A whole-process targeted molecule and its application in constructing a drug delivery system

Abstract

The present invention belongs to the field of pharmacy and relates to a whole-process targeting molecule, specifically to a whole-process targeting molecule with targeting ability to brain capillary endothelial cells (across the blood-brain barrier), tumor neovascular endothelial cells (across the blood-tumor barrier), tumor-mimicking blood vessels, tumor cells, and tumor stem cells, as well as the uses of its modified drug complexes and drug delivery systems in tumor diagnosis and targeted therapy. The whole-process targeting molecule of the present invention and the drug delivery system constructed therefrom can target and deliver the carried imaging molecules, therapeutic drugs, and nano-drug delivery systems to intracranial tumor tissues, or target and deliver them to peripheral tumor tissues with characteristics of brain metastasis, significantly improving the tumor diagnosis and treatment effects. This whole-process targeting molecule can mediate the crossing of imaging molecules, therapeutic drugs, and nano-drug delivery systems across the blood-brain barrier and / or across the blood-tumor barrier, and is used for the diagnosis and treatment of brain tumors and peripheral tumors with characteristics of brain metastasis.

Description

Technical Field

[0001] The present invention belongs to the field of pharmacy, and relates to a whole-process targeting molecule and its application in the construction of a drug delivery system. Specifically, it relates to a whole-process targeting molecule and its application in the construction of a drug delivery system for tumor imaging diagnosis and targeted therapy. In particular, it relates to a whole-process targeting molecule that targets brain capillary endothelial cells (across the blood-brain barrier), tumor neovascular endothelial cells (across the blood-tumor barrier), tumor mimic vessels, tumor cells, and tumor stem cells, as well as the use of its modified drug complexes and drug delivery systems in tumor diagnosis and targeted therapy. Background Art

[0002] The prior art discloses that tumors have become a serious threat to human life and health, with the mortality rate ranking first among all diseases. Among them, the incidence and mortality of primary brain tumors are both in the top 10 of the tumor rankings in China, and glioblastoma accounts for about 45% of primary brain tumors. The median survival of its patients is less than 16 months, posing a great harm. 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 generation of multi-drug resistance. Therefore, in recent years, active targeting has become an important strategy to improve the targeting efficiency of brain tumor tissues. The active targeting strategy mainly targets receptors or transporters highly expressed in brain tumor tissues, and uses corresponding ligands with specific recognition and binding abilities to the receptors or transporters to deliver drugs or drug delivery systems to brain tumor tissues or cells. Most of the current ligands only target a certain receptor or transporter and a certain cell. However, brain tumor tissues not only have tumor cells, but also tumor stem cells, tumor mimic vessels, brain capillaries and their blood-brain barrier (BBB), tumor neovessels and their blood-tumor barrier (BTB), etc. In the early stage of brain tumor occurrence, the BBB remains intact and restricts the entry of drugs into the brain, making about 98% of small molecule chemotherapy drugs and almost 100% of macromolecular drugs such as proteins unable to penetrate the BBB and enter the brain, resulting in almost ineffective drug treatment; as the tumor develops, tumor neovascularization occurs, but the brain tumor neovessels are relatively dense and have poor permeability compared to peripheral tumors. The formed BTB becomes the main obstacle to drug delivery, and the BBB still exists in the glioblastoma infiltration area, which also hinders drug transport; at the same time, 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 high tolerance to drug treatment and are prone to recurrence of glioblastoma. In view of this, it is crucial to select a targeting molecule with better ability to cross the BBB and BTB and better affinity for brain tumor cells.

[0003] Based on the current state of the art, the inventors of the present application further modified the existing targeting molecules. Using the principle of molecular fusion, a brain targeting molecule and a tumor targeting molecule were covalently linked to form a whole-process targeting molecule, enabling it to have a whole-process targeting function for the growth and development of brain tumors or peripheral tumors with brain metastasis characteristics. At the same time, a diagnostic and therapeutic drug complex modified with the whole-process targeting molecule, a modified polymer carrier material, and the drug delivery system constructed therefrom were built, so as to more effectively exert the functions of imaging diagnosis and targeted therapy for brain tumors or peripheral tumors with brain metastasis characteristics. Summary of the Invention

[0004] The object of the present invention is to provide a whole-process targeting molecule and its application in the construction of a drug delivery system based on the current state of the art. Using the principle of molecular fusion, a brain targeting molecule and a tumor targeting molecule are covalently linked to form a whole-process targeting molecule, enabling it to have a whole-process targeting function for the growth and development of brain tumors or peripheral tumors with brain metastasis characteristics. At the same time, a diagnostic and therapeutic drug complex modified with the whole-process targeting molecule, a modified polymer carrier material, and the drug delivery system constructed therefrom are built, effectively exerting the functions of imaging diagnosis and targeted therapy for brain tumors or peripheral tumors with brain metastasis characteristics.

[0005] The whole-process targeting molecule of the present invention, which targets brain capillary endothelial cells (across the BBB), tumor neovascular endothelial cells (across the BTB), tumor mimic blood vessels, tumor cells, and tumor stem cells, is formed by covalently linking a brain targeting molecule and a tumor targeting molecule; further, imaging molecules, therapeutic drugs, and polymer carrier materials are modified with the whole-process targeting molecule to construct a targeting molecule-drug complex and a nano-drug delivery system modified with the targeting molecule, which can improve the targeted diagnosis and treatment effects of drugs on the brain, brain tumors, or peripheral tumors with brain metastasis characteristics.

[0006] Specifically, using the principle of molecular fusion, the present invention prepares a whole-process targeting polypeptide molecule by covalently linking a brain targeting molecule and a tumor targeting molecule, enabling it to have the targeting capabilities of both molecules simultaneously, and capable of exerting a whole-process targeting effect on the growth and development of tumors, especially brain tumors, 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.

[0007] The targeting molecules such as small molecules, polypeptide molecules, or protein molecules involved in the present invention that cross the blood-brain barrier include: p-hydroxybenzoic acid (pHA) and its derivatives, fatty acids, especially myristic acid (MC) and its derivatives, D8 polypeptide, WSW polypeptide, D WSW polypeptide, TGN polypeptide, D TGN polypeptide, CDX polypeptide, D CDX polypeptide, T7 polypeptide, and DPolypeptides such as T7 polypeptide and its derivatives, proteins such as transferrin, lactoferrin and their derivatives. The sequences of each polypeptide are shown in the attached table (Table 1 is the polypeptide amino acid sequence table).

[0008] The targeting molecules such as polypeptide molecules or protein molecules that cross the blood-tumor barrier involved in the present invention include: VAP polypeptide, cVAP polypeptide, S VAP polypeptide, D VAP polypeptide, A7R polypeptide, cA7R polypeptide, D A7R polypeptide, RGD polypeptide, Stapled-RGD polypeptide, RW polypeptide, mn polypeptide, RAP12 polypeptide and D Polypeptides such as RAP12 polypeptide and their derivatives. The sequences of each polypeptide can be seen in the attached table of the specification (Attachment 1 - Polypeptide amino acid sequence table).

[0009] The whole-process targeting molecules designed in the present invention can form modified imaging molecular complexes, therapeutic drug complexes, and polymer carrier material complexes by introducing active functional groups into the molecules.

