Preparation method and application of targeting chimera

Abstract

The invention provides a preparation method and application of a targeting chimera, and the preparation method comprises the following steps: lactoferrin introduces azido sugar into surface glycosyl through glycometabolism labeling, and DNA enzyme is subjected to conjugate modification through a coupling agent; the modified DNA enzyme, lactoferrin with surface glycosyl introduced and chloroquine are subjected to a click chemical reaction, and the targeted chimera is obtained; compared with the existing immune activator, the NucleOTAC chimera obtained by the invention can be used as the immune activator to significantly enhance the bioavailability and focus targeted enrichment capacity of DNA enzyme, prevent the main configuration B-type DNA of eDNA in NETs from being converted into Z-type DNA, destroy the immune escape mechanism of bacteria, reactivate the extracellular sterilization network of neutrophil and activate the body immunity; the method is simple and easy to implement, the preparation process is simple, the property is stable, efficient hydrolysis damage of a tuberculosis biological membrane can be achieved, and host anti-tuberculosis immunotherapy is activated.

Description

Technical Field

[0001] The present invention relates to the technical field of infection immunotherapy, and in particular to a preparation method and application of a targeted chimera. Background Art

[0002] Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis and spread by aerosols. After invading the human body, it can infect the body's macrophages and multiply in large numbers, causing inflammation and damaging the body's immune system. According to statistics from the World Health Organization, there are 9.9 million new tuberculosis patients worldwide each year, of which about 500,000 tuberculosis patients are resistant to rifampicin, and about 78% of these patients are multidrug-resistant tuberculosis (MDR-TB) patients. In vitro drug sensitivity tests have confirmed that patients who are resistant to two or more first-line anti-tuberculosis drugs (such as isoniazid, rifampicin, etc.) are diagnosed as MDR-TB patients. Drug-resistant tuberculosis usually requires more expensive and highly toxic drugs, higher drug treatment doses, and longer treatment cycles, which can easily lead to poor tolerance of treatment regimens, poor treatment effects, and many serious adverse reactions, and bring heavy economic burdens to patients. It is a major challenge facing public health security in my country and even the world. Therefore, how to develop safe and efficient drug-resistant tuberculosis treatment regimens by activating host natural immunity is a key issue to promote the treatment effect of drug-resistant tuberculosis and curb the global spread of tuberculosis.

[0003] During tuberculosis infection, host immune responses mediated by neutrophils and macrophages are critical for infection control. Neutrophils, as the first line of defense, are rapidly recruited to the site of infection, where they undergo NETosis and release neutrophil extracellular traps (NETs) that capture and immobilize Mtb. These NETs are composed of DNA and peroxidase, an antimicrobial protein. The reticular DNA structure captures bacteria through charge interactions, and the peroxidase then kills the captured bacteria. In addition, chemokines released by neutrophils, such as TNF-α, IL-1β, IL-10, and IL-6, can recruit other immune cells, including macrophages, to the site of infection. NETs act as pseudo-opsonins to enhance phagocytic activity, thereby promoting the peroxidase of NETs by macrophages, which effectively kills the ingested bacteria. However, bacteria can convert the eDNA in NETs from B-DNA to Z-DNA, thereby inhibiting the ability of NETs to capture bacteria and hindering the phagocytosis of macrophages, resulting in bacterial immune escape and the immune bactericidal function of macrophages being blocked. Therefore, efficient degradation of Z-DNA is crucial for regulating the host immune response to drug-resistant Mycobacterium tuberculosis.

[0004] Targeting chimeras (TACs) represent a class of sophisticated molecular tools that can degrade specific substances, providing a powerful platform for the removal of pathogenic factors. For example, Proteolysis Targeting Chimeras (PROTACs) utilize the ubiquitin-proteasome system to dismantle target proteins, while RNase Recruiting Chimeras (RIBOTACs) utilize RNA quality control pathways to neutralize toxic transcripts.

[0005] However, in the prior art, Mycobacterium tuberculosis can escape immunity in the body in a variety of ways. The instability, short-term effect, low utilization, and poor immune activation of conventional immune activators in anti-tuberculosis treatment are problems that have not been solved.

[0006] Therefore, the related technologies in the prior art regarding targeted chimeras that can be used as immune activators still need to be further improved. Summary of the invention

[0007] In view of this, the present invention proposes a method for preparing and applying a targeted chimera, which solves the technical problems of stability, short-term effectiveness, low utilization, and poor immune activation of immune activators in the application of anti-tuberculosis treatment in the prior art.

