Rational Engineering Of Ferritin Nanocages For Targeted Therapy Of Osteoarthritis

OBJECTIVE:Intra-articular(IA)drug delivery to treat osteoarthritis(OA)is limited by the short retention time of drugs in the joints due to poor specific targeting and non-responsiveness in the acidic environment.Here we present a biomimetic approach to develop a smart bioresponsive ferritin nanocages that comprised of inner-loaded metformin and surface exposed cartilage-targeting peptide.Methods:The first part:Firstly the fabrication CT-Fn nanocages CT/Fn nanocarriers,extraction and purification of the proteins were detected.The relative characterizations were investigated:the molecular weight of the protein subunits was verified by SDS-PAGE gel electrophoresis;the morphology and distribution of CT-Fn nanocage were observed by scanning electron microscopy;the average diameter and potential of the carrier were investigated by Zetasizer analyzer;The spectrophotometer performs full-wavelength scanning to observe the change of ultraviolet absorption;the drug was loaded according to the self-assembly property of ferritin and the drug loading of the nanocage is determined by HPLC;finally,to simulates the in vivo environment,dialysis were placed to carries out the experiment of in vitro release.The second part:firstly treated mouse chondrocytes with IL-1? as an in vitro OA model,the toxicity of the vector was detected by CCK8,then CT-Fn/Met t nanocages were labeled with cy5.5 to follow their cellular uptake,and type ? collagen,the target of CT-Fn,was detected by immunofluorescence assay.Subsequently,cell viability was detected by CCK-8,FDA/PI staining;PCR and immunofluorescence were used to detect the expression of related inflammatory genes.The third part:The retention time of cy5.5-labeled nanocage in mouse joints was observed by fluorescence scanning and the drug concentration in articular cartilage and joint fluid was detected by HPLC.The mouse arthritis model was induced by intra-articular injection of papain.After successful model establishment,the CT-Fn/Met drug-loading system was injected into the joint cavity,and the relevant control group was set up.Mouse joints were taken and taken at week 2 and week 6.The knee joint femur and tibia of the mouse were photographed and grossly scored.The samples were embedded in paraformaldehyde,and the samples were embedded in sections.HE staining,safranin solid staining and immunohistochemical staining were used to observe the joint repair.RESULTS:We successfully linked the cartilage targeting peptide(WRYGRL)to the surface of each subunit of ferritin and the CT-Fn was a uniformly distributed spherical nanocage.The CT-Fn nanocage has a diameter of about 20 nm,zeta potential of-25.65±2.13 mV and drug loading of 20.46%,In the in vitro release experiment,the drug release of CT-Fn/Met in the acidic environment(pH=6.0)was significantly higher than that in the neutral environment(pH=7.4).In vitro cytotoxicity test results showed that CT-Fn treated chondrocytes had almost no toxic effects between 25 and 100 ?g/mL.Compared to Fn/Met,chondrocytes showed higher cellular uptake of CT-Fn/Met and localized type II collagen in the extracellular matrix.In in vitro inflammation studies,we found that CT-Fn/Met can significantly inhibit IL-1?-induced inflammatory response,enhance chondrocyte viability,reduce the expression of inflammation-related factors MMP-1,MMP-13 and IL-6,and increase Col-II expression.In the arthritis model of mice,CT-Fn/Met can stay in the joint cavity for up to 3 weeks without the modified Fn/Met residence time of 2 weeks.At the same time,the OARSI scores of the joints injected with CT-Fn/Met were significantly increased and the expression of inflammatory factors was decreased.Conclusion:In summary,we successfully fabricated a CT-Fn/Met drug delivery system by using Fn nanocages modified with cartilage-targeting peptides as carriers of Met for IA drug delivery based on IL-1?-induced chondrocytes and a papain-induced OA model.Biocompatible CT-Fn/Met specifically bound to the type II collagen of cartilage and effectively reduced synovial inflammation in joints by prolonging the retention time.It is suitable for the acidic microenvironment associated with OA inflammation.Our findings demonstrate the potential future clinical applicability of CT-Fn/Met for OA treatment.