Preparation method and application of directional degradation liquid
By using natural protein carriers to react with Michael addition of DNA enzymes and chloroquine, a directed degradation solution was prepared, which solved the short-acting and low-utility problems of DNA enzymes in biofilm treatment, and achieved efficient destruction of bacterial biofilms and improvements. Effects of drug sensitivity.
Patent Information
- Application Number
- CN202411659601.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-19
- Publication Date
- 2025-02-14
AI Technical Summary
In the prior art, the short-acting, low utilization, lack of targeting and poor Z-DNA hydrolysis ability of DNA enzymes in biofilm therapy applications are difficult to effectively destroy bacterial biofilms.
A natural protein such as lactoferrin is used as a carrier, and after reducing the reducing agent, Michael addition reaction occurs with the modified DNA enzyme and chloroquine modified by the coupling agent to prepare a directional degradation solution. This method improves the bioavailability of DNA enzymes and the biofilm hydrolysis efficacy through electrostatic interaction and sonication.
It significantly enhances the bioavailability of DNA enzymes and the lesion-directed enrichment ability, can effectively destroy bacterial biofilms, improve bacterial sensitivity to drugs, and enhance the effect of anti-tuberculosis drugs.
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Figure CN119424669A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of infection treatment, and in particular to a preparation method and application of a directional degradation liquid. Background Art
[0002] After bacteria adhere and colonize on biological or non-biological surfaces, they rely on the cross-linking of non-water-soluble extracellular polymeric substances (EPS) such as proteins, polysaccharides, and extracellular DNA (eDNA) synthesized by themselves to form a dense biofilm. This structure not only provides physical protection for the bacterial community, but also greatly reduces their sensitivity to antibiotics and other drugs, which is one of the main reasons why chronic infections are difficult to cure and antibiotic treatment fails.
[0003] First, the physical barrier formed by the biofilm limits the penetration of antibiotics, making it difficult for drugs to reach effective bactericidal concentrations. The bacteria inside it are in a dormant or slow metabolic state, and their sensitivity to antibiotics is also reduced. In addition, there may be different microenvironments inside the biofilm, such as differences in oxygen concentration and pH value, which may also affect the activity of antibiotics. Secondly, bacteria in the biofilm can quickly exchange resistance genes through horizontal gene transfer, accelerating the spread of resistant strains. This gene exchange mechanism enables the bacterial population in the biofilm to quickly adapt and resist external antibiotic pressure. Furthermore, the presence of biofilms enhances the resistance of bacteria to antibiotics. Even under the action of antibiotics, bacteria inside the biofilm may survive by forming persistent cells, which can survive under harsh conditions and resume normal growth in the future. Studies have shown that the presence of biofilms not only increases the adaptive resistance of bacteria to antimicrobial drugs by 1,000 times, but also hinders the infiltration of immune cells to resist damage to the host immune system. The latest research has found that Mycobacterium tuberculosis can form biofilms in vivo and in vitro, which may be the main reason for the stubborn infection of Mycobacterium tuberculosis and drug-resistant tuberculosis, and hinders the treatment of clinical tuberculosis infection. eDNA in EPS is the extracellular DNA that bacteria actively secrete or release by lysis after death, and is also the most critical structural skeleton component in the biofilm formation process. DNase has been approved by the FDA for the degradation of eDNA in Mtb biofilms, but faces limitations due to insufficient biofilm penetration and incomplete eDNA degradation. At the same time, eDNA includes Z-type and B-type DNA (B-DNA). It is worth noting that Z-type DNA (Z-DNA) that resists DNase degradation accumulates as the biofilm matures. The steric hindrance of Z-DNA significantly hinders DNase from approaching and cutting eDNA. This Z-type DNA confers resistance to the biofilm structure to DNase, thereby greatly promoting the persistence of bacterial biofilms that rely on eDNA. Overcoming the challenges of degrading Z-DNA and eradicating established biofilms are essential for combating refractory tuberculosis infections.