[0008] The technical solution of the present invention is achieved in this way:

[0009] The present invention develops a NucleOTAC technology, i.e., a nano nucleic acid degradation targeted chimera, which combines an eDNA binding ligand such as (lactoferrin) with a self-carrying nuclease equipped with a DNA enzyme; the targeted eDNA binding is synergistic with the inherent clearance mechanism. As a nucleic acid degradation targeted chimera, the NucleOTAC technology embeds the DNA enzyme into the Z-DNA of NETs and converts the nuclease-resistant Z-DNA into degradable B-DNA, thereby achieving effective Z-DNA degradation and reactivation of innate immunity.

[0010] In one aspect, the present invention provides a targeting chimera, comprising the following steps:

[0011] Lactoferrin was labeled with sugar metabolism to introduce azido sugars into the surface sugar moieties, and DNA enzymes were conjugated and modified by coupling agents;

[0012] The modified DNA enzyme undergoes a click chemical reaction with lactoferrin introduced with surface sugar groups and chloroquine to obtain the targeted chimera.

[0013] On the basis of this technical solution, it is further preferred that the following steps are specifically included:

[0014] Lactoferrin and azido sugar are placed at 20-25° C. for 20-30 minutes, ultrafiltered and centrifuged, and diluted after removing azido sugar. At the same time, DNA enzyme and coupling agent are placed at 20-25° C. for 20-30 minutes for conjugation modification. The modified DNA enzyme and chloroquine are added to the lactoferrin solution to obtain a mixed solution, which is ultrasonicated at 0-4° C. and a solvent is added dropwise to obtain the targeted chimera.

[0015] On the basis of this technical solution, further preferably, the azido sugar includes Ac4GalNAz.

[0016] On the basis of this technical solution, further preferably, the coupling agent includes dibenzocyclooctyne-tetraethylene glycol-active ester (DBCO-PEG4-NHS ester).

[0017] On the basis of this technical solution, further preferably, the mass ratio of the modified DNA enzyme to chloroquine is 4-5:1.

[0018] On the basis of this technical solution, further preferably, the solvent includes anhydrous ethanol.

[0019] On the basis of this technical solution, further preferably, the volume ratio of the anhydrous ethanol to the mixed liquid is 1.5-2:1.

[0020] On the basis of this technical solution, further preferably, the conditions of the ultrafiltration centrifugation include a rotation speed of 4000 rpm, centrifugation for 10-15 min, and centrifugation 2-3 times.

[0021] On the other hand, the present invention also provides the use of the targeted chimera obtained by the method for preparing the targeted chimera in the first aspect in anti-tuberculosis immune activation.

[0022] More preferably, the targeted chimera obtained by the method for preparing the targeted chimera is used in DNA conformational transformation.

[0023] Compared with the prior art, the preparation method and application of the targeted chimera provided by the present invention have the following beneficial effects:

[0024] The NucleOTAC chimera proposed by the present invention, wherein lactoferrin introduces Ac4GalNAz into the surface sugar group through sugar metabolism labeling, presenting a reactive azo group; and the DNA enzyme is modified by DBCO-PEG4-NHS conjugation to introduce a dibenzocyclooctane (DBCO) group; finally, the NucleOTAC chimera is obtained by a click chemistry reaction between lactoferrin presented by the azo group and the DBCO-modified DNA enzyme; the chimera can be an anti-tuberculosis immune activator, which can greatly improve the hydrolysis and clearance efficiency of Z-type NETs and enhance the host's anti-tuberculosis immunity.

[0025] In addition, the present invention prepares NucleOTAC chimeras with targeted NET degradation function through click chemistry reaction. On the one hand, the DNA chimera chloroquine is used to reverse the NET configuration to type B NET (DNA sensitive). On the other hand, based on the enzymatic hydrolysis ability of DNA enzyme, NET is efficiently disintegrated.

[0026] The NucleOTAC chimera of the present invention significantly enhances the bioavailability and lesion-targeted enrichment ability of DNA enzymes, hinders the transformation of B-type DNA, the main configuration of eDNA in NETs, ​​into Z-type DNA, destroys the immune escape mechanism of bacteria, reactivates the extracellular bactericidal network of neutrophils, and activates the body's immunity; the method is simple and easy, the preparation process is simple, the properties are stable, and efficient hydrolysis and destruction of tuberculosis biofilms can be achieved, and host anti-tuberculosis immunotherapy can be activated. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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.

