Whole-process targeting polypeptide, drug compound thereof, drug delivery system and application of whole-process targeting polypeptide

Abstract

The invention provides a whole-process targeting polypeptide, a drug compound thereof, a drug delivery system and application of the whole-process targeting polypeptide. The whole-process targeting polypeptide comprises pHA and VAP which are covalently connected through a bridging structure; the VAP is LVAP, a reverse sequence polypeptide of the LVAP, DVAP or SVAP; wherein the amino acid sequence of the LVAP is as shown in SEQ ID NO: 1; the SVAP is a D-configuration polypeptide of the LVAP; the DVAP is a D-configuration polypeptide of the LVAP inverted sequence polypeptide; the bridging structure is amino-monoethylene glycol-carboxylic acid or a derivative thereof. The whole-process targeting polypeptide not only can cross a blood-brain barrier, target tumor neovascularization, cross a blood-tumor barrier, target tumor mimicry vessels, tumor cells and stem cells thereof, but also has better tumor cell affinity, anti-tumor treatment and in-vivo safety effects. Meanwhile, the drug compound or the drug delivery system has a synergistic effect after being combined with a chemotherapeutic drug, and the anti-tumor drug effect can be remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of pharmacy, and relates to a whole-process targeting polypeptide, a drug complex thereof, a drug delivery system and an application thereof in tumor targeting diagnosis and treatment. Background Art

[0002] Traditional chemotherapy, as the main means of drug treatment for brain tumors, has defects such as poor selectivity for tumor tissues, high toxicity, narrow therapeutic window, and easy multidrug resistance. In addition, the amount of chemotherapy drugs entering the brain is also very limited. In order to overcome the limitations of traditional treatment methods, active targeting has become an important strategy to improve the efficiency of brain tumor tissue targeting in recent years.

[0003] In addition to tumor cells, brain tumor tissues also contain tumor stem cells, tumor-mimicking blood vessels, brain capillaries, blood-brain barrier (BBB), tumor angiogenesis, and blood-tumor barrier (BTB). In the early stages of brain tumors, the BBB remains intact and restricts the entry of drugs into the brain, making it impossible for about 98% of small-molecule chemotherapy drugs and almost 100% of macromolecular drugs such as proteins to enter the brain through the BBB, resulting in almost ineffective drug treatment. As the tumor develops, the BBB is partially destroyed and tumor angiogenesis occurs, but the brain tumor angiogenesis is relatively denser and less permeable than peripheral tumors, and the formed BTB becomes the main obstacle to drug delivery. At the same time, the BBB still exists at the edge of the infiltration of brain gliomas and hinders drug transport. In addition, brain tumor stem cells have the characteristics of self-renewal, proliferation, and high tumorigenicity. Although their number in tumor tissues is extremely small, they show a high degree of tolerance to drug treatment, which can easily lead to recurrence of brain gliomas. Therefore, for the active targeting strategy of brain tumors, it is crucial to construct a full-process targeting molecule that has the ability to cross the BBB and BTB and has affinity for brain tumor cells and tumor stem cells.

[0004] Glucose-regulated protein GRP78, also known as immunoglobulin heavy chain binding protein (Bip), is one of the main molecular chaperones of the endoplasmic reticulum and plays an important role in protein folding and endoplasmic reticulum stress response. GRP78 protein expression is significantly increased in many tumor cell lines, solid tumors and human tumor tissue biopsy samples. L VAP (L-type amino acid sequence is SNTRVAP (SEQ ID NO: 1)) and its stable optimized polypeptide D VAP (D-type amino acid sequence D P D A D V D R D T D N D S (SEQ ID NO: 3)), LVAP reverse sequence polypeptide (L-type amino acid sequence is PAVRTNS (SEQ ID NO: 2)) and its stable optimized polypeptide S VAP (D-type amino acid sequence D S D N D T D R D V D A D P (SEQ ID NO: 4))) is a 7-peptide that has high binding activity with glucose-regulated protein GRP78 verified by the team of the present invention in the early stage. It can target tumor neovascular endothelial cells and cross the BTB, target tumor cells and tumor stem cells, and has good targeting ability in vivo, but cannot cross the BBB. In addition, recent studies have shown that many clinically used anticancer drugs, such as doxorubicin (DOX), temozolomide (TMZ), 5-fluorouracil, paclitaxel, camptothecin and bortezomib, can induce endoplasmic reticulum stress and increase GRP78 protein expression.

[0005] The benzamide derivative p-hydroxybenzoic acid (pHA) is also a target molecule that can target brain capillary endothelial cells and penetrate the BBB, which was verified by the team of the present invention in the early stage. In addition, after the team of the present invention further covalently linked pHA to VAP, it was initially shown that it has the function of targeting brain capillary endothelial cells (across the BBB), tumor neovascular endothelial cells (across the BTB), tumor mimic blood vessels, tumor cells and tumor stem cells. Summary of the invention

[0006] In order to solve the problem of covalently linking two target molecules to achieve multifunctional targeting, the present invention provides a whole-process targeting polypeptide, its drug complex, drug delivery system and its application. For the realization of multifunctional targeting, the bridging structure often plays a very critical role. Therefore, the present invention designs amino-monoethylene glycol-carboxylic acid or its derivatives (AOHX) as a bridging structure to covalently link pHA and VAP, obtains a whole-process targeting polypeptide, and constructs a drug complex of the targeting polypeptide and a modified nano drug delivery system. The whole-process targeting polypeptide not only has the whole-process targeting function of targeting brain capillary endothelial cells (across BBB), tumor neovascular endothelial cells (across BTB), tumor mimic blood vessels, tumor cells and tumor stem cells, but also shows better tumor cell affinity, and the drug complex constructed by it obtains better anti-tumor therapeutic effect and unexpected effects such as in vivo safety. In addition, the drug complex of the whole-process targeting polypeptide or the modified drug delivery system is used in combination with chemotherapeutic drugs, which has a synergistic effect and can further enhance the anti-tumor efficacy.

[0007] In order to solve the above technical problems, the first aspect of the present invention provides a whole-process targeting polypeptide, wherein the whole-process targeting polypeptide comprises pHA and VAP covalently linked by a bridging structure; the VAP is L VAP, L VAP reverse peptide, D VAP or S VAP;

[0008] in, L The amino acid sequence of VAP is shown in SEQ ID NO: 1; S VAP is L D-configuration peptide of VAP; D VAP is L The D-configuration peptide of the VAP reverse peptide;

[0009] The bridging structure is amino-monoethylene glycol-carboxylic acid or its derivatives, having the following general formula:

[0010] Where n is a natural number.

[0011] In some preferred embodiments of the present invention, n is 1-20, for example, 1.

[0012] In order to solve the above technical problems, the second aspect of the present invention provides a targeted diagnostic polypeptide complex, wherein the targeted diagnostic polypeptide complex is formed by connecting the full-process targeting polypeptide as described in the first aspect of the present invention and an imaging molecule.

[0013] In some embodiments of the present invention, the imaging molecule is an optical imaging molecule, a magnetic resonance imaging agent and / or a radionuclide imaging agent.

[0014] In some preferred embodiments of the present invention, the optical imaging molecule is selected from the following group:

[0015] (1) one or more of the fluorescent probe molecules FITC, FAM, 6-TET, 5-TAMRA, HEX and 6-JOE;

[0016] (2) one or more of the near-infrared dye molecules Cy3, Cy3.5, Cy5, Cy5.5, Cy7, IR783, IR820, DiR, DiD, BODIPY630 / 650-X, BODIPY650 / 665-X, BODIPY665 / 676, TO-PRO-3 and TO-PRO-5;

[0017] (3) one or more of the chemiluminescent molecules luminol, isoluminol, AMPPD, CSPD, CDP-star, lucigenin and Raman probe molecules;

[0018] The magnetic resonance imaging agent is a chelate of Gd magnetic resonance material;

[0019] 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 Chelated radionuclides for Tb imaging.

[0020] In some more preferred embodiments of the present invention, the magnetic resonance imaging agent or radionuclide imaging agent is composed of a bifunctional chelator and a nuclide for magnetic resonance imaging or a radionuclide for imaging; wherein the bifunctional chelator 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 and NS3.

