CN113171468A - Whole-process targeting molecule and application thereof in construction of drug delivery system - Google Patents

Whole-process targeting molecule and application thereof in construction of drug delivery system Download PDF

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CN113171468A
CN113171468A CN202011548622.XA CN202011548622A CN113171468A CN 113171468 A CN113171468 A CN 113171468A CN 202011548622 A CN202011548622 A CN 202011548622A CN 113171468 A CN113171468 A CN 113171468A
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targeting molecule
tumor
molecule
polypeptide
vap
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陆伟跃
周建芬
柴芝兰
吴荪奕
谢操
王浩
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Fudan University
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Abstract

The invention belongs to the field of pharmacy, relates to a whole-process targeting molecule, and particularly relates to a whole-process targeting molecule which can target brain capillary endothelial cells (cross blood-brain barrier), tumor neovascular endothelial cells (cross blood-tumor barrier), tumor mimicry blood vessels, tumor cells and tumor stem cells, and applications of a modified drug compound and a drug delivery system thereof in tumor diagnosis and targeted therapy. The whole-process targeting molecule and the drug-loading system constructed by the whole-process targeting molecule can deliver the carried image molecule, the therapeutic drug and the nano drug-loading system to the brain tumor tissue in a targeted manner or deliver the molecules to the peripheral tumor tissue with the brain metastasis characteristic in a targeted manner, thereby obviously improving the tumor diagnosis and treatment effects. The whole-process targeting molecule can mediate cross-blood-brain barrier and/or cross-blood-tumor barrier of imaging molecules, therapeutic drugs and nano drug-carrying systems, and is used for diagnosis and treatment of brain tumors and peripheral tumors with brain metastasis characteristics.

Description

Whole-process targeting molecule and application thereof in construction of drug delivery system
Technical Field
The invention belongs to the field of pharmacy, relates to a whole-process targeting molecule and application thereof in construction of a drug delivery system, in particular to a whole-process targeting molecule and application thereof in construction of a drug delivery system for tumor image diagnosis and targeted therapy, and particularly relates to a whole-process targeting molecule which has the functions of targeting brain capillary endothelial cells (cross blood-brain barrier), tumor neovascular endothelial cells (cross blood-tumor barrier), tumor mimicry blood vessels, tumor cells and tumor stem cells, and a modified drug compound and a drug loading system thereof in tumor diagnosis and targeted therapy.
Background
The prior art discloses that tumors are serious life and health threatening diseases for human beings, and the mortality rate is high and the first mortality rate of all diseases is high. The morbidity and mortality of the brain primary tumor are in the first 10 of the Chinese tumor leaderboard, the brain glioma accounts for about 45 percent of the brain primary tumor, the median survival time of the patient is less than 16 months, and the hazard is very high. The traditional chemotherapy as the main means of brain tumor drug therapy has the defects of poor selectivity to tumor tissues, high toxicity, narrow therapeutic window, easy generation of multi-drug resistance and the like. Therefore, to overcome the limitations of traditional therapeutic approaches, active targeting has become an important strategy to improve the targeting efficiency of brain tumor tissues in recent years. The active targeting strategy mainly aims at a receptor or a transporter highly expressed in the brain tumor tissue, and utilizes a corresponding ligand which has specific recognition and binding capacity with the receptor or the transporter to deliver a drug or a drug-carrying system to the brain tumor tissue or cells. Most of the current ligands only target a certain receptor or transporter and a certain cell, however, the brain tumor tissue not only has tumor cells, but also has tumor stem cells, tumor mimicry blood vessels, brain capillaries and blood-brain barrier (BBB) thereof, tumor neovascularization and blood-tumor barrier (BTB) thereof, and the like. In the early stage of brain tumor, BBB still remains intact and limits the drugs from entering the brain, so that about 98 percent of small-molecule chemotherapy drugs and almost 100 percent of macromolecular drugs such as proteins and the like can not enter the brain through the BBB, and the drug therapy is almost ineffective; with the occurrence and development of tumors, tumor neovascularization is generated, but brain tumor neovascularization is relatively dense and has poor permeability compared with peripheral tumors, the formed BTB becomes a main obstacle of drug delivery, and BBB still exists in a brain glioma infiltration area and also hinders drug transfer; meanwhile, the brain tumor stem cells have the characteristics of self-renewal, proliferation and high tumorigenicity, and although the number of the brain tumor stem cells in tumor tissues is very small, the brain tumor stem cells show high tolerance to drug treatment and are easy to cause the recurrence of brain glioma. Therefore, it is important to select a targeting molecule with better crossing ability to BBB and BTB and better affinity to brain tumor cells.
Based on the current situation of the prior art, the inventor further reforms the existing targeting molecules, and utilizes the molecule fusion principle to covalently connect the brain targeting molecules and the tumor targeting molecules into the overall process targeting molecules, so that the brain targeting molecules have the overall process targeting function for the growth and development of brain tumors or peripheral tumors with the brain metastasis characteristics. Meanwhile, a diagnosis and treatment drug compound modified by targeting molecules in the whole process, a modified high-molecular carrier material and a drug-loading system constructed by the compound are constructed, so that the effects of image diagnosis and targeted treatment on brain tumors or peripheral tumors with brain metastasis characteristics are more effectively exerted.
Disclosure of Invention
The invention aims to provide a whole-process targeting molecule and application thereof in construction of a drug delivery system based on the current state of the prior art. The invention utilizes the molecule fusion principle to covalently connect the brain targeting molecule and the tumor targeting molecule into the whole-process targeting molecule, so that the brain targeting molecule has the whole-process targeting function for the growth and development of the brain tumor or the peripheral tumor with the brain metastasis characteristic. Meanwhile, a diagnosis and treatment drug compound modified by targeting molecules in the whole process, a modified high-molecular carrier material and a drug-loading system constructed by the compound are constructed, so that the effects of image diagnosis and targeted treatment on brain tumors or peripheral tumors with brain metastasis characteristics are effectively exerted.
The whole-process targeting molecule with targeting brain capillary endothelial cells (spanning BBB), tumor neovascular endothelial cells (spanning BTB), tumor mimicry blood vessels, tumor cells and tumor stem cells is formed by covalently connecting the brain targeting molecule and the tumor targeting molecule; further, the whole process of targeting molecules is used for modifying image molecules, therapeutic drugs and high molecular carrier materials to construct targeting molecule-drug compounds and targeting molecule modified nano drug delivery systems, so that the targeted diagnosis and treatment effect of the drugs on brain and brain tumors or peripheral tumors with brain metastasis characteristics can be improved.
Specifically, the invention utilizes the principle of molecular fusion to prepare the whole-process targeting polypeptide molecule by covalently connecting the brain targeting molecule and the tumor targeting molecule, so that the polypeptide molecule has the targeting capability of two molecules simultaneously, and can play a whole-process targeting role in the growth and development of tumors, particularly brain tumors, such as targeting brain capillary endothelial cells (spanning BBB), tumor neovascular endothelial cells (spanning BTB), tumor mimicry blood vessels, tumor cells and tumor stem cells.
The targeting molecules such as small molecules, polypeptide molecules or protein molecules and the like which cross the blood-brain barrier comprise: p-hydroxybenzoic acid (pHA) and derivatives thereof, fatty acids, particularly myristic acid (MC) and derivatives thereof, D8 polypeptide, WSW polypeptide, and mixtures thereof,DWSW polypeptide, TGN polypeptide,DTGN polypeptides, CDX polypeptides,DCDX polypeptides, T7 polypeptides andDpolypeptides such as T7 polypeptide andits derivatives, proteins such as transferrin and lactoferrin, and their derivatives. The polypeptide sequences are shown in the attached table (table 1 is a polypeptide amino acid sequence table).