[0004] However, in the prior art, the shortcomings of DNA enzymes in biofilm treatment applications, such as short-term effectiveness, low utilization, lack of targeting, and poor Z-DNA hydrolysis ability, are problems that have not been solved.
[0005] Therefore, the related technology of directional degradation liquid for destroying bacterial biofilm in the prior art still needs to be further improved. Summary of the invention
[0006] In view of this, the present invention proposes a preparation method and application of a directional degradation liquid, which solves the technical problems of short-term effectiveness, low utilization, lack of targeting and poor Z-DNA hydrolysis ability of DNA enzymes in biofilm treatment applications in the prior art.
[0007] The technical solution of the present invention is achieved in this way:
[0008] In one aspect, the present invention provides a directional degradation solution, comprising the following steps:
[0009] The natural protein is used as a carrier, and after being reduced by a reducing agent, a Michael addition reaction is carried out with DNA enzyme modified by a coupling agent and chloroquine to obtain the directional degradation solution.
[0010] On the basis of this technical solution, it is further preferred that the method specifically comprises the following steps:
[0011] The natural protein is used as a carrier, reacted with a reducing agent at 20-25°C, diluted after removing the reducing agent, and mixed with a DNA enzyme and a coupling agent at the same time, incubated at 0-4°C overnight, mixed with the reduced natural protein and chloroquine to obtain a protein mixture, ultrasonicated, and a solvent is added dropwise for precipitation. After centrifugation and filtration, the directional degradation solution is obtained.
[0012] On the basis of this technical solution, further preferably, the natural protein includes one of lactoferrin, transferrin, lactoglobulin or hemoglobin.
[0013] On the basis of this technical solution, further preferably, the reducing agent includes at least one of glutathione, TCEP, dithiothreitol, cysteine, homocysteine or tris(2-carboxyethyl)phosphine.
[0014] On the basis of this technical solution, further preferably, the coupling agent is maleimide-tetraethylene glycol-succinimidyl acrylate (MAL-PEG4-NHS).
[0015] On the basis of this technical solution, further preferably, the mass ratio of the natural protein to the chloroquine is 4-5:1.
[0016] On the basis of this technical solution, it is further preferred that the solvent is anhydrous ethanol and chloroform, and the volume ratio of anhydrous ethanol, chloroform and the protein mixture is 1.5-2:0.05:1.
[0017] On the basis of this technical solution, further preferably, the centrifugal conditions are a rotation speed of 4000 rpm and centrifugation for 10-15 min.
[0018] On the other hand, the present invention also provides the use of the directional degradation liquid obtained by the preparation method of the directional degradation liquid described in the first aspect in degrading bacterial biofilm.
[0019] On the basis of this technical solution, further preferably, the present invention also provides the use of the directional degradation liquid obtained by the preparation method of the directional degradation liquid described in the first aspect in improving the drug sensitivity of bacteria.
[0020] Compared with the prior art, the preparation method and application of the directional degradation liquid provided by the present invention have the following advantages:
[0021] Beneficial effects:
[0022] By using the natural cationic glycoprotein lactoferrin as a carrier, the surface charge distribution of lactoferrin under physiological conditions is mostly positive, and it can be quickly and directionally enriched in the bacterial biofilm carrying a large amount of negative charge through electrostatic interaction; under the action of Michael addition reaction, lactoferrin is combined with DNA enzyme and chloroquine to form a directional degradation liquid, and the nucleic acid intercalator chloroquine is used to maintain the eDNA configuration in the biofilm as type B (DNA sensitive), and based on the enzymatic hydrolysis ability of DNA enzyme, the bacterial biofilm is efficiently disintegrated, thereby improving the drug sensitivity of bacteria.
