Nanoparticle material with effect of treating IDD as well as preparation method and application of nanoparticle material

Abstract

The invention relates to the field of biological medicine, in particular to a nanoparticle material with an IDD treatment effect and a preparation method and application thereof. The invention provides a cell-like nanoparticle material, which is named as HIF1A (at) NNP, is formed by wrapping plasmid pHIF1A with NNP, contains a membrane component of NPCs, and realizes overexpression of HIF1A. The plasmid pHIF1A is encapsulated in the NNP to form the HIF1A-coated NNP, the proliferation of blood vessels, nerve growth or polarization of macrophages is not obviously influenced, which indicates that the targeting effect of the HIF1A-coated NNP is very specific, and other IVD affiliated cells are hardly influenced; the HIF1A at NNP can effectively inhibit apoptosis and aging of NPCs, and can also regulate the inflammation microenvironment; meanwhile, the effect of the HIF1A-coated NNP is equivalent to that of an existing transfection reagent, and the toxicity to cells is lower.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a nanoparticle material having the efficacy of treating IDD, and a preparation method and application thereof. Background Art

[0002] The intervertebral disc (IVD) is an essential component of the spine, absorbing shock and providing flexibility. However, due to aging or injury, the disc may degenerate, leading to disc herniation or other spinal disorders. Intervertebral disc degeneration (IDD), occurring in 30% of adults worldwide, is one of the most common clinical conditions with manifestations involving the lower back. Treatments for IDD and its associated low back pain include physical therapy, administration of nonsteroidal anti-inflammatory drugs, epidural injections, and surgery; however, most of these treatments are aimed at relieving pain rather than repairing tissue, while methods for IVD regeneration are rather limited with relatively poor outcomes. Therefore, there is an urgent need to develop new therapeutic strategies for IDD.

[0003] Hypoxia-inducible factor 1α (HIF1A) mediates IVD regeneration by promoting autophagy activation, providing a new direction to overcome the current challenges in treating disc degeneration and low back pain. HIF1A plays an important role in disc homeostasis. Under normoxic conditions, HIF1A is hydroxylated by proline hydroxylases, leading to its degradation. However, under hypoxic or inflammatory conditions, HIF1A is stabilized and translocated to the nucleus and activated in a process involving: transcription of various genes involved in extracellular matrix synthesis, angiogenesis, and cell survival. Stabilization of HIF1A has been shown to promote proliferation of nucleus pulposus cells (NPCs) and synthesis of proteoglycans, which are key components of the disc matrix. Therefore, therapeutic strategies targeting HIF1A signaling are promising for IVD regeneration and the treatment of degenerative disc disease.

[0004] The normal lumbar IVD is avascular. However, during IVD degeneration, vascularization occurs, accompanied by matrix disorganization and increased clefts. Blood vessels growing within the annulus fibrosus (AF) and nucleus pulposus (NP) tissues promote the infiltration of inflammatory cells and the growth of nerve fibers. The increased infiltration of inflammatory cells forms a chronic inflammatory microenvironment, leading to neuroinflammation and sensitization of nerve fibers. Strategies based on HIF1A activation may aggravate inflammation and pain input in IDD. For example, HIF1A plays a key role in angiogenesis and mediates vascularization during IVD by promoting the expression of vascular endothelial growth factor (VEGF). In addition, HIF1A induction enhances axonal regeneration, which may promote the growth of sensory nerves in IVD. In addition, HIF1A-mediated shift to glycolysis reprograms macrophages into an M1-like phenotype, promoting the secretion of proinflammatory factors. Therefore, HIF1A activation in IVD may increase vascularization, nerve growth, and neuroinflammation, leading to the aggravation of IDD and discogenic pain. Currently, HIF1A strategies based on drugs, gene therapy, or other approaches may produce many side effects due to widespread HIF1A activation in various cell types in the IVD and fail to promote ideal IVD regeneration. Therefore, strategies that specifically promote HIF1A activation in NPCs are needed to avoid side effects.

[0005] Currently, there are several methods to deliver exogenous plasmids into cells, such as viral vectors, physical methods, lipid transfection, and electroporation. Viral vectors and lipid transfection are the two most commonly used methods for gene editing; viral vectors such as adenovirus, lentivirus, and adeno-associated virus can effectively deliver DNA into dividing and non-dividing cells; lipofection uses lipid-based transfection reagents to encapsulate DNA to promote cellular uptake. Although these transfection methods are widely used, they have limitations and side effects. Viral vectors are expensive and difficult to produce, and may also trigger immune responses. Lipid-based reagents are toxic to certain cell types. In addition, compared with cell lines, the transfection efficiency of viral vectors and lipid transfection for primary cells is generally less satisfactory. Therefore, the development of safe, efficient, and versatile gene delivery methods remains an important goal in biomedical research and clinical applications.

[0006] At present, the vast majority of strategies reported worldwide (including anti-aging drugs, nanoparticles, and hydrogels) focus on the intervention of cell senescence in the intervertebral disc, while research on various cell types in IVD tissues, especially the effects of materials on blood vessels and nerves, is rare, which leads to insufficient evaluation of potential side effects and difficulties in clinical application. Therefore, there is an urgent need to develop new and precise IDD treatment strategies. Summary of the invention

[0007] The purpose of the present invention is to provide a nanoparticle material having the efficacy of treating IDD and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The nanoparticle material provided by the present invention can effectively treat IDD and can be prepared into a drug for treating IDD and promoting spinal healing.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a nanoparticle material with efficacy in treating IDD. The nanoparticle material comprises a nucleus pulposus cell membrane encapsulating plasmid pHIF1A.

[0010] The present invention provides a method for preparing the above-mentioned nanoparticle material, comprising the following steps:

[0011] After extrusion of the nucleus pulposus cell membrane, vesicles were obtained;

[0012] The vesicles and the plasmid pHIF1A are mixed and extruded to obtain the nanoparticle material.

[0013] Preferably, the mass ratio of the vesicle to the plasmid pHIF1A is (100-200):1.

[0014] Preferably, the method for extracting the nucleus pulposus cell membrane comprises the steps of digesting the nucleus pulposus cells, first centrifuging, culturing, ultrasonicating and second centrifuging to obtain the nucleus pulposus cell membrane.

[0015] Preferably, the digestive enzyme used in the digestion is type II collagenase;

[0016] And / or, the digestion time is 20 minutes and the temperature is 30-37°C.

[0017] Preferably, the centrifugal force of the first centrifugation is 150-200×g, and the time is 5-8 min.

