Application Of Carbon Dot-Prussian Blue Composite Nanomaterials In The Treatment Of Intervertebral Disc Degeneration

Background:Intervertebral disc degeneration（IDD）is one of the main causes of low back pain and seriously affects the quality of life of patients.Among the existing clinical treatment methods,the effect of conservative treatment is often not ideal.Surgical treatment carries high risks,and in severe cases,it can cause nerve damage,resulting in dysfunction or even paralysis.Currently,the mechanism of intervertebral disc degeneration is unclear,and therapeutic targets are limited.Therefore,it is extremely urgent to explore new mechanisms for the treatment of intervertebral disc degeneration and find new therapeutic targets.Studies have shown that oxidative stress caused by excessive production of Reactive oxygen species（ROS）can accelerate intervertebral disc degeneration by affecting intervertebral disc cell senescence,inflammation,autophagy and DNA methylation.Antioxidant stress may be an effective therapeutic target for delaying intervertebral disc degeneration.Therefore,this study focused on the mechanism of oxidative stress of intervertebral disc,based on clinical observation and histological characteristics,combined with basic research methods,designed and prepared a new composite nanomaterial.And through testing material characterization and in vitro and in vivo experiments,to verify the effect of the nanocomposite in delaying intervertebral disc degeneration by scavenging ROS and resisting oxidative stress.Part 1:Imaging features of patients with lumbar disc degeneration and analysis of oxidative stress-related indicatorsObjective:A large number of studies have shown that oxidative stress is closely related to intervertebral disc degeneration,and we speculate that the level of oxidative stress in human intervertebral disc nucleus pulposus may also change with the degeneration process.To verify this hypothesis,this study analyzed the MRI imaging characteristics of intervertebral discs in different populations,and detected the ROS level in the surgical specimens of corresponding intervertebral disc nucleus pulposus tissues.To investigate the changes of imaging characteristics of human intervertebral disc with age and the correlation between the level of ROS in tissues and degeneration grade.Thus,the feasibility of targeting oxidative stress to delay intervertebral disc degeneration was verified.Methods:①Retrospective analysis of lumbar spine MRI images of different populations,and the included populations were divided into young,middle-aged,and elderly age groups.Each age group was divided into people with lumbar disc herniation and normal people.Using image-j software to analyze and compare the signal intensity on MRI T2WI of the intervertebral disc nucleus pulposus in different groups.②Preoperative MRI Pfirrmann gradeⅢ and V specimens were collected.After frozen sections were made,superoxide anion fluorescence probe（DHE）was used to stain the sections.Results:①MRI analysis results:The signal intensity of L4-5（lumbar 4-5）intervertebral disc nucleus pulposus MRI T2WI in young,middle-aged,and elderly people without lumbar disc herniation showed a decreasing trend with age.Compared with the intervertebral disc morphology in the same age group,the signal intensity of the intervertebral disc nucleus pulposus in the patients with intervertebral disc herniation was significantly decreased.②The frozen sections of Pfirrmann grade Ⅲ and Ⅴ showed positive DHE staining,and the surface density of DHE staining of Pfirrmann grade Ⅴ degeneration tissue was higher.Conclusion:Our study demonstrates the radiographic features of intervertebral discs in different age groups and patients with lumbar disc herniation.MRI T2WI signal intensity analysis showed that the water content of intervertebral disc nucleus pulposus decreased gradually with age,MRI T2WI signal intensity analysis showed that the water content of the intervertebral disc nucleus pulposus gradually decreased with age,which was more obvious in patients with lumbar disc herniation.The results of DHE staining indicated that there were different levels of oxidative stress in the degenerated tissue.The clinical data analysis further proved the feasibility of the strategy of anti-oxidative stress delaying intervertebral disc degeneration.Part 2:Preparation,characterization and biocompatibility detection of carbon dot-Prussian blue nanozymeObjective:Currently,natural or synthetic antioxidant drugs used in clinical practice are inefficient and lack target specificity in vivo.Therefore,it is necessary to develop methods that can effectively inhibit oxidative stress and delay tissue degeneration.With the development of nanomedicine,nano-particles with enzyme like activity have gradually come into view because of their high catalytic effect.Carbon-based nanozymes,metal nanozymes and biogenic nanozymes have been applied in different disease models with satisfactory results.Based on our previous research results,we synthesized a novel composite nanomaterial:PEI-600-Fe C-dots Prussian Blue nanoparticles（CD-PBs）.In this chapter,the preparation,physicochemical properties and biocompatibility of CD-PBs will be deeply analyzed.Methods:①CD-PBs nanocomposites were prepared,and their microstructure,surface charge and elemental composition were characterized by Transmission Electron Microscope（TEM）,Malvin particle analyzer and photoelectron spectroscopy（XPS）.