CN110201160B - Preparation method of zirconium metal organic framework compound - Google Patents

Preparation method of zirconium metal organic framework compound Download PDF

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CN110201160B
CN110201160B CN201910420757.9A CN201910420757A CN110201160B CN 110201160 B CN110201160 B CN 110201160B CN 201910420757 A CN201910420757 A CN 201910420757A CN 110201160 B CN110201160 B CN 110201160B
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solution
fam
ala
powder
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CN110201160A (en
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刘建强
缪传康
潘莹
罗志董
李宝红
丁琼洁
彭新生
郑明彬
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Guangdong Medical University
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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
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • 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
    • A61K47/02—Inorganic 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • 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 preparation method of a zirconium metal organic framework compound, which comprises the following steps: 1) preparation of UIO-66-NH2(ii) a 2) Preparing a compound E; 3) preparing a compound H; 4) preparing compound L/compound P; the compound E is UIO-66-NH-FAM, the compound H is ALA/UIO-66-NH-FAM, the compound L is ALA/UIO-66-NH-FAM/MTA, and the compound P is ALA/UIO-66-NH-FAM/CP 1.

Description

Preparation method of zirconium metal organic framework compound
Technical Field
The invention relates to the technical field of biological medicines, in particular to a preparation method of a zirconium metal organic framework compound.
Background
The increasing global risk of cancer has become the first killer threatening the health of human beings. Currently, the most used treatment methods in the clinic are surgery, chemotherapy and also radiotherapy. Chemotherapy causes a range of acute and chronic organ toxicities. Since many chemotherapeutic drugs and their metabolites are excreted through renal tubular epithelial cells, the kidneys are easily damaged by chemotherapeutic drugs. Thus, the use of chemotherapeutic agents is sometimes limited by their nephrotoxic side effects. Chemotherapy-related kidney damage often results in inadequate cancer treatment because renal dysfunction requires the clinician to reduce the dose of chemotherapy to avoid further kidney damage. Kidney damage can also cause other adverse complications such as water and nitrogenous waste retention, electrolyte disturbance, decreased immunity, etc. The development of effective drug carriers, the reduction of the inherent adverse reactions of chemotherapeutic drugs and the improvement of the treatment effect become one of the key problems for treating cancers. Currently, drug nanocarriers, including polymersomes, micelles, polymeric nanoparticles, inorganic nanoparticles and hybrid porous solids, have been developed to improve drug accumulation in tumor regions for enhanced permeability and retention effects. Among them, nano Metal Organic Frameworks (MOFs) are attracting attention as drug nanocarriers with high drug loading, high pore volume, large surface area and adjustable pore size within the framework.
Pemetrexed (MTA) is an antifolate preparation structurally containing a pyrrole pyrimidine group as a core, and inhibits cell replication by destroying normal folate-dependent metabolic processes in cells, thereby inhibiting tumor growth; however, the dose of pemetrexed that can enter cancer cells is limited, which limits the anticancer effect of pemetrexed.
5-aminolevulinic acid (ALA) is an endogenous biochemical substance and generates protoporphyrin IX (PP IX) with strong photosensitivity through delta-aminolevulinic acid dehydratase and a series of enzymatic actions, and exogenous 5-aminolevulinic acid can bypass a feedback inhibition system of free hemoglobin concentration in a human body to enable cells to synthesize and gather enough protoporphyrin IX; irradiating the cancer tissue containing PP IX by using light of 420-640 nm to generate singlet oxygen in the energy transfer process, wherein the singlet oxygen can damage cancer cells when the singlet oxygen is gathered to a certain concentration; on the other hand, after light irradiation, protoporphyrin IX is excited to directly act with biological molecules or energy is transferred to oxygen and water to form free radicals, and a series of chain reactions of the biological molecules are caused through the free radicals to cause cancer cells to die, so that the anti-cancer effect is achieved, however, 5-aminolevulinic acid can not only destroy cancer tissues, but also has no selective damage to normal tissues around the cancer tissues, and has side effects on human bodies.
In view of the above, it would be desirable to develop a compound that can deliver pemetrexed and 5-aminolevulinic acid directly into cancer cells.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a preparation method of a zirconium metal organic framework compound aiming at the defects in the prior art, the preparation method has the advantages of easily available raw materials, simple process, low cost and the like, and the compound prepared by the preparation method can effectively improve the cell delivery capacity of an anticancer drug and enhance the anticancer effect of the anticancer drug.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a preparation method of a zirconium metal organic framework compound comprises the following steps:
1) preparation of UIO-66-NH2: dissolving zirconium chloride in N, N-dimethylformamide to obtain a solution A; dissolving amino terephthalic acid in N, N-dimethylformamide to obtain a solution B; putting the solution A, the solution B and acetic acid into a reaction bottle, uniformly mixing, putting the reaction bottle into an oven at the temperature of 80-100 ℃ for 10-14h, and cooling to room temperature to obtain a suspension C; centrifuging the suspension C for 10-20min at the rotating speed of 12000-14000 r/min, discarding the supernatant to obtain a precipitate D, cleaning the precipitate D with ethanol for 3 times, soaking the precipitate D with methanol for three days, replacing new methanol every 24 hours during the soaking period, and finally drying the precipitate in an oven at the temperature of 70-90 ℃ overnight to obtain UIO-66-NH2Powder;
2) system for makingPreparation of Compound E: the UIO-66-NH prepared by the step 1)2Ultrasonically dispersing the powder in water, then adding 5-carboxyl fluorescein, 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and 1-hydroxybenzotriazole, uniformly mixing, and stirring for 14-18h under a dark condition to obtain a suspension F; centrifuging the suspension F for 10-20min at the rotating speed of 12000-14000 r/min, and discarding the supernatant to obtain a precipitate G; gradient-cleaning the precipitate G with mixed solution of dimethyl sulfoxide and water until the supernatant turns into colorless solution, repeatedly ultrasonically cleaning with ultrapure water for 5 times, and freeze drying to obtain orange yellow powder, i.e. compound E powder;
3) preparation of compound H: ultrasonically dispersing the compound E powder obtained in the step 2) in water to obtain a solution I; then dissolving 5-aminoketone valerate hydrochloride in water to obtain a solution J; then uniformly mixing the solution I and the solution J, stirring for 2-4 days under the condition of keeping out of the sun, centrifuging for 10-20min at the rotating speed of 12000-14000 r/min, and discarding the supernatant to obtain a precipitate K; washing the precipitate K with water for 2 times, and freeze-drying to obtain compound H powder;
4) preparation of compound L: dissolving pemetrexed disodium in water to obtain a pemetrexed disodium solution; dispersing the compound H powder obtained in the step 3) in water, and then respectively adding 0.1 mL, 0.25 mL, 0.5 mL, 0.75 mL and 1 mL of pemetrexed disodium solution to obtain a mixed solution M; adding water into the mixed solution M to change the volume of the mixed solution M into 5mL to obtain a suspension N; stirring the suspension N for 8-12min, centrifuging for 5-15min at the rotating speed of 12000-14000 r/min, and removing the supernatant to obtain a precipitate O; washing the precipitate with water for 3 times, collecting supernatant obtained after each centrifugation, and freeze-drying to obtain compound L powder.
As a preferred embodiment, step 4) prepares compound P: dispersing the compound H powder obtained in the step 3) in N, N-dimethylformamide, adding pemetrexed, stirring for 1-2min, then adding a zirconium chloride aqueous solution, heating for 1-2min at the temperature of 90-110 ℃, stirring violently in the heating process, centrifuging for 5-15min at the rotating speed of 12000-14000 r/min, and removing the supernatant to obtain a precipitate Q; the precipitate Q was washed with water 3 times, cooled and dried to obtain compound P powder.
As a preferable mode, the solid-to-liquid ratio of zirconium chloride to N, N-dimethylformamide in the step 1) is 7:1, the solid-to-liquid ratio of aminoterephthalic acid to N, N-dimethylformamide is 55:1, the volume ratio between the solution A and the solution B is 3:1, and the ratio of the volume of acetic acid to the total volume of the solution A and the solution B is 1: 20.
As a preferred embodiment, UIO-66-NH is used in step 2)2The solid-liquid ratio of the powder to the water is 2:1, 5-carboxyl fluorescein, the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the 1-hydroxybenzotriazole is 1:1:1, 5-carboxyl fluorescein to UIO-66-NH2The mass ratio of the powder is 1:5, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to UIO-66-NH2The mass ratio of the powder is 1:5, 1-hydroxybenzotriazole to UIO-66-NH2The mass ratio of the powders was 1: 5.
As a preferable embodiment, the solid-to-liquid ratio of compound E powder to water in step 3) is 5: the solid-to-liquid ratio of the 3, 5-aminolevulinic acid hydrochloride to the water is 25: 2.
As a preferred embodiment, the compound E described in step 2) is UIO-66-NH-FAM and the compound H described in step 3) is ALA/UIO-66-NH-FAM.
As a preferable scheme, the concentration of the pemetrexed disodium solution in the step 4) is 10mg/mL, the solid-to-liquid ratio of the compound H powder to water is 5:1, and the volume of the water for washing the precipitate O is 4 mL.
