A circadian rhythm-regulating hydrogel microsphere and its preparation method and application

Abstract

The present invention provides a circadian rhythm regulating hydrogel microsphere and its preparation method and application. The preparation method of the circadian rhythm regulating hydrogel microsphere comprises the following steps: (1) dissolving lecithin, cholesterol and DSPE-PEG-PBA in an organic solvent, and preparing a liposome by the thin film method; (2) mixing the melatonin solution with the product obtained in step (1), and performing ultrasonic treatment to obtain a melatonin-loaded liposome; (3) preparing polyvinyl alcohol microspheres by the air microfluidics technique, and then reacting with a crosslinking agent TSPBA to prepare polyvinyl alcohol hydrogel microspheres; (4) mixing the product obtained in step (2) with the product obtained in step (3) in a TSPBA solution to obtain the circadian rhythm regulating hydrogel microsphere. The hydrogel microsphere material constructed by the present invention shows through cell experiments that it can activate the PI3K / AKT pathway in NPCs, regulate the intrinsic circadian rhythm, and promote the secretion of ECM; in vivo experiments confirm that it can well regulate the circadian rhythm of NPCs to significantly promote intervertebral disc regeneration.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to a circadian rhythm regulating hydrogel microsphere and its preparation method and application. Background Art

[0002] Circadian rhythm disorders during tissue degeneration are considered to be potential pathogenesis of various chronic diseases, such as intervertebral disc degeneration (IVDD). The relationship between IVDD and circadian rhythm has long been established. Mainly, the abnormal body circadian rhythm accelerates the process of intervertebral disc degeneration, triggering and aggravating the onset of low back pain. Recent studies have shown that the intervertebral disc has an intrinsic circadian rhythm, independent of the body's circadian rhythm. Intrinsic circadian rhythm disorders can be observed in degenerated intervertebral discs, and in vitro interleukin stimulation of nucleus pulposus cells (NPCs) can disrupt the circadian rhythm of NPCs. These results indicate that the intrinsic circadian rhythm of the intervertebral disc plays an important role in intervertebral disc degeneration.

[0003] Circadian rhythm disorders are considered to be potential pathogenesis of multiple diseases, including many skeletal muscle diseases. With the aging of the population and the fast-paced society, circadian rhythm disorders are becoming more and more common. Therefore, there is an urgent need to explore new treatment strategies based on circadian rhythm regulation.

[0004] Research reports have shown that melatonin (Mel) can be used to reshape the disordered circadian rhythm. However, the treatment strategy of using melatonin to regulate the cellular circadian rhythm is limited by the short drug half-life and the complexity of the in vivo microenvironment.

[0005] Subsequent researchers have tried to construct a suitable drug delivery system to load circadian rhythm regulating drugs for improving the curative effect, targeting pathological cells, precisely regulating the cellular circadian rhythm, thereby promoting the recovery of cellular physiological functions and promoting tissue regeneration. However, the drug delivery system related to melatonin can only show good regulatory effects in vitro, and its in vivo treatment effect has always been poor.

[0006] In recent years, light-responsive biomaterials have become a potential drug delivery strategy. This strategy can coordinate drug release with changes in environmental light, thereby maximizing the drug efficacy. However, light is an uncontrollable and constantly changing state of the external environment. It is difficult for external environmental conditions to reach deep tissues, which limits the clinical application of light-responsive biomaterials.

[0007] Therefore, materials for regulating the cell biological clock should focus more on the changes in the tissue microenvironment. In tissues, cells have physiological patterns controlled by the biological clock, and the changes in the local tissue microenvironment are also closely related to the cell biological clock. Core biological clock genes, including aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) / circadian locomotor output cycles kaput protein (clock) in the brain and muscle, may directly control the expression and activity of various intracellular enzymes, thereby affecting the physiological functions of cells. However, existing materials for regulating the cell biological clock cannot achieve good effects in vivo. For example, the drug delivery system loaded with melatonin cannot exert the corresponding biological clock regulation function in vivo.

[0008] Therefore, how to provide a new drug delivery system for loading melatonin, which can fully exert the effect of melatonin in regulating biological clock disorders, so that it can also have good effects in vivo, with a view to using it for the treatment of intervertebral disc degeneration, has become an urgent technical problem to be solved. Summary of the Invention

[0009] The present invention is to solve the above technical problems, and thus provides a biological clock regulating hydrogel microsphere and its preparation method and application. The technical purpose of the present invention is to provide a hydrogel microsphere for loading melatonin, to solve the problem that the existing melatonin drug delivery system cannot exert curative effects in vivo, resulting in poor treatment effects on intervertebral disc degeneration.

[0010] The present invention first provides a preparation method of a biological clock regulating hydrogel microsphere, including the following steps:

[0011] (1) Dissolve lecithin, cholesterol and DSPE-PEG-PBA in an organic solvent, and prepare liposomes by the thin film method;

[0012] (2) Mix the melatonin solution with the product obtained in step (1), and perform ultrasonic treatment to obtain melatonin-loaded liposomes;

[0013] (3) Prepare polyvinyl alcohol microspheres by air microfluidics technology, and then react with the cross-linking agent TSPBA to prepare polyvinyl alcohol hydrogel microspheres;

[0014] (4) Mix the product obtained in step (2) with the product obtained in step (3) in a TSPBA solution to prepare a biological clock regulating hydrogel microsphere.

[0015] The present invention uses air microfluidics technology and chemical cross-linking of polyvinyl alcohol (PVA) and phenylboronic acid (PBA) to prepare circadian clock regulatory microspheres (ClockMPs) that can effectively activate the intrinsic circadian clock of nucleus pulposus cells in intervertebral disc degeneration (IVDD) and improve the physiological function of NPCs for intervertebral disc regeneration. In vitro experiments confirmed that ClockMPs can promote the binding of BMAL1 and CLOCK proteins, scavenge reactive oxygen species (ROS), and maintain a stable microenvironment for the circadian clock. In addition, ClockMPs can regulate the expression of core circadian clock genes, reshape the intrinsic circadian clock, and promote extracellular matrix synthesis by activating the PI3K / AKT pathway in NPCs. In vivo experiments demonstrated that ClockMPs can significantly promote intervertebral disc regeneration by regulating the circadian clock of NPCs and well exert the function of melatonin in regulating the circadian clock in vivo.

[0016] However, the research of the present invention shows that melatonin alone or melatonin loaded on other carriers cannot exert the effect of melatonin in regulating the circadian clock in vivo.

[0017] Furthermore, the weight ratio of lecithin, cholesterol, and DSPE-PEG-PBA in step (1) is 6.9:2.9:2.

[0018] Furthermore, the organic solvent in step (1) includes at least one of chloroform, methanol, ethanol, and dichloromethane.

[0019] Furthermore, the concentration of the melatonin aqueous solution in step (2) is 0.233 g / mL, and the volume ratio of the melatonin aqueous solution to the organic solvent is 1:1.5.

[0020] Furthermore, the conditions of the ultrasound in step (2) are: 40% power, ultrasound working for 2 s, stopping for 1 s, and the total ultrasound working time is 7 min.