②Detection of enzyme-like activity:Total SOD activity detection kit（NBT method）,dissolved oxygen determination and hydroxyl radical scavenging ability detection kit were used to detect Superoxide Dismutase（SOD）,Catalase（CAT）enzyme-like activities and hydroxyl radical scavenging ability of CD-PBs.③Cell Counting Kit-8（CCK-8）and cell live/dead staining were used to test the biocompatibility of CD-PBs.Results:①The particle size of CD is about 7-12 nm,and the particle size of CD-PBS nanocomposite formed after in-situ synthesis of PB on CD surface is about 40-50 nm,and the zeta potential decreases from 27.42±0.26 mV to 11.97±0.38 mV.②The surface of CD-PBS has obvious Fe-C peak,Fe-N peak and C-N peak,indicating the characteristic peak of Prussian blue on the surface.③Compared with pure PEI-600-Fe C-dots,CD-PBS nanocomposites have higher SOD and CAT activity and hydroxyl radical scavenging ability.④The water solubility of CD-PBs nanocomposite was good,and there was no significant difference in cytotoxicity between CD-PBs nanocomposite and control group when the concentration was below 200 μg/mL（P>0.05）.Conclusion:PEI-600-Fe C-dots were prepared by hydrothermal method,and Prussian blue was in situ synthesized on the surface of PEI-600-Fe C-dots to form core-shell structure complex nanoenzyme CD-PBs.The particle size,surface charge and elemental composition were tested to verify the successful synthesis of the composite structure,and it was proved that it had higher enzvme-like activity and good biocomnatibility.Part 3:Antioxidative stress of C-dots Prussian blue nanozyme delays intervertebral disc degenerationObjective:This part of the study intends to verify the antioxidant stress-anti-aging effect of the prepared nanocomposite in the model of intervertebral disc degeneration by scavenging ROS in vivo and in vitro experiments,so as to achieve the purpose of delaying intervertebral disc degeneration.Methods:①In vitro experiment:The primary rat Nucleus pulposus cells（NPCs）were extracted and cultured for cell identification.NPCs treated with H2O2 induced premature senescence.Superoxide anion probe（DHE）,Mito-Tracker Red CMXRos probe,immunofluorescence staining and RT-qPCR were used to detect the antioxidant performance of CD-PBs in vitro and its effects on catabolism synthesis,inflammatory related proteins and gene expression.Senescence of nucleus pulposus cells was detected by SA-β-Gal staining kit.③In vivo experiment:The model of intervertebral disc degeneration was established by caudal puncture in rats.The protective effect of CD-PBS on intervertebral disc injury was verified by X-ray and MRI imaging examination of the caudal vertebra of rats and paraffin section staining of intervertebral disc tissue samples of rats 8 weeks after puncture and treatment.Results:①In vitro experiments:the average fluorescence intensity of DHE staining in nucleus pulposus cells treated with H2O2 was 6.12 fold that of the blank control group（P<0.05）,while CD-PBs could significantly reduce the intracellular ROS level（1.99 fold）.After H2O2 treatment,the average fluorescence intensity of nucleus pulposus cells mitochondria staining decreased by about 66%compared with blank control group,and only decreased by about 38%in CD-PBS pretreatment group（P<0.05）.CD-PBs could maintain the expression balance of syncatabolic related genes and inhibit the expression of inflammatory factor TNF-α.The positive rate of nucleus pulposus senescence staining increased from 7.15±1.79%（blank control group）to 31.09±3.32%after H2O2 treatment,while decreased to 11.49±1.54%in CD-PBS treatment group（P<0.05）.②In vitro experiments:X-ray and MRI image measurements and tissue staining results indicated that the intervertebral disc height index,nucleus pulposus signal intensity and histological score of cD-PBS treatment group were significantly improved compared with the degeneration group.Conclusion:A composite nanomaterial CD-PBs targeting oxidative stress was designed and prepared by us.In vitro and in vivo experiments have proved that CD-PBs has high enzyme-like activity and good biocompatibility,which can remove excessive ROS in nucleus pulposus cells,maintain mitochondrial homeostasis and matrix synthesis catabolism balance,and inhibit inflammation.CD-PBs has the effect of anti-oxidative stress and anti-aging,thus effectively protecting the intervertebral disc tissue and delaying the progression of degeneration.