Preferably, the solid-to-liquid ratio of the compound H powder to the N, N-dimethylformamide in the step 4) is 1:2, the mass ratio of the pemetrexed to the compound H powder is 1:2, the concentration of the zirconium chloride aqueous solution is 25mg/mL, and the volume ratio of the zirconium chloride aqueous solution to the N, N-dimethylformamide is 1: 50.
As a preferable scheme, the compound L is ALA/UIO-66-NH-FAM/MTA, and the ALA/UIO-66-NH-FAM/MTA is applied to the aspects of targeted delivery, control and release of anticancer drugs to enhance the anticancer effect of the anticancer drugs.
As a preferable scheme, the compound P is ALA/UIO-66-NH-FAM/CP1, and the ALA/UIO-66-NH-FAM/CP1 is applied to the aspects of targeted delivery, control and release of anticancer drugs and enhances the anticancer effect of the anticancer drugs.
The invention has the beneficial effects that: the preparation method has the advantages of easily obtained raw materials, simple process, low cost and the like; the compound prepared by the preparation method can deliver pemetrexed and 5-aminolevulinic acid to cancer cells in a targeted manner, effectively improve the delivery capacity of the cancer cells of the pemetrexed and 5-aminolevulinic acid, and enhance the anticancer effect.
Drawings
FIG. 1 shows UIO-66-NH2Simulated, UIO-66-NH2UIO-66-NH-FAM and ALA/UIO-66-NH-FAM;
FIG. 2 is UIO-66-NH2X-ray diffraction patterns of CP1, ALA/UIO-66-NH-FAM/FA, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP 1;
FIG. 3 is UIO-66-NH2UIO-66-NH-FAM, 5-aminolevulinic acid (ALA), 5-carboxyfluorescein (5-FAM), UIO-66-NH-FAM, and ALA/UIO-66-NH-FAM;
FIG. 4 is an infrared spectrum of Folic Acid (FA), pemetrexed (MTA), CP1, ALA/UIO-66-NH-FAM/FA, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP 1;
FIG. 5 is a thermogravimetric plot of 5-aminolevulinic acid (ALA), Folic Acid (FA), pemetrexed (MTA), 5-carboxyfluorescein (5-FAM), UIO-66-NH-FAM and ALA/UIO-66-NH-FAM;
FIG. 6 shows CP1, UIO-66-NH2Thermogravimetric analysis of ALA/UIO-66-NH-FAM/FA, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP 1;
FIG. 7 shows UIO-66-NH2And TEM images of ALA/UIO-66-NH-FAM/CP 1;
FIG. 8 is an SEM image of CP 1;
FIG. 9 is UIO-66-NH2SEM picture of (1);
FIG. 10 is an SEM image of UIO-66-NH-FAM;
FIG. 11 is an SEM photograph of ALA/UIO-66-NH-FAM/FA;
FIG. 12 is an SEM picture of ALA/UIO-66-NH-FAM/MTA;
FIG. 13 is an SEM photograph of ALA/UIO-66-NH-FAM/CP 1;
FIG. 14 is UIO-66-NH2The particle size distribution map of (a);
FIG. 15 is a graph showing the particle size distribution of ALA/UIO-66-NH-FAM/FA;
FIG. 16 is a graph showing the particle size distribution of ALA/UIO-66-NH-FAM/MTA
FIG. 17 is a particle size distribution diagram of CP 1;
FIG. 18 is a graph showing the particle size distribution of ALA/UIO-66-NH-FAM/CP 1;
FIG. 19 is UIO-66-NH2And a nitrogen adsorption-desorption profile of ALA/UIO-66-NH-FAM;
FIG. 20 is a graph showing nitrogen adsorption-desorption profiles of ALA/UIO-66-NH-FAM/FA, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP 1;
FIG. 21 is a graph of cumulative release rate of pemetrexed (MTA) versus time for CP 1;
FIG. 22 is a graph of the cumulative release rate of pemetrexed (MTA) versus time for ALA/UIO-66-NH-FAM/MTA;
FIG. 23 is a graph showing the fluorescence of A549 cells, Hela cells and KB cells after incubation for 30min in a medium containing ALA/UIO-66-NH-FAM, ALA/UIO-66-NH-FAM/FA and ALA/UIO-66-NH-FAM/MTA, respectively;
FIG. 24 is a graph showing the fluorescence of A549 cells, Hela cells and KB cells after incubating the cells for 60min in a medium containing ALA/UIO-66-NH-FAM, ALA/UIO-66-NH-FAM/FA and ALA/UIO-66-NH-FAM/MTA, respectively;
FIG. 25 is a graph showing the fluorescence of A549 cells, Hela cells and KB cells after incubation for 200min in a medium containing ALA/UIO-66-NH-FAM, ALA/UIO-66-NH-FAM/FA and ALA/UIO-66-NH-FAM/MTA, respectively;
FIG. 26 is a graph showing fluorescence intensity after incubating A549 cells in a medium, a medium containing ALA/UIO-66-NH-FAM/FA, and a medium containing ALA/UIO-66-NH-FAM/MTA, respectively, for 30 min;
FIG. 27 is a graph showing fluorescence intensity after incubating Hela cells in a medium, a medium containing ALA/UIO-66-NH-FAM/FA, and a medium containing ALA/UIO-66-NH-FAM/MTA, respectively, for 30 min;
FIG. 28 is a graph showing fluorescence intensity after incubation of KB cells for 30min in medium, medium containing ALA/UIO-66-NH-FAM/FA, and medium containing ALA/UIO-66-NH-FAM/MTA, respectively;
FIG. 29 is a graph showing fluorescence intensity curves of A549 cells, Hela cells and KB cells after incubation for 30min in a medium containing ALA/UIO-66-NH-FAM/MTA;
FIG. 30 is a graph showing fluorescence intensity after incubating A549 cells in a medium, a medium containing ALA/UIO-66-NH-FAM/FA, and a medium containing ALA/UIO-66-NH-FAM/MTA, respectively, for 120 min;
FIG. 31 is a graph showing fluorescence intensity after incubating Hela cells in culture medium, in culture medium containing ALA/UIO-66-NH-FAM/FA, and in culture medium containing ALA/UIO-66-NH-FAM/MTA, respectively, for 120 min;
FIG. 32 is a graph showing fluorescence intensity after incubation of KB cells for 120min in medium, medium containing ALA/UIO-66-NH-FAM/FA, and medium containing ALA/UIO-66-NH-FAM/MTA, respectively;
FIG. 33 is a graph showing fluorescence intensity curves of A549 cells, Hela cells and KB cells after incubation for 120min in a medium containing ALA/UIO-66-NH-FAM/MTA;
FIG. 34 is a graph showing the cell activities of A549 cells, Hela cells and KB cells after 24h and 48h of incubation in the absence of light at different concentrations of MTA, CP1, ALA/UIO-66-NH-FAM/FA, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP 1;
FIG. 35 is a graph showing the cell viability of A549 cells, Hela cells and KB cells after incubation for 24h and 48h with laser radiation having a wavelength of 660 nm at various concentrations of ALA, ALA/UIO-66-NH-FAM/FA, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP 1;
FIG. 36 is a graph showing the cell activity of LO2 cells after 24h, 48h, and 72h incubation in different concentrations of ALA, ALA/UIO-66-NH-FAM/FA, ALA/UIO-66-NH-FAM/MTA, and ALA/UIO-66-NH-FAM/CP 1.
Detailed Description
The structural and operational principles of the present invention are explained in further detail below with reference to the accompanying drawings.
Example 1
Preparation of zirconium metal organic framework compound ALA/UIO-66-NH-FAM/MTA
1) Preparation of UIO-66-NH2: dissolving 210 mg of zirconium chloride in 30 mL of N, N-dimethylformamide to obtain a solution A; dissolving 550 mg of aminoterephthalic acid in 10 mL of N, N-dimethylformamide to obtain a solution B; putting 3 mL of the solution A, 1 mL of the solution B and 200 muL of acetic acid into a reaction bottle, uniformly mixing, putting the reaction bottle into an oven at the temperature of 80 ℃ for 14h, and cooling to room temperature to obtain a suspension C; centrifuging the suspension C at 12000r/min for 20min, removing supernatant to obtain precipitate D, cleaning the precipitate D with ethanol for 3 times, soaking in methanol for three days, replacing new methanol every 24 hr during soaking, and drying in an oven at 70 deg.C overnight to obtain UIO-66-NH2Powder;
2) preparation of UIO-66-NH-FAM: 100mg of UIO-66-NH prepared in step 1)2Ultrasonically dispersing the powder in 50 mL of water, then adding 20 mg of 5-carboxyl fluorescein, 20 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 20 mg of 1-hydroxybenzotriazole, uniformly mixing, and stirring for 14h under a dark condition to obtain a suspension F; centrifuging the suspension F for 20min at the rotating speed of 12000r/min, and removing the supernatant to obtain a precipitate G; gradient-cleaning the precipitate G with mixed solution of dimethyl sulfoxide and water until the supernatant turns into colorless solution, repeatedly ultrasonically cleaning with ultrapure water for 5 times, and freeze drying to obtain orange yellow powder, i.e. UIO-66-NH-FAM powder;
3) preparation of ALA/UIO-66-NH-FAM: ultrasonically dispersing 200mg of UIO-66-NH-FAM powder obtained in the step 2) in 30 mL of water to obtain a solution I; then 250mg of 5-aminolevulinic acid hydrochloride was dissolved in 20 mL of water to give solution J; then uniformly mixing the solution I and the solution J, stirring for 2 days under the condition of keeping out of the sun, centrifuging for 10min under the condition that the rotating speed is 14000r/min, and discarding the supernatant to obtain a precipitate K; washing the precipitate K with water for 2 times, and freeze drying to obtain ALA/UIO-66-NH-FAM powder;
4) preparation of ALA/UIO-66-NH-FAM/MTA: dissolving 200mg of pemetrexed disodium in 20 mL of water to obtain a pemetrexed disodium solution; dispersing 10mg of ALA/UIO-66-NH-FAM powder obtained in the step 3) in 2 mL of water, and then respectively adding 0.1 mL, 0.25 mL, 0.5 mL, 0.75 mL and 1 mL of pemetrexed disodium solution with the concentration of 10mg/mL to obtain a mixed solution M; adding water into the mixed solution M to change the volume of the mixed solution M into 5mL to obtain a suspension N; stirring the suspension N for 8min, centrifuging for 5min at the rotation speed of 14000r/min, and removing the supernatant to obtain a precipitate O; washing the precipitate with water for 3 times, collecting supernatant obtained after each centrifugation, and freeze-drying to obtain ALA/UIO-66-NH-FAM/MTA powder.