The targeting molecules such as polypeptide molecules or protein molecules and the like of the cross blood-tumor barrier comprise: VAP polypeptide, cVAP polypeptide,SVAP polypeptides,DVAP polypeptide, A7R polypeptide, cA7R polypeptide,DA7R polypeptide, RGD polypeptide, staged-RGD polypeptide, RW polypeptide, mn polypeptide, RAP12 polypeptide andDRAP12 polypeptide and its derivatives. The polypeptide sequences are shown in the attached table of the specification (attached table 1-polypeptide amino acid sequence table).
The whole process targeting molecule designed by the invention can construct the modified imaging molecule compound, the therapeutic drug compound and the polymer carrier material compound by introducing active functional groups into the molecule.
After cysteine is introduced into the whole process targeting molecule designed by the invention, thiol in the molecule reacts with maleimide functional optical imaging molecules (such as fluorescent probe molecules FITC, FAM, 6-TET, 5-TAMRA, HEX, 6-JOE and the like, 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 and the like, chemiluminescent molecules such as luminol, isoluminol, AMPPD, CSPD, CDP-star, lucigenin and the like, Raman probe molecules and the like) TO form a compound.
The whole process targeting molecule designed by the invention can be used with magnetic resonance imaging agent (such as chelate of nuclide for magnetic resonance imaging such as Gd) or radionuclide imaging agent (such as Gd)18F、32P、35S、64Cu、67/68Ga、75Se、89Zr、86Y、99mTc、111/111mIn、123/125I、177Lu、149/161Chelates of imaging radionuclides such as Tb), or chelates with therapeutic radionuclides (e.g. Tb)90Y、131I、152/155Tb、153Sm、177Lu、186/188Re、211At、212/213Bi、212Pb、225Ac、227Th, etc.) to form a complex, wherein the chelate is composed of a bifunctional chelating agent and a radionuclide for magnetic resonance imaging, or a bifunctional chelating agent and a radionuclide for therapy. Bifunctional chelating agents in the chelate include DOTA, DOTAGA, NOTA, NOTAGA, NOTADA, DTPA, TETA, CB-TE2A, Cyclam, DFO, MAG3, EC, EDTA, DADT, HYNIC, CE-DTS, NS3 and the like.
The whole-process targeting molecule modified drug designed by the invention comprises a pH-sensitive hydrazone bond formed by reacting maleimide and hexylhydrazine derivatives (related to drugs containing ketone or aldehyde groups such as adriamycin and epirubicin), or a disulfide bond formed by reacting 3- (2-pyridinedithiol) propionic acid derivatives (related to drugs containing hydroxyl or amino groups such as paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine and vinorelbine), or a pH-sensitive borate formed by reacting dopamine and boric acid groups in drugs (related to drugs containing boric acid groups such as bortezomib), or an amide bond formed directly by solid phase synthesis (related to polypeptide drugs such as p53 activated peptide, melittin, scorpion peptide and antibacterial peptide), or covalent or non-covalent linkage (related to rituximab, scorpion peptide and antibacterial peptide), or covalent or non-covalent linkage (related to polypeptide drugs such as rituximab, Bevacizumab, trastuzumab, cetuximab, pertuzumab, ipilimumab, nivolumab, PD-L1 monoclonal antibody and other antibody drugs and antibody fragment combinations thereof which are modified by genetic engineering means, including Fab fragments, single domain antibodies, Fv fragments, single-chain antibodies, bivalent small molecule antibodies, micro-antibodies, nano-antibodies and the like).
After cysteine is introduced into the whole-process targeting molecule designed by the invention, the whole-process targeting molecule is modified on high-molecular carrier materials such as polyethylene glycol-distearoyl phosphatidyl ethanolamine (PEG-DSPE) containing maleimide functional groups, polyethylene glycol-polylactic acid (PEG-PLA), polyethylene glycol-lactic glycolic acid copolymer (PEG-PLGA) and polyethylene glycol-polycaprolactone (PEG-PCL) and the like, and is used for constructing nano drug-carrying systems such as liposome, micelle, disc, nanoparticle and the like modified by the whole-process targeting molecule.
After cysteine is introduced into the whole-process targeting molecule designed by the invention, the whole-process targeting molecule is modified on targeting materials such as polyethylene glycol-Biotin (PEG-Biotin) containing maleimide functional groups and the like, and is used for constructing a biomembrane-coated nano drug delivery system modified by the whole-process targeting molecule.
The whole-process targeting molecule modified nano drug delivery system designed by the invention is used for encapsulating anthracyclines such as adriamycin and epirubicin, taxanes such as taxol, docetaxel and cabazitaxel, camptothecin drugs such as camptothecin, hydroxycamptothecin, 9-nitrocamptothecin and irinotecan, vinblastine drugs such as vincristine and vinorelbine, platinum drugs such as cisplatin, carboplatin, oxaliplatin and miboplatin, proteasome inhibitors such as bortezomib and carfilzomib, molecular targeting drugs such as parthenolide, molecular targeting drugs such as trametinib, imatinib, nilotinib, dasatinib, everolimus, lotinib, sunitinib, sorafenib, ibrutinib, regorafenib, williamib and olaparib, and molecular targeting drugs such as p53 activating peptide, melittin, scorpion venom peptide, and the like, Polypeptide drugs such as antibacterial peptide, and antibody drugs such as rituximab, bevacizumab, trastuzumab, cetuximab, pertuzumab, ipilimumab, nivolumab, PD-L1 monoclonal antibody, and antibody fragment combinations (including Fab fragment, single domain antibody, Fv fragment, single-chain antibody, bivalent small molecule antibody, micro-antibody, nano-antibody, and the like) modified by genetic engineering means, and the like, are entrapped90Y、131I、152/155Tb、153Sm、177Lu、186/188Re、211At、212/213Bi、212Pb、225Ac、227Th and the like.
The designed full-process targeting molecule modified nano drug delivery system is used for encapsulating optical imaging molecules (such as fluorescent probe molecules FITC, FAM, 6-TET, 5-TAMRA, HEX, 6-JOE and the like, 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 and the like, and chemiluminescent substances luminol, isoluminol, AMPPD, CSPD, CDP-star, lucigenin, etc., Raman probe molecules, magnetic resonance imaging agent (e.g., chelate of magnetic resonance material such as Gd), radionuclide imaging agent (e.g., chelate of magnetic resonance material such as Gd), etc.), and radionuclide imaging agent (e.g., Raman probe molecules)18F、32P、35S、64Cu、67/68Ga、75Se、89Zr、86Y、99mTc、111/111mIn、123/125I、177Lu、149/161Chelates of imaging radionuclides such as Tb).
The whole-process targeting molecule designed by the invention is used for mediating drugs or nano drug delivery systems to cross BBBs and BTBs, targeting tumor neovascularization vessels, tumor mimicry vessels, tumor cells and tumor stem cells, and being used for targeted diagnosis and treatment of brain and brain tumors or peripheral tumors with brain metastasis characteristics.
The method provided by the invention is used for designing and preparing the whole-process targeting molecule pHA-VAP and the modified drug compound and the nano drug-carrying system thereof, and comprises the following steps:
1. preparation of pHA-VAP and its fluorescent marker (pHA-VAP-Cy7)
Preparing pHA-VAP by adopting a solid-phase synthesis method; synthesizing pHA-VAP-Cy7 by Michael addition reaction of maleimide group and sulfhydryl group; HPLC and MS characterize the structure.
2. preparation of pHA-VAP-imaging agents
Synthesizing pHA-VAP-DTPA by Michael addition reaction of maleimide group and sulfhydryl group, chelating Gd or99mTc to obtain pHA-VAP-DTPA-Gd or pHA-VAP-DTPA-99mTc。
3. Evaluation of in vitro and in vivo targeting ability of pHA-VAP
The in vitro affinity of pHA-VAP-Cy7 for Brain Capillary Endothelial Cells (BCEC), umbilical vein endothelial cells (HUVEC) and model tumor cells (e.g., glioma brain cells U87) was examined.
The distribution of the tumor in animals at each time point was examined by tail vein injection of pHA-VAP-Cy7 into normal mice and nude mice loaded with U87 subcutaneous transplantation tumor and U87 intracranial orthotopic tumor model.