[0023] Lactoferrin in the system, a nanocarrier with natural cationic properties and iron chelating properties, uses the principle of electrostatic adsorption to efficiently deliver drugs to the biofilm area. At the same time, through the unique iron chelating sites in its structure, it captures iron ions in the environment to prevent the bacterial iron metabolism process, ultimately leading to bacterial iron deficiency death; the directional degradation system can quickly target and enrich the bacterial biofilm site through electrostatic interactions, and then use the function of the DNA chimera chloroquine to maintain the eDNA configuration of the bacterial biofilm site in the B-type DNA sensitive to DNA enzymes, and finally efficiently disintegrate the bacterial mycobacterial biofilm based on the enzymatic hydrolysis performance of DNA enzymes. The directional degradation liquid can promote the penetration of antibacterial drugs by destroying the strong physical barrier of the biofilm of Mycobacterium tuberculosis, effectively kill the growth of drug-resistant bacteria, and enhance the sensitivity of anti-tuberculosis drugs.
[0024] The preparation method provided by the invention has simple process and stable properties, and can realize directional degradation and removal of bacterial biofilm. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0026] Figure 1 This is a characterization data diagram of the directional degradation liquid obtained in Example 1 of the present invention;
[0027] Figure 2 This is a performance effect diagram of the directional degradation liquid for degrading biofilm in Application Example 1 of the present invention;
[0028] Figure 3 This is a diagram showing the effect of using the directional degradation liquid in Application Example 2 of the present invention to improve bacterial drug sensitivity. DETAILED DESCRIPTION
[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0030] The invention provides a preparation method and application of a directional degradation liquid. Natural protein is used as a carrier, and after being reduced by a reducing agent, Michael addition reaction occurs with DNA enzyme modified by a coupling agent and chloroquine to obtain the directional degradation liquid. The disulfide bonds of the natural protein are mainly opened by the reducing agent, while the DNA enzyme is modified by MAL-PEG4-NHS conjugation, and then the directional degradation liquid is obtained by Michael addition reaction between the protein with a thiol group and the MAL-modified DNA enzyme, which can greatly improve the bioavailability and biofilm hydrolysis efficiency of the DNA enzyme.
[0031] Example 1
[0032] A method for preparing a directional degradation liquid comprises the following steps:
[0033] The natural protein is used as a carrier, reacted with a reducing agent at 20-25°C, diluted after removing the reducing agent, and mixed with a DNA enzyme and a coupling agent at the same time, incubated at 0-4°C overnight, mixed with the reduced natural protein and chloroquine to obtain a protein mixture, ultrasonicated, and a solvent is added dropwise for precipitation. After centrifugation and filtration, the directional degradation solution is obtained.
[0034] In this embodiment, specifically, the natural protein includes one of lactoferrin, transferrin, lactoglobulin or hemoglobin, more preferably lactoferrin.
[0035] In this embodiment, specifically, the reducing agent includes at least one of glutathione, TCEP, dithiothreitol, cysteine, homocysteine or tris(2-carboxyethyl)phosphine.
[0036] In this embodiment, specifically, the coupling agent is MAL-PEG4-NHS.
[0037] In this embodiment, specifically, the mass ratio of the natural protein to the chloroquine is 4-5:1.
[0038] In this embodiment, specifically, the solvent is anhydrous ethanol and chloroform, and the volume ratio of anhydrous ethanol, chloroform and the protein mixture is 1.5-2:0.05:1.
[0039] In this embodiment, specifically, the centrifugal conditions are a rotation speed of 4000 rpm and centrifugation for 10-15 min.
[0040] Example 2
[0041] A method for preparing a directional degradation liquid comprises the following steps:
[0042] 20.0 mg lactoferrin and 1 mM TCEP were added to 2 mL deionized water and placed in a shaker at 20°C for 30 min. Ultrafiltration centrifugation was performed at 4000 rpm for 10 min for a total of 3 times to remove excess TCEP. The lactoferrin concentration was determined using nanodrop and diluted to 5 mg / mL.
