Preparation method of nanoparticles and application of nanoparticles in relieving intervertebral disc degeneration

Abstract

The invention relates to the technical field of gene therapy, and discloses a nano-particle preparation method and application thereof in relieving intervertebral disc degeneration, a special nano-particle delivery system is constructed, the nano-particles are functionalized through a nucleus pulposus cell specific aptamer and can be directly combined with transmembrane protein FasL on the nucleus pulposus cell surface, and the targeting property of the nano-particles is improved. The transmission of the nanoparticles to the nucleus pulposus cells is accelerated. And finally, the miRNA is combined with miR-150-5p to form therapeutic nanoparticles, and stable packaging of the miRNA is ensured by optimizing the weight ratio. The nanoparticle preparation method is simple and convenient, the material biocompatibility is good, polylactic acid-co-glycolic acid (PLGA) and polylysine (PLL) are approved by American Food and Drug Administration (FDA) to be used for human drug delivery, and favorable conditions are provided for clinical transformation. Besides, a series of in-vivo and in-vitro experiments prove that the nano-particle delivery system can remarkably reduce cell apoptosis and increase generation of extracellular matrixes, and an innovative strategy based on the nanotechnology is provided for precise treatment of intervertebral disc degeneration.

Description

Technical Field

[0001] The present invention relates to the technical field of gene therapy, and in particular to a method for preparing nanoparticles and an application thereof in alleviating intervertebral disc degeneration. Background Art

[0002] At present, gene therapy, as an emerging method, is gradually moving from theoretical exploration to clinical application in the field of intervertebral disc degeneration (IDD). Intervertebral disc degeneration is a common cause of low back pain and dysfunction, which seriously affects the quality of life of patients. Among them, microRNAs (miRNAs), as an important class of gene regulatory molecules, have shown great potential in the treatment of IDD because they can accurately regulate the expression of target genes. However, miRNA therapy faces two major challenges in practical applications: one is that the short half-life of miRNA itself limits its duration of action in the body; the other is the low efficiency of cellular uptake, which is mainly attributed to the lack of an efficient delivery system.

[0003] In response to these challenges, the present invention focuses on using nanoparticles with nucleus pulposus cell (NPC) specificity as a carrier to deliver miR-150-5p. MiR-150-5p is a specific miRNA that has been shown to have an important impact on the progression of IDD. The polymer nanoparticles modified with precisely designed NPC-specific ligands aim to solve the problems of biological stability and cellular uptake encountered in traditional miRNA delivery, thereby enhancing the accumulation and effect of miR-150-5p at the target site.

[0004] In order to ensure efficient and specific delivery, the present invention uses an innovative cell system SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology to screen out NPC-specific aptamers. By optimizing asymmetric PCR conditions, a stable and cost-effective EY3 aptamer was obtained, which can efficiently bind to NPC and be integrated into the surface of nanoparticles, significantly improving the enrichment and functional expression of miR-150-5p in target cells. In addition, the experiment also revealed the core mechanism of the FBXW11 / TAK1 / NF-κB signaling pathway in the progression of miR-150-5p-mediated IDD through liquid chromatography tandem mass spectrometry (LC-MS / MS) and co-immunoprecipitation (Co-IP) technology, further confirming the effectiveness of this delivery strategy.

[0005] In summary, the NPC-specific nanoparticle delivery system proposed in the present invention aims to overcome the limitations of miRNA in the prior art for the treatment of IDD, and provides an innovative and effective treatment method, which is expected to bring new breakthroughs in the treatment of intervertebral disc degeneration diseases. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the deficiencies of the prior art, the present invention provides a method for preparing nanoparticles and application thereof in alleviating intervertebral disc degeneration, thereby solving the technical problems raised in the above-mentioned background technology.

[0008] (II) Technical solution

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A method for preparing nanoparticles comprises the following steps:

[0011] S1: Select appropriate polymer materials to prepare nanoparticle matrix;

[0012] S2: Immobilize the nucleus pulposus cell (NPC)-specific aptamer on the surface of nanoparticles by chemical coupling or physical adsorption method;

[0013] S3: Under appropriate conditions, miR-150-5p is loaded into the nanoparticles;

[0014] S4: Purify miR-150-5p-loaded NPC-specific nanoparticles by centrifugation, dialysis or filtration.

[0015] As a preferred technical solution of the present invention, the high molecular polymer material is a biodegradable polymer, preferably polylactic acid-co-glycolic acid (PLGA), polyethylene glycol (PEG), polylysine (PLL) and a combination thereof.

[0016] The nanoparticles have good biocompatibility, stable miR-150-5p loading capacity and efficient NPC targeting performance. The medical uses of the nanoparticle products are particularly for improving the symptoms of patients with intervertebral disc degeneration, improving the quality of life, and delaying or reversing the progression of intervertebral disc degeneration.

