CN117442750A - Methotrexate-mediated tumor targeting metal organic framework pharmaceutical composition, and preparation method and application thereof - Google Patents

Methotrexate-mediated tumor targeting metal organic framework pharmaceutical composition, and preparation method and application thereof Download PDF

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CN117442750A
CN117442750A CN202210842434.0A CN202210842434A CN117442750A CN 117442750 A CN117442750 A CN 117442750A CN 202210842434 A CN202210842434 A CN 202210842434A CN 117442750 A CN117442750 A CN 117442750A
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mtx
organic framework
zif
methotrexate
peg
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高钟镐
吴昊
黄伟
刘艳红
赵鹤鸣
金明姬
陈丽青
王启明
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Institute of Materia Medica of CAMS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The invention discloses a methotrexate-mediated tumor targeting type metal organic framework pharmaceutical composition, and a preparation method and application thereof. The MTX-PEG-COOH tumor targeting drug conjugate is obtained by taking methotrexate with antitumor activity and active targeting function as a material and covalently combining the methotrexate with PEG. Then modifying the compound into a metal organic framework ZIF-8 through interaction of positive and negative charges to prepare the methotrexate-mediated tumor-targeted metal organic framework pharmaceutical composition (MTX/ZIF-8). The preparation process of MTX/ZIF-8 is simple, low in cost, good in repeatability and high in batch-to-batch consistency. The MTX/ZIF-8 can be used for tumor targeted drug delivery system, and the chemotherapeutic drug is encapsulated by a simple single-step encapsulation method, so that the MTX/ZIF-8 has the advantages of high drug loading rate, good stability and responsive drug release. In addition, when the MTX/ZIF-8 is coated with the chemotherapeutic drugs, the advantages of dual mechanism synergistic anti-tumor effect can be exerted, so that the enhanced anti-tumor effect is realized.

Description

Methotrexate-mediated tumor targeting metal organic framework pharmaceutical composition, and preparation method and application thereof
Technical Field
The invention relates to the technical field of medicines, in particular to a methotrexate-mediated tumor active targeting type metal organic framework pharmaceutical composition, and a preparation method and application thereof.
Background
Malignant tumor is a global public health problem, and the incidence rate is increased year by year, so that the human health is seriously endangered. Chemotherapy is one of the common methods of treating tumors, with irreplaceable effects. However, chemotherapeutic drugs generally have the disadvantages of poor water solubility, poor tumor targeting, limited bioavailability and the like. In addition, the chemotherapeutic drugs can inhibit the growth of tumor cells and normal cells, and cause serious toxic and side effects, so that patients cannot tolerate the chemotherapeutic drugs, which greatly limits the clinical application of the chemotherapeutic drugs. Therefore, how to reduce the toxic and side effects of the chemotherapy drugs, improve the bioavailability and enhance the anti-tumor curative effect becomes a key problem to be solved urgently.
In recent years, with the development of molecular biology, the design of an intelligent tumor targeted drug delivery system has attracted extensive attention from researchers. The tumor targeting drug delivery system can specifically deliver the chemotherapeutic drugs to tumor tissues, reduce the damage to normal tissues and realize the efficacy of synergism and attenuation. Tumor targeted drug delivery systems can be classified into passive targeted drug delivery systems, physicochemical targeted drug delivery systems, and active targeted drug delivery systems according to different mechanisms of action. The tumor active targeting drug delivery system modifies a corresponding ligand or antibody on a drug carrier, and realizes high-specificity targeting on tumor cells through specific combination between the ligand and a receptor or between the antibody and an antigen, so that a chemotherapeutic drug is actively delivered to the tumor cells to exert drug effects. Common receptors on the surface of tumor cells are folate receptors, asialoglycoprotein receptors, transferrin receptors, and the like. Among them, active targeting drug delivery systems mediated by folate receptors are widely appreciated by researchers due to their high selectivity for tumor tissue.