[0010] After introducing cysteine into the whole-process targeting molecules designed in the present invention, the sulfhydryl groups in the molecules react with maleimide-functionalized optical imaging molecules (such as fluorescent probe molecules FITC, FAM, 6-TET, 5-TAMRA, HEX, 6-JOE, etc., near-infrared dyes such as Cy3, Cy3.5, Cy5, Cy5.5, Cy7, IR783, IR820, DiR, DiD, BIDIPY630 / 650-X, BIDIPY650 / 665-X, BIDIPY665 / 676, TO-PRO-3, TO-PRO-5, etc., chemiluminescent substance molecules luminol, isoluminol, AMPPD, CSPD, CDP-star, lucigenin, etc., Raman probe molecules, etc.) to form complexes.

[0011] The whole-process targeting molecules designed in the present invention, with magnetic resonance imaging agents (such as chelates of radionuclides for magnetic resonance imaging such as Gd), or with radionuclide imaging agents (such as 18 F, 32 P, 35 S, 64 Cu, 67 / 68 Ga, 75 Se, 89 Zr, 86 Y, 99m Tc, 111 / 111m In, 123 / 125 I, 177 Lu, 149 / 161 Chelates of radionuclides for imaging such as Tb), or with chelates of therapeutic radionuclides (such as 90Y, 131 I, 152 / 155 Tb, 153 Sm, 177 Lu, 186 / 188 Re, 211 At, 212 / 213 Bi, 212 Pb, 225 Ac, 227 chelates of therapeutic radionuclides such as Th) to form a complex, wherein the chelate is composed of a bifunctional chelating agent and a nuclide for magnetic resonance imaging, or a bifunctional chelating agent and a radionuclide for imaging, or a bifunctional chelating agent and a therapeutic radionuclide. The bifunctional chelating agents in the chelate include DOTA, DOTAGA, NOTA, NOTAGA, NODA, DTPA, TETA, CB-TE2A, Cyclam, DFO, MAG3, EC, EDTA, DADT, HYNIC, CE-DTS, NS3, etc.

[0012] The whole-process targeted molecular modified drug designed by the present invention includes the formation of a pH-sensitive hydrazone bond through the reaction of a maleimide hexahydrazide derivative (involving drugs containing keto or aldehyde groups such as doxorubicin and epirubicin), or the formation of a disulfide bond through the reaction of a 3-(2-pyridyldithio)propionic acid derivative (involving drugs containing hydroxyl or amino groups such as paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine, and vinorelbine), or the formation of a pH-sensitive borate ester through the reaction of dopamine with a boric acid group in the drug (involving drugs containing a boric acid group such as bortezomib), or the direct formation of an amide bond through solid-phase synthesis (involving polypeptide drugs such as p53 activating peptide, melittin, scorpion venom peptide, and antimicrobial peptide), or the synthesis of a whole-process targeted molecule-drug complex through covalent or non-covalent connection (involving antibody drugs such as rituximab, bevacizumab, trastuzumab, cetuximab, pertuzumab, ipilimumab, nivolumab, PD-L1 monoclonal antibody, and their combinations of antibody fragments modified by genetic engineering means, including Fab fragments, single-domain antibodies, Fv fragments, single-chain antibodies, bivalent small molecule antibodies, microantibodies, nanobodies, etc.).

[0013] After introducing cysteine into the whole-process targeted molecule designed by the present invention, it is modified on high molecular carrier materials such as polyethylene glycol-distearoyl phosphatidylethanolamine (PEG-DSPE) with maleimide functional groups, polyethylene glycol-polylactic acid (PEG-PLA), polyethylene glycol-polylactic acid-glycolic acid copolymer (PEG-PLGA), polyethylene glycol-polycaprolactone (PEG-PCL), etc., for the construction of nano-drug delivery systems such as liposomes, micelles, disks, and nanoparticles for whole-process targeted molecular modification.

[0014] After introducing cysteine into the whole-process targeting molecule designed in the present invention, it is modified on targeting materials such as polyethylene glycol-biotin (PEG-Biotin) containing maleimide functional groups, and is used for constructing a biomembrane-coated nano-drug delivery system with whole-process targeting molecule modification.

[0015] The nano-drug delivery system modified with the whole-process targeting molecule designed in the present invention is used to encapsulate anthracycline drugs such as doxorubicin and epirubicin, taxane drugs such as paclitaxel, docetaxel, and cabazitaxel, camptothecin drugs such as camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, and irinotecan, vinca alkaloid drugs such as vincristine and vinorelbine, platinum drugs such as cisplatin, carboplatin, oxaliplatin, and miboplatin, proteasome inhibitors such as bortezomib and carfilzomib, lactone drugs such as parthenolide, molecular targeting drugs such as trametinib, imatinib, nilotinib, dasatinib, everolimus, erlotinib, sunitinib, sorafenib, ibrutinib, regorafenib, vemurafenib, olaparib, etc., polypeptide drugs such as p53 activation peptide, melittin, scorpion venom peptide, antimicrobial peptide, etc., antibody drugs such as rituximab, bevacizumab, trastuzumab, cetuximab, pertuzumab, ipilimumab, nivolumab, PD-L1 monoclonal antibody, etc. and their combinations of antibody fragments modified by genetic engineering means (including Fab fragments, single-domain antibodies, Fv fragments, single-chain antibodies, bivalent small molecule antibodies, microantibodies, and nanobodies, etc.), and encapsulate 90 Y, 131 I, 152 / 155 Tb, 153 Sm, 177 Lu, 186 / 188 Re, 211 At, 212 / 213 Bi, 212 Pb, 225 Ac, 227 chelates of therapeutic radionuclides such as Th.

[0016] The nano-drug delivery system with whole-process targeted molecular modification designed by the present invention is used for encapsulating optical imaging molecules (such as fluorescent probe molecules FITC, FAM, 6-TET, 5-TAMRA, HEX, 6-JOE, etc., near-infrared dyes Cy3, Cy3.5, Cy5, Cy5.5, Cy7, IR783, IR820, DiR, DiD, BIDIPY630 / 650-X, BIDIPY650 / 665-X, BIDIPY665 / 676, TO-PRO-3, TO-PRO-5, etc., chemiluminescent substances luminol, isoluminol, AMPPD, CSPD, CDP-star, lucigenin, etc., Raman probe molecules), encapsulating magnetic resonance imaging agents (such as chelates of magnetic resonance substances such as Gd), and encapsulating radionuclide imaging agents (such as 18 F, 32 P, 35 S, 64 Cu, 67 / 68 Ga, 75 Se, 89 Zr, 86 Y, 99m Tc, 111 / 111m In, 123 / 125 I, 177 Lu, 149 / 161 chelates of radionuclides for imaging such as Tb).

[0017] The whole-process targeted molecule designed by the present invention is used to mediate drugs or nano-drug delivery systems to cross the BBB and BTB, target tumor neovessels, tumor mimic vessels, tumor cells and tumor stem cells, and is used for targeted diagnosis and treatment of the brain, brain tumors or peripheral tumors with brain metastasis characteristics.

[0018] Using the method provided by the present invention, the whole-process targeted molecule pHA-VAP and its modified drug complex and nano-drug delivery system are designed and prepared, including:

[0019] 1. Preparation of pHA-VAP and its fluorescent label (pHA-VAP-Cy7)

[0020] pHA-VAP is prepared by solid-phase synthesis method; pHA-VAP-Cy7 is synthesized by Michael addition reaction of maleimide group and sulfhydryl group; its structure is characterized by HPLC and MS.

