FIG. 8 shows the HPLC and ESI-MS spectra of S VAP-Fluorescein:
  • FIG. 9 shows the HPLC and ESI-MS spectra of L VAP-Fluorescein:
  • FIG. 10 shows the HPLC and ESI-MS spectra of D VAP-Cy7:
  • FIG. 11 shows the HPLC and ESI-MS spectra of S VAP-Cy7:
  • Figure 12 shows the HPLC and ESI-MS spectra of L VAP-Cy7
  • FIG. 13 shows the HPLC and ESI-MS spectra of D VAP-DOX:
  • ESI-MS 1599.6, in accordance with the theoretical molecular weight.
  • Figure 14 shows the 1 H-NMR spectrum of D VAP-PEG 3000 -PLA 2000 , S VAP-PEG 3000 -PLA 2000 and L VAP-PEG 3000 -PLA 2000 :
  • the nuclear magnetic spectrum of Mal-PEG-PLA showed a maleimide peak at 6.7 ppm, while the peak disappeared in the nuclear magnetic spectrum of VAP-PEG-PLA, indicating that the maleimide group in Mal-PEG-PLA has reacted. complete.
  • FIG. 15 shows the serum stability of D VAP, S VAP and L VAP:
  • the ordinate of the graph is the residual percentage of intact polypeptide. It can be seen that the stability of D VAP and S VAP in 50% mouse serum is significantly higher than that of L VAP. After incubation for 2 h, L VAP is completely degraded, and D VAP and S VAP are hardly degraded.
  • Figure 16 shows the binding activity of D VAP, S VAP and L VAP to GRP78:
  • the binding activities of D VAP, S VAP and L VAP were similar to those of GRP78, slightly weaker than L VAP, and the K D values were 4.010 ⁇ M and 5.223 ⁇ M, 2.696 ⁇ M, respectively.
  • the dissociation patterns of the three peptides were similar, with Kd values of 0.02091 1/s, 0.02826 1/s, and 0.01898 1/s, respectively.
  • FIG. 17 shows the intracellular distribution of GRP78:
  • GRP78 is widely distributed on the cell membrane and in cells. There was no distribution on the membrane of normal cell HEK293 cells, and a small amount of GRP78 was distributed in the membrane.
  • Figure 18 shows the uptake of Fluorescein marker polypeptide by glioma cell line U87:
  • Figure A and Figure B show the results of laser confocal photographs and flow cytometry after 4 hours of exposure of Fluorescein-labeled D VAP, S VAP and L VAP to U87 cells. It can be seen that the uptake of D VAP, S VAP and L VAP by U87 cells is significantly higher than that of free fluorescein, and there is no significant difference in the uptake of D VAP and S VAP, which is slightly weaker than L VAP.
  • Figure 19 shows the uptake of Fluorescein-labeled polypeptide by HUVEC in umbilical vein endothelial cells:
  • Figure A and Figure B show the results of laser confocal photographs and flow cytometry after 4 hours of exposure of Fluorescein-labeled D VAP, S VAP and L VAP to HUVEC cells. It can be seen that the uptake of D VAP, S VAP and L VAP by HUVEC cells is significantly higher than that of free fluorescein, and there is no significant difference in the uptake of D VAP and S VAP, which is slightly weaker than L VAP.
  • Figure 20 shows U87 tumor sphere uptake by Fluorescein labeled polypeptide and VAP-Micelle/C6:
  • the picture shows the uptake of each of the Fluorescein-labeled peptides and VAP micelles by the U87 tumor sphere. It can be seen from the figure that each of the Fluorescein-labeled peptides and VAP micelles can be well taken up by the U87 tumor sphere, which is significantly different from the target-free micelles and FAM. .
  • FIG. 21 shows the D VAP, S VAP, and L VAP competitive inhibition experiments:
  • Figure 22 shows the distribution of subcutaneous xenografts of Cy7-labeled polypeptides:
  • Figure A shows the results of in vitro tumor image distribution after injection of Cy7-labeled peptide for 1 hour in nude mice bearing U87 subcutaneous xenografts;
  • Figure B shows the semi-quantitative results of fluorescence at various time points after administration;
  • Figure C shows the tumors and organs in vitro. Fluorescence distribution image;
  • Figure D is the semi-quantitative result of in vitro tumor fluorescence.
  • the accumulation of Cy7-labeled D VAP, S VAP and L VAP in tumors was significantly higher than that of free Cy7 (***p ⁇ 0.001), and tumor targeting The effect is as follows: D VAP ⁇ S VAP> L VAP.
  • Figure 23 shows the D VAP-DOX subcutaneous tumor inhibition assay:
  • Panel A is a graph showing the tumor volume of each group in nude mice as a function of time. Compared with the saline group, each of the administration groups inhibited tumor growth. The same dose of D VAP-DOX was significantly better than DOX and MAL-DOX, which was significantly better than RGD-DOX.
  • Figure B shows the results of statistical analysis after removing the tumor tissue from the nude mice.
  • Figure C is a photograph of the isolated tumor tissue. It can be seen that the tumor size and tumor weight of the same dose D VAP-DOX group were significantly lower than those of the other groups.
  • Figure 24 shows the survival curves of nude mice in the D VAP-DOX epitope glioma model:
  • the mean survival times of the PBS group, the DOX group, the MAL-DOX group, the D VAP-DOX group (high, medium, low) and the D VAP group were 36, 36, 48, 58, 52, 48.5, and 45.5 days, respectively.
  • the results showed that compared with the other groups, the survival time of D VAP-DOX prolonged glioma model nude mice was the most significant, and the lowest dose could achieve the effect of 4 times the dose of MAL-DOX anti-cerebral glioma.
  • Figure 25 shows the pharmacokinetic profile and distribution of mice in D VAP-DOX:
  • Figure A shows the pharmacokinetic curve
  • Figure B shows the distribution of major organs in the body.
  • the results showed that the AUC of D VAP-DOX was significantly lower than that of MAL-DOX, but it was significantly higher than that of free DOX.
  • D VAP-DOX can significantly reduce the distribution of drugs in the heart, suggesting that D VAP-DOX may reduce the side effects of doxorubicin on the heart.
  • Figure 26 shows the subcutaneous intratumoral distribution of DiR-loaded VAP micelles:
  • Figure A shows the in vivo fluorescence distribution image after 24 hours of tail vein injection. From left to right, the PBS group, mPEG-Micelle/DiR, L VAP-Micelle/DiR, D VAP-Micelle/DiR and S VAP-Micelle/DiR .
  • Figure B is a fluorescence distribution image of an organ. The results indicate that D VAP or S VAP modified micelles are better targeted to the tumor site.
  • Figure 27 shows the particle size of the loaded paclitaxel micelles
  • the picture shows the particle size of each paclitaxel micelle. As can be seen from the figure, there is no significant difference in the size of the micelles everywhere.
  • Figure 28 shows the activity curves of anti-U87 cells and HUVEC cells in vitro loaded with paclitaxel micelles:
  • Figure A and Figure B show the activity curves of mPEG-Micelle/PTX, D VAP-Micelle/PTX, S VAP-Micelle/PTX, L VAP-Micelle/PTX and Taxol anti-U87 cells and HUVEC cells, respectively.
  • Figure A shows U87 After ICH culture for 72h, the IC 50 was 0.73, 0.09, 0.12, 0.59 and 0.75 ⁇ M, respectively.
  • the four micelles could inhibit the growth of U87 cells in vitro, among which D VAP-Micelle/PTX, S VAP-Micelle/ The in vitro activities of PTX were 6.56 and 4.92 times of L VAP-Micelle/PTX, respectively.
  • Figure B shows that the IC 50 of HUVEC cells were 0.52, 0.04, 0.04, 0.23 and 0.74 ⁇ M after 72 h of culture for 4 h, respectively.
  • the bundles inhibited the growth of HUVEC cells in vitro, and the in vitro activities of D VAP-Micelle/PTX and S VAP-Micelle/PTX were 5.75 of L VAP-Micelle/PTX.
  • Figure 29 shows inhibition of neovascularization by paclitaxel-loaded micelles in vitro:
  • the picture shows the inhibition images of mPEG-Micelle/PTX, D VAP-Micelle/PTX, S VAP-Micelle/PTX, L VAP-Micelle/PTX and Taxol on the in vitro model of neovascularization, compared to L VAP-Micelle/PTX, D VAP-Micelle/PTX and S VAP-Micelle/PTX inhibit the formation of neovascularization more significantly.
  • Figure 30 shows the inhibition of mimic angiogenesis by paclitaxel-loaded micelles in vitro:
  • the picture shows the inhibition of mPEG-Micelle/PTX, D VAP-Micelle/PTX, S VAP-Micelle/PTX, L VAP-Micelle/PTX and Taxol on the mimic vascular in vitro model compared to L VAP-Micelle/PTX, D VAP-Micelle/PTX and S VAP-Micelle/PTX inhibit the formation of mimetic blood vessels more significantly.
  • Figure 31 shows the subcutaneous tumor inhibition experiment with paclitaxel micelles:
  • Figure 33 shows the CD31/PAS double staining results
  • Example 1 VAP, VAP-Fluorescein, VAP-Cy7, VAP-drug, Synthesis and characterization of VAP-PEG-PLA
  • D the VAP in the D configuration amino acids consisting of (sequence D P D A D V D R D T D N D S), D VAP-Cys ( sequence D C D P D A D V D R D T D N D S) and S VAP (sequence is D S D N D T D R D V D A D P), S VAP-Cys (sequence is D S D N D T D R the VAP L (sequence SNTRVAP) D V D a D P D C) and the amino acid L-configuration and composed of L VAP-Cys (sequence SNTRVAPC).
  • the Boc solid phase peptide synthesis method is used to sequentially insert amino acids on the PAM resin in sequence, and the reaction is carried out by using HBTU/DIEA as a condensing agent and TFA as a deprotecting agent.
  • the resin was cut with hydrogen fluoride containing P-cresol, and stirred for 1 hour in an ice bath.
  • the hydrogen fluoride in the tube was removed under reduced pressure, and the precipitate was washed three times with ice diethyl ether. The precipitate was redissolved in 20% acetonitrile, and the filtrate was collected and then evaporated to give a crude peptide solution.
  • the crude peptide was isolated and purified by acetonitrile/water (containing 0.1% TFA) system.
  • the purity and molecular weight (Mw) of D VAP, D VAP-Cys, S VAP, S VAP-Cys, L VAP and L VAP-Cys were characterized by HPLC and ESI-MS. HPLC spectra and mass spectra are shown in Figures 1, 2, 3, 4, 5 and 6.
  • D VAP-Cys, S VAP-Cys or L VAP-Cys obtained in the above step were dissolved in 0.1 M PBS solution (pH 7.2), and Fluorescein-5-maleimide was dissolved in DMF.
