Nano material based on metal organic framework and artemisinin compound as well as preparation method and application of nano material

Abstract

The invention provides a nano material based on a metal organic framework and an artemisinin compound as well as a preparation method and application of the nano material, and relates to the technical field of biological medicines. The preparation method comprises the following steps: dissolving zinc nitrate hexahydrate in water to obtain a solution A; dissolving 2-methylimidazole in water to obtain a solution B; and preparing an artemisinin compound solution, mixing the artemisinin compound solution with the solution A, adding the solution B, stirring, reacting, centrifuging, taking precipitate, and washing to obtain the nano material based on the metal organic framework and the artemisinin compound. Based on good pH responsiveness of MOF, in combination with a microbial survival acid environment, the MOF is taken as a drug delivery carrier, ART with antibacterial activity and derivatives thereof are loaded, a novel nano-drug delivery system is synthesized, a target drug is delivered to an infected focus in a targeted manner, ART and thallus iron ions are decomposed and released, a large amount of ROS is generated, and the infected focus is removed.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and more specifically, to nanomaterials based on metal-organic frameworks and artemisinin compounds, as well as preparation methods and applications thereof. Background Art

[0002] Metal Organic Frameworks (MOFs) nanomaterials are a type of organic-inorganic hybrid material with a periodic network structure. They are centered on inorganic metals and connected to organic ligands through self-assembly. Their advantages of high porosity, large specific surface area, and adjustable structure have led to their rapid development in the past two decades. As a drug carrier, this nanomaterial can achieve the goal of sustained / controlled release of drugs and targeted drug delivery as a responsive smart carrier. The metal central ion is Zn 2+ 、Fe 3+ and Ag + Some metal ions have antibacterial effects, such as Zn 2+ 、Ag + Among them, iron ions can react with ART and its derivatives to produce ROS through Fenton reaction, which has pharmacological effects on microbial pathogens, tumors, etc. in a chemical kinetic manner.

[0003] Artemisinin (ART) is a sesquiterpene lactone compound containing a peroxide group isolated and extracted by Chinese scientist Tu Youyou from Artemisia annua. It is widely used to significantly fight malaria. Based on the basic structure of ART, a series of active derivatives such as dihydroartemisinin (DHA), artesunate (ARS), artemether and arteether that retain the peroxide group were obtained. With the in-depth study of artemisinin and its derivatives, its pharmacological effects are no longer limited to malaria, and have shown good pharmacological effects in many aspects such as anti-tumor, microbial infection, and immune regulation. However, artemisinin and its derivatives have disadvantages such as poor water solubility, low bioavailability, short maintenance time of effective blood drug concentration and rapid excretion, which limit their clinical use. Summary of the Invention

[0004] The purpose of this application is to provide nanomaterials based on metal organic frameworks and artemisinin compounds, their preparation methods and their applications as drug delivery systems. By encapsulating artemisinin derivatives in MOF materials to prepare ART-MOFNPs drug delivery systems, the systems have obvious effects against Gram-positive bacteria, Gram-negative bacteria, and especially in antifungal activity.

[0005] In order to solve the above technical problems, the technical solutions adopted in this application are:

[0006] In a first aspect, the present application provides a method for preparing a nanomaterial based on a metal organic framework and an artemisinin-based compound, comprising the following steps:

[0007] S1. Dissolve zinc nitrate hexahydrate in water to obtain solution A; dissolve 2-methylimidazole in water to obtain solution B;

[0008] S2. Prepare an artemisinin compound solution, mix it with the above solution A, then add solution B, stir to react, and then take the precipitate and wash it to obtain a nanomaterial based on the metal organic framework and the artemisinin compound.

[0009] In a second aspect, the present application provides a nanomaterial based on a metal organic framework and an artemisinin compound prepared by any of the above methods.

[0010] The third aspect is the application of nanomaterials based on metal-organic frameworks and artemisinin compounds in the field of drug delivery.

