CN114948858A - Construction method and application of prodrug antibacterial system based on ATP activation - Google Patents
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Abstract
The invention relates to a construction method and application of an ATP (adenosine triphosphate) -activation-based prodrug antibacterial system. The method uses hydrogels to provide protection and reaction space for the prodrug and its activating enzyme; and the prodrug and the activating enzyme thereof are spatially separated by utilizing the encapsulation effect of MOFs; meanwhile, the response of Adenosine Triphosphate (ATP) of MOFs is utilized to play a role in producing toxic small molecules on demand. The prodrug can activate the catalytic action of enzyme to realize the high-efficiency production of toxic small molecules in one pot, and the produced high-toxicity medicine can realize effective transportation through a hydrogel wall. The pharmacokinetics of the ATP-activated prodrug antibacterial system of the invention shows that the ATP-activated prodrug antibacterial system has high-efficiency controllable drug action.
Description
Technical Field
The invention relates to a construction method of a prodrug antibacterial system based on ATP activation, and controllable and efficient antibacterial action is realized in practical application.
Background
Enzyme-activated prodrug therapy is an effective strategy that is widely used in the cancer or antibacterial fields. The diversity and specificity of biological enzymes are utilized to convert nontoxic prodrugs into highly toxic drugs at specific time and place. The most common prodrug activation strategy is to use an enzyme that is overexpressed in the cellular microenvironment. However, this approach is often limited by insufficient endogenous enzymes in the target tissue region, so that the prodrug is slowly and insufficiently activated, which affects the actual therapeutic effect. In addition, not all prodrugs can be activated by endogenous enzymes. Therefore, efforts have been made to develop various carriers to achieve highly effective enzyme prodrug therapy by co-delivering exogenous enzymes and prodrugs to the target site. However, this manipulation often leads to different spatiotemporal distributions of the enzyme and prodrug in the target region due to their different accumulation and release behavior in the carrier. At the same time, simultaneous encapsulation of the enzyme and prodrug on a single carrier easily leads to premature activation of the prodrug. Therefore, the reasonable design of the prodrug system ensures that the prodrug system has a response treatment effect of distinguishing healthy tissues from diseased tissues, and has extremely important significance.
In order to maximize the efficacy of these stimulus-responsive systems, it is important to design a reasonable stimulation pathway to achieve therapeutic goals. Among them, Adenosine Triphosphate (ATP), a biomolecule in cells, has been widely used for reactive therapy, and has wide applicability. ATP molecules are the monetary unit of energy transfer within cells and are known to be present in the cytoplasm of cells at levels as high as 1 x 10 -2 M, ATP content in extracellular fluid of about 0.5 x 10 -3 And M. In addition, studies have found that ATP is also upregulated to varying degrees in cancerous or bacterially infected tissues. Therefore, after determining a specific stimulus, it is usually necessary to carefully design and synthesize a degradable nanomaterial or functional module to have a stimulus sensitivity of a specific reaction pathway.
Metal Organic Frameworks (MOFs), as a new class of biological nanocarriers, have unique properties such as high porosity, adjustable pore structure, stimuli responsiveness and controlled function, and have attracted much attention in recent years. In particular, the abundant metal nodes and organic functional groups of MOFs can provide an ultra-high-capacity intrinsic anchor point for capturing drugs. Compared with traditional templates, the MOFs can effectively carry drugs, release of the drugs occurs under controlled conditions, and the MOFs are candidate materials for preparing multifunctional and complicated carriers. However, the potential disadvantages of MOFs, such as poor water stability and biocompatibility, limit their application in biomedicine, and in order to increase their practical value, membrane structures, core-shell structures and MOFs particle-loaded hydrogel composites have been developed. Based on the characteristics of the three-dimensional porous structure, the biocompatibility microenvironment and the like of the hydrogel, the long-term immobilization of macromolecules such as MOFs particles, biological enzymes and the like can be realized, and a place required by enzyme reaction is provided. However, in the recently developed hydrogels containing MOFs particles, the macroscopic volume of the hydrogel lacks flexibility and versatility in specific delicate applications. Furthermore, the random encapsulation and distribution of MOFs in hydrogels makes them difficult to use in practical applications where controlled and programmable release is required. These problems greatly limit their biomedical applications. Therefore, it is still desirable to create hydrogel/MOFs composites with engineered morphology and dynamic responsiveness for the construction of prodrug systems.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for constructing an ATP activated prodrug antibacterial system. The method has the characteristics of prodrug and enzyme separation distribution, ATP activation according to needs and the like, and can controllably and efficiently produce toxic small molecules.
