A Novel Cargo Delivery System-AnCar-Exo^LaIMTS Ameliorates Arthritis Via Specifically Targeting Pro-inflammatory Macrophages

Purpose:Macrophages are heterogenic phagocytic cells which play distinct roles in multiple physiological and pathological processes.Targeting different types of macrophages has shown potent therapeutic effects in many diseases.Although many approaches have been developed to target anti-inflammatory macrophages,there are few researches on targeting pro-inflammatory macrophages partially because of their non-specificity phagocytosis.In this study,we constructed a novel recombinant protein that could be anchored on exosome membrane with the purpose of targeting pro-inflammatory macrophages via antigen recognition,which we named it as AnCar-Exo^LaIMTS.Our data indicate that the phagocytosis efficiencies of pro-inflammatory macrophages for different AnCar-Exo^LaIMTS show obvious differences.In the existing candidate range,the AnCar-Exo^LaIMTS3 has the best targeting ability to pro-inflammatory macrophages in vitro and in vivo.Moreover,the optimal AnCar-Exo^LaIMTS3 can efficiently deliver therapeutic cargo to pro-inflammatory macrophages and inhibit the synovial inflammatory response via downregulation of HIF-1αlevel,thus ameliorating the severity of arthritis in vivo.Collectively,our work established a novel gene/drug delivery system that can specifically targeting pro-inflammatory macrophages,which may be beneficial for the treatments of arthritis and other inflammatory diseases.Methods:1.Plasmid ConstructionThree repetitive pro-inflammatory macrophage-targeting sequences（1-10）or random sequences were ingeniously designed in the extracellular region between the signal peptide and mature peptide of human LAMP2B protein.These sequences were connected through flexible polypeptide linkers（GGGGS）,while a green fluorescent protein tag was added to the carboxyl terminus of the recombinant LAMP2B protein.The sequence encoding the recombinant protein was inserted into the PLVX-Puro plasmid,between Xho I and Bam HI restriction enzyme sites.Subsequently,we transiently transfected HEK293T cells to generate lentiviruses.The lentiviral packaging materials included the p SAX2 packaging vector and the p MD2G envelope vector.After 48 hours of transfection in HEK-293T cells,the lentiviral supernatant was collected.2.Establishment of Stable Cell LineHEK293T cells were cultured in 6-well plates at a density of 30%-50%and incubated for the first day.In the afternoon of the second day,lentiviruses containing the gene for puromycin resistance were added to the HEK293T cells at a multiplicity of infection（MOI）of 5.After reaching a cell density of 80%or higher,the complete culture medium was replaced with fresh complete medium containing 2μg/ml of puromycin.When no cell death was observed in the 6-well plate by the fifth day,the concentration of puromycin was reduced to 0.5μg/ml.The mixed clone cells were then dissociated using 0.05%trypsin and counted.Sixty cells cultured in 10 ml of complete medium were seeded into a 96-well plate.After 10 days,the stable monoclonal cell line was passaged and expanded until a sufficient number of cells were obtained to generate exosomes.3.Preparation and Characterization of ExosomesFirst,the supernatant was centrifuged at 300g for 10 minutes to remove dead cells.Next,the supernatant was transferred to a new centrifuge tube and centrifuged at 2000g for10 minutes to remove cellular debris.Then,the supernatant was transferred to another centrifuge tube and centrifuged at 10,000g for 10 minutes to remove microvesicles.Fourth,the supernatant transferred to a new centrifuge tube was filtered through a 0.22-μm filter（Steritop;Millipore）to remove apoptotic bodies.Fifth,the supernatant was centrifuged at100,000g for 90 minutes to obtain exosomes.Finally,the exosomes were either treated with RIPA buffer containing protease inhibitors or resuspended in 200μl of DPBS and stored at-80°C for further use.The exosomes were then characterized using four methods,including Nanosight,transmission electron microscopy,Western blotting,and flow cytometry（Image Stream X Mark II）,to identify their characteristics.4.Isolation and Culture of Primary CellsPrimary chondrocytes were isolated from the articular cartilage of young mice.The cartilage cells were isolated under aseptic