[0011] The working principle of the nanomaterials based on metal organic frameworks and artemisinin compounds (ART-MOF NPs) prepared in this application in the field of drug delivery is as follows: Figure 1 ( Figure 1 A: ART and its derivatives are loaded into MOF materials to construct a drug delivery system that has no effect on normal tissue cells and attaches to low-pH bacterial and fungal infection sites. Figure 1 B: ART-MOF NPs are internalized at the lesion site, releasing ART to exert pharmacological effects, and at the same time undergo a Fenton reaction with intracellular iron ions, releasing a large amount of ROS to produce a toxic reaction) as shown: Most bacteria and fungi can adapt to survival in a slightly acidic environment. This application is based on the good pH responsiveness of MOF and the acidic environment in which microorganisms survive. MOF is used as a drug delivery carrier to load ART and its derivatives with antibacterial activity to synthesize a new nanodrug delivery system ART-MOF NPs, which targets the delivery of target drugs to the infected lesions, decomposes and releases ART to exert pharmacological effects, and at the same time reacts with the iron ions in the bacterial cells to produce a large amount of ROS, clearing the infected lesions, allowing artemisinin and its derivatives to exert the maximum pharmacological effects while reducing damage to other tissue cells.

[0012] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0013] The pharmacological mechanism of artemisinin and its derivatives in antimalarial and antitumor activities is through a Fenton reaction with pathogenic iron ions, generating reactive oxygen species (ROS) to exert their pharmacological effects. In antibacterial research, they have demonstrated bactericidal effects, enhanced antimicrobial sensitization, and the reversal of drug-resistant bacteria. MOF nanomaterials enhance the solubility of artemisinin and its derivatives, and the metal ions they contain possess a certain bactericidal effect. The preparation of ART-MOF NPs drug delivery systems significantly increases the minimum inhibitory concentration (MIC) of artemisinin and its derivatives.

[0014] Bacterial and fungal resistance rates are increasing. This invention combines the intelligent responsiveness of MOF materials with the antibacterial effects of artemisinin and its derivatives to reduce the clinical use of antibacterial drugs, reduce the incidence of drug-resistant bacteria, and explore new treatment options for infectious diseases. The excellent antifungal efficacy of the ART-MOF NPs drug delivery system allows for the preparation of topical formulations for common fungal infection sites such as the mouth, skin, and vagina. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 Schematic diagram of the working principle of ART-MOF NPs as drug delivery materials for this application;

[0017] Figure 2 This is a SEM scanning electron microscope image of the water-based MOF support prepared in Example 1 of the present application;

[0018] Figure 3 This is a SEM scanning electron microscope image of the water-based MOF carrier after adjusting the particle size and morphology in Example 1 of the present application;

[0019] Figure 4 This is a SEM image of the ART-MOF NPs prepared in Example 2 of the present application;

[0020] Figure 5 This is the particle size distribution diagram of ART-MOF NPs in Example 2 of this application;

[0021] Figure 6 This is a SEM scanning electron micrograph of ART-MOF NPs after adjusting the particle size and morphology in Example 2 of the present application;

[0022] Figure 7This is the particle size distribution diagram of ART-MOF NPs after adjusting the particle size morphology in Example 2 of this application;

[0023] Figure 8 This is the infrared characterization image of ART-MOF NPs in Example 2 of the present application;

[0024] Figure 9 This is a graph showing the inhibition rate of ART-MOF NPs against Gram-positive bacteria in Example 3 of the present application;

[0025] Figure 10 This is a graph showing the inhibition rate of ART-MOF NPs against Gram-negative bacteria in Example 4 of the present application;

[0026] Figure 11 This is a graph showing the inhibition rate of ART-MOF NPs against Candida tropicalis in Example 5 of the present application;

[0027] Figure 12 This is a graph showing the inhibition rate of ART-MOF NPs against Candida glabrata in Example 5 of the present application;