[0021] In order to solve the above technical problems, the third aspect of the present invention provides a targeted therapeutic polypeptide complex, wherein the targeted therapeutic polypeptide complex is formed by connecting the full-process targeting polypeptide as described in the first aspect of the present invention and a therapeutic drug.

[0022] In some preferred embodiments of the present invention, the whole-process targeting polypeptide is connected or condensed with the therapeutic drug through a pH-sensitive hydrazone bond, a pH-sensitive boronate bond or a disulfide bond or directly.

[0023] In some embodiments of the present invention, the therapeutic drug is selected from one or more of the following: tumor chemotherapy drugs, anti-tumor stem cell drugs, molecular targeted drugs, polypeptide drugs, antibody drugs or therapeutic radionuclide chelates.

[0024] In some preferred embodiments of the present invention, the tumor chemotherapy drug is selected from the following group:

[0025] (1) one or more anthracyclines such as doxorubicin and epirubicin;

[0026] (2) one or more taxane drugs such as paclitaxel, docetaxel, and cabazitaxel;

[0027] (3) Camptothecin drugs such as one or more of camptothecin, hydroxycamptothecin, 9-nitrocamptothecin and irinotecan;

[0028] (4) one or more vinca alkaloids such as vinblastine and vincristine;

[0029] (5) one or more proteasome inhibitors such as bortezomib;

[0030] The anti-tumor stem cell drugs are selected from lactone anti-tumor drugs, such as parthenolide and its derivatives;

[0031] The molecular targeted drugs are selected from one or more of the following: trametinib, imatinib, nilotinib, dasatinib, everolimus, erlotinib, sunitinib, sorafenib, ibrutinib, regorafenib, vemurafenib and olaparib;

[0032] The polypeptide drug is a p53 activation peptide;

[0033] The antibody drugs are selected from one or more of the following: rituximab, bevacizumab, trastuzumab, cetuximab, pertuzumab, ipilimumab, nivolumab and PD-L1 monoclonal antibody, and combinations of antibody fragments modified by genetic engineering, including Fab fragments, single domain antibodies, Fv fragments, single chain antibodies, bivalent small molecule antibodies, mini antibodies, and nano antibodies;

[0034] 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 and 227 Chelated Th therapeutic radionuclides.

[0035] In some more preferred embodiments of the present invention, the therapeutic radionuclide chelate is composed of a bifunctional chelator and a therapeutic radionuclide, wherein the bifunctional chelator 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 and NS3.

[0036] In order to solve the above technical problems, the fourth aspect of the present invention provides a targeting polypeptide polymer carrier material, which is formed by connecting the full-process targeting polypeptide as described in the first aspect of the present invention and a polyethylene glycol-complex; wherein the complex is a lipophilic material molecule or a hydrophilic ligand molecule.

[0037] In some preferred embodiments of the present invention, the lipophilic material molecule is selected from one or more of the following: phospholipids, polylactic acid, lactic acid-glycolic acid copolymer and polycaprolactone; and the hydrophilic ligand molecule is biotin.

[0038] In order to solve the above technical problems, the fifth aspect of the present invention provides a targeted drug delivery system, which includes the targeted polypeptide polymer carrier material and excipients as described in the fourth aspect of the present invention; wherein, when the polyethylene glycol-complex is polyethylene glycol-phospholipid, the targeted drug delivery system is a liposome delivery system, a micelle delivery system or a disc delivery system; when the polyethylene glycol-complex is polyethylene glycol-polylactic acid, polyethylene glycol-lactic acid copolymer or polyethylene glycol-polycaprolactone, the targeted drug delivery system is a micelle delivery system or a nanoparticle delivery system; when the polyethylene glycol-complex is polyethylene glycol-biotin, the targeted drug delivery system is a biomembrane-coated nano drug delivery system.

[0039] In some preferred embodiments of the present invention, the auxiliary material includes one or more of cholesterol, phospholipids, polyethylene glycol-phospholipids, polyethylene glycol-polylactic acid, polyethylene glycol-lactic acid copolymer and polyethylene glycol-polycaprolactone.

[0040] In some embodiments of the present invention, the targeted drug delivery system is used to encapsulate a drug, and the drug is a diagnostic drug or a therapeutic drug.

[0041] In some preferred embodiments of the present invention, the diagnostic drug is an imaging molecule defined in the targeted diagnostic polypeptide complex as described in the second aspect of the present invention; the therapeutic drug is a therapeutic drug defined in the targeted therapeutic polypeptide complex as described in the third aspect of the present invention.

[0042] In order to solve the above technical problems, the sixth aspect of the present invention provides a drug combination for combined use, wherein the drug combination comprises a chemotherapeutic drug and the targeted therapeutic polypeptide complex as described in the third aspect of the present invention or the targeted drug delivery system as described in the fifth aspect of the present invention;

[0043] In some preferred embodiments of the present invention, the chemotherapy drug is selected from one or more of doxorubicin, epirubicin, temozolomide, paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine, bortezomib, carfilzomib, and 5-fluorouracil;

[0044] In some more preferred embodiments of the present invention, the drug combination is administered by first administering the chemotherapy drug, and then administering the targeted therapeutic polypeptide complex as described in the third aspect of the present invention or the targeted drug delivery system as described in the fifth aspect of the present invention after a certain period of time; the certain period of time is preferably 24-48 hours.

[0045] In order to solve the above technical problems, the seventh aspect of the present invention provides the use of the full-process targeting polypeptide as described in the first aspect of the present invention, the targeted diagnostic polypeptide complex as described in the second aspect of the present invention, the targeted therapeutic polypeptide complex as described in the third aspect of the present invention, the targeted polypeptide polymer carrier material as described in the fourth aspect of the present invention, the targeted drug delivery system as described in the fifth aspect of the present invention and / or the drug combination as described in the sixth aspect of the present invention in the preparation of imaging diagnostic drugs, tracer drugs and / or targeted therapeutic drugs for tumors such as brain tumors or peripheral tumors.

[0046] Specifically include:

[0047] 1. Preparation of whole-process targeting peptides and their fluorescent markers

[0048] A solid phase synthesis method was used to prepare a fully targeted peptide in which pHA and VAP were covalently linked by amino-monoethylene glycol-carboxylic acid or its derivatives. A fully targeted peptide-Fluorescein complex was synthesized by Michael addition reaction of maleimide group and thiol group. Its structure was characterized by HPLC and MS.

[0049] 2. Evaluation of the in vitro targeting ability of the whole process targeting peptide

[0050] The in vitro affinity of the whole-process targeted peptide-modified fluorescein covalently linked pHA and VAP with amino-monoethylene glycol-carboxylic acid or its derivatives to brain capillary endothelial cells (BCEC), umbilical vein endothelial cells (HUVEC) and model tumor cells (such as brain glioma cells U87) was investigated and compared with the targeted peptide-modified fluorescein covalently linked pHA and VAP with aminocaproic acid.

[0051] 3. Preparation of drug complexes targeting peptides throughout the process

[0052] After the introduction of cysteine, the whole process targeting peptide covalently linked pHA and VAP with amino-monoethylene glycol-carboxylic acid or its derivatives reacts with the maleimidohydrazine derivative on the drug to form a peptide-drug complex containing a pH-sensitive hydrazone bond, wherein the drugs involved include doxorubicin, epirubicin and other drugs containing ketone or aldehyde groups.

[0053] After the introduction of cysteine, the whole process targeting peptide that covalently links pHA and VAP with amino-monoethylene glycol-carboxylic acid or its derivatives reacts with the 3-(2-pyridyldithiol)propionic acid derivative on the drug to form a disulfide bond-containing peptide-drug complex, wherein the drugs involved include paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine, vinorelbine and other drugs containing hydroxyl or amino groups.

[0054] The whole process targeting peptide that covalently links pHA and VAP with amino-monoethylene glycol-carboxylic acid or its derivatives is modified with dopamine and then reacts with the boronic acid group on the drug to form a peptide-drug complex containing pH-sensitive boronic acid lipids, wherein the drugs involved include drugs containing boronic acid groups such as bortezomib.