[0021] Furthermore, the concentrations of TSPBA and polyvinyl alcohol in step (3) are 5-10 wt% TSPBA and 5-10 wt% PVA, and the volume ratio of the two is 5-10% TSPBA:5%-10% PVA = 1:0.25-2.

[0022] Furthermore, the operation of preparing PVA microspheres by the air microfluidic technology in step (3) is as follows: Inject a 7.5% (w / v) PVA aqueous solution into the inner layer of the device, connect a nitrogen gas stream (N2) to the outer layer, with the N2 velocity being 1 L / min. Cut the PVA solution into uniform droplets by the principle of gas cutting, collect the PVA hydrogel microspheres using absolute ethanol, and obtain precursor microspheres through the principle of solvent exchange. Scoop up the precursor microspheres from absolute alcohol using a 70-mesh tissue filter for standby. Pour the precursor microspheres into the TSPBA solution to crosslink them to form ROS-responsive PVA hydrogel microspheres.

[0023] The second object of the present invention is to provide a circadian rhythm-regulating hydrogel microsphere prepared by the method as described above.

[0024] The third object of the present invention is to provide the application of the above circadian rhythm-regulating hydrogel microsphere, which is to prepare the hydrogel into a drug for treating intervertebral disc degeneration. Specifically, it is to prepare the hydrogel into a drug for promoting intervertebral disc regeneration.

[0025] Furthermore, the application of the present invention also includes preparing the hydrogel microsphere into a drug for regulating the circadian rhythm.

[0026] The beneficial effects of the present invention are as follows:

[0027] The present invention constructs a hydrogel microsphere material (ClockMPs), which can directly regulate the circadian rhythm of NPCs cells to promote the regeneration of the intervertebral disc. ClockMPs have ROS responsiveness, ROS scavenging ability, and anti-leakage characteristics. Cell experiments show that ClockMPs can activate the PI3K / AKT pathway in NPCs, regulate the inherent circadian rhythm, and promote the secretion of ECM; in vivo experiments confirm that ClockMPs can well regulate the circadian rhythm of NPCs to significantly promote intervertebral disc regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the mechanism of the circadian rhythm-regulating material of the present invention; A) Preparation of ClockMPs; B) ClockMPs promote intervertebral disc regeneration by regulating the circadian rhythm of NPCs.

[0029] Figure 2For the preparation and evaluation of ClockMPs; A) The process of preparing ClockMPs; B) TEM image of Mel@Lipo; C) DLS results of Mel@Lipo; D) Image of ClockMPs under the microscope; E) Size distribution histogram of ClockMPs; F) SEM image of MPs; G) SEM image of ClockMPs; H) Degradation of MPs; I) Scavenging of PTIO by ClockMPs; J) Drug release profiles of ClockMPs and Mel@Lipo; K) In vivo imaging of DiR, DiR@Lipo, and DiR@MPs at 0, 3, 6, and 9 days after intervertebral injection; L) Changes in fluorescence intensity of DiR, DiR@Lipo, and DiR@MPs over time; (n = 3).

[0030] Figure 3 For RNA-seq analysis of the H2O2 group and the ClockMPs group; A - C) Screening of differentially expressed genes between the H2O2 group and the ClockMPs group, and gene ontology (GO) analysis from three aspects: (A) cellular component, (B) biological process, and (C) molecular function; D) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes between the H2O2 group and the ClockMPs group; E - F) Gene set enrichment analysis of differentially expressed genes between the H2O2 group and the ClockMPs group.

[0031] Figure 4 For the regulation of biological clock and physiological functions of npc by ClockMPs; A) Profiles of clock gene expression changes within 24 h in the Control group, H2O2 group, MPs group, Mel group, and ClockMPs group; B) Comprehensive diagram of the change profiles; C) Western blot results of CLOCK, BMAL1, and RORα; D) Relative gray scale of CLOCK protein bands in the five groups; E) Relative gray scale of BMAL1 protein bands in the five groups; F) Relative gray scale of rorα protein bands in the five groups; G) Western blot results of PI3K, p - PI3K, AKT, and p - AKT; H) Relative gray scale of PI3K protein bands in the five groups; I) Relative gray scale of p - PI3K protein bands in the five groups; J) Relative gray scale of AKT protein bands in the five groups; K) Relative gray scale of p - AKT protein bands in the five groups; L) Western blot results of type II collagen and aggrecan; M) Relative gray scale of type II collagen bands in the five groups; N) Relative gray scale of aggrecan bands in the five groups; (n = 3, * indicates P < 0.05, ** indicates P < 0.01, compared with the control group; # indicates P < 0.05, ## indicates P < 0.01, compared with the H2O2 group; $ indicates P < 0.05, $$ indicates P < 0.01, compared with the MPs group; % indicates P < 0.05, %% indicates P < 0.01, compared with the Mel group).

[0032] Figure 5 For ClockMPs to be able to reverse the degeneration of NPCs; A) Representative double immunofluorescence staining of Sox9 and Phalloidin in NPCs; B) Representative double immunofluorescence staining of type II collagen and Phalloidin in NPCs; C) Proportion of Sox9-positive cells in different groups; D) Fluorescence intensity of Sox9 in different groups; E) Fluorescence intensity of type II collagen in different groups; (n = 3, * indicates P < 0.05, ** indicates P < 0.01, compared with the control group; # indicates P < 0.05, ## indicates P < 0.01, compared with the H2O2 group; $ indicates P < 0.05, $$ indicates P < 0.01, compared with the MPs group; % indicates P < 0.05, %% indicates P < 0.01, compared with the Mel group).

[0033] Figure 6 For the radiological results of animal experiments; A) IVDD modeling scheme and treatment; B) X-ray images; C) Changes in the profile of IDH over time; D) Changes in IDH in the healthy group, IVDD group, MPs group, Mel group, and ClockMPs group within 1 week; E) Changes in IDH in the five groups at 4 weeks; F) Changes in IDH in the five groups at 8 weeks; G) Intervertebral disc MRI images; H) Changes in gray values in each group at -1 week, 1 week, 4 weeks, and 8 weeks after surgery; (n = 5, * indicates P < 0.05, ** indicates P < 0.01, compared with the healthy group; # indicates P < 0.05, ## indicates P < 0.01, compared with the IVDD group; $ indicates P < 0.05, $$ indicates P < 0.01, compared with the MPs group; % indicates P < 0.05, %% indicates P < 0.01, compared with the Mel group).

[0034] Figure 7 For the histological results of animal experiments; A) H&E staining of each group at 1, 4, and 8 weeks after surgery; B) Masson staining of each group at 1, 4, and 8 weeks after surgery; C) Histological grading of each group at 1 week; D) Histological grading of each group at 4 weeks; E) Histological grading of each group at 8 weeks; (n = 5, * indicates P < 0.05, ** indicates P < 0.01, compared with the healthy group; # indicates P < 0.05, ## indicates P < 0.01, compared with the IVDD group; $ indicates P < 0.05, $$ indicates P < 0.01, compared with the MPs group; % indicates P < 0.05, %% indicates P < 0.01, compared with the Mel group).