[0028] Figure 13 This is a curve diagram of the inhibition rate of ART-MOF NPs against Aspergillus terreus in Example 5 of the present application;

[0029] Figure 14 This is a graph showing the inhibition rate of ART-MOF NPs against Cryptococcus neoformans in Example 5 of the present application;

[0030] Figure 15 This is a diagram showing the effect of different concentrations of ART-MOF NPs on the morphology of Candida tropicalis in Example 6 of the present application;

[0031] Figure 16 This is a diagram showing the effect of different concentrations of ART-MOF NPs on the morphology of Cryptococcus neoformans in Example 6 of the present application;

[0032] Figure 17 This is a graph showing the effects of different experimental groups on fungal-infected animal hair in Example 7 of this application;

[0033] Figure 18 This is a graph showing the effects of different experimental groups on lung inflammation in fungal-infected animals in Example 7 of this application;

[0034] Figure 19 These are images of lung tissue damage in fungus-infected animals in different experimental groups in Example 7 of this application. DETAILED DESCRIPTION

[0035] To make the purpose, technical solutions and advantages of the examples of the present application clearer, the technical solutions in the examples of the present application will be described clearly and completely below. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0036] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present application will be described in detail below with reference to specific embodiments.

[0037] The preparation method of nanomaterials based on metal organic frameworks and artemisinin compounds comprises the following steps:

[0038] S1. Dissolve zinc nitrate hexahydrate in water to obtain solution A; dissolve 2-methylimidazole in water to obtain solution B;

[0039] S2. Prepare an artemisinin compound solution, mix it with the above solution A, then add solution B, stir to react, and then take the precipitate and wash it to obtain a nanomaterial based on the metal organic framework and the artemisinin compound.

[0040] In some embodiments of the present application, the concentration of the solution A is 50-100 mg / mL, preferably 75 mg / mL; the concentration of the solution B is 200-300 mg / mL, preferably 225 mg / mL.

[0041] In some embodiments of the present application, the concentration of the artemisinin compound solution is 10 mg / mL.

[0042] In some embodiments of the present application, the above-mentioned artemisinin compounds include artemisinin and artemisinin derivatives, and the artemisinin derivatives are dihydroartemisinin, artemether, arteether or artesunate.

[0043] In some embodiments of the present application, the mass ratio of the artemisinin compound solution, solution A and solution in the above step S2 is 1:(5-10):(20-30).

[0044] In some embodiments of the present application, the stirring in the above step S2 is carried out at room temperature and 800-1200 rpm for 10-15 minutes.

[0045] In some embodiments of the present application, the washing in the above-mentioned step S2 is specifically to add deionized water, stir and wash at a speed of 10000-13000 rpm and a temperature of 5°C for 10-15 minutes, and the number of washing times is 3 times.

[0046] A nanomaterial based on a metal organic framework and an artemisinin compound is prepared by any of the above methods.

[0047] Application of metal-organic frameworks and artemisinin-based nanomaterials in drug delivery.

[0048] The features and performance of the present application are further described in detail below with reference to the embodiments.

[0049] Example 1

[0050] In this embodiment, a water-based metal organic framework MOF is prepared, and the steps are as follows:

[0051] Solution A preparation: Accurately weigh 1.5 g zinc nitrate hexahydrate and dissolve it in 20 mL deionized water until fully dissolved.

[0052] Solution B preparation: Accurately weigh 45 g of 2-methylimidazole and dissolve it in 200 mL of deionized water until fully dissolved.

[0053] Take 2mL of solution A and slowly drop it into 10mL of solution B, stir at 800rpm for 10min at room temperature, centrifuge at 5℃, 11000rpm, centrifuge for 10min, and wash repeatedly 3 times to fully remove the unreacted raw materials to obtain a water-based MOF carrier. Its SEM scanning electron microscope image is as follows: Figure 2 As shown, it can be observed that the nanoparticles are poorly dispersed at this time, and have different sizes and shapes, with a hexadecagonal morphology.

