Bimodal nucleic acid removal system for treating intervertebral disc degenerative diseases and preparation and application methods thereof

Abstract

The invention discloses a bimodal nucleic acid removal system for treating an intervertebral disc degenerative disease as well as a preparation method and an application method of the bimodal nucleic acid removal system. A nucleus pulposus cell membrane vesicle is used for entrapping a small molecule mitochondrial membrane stabilizer, and the engineered nano vesicle is prepared. The preparation method comprises the following steps: mixing a polyamidoamine (PAMAM) dendritic polymer with a hyaluronic acid solution, adding the PAMAM dendritic polymer, and carrying out Schiff alkali reaction crosslinking to form hydrogel, so as to obtain the bimodal nucleic acid removal system. The hydrogel is combined with negative cfDNA released in tissue gaps through positive charges, inflammatory nucleic acid in the tissue microenvironment is efficiently removed, polarization of M1 type macrophages and release of inflammatory factors are inhibited, and the local immune microenvironment is improved. The engineered nanovesicles are used for targeted delivery of a small-molecule mitochondrial membrane stabilizer into nucleus pulposus cells, DNA leakage caused by increase of permeability of mitochondrial outer membranes is inhibited, the cell steady state is recovered, cell function degeneration is delayed, and inflammatory response is inhibited from two aspects of intracellular sources and tissue external environments through synergistic effects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical configuration bodies, and relates to a targeted delivery material, in particular to a dual-mode nucleic acid removal system for treating intervertebral disc degenerative disease and a preparation and application method thereof. BACKGROUND

[0002] Intervertebral disc degeneration (IVDD) is one of the main causes of chronic low back pain. The intervertebral disc is composed of a central nucleus pulposus, an external annulus fibrosus, and a cartilage endplate between adjacent vertebral bodies, among which the nucleus pulposus plays a key role in maintaining the structure and function of the intervertebral disc. The aging of nucleus pulposus cells is considered to be a key driver of the occurrence and development of IVDD. Aging cells secrete senescence-associated secretory phenotype (SASP) factors IL-6, IL-8, and TNF-α, which promote local inflammatory reactions and destroy the stability of the extracellular matrix (ECM), further exacerbating tissue degeneration. Studies have shown that increased mitochondrial outer membrane permeability leads to the release of mitochondrial DNA (mtDNA) into the cytoplasm, which activates the cGAS-STING signaling pathway, inducing the expression of inflammatory factors and exacerbating local immune inflammatory reactions.

[0003] In addition, with the apoptosis or necrosis of aging nucleus pulposus cells, extracellular free DNA (cfDNA) is released into the tissue microenvironment. As a ligand for pattern recognition molecules, cfDNA activates Toll-like receptor 9 (TLR9) on macrophages, triggering an inflammatory cascade and promoting further degeneration of nucleus pulposus cells. Current clinical treatments for IVDD mainly rely on painkillers or surgical intervention, but these methods are mainly aimed at relieving symptoms and lack targeted regulation of disease pathogenesis, making it difficult to achieve long-term therapeutic effects. SUMMARY

[0004] To overcome the shortcomings of the prior art, the present application provides a dual-mode nucleic acid removal system for treating intervertebral disc degenerative disease and a preparation and application method thereof. Based on the combined strategy of simultaneously regulating mtDNA leakage and removing extracellular cfDNA, the system can synergistically block the inflammatory cascade induced by intracellular and extracellular nucleic acids, fundamentally blocking the chronic inflammatory process related to IVDD, and providing a new means for delaying or reversing nucleic acid-related inflammatory diseases such as IVDD.

[0005] The dual-mode nucleic acid removal system for treating intervertebral disc degenerative disease comprises mixed engineered nanovesicles and PAMAM cross-linked hyaluronic acid hydrogel. The engineered nanovesicles are used to target nucleus pulposus cells and inhibit increased mitochondrial outer membrane permeability. The PAMAM cross-linked hyaluronic acid hydrogel is used to bind extracellular free DNA in the tissue space.

[0006] As preferred, the engineered nanovesicle is a nucleus pulposus cell membrane vesicle encapsulating a small molecule mitochondrial membrane stabilizer.

[0007] As preferred, the small molecule mitochondrial membrane stabilizer is a Bax pathway inhibitor (BAI1).

[0008] As preferred, the diameter of the engineered nanovesicle is less than 200 nm.

