Preparation method and application of nano protein

Abstract

The invention provides a preparation method and application of nano protein, and the preparation method comprises the following steps: adopting lactoferrin as a carrier, reducing by using a reducing agent, stirring with DNA enzyme and a DNA chimeric agent, and obtaining the nano protein by using a nano precipitation method, the preparation method comprises the following steps: by taking lactoferrin as a nano-carrier, opening a folding structure of the lactoferrin by utilizing glutathione, then adding chloroquine and DNA enzyme, and finally, accelerating the reformation of a disulfide bond of the lactoferrin by stirring. On one hand, a DNA chimeric agent chloroquine is utilized to maintain the eDNA configuration in the biological membrane to be B type, and on the other hand, the mycobacterium tuberculosis biological membrane is efficiently disintegrated based on the enzymatic hydrolysis capability of DNA enzyme; the nano protein can efficiently grab iron ions at a focus part based on the iron chelation characteristic of the nano protein, so that bacteria die due to iron deficiency, and the bioavailability of DNA enzyme and the hydrolysis efficiency of a biological membrane can be improved to a great extent.

Description

Technical Field

[0001] The present invention relates to the technical field of biological infection treatment, and in particular to a preparation method and application of nano protein. Background Art

[0002] Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis and transmitted through aerosols. After invading the human body, it can infect the body's macrophages and multiply in large numbers. In order to create a stable living environment, tuberculosis bacteria will secrete an extracellular polymer matrix (eDNA, proteins, polysaccharides, etc.) after adhering to the surface of the tissue, wrapping itself up to form a dense biofilm. Studies have shown that the presence of biofilms not only increases the bacteria's adaptive resistance to antimicrobial drugs by 1,000 times, but also hinders the infiltration of immune cells to resist damage to the host's immune system, causing stubborn infection with Mycobacterium tuberculosis and drug-resistant tuberculosis, which is the main reason for hindering the treatment of clinical tuberculosis infection.

[0003] DNA enzyme can effectively degrade DNA. It can cleave single-stranded and double-stranded DNA into oligonucleotides with 5'-phosphorylated and 3'-hydroxylated ends. It is one of the potential drugs approved by the US Food and Drug Administration (FDA) to destroy eDNA and remove biofilm. Studies have shown that DNA enzyme-mediated eDNA degradation does prevent the formation of biofilm. However, further studies have shown that biofilm eDNA has two spatial configurations: B type (DNA enzyme sensitive) and Z type (DNA enzyme resistant). Due to the presence of DNABII protein in bacteria that binds and bends DNA with high affinity, the natural B type can be driven to Z type, resulting in Z type eDNA as the dominant configuration in mature biofilms, which has become a technical bottleneck in anti-biofilm treatment research. Chloroquine, as a DNA chimera, can be inserted into DNA fragments to inhibit the conversion of eDNA configuration to Z type DNA, destroy the structural rigidity of mature biofilms and release bacteria in biofilms, which can achieve the destruction of tuberculosis resistance mechanisms and the improvement of the effect of anti-tuberculosis drugs. On the other hand, due to the lack of effective lesion-targeting vectors, the bioavailability of DNA enzymes in the body will be greatly reduced, which greatly limits its application in the treatment of clinical biofilm 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 technologies of nanoproteins for destroying bacterial biofilms in the prior art still need to be further improved. Summary of the invention

[0006] In view of this, the present invention proposes a method for preparing and applying a nanoprotein, 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 nanoprotein comprising the following steps:

[0009] Lactoferrin is used as a carrier, reduced by a reducing agent, and then stirred with DNA enzyme and DNA chimera to obtain the nano protein by a nano precipitation method.

[0010] On the basis of this technical solution, it is further preferred that the method specifically comprises the following steps:

[0011] Lactoferrin is used as a carrier, placed with a reducing agent at 20-25°C for 0.5-1h, centrifuged, the reducing agent is removed, and then diluted, DNA enzyme and DNA chimera are added, mixed with the reduced lactoferrin, stirred to obtain a mixed solution, the pH is adjusted to 7.5-8, a solvent is added for precipitation, ultrafiltration centrifugation and filtration are performed to remove the solvent and free DNA enzyme and DNA chimera to obtain the nanoprotein.

