Preparation method of pH (Potential of Hydrogen) and light dual-response drug-loaded antibacterial nano material for plants

Abstract

The invention discloses a preparation method of a pH and light dual-response drug-loaded antibacterial nano-material for plants. The preparation method specifically comprises the following steps: loading a drug into polyphenol hybridized zinc-based metal organic framework nano-particles in situ through stirring to obtain drug-loaded polyphenol hybridized ZIF-8 nano-particles; a calcium peroxide coating is synthesized in situ on the surface of the prepared polyphenol hybridized ZIF-8 nano-particle loaded with the medicine to obtain the pH and light dual-response medicine-loaded antibacterial nano-material for the plant. According to the invention, the photocatalytic performance and drug loading capacity of ZIF-8 are improved, so that the actual application range of the polyphenol hybrid nano material is wider; the preparation method is simple, low in cost and price, free of complex conditions and suitable for large-scale production.

Description

Technical Field

[0001] The invention belongs to the field of biochemistry, and in particular relates to a method for preparing a plant-based drug-carrying antibacterial nanomaterial with dual responses to pH and light. Background Art

[0002] Bacterial pathogens proliferate rapidly in soil and colonize plants, resulting in a significant reduction in food production that cannot meet the growing demand. In addition, compared with fungicides, relatively few products targeting plant bacterial diseases are sold, so there is still a lack of effective bacterial disease management strategies in agricultural production. Currently, many traditional fungicides, especially some amphiphilic compounds, have low bioavailability due to the effective permeability barriers of bacteria and fungi. In addition, microenvironments such as soil acidification, high temperature, and humidity can cause outbreaks of bacterial wilt in crops such as tomatoes and peppers. Even more unfortunately, the widespread use of antibiotics such as copper ions and streptomycin has also led to the development of bacterial resistance and horizontal gene transfer, resulting in environmental pollution and the crisis of antibiotic resistance. Therefore, there is an urgent need to develop effective technologies to address these huge global challenges.

[0003] Traditional ZIF-8, as a photocatalytic antibacterial agent, is still subject to many limitations in antibacterial applications. First, similar to pure MOFs, its rapid electron-hole recombination leads to poor photocatalytic effect; second, its poor stability limits its long-term stable release of drugs; third, the lack of binding groups on the surface leads to low drug loading rate and lack of adhesion to bacteria. These existing defects limit the further application of ZIF-8 in antibacterial applications. Therefore, it is of certain practical significance to develop efficient photocatalytic ZIF-8 and improve its antibacterial and drug loading properties. Summary of the invention

[0004] In order to overcome the defects of the above-mentioned technology, the present invention provides a method for preparing a plant-based drug-loaded antibacterial nanomaterial with dual response to pH and light.

[0005] The method for preparing a plant-based drug-carrying antibacterial nanomaterial with dual response to pH and light of the present invention comprises the following steps:

[0006] Step A: The drug is loaded into the polyphenol hybridized ZIF-8 nanoparticles in situ by stirring to obtain drug-loaded polyphenol hybridized ZIF-8 nanoparticles.

[0007] A1: Dissolve zinc salt and 2-methylimidazole in buffer solution.

[0008] A2: Add polyphenols and drugs to the solution prepared in A1, mix well and react. After the reaction, centrifuge at 10,000 rpm for 5 minutes to obtain drug-loaded polyphenol hybrid ZIF-8 nanoparticles.

[0009] Step B: In situ synthesizing a calcium peroxide coating on the surface of the drug-loaded polyphenol hybrid ZIF-8 nanoparticles prepared in step A to obtain a pH and light dual-responsive drug-loaded antibacterial nanomaterial for plants.

[0010] B1: Add the drug-loaded polyphenol hybrid ZIF-8 nanoparticles and calcium salt obtained in step A into anhydrous ethanol.

[0011] B2: Add ammonia water and hydrogen peroxide solution respectively under stirring to initiate the reaction. After the reaction is completed, wash with anhydrous ethanol several times to obtain a plant-based drug-loaded antibacterial nanomaterial that is dual-responsive to pH and light.