  • the reaction was stopped after the reaction of D VAP-Cys, S VAP-Cys or L VAP-Cys was completed, and the liquid phase was purified, and purified by acetonitrile/water (containing 0.1% TFA) system. Freeze-dried D VAP-Fluorescein, S VAP-Fluorescein or L VAP-Fluorescein pure product.
  • HPLC charts and mass spectra are shown in Figures 7, 8, and 9.
  • Maleimide-DTPA is dissolved in DMF, and mixed with D VAP-Cys, S VAP-Cys or L VAP-Cys in PBS solution, prepared for liquid phase purification, freeze-dried to obtain D VAP-DTPA, S VAP-DTPA or Pure L VAP-DTPA, chelated Gd or 99m Tc gives VAP-DTPA-Gd or VAP-DTPA- 99m Tc.
  • VAP-adriamycin-based drug as a VAP-linked ketone- or aldehyde-based drug was prepared.
  • 9.4 mg of thiolated VAP ( D VAP or S VAP or L VAP) polypeptide was dissolved in phosphate 3 mL buffer (0.1 mM, pH 7.0) and 10 times the molar amount of tris(2-carboxyethyl)phosphine (TCEP) was added. Stir at 20 ° C for 20 min. Then, a 4-fold molar amount of doxorubicin 6-maleimido hexanide derivative (MAL-DOX) was added and reacted at room temperature in the dark for 1 h. The reaction solution was purified by preparative liquid phase, and lyophilized to obtain L VAP or D VAP or S VAP-adriamycin complex, and the structure was characterized by HPLC and MS. The results are shown in FIG.
  • the paclitaxel 3-(2-pyridinium dimercapto)propionic acid derivative was dissolved in 5 mL of DMF, and 1.5 times the molar amount of VAP-Cys was dissolved in PBS/DMF. The pH of the solution was maintained at 4-5, and paclitaxel 3-(2-pyridine was added. The dimercapto)propionic acid derivative was added dropwise to the thiol-polypeptide solution, reacted at room temperature for 6 h, and purified by preparative liquid phase to obtain a polypeptide-paclitaxel complex.
  • VAP-bortezomib complex as a VAP-linked drug containing a boronic acid group: the amino acid is sequentially inserted into the resin according to the synthesis of VAP, and all amino acid residues of the polypeptide are inserted, and the end of the trifluoroacetic acid is removed. Boc protection. A DMF solution containing 3 times the molar amount of succinic anhydride and DIEA was added and reacted at room temperature for 30 min. After washing the resin, dopeamine was protected by adding 5-fold molar amount of trimethylchlorosilane, and reacted with HBTU/DIEA as a condensing agent for 1 hour at room temperature.
  • the resin was cut with HF and purified by preparative HPLC to obtain a polypeptide-dopamine derivative.
  • the polypeptide-bortezomib complex is obtained by mixing the polypeptide-dopamine derivative with bortezomib in a molar ratio of 1:1 in a buffer of pH 7.4.
  • VAP-PMI fusion polypeptide as a VAP-linked polypeptide drug: directly by a solid phase polypeptide synthesis method, the specific method is: after determining the VAP-PMI polypeptide sequence, the amino acid is sequentially inserted in the same manner as the preparation of VAP, and the HF is obtained.
  • the VAP-PMI fusion polypeptide was obtained after cleavage and purification.
  • the synthesis of the membrane material is achieved by the reaction of the free sulfhydryl group of the polypeptide with the maleimide contained in the Mal-PEG-PLA.
  • 40 mg of Mal-PEG-PLA was dissolved in 5 mL of acetonitrile, rotary evaporated to form a membrane, and 3 mL of PBS (pH 8.0, 0.2 M) was added to hydrate at 37 ° C to form micelles, and 9.6 mg of VAP-Cys was added and reacted overnight for 8 hours.
  • the reaction was detected by HPLC. Excess VAP-Cys was removed by dialysis, lyophilized, and characterized by 1 H-NMR ( Figure 14).
  • D VAP, S VAP and L VAP were formulated into 1 mg/mL aqueous solution, 0.1 mL was added to 0.9 mL of 25% mouse serum, and incubated at 37 ° C, 100 ⁇ L reaction was taken at 0, 15 min, 0.5, 1, 2 and 4 h, respectively.
  • the solution was precipitated by adding 20 ⁇ L of trichloroacetic acid (TCA), and allowed to stand at 4 ° C for 20 min, centrifuged at 12,000 rpm for 10 min, and 20 ⁇ L of the supernatant was taken for HPLC analysis. Serum stability results (Figure 15) indicate that D VAP and S VAP have better serum stability than L VAP.
  • Pre-binding analysis was performed by the biacore system, and pH 5.0 was selected as the optimal GRP78 to bind the pH to the CM5 chip.
  • Recombinant human GRP78 was coupled to the CM5 chip and the RU value reached the target value.
  • D VAP, S VAP, and L VAP were respectively configured as sample solutions having concentrations of 0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 ⁇ M.
  • the injection activity of D VAP, S VAP and L VAP from the protein was analyzed by Biacore T200 Evaluation software software, and the K D value and Kd value were calculated respectively ( FIG. 16 ).
  • Monolayer cultured glioma cells (U87 cells) in logarithmic growth phase were digested with 0.25% trypsin, and mixed with DMEM medium containing 10% fetal bovine serum to prepare a single cell suspension.
  • 1 ⁇ 10 5 cells per well were seeded in a 12-well culture plate at a volume of 1 mL per well.
  • the culture plate was transferred to a carbon dioxide incubator, and cultured at 37 ° C, 5% CO 2 and saturated humidity for 24 hours, with 10%.
  • the DMEM medium of fetal bovine serum was prepared into a solution of FAM, D VAP-Fluorescein, S VAP-Fluorescein and L VAP-Fluorescein at a concentration of 5 ⁇ M.
  • the culture medium in the culture plate was aspirated, and the above solution was added thereto, and incubated at 37 ° C for 4 hours. Discard the supernatant. Wash the cells three times with PBS solution, fix the cells with formaldehyde fixative, and stain the cells with DAPI. After laser confocal observation, the photos of cell internalization are shown in Figure 18A. After washing three times with PBS, flow cytometry analysis is performed. As shown in 18B.
  • Fig. 19A Human umbilical vein endothelial cells (HUVEC cells) cultured in a single layer in the logarithmic growth phase were tested as above, and the internalization photographs are shown in Fig. 19A, and the results of flow cytometry analysis are shown in Fig. 19B.
  • a 2% low molecular weight agarose solution was added to a 48-well plate, 150 ⁇ L per well, and allowed to cool and coagulate at room temperature. Each well was inoculated with 400 ⁇ L of U87 cell suspension at a cell density of 2 ⁇ 10 3 /well. The tumor spheres were formed by incubating in a carbon dioxide incubator for 7 days at 37 ° C, 5% CO 2 and saturated humidity.
  • a solution of FITC, D VAP-Fluorescein, S VAP-Fluorescein and L VAP-Fluorescein at a concentration of 5 ⁇ M was prepared in DMEM medium containing 10% fetal bovine serum.
  • the culture solution in the culture plate was aspirated, and the above solution was separately added, and the solution was incubated at 37 ° C for 4 hours.
  • the supernatant was aspirated, washed three times with PBS, fixed for 15 minutes with paraformaldehyde, and then placed under a confocal microscope. The photograph is shown in Fig. 20A. .
  • the 9 groups of 3 to inspect the inner tube between competing VAP inhibition packet blank control group L VAP-Fluorescein, D VAP -Fluorescein, S VAP-Fluorescein, L VAP-Fluorescein (+ L VAP), L VAP-Fluorescein (+ D VAP), L VAP-Fluorescein (+ S VAP), D VAP-Fluorescein (+ L VAP), D VAP-Fluorescein (+ D VAP), D VAP-Fluorescein (+ S VAP), S VAP-Fluorescein (+ L VAP), S VAP-Fluorescein (+ D VAP), S VAP-Fluorescein (+ S VAP), U87 cells were trypsinized and transferred to EP tubes, and PBS was washed three times to remove trypsin.
  • the prepared peptide solution (non-fluorescent label) was also placed at 4 ° C for low temperature pretreatment, and then the peptide solution was mixed with the cell suspension for 2 h to saturate the receptor protein on the cell surface, and then added.
  • the fluorescein-labeled polypeptide solution was washed 3 times with PBS at 4 ° C, and the cell uptake was measured by flow cytometry (as shown in Figure 21).
  • Example 6 VAP in vivo tumor targeting validation
  • a subcutaneous tumor animal model was constructed, and the U87 cells in the logarithmic growth phase were trypsinized, adjusted to a cell concentration of 3 ⁇ 10 7 cells/mL, and inoculated with 100 ⁇ L to the right abdomen of the nude mice, subcutaneously, and then inoculated. SPF grade, the tumor size was observed regularly. When the tumor size was 200 mm 3 , the tumor-bearing nude mice without necrosis and regular tumor shape were screened and tested in groups. Cy7, D VAP-Cy7, S were administered at a dose of 0.15 ⁇ mol/ L. VAP-Cy7 and L VAP-Cy7 solutions were injected into the tumor-bearing nude mouse model through the tail vein. After 2 hours, the nude mice were sacrificed, the tumor was removed, and the fluorescence distribution of the tumor was detected by a living imager (as shown in Fig. 22A). Quantitative calculation (as shown in Figure 22B).
  • Example 7 D VAP-DOX in vivo pharmacodynamics and pharmacokinetic test
  • the U87 subcutaneous tumor animal model was constructed and tested in groups when the tumor size was 100 mm 3 .
  • the subcutaneous tumor model rats were injected with normal saline, DOX, MAL-DOX (containing high and low doses) and D VAP-DOX (containing high and low doses) and RGD-DOX (integrin receptor ligand c (RGDyK).
  • 100 ⁇ l each of the polypeptide drug complexes prepared in the same manner as MAL-DOX according to VAP-DOX.
  • the total dose of DOX was 1.25 mg/kg, and the others were 2.5 mg/kg, which were divided into five doses, and the interval between each dose was two days.
  • the long diameter (a) and short diameter (b) of the tumor were measured by vernier calipers the next day.
  • the tumor volume of each group of nude mice was calculated according to the formula, and the curve of tumor volume with time was plotted, and the statistical difference of each group was calculated. Calculate the tumor volume according to the following formula:
  • V tumor volume 0.5 (a ⁇ b 2 )
  • U87 in situ glioma model nude mice were constructed: U87 cells in logarithmic growth phase were inoculated, and each nude mouse was inoculated with 5 ⁇ 10 5 cells (dispersed in 5 ⁇ L of PBS buffer). After anesthetizing the nude mice, they were fixed with a stereotaxic apparatus, and the cells were inoculated into the right part of the striatum (0.6 mm in front of the anterior iliac crest, 1.8 mm in the lateral direction, and 3 mm in depth). The status of naked mice was observed regularly. The tail vein was injected with PBS, DOX, MAL-DOX, D VAP, D VAP-DOX (including high, medium and low doses).