[0018] Preferably, the culture temperature is 37°C and the volume percentage of CO2 is 5%;

[0019] And / or, the culture medium used in the culture is based on DMEM / F-12 culture medium and also includes 10% fetal bovine serum and 1% penicillin-streptomycin.

[0020] Preferably, the second centrifugation includes initial centrifugation and repeated centrifugation;

[0021] The centrifugal force of the initial centrifugation is 20000×g, and the time is 25 min;

[0022] The centrifugal force of the multiple centrifugation is 100000×g, and the time is 35 min.

[0023] The present invention provides the use of the above-mentioned nanoparticle material in preparing a medicine for treating IDD and / or promoting spinal healing.

[0024] The present invention provides a medicine for treating IDD and / or promoting spinal healing, wherein the medicine comprises the above-mentioned nanoparticle material.

[0025] The present invention discloses the following technical effects:

[0026] The present invention provides a cell-mimicking nanoparticle material, named HIF1A@NNP, which is composed of a nucleus pulposus cell membrane (NPC membrane, NNP) encapsulating a plasmid pHIF1A, which contains the membrane components of NPCs and achieves overexpression of HIF1A. At the same time, compared with traditional exogenous plasmid delivery methods (e.g., liposomes and lentiviruses), it has minimal impact on cell viability. In a specific embodiment of the present invention, the targeting ability of HIF1A@NNP on NPCs and the HIF1A transfection effect were evaluated, and the anti-apoptosis, anti-aging and anti-inflammatory effects of HIF1A@NNP were evaluated in vitro and in vivo. The results showed that: HIF1A@NNP can be specifically taken up by NPCs, providing a strategy for the targeted delivery of genetic drugs; the activation of HIF1A significantly promoted vascularization, axon growth and M1 polarization, and the encapsulation of plasmid pHIF1A in NNP to form HIF1A@NNP did not significantly affect the proliferation of blood vessels, nerve growth or polarization of macrophages, which shows that the targeting effect of HIF1A@NNP is very specific and has almost no effect on other IVD-affiliated cells; HIF1A@NNP can effectively inhibit the apoptosis and aging of NPCs, and can also regulate the inflammatory microenvironment; at the same time, HIF1A@NNP is comparable to existing transfection reagents and has lower cytotoxicity.

[0027] Moreover, the HIF1A@NNP (biomimetic nanoparticles) provided by the present invention has the advantages of easy preparation and storage, effective transfection of HIF1A and specific NPC targeting ability, and has become a promising tool for treating IDD and promoting spinal healing; at the same time, due to the excellent cell targeting effect of HIF1A@NNP, NNP can be used as a new tool to study gene function by achieving precise regulation of genes in specific cells. This new tool can eliminate the effects of traditional genome editing methods (such as viruses and liposomes) on non-target cell gene changes, especially for in vivo studies. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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 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.

[0029] Figure 1 The preparation and characteristics of HIF1A@NNP; A is a schematic diagram of HIF1A@NNP; B is the evaluation of the encapsulation efficiency of HIF1A@NNP by flow cytometry, the plasmid pHIF1A is stained with PI, and the NNP is stained with DiO; C is the NP cell lysate analyzed by Western blotting (Cell lysate), representative protein bands of typical membrane markers in NNP and HIF1A@NNP; D is the transmission electron microscopy image of NNP and HIF1A@NNP, the scale bar is 100μm; E is the surface zeta potential (ζ, mv) of NNP and HIF1A@NNP measured by dynamic light scattering; F is NPCs treated with different concentrations of NNP or HIF1A@NNP, and the cell viability was detected by CCK8 experiment, the data are expressed as mean ± standard deviation (biological replicates, n=3), ns means no significant difference; *P<0.05; ****P<0.0001; NPC is nucleus pulposus cell, NNP is nucleus pulposus cell membrane, HIF1A@NNP is nanoparticle of NNP loaded with plasmid pHIF1A (nanoparticle material with therapeutic effect of IDD);

[0030] Figure 2 HIF1A@NNP specifically targets NPCs in vitro; A is a schematic diagram of flow cytometry analysis of the targeting effect of HIF1A@NNP, AFCs are stained with DiI, endothelial cells are stained with DiD, neurons are stained with Hoechst 33342, NPCs are stained with Hoechst 33342 and DiI, and macrophages are stained with Hoechst 33342 and DiD; B is flow cytometry evidence showing the uptake of HIF1A@NNP by different cells; NPC is a nucleus pulposus cell, NNP is a nucleus pulposus cell membrane, and HIF1A@NNP is a NNP nanoparticle loaded with plasmid pHIF1A (nanoparticle material with therapeutic efficacy for IDD);

[0031] Figure 3 NPCs were treated with different concentrations of Lipo3000, Lenti-HIF1A or HIF1A@NNP, and cell viability was detected by CCK8 assay; all groups were compared with group 0, and the data were expressed as mean ± SD (biological replicates, n ≥ 3); ns means no significant difference; **P < 0.01; ***P < 0.001; ****P < 0.0001;

[0032] Figure 4The expression level of HIF1A in NPCs treated with Lipo3000, Lenti-HIF1A or HIF1A@NNP was detected by western blotting; A is the western blotting detection graph; B is the bar chart of the relative expression level of HIF1A, and the data are expressed as mean ± standard deviation (biological replicates, n≥3), ns means no significant difference; **P<0.01;

[0033] Figure 5 The effects of different treatments on the migration ability, angiogenesis, DRG axon growth and macrophage polarization of HUVECs; A is the detection of the tubular formation ability of HUVECs by the tubular formation experiment (left figure), and the quantitative results of tubular branching and total tubular length are shown in the right figure, with a scale bar of 200 μm; B is the detection of the migration ability of HUVECs by the scratch test (left figure), and the results of the quantitative migration area are shown in the right figure, with a scale bar of 100 μm; C is a representative immunofluorescence image of neurofilament L in DRGs (left figure) and a statistical analysis based on Sholl analysis (right figure) ), scale bar is 200 μm; D is quantitative analysis of M1 and M2 markers based on RT-qPCR, and the data are presented as mean ± SD (biological replicates, n = 3); ns is not significant; *P < 0.05; **P < 0.01; ***P < 0.001; HUVEC is human umbilical vein endothelial cells, CTRL is the control group (the group not treated with nanoparticles), NNP is the nucleus pulposus cell membrane, HIF1A is the plasmid pHIF1A, and HIF1A@NNP is the nanoparticle of NNP loaded with plasmid pHIF1A (nanoparticle material with therapeutic efficacy for IDD);