Example 2
Preparation of zirconium metal organic framework compound ALA/UIO-66-NH-FAM/MTA
1) Preparation of UIO-66-NH2: dissolving 210 mg of zirconium chloride in 30 mL of N, N-dimethylformamide to obtain a solution A; dissolving 550 mg of aminoterephthalic acid in 10 mL of N, N-dimethylformamide to obtain a solution B; putting 3 mL of the solution A, 1 mL of the solution B and 200 muL of acetic acid into a reaction bottle, uniformly mixing, putting the reaction bottle into an oven at the temperature of 100 ℃ for 10 hours, and cooling to room temperature to obtain a suspension C; centrifuging the suspension C at 14000r/min for 10min, removing supernatant to obtain precipitate D, cleaning the precipitate D with ethanol for 3 times, soaking in methanol for three days, replacing new methanol every 24h during soaking, and drying in an oven at 90 deg.C overnight to obtain UIO-66-NH2Powder;
2) preparation of UIO-66-NH-FAM: 100mg of UIO-66-NH prepared in step 1)2Ultrasonically dispersing the powder in 50 mL of water, then adding 20 mg of 5-carboxyl fluorescein, 20 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 20 mg of 1-hydroxybenzotriazole, uniformly mixing, and stirring for 18h under a dark condition to obtain a suspension F; centrifuging the suspension F for 10min at the rotation speed of 14000r/min, and removing the supernatant to obtain a precipitate G; by mixing dimethyl sulfoxide with waterCleaning the precipitate G with solution gradient until the supernatant turns into colorless solution, repeatedly ultrasonically cleaning with ultrapure water for 5 times, and freeze drying to obtain orange yellow powder, i.e. UIO-66-NH-FAM powder;
3) preparation of ALA/UIO-66-NH-FAM: ultrasonically dispersing 200mg of UIO-66-NH-FAM powder obtained in the step 2) in 30 mL of water to obtain a solution I; then 250mg of 5-aminolevulinic acid hydrochloride was dissolved in 20 mL of water to give solution J; then uniformly mixing the solution I and the solution J, stirring for 4 days under the condition of keeping out of the sun, centrifuging for 20min under the condition that the rotating speed is 12000r/min, and discarding supernatant liquid to obtain a precipitate K; washing the precipitate K with water for 2 times, and freeze drying to obtain ALA/UIO-66-NH-FAM powder;
4) preparation of ALA/UIO-66-NH-FAM/MTA: dissolving 200mg of pemetrexed disodium in 20 mL of water to obtain a pemetrexed disodium solution; dispersing 10mg of ALA/UIO-66-NH-FAM powder obtained in the step 3) in 2 mL of water, and then respectively adding 0.1 mL, 0.25 mL, 0.5 mL, 0.75 mL and 1 mL of pemetrexed disodium solution with the concentration of 10mg/mL to obtain a mixed solution M; adding water into the mixed solution M to change the volume of the mixed solution M into 5mL to obtain a suspension N; stirring the suspension N for 12min, centrifuging for 15min at the rotation speed of 13000r/min, and removing the supernatant to obtain a precipitate O; washing the precipitate with water for 3 times, collecting supernatant obtained after each centrifugation, and freeze-drying to obtain ALA/UIO-66-NH-FAM/MTA powder.
Example 3
Preparation of zirconium metal organic framework compound ALA/UIO-66-NH-FAM/MTA
1) Preparation of UIO-66-NH2: dissolving 210 mg of zirconium chloride in 30 mL of N, N-dimethylformamide to obtain a solution A; dissolving 550 mg of aminoterephthalic acid in 10 mL of N, N-dimethylformamide to obtain a solution B; putting 3 mL of the solution A, 1 mL of the solution B and 200 muL of acetic acid into a reaction bottle, uniformly mixing, putting the reaction bottle into an oven at the temperature of 90 ℃ for 12 hours, and cooling to room temperature to obtain a suspension C; centrifuging the suspension C at 13000r/min for 15min, removing supernatant to obtain precipitate D, cleaning the precipitate D with ethanol for 3 times, soaking in methanol for three days, and replacing every 24h during soakingFresh methanol, and drying at 80 deg.C overnight to obtain UIO-66-NH2Powder;
2) preparation of UIO-66-NH-FAM: 100mg of UIO-66-NH prepared in step 1)2Ultrasonically dispersing the powder in 50 mL of water, then adding 20 mg of 5-carboxyl fluorescein, 20 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 20 mg of 1-hydroxybenzotriazole, uniformly mixing, and stirring for 16h under a dark condition to obtain a suspension F; centrifuging the suspension F for 15min at the rotation speed of 13000r/min, and removing the supernatant to obtain a precipitate G; gradient-cleaning the precipitate G with mixed solution of dimethyl sulfoxide and water until the supernatant turns into colorless solution, repeatedly ultrasonically cleaning with ultrapure water for 5 times, and freeze drying to obtain orange yellow powder, i.e. UIO-66-NH-FAM powder;
3) preparation of ALA/UIO-66-NH-FAM: ultrasonically dispersing 200mg of UIO-66-NH-FAM powder obtained in the step 2) in 30 mL of water to obtain a solution I; then 250mg of 5-aminolevulinic acid hydrochloride was dissolved in 20 mL of water to give solution J; then uniformly mixing the solution I and the solution J, stirring for 3 days under the condition of keeping out of the sun, centrifuging for 15min under the condition that the rotating speed is 13000r/min, and discarding the supernatant to obtain a precipitate K; washing the precipitate K with water for 2 times, and freeze drying to obtain ALA/UIO-66-NH-FAM powder;
4) preparation of ALA/UIO-66-NH-FAM/MTA: dissolving 200mg of pemetrexed disodium in 20 mL of water to obtain a pemetrexed disodium solution; dispersing 10mg of ALA/UIO-66-NH-FAM powder obtained in the step 3) in 2 mL of water, and then respectively adding 0.1 mL, 0.25 mL, 0.5 mL, 0.75 mL and 1 mL of pemetrexed disodium solution with the concentration of 10mg/mL to obtain a mixed solution M; adding water into the mixed solution M to change the volume of the mixed solution M into 5mL to obtain a suspension N; stirring the suspension N for 10min, centrifuging for 10min at the rotation speed of 12000r/min, and removing the supernatant to obtain a precipitate O; washing the precipitate with water for 3 times, collecting supernatant obtained after each centrifugation, and freeze-drying to obtain ALA/UIO-66-NH-FAM/MTA powder.
Example 4
Preparation of zirconium metal organic framework compound ALA/UIO-66-NH-FAM/CP1
1) Preparation of UIO-66-NH2: dissolving 210 mg of zirconium chloride in 30 mL of N, N-dimethylformamide to obtain a solution A; dissolving 550 mg of aminoterephthalic acid in 10 mL of N, N-dimethylformamide to obtain a solution B; putting 3 mL of the solution A, 1 mL of the solution B and 200 muL of acetic acid into a reaction bottle, uniformly mixing, putting the reaction bottle into an oven at the temperature of 80 ℃ for 14h, and cooling to room temperature to obtain a suspension C; centrifuging the suspension C at 12000r/min for 20min, removing supernatant to obtain precipitate D, cleaning the precipitate D with ethanol for 3 times, soaking in methanol for three days, replacing new methanol every 24 hr during soaking, and drying in an oven at 70 deg.C overnight to obtain UIO-66-NH2Powder;
2) preparation of UIO-66-NH-FAM: 100mg of UIO-66-NH prepared in step 1)2Ultrasonically dispersing the powder in 50 mL of water, then adding 20 mg of 5-carboxyl fluorescein, 20 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 20 mg of 1-hydroxybenzotriazole, uniformly mixing, and stirring for 14h under a dark condition to obtain a suspension F; centrifuging the suspension F for 20min at the rotating speed of 12000r/min, and removing the supernatant to obtain a precipitate G; gradient-cleaning the precipitate G with mixed solution of dimethyl sulfoxide and water until the supernatant turns into colorless solution, repeatedly ultrasonically cleaning with ultrapure water for 5 times, and freeze drying to obtain orange yellow powder, i.e. UIO-66-NH-FAM powder;
3) preparation of ALA/UIO-66-NH-FAM: ultrasonically dispersing 200mg of UIO-66-NH-FAM powder obtained in the step 2) in 30 mL of water to obtain a solution I; then 250mg of 5-aminolevulinic acid hydrochloride was dissolved in 20 mL of water to give solution J; then uniformly mixing the solution I and the solution J, stirring for 2 days under the condition of keeping out of the sun, centrifuging for 10min under the condition that the rotating speed is 14000r/min, and discarding the supernatant to obtain a precipitate K; washing the precipitate K with water for 2 times, and freeze drying to obtain ALA/UIO-66-NH-FAM powder;
4) preparation of ALA/UIO-66-NH-FAM/CP 1: dispersing 5mg of ALA/UIO-66-NH-FAM powder obtained in the step 3) in 10 mL of N, N-dimethylformamide, adding 10mg of pemetrexed, stirring for 2min, then adding 200uL of a zirconium chloride aqueous solution with the concentration of 25mg/mL, heating for 2min at the temperature of 90 ℃, violently stirring in the heating process, centrifuging for 5min at the rotation speed of 14000r/min, and discarding the supernatant to obtain a precipitate Q; washing the precipitate Q3 times with water, cooling and drying to obtain ALA/UIO-66-NH-FAM/CP1 powder.