[0055] The whole process targeting peptide covalently linked pHA and VAP with amino-monoethylene glycol-carboxylic acid or its derivatives is directly condensed with polypeptide drugs through solid phase synthesis, wherein the drugs involved include polypeptide drugs such as p53 activation peptide, antimicrobial peptide, polypeptide toxin, etc.

[0056] The whole-process targeting polypeptide covalently linked pHA and VAP with amino-monoethylene glycol-carboxylic acid or its derivatives is modified by random sites (activating the free amino groups in the antibody and then covalently linking it with the whole-process targeting polypeptide) or fixed sites (non-covalently linking the whole-process targeting polypeptide to the antibody through affinity coupling) to obtain an antibody complex modified with the whole-process targeting polypeptide. The drugs involved include rituximab, bevacizumab, trastuzumab, cetuximab, pertuzumab, ipilimumab, nivolumab, PD-L1 monoclonal antibody and other antibody drugs and antibody fragment combinations modified by genetic engineering means (including Fab fragments, single domain antibodies, Fv fragments, single chain antibodies, bivalent small molecule antibodies, mini antibodies, nano antibodies, etc.).

[0057] The whole process targeting polypeptide covalently linked pHA and VAP with amino-monoethylene glycol-carboxylic acid or its derivatives after the introduction of cysteine ​​reacts with the maleimide group on the bifunctional chelator to obtain a targeting polypeptide modified radionuclide chelate, wherein the therapeutic radionuclide chelate involved includes 90 Y. 131 I. 152 / 155 Tb, 153 Sm, 177 Lu, 186 / 188 Re, 211 At 212 / 213 Bi, 212 Pb, 225 Ac and 227 Th.

[0058] 4. Determination of the solubility of the whole process targeting peptide-doxorubicin complex

[0059] The solubility of the whole process targeting peptide-doxorubicin complex in which pHA and VAP were covalently linked by amino-monoethylene glycol-carboxylic acid or its derivatives in PBS was investigated and compared with the targeting peptide-doxorubicin complex in which pHA and VAP were covalently linked by aminocaproic acid.

[0060] 5. Evaluation of the in vitro cellular uptake effect of the whole-process targeted peptide-doxorubicin complex

[0061] The uptake of the whole-process targeting peptide-doxorubicin complex in which pHA and VAP were covalently linked by amino-monoethylene glycol-carboxylic acid or its derivatives was investigated in U87 tumor cells and RAW264.7 cells, and compared with the targeting peptide-doxorubicin complex in which pHA and VAP were covalently linked by aminocaproic acid.

[0062] 6. In vivo safety study of the whole-process targeted peptide-doxorubicin complex

[0063] Normal mice were injected with DOX and the whole process targeting peptide-doxorubicin complex covalently linked to pHA and VAP by amino-monoethylene glycol-carboxylic acid or its derivatives, respectively, through the tail vein to investigate the maximum tolerated dose (MTD).

[0064] 7. Pharmacokinetic study of the whole-process targeted peptide-doxorubicin complex

[0065] The whole-process targeting peptide-doxorubicin complex in which pHA and VAP are covalently linked by amino-monoethylene glycol-carboxylic acid or its derivatives is injected into the tail vein of mice. Blood is collected at different time points to detect the concentration of doxorubicin, and the pharmacokinetic behavior and in vivo distribution are investigated. The complex is compared with the targeting peptide-doxorubicin complex in which pHA and VAP are covalently linked by aminocaproic acid.

[0066] 8. In vivo efficacy test of the whole process targeted peptide-doxorubicin complex

[0067] The whole-process targeted peptide-doxorubicin complex in which pHA and VAP were covalently linked by amino-monoethylene glycol-carboxylic acid or its derivatives was administered through the tail vein of nude mice bearing U87 orthotopic tumor model. The median survival time was used as an indicator to evaluate its in vivo antitumor effect and compared with the targeted peptide-doxorubicin complex in which pHA and VAP were covalently linked by aminocaproic acid.

[0068] 9. Dose- and time-dependence of chemotherapy drugs in upregulating tumor GRP78 protein expression

[0069] Western Blot and immunofluorescence were used to investigate the expression of GRP78 protein in tumor cells or tumor sites after administration of different doses of DOX, TMZ and different time periods of TMZ.

[0070] 10. Effect of pre-administration of chemotherapy drugs on the targeting effect of drug complexes of whole-process targeted peptides in vivo and in vitro

[0071] The uptake of whole-process targeting peptide-modified fluorescein, which was covalently linked to pHA and VAP by amino-monoethylene glycol-carboxylic acid or its derivatives, by model tumor cells U87 after pre-administration of TMZ was investigated.

[0072] The uptake of the whole-process targeting peptide-doxorubicin complex covalently linked with pHA and VAP by amino-monoethylene glycol-carboxylic acid or its derivatives by the model tumor cells U87 after pre-administration of TMZ was investigated.

[0073] Nude mice bearing an orthotopic brain glioma model were pre-administered with TMZ and then intravenously injected with a whole-process targeting peptide-modified fluorescein covalently linked with pHA and VAP by amino-monoethylene glycol-carboxylic acid or its derivatives. The distribution of fluorescein in the brain and normal organs of tumor-bearing nude mice was investigated by in vivo imaging.

[0074] 11. In vivo evaluation of the anti-brain tumor efficacy of TMZ combined with whole-course targeted peptide-doxorubicin complex

[0075] After TMZ was pre-administered orally to nude mice bearing U87 orthotopic tumor model, a whole-process targeting peptide-doxorubicin complex covalently linked with pHA and VAP by amino-monoethylene glycol-carboxylic acid or its derivatives was intravenously injected. The anti-brain tumor effect in vivo was evaluated using the median survival time as an indicator compared with the administration of normal saline, TMZ or whole-process targeting peptide-doxorubicin complex alone.

[0076] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0077] The reagents and raw materials used in the present invention are commercially available.

[0078] The positive and progressive effect of the present invention is that pHA is covalently linked to VAP through amino-monoethylene glycol-carboxylic acid or its derivatives, and the obtained polypeptide not only has the whole process targeting function of crossing the blood-brain barrier, targeting tumor angiogenesis and crossing the blood-tumor barrier, targeting tumor mimic vessels and tumor cells, but also shows better tumor cell affinity, and the constructed drug complex obtains unexpected effects such as better anti-tumor therapeutic effect and in vivo safety. At the same time, the drug complex or modified drug delivery system of the whole process targeted polypeptide has a synergistic effect after combined use with chemotherapeutic drugs, which can significantly improve the anti-tumor efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 HPLC and ESI-MS spectra of pHA-AOHX-VAP-Cys.

[0080] Figure 2 HPLC and ESI-MS spectra of pHA-AOHX-VAP-Fluorescein.

[0081] Figure 3 The figure shows the uptake of pHA-VAP with different bridging structures labeled with Fluorescein by primary brain capillary endothelial cells BCEC; the figure shows the quantitative (Figure A) and qualitative (Figure B) results of flow cytometry fluorescence detection of pHA-AHX-VAP and pHA-AOHX-VAP labeled with Fluorescein after incubation with BCEC cells for 4 hours.

[0082] Figure 4 The figure shows the uptake of pHA-VAP with different bridge structures labeled with Fluorescein by umbilical vein endothelial cells HUVEC; the figure shows the quantitative (Figure A) and qualitative (Figure B) results of flow cytometry fluorescence detection of pHA-AHX-VAP and pHA-AOHX-VAP labeled with Fluorescein after incubation with HUVEC cells for 4 hours.

[0083] Figure 5 The figure shows the uptake of pHA-VAP with different bridging structures labeled with Fluorescein by brain glioma cells U87; the figure shows the quantitative (Figure A) and qualitative (Figure B) results of flow cytometry fluorescence detection of pHA-AHX-VAP and pHA-AOHX-VAP labeled with Fluorescein after incubation with U87 cells for 4 hours.

[0084] Figure 6 HPLC and ESI-MS spectra of pHA-AOHX-VAP-DOX.

[0085] Figure 7 The solubility of pHA-VAP-doxorubicin complexes with different bridging structures in PBS was determined.

[0086] Figure 8 The uptake of pHA-VAP-doxorubicin complexes with different bridging structures by U87 cells.