[0054] Take 1 mL of solution A and slowly drip into 10 mL of solution B. Use DMF to adjust the particle size and morphology. Stir at room temperature at 800 rpm for 10 min. Centrifuge at 5 ° C, 11000 rpm, centrifuge for 10 min. Repeat washing 3 times to fully remove unreacted raw materials to obtain a water-based MOF carrier. Its SEM scanning electron microscope image is as follows: Figure 3 As shown, it can be observed that the nanoparticles are well dispersed, uniform in morphology, and are round in shape.

[0055] Example 2

[0056] A nanomaterial based on a metal organic framework and an artemisinin compound, referred to as ART-MOFNPs in this application, is prepared by:

[0057] S1. Accurately weigh 1.5 g of zinc nitrate hexahydrate and dissolve it in 20 mL of deionized water until fully dissolved to prepare solution A. Accurately weigh 45 g of 2-methylimidazole and dissolve it in 200 mL of deionized water until fully dissolved to prepare solution B.

[0058] S2. Prepare an initial ART concentration of 10 mg / mL, take 1 mL of it and mix it with solution A, and slowly drop 10 mL of solution B. Stir at 800 rpm for 10 minutes at room temperature. After the reaction is complete, add deionized water at 11000 rpm for 10 minutes and wash three times at 5°C to obtain the ART-MOF NPs drug delivery system. Its SEM scanning electron microscopy image is shown below. Figure 4 The size of the synthesized drug nanoparticles is about 90nm Figure 5 , nanomedicines were synthesized after adding DMF to adjust the particle size Figure 6 , the particle size is reduced to 30nm Figure 7 .

[0059] The infrared characterization of ART-MOF NPs clearly shows the synthesis structure. Figure 4 , the infrared spectrum shows the ART characteristic peak 850-900cm -1 (Peroxide OO), 1100-1250cm -1 (CO stretching vibration), 1400-1600 cm -1 (benzene ring skeleton); The characteristic peak of MOF synthesized with water as solvent is 3200-3600cm -1 (OH bending vibration), 1600-1800 cm -1 (C=N stretching vibration), ART-MOF NPs retain the characteristic peaks of ART and MOF, and ARS encapsulates MOF at 1700 cm -1 The peak becomes narrower.

[0060] Example 3

[0061] This example explores the effect of ART-MOF NPs on the inhibition rate of Gram-positive bacteria.

[0062] In this implementation plan, the 2020 American Clinical and Laboratory Standards Institute (CLSI) drug sensitivity standards were first used as a reference, and the clinical isolated strains were derived from the laboratory department of Mianyang 404 Hospital.

[0063] Gram-positive cocci were methicillin-resistant Staphylococcus aureus (MRSA), and the bacterial concentration was adjusted to 1×10 5 CFU / mL, inoculated into a 96-well sterile culture plate, added nanomedicine to the well containing bacterial culture, diluted in series according to solubility, incubated in a 37°C incubator for 24 hours, read the positive and negative control wells, the negative control wells were clear, and the positive control wells were turbid. The MIC of the drug against bacteria is the lowest drug concentration that inhibits the visible growth of bacteria. The antibacterial rate of the drug ART-MOF NPs was calculated according to the formula, and the curve was drawn. The test results are shown in Figure 2. Figure 9As shown in the curve, ART and MOF alone have no antibacterial effect, while ART-MOF NPs achieve a 50% inhibition rate against MRSA at 150ug / mL and a 100% inhibition rate at 300ug / mL.

[0064] Example 4

[0065] This example explores the effect of ART-MOF NPs on the inhibition rate of Gram-negative bacteria.

[0066] In this implementation plan, the 2020 American Clinical and Laboratory Standards Institute (CLSI) drug sensitivity standards were first used as a reference, and the clinical isolated strains were derived from the laboratory department of Mianyang 404 Hospital.