[0021] 2. Preparation of pHA-VAP-imaging agent

[0022] pHA-VAP-DTPA is synthesized by Michael addition reaction of maleimide group and sulfhydryl group, and chelates Gd or 99mTc-labeled pHA-VAP-DTPA-Gd or pHA-VAP-DTPA- 99m Tc.

[0023] 3. Evaluation of the in vitro and in vivo targeting ability of pHA-VAP

[0024] Investigate the in vitro affinity of pHA-VAP-Cy7 for brain capillary endothelial cells (BCEC), umbilical vein endothelial cells (HUVEC), and model tumor cells (such as glioma cell line U87).

[0025] By tail vein injection of pHA-VAP-Cy7 into normal mice and nude mice bearing subcutaneous U87 xenografts or intracranial U87 orthotopic tumors, investigate its distribution in animals at various time points.

[0026] 4. Preparation of pHA-VAP-drug complexes

[0027] After introducing cysteine, pHA-VAP reacts with maleimide hexahydrazide derivatives on the drug to form a polypeptide-drug complex containing a pH-sensitive hydrazone bond. The drugs involved include doxorubicin, epirubicin, and other drugs containing keto or aldehyde groups.

[0028] After introducing cysteine, pHA-VAP reacts with 3-(2-pyridyldithio)propionic acid derivatives on the drug to form a polypeptide-drug complex containing a disulfide bond. The drugs involved include paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine, vinorelbine, and other drugs containing hydroxyl or amino groups.

[0029] pHA-VAP is modified with dopamine and then reacts with boric acid groups on the drug to form a polypeptide-drug complex containing a pH-sensitive borate ester. The drugs involved include bortezomib and other drugs containing boric acid groups.

[0030] pHA-VAP is directly condensed with polypeptide drugs through solid-phase synthesis. The drugs involved include p53 activation peptides, antimicrobial peptides, polypeptide toxins, and other polypeptide drugs.

[0031] pHA-VAP is modified to obtain an antibody complex modified with a targeting functional molecule through random site modification (activating the free amino groups in the antibody and then covalently linking them to pHA-VAP) or site-directed modification (non-covalently linking the targeting molecule to the antibody through an affinity coupling reaction). The drugs involved include antibody drugs such as rituximab, bevacizumab, trastuzumab, cetuximab, pertuzumab, ipilimumab, nivolumab, PD-L1 monoclonal antibody, and their combinations of antibody fragments modified by genetic engineering means (including Fab fragments, single-domain antibodies, Fv fragments, single-chain antibodies, bivalent small molecule antibodies, microantibodies, nanobodies, etc.).

[0032] 5. Evaluation of the anti-tumor effects of pHA-VAP-Doxorubicin in vitro and in vivo

[0033] The pHA-VAP-Doxorubicin complex (pHA-VAP-DOX) was obtained by condensing pHA-VAP conjugated with cysteine and the maleimide hexahydrazide derivative (MAL-DOX) on doxorubicin. The in vitro growth inhibitory effects of pHA-VAP-DOX on U87 cells and HUVEC cells were investigated by the MTT method; the in vivo anti-tumor effects were evaluated by intravenous injection into nude mice bearing an orthotopic U87 intracranial tumor model, with the median survival time as the index.

[0034] 6. Construction and characterization of pHA-VAP-modified nano-drug delivery systems

[0035] First, pHA-VAP-modified polymer materials such as pHA-VAP-PEG-DSPE, pHA-VAP-PEG-PLA, pHA-VAP-PEG-PLGA, pHA-VAP-PEG-PCL, and pHA-VAP-PEG-biotin were prepared. The above-mentioned targeted polymer materials were prepared by introducing cysteine onto pHA-VAP, and the free sulfhydryl group reacted with the maleimide contained in Mal-PEG-DSPE, Mal-PEG-PLA, Mal-PEG-PLGA, Mal-PEG-PCL, Mal-PEG-biotin, etc., that is: Mal-PEG-DSPE, Mal-PEG-PLA, Mal-PEG-PLGA, Mal-PEG-PCL, Mal-PEG-biotin, etc. were dissolved in acetonitrile respectively, rotary evaporated to form a film, and reacted with PBS (pH 8.0) containing sulfhydryl pHA-VAP to obtain pHA-VAP-modified polymer materials.

[0036] Then, a nano-drug delivery system modified with pHA-VAP was constructed. A certain amount of pHA-VAP-PEG-DSPE, mPEG-DSPE, phospholipids, and cholesterol, or pHA-VAP-PEG-DSPE and mPEG-DSPE, or pHA-VAP-PEG-PLA and mPEG-PLA, or pHA-VAP-PEG-PLGA and mPEG-PLGA, or pHA-VAP-PEG-PCL and mPEG-PCL, along with a certain amount of the above-mentioned drugs, were used to construct the corresponding pHA-VAP-modified liposomes, micelles, disks, nanoparticles and other nano-drug delivery systems by methods such as film hydration; after incubating a certain amount of pHA-VAP-PEG-biotin with a biomembrane pre-modified with avidin, the polymer nanoparticles, silicon nanoparticles, nanogels, nanocrystals and other nano-drug delivery systems loaded with the above-mentioned drugs were coated to construct a biomembrane-coated nano-drug delivery system. The particle size and zeta potential of the nano-drug delivery system were characterized by a laser scattering particle size analyzer, and its morphological characteristics were characterized by a transmission electron microscope.

[0037] 7. Evaluation of the in vitro and in vivo targeting ability of the pHA-VAP-modified nano-drug delivery system

[0038] Examine the uptake of the pHA-VAP-modified nano-drug delivery system loaded with tumor therapeutic drugs by BCEC cells, tumor cells (U87 cells, 4T1 cells), and HUVEC cells.

[0039] The pHA-VAP-modified nano-drug delivery system loaded with tumor therapeutic drugs was injected into the tail vein of nude mice with U87 intracranial orthotopic tumor models or balb / c mice with 4T1 breast cancer orthotopic models to examine its tumor distribution at each time point.

[0040] 8. Evaluation of the in vitro and in vivo anti-tumor effects of the pHA-VAP-modified nano-drug delivery system

[0041] The in vitro growth inhibitory effects of the pHA-VAP-modified nano-drug delivery system loaded with tumor therapeutic drugs on tumor cells (U87 cells, 4T1 cells) and HUVEC cells were examined by the MTT method; the pHA-VAP-modified nano-drug delivery system loaded with tumor therapeutic drugs was injected into the tail vein of nude mice with U87 intracranial orthotopic tumor models or balb / c mice with 4T1 breast cancer orthotopic models, and its in vivo anti-tumor effects were evaluated using survival time, tumor inhibition curves, apoptosis of tumor tissue cells, the number of new blood vessels and stem cells as indicators.

[0042] The experimental results of the present invention show that the prepared pHA-VAP simultaneously has the ability of pHA to target brain capillaries and cross the BBB, and the ability of VAP to target tumor neovascular endothelial cells and cross the BTB, target tumor mimic vessels, tumor cells and tumor stem cells. It has good brain and tumor tissue targeting ability and imaging effect in model animals, and shows better brain tumor targeting ability; the drug complex and nano-drug delivery system modified with pHA-VAP show good tumor targeting performance and stronger anti-brain tumor effect.