[0087] Fig. 9 The uptake of pHA-VAP-doxorubicin complexes with different bridging structures by RAW264.7 macrophages.

[0088] Fig.10 It is a maximum tolerated dose (MTD) test; Figure A is the weight change curve of mice in the DOX group at different doses, and Figure B is the weight change curve of mice in the preferred pHA-AOHX-VAP-DOX group at different doses.

[0089] Fig.11The figure shows the efficacy results of the preferred pHA-AOHX-VAP-doxorubicin complex against U87 orthotopic glioma in vivo; the figure shows the survival curve of nude mice in the U87 orthotopic glioma model.

[0090] Fig.12 The preferred pHA-AOHX-VAP-doxorubicin complex has an in vivo inhibitory effect on apoptosis and angiogenesis of U87 glioma cells in situ; the figure shows TUNEL staining of apoptotic tumor cells in the U87 tumor in situ site, CD31 staining of angiogenesis (Figure A) and semi-quantitative results (Figure B), where the white arrow points to the tumor area.

[0091] Fig.13 HPLC and ESI-MS spectra of pHA-AOHX-VAP-Cy5.

[0092] Fig.14 The figure shows the expression of cell membrane GRP78 protein in U87 cells after administration of different concentrations of DOX; The figure shows the Western Blot image and semi-quantitative results of cell membrane GRP78 protein expression in U87 cells after incubation with different concentrations of DOX for 48 hours.

[0093] Fig.15 The figure shows the expression of cell membrane GRP78 protein in U87 cells after administration of different concentrations of pHA-AOHX-VAP-DOX; The figure shows the Western Blot image and semi-quantitative results of cell membrane GRP78 protein expression in U87 cells after incubation with different concentrations of pHA-AOHX-VAP-DOX for 48 hours.

[0094] Fig.16 The figure shows the expression of cell membrane GRP78 protein in U87 cells after being given different concentrations of TMZ; The figure shows the Western Blot image and semi-quantitative results of cell membrane GRP78 protein expression in U87 cells after being incubated with different concentrations of TMZ for 48 hours.

[0095] Fig.17 The figure shows the expression of cell membrane GRP78 protein in U87 cells after incubation with TMZ for different time periods; The figure shows the Western Blot images and semi-quantitative results of cell membrane GRP78 protein expression in U87 cells after being given the same concentration of TMZ (5 μg / mL) for different time periods.

[0096] Fig.18 The figure shows the expression of GRP78 protein in the tumor of nude mice with orthotopic glioma model at different times after TMZ was administered; The figure shows the Western Blot images and semi-quantitative results of GRP78 protein expression in nude mice bearing orthotopic glioma at different times after TMZ (10 mg / mL) was administered orally.

[0097] Fig.19The figure shows the expression of GRP78 protein in the tumor of nude mice with orthotopic glioma model after administration of different concentrations of TMZ; the figure shows the immunofluorescence staining results of GRP78 protein expression in nude mice bearing orthotopic glioma 48 hours after administration of different concentrations of TMZ by gavage (bar=200μm).

[0098] Fig. 20 The figure shows the expression of GRP78 protein in the tumor of nude mice with orthotopic glioma model at different times after TMZ was administered; the figure shows the immunofluorescence staining results of GRP78 protein expression in the tumor site at different times after 10 mg / kg TMZ was administered orally to nude mice bearing orthotopic glioma (bar=200 μm).

[0099] Fig.21 The figure shows the expression of GRP78 protein in normal brain of nude mice with orthotopic glioma model at different times after TMZ administration; the figure shows the immunofluorescence staining results of GRP78 protein expression in normal brain tissue of nude mice bearing orthotopic glioma at different times after oral administration of 10 mg / kg TMZ (bar=200 μm).

[0100] Fig. 22 The figure shows the uptake of pHA-AOHX-VAP-Fluorescein by U87 cells after pre-administration of TMZ; the figure shows the uptake of pHA-AOHX-VAP-modified fluorescein by U87 cells after 48 hours of treatment with 5μg / mL TMZ.

[0101] Fig.23 The figure shows the uptake of pHA-AOHX-VAP-DOX by U87 cells after pre-administration of TMZ; the figure shows the uptake of the drug complex pHA-AOHX-VAP-DOX, a full-process targeted peptide, by U87 cells after treatment with 5 μg / mL TMZ for 48 hours.

[0102] Fig.24 This is the distribution of pHA-AOHX-VAP-Cy5 in the brain of nude mice with an orthotopic glioma model after pre-administration of TMZ.

[0103] Fig.25 Figure 1 is the efficacy of TMZ combined with pHA-AOHX-VAP-DOX in treating orthotopic brain glioma; Figure A shows that TMZ pre-administration combined with pHA-AOHX-VAP-DOX prolongs the median survival time of mice with orthotopic brain glioma model (n=10), and Figure B is the weight change curve of mice treated with TMZ pre-administration combined with pHA-AOHX-VAP-DOX in treating orthotopic brain glioma.

[0104] Fig.26 The figure is the HE section of the mouse brain with orthotopic glioma; the figure shows the HE staining result of the largest cross-section of the mouse brain tumor after the end of drug administration in each group (bar=1mm). DETAILED DESCRIPTION

[0105] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0106] In the present application, AOHX represents amino-monoethylene glycol-carboxylic acid or its derivatives, and AHX represents aminocaproic acid.

[0107] Example 1 Preparation and characterization of the whole process targeting polypeptide and its modified fluorescein and drug complex

[0108] 1. Preparation and characterization of pHA-AOHX-VAP-Cys peptide

[0109] pHA-AOHX-VAP-Cys (sequence: pHA-amino-monoethylene glycol-carboxylic acid-Cys- D P D A D V D R D T D N D S) wherein AOHX represents amino-monoethylene glycol-carboxylic acid.

[0110] Specific method: HBTU / DIEA is used as a condensing agent, 20% piperidine in DMF solution is used as a deprotecting agent, amino acids are added to Fmoc-D-Ser(tBu)-2-Chlorotrityl Resin in the order from the carboxyl end to the amino end of the sequence, and then Fmoc-amino-monoethylene glycol-carboxylic acid and p-hydroxybenzoic acid are added in sequence. After completion, the polypeptide is cut off from the resin, and separated and purified by high performance liquid chromatography in an acetonitrile / water (containing 0.1% TFA) system. The pure polypeptide is characterized by HPLC and mass spectrometry. The results are shown in Figure 1 . Figure 1 Chromatographic method: Chromatographic column (YMC, C18): 150×4.6 mm; Mobile phase A: water (containing 0.1% trifluoroacetic acid), Mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid); Elution program: 2-32 min 5% B-65% B; Flow rate: 0.7 mL / min; Column temperature: 40°C; Detection: UV 214 nm, Retention time: 12.56 min. ESI-MS: 1082.11, consistent with the theoretical molecular weight.

[0111] 2. Synthesis and characterization of pHA-AOHX-VAP-Fluorescein and pHA-AOHX-VAP-Cy5

[0112] The pHA-AOHX-VAP-Cys obtained in the above steps was dissolved in 0.1M PBS solution (pH7.2), and Fluorescein-5-maleimide was dissolved in DMF. After the two were mixed, they were stirred magnetically and monitored by HPLC. After the pHA-AOHX-VAP-Cys reaction was complete, the reaction was stopped, and the preparation liquid was purified. The acetonitrile / water (containing 0.1% TFA) system was used for separation and purification. The pure pHA-AOHX-VAP-Fluorescein was obtained by freeze drying. HPLC spectrum and mass spectrum are shown in Figure 2 . Figure 2 Chromatographic method Figure 1 , retention time: 18.90min. ESI-MS: 1508.56, consistent with the theoretical molecular weight.

[0113] pHA-AOHX-VAP-Cy5 was prepared by the same method as above, and the characterization results are shown in Fig.13 . Fig.13 Chromatographic method Figure 1 , retention time: 26.92min. ESI-MS: 1686.45, consistent with the theoretical molecular weight.

