Engineered Exosome-Mediated Target Delivery Of Kartogenin To Synovial Fluid Mesenchymal Stromal Cells For Cartilage Regeneration

BackgroundOsteoarthritis(OA)is a common degenerative disease of joints.OA is characterized by degradation of articular cartilage,accompanied by aseptic inflammation,degeneration of synovium and meniscus.OA is the main cause of disability among the elderly.Based on epidemiological data in 2019,there are about 250 million patients with OA worldwide.With the increasing aging of the world population,the number of patients suffering from OA is increased each year.OA has caused great suffering to the patients,a heavy economic burden on society,and seriously affecting the development of the family and society.Even OA is a common clinical disease,at present,there are no effective methods for the treatment of OA.For early-stage OA patients,the common treatment methods include weight-control,exercise-change,and physiotherapy.Mid-stage OA patients can receive hyaluronic acid injections and some other drug injections.For the late-stage OA patients,knee replacement surgery is the only method improving the quality of their lives.At present,none treatment methods can reverse the process of OA disease.All the treatment methods can only alleviate the clinical symptoms of patients.The majority of patients were finally treated with knee replacement surgery.However,surgical treatment carried many high risks and complications such as infection and bleeding.And joint replacement prosthesis has a limited life span,which is not suitable for young patients.The number of patients suffering from osteoarthritis has increased year by year,but the effective method of treating osteoarthritis has not yet been found.This situation has caused great pain to patients and posed a great clinical challenge.It is therefore urgent to develop a new method of treatment for OA.Mesenchymal stromal cells(MSCs)are a type of somatic stem cells with high self-renewal potential and multi-lineage differentiation potential.MSCs can be isolated and extracted from a wide range of sources such as bone marrow,umbilical cord,and adipose.Nowadays,MSCs become the "seed cells" with the most powerful in the field of tissue engineering,maybe the "superstar" for OA treatment in the future.Studies showed that MSCs are prone to differentiation into specific tissue as their origination.Therefore,MSCs derived from joint cavity named synovial fluid mesenchymal stromal cells(SF-MSCs)may be the most ideal cell source for cartilage tissue regeneration to treat OA.Patients with OA often suffer from excessive secretion of synovial fluid due to inflammatory stimuli,which causes swelling and pain.Knee joint cavity paracentesis is a common clinical practice,which can extract excess synovial fluid from the knee joint of patients with OA.Previously,the synovial fluid was discarded as medical waste.In this experiment,we used a simple extraction method to efficiently obtain SF-MSCs from synovial fluid which means "Trash to Treasure".However,previous studies have shown that MSCs cannot spontaneously differentiate into cartilage without induction in vitro.Meanwhile,MSCs have poor stability in cartilage differentiation in vivo,and cannot be fully differentiated into hyaline cartilage.The clinical application of MSCs is greatly limited by these disadvantages.The fate of exogenous MSCs in the knee joint still requires further elucidation.Kartogenin(KGN)is an artificially synthesized small molecule compound with strong cartilage formation and cartilage protection.As a stable small molecule,KGN can be stored and transported at room temperature.After KGN enters MSCs,it can efficiently induce MSCs into cartilage differentiation and form hyaline cartilage.The hydrophobic properties of KGN severely hinder it’s engulfed by the cells.In addition,an excessively high dose of KGN is likely to induce excessive hyperplasia of cartilage.It is necessary to develop a novel drug-targeted delivery system that releases KGN to MSCs at proper concentration,and fully perform the MSC chondrogenic differentiation induced by KGN.Exosomes are extracellular vesicles secreted from various cell types with a diameter of 30-200 nm.Phospholipid bilayer structure of exosomes can effectively protect its contents.Exosomes transfer a variety of biological substances such as microRNAs,proteins,from donor cells to recipient cells,thereby exerting intercellular communication.All these advantages make exosomes emerge as a promising drug delivery carrier.Previous studies have