[0012] On the basis of this technical solution, further preferably, the DNA chimera comprises chloroquine.

[0013] On the basis of this technical solution, further preferably, the reducing agent includes at least one of glutathione, tricarboxyethyl phosphate (TCEP), dithiothreitol, cysteine, homocysteine ​​or tris(2-carboxyethyl)phosphine.

[0014] On the basis of this technical solution, further preferably, the mass ratio of the lactoferrin to the reducing agent is 3-5:1.

[0015] On the basis of this technical solution, further preferably, the mass ratio of the DNA enzyme 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 the volume ratio of anhydrous ethanol to the mixed liquid is 1.5-2:1.

[0017] On the basis of this technical solution, further preferably, the ultrafiltration centrifugation conditions are a rotation speed of 4000 rpm, centrifugation for 10-15 min, and centrifugation 2-3 times.

[0018] On the basis of this technical solution, it is further preferred that the ultrafiltration centrifugation is performed using an ultrafiltration tube, and the molecular retention rate of the ultrafiltration tube is 100kD.

[0019] On the other hand, the present invention also provides the use of the nanoprotein obtained by the preparation method of the nanoprotein described in the first aspect in destroying the biofilm of Mycobacterium tuberculosis.

[0020] On the basis of this technical solution, further preferably, the present invention also provides the use of the nanoprotein obtained by the preparation method of the nanoprotein described in the first aspect in killing drug-resistant tuberculosis bacteria.

[0021] Compared with the prior art, the preparation method and application of nanoprotein provided by the present invention have the following beneficial effects:

[0022] Lactoferrin is used as a nanocarrier, and reducing agents such as glutathione and TCEP are used to unfold the folded structure of lactoferrin, then chloroquine and DNA enzyme are added, and finally the reformation of the disulfide bonds of lactoferrin is accelerated by stirring; wherein, the nanoprotein is prepared by the nanoprecipitation method by encapsulating chloroquine and DNA enzyme through disulfide bond reduction; on the one hand, the DNA chimera chloroquine is used to maintain the eDNA configuration in the biofilm as type B (DNA sensitive), and on the other hand, based on the enzymatic hydrolysis ability of DNA enzyme, the Mycobacterium tuberculosis biofilm is efficiently disintegrated.

[0023] Nanoproteins can efficiently grab iron ions from lesions based on their own iron chelating properties, causing bacteria to die due to iron deficiency. After administration of nanoproteins, the bioavailability of DNA enzymes and the efficiency of biofilm hydrolysis can be greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 This is a schematic diagram of the method for preparing the nanoprotein described in Example 1 of the present invention;

[0026] Figure 2 This is a characterization diagram of the nanoprotein obtained in Example 3 of the present invention;

[0027] Figure 3 The biomembrane targeting performance of the nanoprotein in Application Example 1 of the present invention is detected under a laser confocal microscope;

[0028] Figure 4 The biofilm hydrolysis ability of nanoproteins was evaluated by using crystal violet staining experiment in Application Example 1 of the present invention;

[0029] Figure 5 In Application Example 1 of the present invention, a mechanical indentation test is used to evaluate the biofilm destruction ability of nanoproteins. DETAILED DESCRIPTION

[0030] 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.

[0031] The invention provides a preparation method and application of a nano protein. Lactoferrin is used as a carrier, and after being reduced by a reducing agent, the nano protein is obtained by a nano precipitation method with DNA enzyme and chloroquine. The disulfide bond of the lactoferrin is opened mainly by a reducing agent, and the chloroquine and DNA enzyme are encapsulated by disulfide bond reduction. The obtained nano protein can greatly improve the bioavailability of the DNA enzyme and the biofilm hydrolysis efficiency.

[0032] Example 1

[0033] A method for preparing a nanoprotein, such as Figure 1 As shown, the following steps are included:

[0034] Lactoferrin is used as a carrier, placed with a reducing agent at 20-25°C for 0.5-1h, centrifuged, the reducing agent is removed, and then diluted, DNA enzyme and DNA chimera are added, mixed with the reduced lactoferrin, stirred to obtain a mixed solution, the pH is adjusted to 7.5-8, a solvent is added for precipitation, ultrafiltration centrifugation and filtration are performed to remove the solvent and free DNA enzyme and DNA chimera to obtain the nanoprotein.