[0114] 3. Preparation of pHA-AOHX-VAP drug complex

[0115] pHA-AOHX-VAP-doxorubicin was prepared as an example of the whole process of targeting peptides to link ketone or aldehyde drugs. The peptide was dissolved in phosphate buffer (0.1M, pH 7.4), and 10 times the molar amount of tris(2-carboxyethyl)phosphine (TCEP) was added, and stirred at 4°C for 20 minutes. Then 4 times the molar amount of doxorubicin 6-maleimidohexahydrazine derivative was added, and the reaction was carried out at room temperature in the dark for 1 hour. The reaction solution was purified by preparative liquid phase and freeze-dried to obtain pHA-AOHX-VAP-DOX. HPLC spectrum and mass spectrum are shown in Figure 6 . Figure 6 Chromatographic method: Chromatographic column (YMC, C18): 150×4.6 mm; Mobile phase A: water (containing 0.01% formic acid), mobile phase B: acetonitrile (containing 0.01% formic acid); Elution program: 2-32 min 5% B-65% B; Flow rate: 0.7 mL / min; Column temperature: 40°C; Detection: UV214 nm, Retention time: 16.51 min. ESI-MS: 1832.25, consistent with the theoretical molecular weight.

[0116] pHA-AOHX-VAP-paclitaxel is used as an example of a whole-process targeting peptide connected to a drug by a disulfide bond. 200 mg of paclitaxel is dissolved in 10 mL of chloroform, cooled to 0-5°C, 39.99 mg of DCC and 60.4 mg of 3-(2-pyridyldithiol)propionic acid are added successively, and after the addition is completed, the mixture is heated to room temperature and reacted overnight. The reaction solution is filtered and purified by column chromatography (CHCl3 / MeOH=50:1-15:1, V / V elution) to obtain paclitaxel 3-(2-pyridyldithiol)propionic acid derivatives. Paclitaxel 3-(2-pyridyldithiol) propionic acid derivative was dissolved in 5 mL DMF, 1.5 times molar amount of pHA-AOHX-VAP-Cys was dissolved in PBS / DMF, and the pH value of the solution was maintained at 4-5. Paclitaxel 3-(2-pyridyldithiol) propionic acid derivative was added dropwise to the thiol polypeptide solution, reacted at room temperature for 6 hours, and the polypeptide-paclitaxel complex was prepared by lyophilization through the preparation of liquid phase.

[0117] pHA-AOHX-VAP-bortezomib is used as an example of a drug for whole-process targeted peptide nitrogen-terminal modification. Amino acids are sequentially connected to the resin according to the synthesis of the peptide. After all amino acid residues of the peptide are connected, trifluoroacetic acid removes the Boc protection of the nitrogen end. A DMF solution containing 3 times the molar amount of succinic anhydride and DIEA is added and reacted at room temperature for 30 minutes. After washing the resin, 5 times the molar amount of trimethylsilyl chloride is added to protect dopamine, and HBTU / DIEA is used as a condensing agent to react at room temperature for 1 hour. The resin is cut with HF and purified by preparative HPLC to obtain a peptide-dopamine derivative. In a buffer solution of pH 7.4, the peptide-dopamine derivative and bortezomib are mixed in a molar ratio of 1:1 to obtain a peptide-bortezomib complex.

[0118] pHA-AOHX-VAP-PMI is used as an example of a whole-process targeted polypeptide fusion polypeptide drug. It is prepared directly by solid phase polypeptide synthesis, and the specific method is as follows: after determining the pHA-AOHX-VAP-PMI polypeptide sequence, amino acids are sequentially connected in the same way as the preparation of polypeptides, and pHA-AOHX-VAP-PMI is obtained after HF cutting and purification.

[0119] pHA-AOHX-VAP-PD-L1 is used as an example of a fully targeted peptide-modified antibody drug. Sulfo-SMCC (1 mg / mL) was added to the αPDL1 solution, and the mixture was reacted at room temperature for 30 minutes. The excess Sulfo-SMCC was removed by a desalting column. pHA-AOHX-VAP-Cys in PBS was added, mixed, and reacted at 4°C for 12 hours. The mixture was dialyzed twice with pure water (30 minutes each time) in a 14K Da dialysis bag, and the liquid in the dialysis bag was collected to obtain a fully targeted peptide-modified PDL1 antibody complex (pHA-AOHX-VAP-αPDL1).

[0120] pHA-AOHX-VAP-DOTA-Gd was prepared as an example of a whole-process targeted peptide-modified diagnostic or therapeutic nuclide chelate. 0.1 mmol of pHA-AOHX-VAP-Cys was dissolved in PBS at pH 7, and 0.1 mmol of MAL-DOTA was added and stirred for 1 hour. The reaction solution was purified by preparative liquid phase and freeze-dried to obtain pHA-AOHX-VAP-DOTA. The freeze-dried pHA-AOHX-VAP-DOTA was dissolved in an aqueous ammonium acetate solution, and then GdCl3·6H20 was added and stirred at room temperature for 1 hour. The reaction solution was purified by preparative chromatography and freeze-dried to obtain pHA-AOHX-VAP-DOTA-Gd.

[0121] Example 2 In vitro targeting validation of the whole process targeting polypeptide

[0122] 1. In vitro targeting of the whole process targeting peptide to primary brain capillary endothelial cells BCEC

[0123] The brains of 4-week-old SD rats were removed after decapitation, and the cerebral cortex was quickly separated in pre-cooled D-Hanks solution. The meninges and large blood vessels in the brain were rolled off and cut into pieces. After adding collagenase and DNase, the mixture was digested at 37°C for 90 minutes, centrifuged at 1000 rpm for 8 minutes, the supernatant was discarded, and the mixture was transferred to a 20% BSA DMEM solution, centrifuged at 1000g / min at 4°C for 20 minutes, the middle and upper liquid was discarded, the bottom microvessels were transferred to DMEM culture medium, centrifuged at 1000 rpm for 5 minutes, the microvessel segments were resuspended in DMEM culture medium containing 20% ​​fetal bovine serum, inoculated in a 12-well plate, and cultured at 37°C, 5% CO2 and saturated humidity for 24 hours, then changed to endothelial-specific culture medium containing puromycin for further culture for 72 hours, and then changed to endothelial-specific culture medium containing cell growth factor for culture for 72 hours to obtain primary brain capillary endothelial cells. Prepare a fluorescent labeled peptide solution with a fluorescence concentration of 5 μM using DMEM culture medium containing 10% FBS, aspirate the DMEM culture medium in the 12-well plate, add the drug solution, incubate at 37°C for 4 hours, and discard the fluorescein solution. Wash the plate twice with PBS, add trypsin to digest the cells, disperse the cells with DMEM culture medium, centrifuge, discard the supernatant, wash twice with PBS, and finally disperse the cells in each well in 200 μL PBS and measure them by flow cytometry. The results are shown in Figure 3 It can be seen that there is no significant difference in the uptake of pHA-AOHX-VAP and pHA-AHX-VAP by BCEC cells.

[0124] 2. In vitro targeting of the whole-process targeting peptide to umbilical vein endothelial cells (HUVEC)

[0125] HUVEC cells in monolayer culture in the logarithmic growth phase were taken and digested with 0.25% trypsin. Single cell suspension was prepared with DMEM culture medium containing 10% fetal bovine serum. 1×10 cells were added to each well. 5 The cells were inoculated into a 12-well culture plate with a volume of 1 mL per well. The culture plate was moved into a carbon dioxide incubator and cultured for 24 h at 37°C, 5% CO2 and saturated humidity. The same experiment was performed as above. The results of flow cytometric analysis are shown in Figure 4 It can be seen that there is no significant difference in the uptake of pHA-AOHX-VAP and pHA-AHX-VAP by HUVEC cells.

[0126] 3. In vitro targeting of whole-process targeted peptides to glioma cells U87

[0127] The U87 cells in the logarithmic growth phase were cultured in monolayers and the same experiment was performed as above. The results of flow cytometry analysis are shown in Figure 5 It can be seen that the uptake of pHA-AOHX-VAP by U87 cells is significantly higher than that of pHA-AHX-VAP, suggesting that it has better targeting ability to glioma cells.