[0034] Figure 6HIF1A@NNP inhibited the apoptosis of NPCs and regulated the metabolism of extracellular matrix; A is a representative graph of cell apoptosis obtained by flow cytometry analysis after Annexin V / PI staining, and the apoptosis rate is the sum of the proportions of Annexin V+PI- and AnnexinV+PI+ cells; B is a statistical analysis of cell apoptosis; C is a TUNEL experiment in NPCs, green fluorescence represents apoptotic cells, and the scale bar is 100μm; D is a statistical analysis of TUNEL-positive cells in different groups; E is a representative immunofluorescence image of Col2A1, Acan, SOX9, MMP3, MMP13 and Adamts5 in NPCs treated with CTRL, TBHP, TBHP+NNP and TBHP+HIF1A@NNP, and the scale bar is 100μm; F is a representative immunofluorescence image of Col2A1, Acan, SOX9, MMP3, MMP13 and Adamts5 in NPCs. Statistical analysis of the expression of ADAPT3 and Adamts5; G is the cell viability of NPCs treated with CTRL, TBHP, TBHP+NNP and HIF1A@NNP, and the data are presented as mean ± SD (biological replicates, n ≥ 3); ns is not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; CTRL: control group (group not treated with TBHP or nanoparticles), NNP is nucleus pulposus cell membrane, HIF1A@NNP is nanoparticles of NNP loaded with plasmid pHIF1A (nanoparticle material with therapeutic efficacy for IDD), TBHP is tert-butyl hydroperoxide;

[0035] Figure 7 HIF1A@NNP alleviates TBHP-induced NPC cell senescence in vitro; A is a representative protein blot showing the levels of p21 and p16 in NPCs treated with or without TBHP or nanoparticles; B is a quantitative analysis of p21 and p16 expression; C and D are representative images and statistical analysis of cell cycle determination of NPCs treated with or without TBHP or nanoparticles; E and F are representative images and statistical analysis of SA-β-Gal staining of NPCs treated with or without TBHP or nanoparticles (n=3), scale bar is 100μm; G and H are representative images and statistical analysis of SA-β-Gal staining of NPCs treated with or without TBHP or nanoparticles (n=3), scale bar is 100μm; Representative images and statistical analysis of NPCs treated with TBHP or nanoparticles in the comet assay, data are presented as mean ± SD (biological replicates, n ≥ 3); ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, CTRL is the control group (the group not treated with TBHP or nanoparticles), NNP is the nucleus pulposus cell membrane, HIF1A@NNP is the nanoparticle of NNP loaded with plasmid pHIF1A (nanoparticle material with therapeutic efficacy for IDD), TBHP is tert-butyl hydroperoxide;

[0036] Figure 8 HIF1A@NNP inhibits macrophage M1 polarization by regulating SASP; A is the experimental design of the effect of HIF1A@NNP on NPCs-derived SASP-mediated macrophage polarization and migration (conditioned medium was collected from NPCs treated with CTRL (treatment with only PBS added), TBHP, TBHP+NNP, and TBHP+HIF1A@NNP, and co-cultured with macrophages); B is after cells were treated with CTRL, TBHP, TBHP+NNP, and TBHP+HIF1A@NNP, culture supernatants were collected during the treatment period, and the concentrations of IL-1β, IL-6, and TNF-α were measured by ELISA; C is the expression level of IL-1β, IL-6 and TNF-α measured by qPCR; D is macrophages treated with different conditioned media, and the levels of M1 markers (inos, IL-6 and TNF-α) were measured by qPCR; E is macrophages treated with different conditioned media, and the levels of M2 markers (Arg1, CD206 and IL-10) were measured by qPCR; F is the migration ability of macrophages measured by crystal violet staining, the scale bar is 50 μm, and the data are presented as mean ± standard deviation (biological replicates, n = 5); ns is not significant; **P<0.01; ***P<0.001; ****P<0.0001. CTRL: control group (group not treated with TBHP or nanoparticles), NNP nucleus pulposus cell membrane, HIF1A@NNP is a nanoparticle of NNP loaded with plasmid pHIF1A (nanoparticle material with therapeutic efficacy for IDD), TBHP: tert-butyl hydroperoxide;

[0037] Fig. 9HIF1A@NNP alleviates IDD by inhibiting NPCs senescence and M1 macrophage activation in vivo; A is a representative T2-MRI image used to evaluate the relative signal intensity of NP tissue in the Intact group, IDD group, IDD NNP group, and IDD HIF1A@NNP group, and the red arrow indicates the surgical intervertebral disc; B is a quantitative evaluation of the long T2 signal (water retention) in NP tissue; C is a modified Pfirrmann MRI grading; D is a representative histological image of HE and SO staining, with a scale bar of 1 mm; E is the tissue degeneration score of each group; F is a fluorescence image showing the uptake of nanoparticles by NPCs in vivo, NPCs were stained with CD24, and NNPs or HIF1A@NNPs were stained with DiI, with a scale bar of 20 μm; G is a fluorescence image showing the in vivo expression of HIF1A in the four groups, with a scale bar of 50 μm; H is a quantitative evaluation of the fluorescence intensity of HIF1A staining in each group, and the data are presented as mean ± standard deviation (biological replicates, n=5); ns is not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; Intact is the intact group (unmodeled group), NNP is the nucleus pulposus cell membrane, HIF1A@NNP is the nanoparticle of NNP loaded with plasmid pHIF1A (nanoparticle material with therapeutic efficacy for IDD), and IDD is intervertebral disc degeneration. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] Example 1