[0012] Furthermore, the molar mass ratio of the zinc salt to 2-methylimidazole in step A1 is 1:1 to 1:16, and the zinc salt is selected from any one of zinc acetate, zinc chloride, zinc nitrate and zinc sulfate.

[0013] The volume of the buffer solution is 5-30 mL, and the buffer solution is selected from any one of water, phosphate buffer solution, and tris(hydroxymethyl)aminomethane hydrochloride solution.

[0014] Furthermore, in step A2, the concentration of polyphenol is 0.5 mg / mL to 1.5 mg / mL, the concentration of drug is 0.5 mg / mL to 2 mg / mL, and the reaction time is 1 to 3 hours.

[0015] Furthermore, in step B1, the mass ratio of the drug-loaded polyphenol hybrid ZIF-8 nanoparticles to the calcium salt is 1:10 to 1:20, and the calcium salt is selected from any one of calcium chloride, calcium nitrate, calcium bicarbonate, and calcium gluconate;

[0016] The volume of anhydrous ethanol is 5 to 14 mL.

[0017] Furthermore, in step B2, the concentration of aqueous ammonia is 0.1M to 1M, the concentration of hydrogen peroxide is 0.1M to 1M, the volume ratio of aqueous ammonia to hydrogen peroxide is 1:1 to 1:3, and the reaction time is 1 min to 20 min.

[0018] Furthermore, in step A2, the polyphenol is selected from any one of dopamine, tannic acid, caffeic acid, gallic acid, epigallocatechin gallate (EGCG), and syringic acid.

[0019] Furthermore, in step A2, the drug is selected from any one of berberine, apigenin, betaine, curcumin, nitromycin, vancomycin, and allicin.

[0020] The beneficial technical effects of the present invention are:

[0021] (1) The present invention is based on mussel adhesion chemistry, wherein the introduction of polyphenol functional groups enables ZIF-8 to have good dispersibility in aqueous solution; at the same time, after polyphenol hybridization, the photocatalytic performance and drug loading capacity of ZIF-8 are improved, making the practical application range of polyphenol hybrid nanomaterials wider.

[0022] (2) The nanomaterial preparation method of the present invention is simple, low-cost, does not require complicated conditions, and is suitable for mass production.

[0023] (3) The nanomaterials prepared by the present invention have intelligent responsiveness to function, and also have long-term stability and responsive degradation.

[0024] (4) The nanomaterial prepared by the present invention has good biocompatibility and a wide range of applications. It has been proven that it can kill black mold and solanacearum. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The dopamine hybrid ZIF-8 nanomaterial is prepared according to the embodiment of the present invention.

[0026] Figure 2 The invention discloses a berberine-loaded antibacterial nanomaterial with dual response to pH and light, which is prepared in an embodiment of the invention.

[0027] Figure 3 This is an image of the pH and light dual-responsive berberine-loaded antibacterial nanomaterial observed under a transmission electron microscope.

[0028] Figure 4 The drug loading rate and encapsulation rate of the dopamine-hybridized ZIF-8 nanomaterial prepared in this example when different masses of berberine were added.

[0029] Figure 5 The antibacterial effect of the berberine-loaded antibacterial nanomaterial with dual response to pH and light prepared in this example on Botrytis cinerea was monitored by the flat plate coating method.

[0030] Figure 6 The antibacterial effect of the berberine-loaded antibacterial nanomaterial with dual response to pH and light prepared in this embodiment on Ralstonia solanacearum was monitored by the flat plate coating method. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] Embodiment 1:

[0033] Step 1: Drug-loaded polyphenol hybrid ZIF-8 nanoparticles

[0034] Prepare 25 mL of tris(hydroxymethylaminomethane) hydrochloride solution (pH 8.5) containing 0.1 M zinc acetate and 1.6 M 2-methylimidazole; add polyphenol dopamine and drug berberine powder to the above solution to make their concentrations 0.5 mg / mL and 2 mg / mL, respectively, mix well, react for 3 hours, and centrifuge at 10000 rpm for 5 minutes after the reaction to obtain dopamine hybrid ZIF-8 nanoparticles loaded with berberine, freeze-dry and store at low temperature.