[0009] As preferred, the PAMAM cross-linked hyaluronic acid hydrogel is formed by Schiff base reaction between oxidized hyaluronic acid (OHA) and PAMAM dendrimer.

[0010] As preferred, the mass ratio of the oxidized hyaluronic acid to the PAMAM dendrimer is 5:1.

[0011] The preparation method of the bimodal nucleic acid clearance system for treating intervertebral disc degenerative disease comprises the following steps: firstly, extracting nucleus pulposus cell membranes, preparing nanovesicles through membrane extrusion, and then co-incubating with a small molecule mitochondrial membrane stabilizer to obtain the engineered nanovesicles; secondly, mixing the engineered nanovesicles with oxidized hyaluronic acid, and then adding a PAMAM dendrimer solution to generate a PAMAM cross-linked hyaluronic acid hydrogel through a spontaneous Schiff base reaction, thereby obtaining the bimodal nucleic acid clearance system.

[0012] The bimodal nucleic acid clearance system for treating intervertebral disc degenerative disease is applied to the preparation of a drug for nucleic acid-mediated inflammatory diseases, in particular, the preparation of a drug for intervertebral disc degenerative diseases.

[0013] As preferred, the bimodal nucleic acid clearance system for treating intervertebral disc degenerative disease is applied to the preparation of a drug for rheumatoid arthritis and systemic lupus erythematosus.

[0014] The present application has the following beneficial effects: The engineered nanovesicle releases the encapsulated drug in the nucleus pulposus cell, inhibits DNA leakage caused by increased mitochondrial outer membrane permeability, weakens the expression of downstream inflammatory factors, blocks the positive feedback mechanism between nucleus pulposus cell aging and inflammatory response, restores cell homeostasis, delays cell function degradation, and reduces local tissue inflammation level. The PAMAM cross-linked hyaluronic acid hydrogel binds to the negative cfDNA released in the tissue gap through the high-density positive charge of PAMAM, efficiently clears the pro-inflammatory nucleic acid in the tissue microenvironment, blocks the interaction between extracellular free DNA and macrophage Toll-like receptor 9, inhibits M1-type macrophage polarization and inflammatory factor release, and thus improves the local immune microenvironment.

[0015] The bimodal nucleic acid clearance system provides a new combined treatment platform with good biocompatibility and stable drug efficacy, can precisely inhibit inflammatory response from both intracellular source and external tissue environment, shows excellent anti-inflammatory and tissue protection effects, breaks the positive feedback cycle of inflammation-aging-degeneration, significantly delays nucleus pulposus cell aging and reduces ECM degradation, and provides a theoretical basis and engineering solution for IVDD and other nucleic acid related chronic inflammatory diseases. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Particle size and Zeta potential determined in Example 1; Figure 2 Freeze transmission electron microscope image of the engineered nanovesicle in Example 1; Figure 3 Scanning electron microscope image of the bimodal nucleic acid clearance system in Example 1; Figure 4 Rheology analysis results of the bimodal nucleic acid clearance system in Example 1; Figure 5 Mechanical stability analysis results of the bimodal nucleic acid clearance system in Example 1; Figure 6 Tissue dsDNA expression detection results in Example 2; Figure 7 Cytoplasmic DNA content detection results in Example 2; Figure 8 In vivo inflammatory factor expression detection results in Example 2; Figure 9 Nuclear magnetic resonance imaging results in Example 2; Figure 10 Nucleus pulposus tissue extracellular matrix detection results in Example 2. DETAILED DESCRIPTION

[0017] The application will be further explained in conjunction with the accompanying drawings; Example 1 The present embodiment provides a preparation method of a bimodal nucleic acid clearance system for treating intervertebral disc degenerative disease, which comprises mixed engineered nanovesicles and PAMAM cross-linked hyaluronic acid hydrogel, and the specific preparation steps are as follows: Step 1, the intervertebral disc tissue of the tail of healthy SD rats was taken, the nucleus pulposus tissue was isolated under sterile conditions, and was digested with 0.2% collagenase type II for 4-6 hours to obtain primary nucleus pulposus cells. The primary nucleus pulposus cells were inoculated in high glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and were cultured in a 37°C, 5% CO2 incubator. When the primary nucleus pulposus cells grew to 80-90% confluence, they were passaged, and P2-P3 generation nucleus pulposus cells were taken to extract nucleus pulposus cell membranes (NPm). The nucleus pulposus cell membranes were extruded using a polycarbonate membrane with a diameter of 220 nm and repeated 5 times, and the obtained nucleus pulposus cell membrane vesicles were dispersed in a PBS solution.