In order to achieve the purpose, the invention adopts the following technical scheme.
A construction method of an ATP (adenosine triphosphate) activation based prodrug antibacterial system comprises the following steps:
(1) mixing the prodrug and the MOFs to obtain a prodrug @ MOFs;
(2) adding the prodrug @ MOFs and an enzyme into a pre-gel solution to prepare an enzyme/prodrug @ MOFs/pre-gel solution, and promoting the enzyme/prodrug @ MOFs/pre-gel solution to be gelatinized by utilizing a microfluidic technology to obtain an enzyme/prodrug @ MOFs immobilized hydrogel material;
(3) and washing the enzyme/prodrug @ MOFs immobilized hydrogel material to obtain the ATP-activated prodrug antibacterial system.
Further, the step (1) is specifically: the prodrug and the 2-methylimidazole solution are mixed and added with a zinc nitrate solution to react to obtain the prodrug @ MOFs (prodrug @ ZIF-8).
Furthermore, the molar ratio of the zinc nitrate to the 2-methylimidazole is 1: 4.
Further, the prodrug is indole-3-acetic acid (IAA).
Further, the enzyme is horseradish peroxidase (HRP).
Further, the pregel comprises acrylamide, N-methylene-bis-acrylamide, and 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone.
Further, the mass ratio of the acrylamide to the N, N-methylene bisacrylamide to the 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone is 15: 1: 1.
further, the washing refers to washing the enzyme/prodrug @ MOFs immobilized hydrogel material 3 times with water.
The invention also provides application of the ATP-activated prodrug antibacterial line in the antibacterial field.
The invention has the beneficial effects that: the enzyme, prodrug and MOFs immobilization method is simple, mild in condition requirement and easy to realize; the hydrogel can protect the catalytic activity of enzyme and the stability of prodrug @ ZIF-8; the MOFs/hydrogel composite material provides effective space-time distribution and loading for the prodrug and the enzyme, and the prodrug is not activated in advance while the enzyme keeps high activity; under the action of ATP reactive degradation, MOFs regularly disintegrate along with the change of ATP level and release prodrug, and the prodrug is activated in situ in a hydrogel microenvironment by enzyme to generate a large amount of highly toxic drugs, so that the efficient and controllable treatment of bacterial infection is improved.
Drawings
FIG. 1 is a scanning electron micrograph of FAM @ ZIF-8 in the presence or absence of ATP in example 1 of the present invention.
FIG. 2 shows ROS production behavior of HiZP after 0, 2, 5 mM ATP treatment in example 3 of the present invention, respectively.
FIG. 3 is a graph comparing the biocatalytic activities of the IAA @ ZIF-8 + HRP and HiZP systems.
FIG. 4 shows the antibacterial properties of different reaction systems IAA + HRP, IAA @ ZIF-8 + HRP and HiZP.