  • the dose of D VAP-DOX low dose containing DOX was 2.5 mg/kg.
  • the medium dose is 5 mg/kg, the others are 10 mg/kg, and the D VAP dose is the amount of D VAP contained in the high dose of D VAP-DOX, which is given on days 10, 13, 16, 19 and 22 after tumor implantation.
  • Drugs were used to record the survival time of nude mice (Figure 24).
  • ICR mice were injected with 200 ⁇ L of DOX, D VAP-DOX and MAL-DOX (containing DOX 10 mg/kg) in the tail vein, respectively, and whole blood was taken at 1, 5, 15, 30 and 45 min, 1, 2, 4 and 6 h, respectively. 50 ⁇ L, diluted 4-fold in PBS, was measured in fluorescent Ex 485/Em 590, and the drug concentration-time curve was plotted against the in vivo distribution of the drug (Fig. 25).
  • Example 8 Targeting of VAP micelles to U87 tumor spheres in vitro
  • VAP-PEG-PLA 9 mg of mPEG-PLA and 5 ug of coumarin 6 (C6), dissolve in 2 mL of acetonitrile, evaporate at 37 ° C in water bath, decompress ( ⁇ 0.085 MPa), form a film, and dry at room temperature overnight. Hydration was carried out by adding 2 mL of physiological saline, and free coumarin was removed by CL-4B column chromatography to obtain a coumarin 6-coated micelle (VAP-Micelle/C6).
  • 2% of the low molecular weight agarose solution was added to a 48-well plate, 150 ⁇ L per well, and left to cool and solidify at room temperature, and then 400 ⁇ L of U87 cell suspension was inoculated per well to a cell density of 2 ⁇ 10 3 /well. Placed in a carbon dioxide incubator, cultured at 37 ° C, 5% CO 2 and saturated humidity for 7 days to form tumor spheres.
  • Micelle/C6, D was prepared at a concentration of 5 ng/mL in DMEM medium containing 10% fetal bovine serum.
  • VAP Micelle/C6 S VAP Micelle/C6 and L VAP Micelle/C6 solution
  • the culture solution in the culture plate was aspirated, the above solution was added separately, and the solution was incubated at 37 ° C for 4 h, the supernatant was aspirated, and the PBS was washed three times with paraformaldehyde. After fixation for 15 min, DAPI staining was observed under a confocal microscope, and the photograph is shown in Fig. 20B.
  • Example 9 In vivo targeting verification of VAP micelles
  • VAP-Micelle/DiR Di-loaded DiR micelles
  • U87 subcutaneous tumor model nude mice were injected with 100 ⁇ L of PBS, mPEG-Micelle/DiR, D VAP-Micelle/DiR, S VAP-Micelle/DiR and L VAP-Micelle/DiR, respectively, 2, 4, 8 after injection.
  • the nude mice were anesthetized, and the distribution of DiR fluorescence in nude mice was recorded by a living imager and semi-quantitative calculation of fluorescence was performed (as shown in Fig. 26).
  • Example 10 In vitro pharmacodynamic test of paclitaxel-loaded VAP micelles
  • VAP-Micelle/PTX The paclitaxel-loaded VAP micelles (VAP-Micelle/PTX) were prepared by weighing 1 mg of VAP-PEG-PLA, 9 mg of mPEG-PLA and 2 mg of paclitaxel to prepare coumarin 6 micelles. The particle size and distribution are shown in Fig. 27. Shown.
  • U87 cells were seeded in 96-well plates at 4.0 ⁇ 10 3 cells/well. After 24 hours, the culture solution was aspirated, and 200 ⁇ L of a series of concentrations of D VAP-Micelle/PTX, S VAP-Micelle/PTX, L VAP-Micelle/ were added. PTX and mPEG-Micelle/PTX and Taxol were co-cultured for 72 hours. After adding MTT solution for 4 hours, the culture solution was discarded, 150 ⁇ L of DMSO was added, and the mixture was shaken until the purple particles were dissolved. The absorbance was measured at 590 nm with a microplate reader. Cell viability was determined by MTT assay, and cell viability and median lethal dose were calculated (as shown in Figure 28).
  • the U87 subcutaneous tumor animal model was constructed and tested in groups when the tumor size was 100 mm 3 .
  • the subcutaneous tumor model rats were injected with saline, Taxol, L VAP-Micelle/PTX, D VAP-Micelle/PTX, S VAP.
  • the total dose of paclitaxel in the drug-administered group was 25 mg/kg, divided into five times, each time interval was two days, and the long diameter of the tumor was measured with a vernier caliper every other day ( a) and short diameter (b), the tumor volume of each group of nude mice was calculated according to the formula, and the curve of tumor volume with time was plotted, and the statistical difference of each group was calculated.
  • V tumor volume 0.5 (a ⁇ b 2 )
  • the nude mice Eighteen days after the administration (24 days after the inoculation), the nude mice were routinely treated, the subcutaneous tumors were weighed, and statistical differences between the groups were calculated (as shown in Fig. 31).
  • TUNEL Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling

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Abstract

本发明提供了与GRP78蛋白具有高结合活性的高稳定性D构型多肽 DVAP和 SVAP，还提供了L构型多肽 LVAP以及 DVAP、 SVAP修饰的模型药物和高分子载体材料，以及其在肿瘤影像和靶向治疗用递药系统构建中的应用。

Description

VAP多肽及其在制备靶向诊疗肿瘤药物中的应用 技术领域
本发明属药学领域，涉及VAP多肽及其在制药中的应用，具体涉及高度稳定且可靶向葡萄糖调节蛋白GRP78的多功能D构型多肽，及L构型多肽和稳定性D构型多肽的药物复合物和修饰的纳米递药系统，尤其涉及D构型多肽DVAP(D构型氨基酸序列DPDADVDRDTDNDS)和SVAP(D构型氨基酸序列DSDNDTDRDVDADP)，及L构型多肽LVAP(氨基酸序列SNTRVAP)和稳定性D构型多肽的诊断和治疗药物复合物、修饰的高分子载体材料及其所构建的脂质体、聚合物胶束、聚合物圆盘、纳米粒等纳米递药系统，以及在制备肿瘤诊断和靶向治疗药物中的应用。
背景技术
文献报道了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首位。传统的化疗作为肿瘤药物治疗的主要手段，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。因此，为克服传统治疗手段的局限性，近年来，主动靶向成为提高肿瘤组织靶向效率的重要策略。资料公开了主动靶向策略主要针对肿瘤组织中高表达的受体或转运体，利用与特异性受体或转运体具有识别、结合能力的对应配体，将药物或纳米递药系统递送至肿瘤组织或细胞中，常用的对应配体包括单克隆抗体、多肽、核酸适体、小分子化合物等；配体修饰后的药物或纳米递药系统可通过细胞表面受体或转运体与配体的特异性识别、结合、内化，将药物递送至肿瘤组织和细胞内，从而实现药物对肿瘤的主动靶向目标。