4. preparation of pHA-VAP-drug complexes
The pHA-VAP introduced with cysteine reacts with maleimide hexylhydrazine derivative on the medicine to form a polypeptide-medicine compound containing a pH sensitive hydrazone bond, wherein the related medicines comprise medicines containing ketone or aldehyde groups, such as adriamycin and epirubicin.
The pHA-VAP introduced with cysteine reacts with 3- (2-pyridinedimercapto) propionic acid derivatives on the medicine to form a polypeptide-medicine compound containing disulfide bonds, wherein the related medicines comprise medicines containing hydroxyl or amino, such as paclitaxel, docetaxel, cabazitaxel, camptothecin, hydroxycamptothecin, 9-nitrocamptothecin, irinotecan, vincristine, vinorelbine and the like.
The pHA-VAP is modified with dopamine to further react with boric acid groups on the medicine to form a polypeptide-medicine compound containing pH sensitive borate, wherein the related medicine comprises boric acid group-containing medicines such as bortezomib and the like.
The pHA-VAP is directly condensed with polypeptide drugs through solid phase synthesis, wherein the related drugs comprise p53 activation peptide, antibacterial peptide, polypeptide toxin and other polypeptide drugs.
The pHA-VAP is modified by random sites (after the free amino in the antibody is activated, the pHA-VAP is linked covalently) or fixed sites (after the target molecule is linked with the antibody non-covalently through affinity coupling action), so that an antibody compound modified by target functional molecules is obtained, wherein the related drugs comprise rituximab, bevacizumab, trastuzumab, cetuximab, pertuzumab, ipilimumab, nivolumab, PD-L1 monoclonal antibodies and other antibody fragment drugs and antibody fragment combinations (including Fab fragments, single domain antibodies, Fv fragments, single-chain antibodies, bivalent small molecule antibodies, micro antibodies, nano antibodies and the like) modified by genetic engineering means.
5. Evaluation of antitumor Effect of pHA-VAP-Adriamycin Complex in vitro and in vivo
pHA-VAP-doxorubicin compound (pHA-VAP-DOX) obtained by condensing pHA-VAP after cysteine connection and a maleimide-hexylhydrazine derivative (MAL-DOX) on doxorubicin, and the in-vitro growth inhibition effect of pHA-VAP-DOX on U87 cells and HUVEC cells is examined by an MTT method; the in vivo anti-tumor effect of the nude mice is evaluated by the tail vein administration of the nude mice loaded with U87 intracranial orthotopic tumor model by taking the median survival time as an index.
6. Construction and characterization of pHA-VAP modified nano drug delivery system
Firstly, preparing pHA-VAP modified high molecular materials pHA-VAP-PEG-DSPE, pHA-VAP-PEG-PLA, pHA-VAP-PEG-PLGA, pHA-VAP-PEG-PCL, pHA-VAP-PEG-biotin and the like. Cysteine is introduced into pHA-VAP, and free sulfydryl reacts with maleimide contained in Mal-PEG-DSPE, Mal-PEG-PLA, Mal-PEG-PLGA, Mal-PEG-PCL, Mal-PEG-biotin and the like to realize the preparation of the targeting polymer material, namely: dissolving Mal-PEG-DSPE, Mal-PEG-PLA, Mal-PEG-PLGA, Mal-PEG-PCL, Mal-PEG-biotin and the like in acetonitrile respectively, performing rotary evaporation to form a film, and adding PBS (pH8.0) containing sulfhydryl pHA-VAP to react to prepare the pHA-VAP modified polymer material.
And then constructing a nano drug delivery system modified by pHA-VAP. A certain amount of pHA-VAP-PEG-DSPE, mPEG-DSPE, phospholipid 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, and a certain amount of the above drugs are subjected to film forming hydration and other methods to respectively construct corresponding nano drug delivery systems such as pHA-VAP modified liposome, micelle, disc, nanoparticle and the like; after a certain amount of pHA-VAP-PEG-biotin and a biological membrane which is pre-modified with avidin are incubated, polymer nanoparticles, silicon nanoparticles, nanogel, nanocrystals and other nano drug-carrying systems which are loaded with the drugs are coated, and the biological membrane-coated nano drug-carrying system is constructed. The laser scattering particle size instrument characterizes the particle size and the potential of the nano drug-loading system, and the transmission electron microscope characterizes the morphological characteristics.
7. Evaluation of in vivo and in vitro targeting capability of pHA-VAP modified nano drug delivery system
The uptake of the pHA-VAP modified nano drug delivery system coated with tumor therapeutic drugs by BCEC cells, tumor cells (U87 cells and 4T1 cells) and HUVEC cells is examined.
The tail vein injection of a naked mouse with a lotus U87 intracranial in situ tumor model or a balb/c mouse with a 4T1 breast cancer in situ model is used for coating the pHA-VAP modified nano drug-carrying system with the tumor treatment drug, and the distribution of the nano drug-carrying system in the tumor at each time point is inspected.
8. Evaluation of in vivo and in vitro anti-tumor effect of pHA-VAP modified nano drug-loaded system
Examining the in-vitro growth inhibition effect of the pHA-VAP modified nano drug delivery system encapsulating the tumor treatment drug on tumor cells (U87 cells and 4T1 cells) and HUVEC cells by an MTT method; the in vivo anti-tumor effect of a tumor treatment drug-encapsulated pHA-VAP modified nano drug-loaded system is evaluated by tail vein injection of a naked mouse with a U87 intracranial in situ tumor model or a balb/c mouse with a 4T1 breast cancer in situ model by taking survival time, a tumor inhibition curve, tumor tissue cell apoptosis, new vessels, stem cell number and the like as indexes.
The test results of the invention show that: the prepared pHA-VAP has the ability of pHA targeting brain capillaries and crossing BBB, the ability of VAP targeting tumor neovascular endothelial cells and crossing BTB, targeting tumor mimicry blood vessels, tumor cells and tumor stem cells, has good brain and tumor tissue targeting ability and image effect in a model animal body, and shows better brain tumor targeting ability; the PHA-VAP modified drug compound and the nano drug delivery system show good tumor targeting performance and stronger brain tumor resisting effect.
TABLE 1 amino acid sequence listing of polypeptide targeting molecules
Figure BDA0002857172290000071
Drawings
FIG. 1 HPLC and ESI-MS profiles of pHA-VAP-Cys
The chromatographic method comprises the following steps: chromatography column (YMC, C18): 150X 4.6 mm; mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid); elution procedure: 5% B-65% B for 0-45 min; flow rate: 0.7 mL/min; column temperature: 40 ℃; and (3) detection: UV214nm, retention time: and (5) 16 min. ESI-MS: 1080.4, corresponding to the theoretical molecular weight.
FIG. 2 HPLC and ESI-MS profiles of pHA-VAP-Cy7
Chromatography method as above, retention time: and (5) 25 min. ESI-MS: 1750.6, corresponding to the theoretical molecular weight.
FIG. 3 uptake of Cy 7-labeled pHA-VAP by primary brain capillary endothelial cells BCEC
The figure shows the quantitative (left) and qualitative (right) results of flow cytofluorimetric detection after Cy 7-labeled VAP and pHA-VAP are incubated with BCEC cells for 4 h. It can be seen that uptake of pHA-VAP by BCEC cells was significantly higher than that of VAP and free fluorescein.
FIG. 4 uptake of Cy 7-labeled pHA-VAP by umbilical vein endothelial cells HUVEC
The figure shows that Cy 7-labeled VAP and pHA-VAP are incubated with HUVEC cells for 4h, and then flow cytofluorescence detection is carried out for quantitative (left) and qualitative (right) results, so that the uptake of pHA-VAP by U87 cells is obviously higher than that of VAP and free fluorescein.
FIG. 5 shows the uptake of Cy 7-labeled pHA-VAP by brain glioma cells U87
The figure shows that the Cy 7-labeled VAP and pHA-VAP are incubated with U87 cells for 4h, and then flow cytofluorescence detection is carried out on the quantitative (left) and qualitative (right) results, so that the uptake of the pHA-VAP by the U87 cells is obviously higher than that of the VAP and free fluorescein.