Detailed Description
The invention provides a construction method of an ATP (adenosine triphosphate) -activated prodrug antibacterial system, which uses Metal Organic Frameworks (MOFs) with ATP responsiveness as prodrug carriers to be spatially separated from enzymes and simultaneously used as ATP activation units; the prodrug and enzyme loaded hydrogel is constructed using a microfluidic device, providing reaction space for the prodrug and enzyme while preserving their stability. The ATP-activation-based prodrug antibacterial system is also called a hydrogel/metal organic framework composite material of an immobilized enzyme prodrug, and the specific construction steps can be as follows:
1) putting a certain mass of prodrug into 500 mM 2-methylimidazole solution of ligand, uniformly stirring, adding 125 mM zinc nitrate solution of metal salt, and continuously stirring for 3 hours to obtain a composite prodrug @ ZIF-8;
2) adding the prodrug @ ZIF-8 prepared in the step 1) and an enzyme into a pre-gel solution to prepare an enzyme/prodrug @ MOFs/pre-gel solution, and promoting the gelation of the enzyme/prodrug @ MOFs/pre-gel solution by utilizing a microfluidic technology to prepare an enzyme/prodrug @ MOFs immobilized hydrogel material;
3) washing the composite material obtained in the step 2) with water for 3 times to obtain the hydrogel/metal organic framework composite material of the immobilized prodrug and the enzyme.
Wherein the enzyme is horseradish peroxidase (HRP). The prodrug is indole-3-acetic acid (IAA). The pregel comprises acrylamide, N-methylene-bis-acrylamide and 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone. The mass ratio of the acrylamide to the N, N-methylene bisacrylamide to the 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone is 15: 1: 1. the molar ratio of the metal salt to the ligand is 1: 4.
The invention also provides application of the hydrogel/metal organic framework composite material of the immobilized enzyme prodrug in the antibacterial field.
In order that the above objects, features and advantages of the present invention can be more clearly understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments disclosed below.
In the following examples, fluorescein sodium is labeled FAM, indole-3-acetic acid is labeled IAA, horseradish peroxidase is labeled HRP, adenosine triphosphate is labeled ATP, acrylamide is labeled AAm, N, N-methylenebisacrylamide is labeled BIS, 2-hydroxy-4- (2-hydroxyethoxy) -2-methylpropiophenone is labeled PI, a metal organic framework is labeled ZIF-8, Staphylococcus aureus is labeled S. aureus, ZIF-8 of immobilized FAM is labeled FAM @ ZIF-8, and a hydrogel composite material of immobilized HRP and IAA @ ZIF-8 is labeled HiZP.
Example 1
IAA @ ZIF-8 or FAM @ ZIF-8 was prepared according to the following procedure and compared in scanning electron microscopy with/without ATP:
(1) 1 mL of 30 mg/mL IAA or FAM solution was added to 2 mL of 500 mM 2-methylimidazole in water, and 2 mL of 125 mM zinc nitrate in water was added under magnetic stirring;
(2) the resulting mixture was stirred at room temperature for 3 h;
(3) finally washing with PBS with pH = 7.4 for three times to obtain IAA @ ZIF-8 or FAM @ ZIF-8;
(4) respectively putting the obtained IAA @ ZIF-8 or FAM @ ZIF-8 into 0, 2 mM ATP solution for incubation for 30 min;
(5) and washing the incubated IAA @ ZIF-8 or FAM @ ZIF-8 with ultrapure water, and placing the washed product in a scanning electron microscope to observe the morphology change.
Example 2
HiZP was prepared according to the following procedure:
(1) weighing 3 mg/mL HRP, 1.5 mg/mL IAA @ ZIF-8, AAm (15% wt), BIS (1% wt) and PI (1% wt) to prepare 1 mL enzyme/pre-gel mixed solution;
(2) and promoting enzyme/pregel solution gelation according to a microfluidic technology to prepare the HiZP.