葡萄糖调节蛋白GRP78又名免疫球蛋白重链结合蛋白(Bip)，是内质网主要的分子伴侣之一，在蛋白质的折叠以及内质网应激反应中发挥着重要的作用。研究显示，若干肿瘤细胞株，实体瘤以及人癌组织活检样本中GRP78的表达明显升高，研究表明，GRP78蛋白在乳腺癌、肝癌、结肠癌、胃癌等多种肿瘤中均高表达，并且与这些肿瘤的发生、进展、预后和耐药密切相关。最近有研究表明，GRP78在肿瘤细胞中可以转移到细胞膜表面，而在正常细胞的膜表面却没有观察到这种转移；研究还表明GRP78在肿瘤干细胞的调控中发挥关 键作用，通过Cripto/GRP78信号通路调节成体干细胞以及肿瘤干细胞的功能，维持干细胞干性，因此，通过GRP78受体介导途径将药物或纳米递药系统特异性递送到肿瘤组织，对提高药物的肿瘤诊断与治疗效果将具有重要价值。
LVAP(L构型氨基酸序列SNTRVAP)是通过噬菌体展示技术筛选出的一条七肽，研究证明其对GRP78具有高亲和活性的，但迄今尚未见有在肿瘤靶向诊疗方面的研究报道。
基于现有技术的现状，本申请的发明人拟提供VAP多肽在肿瘤靶向诊治中的应用并进一步优化已有多肽的稳定性，达到更好的体内肿瘤靶向效果。
发明内容
本发明的目的是针对现有技术的缺陷，提供VAP多肽及其肿瘤靶向诊治中的应用，并进一步优化已有多肽的稳定性，达到更好的体内肿瘤靶向效果。
本发明的第一方面提供了一种D构型多肽，所述D构型多肽为DVAP和/或SVAP，且所述DVAP的氨基酸序列为DPDADVDRDTDNDS，所述SVAP的氨基酸序列DSDNDTDRDVDADP。
本发明的第二方面提供了一种DVAP和/或SVAP多肽复合物，所述DVAP和/或SVAP多肽复合物为权利要求1所述的DVAP和/或SVAP多肽修饰含有马来酰亚胺基团的影像物质，其中，所述DVAP和/或SVAP多肽复合物的结构为DVAP-X和/或SVAP-X，X为所述影像物质；优选地，所述X选自荧光物质、近红外染料、磁共振影像剂和放射影像剂中的一种或多种；
更优选地，所述荧光物质为Fluorescein，近红外染料为Cy7、IR820、DiR，所述磁共振影像剂为Gd-DTPA，所述放射影像剂99mTc-DTPA。
本发明的第三方面提供了一种L构型的LVAP多肽复合物，所述LVAP多肽复合物为LVAP多肽修饰含有马来酰亚胺基团的影像物质，其中，所述LVAP多肽的氨基酸序列为SNTRVAP，且所述LVAP多肽复合物的结构为LVAP-X，X为所述影像物质；
优选地，所述X选自荧光素、近红外染料、磁共振影像剂和放射影像剂中的一种或多种；
更优选地，所述荧光素为Fluorescein，近红外染料为Cy7、IR820、 DiR，所述磁共振影像剂为Gd-DTPA，所述放射影像剂99mTc-DTPA。
本发明的第四方面提供了一种DVAP和/或SVAP多肽复合物，所述DVAP和/或SVAP多肽复合物为权利要求1所述的DVAP和/或SVAP多肽修饰抗肿瘤药物，其中，所述DVAP和/或SVAP多肽复合物的结构为DVAP-Y和/或SVAP-Y，Y为所述抗肿瘤药物；
优选地，所述抗肿瘤药物选自阿霉素和表阿霉素等蒽环类药物、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类药物、喜树碱和羟基喜树碱和伊立替康等喜树碱类药物、长春新碱和长春瑞滨等长春花碱类药物、硼替佐米和卡非佐米等蛋白酶体抑制剂、小白菊内酯等内酯类药物、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物中的一种或多种；
更优选地：所述抗肿瘤药物选自含酮或醛基的阿霉素或表阿霉素，含羟基或氨基的紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康，含硼酸基团的硼替佐米或卡非佐米，和/或多肽药物p53激活肽、蜂毒肽和蝎毒肽中的一种或多种。
本发明的第五方面提供了一种L构型的LVAP多肽复合物，所述LVAP多肽复合物为LVAP多肽修饰抗肿瘤药物，其中，所述LVAP多肽的氨基酸序列为SNTRVAP，且所述LVAP多肽复合物的结构为LVAP-Y，Y为所述抗肿瘤药物；
优选地，所述抗肿瘤药物选自阿霉素和表阿霉素等蒽环类药物、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类药物、喜树碱和羟基喜树碱和伊立替康等喜树碱类药物、长春新碱和长春瑞滨等长春花碱类药物、硼替佐米和卡非佐米等蛋白酶体抑制剂、小白菊内酯等内酯类药物、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物中的一种或多种；
更优选地：所述抗肿瘤药物选自含酮或醛基的阿霉素或表阿霉素，含羟基或氨基的紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康，含硼酸基团的硼替佐米或卡非佐米，和/或多肽药物p53激活肽、蜂毒肽和蝎毒肽中的一种或多种。
本发明的第六方面提供了一种DVAP和/或SVAP多肽复合物，所 述DVAP和/或SVAP多肽复合物为权利要求1所述的DVAP和/或SVAP多肽修饰高分子载体材料，其中，所述DVAP和/或SVAP多肽复合物的结构为DVAP-聚乙二醇-Z和/或SVAP-聚乙二醇-Z，Z为所述高分子载体材料；
优选地，所述高分子载体材料选自磷脂、聚乳酸、乳酸羟基乙酸共聚物和聚已内酯中的一种或多种。
本发明的第七方面提供了一种L构型的LVAP多肽复合物，所述LVAP多肽复合物为LVAP多肽修饰高分子载体材料，其中，所述，所述LVAP多肽的氨基酸序列为SNTRVAP，且所述LVAP多肽复合物的结构为LVAP-聚乙二醇-Z，Z为所述高分子载体材料；
优选地，所述高分子载体材料选自磷脂、聚乳酸、乳酸羟基乙酸共聚物和聚已内酯中的一种或多种。
本发明的第八方面提供了一种递药系统，所述递药系统包括第六方面或第七方面所述的复合物；优选地，所述递药系统为脂质体递药系统、聚合物胶束递药系统、聚合物圆盘递药系统或纳米粒递药系统。
根据本发明第八方面所述的递药系统，所述递药系统还包括所述DVAP、SVAP和/或LVAP多肽复合物以外的(1)诊断药物和/或(2)抗肿瘤药物；优选地：
所述(1)诊断药物选自荧光物质、近红外染料和磁共振影像剂中的一种或多种，更优选地，所述荧光物质为Fluorescein，所述近红外染料选自Cy7、IR820、DiR，和/或所述磁共振影像剂为Gd-DTPA，和/或
所述(2)抗肿瘤药物选自：阿霉素和表阿霉素等蒽环类药物、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类药物、喜树碱和羟基喜树碱和伊立替康等喜树碱类药物、长春新碱和长春瑞滨等长春花碱类药物、硼替佐米和卡非佐米等蛋白酶体抑制剂、小白菊内酯等内酯类药物、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物中的一种或多种。
本发明第九方面提供了第一方面所述的DVAP和/或SVAP多肽、第二方面至第七方面所述的DVAP、SVAP和/或LVAP多肽复合物、第八方面所述的递药系统在制备用于诊断、示踪和/或治疗肿瘤的药品或医疗产品中的应用，优选地：
所述肿瘤为高表达葡萄糖调节蛋白GRP78肿瘤。
本发明的第十方面提供了一种用于肿瘤诊断和/或靶向治疗的方法，向有需要的受试者给予：
根据第一方面所述的D构型多肽；
根据第二方面至第七方面任一项所述的DVAP、SVAP和/或LVAP
多肽复合物；和/或
第八方面所述的递药系统。
本发明的稳定化D构型VAP多肽，所述D构型VAP多肽为DVAP和/或SVAP，且所述DVAP的氨基酸序列为DPDADVDRDTDNDS，所述SVAP的氨基酸序列DSDNDTDRDVDADP。
具体的，本发明设计并制备了D构型多肽DVAP和/或SVAP，两条多肽均对血清具有高稳定性、与GRP78具有高亲和力。
本发明的D构型多肽或文献报道的L构型多肽(LVAP，氨基酸序列为SNTRVAP)复合物，所述的DVAP、SVAP、LVAP多肽复合物为DVAP、SVAP、LVAP修饰影像物质，其中，所述DVAP、SVAP、LVAP多肽复合物的结构为DVAP-X、SVAP-X和/或LVAP-X，X为所述影像物质。
优选地，所述X选自荧光素、近红外染料、磁共振影像剂和放射影像剂中的一种或多种，更优选地，所述荧光素为Fluorescein，所述近红外染料选自cy7、IR820和DiR中的一种或多种，所述磁共振影像剂为Gd-DTPA，所述放射影像剂99mTc-DTPA。
具体地，本发明的DVAP、SVAP或文献报道的LVAP巯基化后，可利用其分子中巯基与马来酰亚胺功能化荧光物质(如Fluorescein、近红外染料Cy7、IR820、DiR等)、磁共振影像剂Gd-DTPA和放射影像剂99mTc-DTPA，反应而形成复合物。
本发明的DVAP、SVAP或文献报道的LVAP复合物，所述DVAP、SVAP、LVAP多肽复合物为DVAP、SVAP、LVAP修饰抗肿瘤药物，其中，所述DVAP、SVAP、LVAP多肽复合物的结构为DVAP-Y、SVAP-Y和/或LVAP-Y，Y为所述抗肿瘤药物。
优选地，所述抗肿瘤药物选自阿霉素和表阿霉素等蒽环类药物、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类药物、喜树碱和羟基喜树碱和伊立替康等喜树碱类药物、长春新碱和长春瑞滨等长春花碱类药物、硼替佐米和卡非佐米等蛋白酶体抑制剂、小白菊内酯等内酯类药物、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物中的一种或多种。
更优选地：所述抗肿瘤药物选自含酮或醛基的阿霉素或表阿霉素，含羟基或氨基的紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9- 硝基喜树碱、长春新碱、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康，含硼酸基团的硼替佐米或卡非佐米，和/或多肽药物p53激活肽、蜂毒肽和蝎毒肽中的一种或多种。
具体地，本发明的DVAP、SVAP或文献报道的LVAP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、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康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、抗菌肽、多肽毒素等多肽药物)的多肽-药物复合物。
本发明的DVAP、SVAP或文献报道的LVAP复合物，所述DVAP、SVAP、LVAP多肽复合物为DVAP、SVAP、LVAP修饰高分子载体材料，其中，所述DVAP、SVAP、LVAP多肽复合物的结构为DVAP-聚乙二醇-Z、SVAP-聚乙二醇-Z和/或LVAP-聚乙二醇-Z，Z为所述高分子载体材料。
优选地，所述高分子载体材料选自磷脂、聚乳酸、乳酸羟基乙酸共聚物和聚已内酯中的一种或多种。
具体地，本发明的DVAP、SVAP或文献报道的LVAP巯基化后，可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，可用于DVAP、SVAP、LVAP修饰的脂质体、聚合物胶束、聚合物圆盘、纳米粒等纳米递药系统的构建。
本发明的递药系统，所述递药系统包括前述的DVAP、SVAP、LVAP多肽复合物。优选地，所述递药系统为脂质体递药系统、聚合物胶束递药系统、聚合物圆盘递药系统或纳米粒递药系统。
作为优选实施方式，本发明还提供了包括所述DVAP、SVAP、LVAP多肽复合物以外的(1)诊断药物和/或(2)抗肿瘤药物的前述递药系统。
优选地，所述(1)诊断药物选自荧光物质、近红外染料和磁共振影像剂中的一种或多种。更优选地，所述荧光物质为Fluorescein，所述近红外染料选自Cy7、IR820、DiR中的一种或多种，和/或磁共振影像剂为Gd-DTPA。和/或所述(2)抗肿瘤药物选自阿霉素和表阿霉素等蒽环类药物、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类药物、喜树碱和羟基喜树碱和伊立替康等喜树碱类药物、长春新碱和长春瑞滨等长春花碱类药物、硼替佐米和卡非佐米等蛋白酶体抑制剂、小白菊内酯等内酯类药物、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物中的一种或多种。