FIG. 6, in vivo tissue distribution map of Cy 7-labeled pHA-VAP in mouse model of subcutaneous tumor-transplanted with U87
As can be seen, the distribution of pHA-VAP-Cy7 in subcutaneous tumors is increased within 4h and the amount of accumulation is reduced after 24h, compared with free fluorescein and VAP-Cy 7.
FIG. 7 is a tissue distribution diagram of Cy7 labeled pHA-VAP in vivo in U87 brain-loaded tumor-in-situ model mouse
As can be seen, compared with free fluorescein and VAP-Cy7, the distribution of pHA-VAP-Cy7 in-situ tumors is improved within 24h, which indicates that the targeting molecules can cross the blood-brain barrier and the blood-brain tumor barrier and obviously increase the accumulation of the drug at the brain tumor site.
FIG. 8 HPLC and ESI-MS profiles of pHA-VAP-DOX
The chromatographic method comprises the following steps: chromatography column (YMC, C18): 150X 4.6 mm; mobile phase A: water (with 0.01% formic acid), mobile phase B: pure acetonitrile; elution procedure: 5% B-65% B for 0-45 min; flow rate: 0.7 mL/min; column temperature: 40 ℃; and (3) detection: UV214nm, retention time: and (5) 17 min. ESI-MS: 1832.2, corresponding to the theoretical molecular weight.
FIG. 9 pHA-VAP-DOX in vitro anti-U87 cell activity curves
As can be seen, the IC of U87 cells after 72 hours incubation with DOX, MAL-DOX, VAP-DOX or pHA-VAP-DOX, respectively500.06, 1.45, 1.57 and 0.38. mu.M, respectively. The results show that the in vitro antitumor activity of the pHA-VAP modified adriamycin is superior to that of the maleamide adriamycin and the VAP modified adriamycin.
FIG. 10 pHA-VAP-DOX in vitro anti-HUVEC cell Activity Curve
As can be seen, the IC of HUVEC cells after 72 hours incubation with DOX, MAL-DOX, VAP-DOX or pHA-VAP-DOX, respectively500.20, 1.50, 0.70 and 0.30. mu.M, respectively. The results show that the in vitro antitumor activity of the pHA-VAP modified adriamycin is superior to that of the maleamide adriamycin and the VAP modified adriamycin.
FIG. 11 shows the pHA-VAP-DOX anti-U87 in situ glioma survival curves
The figure shows the survival curve of U87 nude mice of in situ glioma model. The median survival time of the model animal is taken as an index, and the survival time of the model animal is prolonged by using physiological saline (the median survival time is 26 days), DOX (the median survival times of 10mg, 20mg and 40mg are respectively 27, 17 and 13 days, and the median survival time of the model nude mouse is shorter due to high toxicity of medium dose and high dose adriamycin), pHA-VAP (the median survival time is 28 days), and pHA-VAP-DOX (the median survival times of 10mg, 20mg and 40mg are respectively 29, 32 and 40 days), and the dosage is dependent.
FIG. 12 is an electron micrograph of a nano drug delivery system of a pHA-VAP modified lipid membrane coated cabazitaxel nanocrystal
As can be seen from the figure, the cabazitaxel nanocrystal (left) is spherical, and the particle size is about 80 nm; the cabazitaxel nanocrystal (middle and right) coated by the pHA-VAP modified lipid membrane is spherical, has an obvious core-membrane structure, and has the particle size of about 100 nm.
FIG. 13 shows particle size characterization of nano drug delivery system of pHA-VAP modified erythrocyte membrane coated docetaxel/parthenolide nano cocrystal
As can be seen from the figure, the particle size of the docetaxel/parthenolide nano eutectic crystal is about 130nm, and the potential is-20 mV; the particle size is about 140nm after the red cell membrane is coated, and the potential is-25 mV; the particle size of the constructed nano drug delivery system is not obviously influenced by the modification of the pHA-VAP, but the potential of the modified nano drug delivery system is increased to-15 mV because the molecule is positively charged.
FIG. 14, HUVEC of umbilical vein endothelial cells, and intake of PHA-VAP modified lipid membrane coated cabazitaxel nanocrystals by breast cancer cells 4T1
As can be seen from the figure, the intake of the PHA-VAP modified lipid membrane coated cabazitaxel nanocrystal by HUVEC cells (left) and 4T1 cells (right) is obviously higher than that of free drug groups, nanocrystal groups and non-target lipid membrane coated cabazitaxel nanocrystal groups.
FIG. 15 uptake of primary brain capillary endothelial cells BCEC, umbilical vein endothelial cells HUVEC, and brain glioma cells U87 into pHA-VAP modified erythrocyte membrane coated docetaxel/parthenolide nanocrystallites
As can be seen from the figure, the uptake of the nano cocrystal (docetaxel and parthenolide) of the drug coated by the pHA-VAP modified erythrocyte membrane is obviously higher than that of the nano cocrystal group and the nano cocrystal group of the drug coated by the untargeted erythrocyte membrane by the BCEC cells (A, B), the HUVEC cells (C, D) and the U87 cells (E, F), and the uptake is equivalent to that of the free drug group.
FIG. 16 shows the in vitro anti-HUVEC, 4T1 cell activity curves of pHA-VAP modified lipid membrane coated cabazitaxel nanocrystal
As can be seen from the figure, after two cells were respectively incubated with free cabazitaxel, cabazitaxel nanocrystal, lipid membrane coated cabazitaxel nanocrystal and pHA-VAP modified lipid membrane coated cabazitaxel nanocrystal for 48 hours, the IC of the cells on HUVEC cells was shown5030.3, 1.86, 1.61 and 1.49nM, respectively (left panel), IC for 4T1500.88, 8.51, 2.93 and 0.06nM, respectively (right panel). The results show that the in vitro anti-tumor effect of the PHA-VAP modified lipid membrane coated cabazitaxel nanocrystal is superior to that of all groups.
FIG. 17 shows in vitro HUVEC and U87 cell activity curves of pHA-VAP modified erythrocyte membrane coated docetaxel/parthenolide nano eutectic
It can be seen from the figure that the two cells are respectively mixed with free docetaxel/parthenolide, docetaxel/parthenolide nano cocrystal, red cell membrane coated docetaxel/parthenolide nano cocrystal, and pHA-VAP modified red cell membrane coated docetaxel/parthenolideAfter 48 hours incubation of the nanocrystallites, the IC of the nanocrystallites on HUVEC cells is shown5023.7, 44.6, 67.0 and 7.0nM (left panel), respectively, IC for U875045.6, 79.9, 90.3 and 3.2nM, respectively (right panel). The results show that the in vitro anti-tumor effect of the docetaxel/parthenolide nano eutectic coated by the red cell membrane modified by the pHA-VAP is superior to that of all groups.
FIG. 18 shows the in vivo tissue distribution map of pHA-VAP modified lipid membrane coated cabazitaxel nanocrystals in 4T 1-carrying breast cancer in situ tumor model mice
As can be seen from the figure, the accumulation amount of the cabazitaxel nanocrystal coated with lipid membranes at different time points at the 4T1 tumor site can be remarkably increased through the modification of pHA-VAP, and the cabazitaxel nanocrystal can be better targeted to the tumor site.
FIG. 19 is a tissue distribution diagram of pHA-VAP modified erythrocyte membrane coated docetaxel/parthenolide nano eutectic in vivo tissue distribution of Holu 87 in situ glioma model mouse
The figure shows that the pHA-VAP modification can obviously improve the accumulation amount of the erythrocyte membrane coated drug nano eutectic (docetaxel (upper figure) and parthenolide (lower figure)) at brain glioma positions at different time points, and the erythrocyte membrane coated drug nano eutectic can be better targeted to the tumor positions.