[0035] Figure 8Immunohistochemical images for animal experiments; A) CLOCK immunohistochemistry in each group at 1, 4, and 8 weeks after surgery; B) Average optical density (AOD) of CLOCK in each group at 1 week; C) AOD of CLOCK in different groups at 4 weeks; D) AOD of CLOCK in different groups at 8 weeks; E) Immunohistochemistry of type II collagen in each group at 1, 4, and 8 weeks after surgery; F) AOD of type I collagen in each group at 1 week; G) AOD of type II collagen in each group at 4 weeks; H) AOD of type II collagen in different groups at 8 weeks; (n = 5, * indicates P < 0.05, ** indicates P < 0.01, compared with the healthy group; # indicates P < 0.05, ## indicates P < 0.01, compared with the IVDD group; $ indicates P < 0.05, $$ indicates P < 0.01, compared with the MPs group; % indicates P < 0.05, %% indicates P < 0.01, compared with the Mel group).

[0036] Figure 9 Steps for the preparation of the crosslinking agent TSPBA and its 1H-NMR spectrum.

[0037] Figure 10 Drug release profiles of ClockMPs and Mel@Lipo within the first 12 h.

[0038] Figure 11 Fluorescence intensity of in vivo imaging at different time points; A) Day 0, B) Day 3, C) Day 6, D) Day 9; (n = 3, * indicates P < 0.05 compared with the DiR group, ** indicates P < 0.01 compared with the DiR group, # indicates P < 0.05 compared with the DiR@Lipo group, # indicates P < 0.01 compared with the DiR@Lipo group).

[0039] Figure 12 Biocompatibility evaluation; A) Representative images of live / dead staining; B) Cell viability in different groups; C) CCK-8 assay in different groups; D) Cell viability profiles in different groups; E) OD450nm profiles in different groups; (n = 3, * indicates P < 0.05 compared with the control group, ** indicates P < 0.01 compared with the control group, # indicates P < 0.05 compared with the H2O2 group, ## indicates P < 0.01 compared with the H2O2 group, $ indicates P < 0.05 compared with the MPs group, $$ indicates P < 0.05 compared with the Mel group, %% indicates P < 0.01 compared with the Mel group).

[0040] Figure 13were: A) The relative expression levels of the CLOCK gene in 5 groups; B) The relative expression levels of the BMAL1 gene in five groups; C) The relative gene expression levels of RORα in five groups; (n = 3, * indicates P < 0.05 compared with the control group, ** indicates P < 0.01, # indicates P < 0.05 compared with the H2O2 group, ## indicates P < 0.01, $ indicates P < 0.05 compared with the MPs group, $$ indicates P < 0.01, % indicates P < 0.05 compared with the Mel group, %% indicates P < 0.01). Figure 14 were: A) The gray value profiles of different populations; B) The histological grades of different groups; (n = 5, ** indicates P < 0.01, NS indicates no significant difference).

[0041] Figure 15 were: A) The immunohistochemical conditions of BMAL1 in each group at 1, 4, and 8 weeks after surgery; B) The average optical density (AOD) of BMAL1 in each group at 1 week; C) The AOD of BMAL1 in each group at 4 weeks; D) The AOD of BMAL1 in different groups at 8 weeks; (n = 5, * indicates P < 0.05, ** indicates P < 0.01 compared with the healthy group, # indicates P < 0.05 compared with the IVDD group, ## indicates P < 0.01, $ indicates P < 0.05 compared with the MPs group, $$ indicates P < 0.01, % indicates P < 0.05 compared with the Mel group, %% indicates P < 0.01).

[0042] Figure 16 were the change curves of AOD with time for each group; A) The change in AOD of the clock; B) The change in AOD of BMAL1; C) The change in AOD of type II collagen. Detailed implementation manners

[0043] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be specifically described below in conjunction with embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still fall within the protection scope of the present invention.

[0044] Embodiment 1

[0045] All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Beijing Shengjihe Experimental Animal Technology Co., Ltd. (P2020109). All procedures were performed to minimize pain, and all surgeries were carried out under anesthesia.

[0046] I. Experimental methods

[0047] (I) Preparation of Mel@Lipo liposomes

[0048] (1) Prepared by the thin film method. 6.9 mg of lecithin, 2.9 mg of cholesterol, and 2 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-phenylboronic acid (DSPE-PEG-PBA) were dissolved in 1 mL of chloroform. Then the mixture was transferred to a 5 mL round-bottom flask, and the flask was placed in a 37 °C water bath and tilted and rotated to evaporate the solvent to form a uniform lipid film (Lipo).

[0049] (2) 2.33 mg of melatonin (Mel) was dissolved in 10 mL of double-distilled water (ddH2O). Then 1.5 mL of the solution was slowly added to the round-bottom flask and then treated with an ultrasonic cell disruptor (Voshin, China) for 7 minutes to prepare Mel-loaded liposomes (Mel@Lipo).

[0050] DiR was used to replace Mel, and DiR-loaded liposomes (DiR@Lipo) were constructed according to the above steps.

[0051] The morphology of the liposomes was observed by transmission electron microscopy (TEM, Tecnai G2 Spirit Biotwin), and the particle size distribution of the liposomes was detected using a dynamic light scattering analyzer (DLS, Malvern Instruments Ltd., UK).

[0052] (II) Preparation of MPs and ClockMPs

[0053] Polyvinyl alcohol (PVA) microspheres were prepared by air-microfluidic technology. An air microfluidic device was constructed using a coaxial needle. The inner layer of the device was filled with a PVA (7.5% w / v) solution, and the outer layer was connected to a nitrogen flow rate (1 L / min). The PVA hydrogel precursor microspheres were collected in ethanol. The precursor microspheres were fished out of the alcohol using a 70-mesh filter. Based on previous studies (H. Ruan, Q. Hu, D. Wen, Q. Chen, G. Chen, Y. Lu, J. Wang, H. Cheng, W. Lu, Z. Gu, Adv Mater 2019, 31, e1806957.), a crosslinking agent N1-(4-boronophenyl)-N3-(4-boronophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine (TSPBA) linker was synthesized, and proton nuclear magnetic resonance (1H-NMR, Bruker, USA) was used to determine the molecular structure of TSPBA (as Figure 9 )

[0054] The precursor microspheres were added to the TSPBA solution to crosslink and form PVA hydrogel microspheres (MPs) that generate reactive oxygen species (ROS).

[0055] The precursor microspheres and Mel@Lipo were added to the TSPBA solution to prepare the circadian rhythm regulating hydrogel microspheres (ClockMPs).

[0056] By adding the precursor microspheres and DiR@Lipo to the TSPBA solution, the MPs loaded with DiR (DiR@MPs) were constructed, and then the cross-linked hydrogel microspheres were rinsed three times with ddH2O to remove the excess TSPBA.