Example 5
Preparation of zirconium metal organic framework compound ALA/UIO-66-NH-FAM/CP1
1) Preparation of UIO-66-NH2: dissolving 210 mg of zirconium chloride in 30 mL of N, N-dimethylformamide to obtain a solution A; dissolving 550 mg of aminoterephthalic acid in 10 mL of N, N-dimethylformamide to obtain a solution B; putting 3 mL of the solution A, 1 mL of the solution B and 200 muL of acetic acid into a reaction bottle, uniformly mixing, putting the reaction bottle into an oven at the temperature of 100 ℃ for 10 hours, and cooling to room temperature to obtain a suspension C; centrifuging the suspension C at 14000r/min for 10min, removing supernatant to obtain precipitate D, cleaning the precipitate D with ethanol for 3 times, soaking in methanol for three days, replacing new methanol every 24h during soaking, and drying in an oven at 90 deg.C overnight to obtain UIO-66-NH2Powder;
2) preparation of UIO-66-NH-FAM: 100mg of UIO-66-NH prepared in step 1)2Ultrasonically dispersing the powder in 50 mL of water, then adding 20 mg of 5-carboxyl fluorescein, 20 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 20 mg of 1-hydroxybenzotriazole, uniformly mixing, and stirring for 18h under a dark condition to obtain a suspension F; centrifuging the suspension F for 10min at the rotation speed of 14000r/min, and removing the supernatant to obtain a precipitate G; gradient-cleaning the precipitate G with mixed solution of dimethyl sulfoxide and water until the supernatant turns into colorless solution, repeatedly ultrasonically cleaning with ultrapure water for 5 times, and freeze drying to obtain orange yellow powder, i.e. UIO-66-NH-FAM powder;
3) preparation of ALA/UIO-66-NH-FAM: ultrasonically dispersing 200mg of UIO-66-NH-FAM powder obtained in the step 2) in 30 mL of water to obtain a solution I; then 250mg of 5-aminolevulinic acid hydrochloride was dissolved in 20 mL of water to give solution J; then uniformly mixing the solution I and the solution J, stirring for 4 days under the condition of keeping out of the sun, centrifuging for 20min under the condition that the rotating speed is 12000r/min, and discarding supernatant liquid to obtain a precipitate K; washing the precipitate K with water for 2 times, and freeze drying to obtain ALA/UIO-66-NH-FAM powder;
4) preparation of ALA/UIO-66-NH-FAM/CP 1: dispersing 5mg of ALA/UIO-66-NH-FAM powder obtained in the step 3) in 10 mL of N, N-dimethylformamide, adding 10mg of pemetrexed, stirring for 1.5min, then adding 200uL of a zirconium chloride aqueous solution with the concentration of 25mg/mL, heating for 1.5min at the temperature of 110 ℃, stirring vigorously in the heating process, centrifuging for 10min at the rotating speed of 12000r/min, and discarding the supernatant to obtain a precipitate Q; washing the precipitate Q3 times with water, cooling and drying to obtain ALA/UIO-66-NH-FAM/CP1 powder.
Example 6
Preparation of zirconium metal organic framework compound ALA/UIO-66-NH-FAM/CP1
1) Preparation of UIO-66-NH2: dissolving 210 mg of zirconium chloride in 30 mL of N, N-dimethylformamide to obtain a solution A; dissolving 550 mg of aminoterephthalic acid in 10 mL of N, N-dimethylformamide to obtain a solution B; putting 3 mL of the solution A, 1 mL of the solution B and 200 muL of acetic acid into a reaction bottle, uniformly mixing, putting the reaction bottle into an oven at the temperature of 90 ℃ for 12 hours, and cooling to room temperature to obtain a suspension C; centrifuging the suspension C at 13000r/min for 15min, removing supernatant to obtain precipitate D, cleaning the precipitate D with ethanol for 3 times, soaking in methanol for three days, replacing new methanol every 24h during soaking, and drying in an oven at 80 deg.C overnight to obtain UIO-66-NH2Powder;
2) preparation of UIO-66-NH-FAM: 100mg of UIO-66-NH prepared in step 1)2Ultrasonically dispersing the powder in 50 mL of water, then adding 20 mg of 5-carboxyl fluorescein, 20 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 20 mg of 1-hydroxybenzotriazole, uniformly mixing, and stirring for 16h under a dark condition to obtain a suspension F; centrifuging the suspension F for 15min at the rotation speed of 13000r/min, and removing the supernatant to obtain a precipitate G; gradient washing the precipitate G with a mixture of dimethyl sulfoxide and water until the supernatant becomes a colorless solution, and purifying with ultrapure waterRepeatedly ultrasonically cleaning with water for 5 times, and freeze drying to obtain orange powder, i.e. UIO-66-NH-FAM powder;
3) preparation of ALA/UIO-66-NH-FAM: ultrasonically dispersing 200mg of UIO-66-NH-FAM powder obtained in the step 2) in 30 mL of water to obtain a solution I; then 250mg of 5-aminolevulinic acid hydrochloride was dissolved in 20 mL of water to give solution J; then uniformly mixing the solution I and the solution J, stirring for 3 days under the condition of keeping out of the sun, centrifuging for 15min under the condition that the rotating speed is 13000r/min, and discarding the supernatant to obtain a precipitate K; washing the precipitate K with water for 2 times, and freeze drying to obtain ALA/UIO-66-NH-FAM powder;
4) preparation of ALA/UIO-66-NH-FAM/CP 1: dispersing 5mg of ALA/UIO-66-NH-FAM powder obtained in the step 3) in 10 mL of N, N-dimethylformamide, adding 10mg of pemetrexed, stirring for 1min, then adding 200uL of a zirconium chloride aqueous solution with the concentration of 25mg/mL, heating for 1min at the temperature of 100 ℃, violently stirring in the heating process, centrifuging for 15min at the rotation speed of 13000r/min, and discarding the supernatant to obtain a precipitate Q; washing the precipitate Q3 times with water, cooling and drying to obtain ALA/UIO-66-NH-FAM/CP1 powder.
In the above examples, example 3 and example 6 were the most preferred embodiments, and the zirconium organometallic framework compound ALA/UIO-66-NH-FAM/MTA used in the experiments in the following examples was prepared from example 3, while the zirconium organometallic framework compound ALA/UIO-66-NH-FAM/CP1 was prepared from example 6.
Comparative example 1
Preparation of Compound CP1
1) Weighing 10mg of pemetrexed, dissolving the pemetrexed in 10 ml of N, N-dimethylformamide, adding 250 uL of 20 mg/ml zirconium chloride aqueous solution, heating in a water bath at 100 ℃, stirring for 1min, cooling to room temperature, centrifuging at a rotation speed of 12000r/min for 10min, and removing supernatant to obtain a precipitate;
2) washing the precipitate obtained in step 1) with ethanol for 3 times, and freeze-drying to obtain CP1 powder.