Example 3
Bound Rh6G-I - The ROS activity test of (1) records the intensity corresponding to the maximum emission peak by using a fluorescence spectrophotometer so as to detect the drug activity of the HiZP under the action of different ATP. The method comprises the following specific steps:
(1) phosphate (PBS) with pH = 7.4 is used as a buffer solution, the total volume is 100 μ L, the reaction temperature is 37 ℃, and the reaction time is 60 min;
(2) using 3 0.5mL centrifuge tubes which are respectively numbered as a, b and c;
(3) the preparation method of the solution in the centrifuge tube a comprises the following steps: 0 μ L ATP (40 mM), 10 μ L IK (20%) and 10 μ L HiZP mix in 80 μ L PBS buffer pH = 7.4;
(4) the preparation method of the solution in the centrifuge tube b comprises the following steps: 5 μ L ATP (40 mM), 10 μ L IK (20%) and 10 μ L HiZP mix in 75 μ L PBS buffer pH = 7.4;
(5) the preparation method of the solution in the centrifuge tube c comprises the following steps: 12.5 μ L ATP (40 mM), 10 μ L IK (20%) and 10 μ L HiZP mix in 67.5 μ L PBS buffer pH = 7.4;
(6) placing 3 centrifugal tubes at the reaction temperature of 37 ℃ for dark reaction for 60 min;
(7) then 5 uL of 200 mM Rh6G solution was added, shaken, and the relative fluorescence intensities of the solutions were measured separately using a fluorescence spectrophotometer.
The results of the assay are shown in FIG. 2, with increasing ATP concentration in the HiZP dispersion, resulting in Rh6G-I - The fluorescence intensity at 550 nm was gradually quenched. The immobilized IAA @ ZIF-8 of the hydrogel microsphere is shown to be disintegrated under the action of ATP, so that the IAA is released and activated. This is mainly due to the permeability of the hydrogel itself, which allows free entry of ATP, and the water environment inside it to some extent determines the ability of HRP to efficiently activate IAA to generate ROS.
Example 4
The biocatalytic activities of the free IAA @ ZIF-8 + HRP and HiZP systems were compared and the intensity corresponding to the maximum emission peak was then recorded using a fluorescence spectrophotometer. The specific method comprises the following steps:
(1) phosphate (PBS) with pH = 7.4 is used as a buffer solution, the total volume is 100 μ L, the reaction temperature is 37 ℃, and the reaction time is 60 min;
(2) respectively adding 10 mu L of HiZP and 10 mu L of IAA @ ZIF-8 + HRP mixed solution with equal concentration into 2 centrifuge tubes of 0.5mL and 2 centrifuge tubes;
(3) after 12.5. mu.L of ABTS (40 mM) substrate is added into each test tube and reacted in the dark, 5. mu.L of 200 mM Rh6G solution is added and shaken up, and the relative fluorescence intensity of the solution is measured by a fluorescence spectrophotometer respectively;
the detection result is shown in figure 3, compared with the mixed IAA @ ZIF-8 + HRP system, the construction of the HiZP system has higher toxic molecule conversion activity. It is shown that IAA can be released from nanocarriers under ATP induction and achieve high efficiency IAA prodrug activation and ROS release in subsequent processes.
Example 5
Different reaction systems: the antibacterial properties of IAA + HRP, IAA @ ZIF-8 + HRP and HiZP were compared, and then cultured using an agar plate, and the number of colonies was counted. The specific method comprises the following steps:
(1) taking a broth culture medium of 25 g/L as a solution, wherein the total volume is 2.2 mL, the reaction temperature is 37 ℃, and the reaction time is 30 h;
(2) using 3 glass tubes with 5mL, and numbering a, b and c respectively;
(3) the preparation method of the solution in the glass tube a comprises the following steps: 50 μ L of HiZP in 2.15 mL of 1X 10 8 (CFU)/mL s. aureus in broth solution;
(4) the preparation method of the solution in the glass tube b comprises the following steps: 50 μ L of mixed solution of IAA + HRP of the same concentration in 2.15 mL of 1X 10 8 (CFU)/mL s. aureus in broth solution;
(5) the preparation method of the solution in the glass tube c comprises the following steps: 50 mu.L of mixed solution of IAA @ ZIF-8 + HRP with the same concentration in 2.15 mL of 1X 10 8 (CFU)/mL s. aureus in broth solution;
(6) placing 3 glass tubes in a wet incubator at 37 ℃ for 30 hours;
(7) subsequently, the cells were cultured on an agar plate, and the number of colonies was counted.