Folate receptor is expressed at high levels in many tumors such as breast, ovarian, renal and colon cancers, but is hardly expressed in most normal tissues. The chemical structure of Methotrexate (MTX) is highly similar to folic acid, belongs to folic acid derivatives, can be specifically combined with folic acid receptor expressed by tumor cells and realizes excellent cancer cell targeting effect. MTX is therefore often used as a tumor targeting ligand for the design of various intelligent tumor active targeting drug delivery systems. Notably, MTX has a greater affinity for folate receptors than folate, more favorable for targeting into tumor cells. In addition to acting as a targeting ligand, methotrexate (MTX) is also a common drug in clinical treatment of solid tumors, lymphomas and hematological tumors. MTX promotes tumor cell death by competitively inhibiting the activity of dihydrofolate reductase, leading to inhibition of DNA, RNA synthesis and cell proliferation. Therefore, the active targeting drug delivery system based on the methotrexate mediated tumor not only endows the active targeting effect of the drug carrier on the tumor, but also can be used as an anti-tumor drug to induce tumor apoptosis, and has very broad application prospect.
MTX mediated active tumor drug delivery system mainly realizes the purpose of targeting tumor tissues by chemotherapeutic drugs in two ways. The first is to directly couple MTX with a chemotherapeutic drug to prepare a drug conjugate. The second is to modify MTX to the drug delivery vehicle surface. Because most chemotherapeutic drugs have low water solubility under physiological conditions, the water solubility of the drug can be further reduced after the drug is coupled with MTX to form a drug conjugate, so that the bioavailability of the chemotherapeutic drug can be reduced. Modification of MTX to a drug delivery vehicle therefore shows further advantages. MTX modifiable drug delivery vehicles are liposomes, micelles, nanoparticles, metal organic frameworks, and the like. Among them, metal organic framework Materials (MOFs) have been attracting attention by researchers due to their porosity, large specific surface area, and high capacity to load chemotherapeutic drugs. Zeolite imidazole ester skeleton-8 (Zeolitic imidazolate frameworks-8, ZIF-8) is one of the most representative MOFs materials, and is a tetrahedral structure formed by N atoms in dimethylimidazole and metal Zn ions connected by coordination bonds. Compared with the traditional drug delivery carrier, ZIF-8 has excellent chemical stability and physical stability, and can avoid premature ejaculation or burst release of the chemotherapeutic drug in the blood circulation process. In addition, ZIF-8 has sensitive pH sensitivity, and the structure has high stability under neutral physiological condition, and can be decomposed in endosome/lysosome environment of tumor cells, thereby realizing controllable and accurate release of loaded medicine at tumor sites and meeting the requirements of the novel nano medicine carrying system on multiple aspects of medicine carrying capacity, stability, medicine release and the like. Therefore, the metal organic framework material ZIF-8 is selected as a carrier material of the chemotherapeutic drug to construct a nano drug-carrying system, so that the advantages of attenuation and synergy can be highlighted.
Disclosure of Invention
The invention solves the technical problems of poor tumor targeting, large toxic and side effects and the like commonly existing in the existing chemotherapeutic drugs, and provides a methotrexate-mediated tumor targeting type metal-organic framework pharmaceutical composition, a preparation method and application thereof. The invention adopts methotrexate with anti-tumor activity and active targeting function as a material, and the methotrexate is covalently combined with PEG to obtain MTX-PEG-COOH tumor targeting drug conjugate. Then modifying the compound into a metal organic framework through interaction of positive and negative charges to prepare the methotrexate-mediated tumor-targeted metal organic framework pharmaceutical composition (MTX@ZIF-8). The composition can not only be used for resisting tumors independently, but also be used as a tumor active drug delivery system for delivering chemotherapeutic drugs, and the purposes of synergism and toxicity reduction are achieved through the synergistic effect of double mechanisms.
Specifically, the invention is realized by the following technical scheme:
in a first aspect of the present invention, a methotrexate-carboxylated polyethylene glycol conjugate (MTX-PEG-COOH) having both antitumor activity and active targeting function is provided for the first time, wherein Methotrexate (MTX) can specifically bind to a folate receptor expressed in tumor cells, and excellent mediated cancer cell active targeting effect is provided. Meanwhile, MTX can also be used as an anti-tumor drug to promote apoptosis of tumor cells. The structure of the methotrexate-carboxylated polyethylene glycol conjugate MTX-PEG-COOH is shown in (I). One end of the polyethylene glycol molecular chain is connected with methotrexate, and the other end of the molecular chain is connected with carboxyl; wherein the polyethylene glycol has a polymerization degree n of 5 to 450, preferably 8 to 230, more preferably 18 to 115, still more preferably 25 to 55, still more preferably 30 to 50, most preferably 40 to 45, still most preferably 45.R is a carboxylic acid group.