Comparative example 2
Preparation of zirconium metal organic framework compound ALA/UIO-66-NH-FAM/FA
1) Preparation of UIO-66-NH2: dissolving 210 mg of zirconium chloride in 30 mL of N, N-dimethylformamide to obtain a solution A; dissolving 550 mg of aminoterephthalic acid in 10 mL of N, N-dimethylformamide to obtain a solution B; putting 3 mL of the solution A, 1 mL of the solution B and 200 muL of acetic acid into a reaction bottle, uniformly mixing, putting the reaction bottle into an oven at the temperature of 90 ℃ for 12 hours, and cooling to room temperature to obtain a suspension C; centrifuging the suspension C at 13000r/min for 15min, removing supernatant to obtain precipitate D, cleaning the precipitate D with ethanol for 3 times, soaking in methanol for three days, replacing new methanol every 24h during soaking, and drying in an oven at 80 deg.C overnight to obtain UIO-66-NH2Powder;
2) preparation of UIO-66-NH-FAM: 100mg of UIO-66-NH prepared in step 1)2Ultrasonically dispersing the powder in 50 mL of water, then adding 20 mg of 5-carboxyl fluorescein, 20 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 20 mg of 1-hydroxybenzotriazole, uniformly mixing, and stirring for 16h under a dark condition to obtain a suspension F; centrifuging the suspension F for 15min at the rotation speed of 13000r/min, and removing the supernatant to obtain a precipitate G; gradient-cleaning the precipitate G with mixed solution of dimethyl sulfoxide and water until the supernatant turns into colorless solution, repeatedly ultrasonically cleaning with ultrapure water for 5 times, and freeze drying to obtain orange yellow powder, i.e. UIO-66-NH-FAM powder;
3) preparation of ALA/UIO-66-NH-FAM: ultrasonically dispersing 200mg of UIO-66-NH-FAM powder obtained in the step 2) in 30 mL of water to obtain a solution I; then 250mg of 5-aminolevulinic acid hydrochloride was dissolved in 20 mL of water to give solution J; then uniformly mixing the solution I and the solution J, stirring for 3 days under the condition of keeping out of the sun, centrifuging for 15min under the condition that the rotating speed is 13000r/min, and discarding the supernatant to obtain a precipitate K; washing the precipitate K with water for 2 times, and freeze drying to obtain ALA/UIO-66-NH-FAM powder;
4) preparation of ALA/UIO-66-NH-FAM/FA: weighing 200mg of folic acid, placing the folic acid in a beaker, adding a proper amount of sodium hydroxide solution with the concentration of 0.1 mol/L for dissolution to obtain a solution R, transferring the solution R into a 20 mL volumetric flask for constant volume, and preparing a sodium folate solution with the concentration of 10 mg/mL. Dispersing 10mg of ALA/UIO-66-NH-FAM powder obtained in the step 3) in 2 mL of water, then respectively adding 10mg/mL of sodium folate solutions of 0.1 mL, 0.25 mL, 0.5 mL, 0.75 mL and 1 mL, and adding water to make the volume of the sodium folate solution to be 5mL to obtain a suspension S; stirring the obtained suspension S for 10min, and centrifuging at the rotating speed of 12000r/min for 10min to obtain a precipitate T; the precipitate T was washed 3 times with 4mL of water while the supernatant obtained after each centrifugation was collected and lyophilized to give ALA/UIO-66-NH-FAM/FA powder.
The experimental compound CP1 was prepared from comparative example 1, and the compound ALA/UIO-66-NH-FAM/FA was prepared from comparative example 2 in the following examples.
Example 7
X-ray diffraction experiments
As shown in FIG. 1, UIO-66-NH2Simulated, UIO-66-NH2UIO-66-NH-FAM and ALA/UIO-66-NH-FAM;
as shown in FIG. 2, UIO-66-NH2X-ray diffraction patterns of, CP1, ALA/UIO-66-NH-FAM/FA, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP 1.
Example 8
Infrared spectroscopy experiment
As shown in FIG. 3, UIO-66-NH2UIO-66-NH-FAM, 5-aminolevulinic acid (ALA), 5-carboxyfluorescein (5-FAM), UIO-66-NH-FAM, and ALA/UIO-66-NH-FAM;
as shown in FIG. 4, the infrared spectra of Folic Acid (FA), pemetrexed (MTA), CP1, ALA/UIO-66-NH-FAM/FA, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP 1.
Example 9
Thermogravimetric analysis
As shown in FIG. 5, thermogravimetric analysis of 5-aminolevulinic acid (ALA), Folic Acid (FA), pemetrexed (MTA), 5-carboxyfluorescein (5-FAM), UIO-66-NH-FAM and ALA/UIO-66-NH-FAM;
as shown in FIG. 6, CP1, UIO-66-NH2Thermogravimetric analysis of ALA/UIO-66-NH-FAM/FA, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP 1.
Among them, 5-aminolevulinic acid (ALA), 5-carboxyfluorescein (5-FAM), Folic Acid (FA), and pemetrexed (MTA) in examples 8 and 9 were obtained from commercial sources, as manufactured by Sahn's chemical technology (Shanghai) Co., Ltd
Example 10
Shown in FIG. 7 as UIO-66-NH2And TEM image of ALA/UIO-66-NH-FAM/CP1, wherein A is UIO-66-NH2B is a TEM image of ALA/UIO-66-NH-FAM/CP 1.
Example 11
Shown in FIGS. 8-13 as CP1 and UIO-66-NH, respectively2UIO-66-NH-FAM, ALA/UIO-66-NH-FAM/FA, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP 1.
Example 12
Dynamic Scattering experiment
As can be seen from FIGS. 14 to 18, UIO-66-NH2The particle sizes of ALA/UIO-66-NH-FAM/FA and ALA/UIO-66-NH-FAM/MTA were smaller than those of CP1 and ALA/UIO-66-NH-FAM/CP 1.
Example 13
Nitrogen adsorption and desorption experiment
As can be seen from FIGS. 19 and 20, UIO-66-NH2The adsorption amount of nitrogen is highest, and the adsorption amount of ALA/UIO-66-NH-FAM on nitrogen is higher than that of UIO-66-NH2The adsorbed amount of nitrogen gas was small, and was larger than that of ALA/UIO-66-NH-FAM/FA, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP1, while that of ALA/UIO-66-NH-FAM/FA, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP1 was almost the same.
Example 14
Pemetrexed release assay
The experimental steps are as follows:
1) weighing 2 parts of 5mg CP1 powder, filling the two parts of 5mg CP1 powder into two dialysis bags, clamping two ends of each dialysis bag by a clamp, then respectively putting the two dialysis bags filled with the CP1 powder into a beaker filled with 250 mL of PBS buffer solution with pH =7.4 and a beaker filled with 250 mL of ultrapure water, then putting the two dialysis bags into a water bath kettle at the temperature of 37 ℃, stirring the two dialysis bags at the rotating speed of 300 r/min in a water bath process, respectively taking out 2 mL of solution in each dialysis bag at regular intervals, correspondingly adding the same amount of PBS buffer solution and the ultrapure water to obtain a first sample solution, measuring the absorbance of the first sample solution by using an ultraviolet spectrophotometer, calculating the release rate of the pemetrexed (MTA) according to the measured absorbance of the first sample solution, and drawing a relation curve of the cumulative release rate of the pemetrexed (MTA) and the time;
2) weighing 2 parts of 10mg ALA/UIO-66-NH-FAM/MTA powder, filling the two parts into two dialysis bags, clamping two ends of each dialysis bag by a clamp, then respectively putting the dialysis bags filled with the ALA/UIO-66-NH-FAM/MTA powder into a beaker filled with 250 mL PBS buffer solution with pH =7.4 and a beaker filled with 250 mL ultrapure water, then putting the two parts into a water bath kettle at the temperature of 37 ℃, stirring the two parts at the rotating speed of 300 r/min in the water bath process, respectively taking out 2 mL solutions from the dialysis bags at regular intervals, correspondingly adding the same amount of PBS buffer solution and the same amount of ultrapure water to obtain a second sample solution, measuring the absorbance of the second sample solution by an ultraviolet spectrophotometer, simultaneously taking ALA/UIO-66-NH-FAM as a reference to eliminate the influence of the absorbance after framework degradation, and then calculating the release rate of the pemetrexed (MTA) according to the measured absorbance of the second sample solution, and drawing a curve of the cumulative release rate of the pemetrexed (MTA) and the time.
The experimental results are as follows:
as can be seen from FIGS. 21 and 22, the release rate of pemetrexed (MTA) from ALA/UIO-66-NH-FAM/MTA was significantly faster than that from CP 1.
Example 15
Confocal fluorescence microscope
Preparation of the experiment:
cell preparation: a549 cells (human lung cancer cells), Hela cells (cervical cancer cells) and KB cells (nasopharyngeal cancer cells);
culture medium: 89% of folic acid-free RPMI1640 culture medium, 10% of fetal bovine serum and 1% of streptomycin qing mixed solution;
sample 1: adding ALA/UIO-66-NH-FAM solution on the basis of the culture medium to enable the concentration of ALA/UIO-66-NH-FAM in the culture medium to be 100 mug/mL;
sample 2: adding ALA/UIO-66-NH-FAM/FA solution on the basis of the culture medium to enable the concentration of ALA/UIO-66-NH-FAM/FA in the culture medium to be equal to the ALA/UIO-66-NH-FAM solution with the concentration of 100 mug/mL;
sample 3: adding ALA/UIO-66-NH-FAM/MTA solution on the basis of the culture medium to ensure that the concentration of ALA/UIO-66-NH-FAM/MTA in the culture medium is equal to that of the ALA/UIO-66-NH-FAM solution of 100 mug/mL.
The experimental steps are as follows:
when the cells grew well in the logarithmic growth phase, the cells were digested with trypsin, centrifuged to remove the supernatant, and prepared with a medium to a cell density of 2X 104Single cell suspension at 2X 10/mL4Inoculating cells into a confocal dish at the density of each hole, and culturing for 24 hours in an incubator at the temperature of 37 ℃; then respectively replacing the culture medium in the confocal dish inoculated with the cells with a sample 1, a sample 2 and a sample 3, respectively continuing incubation for 30min, 60min and 200min, sucking out the culture medium after the time is up, slightly washing the culture medium for 3 times by using PBS (phosphate buffer solution), adding 500mL of PBS to obtain a sample, and finally observing the fluorescence condition of the cells in the sample by using a confocal fluorescence microscope, wherein the experimental result is shown in figures 23-25.