FIG. 20 shows the change curve of the tumor volume in situ of anti-4T 1 breast cancer with the PHA-VAP modified lipid membrane coated cabazitaxel nanocrystal
The figure is a curve of the tumor volume of balb/c mice in each group as a function of time. Compared with the PBS group, each administration group has the effect of inhibiting the growth of the tumor. Compared with the cabazitaxel nanocrystal without the target lipid membrane coated with the pHA-VAP, the cabazitaxel nanocrystal modified by the pHA-VAP has significant difference (n is 6), and the in vivo efficacy of the cabazitaxel nanocrystal coated with the pHA-VAP modified lipid membrane is optimal.
FIG. 21, pHA-VAP modified lipid membrane coated cabazitaxel nanocrystal anti-4T 1 breast cancer in situ tumor density comparison chart
Balb/c mice were sacrificed and tumor tissues were taken out, weighed and statistically analyzed, and the tumors of cabazitaxel nanocrystal group coated with the pHA-VAP modified lipid membrane were significantly lower in weight than the other groups (n ═ 6).
FIG. 22 shows the anti-U87 in situ glioma survival curve of the pHA-VAP modified erythrocyte membrane coated docetaxel/parthenolide nano cocrystal
The figure shows the survival curve of each group of U87 nude mice with in situ brain glioma. Single tail vein administration was used, and the survival of model animals was used as an evaluation index (n-10). Compared with PBS (median survival 38 days), free docetaxel/parthenolide (median survival 40.5 days), and red cell membrane-coated docetaxel/parthenolide nanocrystallines (median survival 41.5 days), the survival time of the pHA-VAP modified red cell membrane-coated docetaxel/parthenolide nanocrystallines mice (median survival 77 days) was significantly prolonged (p < 0.001).
FIG. 23 shows the effect of pHA-VAP modified lipid membrane coated cabazitaxel nanocrystals on apoptosis and angiogenesis inhibition of 4T1 breast cancer in-situ tumor cells
The figure shows the staining photographs of 4T1 in-situ tumor angiogenesis inhibiting (upper panel) and tumor cell apoptosis promoting (lower panel) of two cells respectively associated with free cabazitaxel, cabazitaxel nanocrystal, lipid membrane coated cabazitaxel nanocrystal and pHA-VAP modified lipid membrane coated cabazitaxel nanocrystal, wherein the blood vessel (CD31 staining) is brownish red or brown, and the apoptotic cell (TUNEL staining) is green.
FIG. 24 shows the effect of pHA-VAP modified erythrocyte membrane coated docetaxel/parthenolide nano eutectic on apoptosis, angiogenesis inhibition and tumor stem cell killing of U87 in-situ brain glioma
The figure is a photograph of TUNEL staining (green), neovascular CD31 staining (red) and tumor stem cell CD133 staining (red) of apoptotic tumor cells at the U87 orthotopic tumor site, where blue is nuclear DAPI staining. As can be seen from the figure, compared with the other three groups, the docetaxel/parthenolide nano eutectic coated by the pHA-VAP modified erythrocyte membrane can remarkably promote tumor apoptosis, inhibit angiogenesis and kill tumor stem cells.
Detailed Description
The invention will be further understood by reference to the following examples, but is not limited to the scope of the following description.
Example 1
Synthesis and characterization of targeting molecule, targeting molecule-Cys and targeting molecule-Cy 7
1. Synthesis and characterization of targeting molecule and targeting molecule-Cys
The pHA-VAP polypeptide (amino acid sequence p-hydroxybenzoic acid-Ahx-Cys-pavrtns; upper case letter represents L configuration amino acid, lower case letter represents D configuration amino acid) is designed and synthesized by adopting a solid phase polypeptide synthesis method.
The specific method comprises the following steps: firstly, Cys (Trt) -Acp-4-tert-butylbenzoic acid is synthesized by an Fmoc solid-phase polypeptide synthesis method, amino acids are sequentially added to PAM-Fmoc resin in sequence, and HBTU/DIEA is used as a condensing agent and TFA is used as a deprotection agent for reaction. Then activating side chain carboxyl by using N-hydroxysuccinimide (NHS), grafting VAP peptide synthesized by a Boc protection solid phase peptide synthesis method to Cys (trt) -acp-4-tert-butylbenzoic acid, deprotecting 95% TFA to obtain pHA-VAP-Cys, and separating and purifying the crude polypeptide by using an acetonitrile/water (containing 0.1% TFA) system. HPLC and ESI-MS characterize the purity and molecular weight (Mw) of pHA-VAP-Cys. The HPLC and mass spectrogram of pHA-VAP-Cys is shown in figure 1.
2. Synthesis and characterization of targeting molecule-Cy 7
Dissolving the pHA-VAP-Cys obtained in the above step in 0.1M PBS (pH7.2), dissolving Cy 7-maleimide in DMF, mixing the two, magnetically stirring for reaction, monitoring by HPLC, stopping the reaction after the pHA-VAP-Cys reaction is completed, preparing a liquid phase, purifying, and separating and purifying by using an acetonitrile/water (containing 0.1% TFA) system. And freeze-drying to obtain the pure pHA-VAP-Cy7 product. The HPLC chromatogram and the mass spectrogram are shown in figure 2.
Example 2
In vitro cell targeting verification of targeting molecules
1. In vitro targeting of targeting molecule to primary brain capillary endothelial cell BCEC
Taking the brain of a 4-week-old SD rat after decapitation, quickly separating the brain cortex from a precooled D-Hanks solution to obtain cerebral cortex, rolling off meninges and cerebral macrovessels, shearing into pieces, adding collagenase and DNase, digesting the brain for 90 minutes at 37 ℃, centrifuging the brain for 8 minutes at 1000 rpm, discarding supernatant, transferring the supernatant into a DMEM solution containing 20% BSA, centrifuging the brain for 20 minutes at 1000 g/min and 4 ℃, discarding middle and upper layer liquid, transferring bottom capillaries into a DMEM culture solution, centrifuging the brain for 5 minutes at 1000 rpm, resuspending the capillary sections by a DMEM culture solution containing 20% fetal calf serum, and connecting the capillary sections with the DMEM culture solution containing 20% fetal calf serumPlanting in 12-well plate at 37 deg.C and 5% CO2And culturing for 24 hours under the saturated humidity condition, changing the special endothelial culture solution containing puromycin, continuously culturing for 72 hours, and then changing the special endothelial culture solution containing the cell growth factors, and culturing for 72 hours to obtain the primary brain capillary endothelial cells.
Preparing a fluorescence labeling polypeptide solution with the fluorescence concentration of 5 mu M by using a DMEM culture solution containing 10% FBS, sucking out the DMEM culture solution in a 12-hole plate, adding a liquid medicine, incubating for 4 hours at 37 ℃, and removing a fluorescein solution. Washing the plate twice with PBS, adding trypsin to digest the cells, dispersing the cells with DMEM culture solution, centrifuging, discarding the supernatant, washing twice with PBS, dispersing the cells in each well in 200 mu LPBS, and determining with a flow cytometer. The results are shown in FIG. 3.
2. In vitro targeting of targeting molecule to umbilical vein endothelial cell HUVEC
Taking umbilical vein endothelial cell (HUVEC cell) of monolayer culture in logarithmic growth phase, digesting the monolayer culture cell with 0.25% trypsin, preparing single cell suspension with DMEM culture solution containing 10% fetal calf serum, and culturing at a rate of 1 × 10 per well5The cells were seeded in 12-well plates, each well volume 1mL, and the plates were transferred to a carbon dioxide incubator at 37 ℃ with 5% CO2And culturing for 24h under saturated humidity conditions, and performing the same experiment. The flow cytometry analysis results are shown in FIG. 4.
3. In vitro targeting of targeting molecule to glioma cell U87
Glioma cells (U87 cells) were taken from the logarithmic growth phase in monolayer culture, as in the above experiment. The flow cytometry analysis results are shown in FIG. 5.