[0044] 1. Reagents or kits

[0045] RAW 264.7 mouse macrophage cell line and human umbilical vein endothelial cell line were purchased from HyCyte (China); cell membrane green fluorescent probe (DiO), cell membrane red probe (DiI), cell membrane far-infrared fluorescent probe (DiD), Hoechst 33342, 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), and TUNEL apoptosis detection kit were purchased from Yeasen (China); neurotrophic factors nerve growth factor (NGF) and cytarabine were purchased from MedChemExpress (MedChemExpress, New Jersey, USA); apoptotic flow, cell cycle, and cell counting kit-8 (CCK-8) cell viability assay kit was purchased from Vazyme (China); tert-butyl hydroperoxide (TBHP) was purchased from Sigma-Aldrich (St. Louis, MO, USA); complete medium for rat dorsal root ganglion neurons and endothelial cell culture medium were purchased from ProCell (China); phosphate-buffered saline (PBS), Dulbecco's modified Eagle's medium, Dulbecco's modified Eagle's medium / nutrient mixture F-12 (Ham) 1:1 (DMEM / F-12 medium), fetal bovine serum (FBS), and culture medium required for cell culture were purchased from ThermoFisher Scientific (Waltham, MA, USA); ELISA kits were purchased from Jiangsu Meimian Industrial Co., Ltd. (Jiangsu, China); extruder was purchased from Genizer (USA); protein blotting reagents including Easy PAGE gel rapid preparation kit were purchased from Beyotime Biotechnology (China); PCR-related reagents were purchased from Yeasen; paraformaldehyde and goat serum were obtained from Boster (Beijing, China); Triton was obtained from BioSharp (Beijing, China); senescent β-galactosidase staining kit was purchased from Beyotime (China); alkaline comet assay kit was obtained from IPHASE (Beijing, China); mRFP-GFP-LC3 was purchased from Hanbio (Shanghai, China); basement membrane matrix and Transwell chamber plates were purchased from Corning (New York, USA); 29-gauge microsyringes were purchased from Hamilton (Switzerland); hematoxylin and eosin (H&E) and safranin O-fast green (S&O) staining kits were purchased from Solarbio (China).

[0046] 2. Antibodies

[0047] Primary antibodies: anti-CD24 (ABclonal, China, A2207), anti-CD155 (ABclonal, A5753), anti-CD221 (ABclonal, A0243), anti-HIF1A (NOVUS, USA, NB100-105), anti-Col2a1 (ABcam, UK, ab34712), anti-Acan (ABcam, ab216965), anti-Sox9 (ABcam, ab185966);

[0048] Secondary antibodies: anti-rabbit IgG (as 014, ABclonal), 594-conjugated goat anti-rabbit IgG (as 039, ABclonal), 488-conjugated goat anti-rabbit IgG (as 053, ABclonal), and 488-conjugated goat anti-mouse IgG (as 037, ABclonal).

[0049] 3. Extraction of Nucleus Pulposus Cell Membrane (NNP)

[0050] Nucleus pulposus cells (NPCs) were isolated from the caudal vertebrae of 4- to 5-week-old SD rats, and the specific steps were as follows: after collecting nucleus pulposus cells from the intervertebral disc (IVD), the nucleus pulposus cells (NP) were digested with 0.25% type II collagenase for 20 min at 37°C. Subsequently, the digestion was terminated to obtain a mixture; the mixture was centrifuged to obtain a cell pellet, and the centrifugation speed was 150-200×g for 5-8 min. These cell pellets were then cultured in DMEM / F-12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S) and maintained in an incubator at 37°C and 5% CO2. When the NPCs reached 70% to 80% confluence, they were harvested using a cell scraper to obtain a cell suspension, and then ultrasonicated for 30-40s to destroy the cells to obtain a suspension. The suspension was centrifuged at 20,000×g for 25 min at 4°C. The supernatant was collected and centrifuged at 100,000×g for 35 min at 4°C to collect the cell membrane precipitate, i.e., the nucleus pulposus cell membrane, which was then stored at −80°C for subsequent experiments.

[0051] 5. Extraction of annulus fiber cells (AFCs) and dorsal root ganglion neurons (DRG) of rats

[0052] Extraction of annulus fibrosus cells: The annulus fibrosus was separated from the intervertebral disc of SD rats aged 4-5 weeks, minced, digested with 0.2% type II collagenase at 37°C for 6 h, then digested with 0.25% trypsin-EDTA at 37°C for 15 min, and centrifuged at 150-200×g for 5-8 min. The resulting precipitate was AFCs for later use; the resulting cell precipitate was cultured in DMEM / F-12 containing 10% FBS and 1% P / S.

[0053] Extraction of rat dorsal root ganglion neurons: The dorsal root ganglion was isolated from the spine of 2- to 6-day-old SD rat pups, digested with 0.2% type II collagenase at 37°C for 30 min, then digested with 0.25% trypsin-EDTA at 37°C for 20 min, and centrifuged at 150-200×g for 5-8 min. The resulting precipitate was the cell mass, i.e., the rat dorsal root ganglion neurons. The cell mass was cultured in DMEM / F-12 containing 10% FBS and 1% P / S for 1 day, and the rat dorsal root ganglion neurons were further cultured in complete culture medium for rat dorsal root neurons containing 5 μmol / L cytarabine for 2 days.

[0054] 6. Construction of nanoparticle material (HIF1A@NNP) with therapeutic effect on IDD

[0055] The extracted nucleus pulposus cell membrane was sequentially passed through an extruder equipped with a polycarbonate porous membrane with a pore size of 1 μm and 200-400 nm to obtain vesicles of relatively uniform size. The vesicles were mixed with plasmid pHIF1A (plasmid cDNA3.1+-rHIF1A, GeneChem (Shanghai, China)) at a mass ratio of (100-200):1. In this embodiment, the vesicles were mixed with plasmid pHIF1A at a mass ratio of 150:1, and then sequentially passed through a polycarbonate membrane with a pore size of 1 μm and 200 nm to obtain nanoparticles wrapped by nucleus pulposus cell membranes, named HIF1A@NNP.

[0056] 7. Characterization of HIF1A@NNP

[0057] Transmission electron microscopy (TEM) imaging was used, HIF1A@NNPs were negatively stained with 2% phosphotungstic acid, and their morphology was observed using TEM (Hitachi, Japan). The size (diameter) and surface zeta potential of the nanoparticles were measured using a dynamic light scattering detector (Zetasizer Nano ZS90, Malvern Panalytical, UK).

[0058] 8. Cell Counting Kit-8 (CCK-8) Test

[0059] The cytotoxicity was evaluated using the CCK-8 assay, with the following specific steps: Nucleus pulposus cells (NPC) were seeded in a 96-well plate at a density of 8000 cells per well. The cells were then treated with different concentrations (0, 25, 50, 75, and 100 μg / mL) of NNPs and HIF1A@NNPs (the concentration of NNPs was recorded as the concentration of HIF1A@NNPs) for 24 h. Subsequently, the cells were washed three times with PBS buffer and incubated in 100 μL DMEM / F-12 medium containing 10% CCK-8 for 3 h, and the absorbance (OD) at 450 nm was measured using a multimode plate reader (PerkinElmer). 450 ). Cell viability was determined based on absorbance values, and each experiment was repeated at least 3 times, with 0 μg / mL NNP as the control group, which was normalized to 1.