具体地，本发明所设计的DVAP、SVAP、LVAP修饰的纳米递药系统可包载阿霉素和表阿霉素等蒽环类药物、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类药物、喜树碱和羟基喜树碱和伊立替康等喜树碱类药物、长春新碱和长春瑞滨等长春花碱类药物、硼替佐米和卡非佐米等蛋白酶体抑制剂、小白菊内酯等内酯类药物、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物等；也可包载荧光物质、近红外染料和磁共振影像剂，如Fluorescein、Cy7、IR820、DiR、Gd-DTPA等。
本发明还提供了前述的DVAP、SVAP多肽、前述的DVAP、SVAP、LVAP多肽复合物、前述的递药系统在制备用于诊断、示踪和/或治疗肿瘤的药品或医疗产品中的应用。
优选地：
所述肿瘤为高表达GRP78肿瘤。
具体地，本发明第一方面的DVAP、SVAP或文献报道的LVAP可介导药物或纳米递药系统靶向GRP78高表达的细胞及其组织，用于肿瘤的靶向诊断和治疗。
本发明还提供了一种用于诊断、示踪和/或治疗肿瘤的组合产品，所述组合产品包括选自以下的一种或多种成分：前述的DVAP、SVAP、LVAP多肽复合物和前述的递药系统。
优选地，所述组合产品为试剂盒，和/或
所述肿瘤为高表达GRP78肿瘤。
本发明的一种诊断、示踪和/或治疗肿瘤的方法，包括对患有所述肿瘤或疑患有所述肿瘤的患者通过口服或非口服途径给予有效剂量的选自以下的一种或多种物质：前述的DVAP、SVAP、LVAP多肽 复合物、前述的递药系统和前述的组合产品。
优选地，所述肿瘤为高表达GRP78肿瘤；和/或
优选地，所述口服或非口服途径可以为通过口服、注射、贴片、喷雾和其他已知的一种或多种递送给所述患者。所述有效量可以包括对治疗、降低、缓和、减轻、消除或状况的一种或多种症状有效的量，所述状况寻求被治疗，或可选地，所述状况寻求被避免，或另外在所述状况或其效果中产生临床上可确认的有利变化。
本发明提供了前述的DVAP、SVAP、LVAP多肽在制备肿瘤靶向产品中的应用。优选地：所述肿瘤靶向产品用于靶向GRP78高表达的肿瘤；和/或所述肿瘤靶向产品为用于诊断、示踪和/或治疗肿瘤的药品、实验试剂和/或医疗产品。本发明提供了LVAP多肽(SNTRVAP)修饰的药物复合物和修饰的纳米递药系统；同时针对L构型多肽的体内稳定性差，在血液中易降解，可能导致肿瘤靶向能力降低的问题，提供了高度稳定且与GRP78有高度结合活性的D构型多肽靶向分子DVAP(D构型氨基酸序列DPDADVDRDTDNDS)与SVAP(D构型氨基酸序列DSDNDTDRDVDADP)，并构建其药物复合物和修饰的纳米递药系统，能实现肿瘤的靶向诊断和治疗，获得更好的体内肿瘤靶向效果。
具体的，本发明制备了具有高稳定性的D构型多肽DVAP(D构型氨基酸序列DPDADVDRDTDNDS)和SVAP(D构型氨基酸序列DSDNDTDRDVDADP)，并以LVAP(L构型氨基酸序列SNTRVAP)、DVAP和SVAP修饰药物分子和高分子载体材料，构建VAP药物复合物、VAP修饰的纳米递药系统。
本发明中，利用固相多肽合成技术，设计并制备了D构型多肽靶向分子DVAP(D构型氨基酸序列DPDADVDRDTDNDS)与SVAP(D构型氨基酸序列DSDNDTDRDVDADP)，两条多肽均对血清具有高稳定性、与GRP78具有高亲和力。
本发明中，LVAP和所设计的DVAP、SVAP连接半胱氨酸后，可利用其分子中巯基与马来酰亚胺功能化影像物质(荧光物质Fluorescein、近红外染料Cy7、IR820、DiR、磁共振影像剂Gd-DTPA、放射影像剂99mTc-DTPA等)反应而形成复合物。
本发明中，LVAP和所设计的DVAP、SVAP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、喜树碱、羟基喜树碱、9-硝基 喜树碱、长春新碱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、抗菌肽、多肽毒素等多肽药物)的多肽-药物复合物。
本发明中，LVAP和所设计的DVAP以及SVAP连接半胱氨酸后，可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LVAP、DVAP和SVAP修饰的脂质体、聚合物胶束、聚合物圆盘、纳米粒等纳米递药系统的构建。
本发明中，所设计的LVAP、DVAP和SVAP修饰的纳米递药系统包载阿霉素和表阿霉素等蒽环类、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类、喜树碱和羟基喜树碱和伊立替康等喜树碱类、长春新碱和长春瑞滨等长春花碱类、硼替佐米和卡非佐米等蛋白酶体抑制剂类、小白菊内酯等内酯类、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类等抗肿瘤药物；或包载影像物质，如Fluorescein，近红外染料Cy7、IR820、DiR、磁共振影像剂Gd-DTPA等。
本发明所述的DVAP、SVAP和LVAP可介导药物或纳米递药系统靶向葡萄糖调节蛋白GRP78高表达的细胞及其组织，用于肿瘤的靶向诊断和治疗。
本发明提供了DVAP、SVAP制备和性质考察以及上述LVAP、DVAP和SVAP所修饰的药物复合物和纳米递药系统用于制备肿瘤诊疗药物的物质基础；以及进行了LVAP、DVAP和SVAP介导的体内主动靶向的试验，其中包括，
1.VAP、VAP-Cys及其荧光标记物(VAP-Fluorescein、VAP-Cy7)的合成
采用固相合成方法制备LVAP、LVAP-Cys、DVAP、DVAP-Cys、SVAP、SVAP-Cys。通过马来酰亚胺基团与巯基的Michael加成反应合成了LVAP-Fluorescein、DVAP-Fluorescein、SVAP-Fluorescein、LVAP-Cy7、DVAP-Cy7、SVAP-Cy7。HPLC、MS表征结构。
2.VAP的稳定性和受体亲和性评价
从血清稳定性、与葡萄糖调节蛋白GRP78结合能力和与高表达这种蛋白的细胞摄取能力三方面进行DVAP、SVAP性质的考察。将DVAP、SVAP和LVAP分别与小鼠血清在37℃进行孵育，在不同时间点检测多肽的浓度进行稳定性的比较。采用表面等离子共振法评价DVAP、SVAP和LVAP与GRP78的结合能力，比较DVAP-Fluorescein、 SVAP-Fluorescein、LVAP-Fluorescein对GRP78高表达的细胞(如：脐静脉内皮细胞HUVEC)和模型肿瘤细胞(如：脑胶质瘤细胞U87)的体外靶向性，比较体外3D肿瘤球模型对DVAP-Fluorescein、SVAP-Fluorescein、LVAP-Fluorescein的摄取能力。
3.VAP药物复合物的制备
连接半胱氨酸后的LVAP、DVAP和SVAP与药物的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；
连接半胱氨酸后的LVAP、DVAP和SVAP与药物的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、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱等含羟基或氨基的药物；
LVAP、DVAP和SVAP通过修饰上多巴胺进而与药物的硼酸基团反应，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；
LVAP、DVAP和SVAP通过固相合成直接与多肽药物缩合制成融合多肽，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。
4、VAP-阿霉素复合物的体内药效与药动学
连接半胱氨酸后的DVAP与阿霉素马来酰亚胺己肼衍生物(MAL-DOX)缩合得的DVAP-阿霉素复合物(DVAP-DOX)，通过荷U87皮下移植瘤模型裸鼠尾静脉给药，以瘤体积、瘤重和抑瘤率为指标评价其体内抗肿瘤效果；通过荷U87原位瘤模型裸鼠尾静脉给药，以中位生存时间为指标评价其体内抗肿瘤效果；通过小鼠尾静脉注射给药，荧光法定量绘制药物动力学曲线与体内分布。
5.VAP-PEG-PLA胶束递药系统的构建与表征
首先合成LVAP、DVAP和SVAP修饰的高分子材料LVAP-PEG-PLA、DVAP-PEG-PLA和SVAP-PEG-PLA。通过连接半胱氨酸的多肽上的游离巯基与Mal-PEG-PLA所含马来酰亚胺的反应实现材料的合成。将Mal-PEG-PLA溶解在乙腈中，旋转蒸发，成膜，加入含多肽的PBS(pH 8.0)反应制备得到LVAP-PEG-PLA、DVAP-PEG-PLA和SVAP-PEG-PLA；
然后分别制备LVAP、DVAP、SVAP修饰的胶束(LVAP-Micelle、DVAP-Micelle、SVAP-Micelle)。一定量的VAP-PEG-PLA、mPEG-PLA和药物(香豆素C6、DiR或者紫杉醇)采用成膜法制备胶束，激光散射粒度仪表征胶束粒径和粒径分布。
6.VAP-Micelle的体内外肿瘤靶向性评价
考察U87细胞、HUVEC细胞和U87肿瘤球体外模型对LVAP-Micelle/C6、DVAP-Micelle/C6、SVAP-Micelle/C6和mPEG-Micelle/C6的摄取情况；
通过荷U87皮下移植瘤模型裸鼠尾静脉分别注射LVAP-Micelle/DiR、DVAP-Micelle/DiR、SVAP-Micelle/DiR和mPEG-Micelle/DiR，比较不同组在各时间点的肿瘤内分布。
7.VAP-Micelle/PTX的体内抗肿瘤效果评价
通过荷U87皮下移植瘤模型裸鼠尾静脉分别注射LVAP-Micelle/PTX、SVAP-Micelle/PTX、DVAP-Micelle/PTX和mPEG-Micelle/PTX、临床用制剂泰素、生理盐水，以瘤体积、瘤重和肿瘤组织细胞凋亡、新生血管和拟态血管数量为指标评价不同载紫杉醇递药系统的体内抗肿瘤效果。
本发明提供了DVAP、SVAP制备和性质考察以及上述LVAP、DVAP和SVAP所修饰的药物复合物和纳米递药系统用于制备肿瘤诊疗药物的物质基础；本发明的试验结果表明：LVAP、DVAP和SVAP均可介导的体内主动靶向；与LVAP相比，DVAP和SVAP血清中稳定性更好，因而其介导的体内主动靶向效果更优。
附图的简要说明
图1示出了DVAP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：0-30min 5％B-65％B；流速：0.7mL/min；柱温：40℃；检测：UV 214nm，保留时间：11.1min。ESI-MS：743.5，与理论分子量相符合。
图2示出了DVAP-Cys的HPLC和ESI-MS图谱：
色谱方法同上，保留时间：12.0min。ESI-MS：846.5，与理论分子量相符合。
图3示出了SVAP的HPLC和ESI-MS图谱：
色谱方法同上，保留时间：10.8min。ESI-MS：743.5，与理论分子量相符合。
图4示出了SVAP-Cys的HPLC和ESI-MS图谱：
色谱方法同上，保留时间：11.5min。ESI-MS：846.5，与理论分子量相符合。
图5示出了LVAP的HPLC和ESI-MS图谱：
色谱方法同上，保留时间：9.5min。ESI-MS：743.5，与理论分子量相符合。
图6示出了LVAP-Cys的HPLC和ESI-MS图谱，
色谱方法同上，保留时间：9.6min。ESI-MS：846.5，与理论分子量相符合。
图7示出了DVAP-Fluorescein的HPLC和ESI-MS图谱：
色谱方法同上，保留时间：15.3min。ESI-MS：1274.4，与理论分子量相符合。
图8示出了SVAP-Fluorescein的HPLC和ESI-MS图谱：
色谱方法同上，保留时间：16.9min。ESI-MS：1274.4，与理论分子量相符合。
图9示出了LVAP-Fluorescein的HPLC和ESI-MS图谱：
色谱方法同上，保留时间：16.5min。ESI-MS：1274.4，与理论分子量相符合。
图10示出了DVAP-Cy7的HPLC和ESI-MS图谱：
色谱方法同上，保留时间：26.5min。ESI-MS：1535.8，与理论分子量相符合.