conditions and seeded in culture dishes with DMEM/F12 supplemented with 10%fetal bovine serum.Primary fibroblasts were isolated from the synovium of mice.After digestion with type IV collagenase in a sterile environment,the fibroblasts were seeded in culture dishes.Macrophages derived from bone marrow were obtained from the mouse bone marrow in a sterile environment.The cells were suspended in complete culture medium supplemented with 10 ng/ml M-CSF and seeded in a 6-well plate.On the third day,the culture medium was replaced with fresh complete culture medium supplemented with 10 ng/ml M-CSF.5.Flow CytometryBMDMs were seeded in 35 mm confocal culture dishes at a density of 2×10⁵ cells.After polarization into Mφ,pro-inflammatory,and anti-inflammatory macrophages,complete culture medium without exosomes was added,followed by co-incubation with 1×10⁸ AnCar-Exo La^IMTS3 at 37°C for 12 hours.Subsequently,Mφ,pro-inflammatory,and anti-inflammatory macrophages were collected and resuspended in FACS buffer（PBS containing 1%FBS and 1 m M EDTA）.Blank controls and isotype controls were prepared for subsequent analysis.F4/80,CD86,and CD206 antibodies（1:100,Biolegend）were then incubated with the cells at 4°C for 30 minutes.The cells were washed with FACS buffer and centrifuged at 500g for 5 minutes.Finally,50,000 events were collected and analyzed using a flow cytometer（Beckman,Cyto FLEX SRT）.6.In Vivo Tracing of ExosomesCollagenase was injected into the knee joint to induce arthritis.Exosomes were labeled with DID and administered systemically or locally through tail vein or intra-articular injection.Mouse imaging was performed using the Fusion FX Edge system（Vilber Lourmat）.Signal intensity was recorded at 0,1,3,6,12,24,and 48 hours post-exosome injection.Fusion Capt Advance software was utilized for image analysis.For td Tomato-Lys MCre mice,after anesthetizing the mice,they were placed in a 10 cm culture dish containing 3%low melting point agarose to immobilize them.Subsequently,1×10⁹exosomes were injected into the tail vein.The mice were then monitored using a two-photon microscope from 0 to 120 minutes.7.ImmunofluorescenceAppropriate OCT sections（10μm thickness）were selected for immunofluorescence staining.All knee joint samples were sequentially fixed with 4%paraformaldehyde,treated with 0.5M EDTA p H 7.4,dehydrated with 30%sucrose,and embedded.After blocking with Quick Block^TMBlocking Buffer,the sections were incubated overnight at 4°C with primary antibodies such as F4/80,iNOS,CD206.Subsequently,the sections were incubated with fluorescently labeled secondary antibodies at 37°C for 1 hour.Finally,cell nuclei were stained with DAPI.The fluorescence signals in the sections were detected using a live-cell workstation.8.Quantitative PCRAt the end of cell treatment,total RNA was extracted using a total RNA extraction kit according to the instructions.Complementary DNA sequences were synthesized using the Prime Script TM RT reagent kit with g DNA ERASER.Subsequently,RT-PCR was performed using the SYBR Premix Ex Taq TM II reagent kit on an Mx3000PCR instrument.9.Gait AnalysisPrior to conducting the formal gait analysis experiment,all mice were familiarized with the testing environment to ensure their uninterrupted and continuous passage through the Cat Walk gait analysis system,which is based on video recordings.The system consists of an enclosed walkway,fluorescent lighting,high-speed color cameras,and analysis software.Gait data were recorded by the high-speed color cameras.10.X-ray and MRIAll mice were subjected to X-ray imaging using the Faxitron MX-20 system.For MRI,a small animal MRI scanner（Bruker Bio Spec USR 70/20,Germany）was used.After immobilizing the right knee joint with a four-channel coil,the mice underwent T1-weighted imaging（T1WI）and T2-weighted imaging（T2WI）.The scanning parameters were as follows:（1）T1WI（RARE sequence）:RARE factor=4,TR=693.0 ms,TE=6.0 ms,Flip angle=90°,FOV=25 mm×25 mm,Matrix=256×256,slice thickness=0.4 mm,number of slices=8,scan time=10 minutes.