[0043] Table 1 - Amino acid sequence list of polypeptide targeting molecules

[0044] Brief description of the drawings

[0045] Figure 1 HPLC and ESI-MS spectra of pHA-VAP-Cys

[0046] 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: 0 - 45 min 5% B - 65% B; Flow rate: 0.7 mL / min; Column temperature: 40 °C; Detection: UV214 nm, Retention time: 16 min. ESI-MS: 1080.4, consistent with the theoretical molecular weight.

[0047] Figure 2 HPLC and ESI-MS spectra of pHA-VAP-Cy7

[0048] The chromatographic method is the same as above, retention time: 25 min. ESI-MS: 1750.6, consistent with the theoretical molecular weight.

[0049] Figure 3 Uptake of Cy7-labeled pHA-VAP by primary brain capillary endothelial cells BCEC

[0050] The figure shows the quantitative (left) and qualitative (right) results of flow cytometry fluorescence detection after incubating Cy7-labeled VAP and pHA-VAP with BCEC cells for 4 h. It can be seen that the uptake of pHA-VAP by BCEC cells is significantly higher than that of VAP and free fluorescein.

[0051] Figure 4 Uptake of Cy7-labeled pHA-VAP by umbilical vein endothelial cells HUVEC

[0052] The figure shows the quantitative (left) and qualitative (right) results of flow cytometry fluorescence detection after incubating Cy7-labeled VAP and pHA-VAP with HUVEC cells for 4 h. It can be seen that the uptake of pHA-VAP by U87 cells is significantly higher than that of VAP and free fluorescein.

[0053] Figure 5 Uptake of Cy7-labeled pHA-VAP by glioblastoma cell U87

[0054] The figure shows the quantitative (left) and qualitative (right) results of flow cytometry fluorescence detection after incubating Cy7-labeled VAP and pHA-VAP with U87 cells for 4 h. It can be seen that the uptake of pHA-VAP by U87 cells is significantly higher than that of VAP and free fluorescein.

[0055] Figure 6 Tissue distribution map of Cy7-labeled pHA-VAP in mice bearing subcutaneous U87 xenograft tumor model

[0056] As can be seen from the figure, compared with free fluorescein and VAP-Cy7, the distribution of pHA-VAP-Cy7 in subcutaneous tumors increased within 4 h and the accumulation decreased after 24 h.

[0057] Figure 7 Tissue distribution map of Cy7-labeled pHA-VAP in mice bearing intracranial U87 orthotopic tumor model

[0058] As can be seen from the figure, compared with free fluorescein and VAP-Cy7, the distribution of pHA-VAP-Cy7 in orthotopic tumors increased within 24 h, indicating that the targeting molecule can cross the blood-brain barrier and blood-brain tumor barrier and significantly increase the accumulation of drugs at the brain tumor site.

[0059] Figure 8 HPLC and ESI-MS spectra of pHA-VAP-DOX

[0060] Chromatographic method: Chromatographic column (YMC, C18): 150×4.6 mm; Mobile phase A: water (containing 0.01% formic acid), Mobile phase B: pure acetonitrile; Elution program: 0 - 45 min 5% B - 65% B; Flow rate: 0.7 mL / min; Column temperature: 40°C; Detection: UV214 nm, Retention time: 17 min. ESI-MS: 1832.2, which is consistent with the theoretical molecular weight.

[0061] Figure 9 In vitro anti-U87 cell activity curve of pHA-VAP-DOX

[0062] As can be seen from the figure, after incubating U87 cells with DOX, MAL-DOX, VAP-DOX or pHA-VAP-DOX for 72 hours, their IC 50They were 0.06, 1.45, 1.57 and 0.38 μM respectively. The results showed that the in vitro anti-tumor activity of doxorubicin modified by pHA-VAP was superior to that of maleimide doxorubicin and VAP-modified doxorubicin.

[0063] Figure 10 、In vitro anti-HUVEC cell activity curve of pHA-VAP-DOX

[0064] As can be seen from the figure, after HUVEC cells were incubated with DOX, MAL-DOX, VAP-DOX or pHA-VAP-DOX for 72 hours, their IC 50 They were 0.20, 1.50, 0.70 and 0.30 μM respectively. The results showed that the in vitro anti-tumor activity of doxorubicin modified by pHA-VAP was superior to that of maleimide doxorubicin and VAP-modified doxorubicin.

[0065] Figure 11 、Survival curve of pHA-VAP-DOX against U87 orthotopic glioma

[0066] The figure shows the survival curve of nude mice with U87 orthotopic glioma model. Taking the median survival time of the model animals as an index, compared with normal saline (median survival time 26 days), DOX (median survival times of 10 mg, 20 mg, 40 mg were 27, 17, 13 days respectively, and the medium and high doses of doxorubicin were highly toxic and led to shorter survival times of the model nude mice), pHA-VAP (median survival time 28 days), pHA-VAP-DOX (median survival times of 10 mg, 20 mg, 40 mg were 29, 32, 40 days respectively) prolonged the survival time of the model animals, and there was a dose dependence.

[0067] Figure 12 、Electron microscopy photograph of the nano-delivery system of cabazitaxel nanocrystals coated with pHA-VAP-modified lipid membrane

[0068] As can be seen from the figure, cabazitaxel nanocrystals (left) were spherical with a particle size of about 80 nm; cabazitaxel nanocrystals coated with pHA-VAP-modified lipid membrane (middle and right) were spherical with an obvious core-membrane structure and a particle size of about 100 nm.

[0069] Figure 13 、Particle size characterization of the nano-delivery system of docetaxel / parthenolide nanocrystals coated with pHA-VAP-modified erythrocyte membrane

[0070] As can be seen from the figure, the particle size of docetaxel / parthenolide nanocrystals was about 130 nm and the zeta potential was -20 mV; after being coated with erythrocyte membrane, the particle size was about 140 nm and the zeta potential was -25 mV; pHA-VAP modification had no obvious effect on the particle size of the constructed nano-delivery system, but due to the positively charged molecule itself, the zeta potential of the modified nano-delivery system increased to -15 mV.

[0071] Figure 14 Uptake of cabazitaxel nanocrystals coated with pHA-VAP modified lipid membranes by umbilical vein endothelial cells HUVEC and breast cancer cells 4T1

[0072] As can be seen from the figure, the uptake of cabazitaxel nanocrystals coated with pHA-VAP modified lipid membranes by HUVEC cells (left) and 4T1 cells (right) was significantly higher than that of the free drug group, the nanocrystal group, and the cabazitaxel nanocrystals coated with non-target lipid membranes.

[0073] Figure 15 Uptake of docetaxel / parthenolide nanocrystals coated with pHA-VAP modified red blood cell membranes by primary brain capillary endothelial cells BCEC, umbilical vein endothelial cells HUVEC, and glioblastoma cells U87

[0074] As can be seen from the figure, the uptake of drug nanocrystals (docetaxel and parthenolide) coated with pHA-VAP modified red blood cell membranes by BCEC cells (A, B), HUVEC cells (C, D), and U87 cells (E, F) was significantly higher than that of the nanocrystal group and the drug nanocrystals coated with non-target red blood cell membranes, and was comparable to that of the free drug group.

[0075] Figure 16 In vitro anti-HUVEC and 4T1 cell activity curves of cabazitaxel nanocrystals coated with pHA-VAP modified lipid membranes

[0076] As can be seen from the figure, after the two types of cells were incubated with free cabazitaxel, cabazitaxel nanocrystals, cabazitaxel nanocrystals coated with lipid membranes, and cabazitaxel nanocrystals coated with pHA-VAP modified lipid membranes for 48 hours, their IC 50 against HUVEC cells were 30.3, 1.86, 1.61, and 1.49 nM (left figure), respectively, and their IC 50 against 4T1 cells were 0.88, 8.51, 2.93, and 0.06 nM (right figure), respectively. The results showed that the in vitro anti-tumor effect of cabazitaxel nanocrystals coated with pHA-VAP modified lipid membranes was better than that of all groups.

