A Smart-responsive Drug Delivery System Based On Nanomembrance For Osteoarthritis Treatment:An Application Study

OBJECTIVE:Osteoarthritis(OA)is the most common joint degenerative disease with a very high rate of disability.Reactive oxygen species(ROS)has been proved as the major cause of chondrocyte damage and OA development in previous studys.This study focused on the feasibility of construction of ROS-respond drug delivery system(DDS)by polyhexanolide(PCL)and PEGDA-EDT,a thioether polymer according electrospinning technology.We characterized this DDS,and the best performance was selected to use in the study of antioxidant and anti-inflammatory effects in an OA chondrocyte model in vitro.Then the OA SD rat model was used for detecting the function of this DDS in vivo.Methods:First part,the synthesis of nanomembrance DDS.We systhesized PEGDA-EDT via a thiol-ene reaction,then electrospined with PCL adding rGO as the drug carrier for puerarin(Pue).Fourier transform infrared spectroscopy(FT-IR)and Hydrogen nuclear magnetic spectra(1HNMR)were used to detect the PEGDA-EDT polymer.Scanning electron microscope(SEM)and optical microscope were used to observe the microstructure of nanomembrance DDS,and mechanical properties were detected at soon.Then we explored the ability of ROS-respond and drug delivery in vitro.Second part,smart-respond nanomembrance DDS treated OA in vitro.We first selected the best performance of nanomenbrance DDS by MTT,live/dead staining and SEM assays.Then we executed CCK-8 assay,flow cytometry(FCM),live/dead staining,cytoskeleton staining,biochemical detection,qRT-PCR and immunofluorescent staining to evaluate the nanomembrance DDS’ effect of antioxidant and anti-inflammatory in vitro.We established SD rat OA model via anterior cruciate ligament resection(ACLT)for the verification of nanomembrance DDS’ effect in vivo.After nanomenbrance DDS was implanted into OA animal model,the cartilage protection ability of nanomenbrance DDS at 4 and 8 weeks was estimated by gross observation,gross score,HE staining and safranin O-fast green staining.Also the western-blot and immunohistochemical staining was used to explored the antioxidant and anti-inflammatory effect.RESULT:The PEGDA-EDT polymer which has a thioether group had been systhesized improving by FT-IR and 1HNMR.And the FT-IR also improved that the PCL and PEGDA-EDT can be self-electrospin because of no reaction happened between PCL and PEGDA-EDT.And the SEM showed the microstructure of PCL/PEGDA-EDT@rGO-Pue(PPE@rGO-Pue)nanomembrance DDS which constituted by thick(PCL)and thin(PEGDA-EDT)fibers.The diameter of thick fiber was 219.34±58.82 nm and thin was 53.34±16.45nm,also the proportion of thin fiber was increase along with the ratio of PEGDA-EDT.The mechanical properties of nanomembrances DDS showed that the Young’s modulus of them was in the range of 2-10MPa,which mimicked cartilage extracondral matrix.Then drug delivery in vitro of 30%PPE@rGO-Pue nanomambrance was detection,the Pue concentration was 10.02±0.75 ug/mL and 5.80±0.22 ug/mL,cumulative release rate was 50.48±4.61%and 28.90±1.81%in high ROS(1mM H2O2)and low ROS(100μM H2O2)environment respectively.But there was almost no Pue released in non-ROS environment.The result of CCK-8,live/dead staining,GAG detection and FCM showed that PPE@rGO-Pue can reduced cell apoptosis and protected chondrocytes from OA.We found PPE@rGO-Pue could stimulate chondrocytes produced antioxidase including CAT,SOD,GSH to enhance antioxidant ability and reduced inflammatory factor for anti-inflammatory.And the in vivo reaserch result was coincident with in vitro study.Observation of knee cartilage,HE staining,safranin O-fast green staining and immunohistochemical staining certified that PPE@rGO-Pue can effectively protect cartilage in vivo,increase the anti-oxidation ability of chondrocytes,reduce inflammatory factors,inhibit inflammation and promote the repair of OA cartilage damageCONCLUTION:PPE@rGO-Pue was successfully constructed as a smart-respond DDS,which has ROS responsiveness and only releases an effective concentration of Pue in the ROS environment.We showed the anti-oxidant and anti-inflammatory effects of this DDS in vitro and vivo.The smart-respond nanomembrane DDS constructed in this study can provide a new direction for DDS construction,which proves that nanofiber membrane can be used to construct intelligent response DDS,and also provides a new method for OA treatment.