图11示出了SVAP-Cy7的HPLC和ESI-MS图谱：
色谱方法同上，保留时间：26.5min。ESI-MS：1535.8，与理论分子量相符合。
图12示出了LVAP-Cy7的HPLC和ESI-MS图谱，
色谱方法同上，保留时间：26.5min。ESI-MS：1535.8，与理论分子量相符合。
图13示出了DVAP-DOX的HPLC和ESI-MS图谱：
色谱方法除流动相中0.1％三氟乙酸替换成0.1％甲酸外，其他方法同上，保留时间：15.5min。ESI-MS：1599.6，与理论分子量相符合。
图14示出了DVAP-PEG3000-PLA2000、SVAP-PEG3000-PLA2000和LVAP-PEG3000-PLA2000的1H-NMR图谱：
Mal-PEG-PLA的核磁图谱于6.7ppm显示出马来酰亚胺峰，而VAP-PEG-PLA的核磁图谱中该峰消失，显示Mal-PEG-PLA中的马来酰亚胺基团已反应完全。
图15示出了DVAP、SVAP和LVAP的血清稳定性：
图纵坐标为完整多肽的残留百分比，可见DVAP和SVAP在50％小鼠血清中的稳定性显著高于LVAP，孵育2h，LVAP完全降解、DVAP 和SVAP几乎不降解。
图16示出了DVAP、SVAP和LVAP与GRP78结合活性：
DVAP、SVAP和LVAP与GRP78的结合活性相似，稍弱于LVAP，KD值分别为4.010μM和5.223μM、2.696μM。三条多肽的解离模式相似，Kd值分别为0.02091 1/s、0.02826 1/s、0.01898 1/s。
图17示出了GRP78的细胞内分布：
在肿瘤细胞U87细胞上，及脐静脉细胞HUVEC上，GRP78广泛分布于细胞膜上及细胞内。而在正常细胞HEK293细胞膜上未见分布，膜内有少量GRP78分布。
图18示出了脑胶质瘤细胞U87对Fluorescein标记多肽的摄取：
图A和图B分别为Fluorescein标记的DVAP、SVAP和LVAP与U87细胞作用4h后的激光共聚焦照片和流式细胞荧光检测结果。可见U87细胞对DVAP、SVAP和LVAP的摄取明显高于游离荧光素，对DVAP、SVAP的摄取没有明显差异，稍弱于LVAP。
图19示出了脐静脉内皮细胞HUVEC对Fluorescein标记多肽的摄取：
图A和图B分别为Fluorescein标记的DVAP、SVAP和LVAP与HUVEC细胞作用4h后的激光共聚焦照片和流式细胞荧光检测结果。可见HUVEC细胞对DVAP、SVAP和LVAP的摄取明显高于游离荧光素，对DVAP、SVAP的摄取没有明显差异，稍弱于LVAP。
图20示出了Fluorescein标记多肽及VAP-Micelle/C6的U87肿瘤球摄取：
图为各Fluorescein标记多肽及VAP胶束被U87肿瘤球摄取情况，由图可知，各Fluorescein标记多肽及VAP胶束均能很好的被U87肿瘤球摄取，与无靶胶束和FAM有明显差异。
图21示出了DVAP、SVAP和LVAP竞争抑制实验：
可见DVAP、SVAP和LVAP三条多肽能够相互完全抑制，证明三条多肽均结合在GRP78蛋白的同一位点。
图22示出了Cy7标记多肽的皮下移植瘤内分布：
图A为荷U87皮下移植瘤裸鼠尾静脉注射Cy7标记多肽1h后的离体肿瘤影像分布结果；图B为给药后各个时间点荧光半定量结果；图C为离体肿瘤和脏器的荧光分布图像；图D为离体肿瘤荧光半定量结果，Cy7标记的DVAP、SVAP和LVAP在肿瘤内的蓄积均显著高于游离Cy7(***p<0.001)，且肿瘤靶向效果依次为：DVAP≈SVAP>LVAP。
图23示出了DVAP-DOX皮下瘤抑制实验：
图A为各组裸鼠肿瘤体积随时间变化的曲线，与生理盐水组相 比，各给药组对肿瘤生长均有抑制作用。同剂量DVAP-DOX的药效显著优于DOX和MAL-DOX，明显优于RGD-DOX。图B为将裸鼠处死取出肿瘤组织后称重并进行统计分析结果，图C为离体肿瘤组织照片，可见同剂量DVAP-DOX组肿瘤大小与瘤重显著低于其它各组。
图24示出了DVAP-DOX抗原位脑胶质瘤模型裸鼠的生存曲线：
PBS组、DOX组、MAL-DOX组、DVAP-DOX组(高、中、低)和DVAP组的平均生存时间分别为36、36、48、58、52、48.5和45.5天。结果表明，与其余各组相比，DVAP-DOX延长脑胶质瘤模型裸鼠的生存时间效果最显著，其最低剂量即可达到4倍剂量的MAL-DOX抗脑胶质瘤效果。
图25示出了DVAP-DOX的小鼠体内药物动力学曲线与分布：
图A为药动学曲线，图B为体内主要脏器分布。结果表明，DVAP-DOX的AUC虽然仍显著低于MAL-DOX，但比游离DOX有明显增加。而且，从体内主要脏器的分布结果可见，DVAP-DOX可显著降低药物在心脏中的分布，这提示DVAP-DOX可能可以降低阿霉素用药后对心脏的毒副作用。
图26示出了载DiR的VAP胶束的皮下瘤内分布：
图A为尾静脉注射24h后的在体荧光分布图像，从左到右依次为PBS组，mPEG-Micelle/DiR、LVAP-Micelle/DiR、DVAP-Micelle/DiR和SVAP-Micelle/DiR。图B为脏器的荧光分布图像。结果表明DVAP或SVAP修饰的胶束能更好地靶向至肿瘤部位。
图27示出了载紫杉醇胶束的粒径：
图为各紫杉醇胶束的粒径图片。由图可知，各处方胶束大小均无显著差异。
图28示出了载紫杉醇胶束体外抗U87细胞和HUVEC细胞活性曲线：
图A和图B分别为mPEG-Micelle/PTX、DVAP-Micelle/PTX、SVAP-Micelle/PTX、LVAP-Micelle/PTX和泰素抗U87细胞和HUVEC细胞的活性曲线，图A表明U87细胞给药4h培养72h后，其IC50分别为0.73、0.09、0.12、0.59和0.75μM，四种胶束均能抑制U87细胞的体外生长，其中DVAP-Micelle/PTX、SVAP-Micelle/PTX的体外活性分别为LVAP-Micelle/PTX的6.56倍和4.92倍，图B表明HUVEC细胞给药4h培养72h后，其IC50分别为0.52、0.04、0.04、0.23和0.74μM，四种胶束均能抑制HUVEC细胞的体外生长，其中DVAP-Micelle/PTX、SVAP-Micelle/PTX的体外活性均为LVAP-Micelle/PTX的5.75。
图29示出了载紫杉醇胶束体外对新生血管形成的抑制：
图为mPEG-Micelle/PTX、DVAP-Micelle/PTX、SVAP-Micelle/PTX、LVAP-Micelle/PTX和泰素对新生血管体外模型的抑制照片，相比于LVAP-Micelle/PTX，DVAP-Micelle/PTX和SVAP-Micelle/PTX抑制新生血管的形成更显著。
图30示出了载紫杉醇胶束体外对拟态血管形成的抑制：
图为mPEG-Micelle/PTX、DVAP-Micelle/PTX、SVAP-Micelle/PTX、LVAP-Micelle/PTX和泰素对拟态血管体外模型的抑制照片，相比于LVAP-Micelle/PTX，DVAP-Micelle/PTX和SVAP-Micelle/PTX抑制拟态血管的形成更显著。
图31示出了载紫杉醇胶束皮下瘤抑制实验：
图A为各组裸鼠肿瘤体积随时间变化的曲线，与生理盐水组相比，各给药组对肿瘤生长均有抑制作用。DVAP-Micelle/PTX、SVAP-Micelle/PTX与LVAP-Micelle/PTX相比具有极显著性差异(n＝8，***p<0.001)。将裸鼠处死取出肿瘤组织后称重并进行统计分析(图B)，发现DVAP-Micelle/PTX和SVAP-Micelle/PTX组瘤重显著低于LVAP-Micelle/PTX(n＝8，***p<0.001)。
图32示出了TUNEL染色结果：
图为mPEG-Micelle/PTX、DVAP-Micelle/PTX、SVAP-Micelle/PTX、LVAP-Micelle/PTX促进皮下瘤凋亡的TUNEL染色照片(bar＝50μm)，其中凋亡的阳性细胞核呈棕黄色或棕褐色。
图33示出了CD31/PAS双染色结果：
图为mPEG-Micelle/PTX、DVAP-Micelle/PTX、SVAP-Micelle/PTX、LVAP-Micelle/PTX抑制新生血管形成的CD31/PAS双染色照片(bar＝100μm)，其中新生血管细胞核呈棕黄色或棕褐色。
实施发明的最佳方式
通过下述实施例将有助于进一步理解本发明，但本发明不局限于如下描述范围。
实施例1：VAP、VAP-Fluorescein、VAP-Cy7、VAP-药物、 VAP-PEG-PLA的合成与表征
1.DVAP、SVAP和LVAP，DVAP-Cys、SVAP-Cys和LVAP-Cys的合成与表征
采用固相多肽合成法，设计并合成由D构型氨基酸所构成的 DVAP(序列为DPDADVDRDTDNDS)、DVAP-Cys(序列为DCDPDADVDRDTDNDS)和SVAP(序列为DSDNDTDRDVDADP)、SVAP-Cys(序列为DSDNDTDRDVDADPDC)以及L构型氨基酸所构成的LVAP(序列为SNTRVAP)和LVAP-Cys(序列为SNTRVAPC)。
具体方法：以Boc固相多肽合成法，在PAM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。反应完成后，将树脂用含P-cresol的氟化氢进行切割，冰浴搅拌反应1h。反应结束后减压抽去管中氟化氢，冰乙醚沉淀并洗涤沉淀3次，沉淀以20％乙腈重新溶解，收集滤液后旋蒸，得到多肽粗品溶液。多肽粗品用乙腈/水(含0.1％TFA)体系分离纯化。HPLC和ESI-MS表征DVAP、DVAP-Cys、SVAP、SVAP-Cys、LVAP及LVAP-Cys的纯度和分子量(Mw)。HPLC图谱、质谱图见附图1、图2、图3、图4、图5和图6。
2.VAP-Fluorescein与VAP-Cy7的合成与表征
将上述步骤得到的DVAP-Cys、SVAP-Cys或LVAP-Cys溶于0.1M的PBS溶液中(pH7.2)，取Fluorescein-5-maleimide溶于DMF，两者混合后磁力搅拌反应，HPLC监测，待DVAP-Cys、SVAP-Cys或LVAP-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DVAP-Fluorescein、SVAP-Fluorescein或LVAP-Fluorescein纯品。HPLC图谱、质谱图见附图7、8、9。
VAP-Cy7的制备方法同上。HPLC图谱、质谱图见附图10、11、12。
3.VAP-DTPA-Gd与VAP-DTPA-99mTc的制备
maleimide-DTPA溶于DMF，同上与DVAP-Cys、SVAP-Cys或LVAP-Cys溶于的PBS溶液混合搅拌反应，制备液相纯化，冷冻干燥得DVAP-DTPA、SVAP-DTPA或LVAP-DTPA纯品，螯合Gd或99mTc即得VAP-DTPA-Gd或VAP-DTPA-99mTc。