[0043] After lactoferrin is reduced, DNA enzyme is mixed with 0.1 μl MAL-PEG4-NHS, incubated at 4°C overnight, and added dropwise to the reduced lactoferrin solution with chloroquine at a mass ratio of 4:1 to obtain a protein mixture. The protein mixture is sonicated on ice using an ultrasonic probe, with an ultrasonic power of 40 W and an amplitude of 27% for 5 min. While sonicating, 2 times the volume of anhydrous ethanol and 5% volume of chloroform are added dropwise to the protein mixture, and the temperature is maintained at 1 mL / min. After the end of the ultrasound, ultrafiltration and centrifugation are performed at 4000 rpm for 10 min for a total of 3 times to remove free DNA enzyme and chloroquine, thereby obtaining a purified directional degradation solution.
[0044] Example 3
[0045] A method for preparing a directional degradation liquid comprises the following steps:
[0046] 20.0 mg lactoferrin and 1 mM TCEP were added to 3 mL deionized water and placed in a shaker at 25°C for 30 min. Ultrafiltration centrifugation was performed at 4000 rpm for 10 min for a total of 3 times to remove excess TCEP. The lactoferrin concentration was determined using nanodrop and diluted to 5 mg / mL.
[0047] After lactoferrin is reduced, DNA enzyme is mixed with 0.2 μl MAL-PEG4-NHS, incubated overnight at 0°C, and added dropwise to the reduced lactoferrin solution with chloroquine at a mass ratio of 5:1 to obtain a protein mixture. The protein mixture is sonicated on ice using an ultrasonic probe, with an ultrasonic power of 40 W and an amplitude of 27% for 5 min. While sonicating, 1.5 times the volume of anhydrous ethanol and 5% volume of chloroform are added dropwise to the protein mixture, and the temperature is maintained at 1 mL / min. After the end of the ultrasound, ultrafiltration and centrifugation are performed at 4000 rpm for 15 min for a total of 3 times to remove free DNA enzyme and chloroquine, thereby obtaining a purified directional degradation solution.
[0048] Results: The particle concentration was measured by nanodrop, and the DCNP was characterized by a particle size analyzer. The size and surface potential of DCNP were Figure 1 shown.
[0049] Application Example 1
[0050] The application of the directional degradation liquid obtained in Example 3 in degrading bacterial biofilm comprises the following steps:
[0051] Sputum samples from 10 patients with positive Mycobacterium tuberculosis were collected (collected from the Department of Pulmonary Medicine of a hospital, with signed informed consent). The sputum samples used in this embodiment were collected according to the clinical standard sputum collection procedure, with reference to the "China Tuberculosis Prevention and Control Program·Sputum Smear Microscopy Standardized Operation and Quality Assurance Manual", and by visual inspection, yellow, gray, rust, bloody, purulent, thick, and lumpy specimens were accepted as the standard.
[0052] Firstly, the macroscopic destructive effect of the directional degradation solution on biofilm was investigated. Fresh sputum was divided into three equal parts and added to a 24-well plate. They were incubated with PBS, DNase and directional degradation solution for 24 hours respectively. The control group (with PBS), DNase group (with DNase) and directional degradation solution group (with directional degradation solution) were set up to observe the overall destructive effect on sputum.
[0053] Secondly, the degradation of biofilm components by the targeted degradation solution was investigated. After the sputum was incubated with PBS, DNase and the targeted degradation solution for 24 hours, it was quickly frozen in liquid nitrogen and stored at -80°C. 10 μm continuous sections were cut using a freezing microtome, adhered to a slide and stored at -80°C. The sections were then immunostained and observed using a confocal system to investigate the abundance of Z- and B-type DNA.
[0054] Results, such as Figure 2 As shown, the TB sputum solids were completely disintegrated after treatment with the targeted degradation fluid and the overall eDNA in the biofilm present in the sputum was reduced.