[0017] An application of nanoparticles for alleviating intervertebral disc degeneration comprises the following steps:

[0018] S1: Preparation of NPC-specific nanoparticles loaded with miR-150-5p;

[0019] S2: delivering the nanoparticles to an intervertebral disc region of an individual suffering from IDD;

[0020] S3: The nanoparticles are used to release miR-150-5p into NPC to regulate related gene expression, inhibit inflammatory response, and promote intervertebral disc tissue repair, thereby slowing down or preventing the process of intervertebral disc degeneration.

[0021] As a preferred technical solution of the present invention, in S1, the nanoparticles are made of biocompatible polymer materials and are surface-modified with aptamers that can specifically recognize NPC surface markers.

[0022] As a preferred technical solution of the present invention, the aptamer is a single-stranded DNA or RNA sequence targeting a specific receptor on the surface of NPC, which can enhance the recognition and uptake of NPC by nanoparticles.

[0023] As a preferred technical solution of the present invention, the encapsulation rate of the miR-150-5p in the nanoparticles exceeds 80%, ensuring effective release and biological activity after delivery to the target cells.

[0024] As a preferred technical solution of the present invention, in S2, imaging and biochemical indexes are tested on the subjects before and after the delivery of nanoparticles to evaluate the treatment effect and the changes in the degree of intervertebral disc degeneration.

[0025] This treatment approach is suitable for clinical applications in the management of pain caused by disc degeneration, restoration of function, and prevention of further degeneration.

[0026] (III) Beneficial effects

[0027] Compared with the prior art, the present invention provides a method for preparing nanoparticles and its application in alleviating intervertebral disc degeneration, which has the following beneficial effects:

[0028] 1. Improved targeted delivery efficiency: By designing nanoparticles decorated with aptamers specific to nucleus pulposus cells (NPC), precise targeted delivery of miR-150-5p to the nucleus pulposus region of the intervertebral disc is achieved. This specific targeting not only enhances the accumulation of miRNA in target cells, but also reduces potential side effects on surrounding healthy tissues, improving the safety and effectiveness of treatment.

[0029] 2. Improve drug stability and cell uptake: Nanoparticles constructed with optimized polymer materials (such as PLGA, PLL and PEG) not only ensure the stable encapsulation of miRNA, but also promote its efficient release in cells. The biodegradability of PLGA enables miRNA to be released in time in cells and participate in the gene regulation process, while the addition of PLL and PEG improves the stability and biocompatibility of nanoparticles, helping to prolong the circulation time in the body and increase cell uptake.

[0030] 3. Sustained therapeutic effect: The nanoparticle delivery system of the present invention can achieve long-term sustained effects in vivo, reduce the frequency of repeated injections, reduce the risk of physical damage to the intervertebral disc structure (such as the annulus fibrosus and nucleus pulposus), and help maintain the normal structure and function of the intervertebral disc.

[0031] 4. Inhibit cell aging and degeneration: Through the precise regulation of miR-150-5p, the present invention successfully inhibits the aging of NPC and increases the production of extracellular matrix (ECM), thereby delaying the progression of intervertebral disc degeneration. In particular, by inhibiting the ubiquitination of FBXW11 and TAK1 and downregulating the NF-κB signaling pathway, the present invention blocks the progression of IDD at the molecular level.

[0032] 5. Clinical transformation potential: Given that PLGA and PLL have been approved by the U.S. Food and Drug Administration (FDA) for the delivery of multiple drugs in humans, the nanoparticle platform of the present invention has good prospects for clinical transformation and provides a new and promising treatment option for the treatment of IDD.

[0033] In summary, the present invention shows obvious advantages in improving treatment efficiency and safety compared with existing treatment methods through a precise targeted delivery system, improved drug delivery efficiency, sustained therapeutic effect and targeted intervention in the pathological process of IDD. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The figure is a schematic diagram of the process of a nanoparticle system for efficiently delivering miR-150-5p to nucleus pulposus cells to alleviate intervertebral disc degeneration according to the present invention. DETAILED DESCRIPTION

[0035] 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.

[0036] Example 1: Preparation of NPC-specific miR-150-5p nanoparticles

[0037] Material selection and preparation: Polylactic-co-glycolic acid (PLGA) was selected as the base material of nanoparticles due to its biodegradability and good drug loading capacity. At the same time, polyethylene glycol (PEG) was selected for surface modification to enhance blood circulation time. Blank nanoparticles were prepared by conventional emulsification-solvent evaporation method.

[0038] Aptamer design and synthesis: DNA aptamer sequences that can specifically recognize NPC surface antigens are screened using the systematic evolution ligand enrichment technology (SELEX). The screened aptamer sequences are connected to the surface of nanoparticles through chemical reactions to form NPC-specific targeted nanoparticles.