[0057] (III) Morphological observation of MPs and ClockMPs

[0058] The hydrogel microspheres were immediately observed and photographed under an optical microscope (Nikon, Japan) after cross-linking. According to the images of the hydrogel microspheres, the diameters of the hydrogel microspheres were measured manually. After freeze-drying, the microstructures of MPs and ClockMPs were observed by scanning electron microscopy (SEM, Sirion200, USA).

[0059] (IV) Degradation of MPs

[0060] The MPs were added to the phosphate buffer solution (PBS) with or without H2O2 to study the ROS-responsive degradation of MPs. The degradation distribution of MPs was determined by weighing the remaining mass of MPs (sensitivity: 1 / 10000).

[0061] (V) 2-Phenyl-4,4,5,5-tetramethylimidazoline-3-oxyl-1-oxyl (PTIO) scavenging assay

[0062] The PTIO solution was prepared in ddH2O, and the PTIO scavenging experiment was carried out as follows: The samples (x = 0, 40, 80, 120, 160, 200 μL) were added to (200 - x) μL of ddH2O, and then 800 μL of PTIO solution was added. The mixture was incubated at 37 °C for 2 hours, and then the absorbance was measured at 557 nm using a microplate reader (Bio-Rad, USA).

[0063] The calculation formula for the PTIO scavenging rate is:

[0064] PTIO scavenging rate % = (A0 - Ax) / A0 × 100%.

[0065] (VI) Release experiment of Mel

[0066] The ClockMPs were added to the PBS with or without H2O2, and the release of Mel from Mel@Lipo in PBS was used as a control. The release amount of Mel was determined by high performance liquid chromatography (HPLC, Waters, USA).

[0067] (VII) In vivo retention test

[0068] DiR, DiR@Lipo, and DiR@MPs were used to study the in vivo retention effect of MPs. Sprague-Dawley (SD) rats at 8 weeks of age were purchased from Beijing Shengjihe Laboratory Animal Technology Co., Ltd. All animal breeding and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Beijing Shengjihe Laboratory Animal Technology Co., Ltd., and the ethical approval number was P2020109.

[0069] After anesthesia, a 1 mL syringe was used to puncture the intervertebral discs at the 5 / 6, 6 / 7, and 7 / 8 levels of the caudal vertebrae (Co5 / 6, Co6 / 7, and Co7 / 8 respectively), aspirate the nucleus pulposus (NP) tissue and discard it. Then, 100 μL of DiR was injected into Co5 / 6, 100 μL of DiR@Lipo was injected into Co6 / 7, and 100 μL of DiR@MPs was injected into Co7 / 8. In vivo image acquisition and analysis were performed using an in vivo imaging system (CRiMaestro TM in-vivo imaging system; Cri, Hopkinton, MA) at 0, 3, 6, and 9 days after injection.

[0070] (VIII) Extraction of nucleus pulposus cells (NPCs) and oxidative stimulation experiment of NPCs

[0071] The extraction of rat NPCs was referred to the existing literature (W. Chen, H. Chen, D. Zheng, H. Zhang, L. Deng, W. Cui, Y. Zhang, H. A. Santos, H. Shen, Adv Sci (Weinh) 2020, 7, 1902099.). NPCs were cultured at 37 °C, 5% CO2, and 95% air, and the cell culture medium was changed every 72 h. When the cell density reached 80% - 90% of the culture dish, the cells were digested with trypsin for passage. NPCs in the 2 - 4th passage were used for in vitro experiments. NPCs were treated with 100 nM dexamethasone (DEX) for 15 minutes to synchronize the cells. After 24 h of DEX treatment, the cells were stimulated with 100 μM H2O2 for 2 h to induce oxidative stress. The oxidative stress NPCs were randomly divided into 4 groups and treated with PBS, MPs, Mel, and ClockMPs respectively. Completely normal NPCs were used as the normal control group (control).

[0072] (IX) Biocompatibility of ClockMPs

[0073] The survival of NPCs at 1, 2, and 3 days after treatment was detected using the ClockMPs Live / Dead staining kit (LifeTech, USA), and the live / dead staining was performed strictly according to the instructions. The live / dead staining images were observed with a fluorescence microscope (Nikon, Japan). Live and dead cells were counted manually, and the cell survival rate was determined by the ratio of live cells to total cells. The proliferation of NPCs was detected using the Cell Counting Kit-8 (CCK-8). The CCK-8 assay was performed strictly according to the instructions. After incubation for 40 min, the optical density (OD450) at 450 nm was detected using a Microplate Reader (SpectraMax M3).

[0074] (X) RNA-seq

[0075] Total RNA was independently isolated from each sample using Trizol reagent (Invitrogen) and stored at 80 °C. The cDNA library for each RNA sample was created using the TruSeq kit (Illumina, USA) according to the manufacturer's protocol. The library was subjected to quality control using an Agilent 2200 and paired-end sequencing of 150 bp was performed using a Hiseq X Ten. Clean reads were aligned to the rat genome (GRCm38, NCBI) using the Hisat2 software. Gene counts were obtained using HTseq, and gene expression was determined using the RPKM technique. As described previously, gene set enrichment analysis (GSEA) was performed using the GSEA software version 4.1.0 (Broad Institute). 10,000 random permutations of the gene set were used, and genes were ranked using the signal-to-noise ratio metric to assess statistical significance. The fdr-corrected values of q and lt; 0.25 was significant, and the gene set was from the Molecular Signatures Database (MSigDB) version 7.4.

[0076] (XI) Real-time quantitative polymerase chain reaction (RT-qPCR)

[0077] Total cellular RNA was extracted using Trizol reagent (Invitrogen, USA), and reverse transcription polymerase chain reaction (RT-PCR) was performed using the primescript RT-PCR kit (Takara, Japan) to obtain cDNA. RT-qPCR was performed using the SYBR Green RT-qPCR kit (Takara, Japan) and an ABI step1+ real-time PCR system (applied biosystems, USA). All qRT-PCR primers are listed in Table 1.

[0078] Table 1 Primer sequences used for RT-qPCR

[0079]

[0080]

[0081] (XII) Western Blot

[0082] Total cellular proteins were extracted using RIPA buffer (Beyotime, China), and the total cellular proteins were quantified using the bicinchoninic acid (BCA) protein assay (Pierce Scientific, CA). Protein samples were subjected to SDS electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (0.45 μm, Millipore, USA). The PVDF membranes were treated with 5% non-fat milk for 1 hour and then incubated overnight at 4°C with the following primary antibodies: anti-CLOCK antibody (A7265, ab clone, China), anti-BMAL1 antibody (A4714, ab clone, China), anti-RORα antibody (A6971, AB clone, China), anti-PI3K antibody (A4992, AB clone, China), anti-p-PI3K antibody (AF3242, Affinity, USA), anti-AKT antibody (A18675, AB clone, China), anti-p-AKT antibody (AP1208, AB clone, China), anti-CollagenII antibody (ab307674, Abcam, USA), and anti-Aggrecan antibody (ab3778, Abcam, USA). After washing three times with tris-buffered saline with Tween-20 (TBST), the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) for 2 hours. Antigen-antibody complexes were visualized using the enhanced chemiluminescence detection system recommended by the manufacturer, and the signal intensity of protein bands was quantified using ImageJ software.