[0018] The small molecule drug BAI1 was dissolved in DMSO and added to the PBS solution containing the nucleus pulposus cell membrane vesicles. The mixture was placed in an ice bath and subjected to ultrasonic treatment, with 10 seconds of ultrasonic treatment, 5 seconds of interval, and 10 cycles, to promote the loading of BAI1 into the vesicles. Subsequently, ultracentrifugation was performed at a centrifugal force of 12000 g for 1 hour to obtain engineered nanovesicles (CDNV).

[0019] The particle size and Zeta potential of CDNV were determined, and the results are shown in Figure 1 , with an average particle size of 170 nm and a negative Zeta potential. Cryo-TEM was used to observe the morphology and structure, and the results are shown in Figure 2 , with an intact vesicle structure.

[0020] Step 2, aldehyde hyaluronic acid (HA-CHO) was synthesized by sodium periodate oxidation method. HA-CHO was mixed with engineered nanovesicles (CDNV), with a mass percentage of 5 wt%, and an equal volume of 1 wt% fifth-generation PAMAM dendrimer (G5-PAMAM) solution with a primary amine group on the surface was added to spontaneously generate Schiff base reaction at room temperature, generating PAMAM cross-linked hyaluronic acid hydrogel (CDHP), obtaining a bimodal nucleic acid clearance system (CDNV-HP).

[0021] The morphology of the freeze-dried CDNV-HP sample was observed by SEM, as shown in Figure 3 , which can be seen to have a typical porous microstructure.

[0022] Rheological analysis was performed on the CDNV-HP sample, and the results are shown in Figure 4 . When the shear rate was increased from 0.1 s -1 to 1000 s -1 , the gel viscosity decreased significantly, showing a typical shear-thinning behavior, proving its good flowability and strain response ability when injected.

[0023] The mechanical stability of the CDNV-HP sample was tested by strain sweep, and the results are shown in FIG. 6. As shown in FIG. 6, the storage modulus (G') and the loss modulus (G'') remained stable in the range of 30% strain, indicating that the structure was still stable under large elastic deformation. When the strain increased to 80%, G' and G'' intersected, indicating that the skeleton structure began to break down and reached the critical point of mechanical instability. Figure 5

[0024] Example 2 In this example, the bimodal nucleic acid removal system (CDNV-HP) prepared in Example 1 for treating intervertebral disc degenerative disease was applied to the preparation of a drug for intervertebral disc degenerative disease, and the nucleic acid removal effect of CDNV-HP in vivo was verified through animal experiments. The specific steps are as follows: Step 1, 30 SD male rats weighing 250-300 g and 8 weeks old were randomly divided into three groups, 3 rats in each group.

[0025] The first group was the control group, only the caudal vertebra skin was incised without puncture. The second group was the modeling group, the intervertebral disc degeneration model was established at the Co6 / Co7 segment of the caudal vertebra, and no treatment was given. The third group was the treatment group, after establishing the intervertebral disc degeneration model at the Co6 / Co7 segment of the caudal vertebra, CDNV-HP prepared in Example 1 was injected.

[0026] Step 2, double-stranded DNA (dsDNA) immunofluorescence staining technology was used to stain and observe the intervertebral disc tissue sections of the three groups of rats, and the expression of dsDNA in the tissue was detected. As shown in FIG. 7, compared with the modeling group, the fluorescence signal of dsDNA in the nucleus pulposus tissue of the treatment group was significantly weakened, indicating that CDNV-HP could significantly reduce the content of local extracellular cfDNA. Figure 6 The nucleus pulposus cells of the three groups of rats were separated, the cytoplasmic DNA was extracted, and the DNA content in the cytoplasm was quantitatively detected by qPCR. As shown in FIG. 8, the cytoplasmic DNA content of the treatment group was significantly reduced, and CDNV-HP could effectively remove the abnormally accumulated DNA in the cells and inhibit the release of cytoplasmic DNA.

[0027] Figure 7 IL-6 and IL-1β immunofluorescence staining technology was used to stain and observe the intervertebral disc tissue sections of the three groups of rats, and the expression of inflammatory factors in vivo was detected. As shown in FIG. 9, compared with the modeling group, the fluorescence signal of IL-6 and IL-1β in the nucleus pulposus tissue of the treatment group was significantly weakened, indicating that CDNV-HP could significantly inhibit the inflammation of the intervertebral disc.