[0039] miR-150-5p loading: The miR-150-5p molecules are encapsulated into the nanoparticles modified with aptamers by passive loading or charge adsorption. Ultrasonication and dialysis techniques are used to ensure the effective loading of miRNA while maintaining the stability and biological activity of the nanoparticles.

[0040] Example 2: Functional verification of miR-150-5p nanoparticles

[0041] Cell experiment: Human nucleus pulposus cell lines cultured in vitro were selected and treated with blank control group, unmodified nanoparticles loaded with miR-150-5p group and NPC-specific nanoparticles loaded with miR-150-5p group. Real-time fluorescence quantitative PCR (qRT-PCR), Western Blot and immunofluorescence staining were used to evaluate the transfection efficiency of miR-150-5p, the intracellular expression level and its effect on the biological behavior of NPC, including cell senescence and extracellular matrix (ECM) synthesis.

[0042] Animal model establishment: A rat intervertebral disc acupuncture injury model was used to simulate IDD, and the prepared NPC-specific miR-150-5p nanoparticles were administered to the model animals by intradiscal injection. The degree of intervertebral disc degeneration was regularly monitored by imaging methods such as X-ray and magnetic resonance imaging (MRI), and tissue samples were collected for histological analysis and molecular level testing to evaluate the therapeutic effect.

[0043] Safety assessment: Long-term toxicity tests are conducted at the cellular level and in animal models to observe whether the nanoparticles cause significant inflammatory responses, organ damage, or systemic adverse reactions.

[0044] Example 3: Large-scale production and quality control

[0045] Scale-up: NPC-specific miR-150-5p nanoparticles are prepared in batches based on the optimized formulation at laboratory scale using equipment and processes suitable for industrial production to ensure batch-to-batch consistency and product quality.

[0046] Quality control: Each batch of nanoparticles is strictly tested for particle size distribution, drug loading, encapsulation efficiency, in vitro release curve and other indicators to ensure that they meet the preset standards. At the same time, sterility testing and biocompatibility testing are carried out to ensure that the product is safe and effective.

[0047] Through the above-mentioned embodiments, the present invention provides an efficient and highly specific treatment method, which aims to effectively intervene in the process of intervertebral disc degeneration by precisely regulating the expression of miR-150-5p in NPC, and provides a new strategy for the clinical treatment of IDD.

[0048] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing nanoparticles, characterized in that: The following steps are involved: S1: Select appropriate polymer materials to prepare nanoparticle matrix; S2: Immobilize the nucleus pulposus cell (NPC)-specific aptamer on the surface of nanoparticles by chemical coupling or physical adsorption method; S3: Under appropriate conditions, miR-150-5p is loaded into the nanoparticles; S4: Purify miR-150-5p-loaded NPC-specific nanoparticles by centrifugation, dialysis or filtration.

2. The method for preparing nanoparticles according to claim 1, characterized in that: In S1, the high molecular polymer material is a biodegradable polymer, preferably polylactic acid-co-glycolic acid (PLGA), polyethylene glycol (PEG), polylysine (PLL) and a combination thereof.

3. A use of nanoparticles for alleviating intervertebral disc degeneration, comprising a method for preparing nanoparticles according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1: Preparation of NPC-specific nanoparticles loaded with miR-150-5p; S2: delivering the nanoparticles to an intervertebral disc region of an individual suffering from IDD; S3: The nanoparticles are used to release miR-150-5p into NPC to regulate related gene expression, inhibit inflammatory response, and promote intervertebral disc tissue repair, thereby slowing down or preventing the process of intervertebral disc degeneration.

4. The method for using nanoparticles to alleviate intervertebral disc degeneration according to claim 3, characterized in that: In S1, the nanoparticles are made of biocompatible polymer materials and are surface-modified with aptamers that can specifically recognize NPC surface markers.

5. The method for using nanoparticles to alleviate intervertebral disc degeneration according to claim 4, characterized in that: The aptamer is a single-stranded DNA or RNA sequence targeting a specific receptor on the surface of NPC, and can enhance the recognition and uptake of NPC by nanoparticles.

6. The method for using nanoparticles to alleviate intervertebral disc degeneration according to claim 3, characterized in that: The encapsulation rate of the miR-150-5p in the nanoparticles exceeds 80%, ensuring effective release and biological activity after delivery to target cells.

7. The method for using nanoparticles to alleviate intervertebral disc degeneration according to claim 1, characterized in that: In S2, imaging and biochemical indexes are tested on the subjects before and after the delivery of nanoparticles to evaluate the treatment effect and changes in the degree of intervertebral disc degeneration.

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