Application Of N-acetylcysteine-doped Carbon Dots Nanozyme In Intervertebral Disc Degeneration

Background:Low back pain(LBP)is one of the most common clinical symptoms in spinal surgery,and its etiology is relatively complex.One of the most common causes of low back pain is intervertebral disc degenerative disease.At present,the pathogenesis of intervertebral disc degenerative disease is not clear,and the treatment is relatively limited.It is known that intervertebral disc degeneration(IVDD)is related to the disturbance of dynamic balance of reactive oxygen species(ROS)caused by oxidative stress.Antioxidant stress may be one of the effective treatments to delay the progression of intervertebral disc degeneration.As a kind of nanomaterial with similar activity of natural enzyme,nanozyme can effectively regulate the level of ROS in cells.In this study,focusing on the oxidative stress mechanism related to intervertebral disc degeneration,combined with basic research methods,a new carbon dots nanozyme,N-acetylcysteine carbon dots nanozyme(NAC-CDs),was developed by hydrothermal synthesis,and had been proved to be an ideal antioxidant and anti-aging drug.Our results confirmed for the first time that compared with N-acetylcysteine(NAC),NAC-CDs has better biocompatibility,stronger superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GPx)activity and higher total antioxidant capacity.By observing the decrease of ROS in aging nucleus pulposus cells induced by hydrogen peroxide,it was proved that NAC-CDs had strong ability of scavenging free radicals and antioxidation in vitro,and the scavenging rate of toxic ROS was more than 90%.NAC-CDs could also maintain the dynamic balance of mitochondria,inhibit cell senescence,and then inhibit the expression of inflammatory factors,thus rebalancing anabolism and catabolism.In vivo imaging and histological evaluation showed that the intervertebral disc height index(DHI),magnetic resonance imaging(MRI)grade and histological score of rats treated with NAC-CDs were significantly better than those of the degenerative model,which confirmed the therapeutic effect of NAC-CDs on intervertebral disc degeneration.To sum up,our study began with the observed clinical phenomena and histological features of human intervertebral discs,and around the mechanism of oxidative stress of intervertebral disc,a new type of carbon dots nanozyme was prepared.By using the methods of characterization and detection of materials,determination of enzyme-like activity and related experiments in vivo and in vitro,it was proved that NAC-CDs could effectively fight against nucleus pulposus cell senescence by scavenging ROS and eventually delay intervertebral disc degeneration.This new nanomaterial provided a potential strategy for clinical treatment of IVDD.Part 1:Correlation between degeneration degree of lumbar intervertebral disc and ROS level in tissueObjective:Many studies have found that intervertebral disc degeneration is closely related to oxidative stress.We speculate that with the aggravation of degeneration,the level of oxidative stress in the nucleus pulposus of human intervertebral disc also changes.In order to verify this hypothesis,this study analyzed the MRI imaging characteristics of lumbar intervertebral discs in different populations,and detected the level of reactive oxygen species(ROS)in the tissue samples of nucleus pulposus of intervertebral discs in the surgical population,and explored the changes of imaging characteristics of human intervertebral discs with age,as well as the correlation between the degree of degeneration and the level of ROS in tissues,thus verifying the feasibility of targeting anti-oxidative stress to delay degenerative diseases of intervertebral discs.Methods:①Clinical retrospective analysis of MRI image data of lumbar vertebrae in different groups was divided into young,middle-aged and elderly groups,and each age group was divided into normal people and people with lumbar disc herniation.Image-j software was used to analyze the magnetic resonance T2WI signal intensity of the intervertebral disc nucleus pulposus of these people.②For the patients who need surgery,the Pfirrmann grading method of MRI was used to classify the degree of intervertebral disc degeneration according to the grade,and the frozen sections were made by collecting the samples of nucleus pulposus which were classified into grade Ⅱ and grade Ⅳ after operation.Finally,the dihydroethidium(DHE)was used to stain the frozen sections.Results:①MRI analysis results:with the increase of age,the magnetic resonance T2WI signal intensity of intervertebral disc(L4-5)in young,middle-aged and old age group without lumbar disc herniation showed a tendency to decrease.Compared with the shape of intervertebral disc in the same age group,the signal intensity of nucleus pulposus corresponding to the responsible segment of lumbar disc herniation decreased more significantly.②DHE staining was positive in frozen sections of diseased intervertebral disc tissue(Pfirrmann grade Ⅱ and Ⅳ),and the positive rate of DHE staining in degenerative nucleus pulposus of Pfirrman grade Ⅳ was higher than that of Pfirrmann grade Ⅱ 78.7± 10%vs 40.7 ±8.1%,P<0.01).Conclusion:This part of the study shows the imaging differences of intervertebral discs between normal people and people with lumbar disc herniation in different age groups.Magnetic resonance T2WI signal intensity analysis data showed that the water content of nucleus pulposus of intervertebral disc decreases gradually with age,especially in patients with lumbar disc herniation.The results of DHE staining showed that there were different levels of oxidative stress in human intervertebral disc degeneration.Through further analysis of clinical data,it was suggested that the method of anti-oxidative stress to delay intervertebral disc degeneration should be feasible.Part 2:Preparation,characterization and biocompatibility detection of N-acetylcysteine-doped Carbon Dots NanozymeObjective:In terms of clinical treatment,natural or acquired antioxidants are inefficient in vivo,and the lack of targeting specificity limits their effect.Therefore,there is an urgent need to explore new drugs to effectively inhibit oxidative stress and to find new targets for the treatment of intervertebral disc degeneration(IVDD).In the process of the development of nano-medical science and technology,artificial nanozymes with activities similar to natural enzymes have become