shown that the delivery of hydrophobic drugs through exosomes can significantly enhance their solubility and increase their bioavailability.As a "logistics system" between cells,endogenous exosomes can deliver the "goods" accurately and efficiently.Without the "navigation system",exogenous exosomes are often highly enriched in the liver and spleen.No targeting makes the exogenous exosomes unable to effectively deliver drugs.Consequently,there is an urgent need to develop a new method to give exosomes the ability to target MSCs,making exosomes can efficiently deliver drugs into MSCs.This study adopted an engineered construction method with optimizing the SF-MSCs based treatment of OA.By summarizing the basic methods of SF-MSCs extraction,cultivation,and identification,a standard operating procedure is established to SF-MSCs.A new method named ampliative destabilization of the medial meniscus(DMM)is first carried out to established OA model.Using this OA model,the final fate of SF-MSCs after injection into the joint cavity was studied in details.It is the first time to develop an exosome-based drug delivery system for targeting SF-MSCs.The effects of engineered exosomes loaded KGN on the cartilage differentiation ability of SF-MSCs were discussed in detail.The simulation verified the effect of the dual system engineered treatment system EKS formed by the pre-assembly of engineered exosomes and SF-MSCs on early OA.MethodsThe first part:The study of SF-MSCs extraction,cultivation,and identification.The synovial fluid of OA patients was extracted by puncture,and the supernatant was discarded after high-speed centrifugation.The SF-MSCs were cultured using MSCs complete medium.CD105,CD73,CD90,CD45,CD34,CDllb and other stem cell surface markers were identified by flow cytometry.The induction potential of SF-MSCs in osteogenic,adipogenic,and chondrogenic differentiation was identified by adding induction medium.The second part:The study of SF-MSCs distribution in treating OA ratsGenetic engineering method was used to construct EGFP tracer vector,and SF-MSCs were labeled with EGFP tracer vector.Anterior cruciate ligament transection(ACLT)was used to establish the OA model of Sprague Dawley(SD)rat.High intensity treadmill training was used to accelerate the formation of OA models.Develop a new method named ampliative DMM to establish OA models.H&E staining sections were produced to observe the difference between two OA models.SF-MSCs-EGFP were injected into the knee joints of the two OA models.Frozen sections were made to observe the distribution of SF-MSCs in the cartilage layer at 1 day,3 days and 7 days.The third part:Study of engineered exosomes for targeted transfer KGN to SF-MSCs enhancing its chondrogenic ability.E7-lamp2b engineered vector,E7-EGFP-lamp2b engineered tracking vector,and EGFP-lamp2b tracking vector were constructed by genetic engineering methods.Dendritic cells(DC)were transfected with three expression vectors,respectively.Laser scanning confocal microscopy was used to observe the E7-EGFP-lamp2b engineered tracer vector transfected with DC.Western Blot was used to test whether the expression vector E7-lamp2b was successfully transfected with DC.DC-derived engineered exosomes(E7-lamp2b-exo),engineered tracking exosomes(E7-EGFP-lamp2b-exo),and EGFP-traced exosomes(EGFP-lamp2b-exo)were harvested after transfection by ultrahigh speed centrifugation.Transmission Electron Microscope(TEM)and Nanoparticle Tracking Analysis(NTA)were employed characterize the microstructure and the particle size distribution of engineered exosomes.Western Blot detection was used to identify the surface markers of exosomes.KGN was fluorescently labeled with Rhodamine B by chemical synthesis.The obtained compounds were detected by proton spectrum,carbon spectrum and dihydrogen NMR.Incubation,repeated freeze-thaw,and electroporation were used to load KGN into engineered exosomes.A spectrophotometer was used to study the loading and release efficiency of KGN in engineered exosomes.The effect of engineered exosomes delivery on the solubility of KGN was observed by laser scanning confocal microscopy,and the targeting ability of engineered exosomes to SF-MSCs was also observed.Quantitative real time polymerase chain reaction(RT-qPCR)and Western Blot were used to test the ability of SF-MSCs into cartilage differentiation with engineered exosomes loaded KGN promoting.The OA models was established by ampliative DMM method.SF-MSCs were fluorescently labeled with EGFP tracer vector.Engineered exosomes