As shown in fig. 4, the plates containing HiZP achieved almost complete sterilization after 20 h, whereas a significant amount of bacterial survival was observed in s. aureus treated with IAA alone or with a mixture of IAA @ ZIF-8 and HRP. Therefore, HiZP has higher antibacterial activity because the IAA released in the hydrogel matrix is in close contact with HRP, which can provide higher cytotoxic agent concentration. And the antibacterial activity of the IAA and HRP mixture was slightly higher than when the IAA was encapsulated, indicating the controlled state of IAA in ZIF 8. The plates containing the HiZP almost completely prevented colony formation after 20 h compared to the control group, while a large number of colonies were still visible on Luria-bertani (lb) agar treated with IAA alone or with a mixture of IAA @ ZIF-8 and HRP.
In conclusion, the invention provides a novel construction method of an enzyme prodrug antibacterial system, and the HiZP antibacterial material is prepared by utilizing the protection effect of hydrogel on enzyme space conformation, the loading effect of MOFs on the prodrug and the ATP activated release effect, so that the high-efficiency and controllable antibacterial effect is realized. In the antibacterial system, the prodrug has the catalytic action of enzyme, so that the high-efficiency catalytic conversion of small drug molecules in one pot is realized, and the produced high-toxicity drug can be effectively transported through a hydrogel wall.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A construction method of an ATP-activated prodrug antibacterial system comprises the following steps:
(1) mixing the prodrug and the MOFs to obtain a prodrug @ MOFs;
(2) adding the prodrug @ MOFs and an enzyme into a pre-gel solution to prepare an enzyme/prodrug @ MOFs/pre-gel solution, and promoting the enzyme/prodrug @ MOFs/pre-gel solution to be gelatinized by utilizing a microfluidic technology to obtain an enzyme/prodrug @ MOFs immobilized hydrogel material;
(3) and washing the enzyme/prodrug @ MOFs immobilized hydrogel material to obtain the ATP-activation-based prodrug antibacterial system.
2. The method according to claim 1, wherein the step (1) is specifically: the prodrug and the 2-methylimidazole solution are mixed and added with a zinc nitrate solution to react to obtain the prodrug @ MOFs.
3. The method of claim 2, wherein the molar ratio of zinc nitrate to 2-methylimidazole is 1: 4.
4. The method of claim 1, wherein said prodrug is indole-3-acetic acid.
5. The method of claim 1, wherein the enzyme is horseradish peroxidase.
6. The method of claim 1, wherein the pre-gel comprises acrylamide, N-methylenebisacrylamide, and 2-hydroxy-4- (2-hydroxyethoxy) -2-methylpropiophenone.
7. The method according to claim 6, wherein the mass ratio of acrylamide, N-methylene bisacrylamide and 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone is 15: 1: 1.
8. the method of claim 1, wherein said washing is performed by washing said enzyme/prodrug @ MOFs-immobilized hydrogel material 3 times with water.
9. An ATP-activated prodrug based antibacterial system obtainable by a method according to any one of claims 1 to 8.
10. Use of the ATP-activated based prodrug antibacterial line according to claim 9 in the antibacterial field.
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| CN115475244A (en) * | 2022-09-30 | 2022-12-16 | 浙江理工大学 | Metal organic framework nano composite and preparation method and application thereof |
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| 张凤等: "《材料的化学制备与性能测试实验教程》", 中国纺织出版社, pages: 217 - 218 * |
| 翁宇豪: "MOFs/水凝胶仿生催化体系的构筑及其抗菌应用", no. 04, pages 2 - 3 * |
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| CN114958752A (en) * | 2022-05-17 | 2022-08-30 | 西南交通大学 | Multifunctional magnetic nano composite material and preparation method and application thereof |
| CN114958752B (en) * | 2022-05-17 | 2023-10-24 | 西南交通大学 | Multifunctional magnetic nanocomposite and preparation method and application thereof |
| CN115475244A (en) * | 2022-09-30 | 2022-12-16 | 浙江理工大学 | Metal organic framework nano composite and preparation method and application thereof |
| CN115475244B (en) * | 2022-09-30 | 2023-11-03 | 浙江理工大学 | Metal organic framework nano-composite and preparation method and application thereof |
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