[0083] (XIII) Immunofluorescence Staining

[0084] Cells were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.2% triton X-100. Labeling solutions were prepared by mixing phalloidin with anti-Sox9 antibody (ab185966, Abcam, USA) or anti-CollagenII antibody (ab307674, Abcam, USA). Cells were incubated with the labeling solutions overnight at 4°C. Cells were treated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) for 40 min at room temperature. Nuclei were labeled with 2-(4-aminophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Beyotime, China). Labeled cells were observed under a fluorescence microscope. The fluorescence intensity of Sox9 or CollagenII was analyzed using ImageJ software.

[0085] (14) Animal model construction

[0086] All animal husbandry and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Beijing Shengjihe Laboratory Animal Science Co., Ltd., and the ethical approval number was P2020109. Male Sprague-Dawley (SD) rats at 8 weeks of age were purchased from Beijing Shengjihe Laboratory Animal Science Co., Ltd. The rats were placed in cages with 3 rats per cage and had free access to water. The temperature of the cage room was controlled at 22 ± 2 °C. After 1 month of acclimation, 15 three-month-old SD rats (300 - 350 g) were randomly selected for in vivo experiments.

[0087] A rat intervertebral disc degeneration model was established with reference to previous studies [4]. After anesthesia, the Co5 / 6, Co6 / 7, Co7 / 8, and Co8 / 9 were punctured with an 18-gauge needle. The needle reached the center of the nucleus pulposus (NP) and was removed after 30 s. 100 μL of PBS (Co5 / 6), MPs (Co6 / 7), Mel (Co7 / 8), and ClockMPs (Co8 / 9) were injected respectively. The healthy Co4 / 5 disc was used as the control group. Five rats (n = 5) were included in each group at each time point. All operations were performed under sterile conditions.

[0088] (15) Imaging evaluation

[0089] X-ray examination and magnetic resonance imaging (MRI) of the intervertebral discs were performed at 1 week before surgery (-1w), 1 week after surgery (1w), 4 weeks after surgery (4w), and 8 weeks after surgery (8w) respectively. The degeneration of the intervertebral discs was studied by measuring the intervertebral disc height (IDH) of the experimental intervertebral discs in the X-ray images. ImageJ software was used to determine the gray scale of the MRI intervertebral discs, which reflected the water content of the intervertebral discs.

[0090] (16) Histological evaluation

[0091] Disc tissue samples were collected at 1, 4, and 8 weeks after surgery. All samples were collected around noon to ensure that the biological clock was at the same level. All samples were fixed in 4% paraformaldehyde for 48 hours and decalcified in ethylenediaminetetraacetic acid decalcifying solution for 4 weeks. Then they were rinsed with ddH2O overnight, dehydrated with gradient alcohol, cleared with xylene, and embedded in paraffin. The wax blocks were frozen at -20°C overnight. Serial sections with a thickness of 5 μm were made using a Leica tissue slicer from Germany. The sections were baked at 60°C overnight. Hematoxylin-eosin (H&E) staining was used to observe the structural changes of the intervertebral disc. Masson staining was used to observe the remodeling of collagen. For mouse intervertebral disc degeneration, the histological grade of the rat intervertebral disc was determined according to a standardized grading system (I.P. Melgoza, S.S. Chenna, S.T. Tessier, Y. Zhang, S.Y. Tang, T. Ohnishi, E.J. Novais, G.J. Kerr, S. Mohanty, V. Tam, W.CW Chan, C.M. Zhou, Y. Zhang, V.Y. Leung, A.K. Brice, C.A. Séguin, D. Chan, N. Vo, M.V. Risbud, C.L. Dahia, JORSpine 2021, 4(2):e1164.), and the expressions of CLOCK, BMAL1, and CollagenII were detected by immunohistochemistry.

[0092] (XVII) Statistical analysis

[0093] All data were expressed as mean ± standard deviation (SD), and the sample size of all experiments was n≥3. Graphpad Prism 8.0 software was used to draw data visualization images. The data analysis function of Graphpad Prism 8.0 software was used for statistical analysis. One-way ANOVA and t-test were used to compare whether the differences were statistically significant. P<0.05 was considered significantly different, and P≥0.05 was considered not significantly different (NS).

[0094] II. Experimental results (I) Preparation and evaluation of ClockMPs

[0095] PBA-grafted liposomes were prepared by the film-forming method and loaded with melatonin ( Figure 2 Part A in Figure 2 ). Transmission electron microscope (TEM) images showed that the melatonin-loaded liposomes (Mel@Lipo) had a bilayer membrane structure ( Figure 2 Part B in

[0096] ClockMPs were observed to be uniform transparent microspheres under light microscopy, with an average diameter of 215.13±18.60 μm ( Figure 2 Scanning electron microscopy (SEM) images show that both MPs and ClockMPs exhibit a microstructure with dense small pores ( Figure 2 F and G in the middle), a large number of white high-density dots can be seen on the surface of ClockMPs, indicating that liposomes are attached to ClockMPs ( Figure 2 (Section G in the middle).

[0097] In summary, MPs were prepared by air microfluidics and solvent exchange method, and ClockMPs were prepared by PVA-TSPBA-PBA cross-linking method.

[0098] (2) ROS responsiveness test

[0099] There is a close, bidirectional relationship between the cellular circadian clock and ROS. On the one hand, circadian clock disruption affects cellular redox metabolism, interfering with ROS clearance and leading to abnormal ROS accumulation. On the other hand, increased ROS inhibits the activity of key enzymes in the cellular circadian clock, thereby exacerbating circadian disruption. Therefore, during disease progression, repairing tissue aging requires coordinating circadian clock regulation and ROS clearance. Therefore, we explored the interplay between circadian clock microspheres and ROS.

[0100] The degradation profile of MPs was determined under ROS conditions to explore the ROS response ability of MPs ( Figure 2 MPs degraded slowly in PBS, while in H2O2 solution, the degradation rate of MPs was less than 30% within 1 hour. The results showed that the degradation rate of MPs was accelerated in the presence of ROS, which was beneficial for drug release. The scavenging ability of MPs on 2-phenyl-4,4,5,5-tetramethylimidazol-3-oxyl-1-oxyl (PTIO) was investigated by PTIO scavenging experiments ( Figure 2 (Part I). PTIO scavenging experiments demonstrated that MPs possessed robust PTIO scavenging activity, with the scavenging rate proportional to MP concentration. ClockMPs exhibited slightly lower scavenging efficiency than MPs. These results demonstrate that MPs not only respond to ROS by accelerating degradation but also scavenge excess ROS.