[0035] Step 2: pH and light-responsive plant-based drug-loaded antibacterial nanomaterials

[0036] Weigh 4.5 mg of berberine-loaded dopamine hybrid ZIF-8 nanoparticles and 45 mg of calcium chloride and add them to 10 mL of anhydrous ethanol. Add 1 mL of 1 M ammonia water and 1 M hydrogen peroxide solution respectively under stirring to initiate the reaction. After the reaction is completed for 1 min, wash several times with anhydrous ethanol to obtain pH and light dual-responsive berberine-loaded antibacterial nanomaterials.

[0037] Embodiment 2:

[0038] Step 1: Drug-loaded polyphenol hybrid ZIF-8 nanoparticles

[0039] Prepare 20 mL of an aqueous solution containing 0.2 M zinc acetate and 1.6 M 2-methylimidazole; add polyphenol tannic acid and drug apigenin powder to the solution to make their concentrations 1 mg / mL and 3 mg / mL, respectively; mix well and react for 2 h; after the reaction, centrifuge at 10,000 rpm for 5 min to obtain tannic acid hybrid ZIF-8 nanoparticles loaded with apigenin, freeze-dry and store at low temperature.

[0040] Step 2: pH and light-responsive plant-based drug-loaded antibacterial nanomaterials

[0041] Weigh 9 mg of tannic acid hybrid ZIF-8 nanoparticles loaded with apigenin and 45 mg of calcium bicarbonate and add them to 12 mL of anhydrous ethanol. Under stirring, add 1 mL of 0.5 M ammonia water and 0.5 mL of 0.5 M hydrogen peroxide solution respectively to initiate the reaction. After the reaction is completed for 5 minutes, wash several times with anhydrous ethanol to obtain pH and light dual-responsive berberine-loaded antibacterial nanomaterials.

[0042] Embodiment 3:

[0043] Step 1: Drug-loaded polyphenol hybrid ZIF-8 nanoparticles

[0044] 15 mL of tris(hydroxymethylaminomethane) hydrochloride solution (pH 8.5) containing 0.4 M zinc nitrate and 1.6 M 2-methylimidazole was prepared; polyphenol caffeic acid and drug betaine powder were added to the above solution to make their concentrations 1 mg / mL and 0.5 mg / mL, respectively, and mixed well, reacted for 1 hour, and after the reaction was completed, centrifuged at a speed of 10000 rpm for 5 minutes to obtain caffeic acid hybrid ZIF-8 nanoparticles loaded with betaine, which were freeze-dried and stored at low temperature.

[0045] Step 2: pH and light-responsive plant-based drug-loaded antibacterial nanomaterials

[0046] Weigh 4.5 mg of tannic acid hybrid ZIF-8 nanoparticles loaded with apigenin and 90 mg of calcium nitrate and add them to 10 mL of anhydrous ethanol. Under stirring, add 0.5 mL of 0.1 M ammonia water and 1 mL of 0.1 M hydrogen peroxide solution respectively to initiate the reaction. After the reaction is completed for 10 minutes, wash several times with anhydrous ethanol to obtain pH and light dual-responsive berberine-loaded antibacterial nanomaterials.

[0047] Embodiment 4:

[0048] Step 1: Drug-loaded polyphenol hybrid ZIF-8 nanoparticles

[0049] Prepare 25 mL of 0.1 M zinc sulfate and 1.0 M phosphate buffer solution; add polyphenol gallic acid and drug curcumin powder to the above solution to make their concentrations 1.5 mg / mL and 1 mg / mL respectively, mix well, react for 2 hours, centrifuge at 10000 rpm for 5 minutes after the reaction to obtain gallic acid hybrid ZIF-8 nanoparticles loaded with curcumin, freeze-dry and store at low temperature.

