| Background and objective: Intra-articular injection is a very effective strategy for the treatment of Osteoarthritis(OA).However,on the one hand,small molecule drugs are easy to be cleared by the capillaries and lymphatic system around the joint,making their efficacy unable to be fully developed;on the other hand,the efficacy of many monomer drugs with anti-inflammatory effect is greatly reduced due to their poor water solubility.Nanoparticles can stay in the joint for a longer time.At the same time,nanoparticles are easy to be modified then can target a specific position,which can avoid off-target effects during treatment.Formononetin(FMN)is an active ingredient of traditional Chinese medicine.In our experiment,we construct polymer-drug copolymer——PCFMN with FMN,N-hydroxylsuccinimide-polyethylene glycol-carboxyl(NHS-PEG-COOH),and type II collagen targeting peptide(Coll BP),which can self-assemble into PCFMN NPs,We characterize it and study its ability to the treatment osteoarthritis in vitro and in vivo.Method: This study consists of three parts.Part one: the synthesis and characterization of PCFMN NPs.Firstly,Coll BP and FMN were grafted onto NHS-PEG-COOH through amide reaction and esterification reaction under catalysis.1H NMR,UV-Vis,FT-IR,TEM,and DLS were used to characterize PCFMN NPs.Finally,The drug loading and cumulative drug release rate of PCFMN NPs were determined by HPLC.Part two: studying the targeting and anti-inflammatory effects of PCFMN NPs in vitro.Primary chondrocytes of the knee joint were extracted from suckling male Sprague Dawley(SD)rats aged5-7 days.The chondrocytes were transferred to the third generation for the subsequent experiments.The experiment was divided into four groups: blank group(Control group),IL-1β group(OA group),IL-1β+FMN group(FMN group),IL-1β+PCFMN NPs group(PCFMN group).First,the toxicity of FMN and PCFMN NPS to chondrocytes and their protective effects on chondrocytes were detected by MTT assay.Next,The chondrocytes were treated with PCFMN and PFMN(without Coll BP)labeled with DID.According to the intensity of red fluorescence,the cartilage targeting ability of PCFMN NPS was verified.The protective effect of PCFMN NPs on chondrocytes was verified by Hematoxylin-eosin staining.The anti-inflammatory effect of PCFMN NPs was verified by immunofluorescence staining,and the expression of inflammation-related genes and cartilage-related genes were analyzed by q RT-PCR.Part three: studying the targeting and anti-inflammatory effects of PCFMN NPs in vivo.We successfully constructed the OA model in vivo by amputating the anterior cruciate ligament of SD rats for 4 weeks.On the one hand,SD rats of the OA model were divided into PCFMN group and PFMN group.After IA-injection of DID-labeled PCFMN NPs or PFMN,the fluorescence intensity of the knee joints was measured using an in vivo imaging system at corresponding time points(0 d(within 30 min after IA injection),1 d,3d,7 d,19 d).On the other hand,the experiment in vivo is divided into four groups: blank group(Control group),IL-1β group(OA group),IL-1β+FMN group(FMN group),IL-1β+PCFMN NPs group(PCFMN group).The SD rats in the OA group,FMN group,and PCFMN group were injected with saline,FMN,or PCFMN respectively.The knee joints of the corresponding groups were obtained at the 4th week and the 8th week,and the macroscopic evaluation is conducted.The tissue sections were further stained with HE,Safranin O &Fast Green,and immunohistochemical staining,then OARSI scoring was performed.Results: PCFMN NPs were successfully synthesized.The solubility experiment of PCFMN NPs and FMN showed that the solubility of FMN was increased after nanocrystallization.The results of 1H NMR,UV-Vis,and FT-IR indicate the successful synthesis of PCFMN NPs.TEM showed that PCFMN NPs are spherical nanoparticles with uniform size and good dispersion,with an average particle size of about 34 nm.DLS results show that the average hydrodynamic particle size of PCFMN NPs is 211 nm.The drug loading rate of PCFMN NPs measured by HPLC was about 9%.In the medium of p H 7.4 and6.0,the cumulative release rates on the 9th day were 87.5% and 68.4%,respectively.In vitro experiment,The results of MTT show that PCFMN NPs have no obvious toxicity to chondrocytes in the concentration range of0-50μg/m L,but with the concentration increases,FMN has a toxic effect on chondrocytes.When the concentration of FMN is 1.25μg/m L,The viability of chondrocytes is the best.The PCFMN NPs concentration corresponding to FMN is 14μg/m L.Both 1.25μg/m L FMN and 14μg/m L PCFMN NPs have a protective effect on inflammation-induced chondrocytes,between which PCFMN NPs have more protective effects.By the uptaking of chondrocytes,it was observed that the red fluorescence intensity of the PCFMN group was stronger,reflecting the targeting effect of PCFMN NPs.HE staining results show that PCFMN NPs can better protect chondrocytes and maintain the morphology of chondrocytes.The results of MMP-13 immunofluorescence staining show that PCFMN NPs have better anti-inflammatory effects.The q RT-PCR results showed that the expression of cartilage-related gene type II collagen(Col2al)in the PCFMN group and the FMN group was higher than that in the OA group,and the PCFMN group was up-regulated more significantly.The inflammation-related gene(Il-1β,Mmp-3,Mmp-13)expression in the PCFMN group and the FMN group decreased compared with the OA group,and the PCFMN group was down-regulation more obvious,which indicating that PCFMN NPs have well cartilage protection and anti-inflammatory effects.In vivo experiment.The optical imaging results showed that the fluorescence intensity of the PCFMN group was stronger than that of the PFMN group at the same time point,and the fluorescence duration of the PCFMN group was longer.It shows that PCFMN NPs can target cartilage tissue and stay in the joint for a long time.Furthermore,the macroscopic evaluation,HE staining,Safranin O &Fast Green staining,immunohistochemical staining showed that PCFMN NPs can delay the progression of OA.Conclusion: This study shows that PEGylation improves the solubility of FMN.By further modifying with collagen targeting peptide,our synthesized PCFMN NPs nanomedicine can be better targeted to cartilage tissue.With IA injection,it can delay the progress of OA.This kind of targeted nanomedicine may provide a new idea for the clinical treatment of OA. |
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