[0028] Figure 8

[0029] ​​​​Step 3, T2-weighted imaging scanning of the caudal vertebrae of the three groups of rats was performed by nuclear magnetic resonance, and the signal intensity of the Co6 / Co7 segment intervertebral disc was observed and analyzed, as shown in FIG. 6. Figure 9 As can be seen from FIG. 6, the signal intensity of the treatment group is higher than that of the modeling group, and the MRI score is lower.

[0030] The extracellular matrix of the nucleus pulposus tissue of the three groups of rats was detected by immunohistochemical staining, as shown in FIG. 7. Figure 10 As shown in FIG. 7, the expression of type II collagen in the treatment group is significantly enhanced, and the expression of matrix metalloproteinase 3 is down-regulated, indicating that CDNV-HP can promote the repair and reconstruction of the intervertebral disc matrix.

[0031] The above experimental results prove that the dual-mode nucleic acid removal system CDNV-HP proposed in the present application can synergistically remove intracellular DNA and extracellular cfDNA in the nucleus pulposus tissue in vivo, inhibit inflammatory response, and promote matrix remodeling, thereby effectively delaying the intervertebral disc degeneration process.

Claims

1. A bimodal nucleic acid clearance system for the treatment of intervertebral disc degeneration, characterized in that: The bimodal nucleic acid scavenging system is a mixed engineered nanovesicle and PAMAM cross-linked hyaluronic acid hydrogel; the engineered nanovesicle is used for targeting nucleus pulposus cells and inhibiting mitochondrial outer membrane permeability increase; and the PAMAM cross-linked hyaluronic acid hydrogel is used for binding extracellular free DNA in the interstitial space.

2. The dual-mode nucleic acid clearance system for the treatment of intervertebral disc degeneration of claim 1, wherein: The engineered nanovesicle is a nucleus pulposus cell membrane vesicle loaded with a small molecule mitochondrial membrane stabilizer.

3. The dual-mode nucleic acid clearance system for the treatment of intervertebral disc degeneration of claim 2, wherein: The small molecule mitochondrial membrane stabilizer is a Bax pathway inhibitor.

4. The dual-mode nucleic acid clearance system for the treatment of intervertebral disc degeneration according to claim 1 or 2, wherein: The diameter of the engineered nanovesicle is less than 200 nm.

5. The dual-mode nucleic acid clearance system for the treatment of intervertebral disc degeneration of claim 1, wherein: The PAMAM cross-linked hyaluronic acid hydrogel is formed by Schiff base reaction cross-linking of oxidized hyaluronic acid and PAMAM dendrimers.

6. The dual-mode nucleic acid clearance system for the treatment of intervertebral disc degeneration of claim 5, wherein: The mass ratio of the oxidized hyaluronic acid to the PAMAM dendrimers is 5:

1.

7. The method of claim 1-3, 5, 6, wherein the method is for the preparation of a bimodal nucleic acid scavenging system for the treatment of intervertebral disc degeneration. First, nucleus pulposus cell membranes are extracted, nanovesicles are prepared by membrane extrusion, and then are co-incubated with a small molecule mitochondrial membrane stabilizer to obtain the engineered nanovesicle; second, oxidized hyaluronic acid is mixed with the engineered nanovesicle, and a PAMAM dendrimer solution is added, and a PAMAM cross-linked hyaluronic acid hydrogel is generated by spontaneous Schiff base reaction to obtain the bimodal nucleic acid scavenging system.

8. The method for preparing the dual-modality nucleic acid clearance system for treating intervertebral disc degenerative diseases as described in claim 7, characterized in that: The oxidized hyaluronic acid is aldehyde hyaluronic acid.

9. The method of using the dual-mode nucleic acid removal system for treating intervertebral disc degeneration according to any of claims 1-3, 5, 6, wherein: The bimodal nucleic acid scavenging system is used for preparing a drug for nucleic acid-mediated inflammatory diseases.

10. The method of claim 9, wherein the bi-modal nucleic acid depletion system is used to treat intervertebral disc degeneration. The bimodal nucleic acid scavenging system is used for preparing a drug for intervertebral disc degenerative diseases, rheumatoid arthritis or systemic lupus erythematosus.

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