Example 3
In vivo targeting validation of targeting molecules
1. Detection of tissue distribution of pHA-VAP-Cys in nude mice with subcutaneous tumor model of Duck 87
A U87 subcutaneous graft tumor model was constructed. Tail vein injection of fluorescein-labeled pHA-VAP polypeptide with the same dose is respectively carried out, mice are killed 30min and 1, 4 and 24h after injection, blood, heart, liver, spleen, lung, kidney, brain and tumor are taken, weighed, 1mL of distilled water is added, tissue homogenate is carried out, measurement is carried out by an enzyme-labeling instrument, and fluorescence quantification is carried out. The results are shown in FIG. 6.
2. Detection of tissue distribution of pHA-VAP-Cys in nude mouse with in-situ glioma model of Holo U87
The U87 cells in the logarithmic growth phase were resuspended in an appropriate amount of PBS solution at a cell concentration of 1.3 × 108/mL. The nude mice were anesthetized by intraperitoneal injection with 8% chloral hydrate solution, fixed on a brain stereotaxic instrument, and 5 μ L of U87 cell suspension was injected into the striatum brain region to construct a U87 in situ glioma model. Tail vein injection of fluorescein-labeled pHA-VAP polypeptide with the same dose is respectively carried out, mice are killed 30min and 1, 4 and 24h after injection, blood, heart, liver, spleen, lung, kidney, brain and tumor are taken, weighed, 1mL of distilled water is added, tissue homogenate is carried out, measurement is carried out by an enzyme-labeling instrument, and fluorescence quantification is carried out. The results are shown in FIG. 7.
Example 4
Preparation of targeting molecule-drug complexes
Examples of the preparation of a compound pHA-VAP-doxorubicin as targeting molecule for the attachment of a ketone-or aldehyde-containing drug. 9.4mg of the thiolated pHA-VAP was dissolved in 3mL of a phosphate buffer (0.1mM, pH7.4), and an equimolar amount of doxorubicin 6-maleimidocaprohydrazine derivative was added thereto, followed by reaction at room temperature in the dark for 1 hour. Purifying the reaction liquid by using a preparation liquid phase, and freeze-drying to obtain the pHA-VAP-adriamycin compound. The HPLC chromatogram and the mass spectrum are shown in figure 8.
Examples of drugs containing hydroxyl or amino groups linked by disulfide bonds using the pHA-VAP-paclitaxel complex as the targeting molecule. 200mg of paclitaxel was dissolved in 10mL of chloroform, cooled to 0-5 deg.C, added with 39.99mg of DCC and 60.4mg of 3- (2-pyridinedimercapto) propionic acid, and then warmed to room temperature for reaction overnight. The reaction solution is filtered and purified by column chromatography (CHCl3/MeOH 50:1-15:1, V/V elution) to obtain the taxol 3- (2-pyridinedimercapto) propionic acid derivative. Dissolving a paclitaxel 3- (2-pyridinedimercapto) propionic acid derivative in 5mLDMF, dissolving pHA-VAP-Cys with the molar weight being 1.5 times that of the paclitaxel 3- (2-pyridinedimercapto) propionic acid derivative in PBS/DMF, keeping the pH value of the solution at 4-5, dropwise adding the paclitaxel 3- (2-pyridinedimercapto) propionic acid derivative into a sulfhydryl polypeptide solution, reacting for 6 hours at room temperature, preparing a liquid phase, purifying and freeze-drying to obtain the polypeptide-paclitaxel compound.
Example of linking a drug containing a boronic acid group with a pHA-VAP-bortezomib complex as targeting molecule. Amino acids are sequentially inoculated on the resin according to the synthesis of pHA-VAP, and the Boc protection of the nitrogen removal end of trifluoroacetic acid is carried out after all amino acid residues of the polypeptide are inoculated. Adding DMF solution containing 3 times of succinic anhydride and DIEA, and reacting at room temperature for 30 min. After the resin is washed, 5 times of trimethylchlorosilane in molar weight is added to protect dopamine, HBTU/DIEA is used as a condensing agent, and the reaction is carried out for 1 hour at room temperature. The resin was cleaved with HF and purified by preparative HPLC to give the polypeptide-dopamine derivative. Mixing the pHA-VAP-dopamine derivative and the bortezomib in a buffer solution with the pH value of 7.4 according to the molar ratio of 1:1 to obtain the pHA-VAP-bortezomib compound.
An example of linking a polypeptide drug with a pHA-VAP-PMI fusion polypeptide as a targeting molecule. Is directly prepared by a solid phase polypeptide synthesis method, and the specific method comprises the following steps: after the pHA-VAP-PMI polypeptide sequence is determined, amino acids are sequentially accessed according to the same method as the preparation of the pHA-VAP, and the pHA-VAP-PMI fusion polypeptide is obtained after HF cutting and purification.
Example 5
In vitro pharmacodynamic test of targeting molecule modified nano drug delivery system
1. In vitro potency assay of pHA-VAP-DOX on glioma cell U87
Taking U87 cells in logarithmic growth phase, digesting and blowing into single cells by 0.25% trypsin, suspending the cells in DMEM culture solution containing 10% FBS, inoculating the cells in 96-well cell culture plates at the density of 3000 cells per well, keeping 0.2mL of the cells per well, reserving three holes, adding culture solution containing no cells as blank holes, and culturing in a carbon dioxide incubator for 24 hours. Each group of drugs was diluted six-fold in turn with cell culture medium. The cell culture medium in the 96-well plate was aspirated, and 200. mu.L of each well was added. Three more wells were set for each concentration, leaving three wells to which culture medium alone was added as control wells. After culturing for 72 hours, adding 20 mu L of MTT reagent (5mg/mL) into the experimental well, the control well and the blank well, incubating for 4 hours, discarding culture solution in the wells, adding 150 mu L of dimethyl sulfoxide into each well, oscillating to fully dissolve the generated bluish purple crystals, measuring the absorbance (A) of each well at 490nm by using a microplate reader, and calculating the cell survival rate according to the following formula:
survival rate ═ a490 experimental wells-A490 blank hole)/(A490 control well-A490 blank hole)×100％
Survival was plotted against drug concentration log using GraphPadPrism software (fig. 9) and median Inhibitory Concentration (IC) was calculated50)。
2. In vitro potency assay of pHA-VAP-DOX on umbilical vein endothelial cell HUVEC
HUVEC cells were taken in logarithmic growth phase and tested as above. The results are shown in FIG. 10.
Example 6
In vivo pharmacodynamic assay of targeting molecule modified drug complexes
On the 7 th day after the U87 orthotopic tumor animal model is constructed, the mice are randomly divided into 8 groups, 10 mice in each group are injected with DOX, pHA-VAP-DOX, pHA-VAP and physiological saline every two days, the total dose of the adriamycin is respectively 10mg/kg, 20mg/kg and 40mg/kg, and the amount of the polypeptide in the compound is converted into the amount of the polypeptide in pHA-VAP-DOX40 mg/kg. Survival time was recorded for each group of nude mice and survival curves were plotted (fig. 11).
Example 7
Preparation and characterization of targeting molecule modified nano drug delivery system
1. preparation of pHA-VAP modified lipid membrane coated cabazitaxel nanocrystal
The pHA-VAP-PEG-DSPE is synthesized by reacting free sulfydryl of pHA-VAP-Cys with maleimide contained in Mal-PEG-DSPE. Weighing 4mg of cabazitaxel and a proper amount of surfactant TPGS into a 25ml eggplant-shaped bottle, adding a proper amount of dichloromethane to dissolve, and forming a film to hydrate to prepare cabazitaxel nanocrystals with good dispersibility. Spin-drying appropriate amount of liposome membrane (molar ratio: HSPC: Chol: DSPE-PEG 2000: 50:45:5), adding nanocrystalline solution, hydrolyzing phospholipid membrane at 65 deg.C, and performing ultrasonic treatment with probe for 10min (120W). The morphology of the sample is observed by uranium acetate negative staining electron microscopy, and the result is shown in figure 12.