[0028] Figure 1 This is a characterization diagram of the NucleOTAC chimera particles in Example 4 of the present invention;

[0029] Figure 2 This is a diagram showing the DNA configuration effect of NucleOTAC chimera transforming NETs in Application Example 1 of the present invention;

[0030] Figure 3 This is a graph showing the therapeutic effect of NucleOTAC chimera immune activation in Application Example 2 of the present invention. DETAILED DESCRIPTION

[0031] 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.

[0032] The present invention provides a preparation method and application of a NucleOTAC chimera, wherein azido sugar is introduced into surface glycosylation of lactoferrin through sugar metabolism labeling, and DNA enzyme is conjugated and modified through a coupling agent; the modified DNA enzyme undergoes a click chemistry reaction with the lactoferrin with introduced surface glycosylation and chloroquine to obtain the NucleOTAC chimera, which can be an anti-tuberculosis immune activator, can greatly improve the hydrolysis and clearance efficiency of Z-type NET, and enhance the host's anti-tuberculosis immunity.

[0033] Example 1

[0034] A method for preparing a NucleOTAC chimera comprises the following steps:

[0035] Lactoferrin was labeled with sugar metabolism to introduce azido sugars into the surface sugar moieties, and DNA enzymes were conjugated and modified by coupling agents;

[0036] The modified DNA enzyme undergoes a click chemical reaction with lactoferrin introduced with surface sugar groups and chloroquine to obtain the targeted chimera.

[0037] NucleOTAC chimeras with targeted NET degradation function were prepared through click chemistry reaction. On the one hand, the DNA chimera chloroquine was used to reverse the NET configuration to type B NET. On the other hand, based on the enzymatic hydrolysis ability of DNA enzyme, NET was efficiently disintegrated.

[0038] In this embodiment, specifically, the following steps are included:

[0039] Lactoferrin and azido sugar are placed at 20-25° C. for 20-30 minutes, ultrafiltered and centrifuged, and diluted after removing azido sugar. At the same time, DNA enzyme and coupling agent are placed at 20-25° C. for 20-30 minutes for conjugation modification. The modified DNA enzyme and chloroquine are added to the lactoferrin solution to obtain a mixed solution, which is ultrasonicated at 0-4° C. and a solvent is added dropwise to obtain the targeted chimera.

[0040] Among them, lactoferrin contains more positively charged arginine and lysine, and its surface charge distribution is mostly positive under physiological conditions. It is quickly enriched in bacterial biofilms carrying a large amount of negative charge through electrostatic interactions.

[0041] In this embodiment, specifically, the azido sugar includes Ac4GalNAz.

[0042] In this embodiment, specifically, the coupling agent includes DBCO-PEG4-NHS ester.

[0043] In this embodiment, specifically, the mass ratio of the modified DNA enzyme to chloroquine is 4-5:1.

[0044] In this embodiment, specifically, the solvent includes anhydrous ethanol.

[0045] In this embodiment, more specifically, the volume ratio of the anhydrous ethanol to the mixed liquid is 1.5-2:1.

[0046] In this embodiment, more specifically, the conditions of the ultrafiltration centrifugation include a rotation speed of 4000 rpm, centrifugation for 10-15 min, and centrifugation 2-3 times.

[0047] Example 2

[0048] A method for preparing a NucleOTAC chimera comprises the following steps:

[0049] 20.0 mg lactoferrin and 40 μM Ac4GalNAz 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 15 min for a total of 3 times to remove excess Ac4GalNAz. The lactoferrin concentration was determined by nanodrop and diluted to 5 mg / mL.

[0050] DNA enzyme was treated with 20 μM DBCO-PEG4-NHS ester at 20° C. for 30 min to obtain DBCO-labeled DNA enzyme; DNA enzyme and chloroquine were added dropwise to the lactoferrin solution with an azo group at a mass ratio of 4:1 at 20° C. to obtain a mixed solution;

[0051] The mixed solution was sonicated at 0-4°C using an ultrasonic probe, with an ultrasonic power of 40 W and an amplitude of 27% for 5 min. During the sonication, 2 volumes of anhydrous ethanol were added dropwise to the mixed solution at a rate of 1 mL / min to obtain the NucleOTAC chimera.

[0052] After the ultrasound, the particle concentration was determined using nanodrop, and finally the particle size distribution and surface potential were measured using a particle size analyzer.