The experimental results are as follows:
as is apparent from FIGS. 23 to 25, the entry ability of ALA/UIO-66-NH-FAM/MTA into A549 cells, Hela cells and KB cells was significantly higher than that of ALA/UIO-66-NH-FAM and ALA/UIO-66-NH-FAM/FA, while that of ALA/UIO-66-NH-FAM/FA into A549 cells, Hela cells and KB cells was higher than that of ALA/UIO-66-NH-FAM.
Example 16
Flow cytometry
Preparation of the experiment:
cell preparation: a549 cells (human lung cancer cells), Hela cells (cervical cancer cells) and KB cells (nasopharyngeal cancer cells);
culture medium: 89% of folic acid-free RPMI1640 culture medium, 10% of fetal bovine serum and 1% of streptomycin qing mixed solution;
ALA/UIO-66-NH-FAM-containing medium: adding ALA/UIO-66-NH-FAM solution on the basis of the culture medium to enable the concentration of ALA/UIO-66-NH-FAM in the culture medium to be 100 mug/mL;
ALA/UIO-66-NH-FAM/FA-containing medium: adding ALA/UIO-66-NH-FAM/FA solution on the basis of the culture medium to enable the concentration of ALA/UIO-66-NH-FAM/FA in the culture medium to be equal to the ALA/UIO-66-NH-FAM solution with the concentration of 100 mug/mL;
media containing ALA/UIO-66-NH-FAM/MTA: adding ALA/UIO-66-NH-FAM/MTA solution on the basis of the culture medium to ensure that the concentration of ALA/UIO-66-NH-FAM/MTA in the culture medium is equal to that of the ALA/UIO-66-NH-FAM solution of 100 mug/mL.
The experimental steps are as follows:
when the cells grew well in the logarithmic growth phase, the cells were digested with trypsin, centrifuged to remove the supernatant, and the cell density was 2X 10 in the medium5Single cell suspension at 4X 10/mL5Inoculating the cells into a 6-well plate at the density of each well, and then placing the plate in an incubator with the temperature condition of 37 ℃ for continuous culture for 24 hours; then respectively replacing the culture medium of the 6-well plate inoculated with the cells with a culture medium, a culture medium containing ALA/UIO-66-NH-FAM/FA and a culture medium containing ALA/UIO-66-NH-FAM/MTA, continuously incubating for 30min and 120min respectively, sucking out the culture medium after the time, flushing the culture medium by PBS buffer solution for 3 times, adding 300 mu L of trypsin free cells, centrifuging to obtain sample cells, dispersing the sample cells into 1 mL of PBS buffer solution to obtain a sample cell solution, and detecting the fluorescence intensity of the sample cell solution by using a flow cytometer, wherein the results are shown in tables 1 and 2, wherein the table 1 shows that A549 cells, Hela cells and KB cells are respectively in the culture medium, the culture medium containing ALA/UIO-66-NH-FAM, The average values of fluorescence intensity after incubation for 30min in the medium containing ALA/UIO-66-NH-FAM/FA and the medium containing ALA/UIO-66-NH-FAM/MTA are shown in Table 2 as the average values of fluorescence intensity after incubation for 120min in the medium containing A549 cells, Hela cells and KB cells, respectively, the medium containing ALA/UIO-66-NH-FAM/FA and the medium containing ALA/UIO-66-NH-FAM/MTA; fluorescence intensity curves are plotted according to tables 1 and 2, as shown in fig. 26 to 33.
TABLE 1
A549 cell Hela cell KB cell
Culture medium 4.94 5.84 5.23
Culture medium containing ALA/UIO-66-NH-FAM 42.3 36.3 38.6
Culture medium containing ALA/UIO-66-NH-FAM/FA 39.6 46.9 41.9
Culture medium containing ALA/UIO-66-NH-FAM/MTA 40.5 47.4 42.9
TABLE 2
A549 cell Hela cell KB cell
Culture medium 2.15 7.28 7.41
Culture medium containing ALA/UIO-66-NH-FAM 53.2 57.4 85.7
Culture medium containing ALA/UIO-66-NH-FAM/FA 50.9 74.5 84.1
Culture medium containing ALA/UIO-66-NH-FAM/MTA 49.8 95.5 112
The experimental results are as follows:
as is clear from Table 1, Table 2 and FIGS. 26 to 33, the fluorescence intensity of A549 cells after incubation in the medium containing ALA/UIO-66-NH-FAM was slightly greater than that after incubation in the medium containing ALA/UIO-66-NH-FAM/FA and in the medium containing ALA/UIO-66-NH-FAM/MTA, while the fluorescence intensity of Hela cells and KB cells after incubation for 120min in the medium containing ALA/UIO-66-NH-FAM/MTA was significantly higher than that of the other cells, which was caused by: the capacity of ALA/UIO-66-NH-FAM/MTA to enter A549 cells was slightly lower than ALA/UIO-66-NH-FAM, while the capacity of ALA/UIO-66-NH-FAM/MTA to enter Hela cells and KB cells was significantly higher than ALA/UIO-66-NH-FAM and ALA/UIO-66-NH-FAM/FA.
Example 17
MTT toxicity test without illumination
Preparation of the experiment:
cell preparation: a549 cells (human lung cancer cells), Hela cells (cervical cancer cells) and KB cells (nasopharyngeal cancer cells);
culture medium: 89% of folic acid-free RPMI1640 culture medium, 10% of fetal bovine serum and 1% of streptomycin qing mixed solution;
ALA/UIO-66-NH-FAM-containing medium: adding ALA/UIO-66-NH-FAM solution on the basis of the culture medium to make ALA/UIO-66-NH-FAM concentration in the culture medium be 5 g/mL, 50 g/mL, 100 g/mL, 250 g/mL and 500 g/mL;
ALA/UIO-66-NH-FAM/FA-containing medium: adding ALA/UIO-66-NH-FAM/FA solution on the basis of the culture medium to ensure that the concentrations of ALA/UIO-66-NH-FAM/FA in the culture medium are 5 g/mL, 50 g/mL, 100 g/mL, 250 g/mL and 500 g/mL of ALA/UIO-66-NH-FAM solution with equivalent concentration;
media containing ALA/UIO-66-NH-FAM/MTA: adding ALA/UIO-66-NH-FAM/MTA solution on the basis of the culture medium to make the concentration of ALA/UIO-66-NH-FAM/MTA in the culture medium be 5 g/mL, 50 g/mL, 100 g/mL, 250 g/mL and 500 g/mL of ALA/UIO-66-NH-FAM solution with equivalent concentration.
Culture medium containing ALA/UIO-66-NH-FAM/CP 1: adding ALA/UIO-66-NH-FAM/CP1 solution on the basis of the culture medium to make the concentration of ALA/UIO-66-NH-FAM/CP1 in the culture medium be 5 g/mL, 50 g/mL, 100 g/mL, 250 g/mL and 500 g/mL of ALA/UIO-66-NH-FAM solution with equivalent concentration;
medium containing MTA: adding MTA solution on the basis of the culture medium to ensure that the concentration of MTA in the culture medium is equal to the equivalent concentration of MTA in the culture medium containing ALA/UIO-66-NH-FAM/MTA;
medium containing CP 1: CP1 solution was added to the medium so that the concentration of CP1 in the medium was equivalent to that of CP1 in the medium containing ALA/UIO-66-NH-FAM/CP 1.
The experimental steps are as follows:
digesting the cells with trypsin when the cells are in good growth in the logarithmic phase, centrifuging to remove the supernatant, and preparing the medium to a cell density of 5X 104Single cell suspension at 5X 10/mL3The cells were seeded in 96-well plates at a density per well and placed in an incubator at 37 ℃ for further 24 h. Then discarding the original culture medium, respectively changing into a culture medium containing MTA, a culture medium containing CP1, a culture medium containing ALA/UIO-66-NH-FAM/FA, a culture medium containing ALA/UIO-66-NH-FAM/MTA and a culture medium containing ALA/UIO-66-NH-FAM/CP1, continuing to culture for 24h and 48h, respectively adding 10 μ L of 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide (MTT) solution with the concentration of 5mg/mL, continuing to culture for 4h, discarding the waste liquid in the pores, adding 100 μ L of dimethyl sulfoxide solution into each pore, placing a shaking table to shake for 10min at the rotation speed of 180r/min, finally, an enzyme-linked immunosorbent assay instrument is used for measuring the absorbance value of lambda =490 nm, data are recorded, and a histogram is drawn by taking the concentration of the drug as the abscissa and the survival rate of the cells as the ordinate, as shown in fig. 34.
The experimental results are as follows:
as can be seen from FIG. 34, various concentrations of MTA, CP1, ALA/UIO-66-NH-FAM/FA, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP1 did not substantially affect the cellular activities of A549 cells under dark conditions; under the condition of keeping out of the light, ALA/UIO-66-NH-FAM/FA at different concentrations does not basically influence the cell activity of Hela cells and KB cells; under the condition of keeping out of light, MTA, CP1, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP1 reduce the cell activity of Hela cells and KB cells to different degrees along with the increase of time and concentration, wherein ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP1 obviously reduce the cell activity of Hela cells and KB cells.