[0077] Figure 17 In vitro anti-HUVEC and U87 cell activity curves of docetaxel / parthenolide nanocrystals coated with pHA-VAP modified red blood cell membranes

[0078] As can be seen from the figure, after the two types of cells were incubated with free docetaxel / parthenolide, docetaxel / parthenolide nanocrystals, docetaxel / parthenolide nanocrystals coated with red blood cell membranes, and docetaxel / parthenolide nanocrystals coated with pHA-VAP modified red blood cell membranes for 48 hours, their IC 50They were 23.7, 44.6, 67.0, and 7.0 nM (left figure), and the IC of U87 50 were 45.6, 79.9, 90.3, and 3.2 nM (right figure). The results showed that the in vitro anti-tumor effect of docetaxel / pyrethrin nanocrystal coated with pHA-VAP modified erythrocyte membrane was better than that of all groups.

[0079] Figure 18 and the tissue distribution map of cabazitaxel nanocrystal coated with pHA-VAP modified lipid membrane in mice with 4T1 breast cancer orthotopic tumor model

[0080] As can be seen from the figure, pHA-VAP modification can significantly increase the accumulation of cabazitaxel nanocrystal coated with lipid membrane at different time points in the 4T1 tumor site, and better target to the tumor site.

[0081] Figure 19 and the tissue distribution map of docetaxel / pyrethrin nanocrystal coated with pHA-VAP modified erythrocyte membrane in mice with U87 orthotopic glioma model

[0082] As can be seen from the figure, pHA-VAP modification can significantly increase the accumulation of drug nanocrystal coated with erythrocyte membrane (docetaxel (upper figure) and pyrethrin (lower figure)) at different time points in the glioma site, and better target to the tumor site.

[0083] Figure 20 and the curve of the change in the volume of 4T1 breast cancer orthotopic tumor treated with cabazitaxel nanocrystal coated with pHA-VAP modified lipid membrane

[0084] The figure shows the curve of the change in the tumor volume of each group of balb / c mice over time. Compared with the PBS group, each administration group had an inhibitory effect on tumor growth. There was a significant difference between the cabazitaxel nanocrystal coated with non-target lipid membrane and that after pHA-VAP modification (n = 6), and the in vivo efficacy of cabazitaxel nanocrystal coated with pHA-VAP modified lipid membrane was the best.

[0085] Figure 21 and the comparison chart of the weights of 4T1 breast cancer orthotopic tumors treated with cabazitaxel nanocrystal coated with pHA-VAP modified lipid membrane

[0086] After sacrificing the balb / c mice and removing the tumor tissues, they were weighed and statistically analyzed. The tumor weight of the group treated with cabazitaxel nanocrystal coated with pHA-VAP modified lipid membrane was significantly lower than that of the other groups (n = 6).

[0087] Figure 22 and the survival curve of U87 orthotopic glioma treated with docetaxel / pyrethrin nanocrystal coated with pHA-VAP modified erythrocyte membrane

[0088] The figure shows the survival curves of nude mice with U87 orthotopic glioma in each group. Single-dose tail vein injection was used, and the survival period of the model animals was taken as the evaluation index (n = 10). Compared with PBS (median survival period: 38 days), free docetaxel / parthenolide (median survival period: 40.5 days), and erythrocyte membrane-coated docetaxel / parthenolide nanocrystal (median survival period: 41.5 days), the survival time of the mice treated with pHA-VAP-modified erythrocyte membrane-coated docetaxel / parthenolide nanocrystal (median survival period: 77 days) was significantly prolonged (***p < 0.001).

[0089] Figure 23 Effect of pHA-VAP-modified lipid membrane-coated cabazitaxel nanocrystal on apoptosis of tumor cells and inhibition of neovascularization in 4T1 breast cancer orthotopic tumor

[0090] The figure shows the staining photographs of the inhibition of neovascularization in 4T1 orthotopic tumor (upper figure) and promotion of tumor cell apoptosis (lower figure) of two kinds of cells treated with free cabazitaxel, cabazitaxel nanocrystal, lipid membrane-coated cabazitaxel nanocrystal, and pHA-VAP-modified lipid membrane-coated cabazitaxel nanocrystal, respectively. Among them, blood vessels (CD31 staining) are brownish red or brown, and apoptotic cells (TUNEL staining) are green.

[0091] Figure 24 Effect of pHA-VAP-modified erythrocyte membrane-coated docetaxel / parthenolide nanocrystal on apoptosis of tumor cells, inhibition of neovascularization, and killing of tumor stem cells in U87 orthotopic glioma

[0092] The figure shows the photographs of TUNEL staining (green) of apoptotic tumor cells, CD31 staining (red) of neovascularization, and CD133 staining (red) of tumor stem cells at the site of U87 orthotopic tumor, where blue is the DAPI staining of cell nuclei. It can be seen from the figure that compared with the other three groups, pHA-VAP-modified erythrocyte membrane-coated docetaxel / parthenolide nanocrystal can significantly promote tumor apoptosis, inhibit neovascularization, and kill tumor stem cells. Detailed implementation mode

[0093] The following examples will help to further understand the present invention, but the present invention is not limited to the following description scope.

[0094] Example 1

[0095] Synthesis and characterization of targeting molecule, targeting molecule-Cys, and targeting molecule-Cy7

[0096] 1. Synthesis and characterization of targeting molecule and targeting molecule-Cys

[0097] Using solid-phase peptide synthesis method, pHA-VAP peptide (amino acid sequence: p-hydroxybenzoic acid - Ahx - Cys - pavrtns; capital letters represent L-configured amino acids, and lowercase letters represent D-configured amino acids) was designed and synthesized.

[0098] Specific method: First, Cys(Trt)-Acp-4-tert-butylbenzoic acid was synthesized by Fmoc solid-phase peptide synthesis method. Amino acids were sequentially attached to PAM-Fmoc resin according to the sequence, and reactions were carried out using HBTU / DIEA as the condensing agent and TFA as the deprotecting agent. Then, the side-chain carboxyl group was activated with N-hydroxysuccinimide (NHS), and the VAP peptide synthesized by Boc-protected solid-phase peptide synthesis method was grafted onto cys(trt)-acp-4-tert-butylbenzoic acid. Deprotection was performed with 95% TFA to obtain pHA-VAP-Cys, and the crude peptide was separated and purified using an acetonitrile / water (containing 0.1% TFA) system. The purity and molecular weight (Mw) of pHA-VAP-Cys were characterized by HPLC and ESI-MS. The HPLC chromatogram and mass spectrum of pHA-VAP-Cys are shown in the appendix Figure 1 。

[0099] 2. Synthesis and Characterization of Targeting Molecule - Cy7

[0100] Dissolve the pHA-VAP-Cys obtained in the above steps in 0.1 M PBS solution (pH 7.2), take Cy7-maleimide and dissolve it in DMF. After mixing the two, react with magnetic stirring and monitor by HPLC. Stop the reaction after the reaction of pHA-VAP-Cys is complete, purify by preparative liquid phase, and separate and purify using an acetonitrile / water (containing 0.1% TFA) system. Freeze-dry to obtain pure pHA-VAP-Cy7. The HPLC chromatogram and mass spectrum are shown in the appendix Figure 2 。