[0035] In this embodiment, specifically, lactoferrin, as a natural cationic monomeric glycoprotein produced by human epithelial cells, can not only efficiently deliver drugs to negatively charged biofilm sites through electrostatic adsorption, but also utilize the special iron chelation sites in its structure to seize iron ions in the biofilm environment to block bacterial iron metabolism, block iron-dependent bacterial anaerobic respiration and antibiotic resistance, and ultimately lead to bacterial iron deficiency death. It is a natural cationic nanocarrier that decomposes biofilms and inhibits bacterial growth.

[0036] In this embodiment, specifically, the reducing agent includes at least one of glutathione, TCEP, dithiothreitol, cysteine, homocysteine ​​or tris(2-carboxyethyl)phosphine. Preferably, the reducing agent is glutathione.

[0037] In this embodiment, specifically, the DNA chimera includes chloroquine. The use of the DNA chimera chloroquine can prevent the conformational transformation ability of eDNA and significantly improve the hydrolysis ability of eDNA in the biofilm.

[0038] In this embodiment, specifically, the mass ratio of the lactoferrin to the reducing agent is 3-5:1, and more preferably, the mass ratio of the lactoferrin to the glutathione is 3.57:1.

[0039] In this embodiment, specifically, the mass ratio of the DNA enzyme to the chloroquine is 4-5:1.

[0040] In this embodiment, specifically, the solvent is anhydrous ethanol, and the volume ratio of anhydrous ethanol to the mixed liquid is 1.5-2:1.

[0041] In this embodiment, specifically, the ultrafiltration centrifugation conditions are a rotation speed of 4000 rpm, centrifugation for 10-15 min, and centrifugation 2-3 times.

[0042] In this embodiment, specifically, the ultrafiltration centrifugation is performed using an ultrafiltration tube, and the molecular retention rate of the ultrafiltration tube is 100 kD.

[0043] Example 2

[0044] A method for preparing a nanoprotein comprises the following steps:

[0045] 20.0 mg lactoferrin and 5.60 mg glutathione were added to 2 mL deionized water, and then placed in a shaker at 20°C for 30 min, and ultrafiltration centrifuged through an ultrafiltration tube (MW = 10 kD) at a speed of 4000 rpm for 10 min, for a total of 3 times to remove excess GSH, and then the lactoferrin concentration was determined using nanodrop and diluted to 5 mg / mL.

[0046] After lactoferrin was reduced, nucleic acid degrading enzyme and chloroquine were added dropwise to the reduced lactoferrin solution at a mass ratio of 4:1, and the pH was adjusted to 7.5 with sodium hydroxide solution. Then, 2 volumes of ethanol solution were added while stirring with a magnetic stirrer at 20°C to precipitate the mixture for 4-8 hours. The particle concentration of the mixture was measured and recorded by nanodrop at 4h and 8h.

[0047] Finally, the product was ultrafiltrated and centrifuged through an ultrafiltration tube at a speed of 4000 rpm for 10 min. The product was centrifuged three times to remove ethanol, free nucleic acid degrading enzymes and chloroquine, and the purified nanoprotein was obtained.

[0048] As a result, the particle size distribution and surface potential were measured by a particle size analyzer.

[0049] Example 3

[0050] A method for preparing a nanoprotein comprises the following steps:

[0051] 20.0 mg lactoferrin and 5.60 mg glutathione were added to 2 mL deionized water, and then placed in a shaker at 20°C for 1 h, and ultrafiltration centrifuged through an ultrafiltration tube (MW = 10 kD) at a speed of 4000 rpm for 15 min, for a total of 3 times to remove excess GSH, and then the lactoferrin concentration was determined using nanodrop and diluted to 5 mg / mL.