2. preparation of pHA-VAP modified erythrocyte membrane coated docetaxel/parthenolide nano eutectic
Collecting male ICR mouse whole blood, centrifuging at 1000 g/min and 4 deg.C for 5min, removing upper layer bloodClearing and cleaning a leucocyte layer, washing lower layer red blood cells by using 1 XPBS, then resuspending the lower layer red blood cells in 0.25 XPBS for 30 minutes at 4 ℃, centrifuging the lower layer red blood cells at 15000 g/min at 4 ℃ for 7 minutes to remove hemoglobin, resuspending the obtained light red blood cell membrane, preserving the light red blood cell membrane in double distilled water, and detecting the concentration of membrane protein by using a BCA kit; weighing 4mg of docetaxel, 1.2mg of parthenolide and a proper amount of surfactant F127 into a 25mL eggplant-shaped bottle, adding a proper amount of methanol to dissolve and form a film, and hydrating to prepare the docetaxel/parthenolide nano eutectic crystal with good dispersibility; mu.L of streptavidin-PEG3400The PBS solution of DSPE (5mg/mL) was incubated with the erythrocyte membrane vesicles obtained from 100. mu.L of whole blood in a water bath at 37 ℃ for 30 minutes to obtain streptavidin-erythrocyte membrane vesicles. Mixing the obtained streptavidin-erythrocyte membrane vesicle and docetaxel/parthenolide nano eutectic, performing ultrasonic treatment to obtain a nano drug delivery system of the erythrocyte membrane coated nanocrystal with the surface modified with the streptavidin, and then adding 100 mu L of biotin-PEG2000And (3) incubating in PBS (0.1mg/mL) of-VAP-pHA for 10 minutes in water bath at 37 ℃ to obtain a nano drug delivery system of the docetaxel/parthenolide nano eutectic coated by the red cell membrane modified by the pHA-VAP. The particle size and potential are characterized in figure 13.
Example 8
In vitro targeting of targeting molecule modified nano drug delivery system
1. Umbilical vein endothelial cell HUVEC and breast cancer cell 4T1 uptake test of pHA-VAP modified lipid membrane coated cabazitaxel nanocrystal
HUVEC and 4T1 cell plating method is as above, DMEM culture solution containing 10% FBS is used for preparing two kinds of cells with corresponding concentrations, free cabazitaxel, cabazitaxel nano-crystal coated by lipid membrane and cabazitaxel nano-crystal coated by pHA-VAP modified lipid membrane, the DMEM culture solution in a 12-hole plate is sucked out, liquid medicine is added, incubation is carried out for 4 hours at 37 ℃, and the liquid medicine is discarded. Washing the plate twice with PBS solution, adding trypsin to digest cells, dispersing the cells with DMEM culture solution, counting, centrifuging, discarding the supernatant, washing twice with PBS, finally dispersing the cells in each hole in 100 mu LPBS, ultrasonically breaking the cells, adding 3 times of volume of methanol to precipitate protein, centrifuging at 10000rpm for 10min, taking the supernatant, and measuring the content of the drug by HPLC. The results are shown in FIG. 14.
2. Uptake test of primary brain capillary endothelial cell BCEC, umbilical vein endothelial cell HUVEC and glioma cell U87 on pHA-VAP modified erythrocyte membrane coated docetaxel/parthenolide nano eutectic
The BCEC cell extraction method and the HUVEC and U87 cell plating method are as above, free docetaxel/parthenolide, docetaxel/parthenolide nano eutectic, red cell membrane coated docetaxel/parthenolide nano eutectic and pHA-VAP modified red cell membrane coated docetaxel/parthenolide nano eutectic with corresponding concentrations are prepared by DMEM culture solution containing 10% FBS, the DMEM culture solution in a 12-hole plate is sucked out, liquid medicine is added, incubation is carried out for 1 hour at 37 ℃, and the liquid medicine is discarded. Washing the plate twice with PBS solution, adding trypsin to digest cells, dispersing the cells with DMEM culture solution, counting, centrifuging, discarding the supernatant, washing twice with PBS, dispersing the cells in each well in 100 mu L PBS, ultrasonically breaking the cells, adding 3 times of volume of methanol to precipitate protein, centrifuging at 10000rpm for 10min, taking the supernatant, and measuring the content of the drug by HPLC. The results are shown in FIG. 15.
Example 9
In vivo targeting verification of targeting molecule modified nano drug delivery system
1. Detection of tissue distribution of pHA-VAP modified lipid membrane coated cabazitaxel nanocrystals in nude mice with 4T1 in-situ breast cancer model
A 4T1 in situ breast cancer model was constructed. Tail vein injection of free cabazitaxel, cabazitaxel nanocrystals, cabazitaxel-coated cabazitaxel nanocrystals and pHA-VAP modified cabazitaxel-coated cabazitaxel nanocrystals with the same dose are respectively carried out, mice are killed 2, 12 and 24 hours after injection, blood, heart, liver, spleen, lung, kidney, brain and tumor are taken, weighed, 1mL of distilled water is added, tissue homogenate is carried out, extraction is carried out, and HPLC quantification is carried out (shown in figure 16).
2. Detection of tissue distribution of pHA-VAP modified erythrocyte membrane coated docetaxel/parthenolide nano eutectic in nude mice of in-situ brain glioma model of Holu 87
And (3) constructing a U87 in-situ brain glioma model. Respectively injecting free docetaxel/parthenolide, docetaxel/parthenolide nano eutectic coated with erythrocyte membrane, pHA-VAP modified docetaxel/parthenolide nano eutectic coated with erythrocyte membrane into tail vein at the same dosage, killing mice at 2 and 12h after injection, respectively, collecting blood, heart, liver, spleen, lung, kidney, brain and brain tumors, weighing, adding 1mL of distilled water, homogenizing tissues, extracting twice with methyl tert-butyl ether, volatilizing, re-dissolving with methanol, and performing HPLC quantification (figure 17).
Example 10
In vitro pharmacodynamic test of targeting molecule modified nano drug delivery system
1. In vitro efficacy test of pHA-VAP modified lipid membrane coated cabazitaxel nanocrystal
HUVEC cells or 4T1 cells were taken in logarithmic growth phase and tested as above. The results are shown in FIG. 18.
2. In vitro efficacy test of pHA-VAP modified erythrocyte membrane coated docetaxel/parthenolide nano eutectic
HUVEC cells or U87 cells were taken in logarithmic growth phase, as in the above experiment. The results are shown in FIG. 19.
Example 11
In vivo pharmacodynamic assay for targeting molecule modified nano drug delivery system
1. In vivo pharmacodynamic test of pHA-VAP modified lipid membrane coated cabazitaxel nanocrystal
The constructed 4T1 in-situ breast cancer animal model. The size of the tumor to be detected is 100mm3The preparation method comprises the steps of grouping, and respectively injecting PBS, free cabazitaxel, cabazitaxel nanocrystal, lipid membrane coated cabazitaxel nanocrystal and pHA-VAP modified lipid membrane coated cabazitaxel nanocrystal into tail veins. The total administration dose of cabazitaxel in the administration group is 16mg/kg, which is divided into 4 times, and the interval between each administration is two days. The long diameter (a) and the short diameter (b) of the tumor were measured with a vernier caliper every other day. And calculating the tumor volume of each group of balb/c mice according to a formula, drawing a curve of the change of the tumor volume along with time, and calculating the statistical difference of each group. Tumor volume was calculated and growth inhibition curves were plotted according to the following formula (fig. 20):
Vtumor volume＝0.5(a×b2)
After 20 days of dosing, all balb/c mice were sacrificed by cervical dislocation, subcutaneous tumors were removed and weighed, and statistical differences were calculated for each group (fig. 21).
2. In vivo pharmacodynamic test of pHA-VAP modified erythrocyte membrane coated docetaxel/parthenolide nano eutectic
And constructing a U87 in-situ glioma animal model. After 10 days of tumor implantation, PBS (pH7.4), free docetaxel/parthenolide, docetaxel/parthenolide nano cocrystal coated with erythrocyte membrane, and docetaxel/parthenolide nano cocrystal coated with pHA-VAP modified erythrocyte membrane are respectively injected into tail vein. The total administration dose of docetaxel is 25mg/kg, the total administration dose of parthenolide is 6mg/kg, and the medicine is administered in a single time. The survival time of the nude mice was recorded (fig. 22).