loaded with Rhodamine B-KGN and SF-MSCs-EGFP assembled to form an engineered-therapy system(EKS).EKS is injected into the knee joint cavity of OA rats.Rats were sacrificed after 1 day,3 days,and 7 days.respectively.Frozen sections were made following the steps in the second part.The motion trajectory of the engineered treatment system EKS was observed under a laser scanning confocal microscope.Cell membrane fluorescent probe DIR(iodide1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide(DIR))was used to label engineered exosomes,and the targeting ability of engineered exosomes in vivo was measured using a small animal living imaging system.In order to test the safety and effectiveness of the engineered treatment system EKS in the treatment of OA,EKS was injected into the knee joint cavity once a week for 3 weeks.After 4 weeks,rat knee joint samples were collected,tissue sections were made.Hematoxylin&Eosin,toluidine blue,immunohistochemical staining,and OARSI histological scores were made to evaluate the therapeutic effect.Rats’organs sections were made to check the safety of engineered treatment system EKS.Results:SF-MSCs were successfully extracted from synovial fluid of OA patients.Flow cytometry results showed that SF-MSCs highly expressed CD105,CD73,and CD90,and lowly expressed CD45,CD 19,CD11b,CD34 and HLA-DR.The standard operating procedures for the extraction,cultivation and identification of SF-MSCs were successfully summarized and established.EGFP tracer vector was successfully constructed and tranfected into SF-MSCs.ACLT combined platform running and ampliative DMM had successfully established OA rat models.The knee cartilage of OA rats was successfully isolated.Frozen sections were successfully made.Frozen section results showed that a small number of SF-MSCs were localized in the cartilage layer at 3 days,and SF-MSCs were widely distributed in the cartilage layer at 7 days.E7-lamp2b engineering vector,E7-EGFP-lamp2b engineering tracer vector,and EGFP-lamp2b tracer vector were successfully constructed.Laser scanning confocal microscopy and Western Blot results indicated that the expression vector was successfully transfected with DC.Engineered exosomes were successfully harvested after transfection by ultrahigh speed centrifugation.Western Blot results showed the high expression of exosomes Lamp2b,which confirmed that the engineered exosomes were successfully constructed.Transmission electron microscopy results and NTA results showed that engineered exosomes exhibited a round or cup-shaped morphology with a diameter of 30-200 nm.Western Blot indicated that engineered exosomes highly expressed exosomal-marker:CD63 and CD81.Taken together,the above results indicated that engineered exosomes met the criteria for exosomal identification.The results of the proton spectrum,carbon spectrum,and dihydrogen NMR showed that KGN was successfully labeled with Rhodamine B.Compared to incubation and repeated freeze-thaw methods,electroporation significantly improved the efficiency of exosomes loaded with KGN.The engineered exosomes can maintain stability after loading KGN,and can realize sustained release of KGN.Compared with the incubation method of KGN and SF-MSCs,the delivery of KGN into SF-MSCs via engineered exosomes is more efficient and can significantly improve the solubility of KGN.Compared with DC-derived exosomes,engineered exosomes enter SF-MSCs more efficiently,showing a good targeting ability to SF-MSCs.The results of RT-qPCR and Western Blot showed that the expression of collagen type II and aggrecan in SF-MSCs significantly increased after pretreatment with engineered exosomes loaded KGN.Engineered exosomes loaded KGN could effectively enhance the differentiation of SF-MSCs into chondrocytes.Laser scanning confocal microscopy results indicated that no EKS fluorescence signal of the engineered treatment system EKS was seen in the cartilage layer at the first day.After 3 days,some of the engineered treatment systems EKS could homing to the cartilage layer of the knee joint.After 7 days,a large number of engineered treatment systems EKS spread over the entire cartilage layer of the knee joint.In vivo imaging results showed that the engineered exosomes still maintained a stable targeting ability in the rat joint cavity.Sections of knee joint and OARSI score results showed that the engineered treatment system EKS was effective method for knee OA therapy.From the visceral sections,no obvious tissue damage could be observed after engineered treatment system EKS injected.