[0060] 9. Evaluation of the encapsulation efficiency of HIF1A@NNP

[0061] The encapsulation efficiency of HIF1A@NNP was evaluated using a flow cytometer (Accuri C6 Plus, BD, New Jersey, USA). Plasmid pHIF1A was stained with propidium iodide (PI) for 10 min in the dark, while NNP was stained with DiO for 10 min in the dark. After the stained plasmid was encapsulated in the nucleus pulposus cell membrane, the proportion of fluorescent nanoparticles emitting dual fluorescence was evaluated by flow cytometry, and the encapsulation efficiency was calculated. The encapsulation efficiency was calculated as follows: Encapsulation efficiency = DiO + PI + % / (DiO + PI - %+DiO + PI + %), among which DiO + PI + % is the ratio of particles labeled by both DiO and PI. + PI - % is the ratio of particles labeled with DiO but not labeled with PI, DiO + PI - %+DiO + PI + % is the proportion of all particles labeled with DiO.

[0062] 10. Selective uptake of HIF1A@NNP by NPC

[0063] Flow cytometry (C6 plus type) was used to characterize the uptake capacity of different cells in the degenerative intervertebral disc for HIF1A@NNP. AFCs were stained with DiI, endothelial cells were stained with DiD, neurons were stained with Hoechst 33342, NPCs were stained with Hoechst 33342 and DiI, and macrophages were stained with Hoechst 33342 and DiD. 5 AFCs, NPCs, macrophages, neurons, and endothelial cells (100 cells) were co-cultured in culture dishes and incubated overnight. Afterwards, the cells were treated with DiO-labeled HIF1A@NNPs at a concentration of 100 μg / mL for 6 h. Subsequently, flow cytometry analysis was performed to detect the co-staining of different dyes and evaluate the uptake of HIF1A@NNPs by different cells.

[0064] In addition, NPCs were cultured in 24-well plates and labeled with DiO, and DiD was used to label HIF1A@NNP. NPCs were incubated with HIF1A@NNP and plasmid pHIF1A at 37°C for 4 h. Subsequently, the cells were fixed with 4% paraformaldehyde and stained with DAPI. Finally, fluorescence microscopy was used for image acquisition and subsequent analysis.

[0065] 11. Western Blot Analysis

[0066] Proteins were extracted using radioimmunoprecipitation (RIPA) lysis buffer, and then separated using a simple PAGE gel rapid preparation kit. After electrophoresis, proteins were transferred to a membrane and blocked with a rapid blocking solution. After incubation with primary and secondary antibodies, detection was performed using an ECL chemiluminescent substrate reagent on a ChemiDocTM MP imaging system (Bio-Rad, USA).

[0067] 12. Flow cytometer

[0068] For flow cytometric analysis of apoptosis, the treated NPCs were transferred to flow tubes and washed three times with PBS. Subsequently, NPCs were incubated with Annexin V-FITC / PI apoptosis detection kit at 4°C in the dark for 30 min before detection by flow cytometry (Accuri C6 Plus, BD).

[0069] For cell cycle flow cytometry analysis, the treated NPCs were transferred to flow tubes and washed three times with PBS. The cells were treated with 70% ethanol overnight and then washed three more times with PBS. Subsequently, NPCs were stained using a cell cycle staining kit for 30 min at room temperature in the dark before analysis.

[0070] 13. TUNEL staining

[0071] After cells were treated and nicked on coverslips by terminal deoxynucleotide maker, images were captured and analyzed using a fluorescence microscope (Olympus, Tokyo, Japan).

[0072] 14. Cell migration analysis

[0073] Transwell chamber plates were used to evaluate the migration ability of cells. The steps were as follows: cells (5 × 10 4 ) were inoculated into the upper chamber of the Transwell chamber. The lower chamber was filled with growth medium containing 10% FBS. After 24 h of treatment, cells that migrated to the other side of the membrane were fixed with 4% paraformaldehyde for 15 min and stained with 1% crystal violet for 30 min. After staining, images were captured and analyzed using an optical microscope (Olympus, Japan).

[0074] 15. Cell Immunofluorescence

[0075] After treatment, cells were fixed with 4% paraformaldehyde for 12 min. After fixation, cells were permeabilized in PBS containing 0.5% Triton for 30 min. Cells were then blocked with goat serum for 1 h and incubated with primary antibodies overnight at 4 °C, followed by incubation with fluorescent secondary antibodies for 1 h at room temperature. After counterstaining with DAPI for 15 min at room temperature, images were acquired by fluorescence microscopy (Olympus, Tokyo, Japan) for subsequent analysis.

[0076] 16. Senescence β-galactosidase staining

[0077] Senescent cells were detected using the senescent β-galactosidase staining kit, as follows: After washing once with PBS, the cells were fixed with β-galactosidase staining fixative for 15 min at room temperature. Subsequently, the cells were washed three times with PBS. Freshly prepared staining working solution was then added, the cells were incubated at 37°C overnight, and the plate was sealed with plastic film to prevent evaporation. Finally, the staining working solution was aspirated, and images were acquired and analyzed using an optical microscope (Olympus, Japan).

[0078] 17. Comet test

[0079] The IPHASE alkaline comet assay kit was used to assess cell DNA damage, and the steps were as follows: the treated cells were separated from the wells of the culture plate to prepare a cell suspension (1×10 5cells / mL). The gel for electrophoresis was prepared and mixed with the cell suspension at a volume ratio of 10:1. Subsequently, 50 μL of the mixture was evenly distributed on the slide and cured in the dark at 4°C for 60 min. Next, the slides were soaked in pre-cooled cell lysis buffer and incubated at 4°C overnight. After washing the slides three times with ultrapure water, they were treated with pre-cooled alkaline electrophoresis solution at 4°C in the dark for 1 h. Electrophoresis was performed for 30 min, followed by dehydration in anhydrous ethanol for 5 min and air drying. Finally, the slides were stained with PI for 10 min in the dark, and images were acquired and analyzed using a fluorescence microscope (Olympus, Japan).

[0080] 18. Enzyme-linked immunosorbent assay (ELISA)

[0081] Cell supernatants were collected and centrifuged at 20,000 × g to remove debris. The treated supernatants were evaluated using an ELISA assay kit according to the manufacturer's instructions and were measured using a multimode plate reader at 450 nm (OD 450 The concentrations of IL-1β, IL-6 and TNF-α in the supernatant were determined by extrapolation from the standard curve.