4.VAP-药物复合物的制备
以VAP-阿霉素复合物制备作为VAP连接含酮或醛基药物的实施例。9.4mg巯基化VAP(DVAP或SVAP或LVAP)多肽溶于磷酸盐3mL缓冲液(0.1mM，pH 7.0)，加入10倍摩尔量的三(2-羧乙基)膦(TCEP)，于4℃搅拌20min。然后加入4倍摩尔量的阿霉素6-马来酰亚胺己肼衍生物(MAL-DOX)，于室温避光反应1h。反应液用制备液相纯化，冷冻干燥得LVAP或DVAP或SVAP-阿霉素复合物，HPLC、MS表征结构，结果见附图13。
以VAP-紫杉醇复合物作为VAP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VAP-硼替佐咪复合物作为VAP连接含硼酸基团药物的实施例：依照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pH7.4的缓冲液中，多肽-多巴胺衍生物与硼替佐咪以摩尔比1:1混合即得多肽-硼替佐咪复合物。
以VAP-PMI融合多肽作为VAP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VAP-PMI多肽序列后，按与制备VAP相同的方法依次接入氨基酸，经HF切割并纯化后得VAP-PMI融合多肽。
5.VAP-PEG-PLA的合成与表征
通过多肽的游离巯基与Mal-PEG-PLA所含马来酰亚胺的反应实现膜材料的合成。将40mg Mal-PEG-PLA溶解在5mL乙腈中，旋转蒸发，成膜，加入3mL PBS(pH8.0，0.2M)在37℃水化形成胶束，8h内加入9.6mg VAP-Cys并反应过夜，HPLC检测反应。过量的VAP-Cys通过透析除去，冻干，1H-NMR表征(图14)。
实施例2:VAP的血清稳定性考察
将DVAP、SVAP及LVAP配成1mg/mL水溶液，取0.1mL加入0.9mL的25％小鼠血清中，37℃孵育，分别于0和15min，0.5、1、2和4h取出100μL反应液，加入20μL三氯乙酸(TCA)沉淀血清中蛋白，4℃静置20min，12000转/分钟离心10min，取上清液20μL进行HPLC分析。血清稳定性结果(图15)表明，DVAP和SVAP具有比LVAP更好的血清稳定性。
实施例3：VAP与葡萄糖调节蛋白GRP78的结合活性实验
通过biacore系统进行预结合分析，选取pH5.0为最佳GRP78与CM5芯片结合pH。将重组人GRP78偶联至CM5芯片上，RU值达到目标值。将DVAP、SVAP及LVAP分别配置成浓度为0.3125、0.625、1.25、2.5、5、10和20μM的样品溶液。从低到高依次进样，用Biacore T200Evaluation software软件分析DVAP、SVAP及LVAP与蛋白的结合活性，并分别计算其KD值及Kd值(图16)。
实施例4:GRP78的细胞内分布考察
选用三种细胞U87，HUVEC,HEK293做GRP78膜定位实验，选用破膜不破膜两种方式考察GRP78在细胞中的分布。将三种细胞分别接种到共聚焦小皿上，用PBS洗三次后用10μM的DIO多聚甲醛溶液固定8min，用1％BSA于37℃封闭30min后用PBS洗三次，加入1％BSA稀释的Anti-GRP78抗体，37℃孵育4h，PBS洗两次，加入1％BSA稀释的二抗，37℃孵育2h，PBS洗两次，每次5min。DAPI染色10min，PBS洗三次，甘油封片，激光共聚焦观察，结果如图17所示。
实施例5:VAP的体外细胞靶向性验证
1.VAP对脑胶质瘤细胞U87的体外靶向性
取对数生长期的单层培养的脑胶质瘤细胞(U87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1×105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％CO2及饱和湿度条件下培养24h后，用含10％胎牛血清的DMEM培养液配制浓度为5μM的FAM、DVAP-Fluorescein、SVAP-Fluorescein及LVAP-Fluorescein溶液，将培养板中的培养液吸出，分别加入上述溶液，37℃孵育4h，吸弃上清液。用PBS溶液洗三次，甲醛固定液固定细胞，DAPI进行细胞核染色后，激光共聚焦观察，细胞内化照片如图18A所示，另用PBS洗三次后，进行流式细胞仪分析，结果如图18B所示。
2.VAP对人脐静脉内皮细胞HUVEC的体外靶向性
取对数生长期的单层培养的人脐静脉内皮细胞(HUVEC细胞)，同上试验，细胞内化照片如图19A所示，流式细胞仪分析结果如图19B所示。
3.VAP对体外U87肿瘤球的靶向性
将2％的低分子琼脂糖溶液趁热加入48孔板中，每孔150μL，室 温放置冷却凝固后，每孔接种400μL U87细胞悬液，细胞密度为2×103个/孔。置于二氧化碳培养箱中，37℃、5％CO2及饱和湿度条件下培养7天即形成肿瘤球。用含10％胎牛血清的DMEM培养液配制浓度为5μM的FITC、DVAP-Fluorescein、SVAP-Fluorescein及LVAP-Fluorescein溶液。将培养板中的培养液吸出，分别加入上述溶液，37℃孵育4h，吸弃上清液，PBS洗三次，多聚甲醛固定15min后，置于共聚焦显微镜下观察，照片如图20A所示。
4.VAP相互竞争抑制试验
设9组，每组3复管进行VAP间相互竞争抑制的考察，分组分别为空白对照组、LVAP-Fluorescein、DVAP-Fluorescein、SVAP-Fluorescein、LVAP-Fluorescein(+LVAP)、LVAP-Fluorescein(+DVAP)、LVAP-Fluorescein(+SVAP)、DVAP-Fluorescein(+LVAP)、DVAP-Fluorescein(+DVAP)、DVAP-Fluorescein(+SVAP)、SVAP-Fluorescein(+LVAP)、SVAP-Fluorescein(+DVAP)、SVAP-Fluorescein(+SVAP)，将U87细胞用胰酶消化后转移到EP管内，PBS洗三次去除胰酶，低温4℃预处理20min，配好的多肽溶液(非荧光标记)也放置在4℃低温预处理，随后将多肽溶液与细胞悬液混合震荡处理2h，使细胞表面的受体蛋白饱和，然后加入荧光素标记的多肽溶液，4℃放置过夜后用PBS洗涤3次，流式测细胞摄取量(如图21所示)。
实施例6:VAP体内肿瘤靶向性验证
首先构建皮下瘤动物模型，将处于对数生长期的U87细胞胰酶消化，调整细胞浓度为3×107个/mL，接种100μL至裸小鼠右背侧近腋部皮下，接种后饲养于SPF级，定期观察肿瘤大小，待肿瘤大小为200mm3时，筛选出无坏死、肿瘤形状规则的荷瘤裸鼠，分组进行试验，以0.15μmoL/只的剂量将Cy7、DVAP-Cy7、SVAP-Cy7及LVAP-Cy7溶液通过尾静脉注入荷瘤裸鼠动物模型体内，2h后处死裸鼠，取出肿瘤，用活体成像仪检测肿瘤的荧光分布(如图22A所示)并进行荧光半定量计算(如图22B所示)。
实施例7： DVAP-DOX体内药效学和药动学试验
1.U87皮下瘤药效试验
构建的U87皮下瘤动物模型，待肿瘤大小为100mm3时，分组进行试验。皮下瘤模型鼠尾静脉分别注射生理盐水、DOX、MAL-DOX(含高、低剂量)和DVAP-DOX(含高、低剂量)以及RGD-DOX(整 合素受体配体c(RGDyK)与MAL-DOX按照VAP-DOX相同方法制备的多肽药物复合物)各100μl。给药组除低剂量含DOX总给药剂量为1.25mg/kg，其它均为2.5mg/kg，分为五次给药，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)。根据公式计算各组裸鼠肿瘤体积，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：
V瘤体积＝0.5(a×b2)
给药21天后，断颈处死所有裸鼠，取出皮下肿瘤称重，并计算各组统计学差异与抑瘤率(附图23)。
2.U87原位瘤药效试验
构建U87原位脑胶质瘤模型裸鼠：取对数生长期的U87细胞，每只裸小鼠接种5×105个细胞(分散于5μL PBS缓冲液中)。裸小鼠麻醉后，用脑立体定位仪固定，细胞接种于纹状体右部(前囟前0.6mm，侧1.8mm，深3mm)。定期观察裸小鼠状态。尾静脉分别注射PBS、DOX、MAL-DOX、DVAP、DVAP-DOX(含高、中、低剂量)，给药组除DVAP-DOX低剂量含DOX总给药剂量为2.5mg/kg，中剂量为5mg/kg，其它均为10mg/kg，DVAP剂量为DVAP-DOX高剂量中所含的DVAP量，分别在肿瘤种植后第10、13、16、19和22天给药，记录裸鼠的生存时间(附图24)。
3.DVAP-DOX体内药动学试验
ICR小鼠分别尾静脉注射200μL DOX、DVAP-DOX和MAL-DOX(其中含DOX为10mg/kg)，于1、5、15、30和45min，1、2、4和6h分别取全血50μL，以PBS稀释4倍，在荧光Ex 485/Em 590测定，绘制药物浓度-时间曲线与药物的体内分布(附图25)。
实施例8：VAP胶束对体外U87肿瘤球的靶向性
1.载香豆素6胶束的制备
称取1mg VAP-PEG-PLA，9mg mPEG-PLA和5ug香豆素6(C6)，溶解在2mL乙腈中，37℃水浴，减压(～0.085MPa)蒸干，成膜，室温真空干燥过夜，加入2mL生理盐水水化，CL-4B柱层析除去游离香豆素，制得包载香豆素6的胶束(VAP-Micelle/C6)。
2.VAP-Micelle/C6胶束体外U87肿瘤球靶向性验证
将2％的低分子琼脂糖溶液趁热加入48孔板中，每孔150μL，室温放置冷却凝固后，每孔接种400μL U87细胞悬液，细胞密度为2×103个/孔。置于二氧化碳培养箱中，37℃、5％CO2及饱和湿度条件下培养7天即形成肿瘤球，用含10％胎牛血清的DMEM培养液配制浓度 为5ng/mL的Micelle/C6、DVAP Micelle/C6、SVAP Micelle/C6及LVAP Micelle/C6溶液，将培养板中的培养液吸出，分别加入上述溶液，37℃孵育4h，吸弃上清液，PBS洗三次，多聚甲醛固定15min后，DAPI染色，置于共聚焦显微镜下观察，照片如图20B所示。
实施例9：VAP胶束体内靶向性验证
1.载DiR胶束的制备
1mg VAP-PEG-PLA，9mg mPEG-PLA和1mg DiR以制备载香豆素6胶束的方法制得载DiR胶束(VAP-Micelle/DiR)。
2.VAP-Micelle/DiR体内靶向性验证
U87皮下瘤模型裸鼠分别尾静脉注射100μL的PBS,mPEG-Micelle/DiR、DVAP-Micelle/DiR、SVAP-Micelle/DiR及LVAP-Micelle/DiR，分别在注射后2、4、8、12及24h时麻醉裸鼠，用活体成像仪记录DiR荧光在裸鼠体内的分布情况并进行荧光半定量计算(如图26所示)。