[0050] Step 2: pH and light-responsive plant-based drug-loaded antibacterial nanomaterials

[0051] 4.5 mg of curcumin-loaded gallic acid hybrid ZIF-8 nanoparticles and 75 mg of calcium chloride were weighed and added to 10 mL of anhydrous ethanol. Under stirring, 0.5 mL of 0.5 M ammonia water and 1.5 mL of 0.5 M hydrogen peroxide solution were added respectively to initiate the reaction. After the reaction was completed for 10 minutes, the mixture was washed several times with anhydrous ethanol to obtain pH and light dual-responsive berberine-loaded antibacterial nanomaterials.

[0052] Embodiment 5:

[0053] Step 1: Drug-loaded polyphenol hybrid ZIF-8 nanoparticles

[0054] Prepare 10 mL of tris(hydroxymethylaminomethane) hydrochloride solution (pH 8.5) containing 0.4 M zinc chloride and 0.4 M 2-methylimidazole; add polyphenol dopamine and drug betaine powder to the above solution to make their concentrations 0.5 mg / mL and 1 mg / mL, respectively, mix well, react for 3 hours, and centrifuge at 10000 rpm for 5 minutes after the reaction to obtain betaine-loaded dopamine hybrid ZIF-8 nanoparticles, freeze-dry and store at low temperature.

[0055] Step 2: pH and light-responsive plant-based drug-loaded antibacterial nanomaterials

[0056] Weigh 4.5 mg of betaine-loaded dopamine hybrid ZIF-8 nanoparticles and 45 mg of calcium nitrate and add them to 15 mL of anhydrous ethanol. While stirring, add 0.5 mL of 0.5 M ammonia water and 1.5 mL of 0.5 M hydrogen peroxide solution respectively to initiate the reaction. After the reaction is completed for 15 minutes, wash several times with anhydrous ethanol to obtain pH and light dual-responsive berberine-loaded antibacterial nanomaterials.

[0057] Embodiment 6:

[0058] Step 1: Drug-loaded polyphenol hybrid ZIF-8 nanoparticles

[0059] Prepare 5 mL of a phosphate buffer solution containing 0.1 M zinc acetate and 1.0 M 2-methylimidazole; add polyphenol dopamine and drug nitromycin powder to the above solution to make their concentrations 0.5 mg / mL and 1 mg / mL, respectively, mix well, react for 3 hours, and after the reaction is completed, centrifuge at a speed of 10000 rpm for 5 minutes to obtain dopamine-loaded nitromycin hybrid ZIF-8 nanoparticles, freeze-dry and store at low temperature.

[0060] Step 2: pH and light-responsive plant-based drug-loaded antibacterial nanomaterials

[0061] 4.5 mg of dopamine hybrid ZIF-8 nanoparticles loaded with nitromycin and 90 mg of calcium gluconate were weighed and added to 10 mL of anhydrous ethanol. Under stirring, 0.5 mL of 1 M ammonia water and 1 mL of 1 M hydrogen peroxide solution were added respectively to initiate the reaction. After the reaction was completed for 10 minutes, the mixture was washed several times with anhydrous ethanol to obtain pH and light dual-responsive nitromycin-loaded antibacterial nanomaterials.

[0062] Embodiment 7:

[0063] Step 1: Drug-loaded polyphenol hybrid ZIF-8 nanoparticles

[0064] Prepare 25 mL of tris(hydroxymethylaminomethane) hydrochloride solution (pH 8.5) containing 0.1 M zinc acetate and 1.6 M 2-methylimidazole; add polyphenol dopamine and drug berberine powder to the above solution to make their concentrations 0.5 mg / mL and 2 mg / mL, respectively, mix well, react for 3 hours, and centrifuge at 10000 rpm for 5 minutes after the reaction to obtain dopamine hybrid ZIF-8 nanoparticles loaded with berberine, freeze-dry and store at low temperature.

