Cartilage-targeting And Dual MMP-13/pH Responsive Theranostic Ferritin Nanoprobes For Osteoarthritis Imaging And Precision Therapy

OBJECTIVE:Osteoarthritis(OA)microenvironment is marked by matrix metalloproteinases-13(MMP-13)overexpression and weak acidity,making it possible to develop dual-stimuli responsive theranostic nanoprobes for both OA diagnosis and therapy.However,current MMP/pH-responsive systems have not been applied in OA and are not suitable for OA because of their poor biocompatibility,poor degradation and non-cartilage-targeting of the responsive probes.Here we designed a novel biocompatible cartilage-targeting and MMP-13/pH-responsive ferritin nanocages(CMFn)loaded with an anti-inflammatory drug(Hydroxychloroquine,HCQ),termed CMFn@HCQ,for OA imaging and therapy.METHODS:The experiment was divided into three parts.The first part,synthesis and properties of CMFn@HCQ.The ferritin nanocage(Fn)surface was first modified with a cartilage targeting peptide(WRYGRL),then the cy5.5-labeled MMP-13 cleavable peptide(MMP-cy5.5)was conjugated to CFn,and then the BHQ-3-labeled CFn(CFn-BHQ)was conjugated to it to form a hybrid ferritin(CMFn)by adjusting the pH,and finally adjusting the pH loading HCQ to form a CMFn@HCQ nanocomposites.Characterization of CMFn with a serioes dectetion.The particle size and stability of material were measured by a laser particle size analyzer(DLS).The morphology of the purified CMFn was measured by a transmission electron microscope(TEM).The molecular weight was measured by gel electrophoresis(SDS-PAGE).The ultraviolet absorption spectrum was measured by a full-wavelength microplate reader.The fluorescence spectra and fluorescence recovery of CMFn were measured by a fluorescence spectrophotometer.The drug loading and the cumulative release amount of HCQ were detected by High performance liquid phase(HPLC).In the second part,the study about the targeting,MMP-13/pH responsiveness and anti-inflammatory activity of the CMFn@HCQ nanocomposites in vitro.Divided into five groups:(1)control group;(2)IL-1? group;(3)IL-1?+HCQ group;(4)IL-1?+MFn@HCQ group;(5)IL-1?+CMFn@HCQ group.The chondrocytes of C57BL6/J suckling mice were subjected to subsequent cell experiments after being treated and cultured.CCK-8 was used to detect the toxic effect of CMFn on chondrocytes.Cells were induced with IL-1? for 1 day,and cell-uptake and Col2a1 immunofluorescence staining were used to study targeting and enzyme responsiveness of CMFn.Chondrocytes were induced with IL-1? for 1,3,and 5 days,respectively,and cell uptake experiments were used to study enzyme responsiveness of CMFn.Finally,RT-qPCR(MMP-13,IL-6,MMP-3 and Col2a1 gene expression),hematoxylin-eosin(HE)staining,MMP-13 and IL-6 immunofluorescence staining were used to detect the anti-inflammatory activity of cells in vitro.The third part,the study about the targeting,MMP-13/pH responsiveness and anti-inflammatory activity of the CMFn@HCQ nanocomposites in vivo.In-vivo Multispectral Imaging Systems was used to detect the response of CMFn to MMP-13 at various time points.High-performance liquid chromatography(HPLC)was used to detect the retention of drugs in the joint cavity at various time points in mice,and to verify the targeting of CMFn in vivo.The samples were taken after 14 days and 42 days of treatment,respectively,for gross view scores.The samples were fixed and sectioned for HE,Safranin O-solid green,MMP-13 immunohistochemical staining and histological scoring to verify the pharmacodynamics of CMFn in vivo.RESULTS:The CMFn@HCQ nanocomposites was successfully synthesized and characterized.CMFn nanocage particle size was about 22 nm,and the structure of which was spherical,uniform in size,good in particle dispersibility.The molecular weight was about 25 kDa.The material has good stability and the volume concentration range is about 22 nm.The UV absorption spectrum confirms the characteristic peaks of cys.5 and BHQ-3,the fluorescence spectrum shows that the fluorescence intensity of CMFn is significantly reduced,which confirms the successful construction of CMFn.The fluorescence recovery experiment confirms the fluorescence of CMFn stimulated by MMP-13,and monitoring of fluorescence intensity over time.The drug loading of CMFn@HCQ was 49%.The vitro release experiments of CMFn@HCQ group showed that the cumulative release of HCQ was only 36%at pH=7.4,and the cumulative release was 59%at PH=5.0.After MMP-13 was added at pH=5.0,the cumulative release of HCQ reached 83%,which proved that CMFn@HCQ is MMP-13/pH responsive system.CMFn has significant anti-inflammatory activity on chondrocytes.CCK-8 results show that CMFn nanocages do not show cytotoxicity at doses of 0 to 120 ?g/mL.Cell uptake experiments indicate that CMFn successfully targets type ? collagen sites produced by chondrocytes,and as the concentration of MMP-13 increases,the fluorescence intensity in CMFn increases gradually,indicating the enzyme response of CMFn in cells.The results of T-qPCR showed that the expression of inflammatory factor markers was down-regulated by 55-60%and the expression of Col2al was up-regulated by 77%after CMFn@HCQ and cells were incubated for 1 day.HE and immunofluorescence staining showed that CMFn@HCQ nanocage can maintain the phenotype of chondrocytes in the presence of IL-1? and inhibit the expression of inflammation factors,such as MMP-13 and IL-6 induced by IL-1?.In-vivo Multispectral Imaging Systems showed that CMFn had strong fluorescence in the OA joint cavity and weak fluorescence in the normal joint,indicating that the fluorescence intensity was positively correlated with the increase of the severity of OA,and the fluorescence retention time of CMFn was longer than MFn.The targeting of CMFn in the joint cavity and the performance of the MMP-13 response were verified.The HPLC results showed that the HCQ concentration in CMFn@HCQ was greater than HCQ and MFn@HCQ at each time point,and the results demonstrated the targeting performance of CMFn in the joint cavity.The therapeutic effect of CMFn on OA mice was verified.The joints of each group were photographed,scored,and finally histologically stained and histologically scores were obtained.The results showed that CMFn@HCQ had the best therapeutic effect.CONCLUSION:In conclusion,we have successfully designed a novel multifunctional nanocomplex drug delivery platform that is both dual-stimuli activatable and cartilage targeting for on-demand prognosis and IA release of therapeutic agents.This system is designed to be responsive to two unique chemical signals of the microenvironment of OA joints.One is the overexpressed MMP-13,which enables the system to achieve MMP-13 responsive fluorescence signal recovery of a NIR dye for fluorescence imaging and diagnosis of OA.Another is the acidity,which is adopted to realize the pH-responsive release of anti-inflammatory drug from the system for OA treatment.Therefore,this system holds promise for precision OA theranostics for clinical applications.