| Background:Low back pain has a severe impact on the quality of life and workability of patients,eventually bringing a heavy economic burden to society.Studies have shown that intervertebral disc degeneration is the most common cause of low back pain.The current treatment options for degenerative disc disease include rest,physical therapy,drug therapy,interventional treatments such as radiofrequency ablation of the disc,and various surgical treatments.Although these treatments are effective in a short period,patients still suffer from a high possibility of long-term recurrent pain.In recent years,biotherapeutics based on biomolecular,cell,and tissue engineering have become the focus of research due to their great potential in restoring the structure of the intervertebral disc.However,despite numerous new biotherapeutics developed by researchers,the influence of insufficient nutrient supply to the intervertebral discs has generally been ignored.Furthermore,the widely recognized disc repair strategies that promote cell proliferation and extracellular matrix anabolic activity further exacerbate the nutritional demands of degenerated discs,thereby reducing the viability of resident disc cells.Therefore,there is need to develop a repair strategy focused on the nutrient restriction of the intervertebral disc,thus providing new insights for the treatment of disc degeneration.Compared with traditional molecular therapies such as proteins,peptides,and growth factors,gene therapy has the potential to cure the root of intervertebral disc degeneration in one injection.Circular RNA（circ RNA）is an emerging class of RNA family members that plays a key role in regulating cellular functions such as proliferation,differentiation,invasion,migration,and metabolism by affecting gene expression at the transcriptional or post-transcriptional level.circ RNAs have the characteristics of abundant species,stable structure,sequence conservation,and cell or tissue specificity,which provide them with extra advantages compared with other RNAs during the regeneration process of the intervertebral disc.However,simple genetic drugs are easily degraded by endogenous enzymes and have low transfection efficiency,significantly lowering their intracellular efficacy,thus requiring aid from artificial gene carriers.Cationic liposomes are widely used as gene carriers due to their ability to package DNA or RNA and adsorb to cell membranes.However,direct injection into the target organ may results in the rapid dispersion and loss of gene vector,making it difficult to target local lesions.Moreover,vector-based expression of nucleic acid biology is transient,thereby not always suitable for studies that require long-term sustained regeneration and repair.In addition,uncontrollable expression of gene drugs may produce excess extracellular matrix leading to pathological fibrosis.Therefore,there is an urgent need to develop an efficient,sustained,and controllable gene delivery system for locally targeted regeneration of the intervertebral disc.Advanced biomaterial-assisted gene carrier delivery allows the control and delivery of gene drugs in a spatiotemporally precise manner and has become an emerging and attractive therapeutic option for targeted regeneration.Monodisperse,uniform,and bioactive hydrogel microspheres prepared by microfluidic synchronous cross-linking technology can reduce tissue damage and achieve precise treatment by minimally invasive injection.In addition,hydrogel microspheres can protect and release the bioactive component encapsulated inside them,and the low local drug concentration and administration frequency can provide safe and effective treatment.Therefore,this study intends to start from the pathological mechanism of nutritional restriction-induced intervertebral disc degeneration,based on the reliable circ RNA targets screened by microarray chip,combined with advanced injectable hydrogel microspheres to construct an ideal targeted delivery system by chemical grafting for the long-term regeneration of degenerative discs.Objectives:1.To screen and identify key circ RNAs in the process of nutritional restriction-induced intervertebral disc degeneration;2.To evaluate the relevant properties of circ RNA silencing gene-engineered injectable hydrogel microspheres;3.To evaluate the in vitro and in vivo repair ability of circ RNA silencing gene-engineered injectable hydrogel microspheres in intervertebral disc degeneration.Methods:Part I:Glucose-deprived culture of nucleus pulposus cells（NPCs）to simulate the nutritionally restricted microenvironment of the intervertebral disc.After culturing the NPCs for respective 0 h,6 h,12 h,and 24 h under the glucose deprivation condition,samples were collected,and the expression of circular RNAs was detected via the circ RNA chips at each time point.The cluster analysis on the time series was performed using the STEM software to screen the differentially expressed circ RNAs that changed continuously with the culture time.Functional enrichment analysis was used to predict the potential functions of consecutive up-and down-regulated circ RNAs.The 5 circ RNAs with the most significant up-regulation and the 5 circ RNAs with the most obvious down-regulation were selected to construct a circ RNA-mi RNA regulatory network and screen the key circ RNAs.q RT-PCR analysis was carried out to detect the expression of key circ RNAs in degenerative nucleus pulposus tissue and NPCs cultured under glucose deprivation.Sanger sequencing and RNase R tolerance experiments were used to verify the structure of key circ RNAs.Western Blot detection was