[0128] Example 3 Saturation Solubility Determination of the Whole-Process Targeted Peptide Drug Complex

[0129] A certain amount of DOX, pHA-AHX-VAP-DOX and pHA-AOHX-VAP-DOX were weighed and dispersed in 100 μL PBS (pH 7.4), incubated at 37°C for 24 h, filtered through a 0.22 μm filter membrane, and then diluted 10-fold and 20-fold, respectively, to detect the concentration of doxorubicin. The results are shown in Figure 7 Free doxorubicin has poor water solubility, with a saturated solubility of 0.22±0.03 mg / mL in PBS buffer at pH 7.4. After forming a covalent complex with the polypeptide, the solubility of doxorubicin is greatly increased due to the hydrophilicity of the polypeptide. The saturated solubility of pHA-AHX-VAP-DOX is 7.09±0.15 mg / mL (based on doxorubicin), and the saturated solubility of pHA-AOHX-VAP-DOX is 17.29±0.43 mg / mL, which are 32 times and 78 times higher than that of the prototype doxorubicin, respectively. From the perspective of solubility, since the use of organic solvents can be avoided, the complex pHA-AOHX-VAP-DOX has better in vivo application prospects, which is conducive to increasing the dosage and improving the therapeutic effect.

[0130] Example 4 Intracellular doxorubicin dosage detection

[0131] Take the monolayer cultured tumor cells U87 and macrophages RAW264.7 in the logarithmic growth phase, digest the monolayer cultured cells with 0.25% trypsin, and prepare a single cell suspension in DMEM culture medium containing 10% fetal bovine serum. 5 Each cell was inoculated into a 6-well culture plate with a volume of 2 mL per well. The culture plate was moved into a carbon dioxide incubator and cultured overnight at 37°C, 5% CO2 and saturated humidity. The culture medium in the culture plate was aspirated and DOX, pHA-AHX-VAP-DOX and pHA-AOHX-VAP-DOX solutions were added respectively. The cells were incubated at 37°C for 4 h and the supernatant was aspirated. After digestion and collection of cells, they were washed three times with PBS solution and counted. After the last wash, the supernatant was aspirated as much as possible and acidified methanol (V 20) was added to the cell pellet. 甲醇 :V 甲酸 =98:2), ultrasonically disrupted cells, centrifuged at 10000 rpm for 10 min, and the supernatant was collected to detect the concentration of doxorubicin. The results are shown in Figure 8 , Fig. 9 .Depend on Figure 8 It can be seen that free doxorubicin can diffuse into cells passively due to direct contact with cells, so the cells take up a large amount of drug. The whole process of targeted peptide-doxorubicin complex is mediated by receptors into cells, among which the uptake of pHA-AOHX-VAP-DOX by U87 cells is significantly higher than that of pHA-AHX-VAP-DOX. Fig. 9 It can be seen that compared with the model tumor cells U87, the uptake of the whole-process targeting peptide-doxorubicin complex by macrophages was greatly reduced, suggesting that it has good targeting specificity.

[0132] Example 5 Maximum Tolerated Dose Experiment

[0133] Male Balb / c mice, 5 mice in a group, were given DOX (5mg / kg; 10mg / kg; 20mg / kg) and pHA-AOHX-VAP-DOX (20mg / kg; 40mg / kg; 60mg / kg; 80mg / kg; 100mg / kg) via tail vein. The weight changes of mice were recorded every day for 14 consecutive days to observe whether there were any deaths of mice. MTD is the dose at which mice did not die. The results are shown in Fig.10 The mice injected with pHA-AHX-VAP-DOX at the same DOX dose died. Fig.10 It can be seen that the MTD of free DOX is about 10 mg / kg, and the MTD of pHA-AOHX-VAP-DOX is about 40 mg / kg, and the in vivo safety is greatly improved.

[0134] Relative weight change = daily weight / initial weight

[0135] Example 6 In vivo pharmacodynamics experiment of the whole process targeted polypeptide drug complex

[0136] Nude mice weighing about 20 g were anesthetized and fixed on a stereotaxic apparatus. 5 μL of U87 cell suspension (5×10 5 The cells were inoculated into the striatum of mice (0.6 mm forward, 1.8 mm to the right, and 3 mm deep from the anterior fontanelle) to construct an orthotopic glioma mouse model. On the 5th day after tumor inoculation, the mice were randomly divided into 4 groups, with 10 mice in each group. Every two days, normal saline, DOX (single dose of 1 mg / kg), and pHA-AOHX-VAP-DOX (single dose of doxorubicin of 1 mg / kg and 3 mg / kg, respectively) were injected into the tail vein. The death time of each group of model mice was recorded, and the survival curve was drawn. The results are shown in Fig.11 Taking the median survival time of the model animals as an indicator, pHA-AOHX-VAP-DOX (median survival time of 39 and 44.5 days at 1 mg / kg and 3 mg / kg, respectively) significantly prolonged the survival time of the model animals compared with the normal saline (median survival time of 32.5 days) and free DOX (median survival time of 35.5 days) groups, and the survival time was dose-dependent.

[0137] On the second day after administration, the tumor-bearing nude mice were killed and the tumor tissues were fixed and made into paraffin sections or frozen sections. The inhibition of neovascularization was detected by CD31 staining, and the promotion of apoptosis was detected by TUNEL staining. The results are shown in Fig.12 .Depend on Fig.12 It can be seen that compared with free doxorubicin, pHA-AOHX-VAP modified doxorubicin can significantly promote tumor apoptosis and inhibit angiogenesis.

[0138] Example 7 Western Blot Detection of GRP78 Protein Expression

[0139] After different treatments, the cells were aspirated for medium, washed twice with pre-cooled PBS, scraped and collected into EP tubes, membrane proteins were extracted using a cell membrane protein extraction kit, and the supernatant was centrifuged at 4°C 12000rpm for use. The total protein concentration was determined by the BCA method, and then 5× loading was added at a ratio of 4:1 and denatured by boiling. The samples were stored at -20°C for use. The samples containing about 40 μg of protein were subjected to 15% polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in TBST containing 5% skim milk powder at room temperature for 1h, and then hybridized with the corresponding primary antibody at 4°C overnight. TBST was washed three times, and then the membrane was incubated with HRP-labeled secondary antibodies at room temperature for 1h and washed three times with TBST. The protein bands were developed and detected on a gel imager using an enhanced chemiluminescence protein blotting detection kit (ECL) according to the instructions.

[0140] After U87 cells were treated with different concentrations of free DOX for 48 h, membrane proteins were extracted and detected according to the above method. The results are shown in Fig.14 .Depend on Fig.14 It can be seen that after U87 cells were treated with different concentrations of DOX, the expression of GRP78 protein on the cell membrane surface was significantly upregulated.

[0141] U87 cells were treated with different concentrations of pHA-AOHX-VAP-DOX (concentration calculated based on doxorubicin) for 48 h, and membrane proteins were extracted and detected according to the above method. The results are shown in Fig.15 .Depend on Fig.15 It can be seen that after U87 cells were treated with different concentrations of pHA-AOHX-VAP-DOX, the expression of GRP78 protein on the cell membrane surface was significantly upregulated.

[0142] After U87 cells were treated with different concentrations of TMZ for 48 h, membrane proteins were extracted and detected according to the above method. The results are shown in Fig.16 .Depend on Fig.16 It can be seen that after U87 cells were treated with different concentrations of TMZ, the expression of GRP78 protein on the cell membrane surface was significantly upregulated.

[0143] After U87 cells were treated with 5 μg / mL TMZ for different time periods, membrane proteins were extracted and detected according to the above method. The results are shown in Fig.17 .Depend on Fig.17 It can be seen that after U87 cells were treated with TMZ, the expression of GRP78 protein on the membrane surface was significantly upregulated starting from 12 hours, and the expression of GRP78 protein on the cell membrane surface was still significantly upregulated until 48 hours.

[0144] Mice bearing orthotopic glioma were intragastrically administered with 10 mg / mL TMZ for different time periods, then the mice were killed, the brain tumors were removed, and membrane proteins were extracted and tested according to the above method. The results are shown in Fig.18 .Depend on Fig.18 It can be seen that after TMZ treatment of nude mice bearing orthotopic brain glioma models, the expression of GRP78 protein in tumor tissue was significantly upregulated 48 hours later, which was 1.45 times that of the control group.