[0067] Gram-negative bacteria were selected from carbapenem-resistant Enterobacteriaceae (CRE), and the bacterial concentration was adjusted to 1×10 5 CFU / mL, inoculated into a 96-well sterile culture plate, added the drug to the well containing the bacterial culture, diluted in series according to the solubility, incubated in a 37°C incubator for 24 hours, read the positive and negative control wells, the negative control wells were clear, and the positive control wells were turbid. The antibacterial rate of the drug ART-MOF NPs was calculated according to the formula, and the curve was drawn. The test results are shown in Figure 2. Figure 10 As shown in the curve, ART and MOF alone have no antibacterial effect, while ART-MOF NPs achieve an inhibition rate of 58.07% against CRE at 300ug / mL, improving the inhibition rate of ART and MOF alone.

[0068] Example 5

[0069] This example explores the effect of ART-MOF NPs on the antibacterial rate of fungi (Candida tropicalis, Candida glabrata, Aspergillus terreus, Cryptococcus neoformans).

[0070] In this implementation plan, the 2020 American Clinical and Laboratory Standards Institute (CLSI) drug sensitivity standards were first used as a reference, and the clinical isolated strains were derived from the laboratory department of Mianyang 404 Hospital.

[0071] The fungal strains selected included Candida tropicalis, Candida glabrata, Aspergillus terreus, and Cryptococcus neoformans. The bacterial concentration was adjusted to 1 × 10 5CFU / mL, inoculated into a 96-well sterile culture plate, added the drug to the fungus culture well, diluted in series according to the solubility, incubated in a 37°C incubator for 24-72 hours, read the positive and negative control wells, the negative control wells were clear, and the positive control wells were turbid. The MIC of the drug against bacteria is the lowest drug concentration that inhibits the visible growth of bacteria. The antibacterial rate of the drug ART-MOF NPs was calculated according to the formula, and the curve was drawn. The test results are shown in Figure 2. Figure 11-14 As shown in the figure, low concentration ART-MOF NPs drug delivery system can significantly improve the antibacterial rate. Specifically:

[0072] Candida tropicalis ( Figure 11 ): 24 hours after drug administration, the ART-MOF NPs drug delivery system achieved an 88.45% inhibition rate at 36.25 μg / mL; ART achieved a 52.96% inhibition rate at 150 μg / mL; MOF alone had no antibacterial effect;

[0073] Candida glabrata ( Figure 12 ): 24 hours after drug administration, the ART-MOF NPs drug delivery system achieved a 74.65% inhibition rate at 18.75 μg / mL; ART achieved a 77.89% inhibition rate at 75 μg / mL; MOF alone had no antibacterial effect;

[0074] Aspergillus terreus ( Figure 13 ): Because soil mold grows slowly, according to the requirements of the drug sensitivity test, the drug was given for 72 hours. The ART-MOF NPs drug delivery system showed a significant antibacterial trend with increasing drug concentration, with an inhibition rate of 70.89% at 75ug / mL; ART greater than 37.5ug / mL had an inhibition rate of 60%, with no antibacterial trend; MOF showed a certain antibacterial effect as the drug action time increased.

[0075] Cryptococcus neoformans ( Figure 14 ): Because Cryptococcus neoformans grows slowly, according to the requirements of the drug sensitivity test, the ART-MOF NPs drug delivery system has a significant antibacterial effect after 72 hours of drug administration. The inhibition rate can reach 57.33% at 9.375ug / mL and 93.57% at 18.75ug / mL. ART has an antibacterial effect of 60%, with no trend. MOF shows a certain antibacterial effect as the drug action time increases.

[0076] Example 6

[0077] This example investigates the effects of ART-MOF NPs on the morphology of fungi (Candida tropicalis and Cryptococcus neoformans).

[0078] Fungal strains including Candida tropicalis and Cryptococcus neoformans were selected. After different concentrations of ART-MOF NPs drug delivery system acted on the fungi, the fungal morphology was observed under a microscope. The results under the microscope are as follows: Figure 15 and Figure 16 .