applied to investigate the expression of extracellular matrix components in the glucose-deprived culture of NPCs after silencing the key circ RNAs.Part II:Preparation of 1,2-dioleoyl-3-trimethylaminopropane/cholesterol/1,2-dioleo yl-sn-glycero-3-phosphoethanolamine（DOTAP/Chol/DOPE）cationic liposomes.Gene-liposome complexes（lipoplexes）with different N/P ratios were prepared according to the ratio of nitrogen atoms of DOTAP to the phosphate of p DNA.Dynamic light scattering（DLS）and transmission electron microscopy（TEM）analysis were used to observe the size,zeta potential,and morphology of cationic liposomes and lipoplexes with different N/P ratios.Gel electrophoresis retardation assay was performed to evaluate the ability of cationic liposomes to polymerize p DNA.Flow cytometry detect the percentage of GFP-positive cells,CCK-8 assay and q RT-PCR were conducted to investigate the transfection efficiency,cytotoxicity,and circ RNA silencing efficiency of lipoplexes with different N/P ratios,respectively,thus determining the optimal transfection ratio.Furthermore,the aqueous methacrylated hyaluronic acid（HAMA）solution can be formed into HAMA droplets by the water-in-oil method via the microfluidic device,which was further condensed,cross-linked,and freeze-dried to obtain HAMA microspheres.Then,the DOTAP/Chol/DOPE cationic liposomes encapsulating circ RNA-silencing genes were grafted into HAMA microspheres through amide bonds to construct a locally injectable circ RNA silencing-hydrogel microsphere.The morphology and internal structure of HAMA microspheres were observed by the inverted phase-contrast microscope and scanning electron microscope（SEM）.The biodegradability of HAMA microspheres was evaluated by the hyaluronidase degradation assay.The successful graft of gene complexes into the microsphere was assessed after the Dil staining with fluorescence microscopy,laser scanning confocal microscopy（LSCM）,environmental scanning electron microscopy（ESEM）,Fourier transform infrared spectroscopy（FTIR）,and X-ray photoelectron spectroscopy（XPS）assays.The amount of p DNA in the supernatant was detected by q RT-PCR to determine the in vitro release kinetics of the gene complexes.Live/dead staining and CCK-8experiments were used to detect the biocompatibility of circ RNA silencing-hydrogel microspheres.The percentage of GFP-positive cells was measured by the flow cytometry method,and q RT-PCR was used to compare the transfection efficacy and circ RNA silencing efficiency of circ RNA silencing-hydrogel microspheres at different time points,respectively.Part III:In vitro experiments were divided into control group,nutrient restricted group（GD）,HAMA microsphere group（MS）,grafted blank gene complex microsphere group（Blank p DNA-lipo@MS）and grafted circ STC2 silencing gene complex microsphere group（psh-circ STC2-lipo@MS）.q RT-PCR and immunofluorescence staining were performed to detect the expression of extracellular matrix-related components at gene and protein levels in each group.50μl of the glucose-free medium was injected via a self-made 1ml syringe needle with limited 5mm depth to establish a rat model of nutritionally limited degeneration of the intervertebral disc.MS,Blank p DNA-lipo@MS and psh-circ STC2-lipo@MS were then injected into the nutrient-restricted intervertebral discs,respectively.only punctured skin was used as the sham-operated group（Sham）.At 4-and 8-weeks post-operation,X-ray and MRI experiments were performed respectively to observe the changes of intervertebral disc height and nucleus pulposus signal intensity in each group.Hematoxylin-eosin and safranin/fast green staining were used to evaluate each group’s disc integrity and collagen content.Immunohistochemical staining was applied to detect the expression of Aggrecan and Collagen II in the nucleus pulposus of rats in each group.Results:Part I:1135（12 h＿vs＿6 h）,7438（24 h＿vs＿12 h）,12521（24 h＿vs＿6 h）,2953（6 h＿vs＿0 h）,3246（12 h＿vs＿0 h）and 13578（24 h＿vs＿0 h）differentially expressed circ RNA were screened out by chip detection and comparison between different time points.STEM software performed time-series clustering analysis on the screened differential circ RNAs and obtained differential circ RNAs that were continuously upregulated and down-regulated with culture time.The functional enrichment analysis results showed that the functions of upregulated circ RNAs were mainly enriched in biological processes such as ion transmembrane transport and regulation of ion transport,as well as pathways such as focal adhesion ECM-receptor interaction and calcium signaling pathway.The functions of down-regulated circ RNAs are mainly enriched in biological processes such as cytoskeleton organization and cell migration,as well as pathways such as cytokine-cytokine receptor interaction and metabolic pathway.Circ RNA-mi RNA regulatory network results showed that circ＿0075062（degree,12）and circ＿0075063（degree,12）are the most connected circ RNAs.In vitro validation results showed that circ＿0075062 was upregulated in degenerated nucleus pulposus tissue and NPCs cultured under glucose deprivation conditions.Sanger sequencing and RNase R tolerance experiments proved that circ＿0075062 is a circular transcript.Western Blot results indicated that silencing the expression of circ＿0075062 could alleviate the glucose deprivation-induced imbalance of extracellular matrix synthesis and degradation in the NPCs.Part II:DLS measurement results showed that DOTAP/Chol/DOPE liposomes were uniform in size and had good dispersibility.After incubation of DOTAP/Chol/DOPE liposomes with p DNA,the particle size of the lipoplexes