In a second aspect of the invention, there is provided for the first time a methotrexate-mediated tumor-targeted metalorganic framework pharmaceutical composition. The pharmaceutical composition is formed by modifying MTX-PEG-COOH conjugate to a metal organic framework ZIF-8 through interaction of positive and negative charges, namely [email protected]@ZIF-8 can be used for resisting tumors independently, and can also be used as a tumor targeting delivery system for encapsulating chemotherapeutic drugs by a simple single-step encapsulation method, wherein the encapsulated chemotherapeutic drugs comprise triptolide, doxorubicin, mitomycin C prodrugs, mitomycin C derivatives, triptolide, docetaxel or hydroxycamptothecin. Triptolide is preferred.
The invention also provides a preparation method of the methotrexate-mediated tumor-targeted metal-organic framework pharmaceutical composition, which comprises the following steps: mixing dimethyl sulfoxide solution containing zinc nitrate hexahydrate with dimethyl imidazole water solution for reaction, and obtaining a metal organic framework material ZIF-8 through centrifugal purification; and dispersing ZIF-8 into the MTX-PEG-COOH solution, uniformly mixing, stirring for reaction, and centrifuging and purifying to obtain a solid, namely the methotrexate-mediated tumor targeting metal organic framework pharmaceutical composition.
The preferred preparation method is as follows:
80mg of zinc nitrate hexahydrate was dissolved in 3ml of dimethyl sulfoxide, and 7ml of an aqueous solution containing 800mg of dimethyl imidazole was added thereto to react for 30 minutes. The reaction solution was centrifuged at 15000rpm/min for 15min in a 2ml centrifuge tube. And then washing with methanol for three times to obtain the metal organic framework material ZIF-8. 10mg of ZIF-8 is dispersed into 5mL of aqueous solution of MTX-PEG-COOH conjugate with the concentration of 2mg/mL, stirred and reacted for 12 hours, and the solid obtained by centrifugal purification is MTX-PEG@ZIF-8.
The invention also provides application and a preparation method of the methotrexate-mediated tumor-targeted metal-organic framework pharmaceutical composition in the aspect of delivering active therapeutic drugs. The composition can be used as a delivery vehicle for liposoluble and hydrophilic chemotherapeutic drugs. The chemotherapeutic agent may be loaded into the pores of the metal organic framework material by a simple single step entrapment process. The preparation method comprises the following steps: adding a chemotherapeutic drug into a dimethyl sulfoxide solution containing zinc nitrate hexahydrate, reacting for a period of time, dropwise adding a dimethyl imidazole water solution under the stirring condition, and after the reaction is finished, obtaining a solid through centrifugal separation, washing and drying. Dispersing the solid into MTX-PEG-COOH solution, mixing, stirring for reaction, centrifugal separation after reaction, washing and drying. The medicine carrying amount of the methotrexate-mediated tumor targeting type metal organic framework medicine composition on the chemotherapeutic medicine is 5-30%.
When the chemotherapeutic drug is triptolide, the preferred preparation method is as follows:
10mg of triptolide and 80mg of zinc nitrate hexahydrate are dissolved in 3ml of dimethyl sulfoxide and stirred for 30 min. 7ml of an aqueous solution containing 800mg of dimethylimidazole was added thereto at room temperature, and the reaction was carried out for 30 minutes. Centrifuge for 15min at 15000 rpm/min. Washing with methanol for three times to obtain TPL@ZIF-8; 10mg of TPL@ZIF-8 is dispersed into 5mL of MTX-PEG-COOH aqueous solution with the concentration of 2mg/mL, stirred and reacted for 12 hours, and the solid obtained by centrifugal purification is MTX@TPL@ZIF-8NPs.