[0101] Example 2

[0102] Verification of the in vitro cell targeting property of the targeting molecule

[0103] 1. In vitro targeting property of the targeting molecule to primary brain capillary endothelial cells BCEC

[0104] After decapitating 4-week-old SD rats, take the brain, quickly isolate the cerebral cortex in pre-cooled D-Hanks solution, roll off the meninges and large blood vessels in the brain, then cut it into pieces. Add collagenase and DNAse and digest at 37 °C for 90 minutes, centrifuge at 1000 revolutions per minute for 8 minutes, discard the supernatant, transfer it to DMEM solution containing 20% BSA, centrifuge at 1000 g per minute at 4 °C for 20 minutes, discard the middle and upper layers of the liquid, transfer the bottom microvessels to DMEM culture medium, centrifuge at 1000 revolutions per minute for 5 minutes, resuspend the microvessel segments with DMEM culture medium containing 20% fetal bovine serum, inoculate them in a 12-well plate, and culture at 37 °C, 5% CO2 Cultivate for 24 hours under saturated humidity conditions, then change to the endothelial cell-specific culture medium containing puromycin and continue to cultivate for 72 hours. After that, change to the endothelial cell-specific culture medium containing cell growth factors and cultivate for 72 hours to obtain primary brain capillary endothelial cells.

[0105] Prepare a fluorescently labeled polypeptide 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 and then centrifuge, discard the supernatant, wash twice with PBS, and finally disperse the cells in each well in 200 μL PBS for measurement by flow cytometry. The results are shown in Figure 3.

[0106] 2. In vitro targeting of the targeting molecule to human umbilical vein endothelial cells (HUVECs)

[0107] Take human umbilical vein endothelial cells (HUVECs) in monolayer culture in the logarithmic growth phase, digest the monolayer culture cells with 0.25% trypsin, and prepare a single-cell suspension with DMEM culture medium containing 10% fetal bovine serum. Inoculate 1×10 5 cells per well into a 12-well culture plate, with a volume of 1 mL per well. Transfer the culture plate to a carbon dioxide incubator and cultivate at 37 °C, 5% CO 2 under saturated humidity conditions for 24 h, and then perform the same experiment as above. The results of flow cytometry analysis are shown in Figure 4.

[0108] 3. In vitro targeting of the targeting molecule to glioma cells U87

[0109] Take glioma cells (U87 cells) in monolayer culture in the logarithmic growth phase and perform the same experiment as above. The results of flow cytometry analysis are shown in the appendix Figure 5 .

[0110] Example 3

[0111] Verification of the in vivo targeting of the targeting molecule

[0112] 1. Detection of the tissue distribution of pHA-VAP-Cys in nude mice bearing subcutaneous U87 tumor xenograft models

[0113] Construct a subcutaneous U87 tumor xenograft model. Inject the same dose of fluorescein-labeled pHA-VAP polypeptide via the tail vein respectively. Sacrifice the mice at 30 min, 1 h, 4 h, and 24 h after injection, take blood, heart, liver, spleen, lung, kidney, brain, and tumor, weigh them, add 1 mL of distilled water, homogenize the tissues, and measure the fluorescence quantitatively with an enzyme-labeled instrument. The results are shown in the appendix Figure 6 .

[0114] 2. Detection of Tissue Distribution of pHA-VAP-Cys in Nude Mice with U87 Orthotopic Glioma Model

[0115] Resuspend U87 cells in the logarithmic growth phase in an appropriate amount of PBS solution at a cell concentration of 1.3×108 / mL. Anesthetize nude mice by intraperitoneal injection of 8% chloral hydrate solution, fix them on a stereotaxic apparatus, and inject 5 μL of U87 cell suspension into the striatum region of the brain to construct an U87 orthotopic glioma model. Inject the same dose of fluorescein-labeled pHA-VAP polypeptide via the tail vein respectively. Sacrifice the mice at 30 min, 1 h, 4 h, and 24 h after injection, collect blood, heart, liver, spleen, lung, kidney, brain, and tumor, weigh them, add 1 mL of distilled water, homogenize the tissues, and measure fluorescence quantification with an enzyme-labeled instrument. The results are shown in the appendix Figure 7 .

[0116] Example 4

[0117] Preparation of Targeting Molecule-Drug Complex

[0118] Take the preparation of pHA-VAP-doxorubicin complex as an example of connecting a drug containing a keto or aldehyde group with a targeting molecule. 9.4 mg of thiolated pHA-VAP is dissolved in 3 mL of phosphate buffer (0.1 mM, pH 7.4), add an equimolar amount of doxorubicin 6-maleimidocaproic acid hydrazide derivative, and react at room temperature in the dark for 1 h. The reaction solution is purified by preparative liquid chromatography and freeze-dried to obtain pHA-VAP-doxorubicin complex. The HPLC chromatogram and mass spectrum are shown in the appendix Figure 8 .

[0119] Take the pHA-VAP-paclitaxel complex as an example of connecting a drug containing a hydroxyl or amino group with a disulfide bond as a targeting molecule. 200 mg of paclitaxel is dissolved in 10 mL of chloroform, cooled to 0 - 5 °C, and 39.99 mg of DCC and 60.4 mg of 3-(2-pyridyldithio)propionic acid are added successively. After adding the materials, raise the temperature to room temperature and react overnight. Filter the reaction solution, and purify it by column chromatography (eluted with CHCl3 / MeOH = 50:1 - 15:1, V / V) to obtain paclitaxel 3-(2-pyridyldithio)propionic acid derivative. Dissolve the paclitaxel 3-(2-pyridyldithio)propionic acid derivative in 5 mL of DMF, dissolve 1.5 times the molar amount of pHA-VAP-Cys in PBS / DMF, and keep the pH value of the solution at 4 - 5. Drop the paclitaxel 3-(2-pyridyldithio)propionic acid derivative into the thiol polypeptide solution and react at room temperature for 6 h. Purify it by preparative liquid chromatography and freeze-dry to obtain the polypeptide-paclitaxel complex

[0120] Example of using the pHA-VAP-bortezomib complex as a targeting molecule to link drugs containing a boronic acid group. According to the synthesis of pHA-VAP, amino acids are sequentially attached to the resin. After all amino acid residues of the polypeptide are attached, the Boc protection at the N-terminus is removed with trifluoroacetic acid. A DMF solution containing 3-fold molar amount of succinic anhydride and DIEA is added and reacted at room temperature for 30 min. After washing the resin, 5-fold molar amount of trimethylchlorosilane is added to protect dopamine, and HBTU / DIEA is used as a condensing agent and reacted at room temperature for 1 h. The resin is cleaved with HF and purified by preparative HPLC to obtain the polypeptide-dopamine derivative. In a buffer solution with pH 7.4, the pHA-VAP-dopamine derivative and bortezomib are mixed at a molar ratio of 1:1 to obtain the pHA-VAP-bortezomib complex.

[0121] Example of using the pHA-VAP-PMI fusion polypeptide as a targeting molecule to link polypeptide drugs. It is directly prepared by solid-phase polypeptide synthesis. The specific method is as follows: After determining the pHA-VAP-PMI polypeptide sequence, amino acids are sequentially attached in the same method as for the preparation of pHA-VAP. After cleavage with HF and purification, the pHA-VAP-PMI fusion polypeptide is obtained.