[0101] In addition, the release profiles of Mel under different administration methods were determined ( Figure 2 The drug release rate of Mel@Lipo was faster than that of ClockMPs, while ClockMPs released even faster in H2O2 solution. ClockMPs released the drug slowly and continuously during the first 12 hours of release, while the addition of H2O2 promoted the release of melatonin from ClockMPs, making its early release rate close to that of liposomes (Figure 10 )。 These results indicate that ClockMPs have effective controlled release of Mel's ros responsiveness.

[0102] In summary, we prepared a ROS-responsive drug delivery system that degrades by responding to the ROS in the local microenvironment, scavenges excess ROS in the microenvironment, and releases drugs under ROS stimulation. (III) Retention results of ClockMPs in vivo

[0103] The duration of action of a drug in the body is related to the residence time of the drug in the body. For some deep tissues, such as the intervertebral disc, drug administration can be achieved by injection. The intervertebral disc is a weight-bearing tissue in the human body. The pressure on the intervertebral disc during drug administration can cause leakage of the intervertebral disc and outflow through the needle track, resulting in a shorter residence time in the body and leading to drug administration failure.

[0104] To evaluate the in vivo retention of ClockMPs, we prepared DiR@Lipo and DiR@MPs using DiR and in vivo imaging fluorescence instead of Mel. DiR, DiR@Lipo, and DiR@MPs were injected into the intervertebral space of rats, and the injected intervertebral space was observed by in vivo imaging at 0, 3, 6, and 9 days after injection. The in vivo images showed that there was a significant loss of the DiR solution alone at 0 d, while the in vivo retention rate of DiR@Lipo or DiR@MPs was significantly increased ( Figure 2 in part K). The fluorescence intensity at each time point was analyzed. At 0 d, the fluorescence intensity of the DiR group was the lowest, while the fluorescence intensities of the DiR@Lipo and DiR@MPs groups were both higher than that of the DiR group ( Figure 11 in part A). From 3 to 9 d, the fluorescence intensity of the DiR@Lipo group gradually decreased and was significantly lower than that of the DiR@MPs group ( Figure 11 in parts B-D). The time-fluorescence intensity curve showed that the fluorescence intensity curve of the DiR@MPs group was higher than the other two groups ( Figure 2 in part L).

[0105] The above results indicate that MPs have a good anti-leakage effect during injection, effectively reduce the possible side effects of drugs, increase the residence of drugs in the body, and thus effectively improve the persistence of drug action. (IV) Regulation of the biological clock by ClockMPs

[0106] The intervertebral disc is composed of the nucleus pulposus, annulus fibrosus and cartilaginous endplate. Intervertebral disc degeneration often begins with nucleus pulposus degeneration. To study the in vitro effect of ClockMPs on the intervertebral disc, we extracted the main NPCs of rats according to previous studies. As the intervertebral disc degenerates, the intervertebral microenvironment deteriorates continuously. Therefore, we stimulated NPCs with H2O2 to simulate the microenvironmental threats that nucleus pulposus cells may face in IVDD and induced the cells to produce oxidative stress responses. Phosphate buffered saline (PBS), MPs, Mel, and ClockMPs were added to treat the oxidative stress NPCs, with normal NPCs as the control group. By detecting the growth, proliferation and expression of related genes and proteins of NPCs, the in vitro effect of ClockMPs on NPCs and its possible molecular mechanism were studied.

[0107] The growth and proliferation of NPCs were detected by live / dead staining and CCK-8 assay. NPCs treated with different treatments were detected by live / dead staining kit on days 1, 2, and 3 respectively. The live / dead staining images showed that the live cells of NPCs under oxidative stress (H2O2 group) grew slowly and the dead cells increased ( Figure 12 in part A). After treatment with MPs or Mel, the growth and proliferation of NPCs did not improve significantly. However, after treatment with ClockMPs, the dead cells were significantly reduced.

[0108] Cell viability was determined by calculating the ratio of live cells to total cells. It was found that the cell viability of the ClockMPs group was better than that of the H2O2 group ( Figure 12 in part B). There was no significant difference in cell viability between the ClockMPs group and the control group, indicating that ClockMPs had a protective effect on NPCs against H2O2-induced oxidative stress. In the time-cell viability spectrum, the ClockMPs group was closest to the Control group, and the others were all lower than the ClockMPs group ( Figure 12 in part D).

[0109] The above results showed that ClockMPs had good biocompatibility, could protect NPCs from oxidative stress, and ultimately promoted the growth and proliferation of NPCs.

[0110] To further study the effect of ClockMPs on the proliferation of NPCs, CCK-8 experiments were carried out on days 1, 2, and 3 respectively ( Figure 12 in part C). At different time points, the number of NPCs in the ClockMPs group was the highest among all groups except the control group. There was no significant difference in the number of NPCs between the ClockMPs group and the control group. The same result was also reflected in the proliferation of NPCs. Except for the control group, the proliferation spectrum of the ClockMPs group was significantly higher than that of other groups ( Figure 12In part E). The results of the CCK-8 experiment verified that ClockMPs could promote the proliferation of NPCs under the oxidative stress microenvironment. In summary, under the oxidative stress microenvironment, ClockMPs could protect NPCs from ROS damage, improve cell viability, and promote the proliferation of NPCs.

[0111] Through RNA-seq detection of cells in the H2O2 group and the ClockMPs group, clarify the possible mechanisms and molecular pathways of the protective effect of ClockMPs on NPCs under the oxidative stress microenvironment. By comparing the changes in the transcriptomes of the two groups, analyze the effects of ClockMPs on NPCs and their possible molecular pathways. Conduct gene ontology (GO) enrichment analysis on these differentially expressed genes. Biological process (BP) represents biological processes completed through multiple molecular activities, cellular component (CC) represents the location where gene products play roles in cell structures, and molecular function (MF) represents the activities of gene products at the molecular level. Cellular components can sometimes determine the functions of molecules, and molecules participate in biological processes according to their functions. The CC enrichment results showed that the differentially expressed genes were mainly localized in extracellular matrix components, including extracellular matrix, outer encapsulating structure, and collagen-containing extracellular matrix ( Figure 3 In part A). The BP enrichment results showed that the differentially expressed genes were involved in the biological processes of extracellular matrix synthesis and secretion, including extracellular matrix organization, extracellular structure organization, and outer encapsulating structure organization ( Figure 3 In part B). The MF enrichment results showed that the molecular functions of the differentially expressed genes were mainly related to extracellular matrix synthesis, including integrin binding, extracellular matrix structural constituent, cell adhesion molecule binding, and collagen binding ( Figure 3 In part C).

[0112] In summary, the GO analysis results showed that ClockMPs could affect the expression of extracellular matrix (ECM)-related genes in oxidative stress NPCs, thereby altering the physiological functions of NPCs.

[0113] Adopt Gene Set Enrichment Analysis (GSEA) to evaluate the distribution trend of genes through phenotypic correlation and evaluate the contribution of genes to phenotypes. GSEA showed that the circadian rhythm, ECM receptor interaction, and focal adhesion pathways were significantly upregulated ( Figure 3 In parts E and F). After treatment with ClockMPs, the circadian rhythm and ECM-related pathways in NPCs were upregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was also performed to study the specific molecular pathways. The KEGG results showed that the differentially expressed genes in the two groups were significantly related to the PI3K-AKT pathway ( Figure 3Part D). In summary, the above results indicate that ClockMPs have a positive effect on the biological clock and physiological functions of NPCs, which may be achieved through the PI3K-AKT pathway.