The invention also provides application of the methotrexate-mediated tumor-targeted metal organic framework pharmaceutical composition (MTX-PEG/ZIF-8) in-vitro drug delivery. The method comprises the following steps:
the MTX-PEG/ZIF-8 coated with the chemotherapeutic drug is diluted with a culture solution to a predetermined concentration. Each concentration was set with 6 duplicate wells, and control and zeroing groups were set. Inoculating a proper amount of breast cancer 4T1 cells in a logarithmic growth phase into a 96-well plate, continuously culturing for 24 hours, replacing the medium with a fresh culture solution containing MTX-PEG@ZIF-8 coated with a chemotherapeutic drug, and continuously culturing for 24 hours or 48 hours respectively. 200 μl of 10% CCK-8 reagent was added to each well. Incubation was continued for 2h, absorbance was measured at 450nm and 650nm was the reference wavelength. Cell viability was calculated as follows:
cell viability (%) = [ (OD experiment-OD zeroing)/(OD control-OD zeroing) ]x100
The invention also provides application of the methotrexate-mediated tumor-targeted metal organic framework pharmaceutical composition (MTX@ZIF-8) in-vivo treatment of brain glioma. The method comprises the following steps:
the method comprises the steps of randomly dividing 4T1 breast cancer mice into 5 groups, carrying out intravenous injection of [email protected] mL of chemotherapeutic medicine, and simultaneously setting physiological saline, free chemotherapeutic medicine, MTX@ZIF-8 and medicine carrying organic metal frameworks without MTX peptide modification as control groups. Once every three days, mice were sacrificed 5 times after dosing, tumors were isolated, photographed and weighed.
The beneficial effects of the invention are as follows:
the methotrexate-mediated tumor targeting type metal organic framework pharmaceutical composition has good tumor tissue targeting capability, biocompatibility and blood circulation function, and can be used as a tumor targeting drug delivery carrier for delivering chemotherapeutic drugs, so that the problems of low bioavailability, poor targeting, large toxic and side effects, low patient tolerance and the like are solved.
The methotrexate-mediated tumor-targeted metal-organic framework pharmaceutical composition prepared by the invention has good entrapment capacity for various chemotherapeutic drugs, and the structure is highly stable under physiological conditions, so that the composition can be decomposed in an endosome/lysosome environment of tumor cells, and the controllable and accurate release of the loaded drugs at tumor positions can be realized.
The methotrexate-mediated tumor-targeted metal organic framework pharmaceutical composition has both anti-tumor activity and active targeting function, and can exert the advantage of dual-mechanism synergistic anti-tumor effect when delivering chemotherapeutic drugs, thereby realizing enhanced anti-tumor effect.
4. The methotrexate-mediated tumor targeting type metal organic framework pharmaceutical composition prepared by the invention has the advantages of simple and rapid preparation process and high stability and repeatability.
Drawings
Fig. 1: MTX-PEG-COOH 1 H-NMR spectrum
Fig. 2: particle size diagram of TPL@ZIF-8
Fig. 3: potential diagram of TPL@ZIF-8
Fig. 4: TPL@ZIF-8 scanning electron microscope image
Fig. 5: particle size diagram of MTX-PEG@TPL@ZIF-8
Fig. 6: potential diagram of MTX-PEG@TPL@ZIF-8
Fig. 7: MTX-PEG@TPL@ZIF-8 scanning electron microscope image
Fig. 8: toxicity of blank Metal organic frameworks ZIF-8 to 4T1 cells
Fig. 9:24h drug-loaded metal organic framework for inhibiting 4T1 cell proliferation
Fig. 10: under the condition that 48h medicine-carrying metal organic framework inhibits 4T1 cell proliferation
Fig. 11: uptake of coumarin 6-labeled metal-organic frameworks in 4T1 cells
Fig. 12: isolated tumor of 4T1 breast cancer-bearing mice
Fig. 13: weight of isolated tumor of 4T 1-bearing breast cancer mice
Detailed Description
The following examples are intended to illustrate the invention and are not intended to be limiting. The present invention will be further illustrated in detail with reference to examples, but the present invention is not limited to these examples and the preparation methods used. Moreover, the present invention may be equivalently replaced, combined, improved, or modified by those skilled in the art in light of the description of the present invention, but are included in the scope of the present invention.