[0122] Example 5

[0123] In vitro pharmacodynamic experiments of targeted molecule-modified nano drug delivery systems

[0124] 1. In vitro pharmacodynamic test of pHA-VAP-DOX on glioma cell U87

[0125] U87 cells in the logarithmic growth phase are digested with 0.25% trypsin and blown into single cells. The cells are suspended in DMEM culture medium containing 10% FBS and seeded in a 96-well cell culture plate at a density of 3000 cells per well, with a volume of 0.2 mL per well. Three wells are reserved to add cell-free culture medium as blank wells and cultured in a carbon dioxide incubator for 24 hours. The drugs in each group are serially diluted six-fold with cell culture medium. The cell culture solution in the 96-well plate is aspirated, and 200 μL of the drug solution with a series of concentrations is added to each well. Three replicates are set for each concentration, and three wells only added with culture medium are reserved as control wells. After culturing for 72 hours, 20 μL of MTT reagent (5 mg / mL) is added to the experimental wells, control wells, and blank wells and incubated for 4 hours. The culture solution in the wells is discarded, 150 μL of dimethyl sulfoxide is added to each well, and after shaking to fully dissolve the generated blue-violet crystals, the absorbance (A) of each well at 490 nm is measured with an enzyme-linked immunosorbent assay (ELISA) reader. The cell survival rate is calculated according to the following formula:

[0126] Survival rate = (A 490实验孔 - A 490空白孔 ) / (A 490对照孔 - A 490空白孔 ) × 100%

[0127] The survival rate was plotted against the logarithm of the drug concentration using GraphPad Prism software (see attachment Figure 9 ), and the half-maximal inhibitory concentration (IC 50 ) was calculated.

[0128] 2. In vitro pharmacodynamic test of pHA-VAP-DOX on human umbilical vein endothelial cells (HUVECs)

[0129] HUVEC cells in the logarithmic growth phase were used for the same test as above. The results are shown in the attachment Figure 10 .

[0130] Example 6

[0131] In vivo pharmacodynamic study of the targeted molecule-modified drug complex

[0132] On the 7th day after establishing the U87 orthotopic tumor animal model, the mice were randomly divided into 8 groups of 10 mice each. DOX, pHA-VAP-DOX, pHA-VAP, and normal saline were injected via the tail vein every two days. The total doses of doxorubicin were 10 mg / kg, 20 mg / kg, and 40 mg / kg, respectively, and the polypeptide was converted to the amount of polypeptide in the pHA-VAP-DOX 40 mg / kg complex. The survival time of nude mice in each group was recorded, and the survival curve was plotted (see attachment Figure 11 ).

[0133] Example 7

[0134] Preparation and characterization of the targeted molecule-modified nano-drug delivery system

[0135] 1. Preparation of pHA-VAP-modified lipid membrane-coated cabazitaxel nanocrystals

[0136] pHA-VAP-PEG-DSPE was synthesized by the reaction of the free sulfhydryl group of pHA-VAP-Cys with the maleimide group of Mal-PEG-DSPE. 4 mg of cabazitaxel and an appropriate amount of the surfactant TPGS were weighed into a 25 ml eggplant-shaped flask, dissolved in an appropriate amount of dichloromethane and then formed a film and hydrated to prepare well-dispersed cabazitaxel nanocrystals. An appropriate amount of lipid membrane material (molar ratio: HSPC:Chol:DSPE-PEG2000 = 50:45:5) was dried by rotation, and the nanocrystal solution was added to hydrate the phospholipid membrane at 65 °C, followed by probe sonication for 10 min (120 W). The morphology was observed by negative staining electron microscopy with uranyl acetate, and the results are shown in the attachment Figure 12 .

[0137] 2. Preparation of pHA-VAP-modified erythrocyte membrane-coated docetaxel / parthenolide nanococrystals

[0138] Collect the whole blood of male ICR mice, centrifuge at 1000 g / min at 4 °C for 5 minutes, discard the upper serum and white blood cell layer, wash the lower layer of red blood cells with 1×PBS, and then resuspend them in 0.25×PBS at 4 °C for 30 minutes. Centrifuge at 15000 g / min at 4 °C for 7 minutes to remove hemoglobin. The obtained light red red blood cell membrane is resuspended and stored in double-distilled water, and its membrane protein concentration is detected by a BCA kit; Weigh 4 mg of docetaxel, 1.2 mg of parthenolide and an appropriate amount of surfactant F127 into a 25 mL eggplant-shaped flask, add an appropriate amount of methanol to dissolve and hydrate the film to prepare well-dispersed docetaxel / parthenolide nanococrystals; Add 40 μL of streptavidin-PEG 3400 -DSPE in PBS solution (5 mg / mL) and the red blood cell membrane vesicles obtained from 100 μL of whole blood were incubated in a 37 °C water bath for 30 minutes to obtain streptavidin-red blood cell membrane vesicles. The obtained streptavidin-red blood cell membrane vesicles were mixed with docetaxel / parthenolide nanococrystals and then sonicated to obtain a nano-delivery system of red blood cell membrane-coated nanocrystals modified with streptavidin on the surface. Then, 100 μL of biotin-PEG 2000 -VAP-pHA in PBS solution (0.1 mg / mL) was added and incubated in a 37 °C water bath for 10 minutes to obtain a nano-delivery system of pHA-VAP-modified red blood cell membrane-coated docetaxel / parthenolide nanococrystals. Particle size and potential characterization are shown in the appendix Figure 13 。

[0139] Example 8

[0140] In vitro targeting of the targeted molecule-modified nano-delivery system

[0141] 1. Uptake test of pHA-VAP-modified lipid membrane-coated cabazitaxel nanocrystals by human umbilical vein endothelial cells HUVEC and breast cancer cells 4T1

[0142] The plating methods of HUVEC and 4T1 cells are as above. The two cells with corresponding concentrations are prepared with DMEM culture medium containing 10% FBS and are respectively mixed with free cabazitaxel, cabazitaxel nanocrystals, lipid membrane-coated cabazitaxel nanocrystals, and pHA-VAP-modified lipid membrane-coated cabazitaxel nanocrystals. The DMEM culture medium in the 12-well plate is aspirated, the drug solution is added, and incubated at 37 °C for 4 hours, and then the drug solution is discarded. Wash the plate twice with PBS solution, add trypsin to digest the cells, disperse the cells with DMEM culture medium and then count, centrifuge, discard the supernatant, wash twice with PBS, and finally disperse the cells in each well in 100 μL of PBS, sonicate to break the cells, add 3 times the volume of methanol to precipitate proteins, centrifuge at 10000 rpm for 10 min, and take the supernatant, and determine the drug content by HPLC. The results are shown in the appendix Figure 14 。

[0143] 2. Uptake experiments of docetaxel / pyrethrin nanocrystal coated with erythrocyte membrane modified by pHA-VAP by primary brain capillary endothelial cells BCEC, umbilical vein endothelial cells HUVEC, and glioma cells U87