3. Detection of apoptosis promotion and angiogenesis inhibition of pHA-VAP modified lipid membrane coated cabazitaxel nanocrystal
On day 2 after completion of the administration, tumor-bearing balb/c mice were sacrificed and tumor tissues were removed for fixation, and paraffin sections or frozen sections were prepared, and angiogenesis inhibition by CD31 staining or apoptosis promotion by TUNEL staining were examined. The results are shown in FIG. 23.
4. Detection of apoptosis promotion, angiogenesis inhibition and tumor stem cell killing of pHA-VAP modified erythrocyte membrane coated docetaxel/parthenolide nano eutectic
Tumor-bearing nude mice were sacrificed and tumor tissues were taken out for fixation on the 10 th day after completion of administration, frozen sections were taken, and apoptosis promotion, neovascularization inhibition and tumor stem cell killing were examined by TUNEL staining, CD31 and CD133 antibody staining. The results are shown in FIG. 24.

Claims (12)

1. The whole-process targeting molecule is characterized in that a brain targeting molecule and a tumor targeting molecule are covalently connected to form a targeting delivery system for mediating an image molecule, a therapeutic drug and a nano drug-loading system;
the brain targeting molecule and the tumor targeting molecule are formed by combining small molecules, polypeptide molecules or protein molecule targeting molecules which have blood-brain crossing barriers and blood-tumor crossing barriers and target tumor mimicry blood vessels, tumor cells and stem cells thereof.
2. The whole process targeting molecule of claim 1, wherein said small molecule, polypeptide molecule or protein molecule targeting molecule across the blood-brain barrier is selected from the group consisting of: p-hydroxybenzoic acid (pHA) and its derivatives, fatty acids such as myristic acid (MC) and its derivatives, D8 polypeptide, WSW polypeptide, and mixtures thereof,DWSW polypeptide, TGN polypeptide,DTGN polypeptides, CDX polypeptides,DCDX polypeptides, T7 polypeptides andDt7 polypeptide and its derivatives, transferrin, lactoferrin and its derivatives.
3. The process-targeting molecule of claim 2, wherein said polypeptide or protein molecule targeting molecule across the blood-tumor barrier is selected from the group consisting of: VAP polypeptide, cVAP polypeptide,SVAP polypeptides,DVAP polypeptide, A7R polypeptide, cA7R polypeptide,DA7R polypeptide, RGD polypeptide, staged-RGD polypeptide, RW polypeptide, mn polypeptide, RAP12 polypeptide andDRAP12 polypeptides and derivatives thereof.
4. The process-targeting molecule of claim 1, wherein said molecule is modified by the introduction of reactive functional groups to form a complex of imaging molecules, a complex of therapeutic agents, and a complex of polymeric carrier materials; wherein, an image molecule X is introduced into the whole process targeting molecule to prepare a whole process targeting molecule-X compound; introducing a therapeutic drug molecule Y into the whole process targeting molecule to prepare a whole process targeting molecule-Y compound; introducing polyethylene glycol-Z compound molecules on the whole process targeting molecules to prepare the whole process targeting molecule-polyethylene glycol-Z compound, wherein Z is lipophilic material molecules or lipophilic drug molecules or hydrophilic ligand molecules.
5. The full-process targeting molecule according to claim 4 wherein in said full-process targeting molecule-X complex, X is an optical imaging molecule such as FITC, FAM, 6-TET, 5-TAMRA, HEX, 6-JOE, near infrared dye molecule Cy3, Cy3.5, Cy5, Cy5.5, Cy7, IR783, IR820, DiR, DiD, BIDIPY630/650-X, BIDIPY650/665-X, BIDIPY665/676, TO-PRO-3, TO-PRO-5, chemiluminescent molecules lumineol, isolumenol, AMPPD, CSPD, CDP-star, lucigenin, Raman probe molecules; magnetic resonance imaging agents, e.g. chelates of Gd magnetic resonance materials, radionuclide imaging agents, e.g. Gd18F、32P、35S、64Cu、67/68Ga、75Se、89Zr、86Y、99mTc、111/111mIn、123/125I、177Lu、149/161Chelates of radionuclides for Tb imaging.
6. The full-process targeting molecule according to claim 5 wherein said magnetic resonance imaging agent and radionuclide imaging agent are comprised of a bifunctional chelator and a radionuclide for magnetic resonance imaging or radionuclide for imaging, wherein the bifunctional chelator comprises DOTA, DOTAGA, NOTA, NOTAGA, NODA, DTPA, TETA, CB-TE2A, Cyclam, DFO, MAG3, EC, EDTA, DADT, HYNIC, CE-DTS, NS3 for chelating nuclides for magnetic resonance imaging or radionuclide for radioimaging.
7. The process-targeting molecule according to claim 4, wherein in said process-targeting molecule-Y complex, Y is an anthracycline of a chemotherapeutic agent for tumors, such as doxorubicin or epirubicin, a taxane, such as paclitaxel, docetaxel or carboplatin, a camptothecin, such as camptothecin, hydroxycamptothecin, 9-nitrocamptothecin or irinotecan, a vinblastine, such as vinblastine or vincristine, a proteasome inhibitor, such as bortezomib; anti-tumor stem cell drugs such as parthenolide and derivatives thereof; molecular targeting drugs such as trametinib, imatinib, nilotinib, dasatinib, everolimus, erlotinib, sunitinib, sorafenib, ibrutinib, regorafenib, vemurafenib, olaparib; polypeptide drugs such as p53 activating peptide; antibody drugs such as rituximab, bevacizumab, trastuzumab, cetuximab, pertuzumab, and ipilimumabThe monoclonal antibody, the monoclonal antibody PD-L1 and the antibody fragment combination which is transformed by genetic engineering comprise Fab fragment, single domain antibody, Fv fragment, single-chain antibody, bivalent small molecule antibody, micro antibody and nano antibody; therapeutic radionuclide chelates such as90Y、131I、152/155Tb、153Sm、177Lu、186/188Re、211At、212/213Bi、212Pb、225Ac、227Chelates of therapeutic radionuclides of Th.
8. The full-process targeting molecule according to claim 5 wherein said therapeutic radionuclide chelate is comprised of a bifunctional chelator and a therapeutic radionuclide, wherein the bifunctional chelator is selected from the group consisting of DOTA, DOTAGA, NOTA, NOTAGA, NODA, DTPA, TETA, CB-TE2A, Cyclam, DFO, MAG3, EC, EDTA, DADT, HYNIC, CE-DTS, NS 3.
9. The process-targeting molecule according to claim 4, wherein said lipophilic material molecule Z is a phospholipid, polylactic acid (PLA), poly (lactic-co-glycolic acid) (PLGA), Polycaprolactone (PCL); the lipophilic drug molecule Z is anthracycline drug, taxane drug, camptothecin drug, vinblastine drug, proteasome inhibitor drug, and lactone antineoplastic drug; the hydrophilic ligand molecule Z is biotin.
10. The full-process targeting molecule of claim 4 wherein said targeting molecule-polyethylene glycol-phospholipid complex is used in the preparation of liposomal drug delivery systems, micellar drug delivery systems, and disc drug delivery systems.
11. The full-process targeting molecule according to claim 4, wherein said full-process targeting molecule-polyethylene glycol-polylactic acid complex, full-process targeting molecule-polyethylene glycol-lactic glycolic acid copolymer complex, full-process targeting molecule-polyethylene glycol-polycaprolactone complex is used for preparing micelle drug delivery system, nanoparticle drug delivery system.
12. The all-process targeting molecule according to claim 8 or 9 wherein said liposomal drug delivery system, micellar drug delivery system, disc drug delivery system, nanoparticle drug delivery system and biofilm coated Nanocarrier drug delivery system are used to entrap diagnostic drugs.
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