[0053] result:

[0054] Example 3

[0055] A method for preparing a NucleOTAC chimera comprises the following steps:

[0056] 30.0 mg lactoferrin and 60 μM Ac4GalNAz were added to 3 mL deionized water and placed in a shaker at 25°C for 20 min. Ultrafiltration centrifugation was performed at 4000 rpm for 10 min, twice to remove excess Ac4GalNAz. The lactoferrin concentration was determined by nanodrop and diluted to 5 mg / mL.

[0057] DNA enzyme was treated with 20 μM DBCO-PEG4-NHS ester at 25° C. for 30 min to obtain DBCO-labeled DNA enzyme; DNA enzyme and chloroquine were added dropwise to the lactoferrin solution with an azo group at a mass ratio of 5:1 at 25° C. to obtain a mixed solution;

[0058] The mixed solution was sonicated at 4°C using an ultrasonic probe, with an ultrasonic power of 40 W and an amplitude of 27% for 5 min. 1.5 times the volume of anhydrous ethanol was added dropwise to the mixed solution while sonicating, and the rate was maintained at 1 mL / min to obtain the NucleOTAC chimera.

[0059] After the ultrasound, the particle concentration was determined using nanodrop, and finally the particle size distribution and surface potential were measured using a particle size analyzer.

[0060] Example 4

[0061] A method for preparing a NucleOTAC chimera comprises the following steps:

[0062] 20.0 mg lactoferrin and 40 μM Ac4GalNAz were added to 2 mL deionized water and placed in a shaker at 22°C for 25 min. Ultrafiltration centrifugation was performed at 4000 rpm for 12 min for a total of 3 times to remove excess Ac4GalNAz. The lactoferrin concentration was determined by nanodrop and diluted to 4 mg / mL.

[0063] DNA enzyme was treated with 20 μM DBCO-PEG4-NHS ester at 22° C. for 25 min to obtain DBCO-labeled DNA enzyme; DNA enzyme and chloroquine were added dropwise to the lactoferrin solution with an azo group at a mass ratio of 4.5:1 at 22° C. to obtain a mixed solution;

[0064] The mixed solution was sonicated at 2° C. using an ultrasonic probe, with an ultrasonic power of 40 W and an amplitude of 27% for 5 min. 1.8 times the volume of anhydrous ethanol was added dropwise to the mixed solution while sonicating, and the rate was maintained at 1 mL / min to obtain the NucleOTAC chimera.

[0065] After the ultrasound, the particle concentration was measured using nanodrop, and finally the particle size distribution and surface potential were measured using a particle size analyzer. The results are as follows: Figure 1 As shown, the NucleOTAC chimera exhibited a cationic surface potential (+17.3 mV) and a size distribution of 25.4 ± 1.5 nm.

[0066] Application Example 1

[0067] The application of the NucleOTAC chimera obtained in Example 2 in the DNA configuration of transforming NETs specifically comprises the following steps:

[0068] The cells were divided into control group (PBS), DNase group (DNase), and NucleOTAC group (NucleOTAC).

[0069] Mycobacterium smegma (M.smeg) biofilms were cultured in 24-well plates for 5 days and treated with PBS, DNase or NucleOTAC. Neutrophils were isolated from human peripheral blood using a neutrophil extraction kit. 5×10 5 neutrophils, and the lower chamber was added with medium containing M.smeg treated with PBS, DNase or NucleOTAC, and the bottom was covered with cell slides; after 3 hours, the cell slides were collected, immunofluorescence staining was performed with B-DNA and Z-DNA specific primary antibodies, and incubated overnight at 4°C. Then, the corresponding secondary antibodies were incubated for 1 hour, and the NETs were observed using a laser confocal microscope.

[0070] result: Figure 2 The figure shows the expression changes of neutrophil extracellular network (NETs) under different treatments. Blue represents B-DNA (easily degraded) and yellow represents Z-DNA (not easily degraded). NucleOTAC chimera treatment can increase the expression of NETs containing B-DNA, indicating that NucleOTAC chimera induces effective neutrophil NETosis; at the same time, it can also reduce the expression of NETs containing Z-DNA, indicating that the chimera can convert Z-type NETs into easily degraded B-type NETs to further activate immunity.