Example 1: preparation of MTX-PEG-COOH
50mg MTX was weighed and dissolved in 10ml DMF, EDC (1.5 eq.) and NHS (1.5 eq.) were added and dissolved completely, reacted for 6h at room temperature, then COOH-PEG-NH was added 2 (1.0 eq) was completely dissolved, reacted at 40℃for 12 hours, and the reaction mixture was concentrated under reduced pressurePouring the mixture into a large amount of glacial ethyl ether for precipitation, repeatedly dissolving the precipitate for 3 times, filtering and collecting the product, and drying in vacuum to obtain the product. MTX-PEG-COOH 1 The H-NMR spectrum is shown in FIG. 1.
Example 2: preparation of metal organic framework ZIF-8 for entrapping triptolide
In a 10ml centrifuge tube, 10mg of triptolide and 80mg of zinc nitrate hexahydrate are dissolved in 3ml of dimethyl sulfoxide, and the mixture is stirred for 30 min. 7ml of an aqueous solution containing 800mg of dimethylimidazole was added thereto at room temperature, and the reaction was carried out for 30 minutes. The reaction solution was centrifuged at 15000rpm/min for 15min in a 2ml centrifuge tube. And then washing with methanol for three times to obtain TPL@ZIF-8.
Physical properties of TPL@ZIF-8 NPs: as shown in FIGS. 2 and 3, the particle diameter was 105.7nm, PDI was 0.079 and zeta potential was 25V as measured by a dynamic light scattering method; the encapsulation rate of the nano particles is 85% and the drug loading rate is 12.8% measured by high performance liquid chromatography. The scanning electron microscope can show that the TPL@ZIF-8 is a dodecahedron with a diamond single side after medicine carrying, which indicates that the medicine carrying does not affect the physical properties of the material, as shown in figure 4.
Example 3: preparation of MTX-PEG@TPL@ZIF-8 of mammary gland targeting metal organic framework
In a 10ml centrifuge tube, 10mgMTX-PEG-COOH was dissolved in 5ml water, ice-bathed for 10min to uniformly disperse it in the aqueous solution, and then 10mgTPL@ZIF-8 was added. After stirring for 12h, the reaction was terminated. The reaction solution was centrifuged at 10000rpm/min for 10min in a 2ml centrifuge tube. And then washing with water for three times to obtain MTX-PEG@TPL@ZIF-8.
Physical properties of MTX-PEG@TPL@ZIF-8 NPs: as shown in FIGS. 5 and 6, the particle diameter was 126.5nm, PDI was 0.055 and zeta potential was 13.3V as measured by the dynamic light scattering method. As shown in fig. 8, the successful connection of the target head is proved by changing the dodecahedron with a diamond-shaped single side into a sphere after the MTX-PEG-COOH is connected through a scanning electron microscope, as shown in fig. 7.
Example 4: cytotoxicity study of materials
The blank nanoparticle solution is diluted with a culture solution to a solution of a predetermined concentration. Each concentration was set with 6 duplicate wells, and control and zeroing groups were set. Inoculating a proper amount of breast cancer 4T1 cells in a logarithmic growth phase into a 96-well plate, continuously culturing for 24 hours, replacing the medium with a fresh culture solution containing ZIF-8 solutions with different blank metal-organic frameworks, and continuously culturing for 24 hours or 48 hours respectively. 200 μl of 10% CCK-8 reagent was added to each well. The incubation was continued for 2h and the absorbance at 450nm was measured to calculate cell viability as follows:
cell viability (%) = [ (OD) Experiment -OD Zeroing )/(OD Control -OD Zeroing )]×100
The results of FIG. 8 show that the activity of the 4T1 cells is above 85% in the determined concentration range of the blank metal-organic framework ZIF-8, which shows that the material is less toxic to cells. The metal-organic framework ZIF-8 provided by the patent has advantages in the aspect of safety.