[0082] 19. Quantitative polymerase chain reaction (qPCR)

[0083] Total RNA was extracted from cells using an RNA extraction kit and then reverse transcribed into cDNA using a reverse transcription reagent. The transcripts of the target genes were detected using SYBR Green quantitative real-time polymerase chain reaction (PCR) SuperMix Plus on a real-time PCR system (Bio-Rad). △△CT Methods The relative expression of target genes was determined, and GAPDH was used as a reference gene for normalization of other genes. The primers used for qPCR are shown in Table 1.

[0084] Table 1 PCR primers used in this experiment

[0085]

[0086] Note: R: rat, M: mouse.

[0087] 20. Autophagy double-labeled adenovirus (mRFP-GFP-LC3)

[0088] Next, mRFP-GFP-LC3 cells were used to assess autophagic flux, and the colocalization of GFP and RFP fluorescence indicated the presence of autophagosomes, while RFP fluorescence alone indicated the presence of autolysosomes. NPCs were transfected with mRFP-GFP-LC3 for 12 h and then cultured in complete medium of rat dorsal root neurons for 1 d. Subsequently, after the cells were treated for 1 d, NPCs were fixed with 4% paraformaldehyde for 15 min and then incubated with DAPI for 15 min at room temperature. Finally, images were captured and analyzed using a confocal microscope (Olympus, Tokyo, Japan), and the number of dots per cell was counted.

[0089] 21. Test tube formation analysis

[0090] After dissolution at 4°C, the basement membrane matrix was dispensed into a 96-well plate at 50 μL / well. The plate was incubated at 37°C for 1 h to allow the gel to solidify. Subsequently, 8,000 human umbilical vein endothelial cells (HUVECs) were cultured in 100 μL of endothelial cell culture medium without FBS and added to the 96-well plate. After 18 h of cell treatment, images were captured and analyzed under an optical microscope (Olympus, Japan). Tube branching and total tube length were quantified using ImageJ software.

[0091] 22. Scratch wound analysis

[0092] HUVECs were seeded in 6-well plates and grown to confluence. A uniform scratch was made on the confluent cell layer using the tip of a P200 pipette. After washing three times with PBS to remove loose cells, the cells were processed and incubated at 37°C. Images were captured and analyzed under an optical microscope (Olympus, Japan) at 0, 12, and 24 h, and measured using ImageJ software.

[0093] 23. Nerve Growth Assessment

[0094] DRGs were isolated from the spinal cords of SD rats at 2-6 days of age, using the same isolation method as step “5. Isolation of annulus fiber cells (AFCs) and sensory neurons (DRG)”. All axons of the DRG were trimmed, and the ganglia were cultured in a culture dish containing rat dorsal root ganglion neuron culture medium containing NGF (20 ng / mL). After cell treatment, the cells were incubated horizontally at 37°C for 3 days. Immunofluorescence staining of DRGs was performed with neurofilament L rabbit pAb (neurofilament L), and images were captured using a fluorescence microscope. ImageJ software was used for quantitative analysis of neuronal complexity (branching) using the “Shaw Analysis” plug-in, in which concentric circles spaced 10 μm apart were drawn to quantify axonal crossings, and the DRG center was manually defined.

[0095] 24. Results

[0096] (1) HIF1A@NNP can improve cell viability

[0097] The cell membrane contains biomolecules and structures that promote targeted cell interactions. In this example, cell membranes were extracted from NPCs and extruded together with plasmid pHIF1A to develop a nanomaterial (HIF1A@NNP) with NNP as the shell and plasmid pHIF1A encapsulated in the core ( Figure 1 A in Figure 1). Plasmid pHIF1A was stained with PI and the cell membrane was stained with DiO before extrusion to generate HIF1A@NNP. Co-staining of PI and DiO was evaluated by flow cytometry, indicating that plasmid pHIF1A was successfully encapsulated within the membrane ( Figure 1 The encapsulation efficiency was calculated to be 88.41% ± 1.91%. Western blot analysis showed that NPC typical membrane markers, such as CD24, CD155, and CD 221, were positive in NNPs and HIF1A@NNPs compared with cell lysates ( Figure 1 Negatively stained transmission electron microscopy images show that after encapsulation of plasmid pHIF1A within the NPC membrane, vesicles of appropriate size were obtained ( Figure 1 Particle size and zeta potential measurements showed that the average sizes of NNP and HIF1A@NNP were approximately 186.80±4.06nm and 187.26±5.51nm, respectively, and the zeta potentials were approximately -41.51±0.90mV and -41.61±1.22mV, respectively. Figure 1 E). CCK-8 assay was used to evaluate the effects of NNP and HIF1A@NNP on NPC proliferation. Figure 1 As shown in F in Figure 3, the results showed that different concentrations of NNP (25, 50, 75, and 100 μg / mL) did not cause a significant increase in cell viability. However, when the HIF1A@NNP concentration reached 50 μg / mL, the viability of NPCs increased, and their proliferation increased significantly at concentrations of 75 and 100 μg / mL. Based on these results, a HIF1A@NNP concentration of 100 μg / mL was finally selected for subsequent experiments.

[0098] (2) HIF1A@NNP specifically targets and engulfs NP cells

[0099] AFCs were stained with DiI, endothelial cells were stained with DiD, neurons were stained with Hoechst 33342, NPCs were stained with Hoechst 33342 and DiI, and macrophages were stained with Hoechst 33342 and DiD. The cells were mixed and treated with HIF1A@NNP stained with DiO for 6 h. Different cells were distinguished by fluorescence, and the positive rate of DiO was recorded (process as shown in Figure 2NPC selectively uptakes HIF1A@NNP. Figure 2 As shown in B. The results showed that the DiO positive rate of endothelial cells was 0.32%, NPCs was 53.7%, neurons was 1.33%, macrophages was 5.12%, AFCs was 1.62%, and HIF1A@NNP specifically targeted and engulfed NP cells. The above results show that HIF1A@NNP has a good effect of targeting nucleus pulposus cells and can specifically present cargo to nucleus pulposus cells.