[0144] The extraction method of BCEC cells and the seeding methods of HUVEC and U87 cells are as above. Free docetaxel / pyrethrin, docetaxel / pyrethrin nanocrystals, docetaxel / pyrethrin nanocrystals coated with erythrocyte membrane, and docetaxel / pyrethrin nanocrystals coated with erythrocyte membrane modified by pHA-VAP were prepared with DMEM culture medium containing 10% FBS at corresponding concentrations. The DMEM culture medium in the 12-well plate was aspirated, the drug solution was added, and incubated at 37 °C for 1 hour, then the drug solution was discarded. The plate was washed twice with PBS solution, the cells were digested with trypsin, dispersed with DMEM culture medium and then counted, centrifuged, the supernatant was discarded, washed twice with PBS, and finally the cells in each well were dispersed in 100 μL PBS. After ultrasonic cell disruption, 3 volumes of methanol were added to precipitate proteins. After centrifugation at 10000 rpm for 10 min, the supernatant was taken, and the drug content was determined by HPLC. The results are shown in the appendix Figure 15 。

[0145] Example 9

[0146] Verification of in vivo targeting of targeted molecule-modified nano-drug delivery systems

[0147] 1. Detection of tissue distribution of cabazitaxel nanocrystals coated with lipid membrane modified by pHA-VAP in nude mice with 4T1 orthotopic breast cancer model

[0148] A 4T1 orthotopic breast cancer model was constructed. Mice were sacrificed at 2, 12, and 24 h after intravenous injection of the same dose of free cabazitaxel, cabazitaxel nanocrystals, cabazitaxel nanocrystals coated with lipid membrane, and cabazitaxel nanocrystals coated with lipid membrane modified by pHA-VAP. Blood, heart, liver, spleen, lung, kidney, brain, and tumor were collected, weighed, 1 mL of distilled water was added, homogenized, extracted, and quantified by HPLC (appendix Figure 16 )

[0149] 2. Detection of tissue distribution of docetaxel / pyrethrin nanocrystals coated with erythrocyte membrane modified by pHA-VAP in nude mice with U87 orthotopic glioma model

[0150] Construct a U87 orthotopic glioma model. Mice were intravenously injected with the same dose of free docetaxel / pyrethrin, docetaxel / pyrethrin nanocrystal coated with erythrocyte membrane, and docetaxel / pyrethrin nanocrystal coated with pHA-VAP modified erythrocyte membrane via the tail vein. The mice were sacrificed at 2 and 12 h after injection, and blood, heart, liver, spleen, lung, kidney, brain, and brain tumors were collected, weighed, added with 1 mL of distilled water, homogenized, extracted twice with methyl tert-butyl ether, dried by evaporation, and re-dissolved in methanol for HPLC quantification (attached Figure 17 )

[0151] Example 10

[0152] In vitro pharmacodynamic study of targeted molecule modified nano-drug delivery system

[0153] 1. In vitro pharmacodynamic study of cabazitaxel nanocrystal coated with pHA-VAP modified lipid membrane

[0154] HUVEC cells or 4T1 cells in the logarithmic growth phase were used for the same experiment as above. The results are shown in the attachment Figure 18

[0155] 2. In vitro pharmacodynamic study of docetaxel / pyrethrin nanocrystal coated with pHA-VAP modified erythrocyte membrane

[0156] HUVEC cells or U87 cells in the logarithmic growth phase were used for the same experiment as above. The results are shown in the attachment Figure 19

[0157] Example 11

[0158] In vivo pharmacodynamic study of targeted molecule modified nano-drug delivery system

[0159] 1. In vivo pharmacodynamic study of cabazitaxel nanocrystal coated with pHA-VAP modified lipid membrane

[0160] A 4T1 orthotopic breast cancer animal model was constructed. When the tumor size reached 100 mm 3 , the animals were grouped and intravenously injected with PBS, free cabazitaxel, cabazitaxel nanocrystal, cabazitaxel nanocrystal coated with lipid membrane, and cabazitaxel nanocrystal coated with pHA-VAP modified lipid membrane via the tail vein. The total dosage of cabazitaxel in the dosing group was 16 mg / kg, divided into 4 times with an interval of two days between each dosing. The long diameter (a) and short diameter (b) of the tumor were measured with a vernier caliper every other day. The tumor volume of each group of balb / c mice was calculated according to the formula, and the change curve of tumor volume over time was plotted to calculate the statistical differences among groups. The tumor volume was calculated according to the following formula and the growth inhibition curve was plotted (attached Figure 20 )

[0161] V 瘤体积 = 0.5(a × b 2)

[0162] Twenty days after administration, all balb / c mice were sacrificed by cervical dislocation, and the subcutaneous tumors were removed and weighed, and the statistical differences between groups were calculated (attached Figure 21 ).

[0163] 2. In vivo pharmacodynamic study of docetaxel / pyrethrin nanocrystal coated with pHA-VAP modified erythrocyte membrane

[0164] An animal model of U87 orthotopic glioma was constructed. Ten days after tumor implantation, PBS (pH 7.4), free docetaxel / pyrethrin at different concentrations, docetaxel / pyrethrin nanocrystal coated with erythrocyte membrane, and docetaxel / pyrethrin nanocrystal coated with pHA-VAP modified erythrocyte membrane were injected into the tail vein respectively. The total dosage of docetaxel was 25 mg / kg, and the total dosage of pyrethrin was 6 mg / kg, with a single administration. The survival time of nude mice was recorded (Figure 22).

[0165] 3. Detection of apoptosis promotion and neovascularization inhibition of cabazitaxel nanocrystals coated with pHA-VAP modified lipid membrane

[0166] On the 2nd day after the completion of drug administration, the tumor-bearing balb / c mice were sacrificed and the tumor tissues were removed for fixation, paraffin sections or frozen sections were made, and the neovascularization inhibition was detected by CD31 staining, or the apoptosis promotion was detected by TUNEL staining. The results are shown in the attached Figure 23 .

[0167] 4. Detection of apoptosis promotion, neovascularization inhibition and tumor stem cell killing of docetaxel / pyrethrin nanocrystal coated with pHA-VAP modified erythrocyte membrane

[0168] On the 10th day after the completion of drug administration, the tumor-bearing nude mice were sacrificed and the tumor tissues were removed for fixation, frozen sections were made, and the apoptosis promotion, neovascularization inhibition and tumor stem cell killing were detected by TUNEL staining, CD31 and CD133 antibody staining. The results are shown in the attached Figure 24 .

Claims

1. Use of a whole-process targeting molecule in the preparation of a targeted delivery system for targeting brain tumors, characterized in that, the whole-process targeting molecule is formed by covalently connecting two parts of molecules, namely a brain targeting molecule and a tumor targeting molecule, and the whole-process targeting molecule mediates the targeted delivery of imaging molecules and therapeutic drugs; wherein, an imaging molecule X is introduced onto the whole-process targeting molecule to obtain a whole-process targeting molecule-X complex, and X is an optical imaging molecule; or a therapeutic drug molecule Y is introduced onto the whole-process targeting molecule to obtain a whole-process targeting molecule-Y complex, and Y is a tumor chemotherapy drug; the brain targeting molecule is p-hydroxybenzoic acid pHA; the tumor targeting molecule is selected from: L-configured polypeptide VAP polypeptide, with the amino acid sequence SNTRVAP; or L-configured VAP reverse polypeptide, with the amino acid sequence PAVRTNS; either D-configured VAP polypeptide S VAP, with the amino acid sequence of D S D N D T D R D V D A D P; The D-configured or VAP reverse polypeptide, with the amino acid sequence being D P D A D V D R D T D N D S; the optical imaging molecules are selected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7; the tumor chemotherapy drugs are selected from doxorubicin, epirubicin, paclitaxel, docetaxel, cabazitaxel, bortezomib.

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