[0114] To further demonstrate the regulatory effect of ClockMPs on the biological clock of NPCs, real-time quantitative polymerase chain reaction (rt-qPCR) was used to detect the gene expression changes of CLOCK within 24 hours. In the control group, the CLOCK expression reached its peak at 12 h and decreased to the baseline at 24 h, forming a regular biological clock ( Figure 4 Part A). In the H2O2 and MPs groups, the increase in CLOCK expression was much less than that in the control group. In addition, the CLOCK expression in the H2O2 group and the MPs group could not be reduced to the baseline level, indicating a disordered biological clock in NPCs.

[0115] In the Mel and ClockMPs group, the expression of CLOCK was similar to that in the control group, showing a trend of first increasing and then decreasing, which indicates that Mel and ClockMPs have an effective regulatory effect on the circadian rhythm of NPCs. From the summary image of the CLOCK expression change spectrum, it can be seen that ClockMPs can protect NPCs from oxidative stress and regulate the disordered biological clock to a normal biological clock ( Figure 4 Part B). The long-term (48 h) expressions of the core biological clock genes CLOCK, BMAL1, and retinoic acid-related orphan receptor α (RORα) were detected by rt-qPCR and Western blot (WB). The WB images showed that the expression trends of CLOCK, BMAL1, and RORα were similar ( Figure 4 Part C).

[0116] The relative gray scale of the protein bands was analyzed to reflect the relative expression of the proteins ( Figure 4 Parts D-F). After H2O2 stimulation, the expressions of CLOCK, BMAL1, and RORα were significantly downregulated. MPs treatment could upregulate the expressions of BMAL1 and RORα, but had no obvious effect on the expression of CLOCK. In addition, Mel and ClockMPs treatment could significantly upregulate the expressions of CLOCK, BMAL1, and RORα. The WB results showed that ClockMPs could effectively upregulate the expressions of CLOCK, BMAL1, and RORα, thereby regulating the biological clock of NPCs.

[0117] The results of rt-qPCR also confirmed the regulation of ClockMPs on the biological clock of MPs ( Figure 13 ). The expressions of PI3K, p-PI3K, AKT, and p-AKT were detected by WB ( Figure 4In part G). The expressions of PI3K, p-PI3K, AKT, and p-AKT were downregulated in the H2O2 group. After treatment with ClockMPs, the expressions of PI3K, p-PI3K, AKT, and p-AKT were all significantly upregulated ( Figure 4 In parts H-K). Therefore, under the oxidative stress microenvironment, ClockMPs can activate the PI3K-AKT pathway in NPCs.

[0118] In summary, during the process of intervertebral disc degeneration, the microenvironment of the intervertebral disc deteriorates, and the increase in ROS disrupts the biological clock of NPCs. ClockMPs can upregulate the expression of biological clock-related genes through the PI3K-AKT pathway, thereby regulating the biological clock of NPCs and promoting intervertebral disc regeneration.

[0119] (V) Improvement of the physiological functions of NPCs by ClockMPs

[0120] Intervertebral disc regeneration depends on the normal physiological functions of NPCs, mainly the secretion of ECM, such as type II collagen and aggrecan. The expressions of type II collagen and aggrecan were detected by WB ( Figure 4 In part L). The expression of type II collagen in NPCs stimulated by H2O2 was lower than that in the control group. After treatment with ClockMPs, the expression of type II collagen was significantly upregulated and there was no significant difference from the control group ( Figure 4 In part M). The expression trend of aggrecan was the same as that of type II collagen ( Figure 4 In part N). The expressions of type II collagen and Aggrecan proteins were higher in the ClockMPs group, indicating that ClockMPs can effectively improve the physiological functions of NPCs.

[0121] The physiological functions of NPCs were detected by immunofluorescence. The sry-related high mobility group box 9 (Sox9) is involved in the physiological functions of NPCs such as differentiation, proliferation, and secretion, and is one of the key markers of NPCs degeneration. The low expression of Sox9 often indicates the degeneration of NPCs. The immunofluorescence images of Sox9 showed that H2O2 downregulated the expression of Sox9 ( Figure 5 In part A). After treatment with ClockMPs, the expression of Sox9 was upregulated. The number of Sox9-positive cells in the ClockMPs group was more than that in the H2O2 group ( Figure 5 In part C). The fluorescence intensity of Sox9 in the ClockMPs group was higher than that in the H2O2 group ( Figure 5 In part D). The immunofluorescence results of Sox9 indicated that ClockMPs can effectively protect NPCs from the stimulation of H2O2 and improve the physiological functions of NPCs. By detecting the immunofluorescence of type II collagen ( Figure 5Part B), to determine the ECM secretion function of NPCs. The fluorescence area of type II collagen in the ClockMPs group was the largest. The fluorescence intensity of type II collagen in ClockMPs was significantly higher than that in the H2O2 group ( Figure 5 Part E). The immunofluorescence results of type II collagen showed that ClockMPs could improve the ECM secretion of NPCs.

[0122] In summary, the physiological function of NPCs plays a key role in intervertebral disc regeneration, but the circadian clock disorder of NPCs caused by the deterioration of the microenvironment restricts the physiological function of NPCs. ClockMPs can improve physiological functions such as ECM secretion by regulating the circadian clock of NPCs, thus providing a potential strategy for intervertebral disc regeneration.

[0123] (VI) Rat ClockMPs intervertebral disc regeneration

[0124] An IVDD animal model was established by acupuncture of the rat caudal intervertebral disc to evaluate the in vivo effect of ClockMPs. This animal model has been verified and can simulate the physiological process of intervertebral disc degeneration. To reduce secondary injuries caused by drug administration, drugs were injected through the same needle tract in the intervertebral space. Different intervertebral discs of the same rat were selected for surgery and treatment to reduce allogeneic variation. One week before surgery (-1w), 1, 4, and 8 weeks after surgery, the caudal intervertebral disc X-ray and magnetic resonance imaging (MRI) were respectively detected to monitor the degeneration or regeneration of the intervertebral disc ( Figure 6 Part A). The intervertebral disc height (IDH) was determined by measuring the distance between two adjacent vertebral bodies in the X-ray image. The change of IDH can reflect the change of intervertebral disc degeneration degree over time. The X-ray image showed that the acupuncture intervertebral disc was significantly degenerated after surgery, indicating that the IVDD rat model was successfully constructed ( Figure 6 Part B). The IDH change curves of the MPs group and the Mel group were higher than those of the IVDD group, but were still significantly lower than those of the healthy group ( Figure 6 Part C). The profile of IDH change in the ClockMPs group was higher than those of the IVDD group, the MPs group, and the Mel group, and there was an obvious recovery trend of IDH after 1w.