[0100] (3) HIF1A@NNP enhances the targeting ability of HIF1A to NPCs

[0101] The differences in cell viability and HIF1A overexpression efficiency between Lipo3000 (manufacturer: Thermofisher, catalog number: L3000015), lentivirus (Lenti-HIF1A, manufacturer: GeneCare, catalog number: Ubc-MCS-3FLAG-SV40-Puro-HIF1A) and HIF1A@NNP were compared. The concentrations of Lipo3000 and lentivirus were set according to the manufacturer's instructions. The results showed that NPCs treated with HIF1A@NNP showed increased viability with increasing concentrations, while Lipo3000 and lenti-HIF1A showed significant cytotoxicity to NPCs at high doses ( Figure 3 In addition, the results of Western blotting showed that when Lipo3000 (7.5 μL / 2×10 5 Compared with lenti-HIF1A (MOI = 50), HIF1A@NNP showed similar efficiency of overexpressing HIF1A ( Figure 4 ). These results suggest that HIF1A@NNPs enhance the targeting ability of HIF1A to NPCs and serve as a better reagent for gene editing.

[0102] At the same time, this example also compares the effects of Lipo3000-mediated plasmid pHIF1A transfection and HIF1A@NNP transfection in different types of cells adjacent to NPCs. The results of the tube formation and scratch experiments in HUVECs showed that Lipo3000-mediated HIF1A overexpression significantly enhanced the angiogenic tube formation and migration ability of HUVECs, while HIF1A@NNP had no significant effect on the tube formation and migration of HUVECs ( Figure 5In parallel, DRGs were also stained with neurofilament L and subsequently subjected to Lipo3000-mediated transfection of plasmid pHIF1A or treatment with NNPs or HIF1A@NNPs, and axonal growth was estimated using fluorescence. Again, HIF1A@NNPs were less efficient in increasing neurite branching and growth compared to Lipo3000-mediated HIF1A overexpression ( Figure 5 C). Compared with the plasmid pHIF1A group, the polarization of macrophages toward M1 and M2 in the HIF1A@NNP group was less obvious ( Figure 5 D). These results indicate that HIF1A@NNPs have minimal effects on the migration ability, angiogenesis, DRG axon growth, and macrophage polarization of HUVECs compared with HIF1A; moreover, HIF1A@NNPs effectively deliver HIF1A to NPCs and have less effects on other cells compared with Lipo3000-mediated transfection of plasmid pHIF1A.

[0103] (4) HIF1A@NNP protects NPCs from harmful microenvironment

[0104] We further investigated the role of HIF1A@NNP in protecting nucleus pulposus cells (NPCs) from harmful microenvironmental effects. NPCs were treated with TBHP, NNP, or HIF1A@NNP for 12 h, and apoptosis and extracellular matrix metabolism of NPCs were evaluated. The apoptosis rate of NPCs in the TBHP-treated group was significantly increased, while HIF1A@NNP inhibited TBHP-induced apoptosis ( Figure 6 A and B in Figure 3). And HIF1A@NNP showed a stronger inhibitory effect on apoptosis than NNP. Similarly, TUNEL staining further confirmed that HIF1A@NNP showed a stronger inhibitory effect on apoptosis than NNP ( Figure 6 C and D in Figure 3). Immunofluorescence analysis showed that HIF1A@NNP downregulated the expression of NPC catabolic proteins (MMP3, MMP13, and Adamts5) and promoted the expression of NPC anabolic proteins (COL2A1, Acan, and SOX9), indicating its ability to enhance matrix synthesis. As expected, HIF1A@NNP showed a stronger ability to promote matrix synthesis than NNP ( Figure 6 In addition, CCK8 experiments showed that TBHP significantly reduced cell viability. Compared with NNP, HIF1A@NNP showed a more obvious ability to enhance cell viability ( Figure 6 G). These results demonstrate the beneficial effects of HIF1A@NNP in improving NPCs survival and extracellular metabolism.

[0105] (5) HIF1A@NNP significantly inhibited the aging pathway

[0106] NPCs were cultured in 10% FBS / DMEM-F12 / 1% double-antibody medium containing TBHP and treated with NNPs or HIF1A@NNPs for 12 h. As expected, HIF1A@NNPs downregulated TBHP-induced p21 and p16 expression, while NNPs had no inhibitory effect on their expression ( Figure 7 In addition, flow cytometry results showed that HIF1A@NN P significantly upregulated the G2 / M cycle cell distribution, indicating reduced cell cycle arrest ( Figure 7 C and D). Meanwhile, HIF1A@NNP reduced the number of SA-β-gal positive cells induced by TBHP ( Figure 7 Comet assay, which assesses cellular DNA damage by quantifying the DNA content in the comet tail. This approach revealed that application of HIF1A@NNP significantly increased the accumulation of DNA damage in single cells ( Figure 7 Overall, these findings demonstrated that pHIF1A@NNPs significantly suppressed the aging pathway, suggesting that the combination of plasmid pHIF1A and NNPs may be an ideal strategy for anti-aging therapy.

[0107] (6) HIF1A@NNP regulates the inflammatory microenvironment by inhibiting SASP

[0108] Macrophage recruitment and M1 polarization play a key role in the progression of IDD. Current views believe that inhibiting macrophage infiltration and M1 polarization is a new approach for the treatment of IDD.

[0109] Conditioned media from NPCs treated with NNPs or HIF1A@NNPs were collected to evaluate their roles in regulating macrophage migration and polarization ( Figure 8 A in the figure). NPCs were stimulated with TBHP to simulate a degenerative environment, and 100 μg / mL of NNPs or HIF1A@NNPs were added to 10% FBS / DMEM-F12 / 1% double-antibody medium for 6 h. Cell supernatants were extracted to detect the senescence-associated secretory phenotype (SASP), and the results showed that compared with the CTRL group with only PBS, the SASP factors in the TBHP group were significantly increased, including IL-1β, IL-6, and TNF-α. When HIF1A@NNPs were added, inflammatory factors in the supernatant were significantly downregulated, while NNPs did not show any inhibitory effect ( Figure 8 Correspondingly, RT-qPCR showed that the levels of il1β, il6, and tnfα mRNA were significantly increased in TBHP-treated NPCs, while the administration of HIF1A@NNP significantly prevented this trend ( Figure 8C). Macrophage polarization phenotype analysis also showed that M1 polarization markers, including iNOS, TNF-α, and IL-6, were upregulated in the TBHP group but downregulated in the HIF1A@NNP group ( Figure 8 D). The relative mRNA expression levels of M2 polarization markers, including ARG1, CD206, and IL-10, were also tested under the same conditions. The analysis showed no significant difference in the expression of M2 polarization markers among the four groups ( Figure 8 Next, we evaluated whether HIF1A@NNPs affect macrophage migration. Figure 8 As shown in Figure F, TBHP increased macrophage migration, which was significantly inhibited by HIF1A@NNP. Therefore, SASP released by aged NPCs exacerbated IDD and low back pain, which not only changed the phenotype of healthy disc cells, but also recruited macrophages into the disc space and promoted their polarization toward the M1 phenotype, while HIF1A@NNP regulated the inflammatory microenvironment by inhibiting SASP.