Example 5: inhibition of cell proliferation study
Diluting the drug-carrying organometallic framework MTX-PEG@ZIF-8, free TPL, TPL@ZIF-8 and MTX-PEG@TPL@ZIF-8 into a solution with a preset concentration by using a culture solution. Each concentration was set with 6 duplicate wells, and control and zeroing groups were set. Inoculating a proper amount of breast cancer 4T1 cells in a logarithmic growth phase into a 96-well plate, continuously culturing for 24 hours, replacing a medium with a fresh culture solution containing an organic metal framework solution, and continuously culturing for 24 hours or 48 hours respectively. 200 μl of 10% CCK-8 reagent was added to each well. Incubation was continued for 2h, absorbance was measured at 450nm and 650nm was the reference wavelength. Cell viability was calculated as follows:
cell viability (%) = [ (OD) Experiment -OD Zeroing )/(OD Control -OD Zeroing )]×100
The results in FIGS. 9 and 10 show that the drug-loaded organometallic framework MTX-PEG@TPL@ZIF-8 inhibited cell proliferation more than free TPL in the range of drug concentration 0.01-10 μg/mL.
Example 6 laser confocal microscope observation of Metal-organic framework Cytometric conditions
And qualitatively observing the uptake condition of the breast cancer 4T1 cells to the metal organic framework by using laser confocal. 4T1 cells were grown at 1.5X10 5 Inoculating the cells/wells into 12-well plate with cell climbing sheet placed at bottom, and culturing at 37deg.C and 5% CO 2 Culturing in incubator for 24 hr, adding free Cou-6, cou-6@ZIF-8, MTX-PEG@Cou-6@ZIF-8 diluted with serum-free culture medium to final Cou-6 concentration of 5 μg/ml, and placing at 37deg.C and 5% CO 2 Culturing in an incubator for 2 hours. After the incubation, the culture solution was discarded and washed twice with PBS, 4% paraformaldehyde was fixed for 15 minutes in the absence of light, after the PBS was washed, nuclei were stained with 1. Mu.g/ml DAPI for 15 minutes, finally after the PBS was washed, the cell slide at the bottom of the 12-well plate was taken out, and placed on a slide glass treated with an anti-fluorescence quencher, and finally, the metal-organic framework was observed by a laser confocal microscope for cell entry.
As shown in FIG. 11, MTX-PEG@Cou-6@ZIF-8 has higher uptake efficiency than other groups, and has obvious time dependence, and the uptake efficiency gradually increases with time.
EXAMPLE 7 in vivo pharmacodynamics evaluation of mammary gland-targeted Metal organic frameworks
Female BALB/c mice of 6 weeks old are randomly divided into 5 groups of 6 mice after 7 days of establishment of a model of the breast cancer mice with 4T1, and MTX-PEG@TPL@ZIF-8, MTX@ZIF-8, TPL@ZIF-8, free TPL and physiological saline are respectively used for tail veins of 0.2mL according to the dosage of 0.4mg/kg of triptolide. Mice were sacrificed by cervical dislocation after 5 doses every three days, and tumors were isolated, as shown in fig. 12 and 13, and the results showed that the tumor weight of the MTX-peg@tpl@zif-8 dose group was smaller than that of the control group, indicating that MTX-peg@tpl@zif-8 had a good therapeutic effect on breast cancer.

Claims (11)

1. A methotrexate-mediated tumor targeting metal organic framework pharmaceutical composition is characterized in that methotrexate with anti-tumor activity and active targeting function is adopted as a material and is covalently combined with PEG to obtain MTX-PEG-COOH tumor targeting drug conjugate; then modifying the compound into a metal organic framework through interaction of positive and negative charges to prepare the methotrexate-mediated tumor-targeted metal organic framework pharmaceutical composition.
2. The methotrexate-mediated tumor-targeted metal-organic framework pharmaceutical composition according to claim 1, wherein the metal-organic framework material is ZIF-8.
3. The methotrexate-mediated tumor-targeted metal-organic framework pharmaceutical composition according to claim 1, wherein the MTX-PEG-COOH tumor-targeted drug conjugate has the structural composition: one end of the polyethylene glycol molecular chain is connected with methotrexate, and the other end of the molecular chain is connected with carboxyl; the polymerization degree n of polyethylene glycol is preferably 8 to 230, more preferably 18 to 115, still more preferably 25 to 55, still more preferably 30 to 50, most preferably 40 to 45, still more preferably 45.
4. A method for preparing the methotrexate-mediated tumor-targeted metal-organic framework pharmaceutical composition of claim 1, which comprises the following steps: mixing dimethyl sulfoxide solution containing zinc nitrate hexahydrate with dimethyl imidazole water solution for reaction, and obtaining a metal organic framework material ZIF-8 through centrifugal purification; and dispersing ZIF-8 into the MTX-PEG-COOH solution, uniformly mixing, stirring for reaction, and centrifuging and purifying to obtain a solid, namely the methotrexate-mediated tumor targeting metal organic framework pharmaceutical composition.