[0055] Application Example 2
[0056] The application of the directional degradation solution obtained in Example 4 to improving the drug sensitivity of bacteria comprises the following steps:
[0057] Fresh sputum was divided into four equal parts and added to a 24-well plate, and an untreated group, a control group, a DNase group, and a directed degradation liquid group were set up. The untreated group and the control group were treated with PBS, and the DNase group and the directed degradation liquid group were added with DNase and directed degradation liquid, respectively, and incubated at 37°C for 24 hours. Subsequently, the control group, the DNase group, and the directed degradation liquid group were incubated with rifampicin for 12 hours. 50 μL of the supernatant was spread on a solid Middlebrook 7H10 agar plate added with 0.5% (w / v) glycerol and 10% (w / v) OADC. The increased drug sensitivity effect was determined by colony forming unit (CFU) analysis. As a result, Figure 3 As shown, intervention with targeted degradation fluid can significantly enhance the therapeutic effect of rifampicin against Mtb. Compared with rifampicin alone, targeted degradation fluid caused a 98% reduction in bacterial load.
[0058] In summary, the present invention provides a preparation method and application of a directional degradation liquid, which is obtained by the Michael addition reaction between a protein with a thiol group and a MAL-modified DNA enzyme, and finally the above mixture is further treated by ultrasound, which can greatly improve the bioavailability and biofilm hydrolysis efficiency of the DNA enzyme. On the one hand, the bioavailability and lesion-oriented enrichment ability of the DNA enzyme are significantly enhanced, and on the other hand, the directional degradation liquid can promote the penetration of antibacterial drugs and enhance the sensitivity of bacteria to drugs by destroying the strong biofilm physical barrier of Mycobacterium tuberculosis.
[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for preparing a directional degradation liquid, characterized in that: The following steps are involved: The natural protein is used as a carrier, and after being reduced by a reducing agent, a Michael addition reaction is carried out with DNA enzyme modified by a coupling agent and chloroquine to obtain the directional degradation solution.
2. The method for preparing the directional degradation liquid according to claim 1, characterized in that: The specific steps include: The natural protein is used as a carrier, reacted with a reducing agent at 20-25°C, diluted after removing the reducing agent, and mixed with a DNA enzyme and a coupling agent at the same time, incubated at 0-4°C overnight, mixed with the reduced natural protein and chloroquine to obtain a protein mixture, ultrasonicated, and a solvent is added dropwise for precipitation. After centrifugation and filtration, the directional degradation solution is obtained.
3. The method for preparing the directional degradation liquid according to claim 1, characterized in that: The natural protein includes one of lactoferrin, transferrin, lactoglobulin or hemoglobin.
4. The method for preparing the directional degradation liquid according to claim 1, characterized in that: The reducing agent includes at least one of glutathione, TCEP, dithiothreitol, cysteine, homocysteine or tris(2-carboxyethyl)phosphine.
5. The method for preparing the directional degradation liquid according to claim 1, characterized in that: The coupling agent is maleimide-tetraethylene glycol-succinimide acrylate.
6. The method for preparing the directional degradation liquid according to claim 2, characterized in that: The mass ratio of the natural protein to the chloroquine is 4-5:
1.
7. The method for preparing the directional degradation liquid according to claim 2, characterized in that: The solvents are anhydrous ethanol and chloroform, and the volume ratio of anhydrous ethanol, chloroform to the protein mixture is 1.5-2:0.05:
1.
8. The method for preparing the directional degradation liquid according to claim 2, characterized in that: The centrifugal conditions are a rotation speed of 4000 rpm and centrifugation for 10-15 min.
9. Use of the directional degradation liquid obtained by the preparation method of the directional degradation liquid according to any one of claims 1 to 8 in degrading bacterial biofilm.
10. Use of the directional degradation liquid obtained by the preparation method of the directional degradation liquid according to any one of claims 1 to 8 in improving bacterial drug sensitivity.