[0110] Example 2 Animal Experiment

[0111] 1. Animal model construction

[0112] All animal experiments performed in this example were approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (No.

[2023] IAC UC 3514). The animals were housed in a ventilated environment with a constant temperature of 22°C and a 12:12h light-dark cycle. The rat model of involution (IDD) was established by acupuncture. After intraperitoneal injection of 0.6% pentobarbital anesthesia, the animals were positioned, and the puncture segment was located and marked at the Co7-8 level using X-ray imaging. SD rats were divided into 4 groups: normal group, IDD group, IDD NNP group, and IDD HIF1A@NNP group. The untreated rat tail IVD was represented as the intact group (i.e., the normal group mentioned above). The IDD experimental model was established by percutaneous puncture at the Co7-8 level of the rat tail using a 20-gauge needle. The needle was inserted to a depth of 6 mm, rotated 360°, held for 30 seconds, and then withdrawn. After the animal model was established, nanomaterials (100 μg / mL, 5 μL) were injected on days 7, 14, and 21. Nanomaterial injection was performed using a 29-gauge microsyringe. After 30 days, the intervertebral discs at the Co7-8 level of the rat tail were harvested, fixed in 4% paraformaldehyde, and decalcified for 1 month. Subsequently, the treated specimens were embedded in paraffin and sectioned for further analysis.

[0113] 2. Magnetic resonance imaging (MRI) analysis

[0114] T2-weighted mid-sagittal sections of the coccygeal spine were obtained using a 3.0T MRI scanner (GE Medical Systems, UK).In this example, the modified Pfirrmann grade and the relative water content of IVDs were evaluated based on MRI images by five spinal surgeons who were blinded to the group assignments.

[0115] 3. Histological evaluation

[0116] Rat tissue sections were stained with hematoxylin and eosin (H&E) and safranin O-fast green (S&O). The histological grading of rat sections was assessed independently by five researchers.

[0117] 4. Immunofluorescence staining

[0118] The disc sections were immersed in PBS containing 0.5% Triton for 30 min to promote permeabilization. The cells were treated with goat serum for 1 h. The sections were then exposed to primary and secondary antibodies and incubated with DAPI for 20 min at room temperature before observation under a fluorescence microscope.

[0119] 5. Statistical analysis

[0120] All experiments were repeated at least three times, and data are expressed as mean standard deviation (SD). Statistical differences between treatment groups were assessed using Student's t-test (two groups) or one-way analysis of variance (ANOVA) (more than two groups). Statistical analysis was performed using SPSS software package (IBM SPSS software package 18.0), and graphical representations were generated using GraphPadPrism 7 software (GraphPad Software Inc., SanDiego, CA). Statistical significance was set at P<0.05.

[0121] 6. Results

[0122] The IDD (degenerative disc disease) model was further established by coccygeal puncture, and NNP and HIF1A@NNP were injected into the intervertebral disc, and then MRI T2-weighted imaging was used to evaluate the severity of intervertebral disc degeneration ( Fig. 9 Figure A). Based on the MRI images, the modified Pfirmmann grading and relative water content of IVDs (intervertebral discs) were evaluated. HIF1A@NNP reversed the impairment of NP (nucleus pulposus) water retention capacity in the IDD group, while NNP alone showed no protective effect ( Fig. 9 (B). The modified Pfirmmann grade, which indicates the severity of degeneration, showed that HIF1A@NNP significantly reduced the degeneration grade, while the therapeutic effect of NNP was limited ( Fig. 9 HE and SO staining showed that HIF1A@NNP significantly alleviated the structural changes caused by puncture, as evidenced by the restoration of volume and vacuolar-like matrix in the IVD ( Fig. 9 In addition, NNPs and HIF1A@NNPs were stained with DiI before injection into the intervertebral disc, and co-staining of DiI and NPCs marker (CD24) was found, indicating that HIF1A@NNPs were taken up by NPCs in vivo ( Fig. 9 F in Figure 1). NNPs can be specifically recognized and engulfed by NPCs, indicating their potential as an effective drug or molecule delivery medium. Next, the delivery efficiency of plasmid pHIF1A by HIF1A@NNP was evaluated. After injection of HIF1A@NNP, the expression of HIF1A in the intervertebral disc tissue was significantly higher than that in the IDD group ( Fig. 9 G and H in ).

[0123] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A nanoparticle material having the efficacy of treating IDD, characterized in that: The nanoparticle material includes a nucleus pulposus cell membrane encapsulating plasmid pHIF1A.

2. The method for preparing the nanoparticle material according to claim 1, characterized in that: The following steps are involved: After extrusion of the nucleus pulposus cell membrane, vesicles are obtained; The vesicles and the plasmid pHIF1A are mixed and extruded to obtain the nanoparticle material.

3. The preparation method according to claim 2, characterized in that: The mass ratio of the vesicle to the plasmid pHIF1A is (100-200):

1.

4. The preparation method according to claim 2, characterized in that: The method for extracting nucleus pulposus cell membranes comprises the steps of digesting nucleus pulposus cells, first centrifuging, culturing, ultrasonicating and second centrifuging to obtain the nucleus pulposus cell membranes.

5. The preparation method according to claim 4, characterized in that: The digestive enzyme used in the digestion is type II collagenase; And / or, the digestion time is 20 minutes and the temperature is 30-37°C.

6. The preparation method according to claim 4, characterized in that: The centrifugal force of the first centrifugation is 150-200×g, and the time is 5-8 min.

7. The preparation method according to claim 4, characterized in that: The culture temperature is 37°C and the volume percentage of CO2 is 5%; And / or, the culture medium used in the culture is based on DMEM / F-12 culture medium and also includes 10% fetal bovine serum and 1% penicillin-streptomycin.

8. The preparation method according to claim 4, characterized in that: The second centrifugation includes initial centrifugation and repeated centrifugation; The centrifugal force of the initial centrifugation is 20000×g, and the time is 25 min; The centrifugal force of the multiple centrifugation is 100000×g, and the time is 35 min.

9. Use of the nanoparticle material according to claim 1 in the preparation of a drug for treating IDD and / or promoting spinal healing.

10. A drug for treating IDD and / or promoting spinal healing, characterized in that: The drug comprises the nanoparticle material of claim 1.

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