Example 18
Light irradiation MTT toxicity test
Preparation of the experiment:
cell preparation: a549 cells (human lung cancer cells), Hela cells (cervical cancer cells) and KB cells (nasopharyngeal cancer cells);
culture medium: 89% of folic acid-free RPMI1640 culture medium, 10% of fetal bovine serum and 1% of streptomycin qing mixed solution;
ALA/UIO-66-NH-FAM-containing medium: adding ALA/UIO-66-NH-FAM solution on the basis of the culture medium to make ALA/UIO-66-NH-FAM concentration in the culture medium be 5 g/mL, 50 g/mL, 100 g/mL, 250 g/mL and 500 g/mL;
ALA/UIO-66-NH-FAM/FA-containing medium: adding ALA/UIO-66-NH-FAM/FA solution on the basis of the culture medium to ensure that the concentrations of ALA/UIO-66-NH-FAM/FA in the culture medium are 5 g/mL, 50 g/mL, 100 g/mL, 250 g/mL and 500 g/mL of ALA/UIO-66-NH-FAM solution with equivalent concentration;
media containing ALA/UIO-66-NH-FAM/MTA: adding ALA/UIO-66-NH-FAM/MTA solution on the basis of the culture medium to ensure that the concentrations of ALA/UIO-66-NH-FAM/MTA in the culture medium are 5 g/mL, 50 g/mL, 100 g/mL, 250 g/mL and 500 g/mL of ALA/UIO-66-NH-FAM solution with equivalent concentrations;
culture medium containing ALA/UIO-66-NH-FAM/CP 1: adding ALA/UIO-66-NH-FAM/CP1 solution on the basis of the culture medium to make the concentration of ALA/UIO-66-NH-FAM/CP1 in the culture medium be 5 g/mL, 50 g/mL, 100 g/mL, 250 g/mL and 500 g/mL of ALA/UIO-66-NH-FAM solution with equivalent concentration;
ALA-containing medium: adding ALA solution on the basis of the culture medium to make ALA concentration in the culture medium equal to ALA concentration in the culture medium containing ALA/UIO-66-NH-FAM.
The experimental steps are as follows:
digesting the cells with trypsin when the cells are in good growth state in logarithmic phase, centrifuging to remove supernatant, and preparing the culture medium into fine cellsCell density of 5 × 104Single cell suspension at 5X 10/mL3The cells were seeded in 96-well plates at a density per well and placed in an incubator at 37 ℃ for further 24 h. Then discarding the original culture medium, respectively replacing with culture medium, ALA-containing culture medium, ALA/UIO-66-NH-FAM/FA-containing culture medium, ALA/UIO-66-NH-FAM/MTA-containing culture medium and ALA/UIO-66-NH-FAM/CP 1-containing culture medium, continuing to culture for 6h, then placing at 660 nm wavelength and 20J/cm laser power density2Irradiating the culture medium in a pulse laser for 15min and continuously culturing for 18 h; another parallel experiment is to continue culturing for 6h based on the above experiment, and then placing at 660 nm wavelength and 20J/cm laser power density2The culture was continued for 18h (i.e., total incubation time was 48 h) with 15min irradiation. Finally, 10 μ L of 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide (MTT) solution with the concentration of 5mg/mL is respectively added, after continuous culture is carried out for 4h, waste liquid in the holes is discarded, 100 μ L of dimethyl sulfoxide solution is added into each hole, a shaking table is placed and shaken for 10min under the condition that the rotating speed is 180r/min, finally, an enzyme linked immunosorbent assay detector is used for measuring the absorbance value of lambda =490 nm, data is recorded, and a bar graph is drawn by taking the drug concentration as the horizontal coordinate and the cell survival rate as the vertical coordinate, as shown in figure 35.
The experimental results are as follows:
comparing the analysis in conjunction with FIG. 34 and FIG. 35, in the light condition, ALA/UIO-66-NH-FAM/FA, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP1 reduced the cell activities of A549 cells, Hela cells and KB cells to different degrees, wherein ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP1 significantly reduced the cell activities of A549 cells, Hela cells and KB cells, and the effect is better as the concentration is higher and the time is longer; and compared with ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP1 under the condition of no illumination, the cell activity of A549 cells, Hela cells and KB cells can be reduced under the condition of illumination.
Example 19
Normal hepatotoxicity test
Preparation of the experiment:
cell preparation: LO2 cell (human normal liver cell)
Culture medium: 89% of folic acid-free RPMI1640 culture medium, 10% of fetal bovine serum and 1% of streptomycin qing mixed solution;
ALA/UIO-66-NH-FAM-containing medium: adding ALA/UIO-66-NH-FAM solution on the basis of the culture medium to make ALA/UIO-66-NH-FAM concentration in the culture medium be 5 g/mL, 50 g/mL, 100 g/mL, 250 g/mL and 500 g/mL;
ALA/UIO-66-NH-FAM/FA-containing medium: adding ALA/UIO-66-NH-FAM/FA solution on the basis of the culture medium to ensure that the concentrations of ALA/UIO-66-NH-FAM/FA in the culture medium are 5 g/mL, 50 g/mL, 100 g/mL, 250 g/mL and 500 g/mL of ALA/UIO-66-NH-FAM solution with equivalent concentration;
media containing ALA/UIO-66-NH-FAM/MTA: adding ALA/UIO-66-NH-FAM/MTA solution on the basis of the culture medium to ensure that the concentrations of ALA/UIO-66-NH-FAM/MTA in the culture medium are 5 g/mL, 50 g/mL, 100 g/mL, 250 g/mL and 500 g/mL of ALA/UIO-66-NH-FAM solution with equivalent concentrations;
culture medium containing ALA/UIO-66-NH-FAM/CP 1: adding ALA/UIO-66-NH-FAM/CP1 solution on the basis of the culture medium to make the concentration of ALA/UIO-66-NH-FAM/CP1 in the culture medium be 5 g/mL, 50 g/mL, 100 g/mL, 250 g/mL and 500 g/mL of ALA/UIO-66-NH-FAM solution with equivalent concentration;
ALA-containing medium: adding ALA solution on the basis of the culture medium to make the ALA concentration in the culture medium equal to the ALA concentration in the culture medium containing ALA/UIO-66-NH-FAM;
medium containing MTA: adding MTA solution on the basis of the culture medium to ensure that the concentration of MTA in the culture medium is equal to the equivalent concentration of MTA in the culture medium containing ALA/UIO-66-NH-FAM/MTA;
medium containing CP 1: CP1 solution was added to the medium so that the concentration of CP1 in the medium was equivalent to that of CP1 in the medium containing ALA/UIO-66-NH-FAM/CP 1.
The experimental steps are as follows:
digesting the cells with trypsin when the cells are in good growth state in the logarithmic phase, centrifuging to remove the supernatant,and the culture medium is prepared to have a cell density of 5X 104Single cell suspension at 5X 10/mL3The cells were seeded in 96-well plates at a density per well and placed in an incubator at 37 ℃ for further 24 h. Then discarding the original culture medium, respectively changing into a culture medium containing MTA, a culture medium containing ALA/UIO-66-NH-FAM/FA, a culture medium containing CP1, a culture medium containing ALA/UIO-66-NH-FAM/MTA and a culture medium containing ALA/UIO-66-NH-FAM/CP1, continuing culturing for 24h, 48h and 72h, finally respectively adding 10 μ L of 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide (MTT) solution with the concentration of 5mg/mL, continuing culturing for 4h, discarding the waste liquid in the hole, adding 100 μ L of dimethyl sulfoxide solution into each hole, placing a shaking table to shake for 10min at the rotation speed of 180r/min, finally, an enzyme-linked immunosorbent assay instrument is used for measuring the absorbance value of lambda =490 nm, data are recorded, and a histogram is drawn by taking the concentration of the drug as the abscissa and the survival rate of the cells as the ordinate, as shown in fig. 36.
The experimental results are as follows:
comparing the analysis in FIG. 35 and FIG. 36, different concentrations of ALA did not substantially affect the cellular activity of LO2 cells; MTA, ALA/UIO-66-NH-FAM/FA, CP1, ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP1 reduce the cell activity of LO2 cells to different degrees along with higher concentration of the medicine and longer time, wherein ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP1 can reduce the cell activity of LO2 cells; the capacity of ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP1 for reducing the cell activity of A549 cells, Hela cells and KB cells is obviously greater than the capacity of ALA/UIO-66-NH-FAM/MTA and ALA/UIO-66-NH-FAM/CP1 for reducing the cell activity of LO2 cells.
The above description is only a preferred embodiment of the present invention, and all the minor modifications, equivalent changes and modifications made to the above embodiment according to the technical solution of the present invention are within the scope of the technical solution of the present invention.