[0079] Candida tropicalis ( Figure 15 ): Complete fungal cells and hyphae were visible in the negative group; 24 hours after administration of ART-MOF NPs, the fungal cells were intact at a concentration of 9.375ug / mL, and no hyphae were seen; at a concentration of 18.75ug / mL, the fungal cells ruptured and the contents began to seep out; as the drug concentration increased, the fungal cells ruptured and no complete fungal cells were seen.

[0080] Cryptococcus neoformans ( Figure 16 ): In the negative group, the complete morphology and capsule of the fungus were visible; 72 hours after administration of ART-MOF NPs, the fungus began to rupture and the contents began to seep out at a concentration of 9.375ug / mL; as the drug concentration increased, no complete morphology of the fungus was seen.

[0081] Example 7

[0082] This example explores the protective effect of ART-MOF NPs on the fur and internal organs of animals infected with fungi.

[0083] Intraperitoneal injection of cyclophosphamide formed immunosuppression in mice. The injection time was 3 days and the dose was 20 mg / kg. Tropical Candida was injected into the tail vein. ART-MOF NPs were given by gavage 24 hours after modeling. The mice were divided into blank group, negative control group and ART-MOF NPs group. The treatment time was 10 days. After modeling, the hair loss of the mice in the negative group was severe, spreading from the neck to the back and to the abdomen. Slight hair loss was observed in the drug-treated group. The ART-MOF NPs group was in a better condition with less hair loss. Figure 17 ; Histopathological sections showed that compared with the negative control group, the ART-MOF NPs drug group could significantly reduce the accumulation of inflammatory factors in lung tissue. Figure 18 Compared with the negative control group, the ART-MOF NPs drug group could significantly reduce the swelling, degeneration and necrosis of hepatocytes. Figure 19 .

[0084] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

Claims

1. A method for preparing nanomaterials based on metal organic frameworks and artemisinin compounds, characterized in that: The following steps are involved: S1. Dissolve zinc nitrate hexahydrate in water to obtain solution A; dissolve 2-methylimidazole in water to obtain solution B; S2. Prepare an artemisinin compound solution, mix it with the above solution A, then add solution B, stir to react, and then take the precipitate and wash it to obtain a nanomaterial based on the metal organic framework and the artemisinin compound.

2. The method for preparing a nanomaterial based on a metal organic framework and an artemisinin compound according to claim 1, characterized in that: The concentration of solution A is 50-100 mg / mL; the concentration of solution B is 200-300 mg / mL.

3. The method for preparing nanomaterials based on metal organic frameworks and artemisinin compounds according to claim 1, characterized in that: The concentration of the artemisinin compound solution is 10 mg / mL.

4. The method for preparing a nanomaterial based on a metal organic framework and an artemisinin compound according to claim 3, characterized in that: The artemisinin compounds include artemisinin and artemisinin derivatives, and the artemisinin derivatives are dihydroartemisinin, artemether, arteether or artesunate.

5. The method for preparing nanomaterials based on metal organic frameworks and artemisinin compounds according to claim 1, characterized in that: In step S2, the mass ratio of the artemisinin compound solution, solution A and solution B is 1:(5-10):(20-30).

6. The method for preparing nanomaterials based on metal organic frameworks and artemisinin compounds according to claim 1, characterized in that: In the step S2, stirring is performed at room temperature at 800-1200 rpm for 10-15 minutes.

7. The method for preparing nanomaterials based on metal organic frameworks and artemisinin compounds according to claim 1, characterized in that: The washing in step S2 is specifically performed by adding deionized water, stirring and washing at a speed of 10000-13000 rpm and a temperature of 5° C. for 10-15 minutes, and the washing times are 3 times.

8. Nanomaterials based on metal organic frameworks and artemisinin compounds, characterized in that: Prepared by the method according to any one of claims 1 to 7.

9. Use of the nanomaterial based on metal organic framework and artemisinin compound according to claim 8 in the field of drug delivery.

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