[0145] Example 8 Immunofluorescence detection of GRP78 protein expression

[0146] After different treatments, nude mice bearing orthotopic glioma were killed, brain tumors were removed, fixed with 4% paraformaldehyde, embedded in paraffin, and sliced ​​at 10 μm. The slices were incubated with GRP78 antibody (1:100) at 4°C overnight, then incubated with FITC-labeled secondary antibody (1:500) for 2 hours, and finally the slices were counterstained with DAPI to show the cell nucleus. After sealing, the slices were observed with a laser confocal microscope.

[0147] Different doses of TMZ were given to mice bearing orthotopic glioma by gavage. Brain tumors were removed and tested 48 hours later according to the above method. The results are shown in the figure. Fig.19 .Depend on Fig.19 It can be seen that the expression of tumor GRP78 protein increased to varying degrees after administration of different concentrations of TMZ, high, medium and low. After comprehensive consideration of safety and efficacy, a dose of 10 mg / kg was selected for subsequent experiments.

[0148] Mice bearing orthotopic glioma were given 10 mg / kg TMZ by gavage. Brain tumors or normal brain tissues were obtained for testing at different time points according to the above method. The results are shown in the figure. Fig. 20 , 21 .Depend on Fig. 20 It can be seen that the expression of GRP78 protein in the tumor site increased significantly 48 hours after administration, which is consistent with the results of Western Blot. Fig.21 It can be seen that there is no significant change in the expression of GRP78 protein in normal brain tissue at different times after administration, indicating that the expression of GRP78 protein in normal tissue is low and will not be upregulated by chemotherapy drugs.

[0149] Example 9 Uptake of Fluorescein Modified with Targeted Peptide by U87 Cells after TMZ Pre-administration

[0150] In vitro cell uptake experiment: monolayer cultured cells in the logarithmic growth phase were taken, digested with 0.25% trypsin, and prepared into single cell suspension with DMEM culture medium containing 10% fetal bovine serum. 5 Cells 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 cultured overnight at 37°C, 5% CO2 and saturated humidity. The culture medium was then replaced with DMEM culture medium containing 5 μg / mL TMZ and 10% fetal bovine serum. After 48 hours, the culture medium in the culture plate was aspirated, and FAM and pHA-AOHX-VAP-Fluorescein solutions were added respectively. The cells were incubated at 37°C for 4 hours, and the supernatant was aspirated and discarded. The cells were washed three times with PBS solution and analyzed by flow cytometry.

[0151] Competitive inhibition experiment: In order to further investigate whether TMZ pre-administration promoted the uptake of the whole process of targeted peptide-modified fluorescein through the GRP78 receptor, the U87 cells pre-administered with TMZ for 48 hours were subjected to competitive inhibition experiments. 5The density of cells / mL was dispersed in a 2mL EP tube, precooled at 4℃ for 30min, and then 100μM VAP solution prepared in cell culture medium was added, incubated at 4℃ for 2h to saturate the receptors, and then pHA-AOHX-VAP-Fluorescein was added and incubated at 4℃ for 12h. Then PBS was washed three times, and new PBS was added to resuspend the cells, and flow cytometry analysis was performed. The results are shown in Fig. 22 .Depend on Fig. 22 It can be seen that TMZ pre-administration for 48 hours can promote the uptake of pHA-AOHX-VAP-modified fluorescein by U87 cells. After TMZ pre-administration, the fluorescence intensity of the pHA-AOHX-VAP-Fluorescein group was 1.35 times that of the control group, which is close to the multiple of TMZ upregulation of GRP78 protein. After TMZ pre-administration, the GRP78 protein on the cell membrane surface was saturated with excessive VAP, and the uptake of pHA-AOHX-VAP-modified fluorescein by U87 cells was significantly reduced, proving that TMZ does promote the uptake of pHA-AOHX-VAP-Fluorescein by U87 cells by upregulating GRP78 protein.

[0152] Example 10 Uptake of the drug complex of the whole-process targeting polypeptide by U87 cells after TMZ pre-administration

[0153] U87 cells were pre-administered with TMZ as described above. After 48 h, the culture medium in the culture plate was aspirated, and DOX and pHA-AOHX-VAP-DOX solutions were added, respectively, and incubated at 37 °C for 4 h, and the supernatant was aspirated. After digestion and collection of cells, they were washed twice with PBS solution and counted. After the last wash, the supernatant was aspirated as much as possible, and acidified methanol (V 甲醇 :V 甲酸 =98:2), ultrasonically disrupted cells, centrifuged at 10000 rpm for 10 min, and the supernatant was collected to detect the concentration of doxorubicin. The results are shown in Fig.23 .Depend on Fig.23 It can be seen that TMZ pre-administration for 48 hours can significantly promote the uptake of pHA-AOHX-VAP-DOX by U87 cells. After pre-administration, the average drug dose of the pHA-AOHX-VAP-DOX group was 1.34 times that of the control group.

[0154] Example 11 Distribution of fluorescein modified with targeted polypeptide in nude mice with orthotopic glioma model after TMZ pre-administration

[0155] Nude mice weighing about 20 g were anesthetized and fixed on a stereotaxic apparatus. 5 μL of U87 cell suspension (8×10 5cells) and inoculated them in the mouse striatum (0.6 mm forward, 1.8 mm to the right, and 3 mm in depth) to construct an orthotopic glioma mouse model. Ten days after tumor inoculation, mice were gavaged with 10 mg / kg TMZ or injected with saline via the tail vein. 48 hours later, mice were given a pHA-AOHX-VAP-Cy5 solution with the same fluorescence intensity via the tail vein. Two hours later, the mice were anesthetized and perfused with saline. Major organs such as the brain, heart, liver, spleen, lungs, and kidneys were collected, and the fluorescence distribution of each organ was observed using a small animal in vivo imaging device. The results are shown in Fig.24 .Depend on Fig.24 It can be seen that pre-administration of TMZ can significantly increase the accumulation of pHA-AOHX-VAP modified fluorescein in the brain. After 2 hours, the mice were killed and perfused, and the tissues were taken for in vitro imaging. Semi-quantitative results showed that the fluorescence intensity of the TMZ pre-administration group was 1.21 times that of the pHA-AOHX-VAP-Cy5 group. The results of in vitro organs showed that pre-administration of TMZ would not change the distribution of pHA-AOHX-VAP modified fluorescein in normal organs, indicating that combined medication would not cause safety risks to major peripheral tissues.

[0156] Example 12 Pharmacological efficacy of TMZ combined with pHA-AOHX-VAP-DOX in the treatment of glioma

[0157] The orthotopic brain glioma model was constructed as described above. The modeling day was set as day 0. TMZ was administered by gavage on days 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21, with a single dose of 10 mg / kg. pHA-AOHX-VAP-DOX was administered through the tail vein on days 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, with a single dose of 3 mg / kg containing doxorubicin. The weight changes of mice were recorded at each administration of pHA-AOHX-VAP-DOX, and the death time of the model mice was recorded. The survival curve was drawn. The results are shown in Fig.25 .Depend on Fig.25 As can be seen from A, the median survival time of the normal saline group was 29 days, that of the pHA-AOHX-VAP-DOX group was 38.5 days, and that of the TMZ group was 48 days. The median survival time of the TMZ pre-administration combined with pHA-AOHX-VAP-DOX group was extended to 62 days, which was significantly different from that of the single drug, reflecting the advantages of the drug combination.

[0158] The Kim Jong-kyun formula was used to evaluate whether TMZ and pHA-AOHX-VAP-DOX in the combination had a synergistic effect. (Among them, E A 、E B is the effect of drug A and drug B when administered alone, E A+BThe effect of A and B combined, here converted by median survival extension rate, median survival extension rate (%) = (median survival of mice in the drug group - median survival of mice in the control group) / median survival of mice in the control group × 100%. q value 0.85-1.15 is additive, greater than 1.15 is synergistic, less than 0.85 is antagonistic), the calculated q value is 1.48, indicating that TMZ and pHA-AOHX-VAP-DOX have a significant synergistic effect.

