Preparation Of High-performance Prussian Blue Nanoparticles And The Basic Research On Treatment Of Osteoarthritis

Prussian blue nanoparticles(PBNPs)have shown great application potentials in the diagnosis and treatment of diseases with the exploration of nanoenzyme effect,reactive oxygen species(ROS)scavenging activity,photoacoustic imaging and magnetic resonance imaging(MRI)capabilities.Generally,the qualities of the nanomaterials have decisive influences on their performances and applications.Therefore,in order to further promote the applications of PBNPs in the biomedical field and overcome the still existing defects including low biological activity and hard control of the quality of the nanoparticles,it is of great significant to develop synthetic techniques for high-performance PBNPs with simple and green preparation process.In this thesis,two types of synthetic techniques of PBNPs were developed to regulate the crystallinity,dispersion uniformity and size of nanoparticles.Meanwhile,we explored the formation mechanism of PBNPs.Then,the key parameters for quality control,stability,catalytic efficiency,ROS scavenging activity and MRI imaging ability of PBNPs were also investigated.Finally,the as-prepared high-performance ultrasmall Prussian blue nanoparticles(USPBNPs)were further used for the treatment of osteoarthritis in rats.The main research concents and results are shown in the following:(1)A magnetic internal heating approach was developed to prepare high-performance PBNPs,and the formation mechanism was explored.The results showed that the half-width of the X-ray diffraction(XRD)peak and the polydispersity index(PDI)of the nanoparticles(PBNPs-IH)fabricated by magnetic internal heating method were 0.23°and 0.015,which were significantly lower than those of the nanoparticles(PBNPs-EH)prepared by conventional external heating method.This result revealed that the crystallinity and dispersion uniformity of PBNPs-IH are improved.Furthermore,the magnetization,longitudinal relaxation rate,peroxidase-like(POD)and catalase-like(CAT)catalytic rates of PBNPs-IH were 64.2emu g^-1 PB,1.47 m M^-1 S^-1,(4.76±0.29)×10^-8 M s^-1 and(3.44±0.11)×10^-6 M s^-1,respectively,which were significantly higher than those of PBNPs-EH(P<0.05).During the formation process of PBNPs-IH,the current intensity played a vital role.Under the condition of 6.5 A,the formation process of the nanoparticles would follow the classical crystallization process,while adjusting to 11.5 A,it would follow the non-classical crystallization process.(2)A facile strategy was developed to prepare USPBNPs(<5 nm)and the formation mechanism was also explored.The results showed that USPBNPs with?3.4 nm in diameter could be successfully obtained by using 75%ethanol/water mixture as the solvent.During the formation process,on the one hand,ethanol could transfer the molecular configuration of polyvinylpyrrolidone(PVP)from the chain to globular,so as to increase the steric hindrance among the primary nanonuclei and prevent PB nuclei from attaching with each other to obtain ultrasmall-sized nanoparticles.On the other hand,ethanol could also reduce the surface energy of the primary nanonuclei and induce a strong coordination between PVP and iron ions,promoting the formation and stabilization of USPBNPs.(3)Subsequently,the key parameters for quality control,stability in different temperatures,p H and solvents,catalytic activities,MRI imaging ability and photothermal performance of USPBNPs were analysed.The results showed that the molecular structure of USPBNPs was Fe₄[Fe(CN)₆]₃ and the maximum absorption wavelength,hydrodynamic size,zeta potential,crystal size,ethanol residue,content of PVP coating and cyanide dissolution were 697.5±2.5 nm,51.0±27.4 nm,-30.9±0.4 m V,3.4±0.5 nm,<5%,15.5±0.8%,<50 ppm,respectively.In addition,USPBNPs presented good stability in the temperature lower than 37?,PBS with p H of 2.0-7.4,physiological saline and cell culture medium.The POD-like catalytic activity,CAT-like catalytic activity and longitudinal relaxation rate of USPBNPs were 465.8 U/mg PB,15.17 U/mg PB and 1.34 m M^-1 S^-1,respectively,which were significantly improved compared to Prussian Blue nanocubes(PBNCs)with larger sizes(P<0.01).Moreover,the ability of USPBNPs to remove·OH was four times higher than that of PBNCs.USPBNPs also displayed an intensive scavenging activity on·OOH.(4)Finally,the biological safety,the ability to remove intracellular ROS andinflammatory factors and the therapeutic effect on osteoarthritis of USPBNPs were evaluated.The results showed that USPBNPs presented negligible effect on cell survival,cytochrome C release,mitochondrial membrane potential,cell membrane permeability,ATP content and oxygen consumption rate under the concentration of10?g/m L.USPBNPs could effectively eliminate intracellular ROS and reduce the m RNA expression levels of IL-6,IL-1?,TNF-?and MMP-13 within the concentration range of 5-10?g/m L,exhibiting an excellent anti-inflammatory activity.In the rat osteoarthritis model,USPBNPs could effectively relieve joint swelling.HE staining and safranine-fast green staining of articular cartilage tissue sections have confirmed that USPBNPs could protect and restore the articular cartilage cells.According to the results of immunohistochemistry research,USPBNPs could reduce the expression of IL-6 and IL-1?,displaying a equivalent therapeutic effect to that of the hydrocortisone for clinical injection.The above results indicated that USPBNPs could effectively eliminate ROS and treat inflammatory diseases.