formed increased significantly.With the gradual increase of N/P ratio,the p DNA was completely encapsulated,and the particle size of lipoplexes gradually decreased until no more changes were observed.The negative charge of p DNA was completely shielded,and the zeta potential also changed from negative to positive.TEM results revealed that DOTAP/Chol/DOPE liposomes had good dispersibility,and most of the particles were regular spherical.After encapsulating p DNA,the particle size increased.The results of gel electrophoresis retardation assay showed that the encapsulation rate and compression effect of DOTAP/CHOL/DOPE liposomes on p DNA increased with the N/P ratio increased.The flow cytometry and CCK-8 experiments demonstrated that with the increment of N/P ratio,the transfection efficiency of DOTAP/Chol/DOPE liposome gradually enhanced,and the lipoplexes showed no obvious toxicity the growth of NPCs.Circ＿0075062 was formed by the back splicing of exon 4 of the STC2 gene,so it was named circ STC2.The results of q RT-PCR showed that compared with the transfection of blank plasmid（Blank p DNA-lipo）,DOTAP/Chol/DOPE liposome transfection of circ STC2 silencing plasmid（psh-circ STC2-lipo）at N/P=3:1 could significantly inhibit the expression of circ STC2.Therefore,N/P=3:1 was chosen as the optimal transfection ratio.Inverted phase contrast microscope observation displayed that HAMA microspheres had uniform size and excellent stability.HAMA microspheres had good dispersibility in an aqueous solution and could smoothly pass through a 1 ml syringe needle.SEM analysis showed that HAMA microspheres were spherical and porous inside.In addition,HAMA microspheres could last for 70 d in a hyaluronidase-simulated in vitro degradation solution until complete degradation.After Dil staining,it was observed via fluorescence microscopy and LSCM that the ungrafted microspheres only had a faint red layer on the surface,while the microspheres of the grafted lipoplexes showed bright red in the interior and on the surface of the pores.At the same time,ESEM revealed that lipoplexes attached to the surface of the microspheres.After processing by the second derivative method,the characteristic peaks of amide bonds were observed at 1659 cm-1（amide I band）and 1558 cm-1（amide II bands）in the FTIR data of the grafted gene complex microspheres.XPS was used to analyze the changes of surface elements before and after grafting with lipoplexes.The presence of phosphorus elements further confirmed the successful grafting of the lipoplexes to the microspheres.The results of sustained-release experiments showed that the circ RNA silencing-hydrogel microsphere system could continuously and stably release p DNA for up to 27 days,and the total release rate exceeded 80%.The NPCs were incubated with MS,Blank p DNA-lipo@MS,and psh-circ STC2-lipo@MS for a predetermined time.Live/dead cell staining,and CCK-8 assay results showed that all MS,Blank p DNA-lipo@MS,and psh-circ STC2-lipo@MS exhibited superior biocompatibility without obvious cell death.Flow cytometry results showed that the circ RNA silencing-hydrogel microsphere system had a high transfection efficiency after co-incubating with NPCs for 72 hours.q RT-PCR results showed that,compared with the Blank p DNA-lipo@MS group,psh-circ STC2-lipo@MS group could significantly inhibit the expression of circ STC2 after co-incubating with NPCs for 72hours.Part III:In vitro results showed that compared with the GD,MS and Blank p DNA-lipo@MS groups,the psh-circ STC2-lipo@MS group promoted the synthesis of Aggrecan and Collagen II and inhibited the secretion of extracellular matrix degradation enzymes ADAMST-4 and MMP-13.The results of X-ray and MRI showed that compared with the GD,MS,and Blank p DNA-lipo@MS groups,the psh-circ STC2-lipo@MS group significantly recovered the intervertebral disc height at 8w,and the signal intensity of the nucleus pulposus was also significantly enhanced,which was close to the sham group.Hematoxylin-eosin staining revealed that the GD,MS,and Blank p DNA-lipo@MS groups showed atrophy of the nucleus pulposus tissue and deformation of the annulus fibrosus at 4 w and 8 w,while the psh-circ STC2-lipo@MS group demonstrated good disc structure and morphology at 8 w.Safranin fast green staining indicated that the collagen content of psh-circ STC2-lipo@MS group was significantly enhanced at 8 w.Immunohistochemical analysis showed that except for the Sham group,the psh-circ STC2-lipo@MS group displayed strong aggrecan and collagen II expressions in the nucleus pulposus at 8w,while the other groups showed weak or almost none no expression.Conclusions:1.Through microarray chip detection,bioinformatics analysis,and in vitro validation,circ＿0075062 was identified as a key circ RNA in the process of nutritional restriction-induced intervertebral disc degeneration.2.HAMA microspheres held good dispersibility,degradability,and injectability and realized the efficient drug loading sustained and stable release of gene complexes through the method of amide bond grafting,which could serve as an ideal gene delivery platform.3.In a nutrient-limited microenvironment,circ RNA silencing-hydrogel microspheres can silence pathological genes in NPCs and regulate the metabolic balance of the extracellular matrix,thereby inhibiting intervertebral disc degeneration.In conclusion,circ RNA silencing-hydrogel microspheres,as a safe,controllable,and effective targeted gene delivery system,provide a new strategy for the prevention and treatment of intervertebral disc degenerative diseases. |
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