[0052] After lactoferrin was reduced, nucleic acid degrading enzyme and chloroquine were added dropwise to the reduced lactoferrin solution at a mass ratio of 5:1, and the pH was adjusted to 8 with sodium hydroxide solution. Then, 1.5 times the volume of ethanol solution was added while stirring with a magnetic stirrer at 25°C to precipitate the mixture for 4-8 hours. The particle concentration of the mixture was measured and recorded by nanodrop at 4h and 8h.

[0053] Finally, the product was ultrafiltrated and centrifuged through an ultrafiltration tube at a speed of 4000 rpm for 15 min and three times to remove ethanol, free nucleic acid degrading enzymes and chloroquine, thereby obtaining purified nanoprotein.

[0054] Results: The particle concentration was measured by nanodrop, and the nanoprotein was characterized by a particle size analyzer. The size and surface potential of the nanoprotein were Figure 2 shown.

[0055] Example 4

[0056] A method for preparing a nanoprotein comprises the following steps:

[0057] 20.0 mg lactoferrin and 5.60 mg glutathione were added to 3 mL deionized water, and then placed in a shaker at 22°C for 0.8 h, and ultrafiltered and centrifuged through an ultrafiltration tube (MW = 10 kD) at a speed of 4000 rpm for 12 min, a total of 3 times to remove excess glutathione, and then the lactoferrin concentration was determined using nanodrop and diluted to 5 mg / mL.

[0058] After lactoferrin was reduced, nucleic acid degrading enzyme and chloroquine were added dropwise to the reduced lactoferrin solution at a mass ratio of 4.5:1, and the pH was adjusted to 7.5 with sodium hydroxide solution. Then, 1.8 times the volume of ethanol solution was added while stirring with a magnetic stirrer at 22°C to precipitate the mixture for 4-8 hours. The particle concentration of the mixture was measured and recorded by nanodrop at 4h and 8h.

[0059] Finally, the product was ultrafiltrated and centrifuged through an ultrafiltration tube at a speed of 4000 rpm for 12 min and three times to remove ethanol, free nucleic acid degrading enzymes and chloroquine, thereby obtaining purified nanoprotein.

[0060] Application Example 1

[0061] Application of the nanoprotein obtained in Example 3 in destroying the biofilm of Mycobacterium tuberculosis

[0062] Fluorescently labeled nanoproteins were co-incubated with bacterial biofilms, and the targeted enrichment and hydrolysis destruction of bacterial biofilms were observed using a laser confocal microscope to analyze the targeted penetration and destructive effects of the nanoprotein solution on the biofilms. At the same time, the crystal violet staining experiment was used to evaluate the hydrolysis performance of the nanoprotein solution on the biofilm by staining the eDNA component in the Mycobacterium tuberculosis biofilm. The mechanical indentation test was used to determine the rigidity and hardness changes of the bacterial biofilm after treatment with the nanoprotein solution, and the destructive ability of the nanoprotein solution on the bacterial biofilm was evaluated.

[0063] The specific steps include:

[0064] The cells were divided into groups: a control group, a DNase group, and a nanoprotein group (DCNP). First, DNase and fluorescein isothiocyanate derivative (FITC-NHS) were stirred at room temperature at a molar ratio of 10:1 for 1 h, and unreacted FITC-NHS was removed by ultrafiltration with deionized water (MW=10 kDa) to obtain FITC-labeled DNase. Then, the FITC-labeled DNase was used to prepare FITC-labeled DCNP according to the preparation scheme of DCNP in Example 4.

[0065] The biofilms of Mycobacterium smegmatis expressing mcheery were cultured in 24-well plates at 37°C for 5 days. The biofilms were then incubated with FITC-DNase or DCNP for 2 hours, washed with PBS, and photographed using a laser confocal microscope.

[0066] The results are as follows Figure 3 As shown, compared with free DNase, DCNP achieved efficient biomembrane enrichment and penetration.

[0067] The Mycobacterium smegmatis biofilm was cultured in a 24-well plate at 37°C for 5 days and then incubated with PBS, DNase or DCNP for 2 hours. The supernatant was removed and crystal violet stain was added for incubation for 30 minutes. The supernatant was then rinsed three times with PBS and the precipitate was dissolved with anhydrous ethanol. The absorbance was measured on a microplate reader.