[0159] Depend on Fig.25 As shown in Figure B, during the administration process, except for the mice in the normal saline group which began to lose weight due to tumor progression on day 21, there was no significant weight loss in the other groups of mice, indicating that the drug combination has good safety.

[0160] On the second day after the administration of pHA-AOHX-VAP-DOX, two nude mice in each group were killed, and brain tissue was obtained, fixed with 4% paraformaldehyde, dehydrated and transparent, embedded in wax, sliced, dewaxed, stained with HE, sealed with coverslip, and observed using a slice scanner. The results are shown in Fig.26 .Depend on Fig.26 It can be seen that the administration of TMZ and pHA-AOHX-VAP-DOX alone can inhibit the growth of orthotopic brain gliomas to a certain extent, while no obvious tumors were found in the brains of mice in the TMZ pre-administration combined with pHA-AOHX-VAP-DOX group, indicating that the drug combination can significantly inhibit the growth of orthotopic brain gliomas.

Claims

1. A whole-process targeting polypeptide, characterized in that: The whole-process targeting polypeptide comprises pHA and VAP covalently linked by a bridging structure; the VAP is L VAP, L VAP reverse peptide, D VAP or S VAP; in, L The amino acid sequence of VAP is shown in SEQ ID NO: 1; S VAP is L D-configuration peptide of VAP; D VAP is L The D-configuration peptide of the VAP reverse peptide; The bridging structure is amino-monoethylene glycol-carboxylic acid or its derivatives, having the following general formula: Wherein n is a natural number, preferably 1-20.

2. A targeted diagnostic polypeptide complex, characterized in that: The targeted diagnostic polypeptide complex is formed by connecting the whole-process targeting polypeptide as claimed in claim 1 with an imaging molecule.

3. The targeted diagnostic polypeptide complex according to claim 2, characterized in that: The imaging molecule is an optical imaging molecule, a magnetic resonance imaging agent and / or a radionuclide imaging agent; Preferably, the optical imaging molecule is selected from the following group: (1) one or more of the fluorescent probe molecules FITC, FAM, 6-TET, 5-TAMRA, HEX and 6-JOE; (2) one or more of the near-infrared dye molecules Cy3, Cy3.5, Cy5, Cy5.5, Cy7, IR783, IR820, DiR, DiD, BODIPY630 / 650-X, BODIPY650 / 665-X, BODIPY665 / 676, TO-PRO-3 and TO-PRO-5; (3) one or more of the chemiluminescent molecules luminol, isoluminol, AMPPD, CSPD, CDP-star, lucigenin and Raman probe molecules; The magnetic resonance imaging agent is a chelate of Gd magnetic resonance material; 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 Chelates of radionuclides for Tb imaging; More preferably, the magnetic resonance imaging agent or radionuclide imaging agent consists of a bifunctional chelator and a nuclide for magnetic resonance imaging or a radionuclide for imaging; wherein the bifunctional chelator 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 and NS3.

4. A targeted therapeutic polypeptide complex, characterized in that: The targeted therapeutic polypeptide complex is formed by connecting the whole-process targeting polypeptide as claimed in claim 1 with a therapeutic drug; Preferably, the whole-process targeting polypeptide is connected or condensed with the therapeutic drug via a pH-sensitive hydrazone bond, a pH-sensitive boronate bond or a disulfide bond or directly.

5. The targeted therapeutic polypeptide complex according to claim 4, characterized in that: The therapeutic drug is selected from one or more of the following: tumor chemotherapy drugs, anti-tumor stem cell drugs, molecular targeted drugs, polypeptide drugs, antibody drugs or therapeutic radionuclide chelates; Preferably, the tumor chemotherapy drug is selected from the following group: (1) one or more anthracyclines such as doxorubicin and epirubicin; (2) one or more taxane drugs such as paclitaxel, docetaxel, and cabazitaxel; (3) Camptothecin drugs such as one or more of camptothecin, hydroxycamptothecin, 9-nitrocamptothecin and irinotecan; (4) one or more vinca alkaloids such as vinblastine and vincristine; (5) one or more proteasome inhibitors such as bortezomib and carfillozomib; The anti-tumor stem cell drugs are selected from lactone anti-tumor drugs, such as parthenolide and its derivatives; The molecular targeted drugs are selected from one or more of the following: trametinib, imatinib, nilotinib, dasatinib, everolimus, erlotinib, sunitinib, sorafenib, ibrutinib, regorafenib, vemurafenib and olaparib; The polypeptide drug is a p53 activation peptide; The antibody drugs are selected from one or more of the following: rituximab, bevacizumab, trastuzumab, cetuximab, pertuzumab, ipilimumab, nivolumab and PD-L1 monoclonal antibody, and combinations of antibody fragments modified by genetic engineering, including Fab fragments, single domain antibodies, Fv fragments, single chain antibodies, bivalent small molecule antibodies, mini antibodies, and nano antibodies; 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 and 227 Th therapeutic radionuclide chelates; More preferably, the therapeutic radionuclide chelate is composed of a bifunctional chelator and a therapeutic radionuclide, wherein the bifunctional chelator 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 and NS3.

6. A targeting polypeptide polymer carrier material, characterized in that: The targeting polypeptide polymer carrier material is formed by connecting the whole-process targeting polypeptide as claimed in claim 1 with a polyethylene glycol-complex; wherein the complex is a lipophilic material molecule or a hydrophilic ligand molecule; Preferably, the lipophilic material molecule is selected from one or more of the following: phospholipids, polylactic acid, lactic acid-glycolic acid copolymer and polycaprolactone; and the hydrophilic ligand molecule is biotin.

7. A targeted drug delivery system, characterized in that: The targeted drug delivery system comprises the targeted polypeptide polymer carrier material and excipients as claimed in claim 6; when the polyethylene glycol-complex in the targeted polypeptide polymer carrier material is polyethylene glycol-phospholipid, the targeted drug delivery system is a liposome drug delivery system, a micelle drug delivery system or a disc drug delivery system; when the polyethylene glycol-complex is polyethylene glycol-polylactic acid, polyethylene glycol-lactic acid copolymer or polyethylene glycol-polycaprolactone, the targeted drug delivery system is a micelle drug delivery system or a nanoparticle drug delivery system; when the polyethylene glycol-complex is polyethylene glycol-biotin, the targeted drug delivery system is a biomembrane-coated nano drug delivery system; Preferably, the auxiliary material comprises one or more of cholesterol, phospholipid, polyethylene glycol-phospholipid, polyethylene glycol-polylactic acid, polyethylene glycol-lactic acid copolymer and polyethylene glycol-polycaprolactone.

8. The targeted drug delivery system according to claim 7, characterized in that: Using the targeted drug delivery system to encapsulate a drug, wherein the drug is a diagnostic drug or a therapeutic drug; Preferably, the diagnostic drug is the imaging molecule defined in the targeted diagnostic polypeptide complex as claimed in claim 3; and the therapeutic drug is the therapeutic drug defined in the targeted therapeutic polypeptide complex as claimed in claim 5.

9. A drug combination for combined use, characterized in that: The drug combination comprises a chemotherapeutic drug and the targeted therapeutic polypeptide complex as described in claims 4 and 5 or the targeted drug delivery system as described in claims 7 or 8; Preferably, the chemotherapy drug is selected from one or more of doxorubicin, epirubicin, temozolomide, paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine, bortezomib, carfilzomib, and 5-fluorouracil; More preferably, the drug combination is administered by first administering the chemotherapy drug, and then administering the targeted therapeutic polypeptide complex as described in claims 4 and 5 or the targeted drug delivery system as described in claims 7 or 8 after a certain period of time; the certain period of time is preferably 24-48 hours.

10. Use of the whole-process targeting polypeptide as described in claim 1, the targeted diagnostic polypeptide complex as described in claims 2 and 3, the targeted therapeutic polypeptide complex as described in claims 4 and 5, the targeted polypeptide polymer carrier material as described in claim 6, the targeted drug delivery system as described in claim 7 or 8 and / or the drug combination as described in claim 9 in the preparation of imaging diagnostic drugs, tracer drugs and / or targeted therapeutic drugs for tumors such as brain tumors or peripheral tumors.

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