5. The preparation method according to claim 4, wherein: the concentration of the zinc nitrate hexahydrate in the dimethyl sulfoxide solution containing the zinc nitrate hexahydrate is 1-100mg/mL, preferably 8mg/mL; the concentration of the dimethyl imidazole water solution is 0.5-1000mg/mL, preferably 80mg/mL; the molar ratio of zinc nitrate hexahydrate to dimethylimidazole is 1:1 to 1:72, preferably 1:36; mixing dimethyl sulfoxide solution containing zinc nitrate hexahydrate with dimethyl imidazole water solution for reaction at room temperature for 10-120min, preferably 30min; the concentration of the MTX-PEG-COOH solution is 0.1-20mg/mL, preferably 2mg/mL; dispersing ZIF-8 into MTX-PEG-COOH solution, mixing well, stirring at room temperature for 6-48h, preferably 12h; dispersing ZIF-8 into MTX-PEG-COOH solution, mixing uniformly, and the concentration of the metal organic framework material is 0.1-40mg/mL, preferably 2mg/mL; the mass ratio of the metal organic framework material to MTX-PEG-COOH is 20:1-1:1, preferably 1:1.
6. Use of a methotrexate-mediated tumor-targeted metal-organic framework pharmaceutical composition according to claim 1 for the preparation of a delivery vehicle as a liposoluble or water-soluble chemotherapeutic drug.
7. The use according to claim 6, characterized in that the entrapped chemotherapeutic agent comprises triptolide, doxorubicin, mitomycin C, a mitomycin C prodrug, a mitomycin C derivative, triptolide, docetaxel or hydroxycamptothecin; preferably, the chemotherapeutic agent is triptolide.
8. Use according to claim 6, wherein the composition is capable of effecting entrapment of the chemotherapeutic agent by a simple one-step entrapment process, the chemotherapeutic agent being loaded into the pores of the metal organic framework material during formation of the metal organic framework material; the method comprises the following steps: adding a chemotherapeutic drug into a dimethyl sulfoxide solution containing zinc nitrate hexahydrate, reacting for a period of time, dropwise adding a dimethyl imidazole aqueous solution under the stirring condition, and after the reaction is finished, obtaining a solid through centrifugal separation, washing and drying; dispersing the solid into MTX-PEG-COOH solution, mixing, stirring for reaction, centrifugal separation after reaction, washing and drying.
9. Use according to claim 8, characterized in that the total concentration of chemotherapeutic agent is 0.1-50mg/mL, preferably 1mg/mL; adding a chemotherapeutic drug into a dimethyl sulfoxide solution containing zinc nitrate hexahydrate, and reacting for 3-120min, preferably 30min at room temperature; the drug loading rate of the composition to the chemotherapeutic drugs is 5% -30%.
10. The use according to claim 8, wherein when the chemotherapeutic agent is triptolide, the steps comprise: 10mg of triptolide and 80mg of zinc nitrate hexahydrate are dissolved in 3ml of dimethyl sulfoxide and stirred for 30min; 7ml of an aqueous solution containing 800mg of dimethylimidazole was added thereto at room temperature, and after 30 minutes of reaction; centrifuging for 15min at a speed of 15000 rpm/min; washing with methanol for three times to obtain TPL@ZIF-8; 10mg of TPL@ZIF-8 is dispersed into 5mL of MTX-PEG-COOH aqueous solution with the concentration of 2mg/mL, stirred and reacted for 12 hours, and the solid obtained by centrifugal purification is MTX-PEG@TPL@ZIF-8NPs.
11. The use of a methotrexate-mediated tumor-targeted metal-organic framework pharmaceutical composition according to claim 1, wherein the treatable tumor type comprises breast, ovarian, renal and colon cancer, preferably breast cancer.
CN202210842434.0A 2022-07-18 2022-07-18 Methotrexate-mediated tumor targeting metal organic framework pharmaceutical composition, and preparation method and application thereof Pending CN117442750A (en)

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