Claims (14)

1. A preparation method of a zirconium metal organic framework compound is characterized by comprising the following steps: the method comprises the following steps:
1) preparation of UIO-66-NH2: dissolving zirconium chloride in N, N-dimethylformamide to obtain a solution A; dissolving amino terephthalic acid in N, N-dimethylformamide to obtain a solution B; putting the solution A, the solution B and acetic acid into a reaction bottle, uniformly mixing, putting the reaction bottle into an oven at the temperature of 80-100 ℃ for 10-14h, and cooling to room temperature to obtain a suspension C; centrifuging the suspension C for 10-20min at the rotating speed of 12000-14000 r/min, discarding the supernatant to obtain a precipitate D, cleaning the precipitate D with ethanol for 3 times, soaking the precipitate D with methanol for three days, replacing new methanol every 24 hours during the soaking period, and finally drying the precipitate in an oven at the temperature of 70-90 ℃ overnight to obtain UIO-66-NH2Powder;
2) preparation of compound E: the UIO-66-NH prepared by the step 1)2Ultrasonically dispersing the powder in water, then adding 5-carboxyl fluorescein, 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and 1-hydroxybenzotriazole, uniformly mixing, and stirring for 14-18h under a dark condition to obtain a suspension F; centrifuging the suspension F for 10-20min at the rotating speed of 12000-14000 r/min, and discarding the supernatant to obtain a precipitate G; gradient-cleaning the precipitate G with mixed solution of dimethyl sulfoxide and water until the supernatant turns into colorless solution, repeatedly ultrasonically cleaning with ultrapure water for 5 times, and freeze drying to obtain orange yellow powder, i.e. compound E powder;
3) preparation of compound H: ultrasonically dispersing the compound E powder obtained in the step 2) in water to obtain a solution I; then dissolving 5-aminoketone valerate hydrochloride in water to obtain a solution J; then uniformly mixing the solution I and the solution J, stirring for 2-4 days under the condition of keeping out of the sun, centrifuging for 10-20min at the rotating speed of 12000-14000 r/min, and discarding the supernatant to obtain a precipitate K; washing the precipitate K with water for 2 times, and freeze-drying to obtain compound H powder;
4) preparation of compound L: dissolving pemetrexed disodium in water to obtain a pemetrexed disodium solution; dispersing the compound H powder obtained in the step 3) in water, and then respectively adding 0.1 mL, 0.25 mL, 0.5 mL, 0.75 mL and 1 mL of pemetrexed disodium solutions to obtain a plurality of different mixed solutions M; adding water into the mixed solution M to change the volume of the mixed solution M into 5mL to obtain a suspension N; stirring the suspension N for 8-12min, centrifuging for 5-15min at the rotating speed of 12000-14000 r/min, and removing the supernatant to obtain a precipitate O; washing the precipitate with water for 3 times, collecting supernatant obtained after each centrifugation, and freeze-drying to obtain compound L powder.
2. The method of preparing a zirconium metal organic framework compound according to claim 1, characterized in that: in the step 1), the solid-to-liquid ratio of zirconium chloride to N, N-dimethylformamide is 7:1, the solid-to-liquid ratio of amino terephthalic acid to N, N-dimethylformamide is 55:1, the volume ratio of the solution A to the solution B is 3:1, and the ratio of the volume of acetic acid to the total volume of the solution A and the solution B is 1: 20.
3. The method of preparing a zirconium metal organic framework compound according to claim 1, characterized in that: UIO-66-NH in step 2)2The solid-liquid ratio of the powder to the water is 2:1, 5-carboxyl fluorescein, the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the 1-hydroxybenzotriazole is 1:1:1, 5-carboxyl fluorescein to UIO-66-NH2The mass ratio of the powder is 1:5, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to UIO-66-NH2The mass ratio of the powder is 1:5, 1-hydroxybenzotriazole to UIO-66-NH2The mass ratio of the powders was 1: 5.
4. The method of preparing a zirconium metal organic framework compound according to claim 1, characterized in that: the solid-liquid ratio of the compound E powder to water in the step 3) is 5: the solid-to-liquid ratio of the 3, 5-aminolevulinic acid hydrochloride to the water is 25: 2.
5. The method of preparing a zirconium metal organic framework compound according to claim 1, characterized in that: the compound E in the step 2) is UIO-66-NH-FAM, and the compound H in the step 3) is ALA/UIO-66-NH-FAM.
6. The method of preparing a zirconium metal organic framework compound according to claim 1, characterized in that: in the step 4), the concentration of the pemetrexed disodium solution is 10mg/mL, the solid-to-liquid ratio of the compound H powder to water is 5:1, and the volume of water for washing the precipitate O is 4 mL.
7. The method of preparing a zirconium metal organic framework compound according to claim 6, wherein: the compound L is ALA/UIO-66-NH-FAM/MTA, and the ALA/UIO-66-NH-FAM/MTA is applied to the aspects of targeted delivery, control and release of anticancer drugs, so that the anticancer effect of the anticancer drugs is enhanced.
8. The method of preparing a zirconium metal organic framework compound according to claim 7, wherein: the compound P is ALA/UIO-66-NH-FAM/CP1, and the ALA/UIO-66-NH-FAM/CP1 is applied to the aspects of targeted delivery, control and release of anticancer drugs, so that the anticancer effect of the anticancer drugs is enhanced.
9. A preparation method of a zirconium metal organic framework compound is characterized by comprising the following steps: the method comprises the following steps:
1) preparation of UIO-66-NH2: dissolving zirconium chloride in N, N-dimethylformamide to obtain a solution A; dissolving amino terephthalic acid in N, N-dimethylformamide to obtain a solution B; putting the solution A, the solution B and acetic acid into a reaction bottle, uniformly mixing, putting the reaction bottle into an oven at the temperature of 80-100 ℃ for 10-14h, and cooling to room temperature to obtain a suspension C; centrifuging the suspension C for 10-20min at the rotating speed of 12000-14000 r/min, discarding the supernatant to obtain a precipitate D, cleaning the precipitate D with ethanol for 3 times, soaking the precipitate D with methanol for three days, replacing new methanol every 24 hours during the soaking period, and finally drying the precipitate in an oven at the temperature of 70-90 ℃ overnight to obtain UIO-66-NH2Powder;
2) preparation of compound E: the UIO-66-NH prepared by the step 1)2Ultrasonically dispersing the powder in water, adding 5-carboxyl fluorescein, 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and 1-hydroxybenzotriazole, mixing, and stirring for 14-18h in dark to obtain the final productTo suspension F; centrifuging the suspension F for 10-20min at the rotating speed of 12000-14000 r/min, and discarding the supernatant to obtain a precipitate G; gradient-cleaning the precipitate G with mixed solution of dimethyl sulfoxide and water until the supernatant turns into colorless solution, repeatedly ultrasonically cleaning with ultrapure water for 5 times, and freeze drying to obtain orange yellow powder, i.e. compound E powder;
3) preparation of compound H: ultrasonically dispersing the compound E powder obtained in the step 2) in water to obtain a solution I; then dissolving 5-aminoketone valerate hydrochloride in water to obtain a solution J; then uniformly mixing the solution I and the solution J, stirring for 2-4 days under the condition of keeping out of the sun, centrifuging for 10-20min at the rotating speed of 12000-14000 r/min, and discarding the supernatant to obtain a precipitate K; washing the precipitate K with water for 2 times, and freeze-drying to obtain compound H powder;
4) preparation of compound P: dispersing the compound H powder obtained in the step 3) in N, N-dimethylformamide, adding pemetrexed, stirring for 1-2min, then adding a zirconium chloride aqueous solution, heating for 1-2min at the temperature of 90-110 ℃, stirring violently in the heating process, centrifuging for 5-15min at the rotating speed of 12000-14000 r/min, and removing the supernatant to obtain a precipitate Q; the precipitate Q was washed with water 3 times, cooled and dried to obtain compound P powder.
10. The method of preparing a zirconium metal organic framework compound according to claim 9, characterized in that: in the step 1), the solid-to-liquid ratio of zirconium chloride to N, N-dimethylformamide is 7:1, the solid-to-liquid ratio of amino terephthalic acid to N, N-dimethylformamide is 55:1, the volume ratio of the solution A to the solution B is 3:1, and the ratio of the volume of acetic acid to the total volume of the solution A and the solution B is 1: 20.
11. The method of preparing a zirconium metal organic framework compound according to claim 9, characterized in that: UIO-66-NH in step 2)2The solid-liquid ratio of the powder to the water is 2:1, 5-carboxyl fluorescein, the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the 1-hydroxybenzotriazole is 1:1:1, 5-carboxyl fluorescein to the 1-hydroxybenzotriazoleUIO-66-NH2The mass ratio of the powder is 1:5, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to UIO-66-NH2The mass ratio of the powder is 1:5, 1-hydroxybenzotriazole to UIO-66-NH2The mass ratio of the powders was 1: 5.
12. The method of preparing a zirconium metal organic framework compound according to claim 9, characterized in that: the solid-liquid ratio of the compound E powder to water in the step 3) is 5: the solid-to-liquid ratio of the 3, 5-aminolevulinic acid hydrochloride to the water is 25: 2.
13. The method of preparing a zirconium metal organic framework compound according to claim 9, characterized in that: the compound E in the step 2) is UIO-66-NH-FAM, and the compound H in the step 3) is ALA/UIO-66-NH-FAM.
14. The method of preparing a zirconium metal organic framework compound according to claim 9, characterized in that: in the step 4), the solid-to-liquid ratio of the compound H powder to the N, N-dimethylformamide is 1:2, the mass ratio of the pemetrexed to the compound H powder is 1:2, the concentration of the zirconium chloride aqueous solution is 25mg/mL, and the volume ratio of the zirconium chloride aqueous solution to the N, N-dimethylformamide is 1: 50.
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