[0065] Step 2: pH and light-responsive plant-based drug-loaded antibacterial nanomaterials

[0066] Weigh 4.5 mg of berberine-loaded dopamine hybrid ZIF-8 nanoparticles and 75 mg of calcium chloride and add them to 14 mL of anhydrous ethanol. While stirring, add 1 mL of 1 M ammonia water and 1 mL of 1 M hydrogen peroxide solution respectively to initiate the reaction. After the reaction is completed for 10 minutes, wash several times with anhydrous ethanol to obtain pH and light dual-responsive berberine-loaded antibacterial nanomaterials.

[0067] Example 8: The operation of this example is basically the same as that of Example 7, except that the berberine described in Example 7 is replaced by apigenin.

[0068] Example 9: The operation of this example is basically the same as that of Example 7, except that the berberine described in Example 7 is replaced by betaine.

[0069] Example 10: The operation of this example is substantially the same as that of Example 7, except that the berberine described in Example 7 is replaced by curcumin.

[0070] Example 11: The operation of this example is basically the same as that of Example 7, except that the berberine described in Example 7 is replaced by vancomycin.

[0071] Example 12: The operation of this example is basically the same as that of Example 7, except that the berberine described in Example 7 is replaced by allicin.

[0072] Example 13: The operation of this example is basically the same as that of Example 7, except that the berberine described in Example 7 is replaced by azomycin.

[0073] Example 14: The operation of this example is basically the same as that of Example 7, except that the dopamine described in Example 7 is replaced by EGCG.

[0074] Example 15: The operation of this example is basically the same as that of Example 7, except that the dopamine described in Example 7 is replaced by syringic acid.

[0075] The dopamine hybrid ZIF-8 nanomaterial prepared in Example 1 is as follows Figure 1As shown in FIG. 1 , the prepared pH and light dual-responsive berberine-loaded antibacterial nanomaterials are as follows Figure 2 shown.

[0076] Figure 3 This is an image of the pH and light dual-responsive berberine-loaded antibacterial nanomaterial observed under a transmission electron microscope. It can be seen that the nanoparticles are spherical, with a particle size of about 110 nm and are evenly dispersed.

[0077] Figure 4 The drug loading rate and encapsulation rate of the prepared dopamine hybridized ZIF-8 nanomaterials when different masses of berberine were added. It can be seen that when the amount of berberine added was 50 mg, the drug loading rate and encapsulation rate of the nanoparticles for berberine were the highest.

[0078] Figure 5 In order to prepare the pH and light dual-responsive berberine-loaded antibacterial nanomaterials, the antibacterial effect on Botrytis cinerea was monitored by the plate coating method. It can be seen that the bacterial circle area of ​​Botrytis cinerea on the plates treated with different concentrations of nanomaterials is reduced.

[0079] Figure 6 In order to prepare pH and light dual-responsive berberine-loaded antibacterial nanomaterials, the antibacterial effect on Ralstonia solanacearum was monitored by the plate coating method. It can be seen that with the increase of the concentration of the nanomaterial, the number of Ralstonia solanacearum colonies decreased.

[0080] In the present invention, due to the conjugated double bond structure of polyphenol itself, electron transfer can be achieved to inhibit the rapid recombination of electron holes in ZIF-8 and improve the photocatalytic performance of ZIF-8. At the same time, polyphenol has abundant phenolic hydroxyl groups that can interact with various structures, has good adhesion, and can improve the loading rate of drugs.

[0081] The calcium peroxide coating of the present invention not only has pH-responsive degradation and release of drugs, but also has a certain encapsulation effect on the drugs to prevent premature leakage, thereby improving the bioavailability of the drugs.