[0071] Application Example 2

[0072] The application of the NucleOTAC chimera obtained in Example 3 in immune activation specifically comprises the following steps:

[0073] Grouping: control group, DNase group, NucleOTAC group (NucleOTAC)

[0074] THP-1 cells were cultured in RPMI 1640 supplemented with 10% bovine serum and 1% penicillin-streptomycin. M0 macrophages were first induced with PMA, and then M1 macrophages were stimulated with LPS and INF-γ. 5×10 5 M1 macrophages were added to the lower chamber, and the mixture of neutrophils and M.smeg treated with PBS, DNase and NucleOTAC chimera in the above application example 1 was added;

[0075] After 12 hours, 50 μL of the supernatant was spread on a solid Middlebrook 7H10 agar plate supplemented with 0.5% (w / v) glycerol and 10% (w / v) OADC. The antibacterial immune activity of the NucleOTAC chimera was determined by colony forming unit (CFU) analysis.

[0076] result: Figure 3 The bactericidal effect of macrophages on bacteria under different treatments. Compared with the DNase group, the survival rate of M. smeg was significantly reduced after NucleOTAC chimera treatment, indicating that the chimera can induce strong immune bactericidal activity.

[0077] As can be seen from the above, NucleOTAC chimera shows significant advantages in activating host anti-tuberculosis immunity, including the following aspects:

[0078] ① Targeted enrichment: NucleOTAC chimeras can target enrichment and penetrate into negatively charged NETs-rich areas through electrostatic interactions, thereby achieving targeted drug delivery in deep areas.

[0079] ② Reverse DNA configuration and enhance enzymatic degradation: NucleOTAC chimera can transform the configuration from Z-type DNA to B-type DNA by embedding into the eDNA double-strand through the function of DNA intercalator chloroquine, significantly reducing the composition of Z-type DNA in NETs and enhancing the hydrolysis efficiency of DNA enzyme on eDNA components.

[0080] ③Immune activation and sterilization: Under the action of NucleOTAC chimera, the Mycobacterium tuberculosis biofilm can inhibit the bacteria's utilization of host-derived eDNA, destroy the bacteria's immune escape mechanism, reactivate the extracellular bactericidal network of neutrophils, and activate the body's immunity.

[0081] In summary, the preparation method and application of a NucleOTAC chimera provided by the present invention adopts the click chemistry method to prepare a NucleOTAC chimera with targeted enrichment and immune activation functions. The obtained NucleOTAC chimera uses the DNA chimera agent chloroquine to prevent the conformational transformation ability of eDNA, significantly improve the hydrolysis ability of eDNA in NETs, ​​and can also destroy the immune escape mechanism of bacteria, reactivate the extracellular bactericidal network of neutrophils, and activate the body's immunity.

[0082] 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 targeted chimera, characterized in that: The following steps are involved: Lactoferrin was labeled with sugar metabolism to introduce azido sugars into the surface sugar moieties, and DNA enzymes were conjugated and modified by coupling agents; The modified DNA enzyme undergoes a click chemical reaction with lactoferrin introduced with surface sugar groups and chloroquine to obtain the targeted chimera.

2. The method for preparing a targeted chimera according to claim 1, characterized in that: The specific steps include: Lactoferrin and azido sugar are placed at 20-25° C. for 20-30 minutes, ultrafiltered and centrifuged, and diluted after removing azido sugar. At the same time, DNA enzyme and coupling agent are placed at 20-25° C. for 20-30 minutes for conjugation modification. The modified DNA enzyme and chloroquine are added to the lactoferrin solution to obtain a mixed solution, which is ultrasonicated at 0-4° C. and a solvent is added dropwise to obtain the targeted chimera.

3. The method for preparing a targeted chimera according to claim 2, characterized in that: The azido sugar includes Ac4GalNAz.

4. The method for preparing a targeted chimera according to claim 2, characterized in that: The coupling agent includes dibenzocyclooctyne-tetraethylene glycol-active ester.

5. The method for preparing a targeted chimera according to claim 2, characterized in that: The mass ratio of the modified DNA enzyme to chloroquine is 4-5:

1.

6. The method for preparing a targeted chimera according to claim 2, characterized in that: The solvent includes anhydrous ethanol.

7. The method for preparing a targeted chimera according to claim 6, characterized in that: The volume ratio of the anhydrous ethanol to the mixed liquid is 1.5-2:

1.

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

9. Use of the targeted chimera obtained by the method for preparing the targeted chimera according to any one of claims 1 to 8 in anti-tuberculosis immune activation.

10. Use of the targeting chimera obtained by the method for preparing the targeting chimera according to any one of claims 1 to 8 in DNA conformational transformation.

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