实施例10：载紫杉醇VAP胶束的体外药效学试验
1.载紫杉醇胶束的制备及表征
称取1mg VAP-PEG-PLA，9mg mPEG-PLA和2mg紫杉醇以制备载香豆素6胶束的方法制得载紫杉醇的VAP胶束(VAP-Micelle/PTX)，粒径与分布如图27所示。
2.VAP-Micelle/PTX体外药效试验
以4.0×103个/孔将U87细胞接种于96孔板，24h后，将培养液吸出，加200μL一系列浓度的DVAP-Micelle/PTX、SVAP-Micelle/PTX、LVAP-Micelle/PTX和mPEG-Micelle/PTX和泰素，共培养72h，后加入MTT溶液继续培养4h，弃去培养液，加入150μL DMSO，振荡至紫色颗粒溶解，用酶标仪在590nm处测定吸光度值，采用MTT法测定细胞存活率，计算细胞存活率和半数致死剂量(如图28所示)。
3.VAP-Micelle/PTX对新生血管形成的抑制试验
取24孔培养板每孔加入50μL基质胶，平铺于24孔板内，37℃培养箱内孵育30min待其凝固，0.25％胰酶消化HUVEC细胞，用含1μM紫杉醇VAP胶束或游离紫杉醇药液的DMEM培养液配成单细胞悬液，以每孔1×105个细胞接种于24孔培养板中，37℃、5％CO2及饱和湿度条件下培养12h后观察血管样结构形成(如图29所示)。
4.VAP-Micelle/PTX对拟态血管形成的抑制试验
取24孔培养板每孔加入50μL基质胶，平铺于24孔板内，37℃ 培养箱内孵育30min待其凝固，0.25％胰酶消化U87细胞，用含1μM紫杉醇VAP胶束或游离紫杉醇药液的DMEM培养液配成单细胞悬液，以每孔1×105个细胞接种于24孔培养板中，37℃、5％CO2及饱和湿度条件下培养12h后观察血管样结构形成(如图30所示)。
实施例11：VAP-Micelle/PTX体内药效学试验
1.VAP-Micelle/PTX体内药效学试验
构建的U87皮下瘤动物模型，待肿瘤大小为100mm3时，分组进行试验，皮下瘤模型鼠尾静脉分别注射生理盐水、泰素、LVAP-Micelle/PTX、DVAP-Micelle/PTX、SVAP-Micelle/PTX和mPEG-Micelle/PTX各100μl，给药组紫杉醇总给药剂量为25mg/kg，分为五次，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)，根据公式计算各组裸鼠肿瘤体积，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：
V瘤体积＝0.5(a×b2)
给药18天后(接种后24天)，常规处理裸鼠，取皮下肿瘤称重，并计算各组统计学差异(如图31所示)。
2.VAP-Micelle/PTX促凋亡试验
荷瘤裸鼠在给药完成后的第14天，处死取出瘤组织进行固定，作冰冻切片，通过TUNEL法检测肿瘤凋亡情况，采用末端脱氧核苷酸转移酶(TDT)介导的dUTP缺口末端标记法(Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling，TUNEL)检测肿瘤细胞的凋亡程度，按步骤：石蜡切片常规脱蜡至水；PBS漂洗3次，每次3min；0.3％H2O2溶液室温处理20min；20μg/mL蛋白酶K 37℃消化20min；PBS漂洗3次，每次3min；每张切片滴加TUNEL混合液(TDT和biotin-dNTP)30μL置于湿盒中37℃孵育60min；阳性结果为细胞核呈棕黄色或棕褐色，细胞核内棕色颗粒阳性即判定为凋亡细胞。在普通光学显微镜下连续观察5个高倍视野计数阳性细胞数，视野内细胞中阳性细胞数所占的百分比为凋亡指数，结果如图32所示。
3.VAP-Micelle/PTX对肿瘤血管抑制试验
荷瘤裸鼠在给药完成后的第14天，处死取出瘤组织固定，石蜡包埋切片，进行CD31免疫组化染色与PAS双染。在普通光学显微镜下连续观察3个高倍视野计数CD31阳性血管数，结果如图33所示。

Claims (11)

  1. 一种D构型多肽，其特征在于，所述D构型多肽为DVAP和/或SVAP，且所述DVAP的氨基酸序列为DPDADVDRDTDNDS，所述SVAP的氨基酸序列DSDNDTDRDVDADP。
  2. 一种DVAP和/或SVAP多肽复合物，其特征在于，所述DVAP和/或SVAP多肽复合物为权利要求1所述的DVAP和/或SVAP多肽修饰含有马来酰亚胺基团的影像物质，其中，所述DVAP和/或SVAP多肽复合物的结构为DVAP-X和/或SVAP-X，X为所述影像物质；
    优选地，所述X选自荧光物质、近红外染料、磁共振影像剂和放射影像剂中的一种或多种；
    更优选地，所述荧光物质为Fluorescein，近红外染料为Cy7、IR820、DiR，所述磁共振影像剂为Gd-DTPA，所述放射影像剂99mTc-DTPA。
  3. 一种L构型的LVAP多肽复合物，其特征在于，所述LVAP多肽复合物为LVAP多肽修饰含有马来酰亚胺基团的影像物质，其中，所述LVAP多肽的氨基酸序列为SNTRVAP，且所述LVAP多肽复合物的结构为LVAP-X，X为所述影像物质；
    优选地，所述X选自荧光素、近红外染料、磁共振影像剂和放射影像剂中的一种或多种；
    更优选地，所述荧光素为Fluorescein，近红外染料为Cy7、IR820、DiR，所述磁共振影像剂为Gd-DTPA，所述放射影像剂99mTc-DTPA。
  4. 一种DVAP和/或SVAP多肽复合物，其特征在于，所述DVAP和/或SVAP多肽复合物为权利要求1所述的DVAP和/或SVAP多肽修饰抗肿瘤药物，其中，所述DVAP和/或SVAP多肽复合物的结构为DVAP-Y和/或SVAP-Y，Y为所述抗肿瘤药物；
    优选地，所述抗肿瘤药物选自阿霉素和表阿霉素等蒽环类药物、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类药物、喜树碱和羟基喜树碱和伊立替康等喜树碱类药物、长春新碱和长春瑞滨等长春花碱类药物、硼替佐米和卡非佐米等蛋白酶体抑制剂、小白菊内酯等内酯类药物、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物中的一种或多种；
    更优选地：所述抗肿瘤药物选自含酮或醛基的阿霉素或表阿霉素，含羟基或氨基的紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9- 硝基喜树碱、长春新碱、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康，含硼酸基团的硼替佐米或卡非佐米，和/或多肽药物p53激活肽、蜂毒肽和蝎毒肽中的一种或多种。
  5. 一种L构型的LVAP多肽复合物，其特征在于，所述LVAP多肽复合物为LVAP多肽修饰抗肿瘤药物，其中，所述LVAP多肽的氨基酸序列为SNTRVAP，且所述LVAP多肽复合物的结构为LVAP-Y，Y为所述抗肿瘤药物；
    优选地，所述抗肿瘤药物选自阿霉素和表阿霉素等蒽环类药物、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类药物、喜树碱和羟基喜树碱和伊立替康等喜树碱类药物、长春新碱和长春瑞滨等长春花碱类药物、硼替佐米和卡非佐米等蛋白酶体抑制剂、小白菊内酯等内酯类药物、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物中的一种或多种；
    更优选地：所述抗肿瘤药物选自含酮或醛基的阿霉素或表阿霉素，含羟基或氨基的紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康，含硼酸基团的硼替佐米或卡非佐米，和/或多肽药物p53激活肽、蜂毒肽和蝎毒肽中的一种或多种。
  6. 一种DVAP和/或SVAP多肽复合物，其特征在于，所述DVAP和/或SVAP多肽复合物为权利要求1所述的DVAP和/或SVAP多肽修饰高分子载体材料，其中，所述DVAP和/或SVAP多肽复合物的结构为DVAP-聚乙二醇-Z和/或SVAP-聚乙二醇-Z，Z为所述高分子载体材料；
    优选地，所述高分子载体材料选自磷脂、聚乳酸、乳酸羟基乙酸共聚物和聚已内酯中的一种或多种。
  7. 一种L构型的LVAP多肽复合物，其特征在于，所述LVAP多肽复合物为LVAP多肽修饰高分子载体材料，其中，所述，所述LVAP多肽的氨基酸序列为SNTRVAP，且所述LVAP多肽复合物的结构为LVAP-聚乙二醇-Z，Z为所述高分子载体材料；
    优选地，所述高分子载体材料选自磷脂、聚乳酸、乳酸羟基乙酸共聚物和聚已内酯中的一种或多种。
  8. 一种递药系统，其特征在于，所述递药系统包括权利要求6或7 所述的复合物；优选地，所述递药系统为脂质体递药系统、聚合物胶束递药系统、聚合物圆盘递药系统或纳米粒递药系统。
  9. 根据权利要求8所述的递药系统，其特征在于，所述递药系统还包括所述DVAP、SVAP和/或LVAP多肽复合物以外的(1)诊断药物和/或(2)抗肿瘤药物；优选地：
    所述(1)诊断药物选自荧光物质、近红外染料和磁共振影像剂中的一种或多种，更优选地，所述荧光物质为Fluorescein，所述近红外染料选自Cy7、IR820、DiR，和/或所述磁共振影像剂为Gd-DTPA，和/或
    所述(2)抗肿瘤药物选自：阿霉素和表阿霉素等蒽环类药物、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类药物、喜树碱和羟基喜树碱和伊立替康等喜树碱类药物、长春新碱和长春瑞滨等长春花碱类药物、硼替佐米和卡非佐米等蛋白酶体抑制剂、小白菊内酯等内酯类药物、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物中的一种或多种。
  10. 权利要求1所述的DVAP和/或SVAP多肽、权利要求2-7中任一项所述的DVAP、SVAP和/或LVAP多肽复合物、权利要求8或9所述的递药系统在制备用于诊断、示踪和/或治疗肿瘤的药品或医疗产品中的应用，优选地：
    所述肿瘤为高表达葡萄糖调节蛋白GRP78肿瘤。
  11. 一种用于肿瘤诊断和/或靶向治疗的方法，其特征在于，向有需要的受试者给予：
    根据权利要求1所述的D构型多肽；
    根据权利要求2-7任一项所述的DVAP、SVAP和/或LVAP多肽复合物；和/或
    权利要求8或9所述的递药系统。
PCT/CN2017/114796 2016-12-07 2017-12-06 Vap多肽及其在制备靶向诊疗肿瘤药物中的应用 WO2018103660A1 (zh)

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