one of the current research focuses because of their efficient catalytic effect.Compared with natural enzymes,nanozymes have attracted much attention because of their relatively higher physical and chemical stability,stronger durability and lower cost in harsh environments.Among all kinds of artificial nanozymes,carbon dots(CDs)are widely used in nanomedicine because of their ultra-small size,excellent adjustable optical properties,suitable water solubility and high biocompatibility.Many literatures have reported the activity of natural enzymes and the ability of scavenging free radicals,which make carbon spots play a good role in anti-oxidative stress.These superior properties make them the preferred nanomaterials for enzyme applications.Therefore,we synthesized a new type of carbon dots nanozyme,N-acetylcysteine carbon dots nanozyme(NAC-CDs).In this chapter,we will study the synthesis,physicochemical properties and biocompatibility of NAC-CDs.Methods:①NAC-CDs were prepared and characterized by TEM transmission electron microscope,Malvern particle analyzer,micro infrared spectrometer and photoelectron spectrometer,respectively.②Detection of enzyme-like activity:the activities of superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GPx)of NAC-CDs were detected.③Determination of total antioxidant capacity of NAC and NAC-CDs.④ Using MTT cell proliferation and cytotoxicity test kit to detect the biocompatibility of NAC-CDs.Results:①Under transmission electron microscope,the particle size of NAC-CDs is about 6-10 nm and the zeta potential is about-3.59 mV.NAC-CDs has low crystallinity and belongs to amorphous structure.NAC-CDs partially retains the structural characteristics of NAC,which contains a variety of hydrophilic functional groups(such as hydroxyl,amino and sulfhydryl),which makes them have good biocompatibility.②Compared with NAC,NAC-CDs has higher activity of SOD,CAT and GPx-like enzymes.③Compared with NAC,NAC-CDs have higher total antioxidant capacity.④There was no significant difference in cytotoxicity between the control group and the NAC-CDs group when the concentration was less than 50 μg/mL.When the concentration was increased to 200 μg/mL,the survival rate of NPCs remained above 85%.Conclusion:NAC-CDs were prepared by hydrothermal synthesis.The successful synthesis of this carbon dots nanozyme was verified by testing its particle size,surface charge,element composition,distribution of functional groups and other factors.Through the determination of all kinds of enzyme activity and the detection of total antioxidant capacity kit,it was confirmed that the nano-material had more efficient natural enzyme-like activity and stronger total antioxidant capacity.In addition,the cell survival rate detected by MTT showed that NAC-CDs had no obvious cytotoxicity and had excellent biocompatibility.Part 3:Antioxidative stress of N-acetylcysteine-doped Carbon Dots Nanozyme delays intervertebral disc degeneration(in vitro and in vivo experiments)Objective:In this part of the study,we plan to carry out in vivo and in vitro experiments to prove that the prepared carbon dots nanozymes inhibit oxidative stress by clearing ROS in the model of intervertebral disc degeneration,so as to achieve the purpose of delaying intervertebral disc degeneration.Methods:①Cell experiment in vitro:the primary nucleus pulposus cells of rats were extracted and cultured,and the cells were identified by immunofluorescence staining and toluidine blue staining.Secondly,NPCs was treated with H2O2 to induce cell aging under oxidative stress.Dihydroethidium(DHE)probe,mitochondrial red fluorescent probe and immunofluorescence staining were used to detect the antioxidant activity of NAC-CDs in vitro and its effects on synthesis and catabolism(Aggrecan,Col-2,MMP9/13),inflammation related(TNF-α,IL-1β)and aging related(p21).Senescence-related β-galactosidase(SAβ-Gal)staining was used to detect the aging status of nucleus pulposus cells.② Animal experiment in vivo:Rat caudal vertebra acupuncture was used to establish the model of intervertebral disc degeneration.The experimental animals were divided into three groups:blank control group(NC group),degeneration group(DC group)and two groups of NAC-CDs treatment group(low concentration and high concentration).X-ray and MRI imaging examination were performed 4 weeks after operation,and then histological staining was performed to verify the effect of NAC-CDs on delaying intervertebral disc degeneration.Results:①Compared with the control group,after H2O2 treatment,the mean fluorescence intensity(MFI)of DHE staining of nucleus pulposus cells increased by 4 times,while NAC-CDs could significantly reduce the level of intracellular ROS,especially in the 3μg/mL NAC-CDs pretreatment group,the MFI decreased to 1.9 times compared with the control group.The MFI degree of mitochondrial staining of NPCs treated with H2O2 decreased by about 64%compared with the blank control group(3 μg/mL).Similarly,after H2O2 treatment,the positive rate of aging staining and the fluorescence intensity of p21 staining of nucleus pulposus cells increased,while the positive rate of aging staining and the fluorescence intensity of p21 staining decreased in varying degrees in the NAC-CDs treatment group.In addition,after H2O2 treatment,the fluorescence intensity of Aggrecan and Col2 staining decreased significantly,while the positive immunostaining intensity of MMP9,MMP13,TNF-α and IL-1β increased.The NAC-CDs treatment group was on the contrary.②In vivo experiments:X-ray and MRI images and histological staining results showed that the intervertebral disc height index,MRI grade and histological score in the NAC-CDs group were significantly improved compared with the degenerative model group.Conclusion:We successfully designed and prepared this novel carbon dots nanozyme(NAC-CDs)for targeted oxidative stress therapy,and confirmed that NAC-CDs had higher enzyme-like activity and good biocompatibility by in vivo and in vitro experiments.It can reduce intracellular excess ROS level,maintain mitochondrial homeostasis and balance anabolic water balance,inhibit the expression of inflammatory factors and exert anti-inflammatory effects.NAC-CDs has the effect of anti-oxidative stress and anti-aging,thus effectively maintaining the intervertebral disc height(DHI),improving the MRI grade and histological morphology of the intervertebral disc,and finally delaying the progress of intervertebral disc degeneration.