[0125] By analyzing the IDH changes at five different time points of the five groups, it was found that the IDH of each group decreased significantly in the first week after acupuncture ( Figure 6 Part D). At the 4th and 8th weeks, the IDH of the ClockMPs group was significantly restored and gradually approached the IDH of the healthy group ( Figure 6 Parts E and F). The X-ray examination results of the intervertebral disc showed that ClockMPs had the effect of restoring IDH and had a good promoting effect on the repair process of intervertebral disc degeneration.

[0126] The restoration of IDH is usually accompanied by an increase in the water content of the intervertebral disc. To evaluate the changes in the water content of the intervertebral disc, we collected MRI images and analyzed the changes in the water content of the intervertebral disc through the gray value at the center of the intervertebral disc. The gray value change curve was determined. The MRI images showed that the intervertebral disc structure in the healthy group was clear and the water content was abundant. In the IVDD group, the boundary of the intervertebral disc was blurred and the water content was low. The degree of intervertebral disc degeneration in other groups was different ( Figure 6 part G). The gray value change curve showed that the healthy group had the highest water content among all groups ( Figure 14 part A). The water content in the ClockMPs group decreased the least and was relatively stable. The water content of the intervertebral disc was reflected by the gray value of the intervertebral disc at different time points. The results showed that the water content in the ClockMPs group was significantly higher than that in the IVDD group ( Figure 6 part H). The above MRI results indicated that ClockMPs had a positive effect on maintaining the water content of degenerated intervertebral discs, thereby improving the local microenvironment and promoting the repair of degenerated intervertebral discs. In summary, the radiological results indicated that ClockMPs could promote the regeneration of rat intervertebral discs.

[0127] Histological sections of rat intervertebral discs were taken at 1, 4, and 8 weeks after surgery. Hematoxylin-eosin staining (H&E) was used to detect the changes in the intervertebral disc structure, and Masson staining was used to detect collagen remodeling and ratio ( Figure 7 parts A and B). The microscopic structure of the intervertebral disc in the healthy group was normal, the nucleus pulposus was intact, and the boundary was clear. In the IVDD group, the nucleus pulposus was absent and the intervertebral disc structure was disordered. In the ClockMPs group, the nucleus pulposus was more intact, and the local microstructure of the intervertebral disc was close to that of the control group. The histological grading of all intervertebral discs was determined according to previous studies. The results showed that at different time points, the histological grading of the ClockMPs group was closest to that of the control group ( Figure 14 part B). There was no obvious improvement in the histological grading of the intervertebral discs in the IVDD group, MPs group, and Mel group during the time progression. Over time, the histological grading of the ClockMPs group gradually improved ( Figure 7 parts C-E). The histological results were consistent with the imaging results, further confirming the positive role of ClockMPs in repairing degenerated intervertebral discs.

[0128] To further investigate the mechanism of intervertebral disc regeneration by ClockMPs in vivo, we performed immunohistochemical analysis on histological sections of intervertebral discs. The biological clock of the intervertebral disc was determined by detecting the expression of CLOCK and BMAL1 ( Figure 8 part A, Figure 15 part A), and the ECM content of the intervertebral disc was determined by detecting the expression of type II collagen ( Figure 8Part E). The average optical density (AOD) of immunohistochemistry was calculated to reflect the relative expression of the protein. The expression of CLOCK in the ClockMPs group at different time points was significantly higher than that in the IVDD group, MPs group, and Mel group ( Figure 8 Parts B-D, Figure 16 Part A). The expression trend of BMAL1 was the same as that of CLOCK ( Figure 15 Parts B-D, Figure 16 Part B). The results were consistent with the in vitro cell experiments, indicating that ClockMPs could repair the degenerated intervertebral disc in vivo by activating the biological clock of the intervertebral disc. In addition, at different time points, the expression level of type II collagen in the intervertebral disc of the ClockMPs group was higher than that in the IVDD group, MPs group, and Mel group ( Figure 8 Parts F-H, Figure 16 Part C). In summary, ClockMPs can regulate the intervertebral biological clock, increase the ECM synthesis of NPCs, and thus promote intervertebral disc regeneration.

Claims

1. A preparation method of a circadian rhythm regulating hydrogel microsphere, characterized in that, It includes the following steps: (1) Dissolve lecithin, cholesterol and DSPE-PEG-PBA in an organic solvent and form a lipid film by rotary evaporation; the weight ratio of lecithin, cholesterol and DSPE-PEG-PBA is 6.9:2.9:2; (2) Mix the melatonin aqueous solution with the product obtained in step (1) and perform ultrasonic treatment to obtain melatonin-loaded liposomes; (3) Prepare polyvinyl alcohol microspheres by air microfluidics technology, and then react with the crosslinking agent TSPBA to prepare polyvinyl alcohol hydrogel microspheres; (4) Mix the product obtained in step (2) with the product obtained in step (3) in a TSPBA solution to prepare circadian rhythm-regulating hydrogel microspheres.

2. The preparation method according to claim 1, wherein The organic solvent described in step (1) includes at least one of chloroform, methanol, ethanol, and dichloromethane.

3. The preparation method according to claim 1, characterized in that, The concentration of the melatonin aqueous solution in step (2) is 0.233 g / mL, and the volume ratio of the melatonin aqueous solution to the organic solution is 1:1.

5.

4. The preparation method according to claim 1, characterized in that, The conditions of the ultrasonic treatment in step (2) are: 40% power, ultrasonic working for 2 s, stopping for 1 s, and the total ultrasonic working time is 7 min.

5. The preparation method according to claim 1, wherein The concentrations of TSPBA and polyvinyl alcohol in step (3) are 5-10 wt% TSPBA and 5-10 wt% polyvinyl alcohol, and the volume ratio of the two is 1:0.25-2.

6. The preparation method according to claim 1, wherein The operation of preparing polyvinyl alcohol microspheres by air microfluidics technology in step (3) is: inject a 7.5% (w / v) polyvinyl alcohol aqueous solution into the inner layer of the device, connect a nitrogen gas flow to the outer layer, the nitrogen gas flow rate is 1 L / min, cut the polyvinyl alcohol solution into uniform droplets by the principle of gas cutting, collect the polyvinyl alcohol hydrogel microspheres with absolute ethanol, obtain precursor microspheres through the principle of solvent exchange, use a 70-mesh tissue filter to fish up the precursor microspheres from absolute alcohol for standby, pour the precursor microspheres into a TSPBA solution, and crosslink them to form ROS-responsive polyvinyl alcohol hydrogel microspheres.

7. The circadian rhythm-regulating hydrogel microspheres prepared by the method according to any one of claims 1-6.

8. Use of the hydrogel microspheres for regulating biological clock according to claim 7, characterized in that, It is to prepare the hydrogel into a drug for treating intervertebral disc degeneration or to prepare the hydrogel into a drug for promoting intervertebral disc regeneration.

9. Use of the hydrogel microspheres for regulating biological clock according to claim 7, characterized in that, It is characterized in that it is to prepare the hydrogel microspheres into a drug for regulating the circadian rhythm.

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