[0082] The present invention targets the microenvironment of pathogenic bacteria in crops, designs a polyphenol hybrid ZIF-8 nanoshell loaded with drugs with photocatalytic properties and a pH-responsive degradable calcium peroxide coating based on the mussel adhesion chemistry and the hydrogen peroxide self-supply mechanism of metal peroxides. The nanomaterial prepared by the present invention can be responsively degraded, has good biocompatibility, and has no side effects. The present invention has the advantages of controllable preparation process, low price, fast reaction speed, etc.

Claims

1. A method for preparing a pH and light dual-responsive drug-carrying antibacterial nanomaterial for plants, characterized in that: The following steps are involved: Step A: loading the drug into the polyphenol hybridized zinc-based metal organic framework ZIF-8 nanoparticles in situ by stirring to obtain the drug-loaded polyphenol hybridized ZIF-8 nanoparticles; A1: Dissolve zinc salt and 2-methylimidazole in buffer solution; A2: adding polyphenols and drugs to the solution prepared in A1, mixing and reacting uniformly, and centrifuging at 10,000 rpm for 5 min after the reaction to obtain drug-loaded polyphenol hybrid ZIF-8 nanoparticles; Step B: In situ synthesizing a calcium peroxide coating on the surface of the drug-loaded polyphenol hybrid ZIF-8 nanoparticles prepared in step A to obtain a pH and light-responsive drug-loaded antibacterial nanomaterial for plants; B1: adding the drug-loaded polyphenol hybrid ZIF-8 nanoparticles and calcium salt obtained in step A into anhydrous ethanol; B2: Add ammonia water and hydrogen peroxide solution respectively under stirring to initiate the reaction. After the reaction is completed, wash with anhydrous ethanol several times to obtain a plant-based drug-loaded antibacterial nanomaterial that is dual-responsive to pH and light.

2. The method for preparing a pH and light dual-responsive plant-based drug-carrying antibacterial nanomaterial according to claim 1, characterized in that: The molar mass ratio of the zinc salt to 2-methylimidazole in step A1 is 1:1 to 1:16, and the zinc salt is selected from any one of zinc acetate, zinc chloride, zinc nitrate, and zinc sulfate; The volume of the buffer solution is 5-30 mL, and the buffer solution is selected from any one of water, phosphate buffer solution, and tris(hydroxymethyl)aminomethane hydrochloride solution.

3. The method for preparing a pH and light dual-responsive plant-based drug-carrying antibacterial nanomaterial according to claim 1, characterized in that: In step A2, the concentration of polyphenol is 0.5 mg / mL to 1.5 mg / mL, the concentration of drug is 0.5 mg / mL to 2 mg / mL, and the reaction time is 1 to 3 hours.

4. The method for preparing a pH and light dual-responsive plant-based drug-carrying antibacterial nanomaterial according to claim 1, characterized in that: In the step B1, the mass ratio of the drug-loaded polyphenol hybrid ZIF-8 nanoparticles to the calcium salt is 1:10 to 1:20, and the calcium salt is selected from any one of calcium chloride, calcium nitrate, calcium bicarbonate, and calcium gluconate; The volume of the anhydrous ethanol is 5 to 14 mL.

5. The method for preparing a pH and light dual-responsive plant-based drug-carrying antibacterial nanomaterial according to claim 1, characterized in that: In step B2, the concentration of aqueous ammonia is 0.1M to 1M, the concentration of hydrogen peroxide is 0.1M to 1M, the volume ratio of aqueous ammonia to hydrogen peroxide is 1:1 to 1:3, and the reaction time is 1 min to 20 min.

6. The method for preparing a pH and light dual-responsive plant-based drug-carrying antibacterial nanomaterial according to claim 3, characterized in that: In step A2, the polyphenol is selected from any one of dopamine, tannic acid, caffeic acid, gallic acid, epigallocatechin gallate (EGCG), and syringic acid.

7. The method for preparing a pH and light dual-responsive plant-based drug-carrying antibacterial nanomaterial according to claim 3, characterized in that: The drug in step A2 is selected from any one of berberine, apigenin, betaine, curcumin, nitromycin, vancomycin and allicin.

Images