[0068] The results are as follows Figure 4As shown, DCNP can significantly reduce the content of biofilm compared with free DNA enzyme. Similarly, the biofilms treated with the above three different treatments were subjected to axial mechanical indentation analysis using a hybrid rheometer, and the data were collected using TRIOS v5 software and converted into stress-strain curves in GraphPad 8.0, where the deformation stress (DS) was represented by Young's modulus.

[0069] The results are as follows Figure 5 As shown, DCNPs showed a significant decrease in deformation stress (DS) and minimum Young's modulus compared to the other treatments.

[0070] It can be seen from the above embodiments that the nanoprotein drugs prepared by the present invention show significant advantages in the treatment of Mycobacterium tuberculosis biofilm. Including but not limited to ① targeted enrichment: through electrostatic interaction, targeted enrichment and penetration into the negatively charged biofilm eDNA can be achieved to achieve directional drug delivery in deep parts. ② Containing DNA configuration transformation and enhancing enzymatic degradation: Nanoproteins can prevent its configuration from B-type DNA to Z-type DNA by embedding the double-stranded eDNA through the function of DNA intercalator chloroquine, significantly reducing the composition of Z-type DNA in mature biofilms, and enhancing the hydrolysis efficiency of DNA enzymes on eDNA components in biofilms. ③ Iron-snatching sterilization: Nanoproteins can be used as a drug for the iron metabolism intervention of Mycobacterium tuberculosis, blocking the iron metabolism of Mycobacterium tuberculosis by competitively chelating iron ions, and achieving effective killing of the growth of drug-resistant tuberculosis bacteria.

[0071] In summary, the present invention provides a method for preparing a nanoprotein and an application thereof. Lactoferrin is used as a carrier and glutathione is used to open the folded structure of lactoferrin. On the one hand, the DNA chimera chloroquine is used to maintain the eDNA configuration in the biofilm as type B. On the other hand, based on the enzymatic hydrolysis ability of DNA enzyme, the Mycobacterium tuberculosis biofilm is efficiently disintegrated.

[0072] 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 nanoprotein, characterized in that: The following steps are involved: Lactoferrin is used as a carrier, reduced by a reducing agent, and then stirred with DNA enzyme and DNA chimera to obtain the nano protein by a nano precipitation method.

2. The method for preparing nanoprotein according to claim 1, characterized in that: The specific steps include: Lactoferrin is used as a carrier, placed with a reducing agent at 20-25°C for 0.5-1h, centrifuged, the reducing agent is removed, and then diluted, DNA enzyme and DNA chimera are added, mixed with the reduced lactoferrin, stirred to obtain a mixed solution, the pH is adjusted to 7.5-8, a solvent is added for precipitation, ultrafiltration centrifugation and filtration are performed to remove the solvent and free DNA enzyme and DNA chimera to obtain the nanoprotein.

3. The method for preparing nanoprotein according to claim 2, characterized in that: The DNA intercalating agent includes chloroquine.

4. The method for preparing nanoprotein according to claim 2, 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 nanoprotein according to claim 4, characterized in that: The mass ratio of the lactoferrin to the reducing agent is 3-5:

1.

6. The method for preparing nanoprotein according to claim 3, characterized in that: The mass ratio of the DNA enzyme to the chloroquine is 4-5:

1.

7. The method for preparing nanoprotein according to claim 2, characterized in that: The solvent is anhydrous ethanol, and the volume ratio of anhydrous ethanol to the mixed liquid is 1.5-2:

1.

8. The method for preparing nanoprotein according to claim 2, characterized in that: The ultrafiltration centrifugation conditions are: a rotation speed of 4000 rpm, centrifugation for 10-15 minutes, and centrifugation 2-3 times.

9. The method for preparing nanoprotein according to claim 8, characterized in that: The ultrafiltration centrifugation is performed using an ultrafiltration tube, and the molecular retention rate of the ultrafiltration tube is 100 kD.

10. Use of the nanoprotein obtained by the method for preparing the nanoprotein according to any one of claims 1 to 9 in destroying the biofilm of Mycobacterium tuberculosis.

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