（2）T2WI（Turbo RARE sequence）:RARE factor=6,TR=3,000 ms,TE=45 ms,Flip angle=90°,FOV=25 mm×25 mm,Matrix=256×256,slice thickness=0.4 mm,number of slices=8,scan time=10 minutes.11.Histological StainingAll knee joint samples were fixed in 4%paraformaldehyde,decalcified in 0.5M EDTA p H 7.4,dehydrated,cleared in xylene,and embedded in paraffin.Paraffin sections（5μm thick）were selected for staining with Hematoxylin and Eosin（H&E）,Safranin O/Fast Green,and Masson’s Trichrome.H&E staining was used to evaluate synovial inflammation based on synovial hyperplasia,sublining cell architecture,and inflammatory infiltrates.The synovial inflammation score was as follows:0-1,no synovitis;2-4,mild synovitis;5-9,severe synovitis.12.Induction of Arthritis and Exosome InjectionTwelve-week-old male C57BL/6J mice were divided into four groups（5 males/group）.After anesthesia with 0.8%pentobarbital,mice in the sham surgery group had the skin over their right knee joint incised without collagenase injection.In the arthritis group,the knee joint was fully exposed,and 10μl of 1 unit of type VII collagenase in Ca Cl2 was injected on day 1 and day 3.The same procedure was applied to the transgenic mice group.Exosomes were injected into the joint cavity at a dose of 1×10⁹ particles per mouse.Results:1.Design and construction of AnCar-LaIMTS plasmidsThe design and construction of the AnCar-LaIMTS vector involve designing and assembling the DNA components necessary for the vector,including the AnCar gene and the LaIMTS region.The DNA fragments encoding these components are cloned into a vector backbone,and the resulting construct is then introduced into host cells through transformation.The presence of the AnCar-LaIMTS vector is confirmed using various molecular biology techniques.2.Characteristics of AnCar-LaIMTS modified exosomesThe characteristics of the AnCar-LaIMTS-modified exosomes were assessed using particle size analysis,electron microscopy,immunoblotting experiments,and nanoparticle flow cytometry.Firstly,in terms of particle size analysis,the exosomes showed a quantity ranging from 1.23×10^11 to 1.92×10^11 particles/ml,with diameters between 30 and 200nm.The majority of exosomes had particle sizes ranging from 99.8 to 158 nm,and the average size ranged from 123.6 to 182.1 nm.Secondly,electron microscopy analysis demonstrated the presence of typical disc-shaped bilayer membrane structures of exosomes,which were isolated from the supernatant.Thirdly,immunoblotting experiments revealed the detection of the fusion protein AnCar and exosomal surface markers,including CD63,CD81,and TSG101,in the exosomes.Lastly,nanoparticle flow cytometry analysis using Image Stream X showed successful loading of GFP into AnCar-Exo^LaRANDOM,AnCar-Exo^LaIMTS1,and AnCar-Exo La^IMTS10,compared to unmodified exosomes(An-Car^LaCTRL).3.Screening and identification of the optimal AnCar-Exo^LaIMTSspecifically targeting pro-inflammatory macrophages in vitroThe ability of the exosomes to target pro-inflammatory macrophages was evaluated using the RAW264.7 macrophage cell line and bone marrow-derived macrophages（BMDMs）.Firstly,fluorescence detection was used to assess the co-expression of F4/80,GFP,and iNOS in RAW264.7 macrophages.It was found that AnCar-Exo LaIMTS1 to AnCar-Exo LaIMTS10 and AnCar-Exo^LaRANDOM exhibited varying degrees of co-localization and expression of F4/80,GFP,and iNOS,indicating different levels of targeting ability of the modified exosomes.Similarly,fluorescence detection was performed in BMDMs to evaluate the co-expression of F4/80,GFP,and iNOS.It was observed that AnCar-Exo LaIMTS1 to AnCar-Exo LaIMTS10 and AnCar-Exo^LaRANDOM showed varying degrees of co-localization and expression of F4/80,GFP,and iNOS in BMDMs,suggesting different levels of targeting ability of the modified exosomes.Importantly,AnCar-Exo LaIMTS3 exhibited the strongest affinity for pro-inflammatory macrophages derived from primary macrophages,as indicated by significantly higher fluorescence intensity compared to other targeted peptide exosomes.4.Evaluating the distribution and potential toxicity of AnCar-Exo^LaIMTS3 in vivoMice injected with AnCar-Exo^LaCTRL and AnCar-Exo^LaIMTS3 exhibited detectable DID fluorescence,while mice injected with PBS did not show any DID fluorescence.The fluorescence intensity of DID in both AnCar-Exo^LaCTRL and AnCar-Exo^LaIMTS3 groups gradually increased after administration,reaching a peak at 3 hours,and then declined and nearly disappeared after 48 hours,indicating similar metabolic rates of AnCar-Exo^LaCTRLand AnCar-Exo^LaIMTS3 in vivo.After studying the distribution of exosomes in different organs of mice 3 hours after systemic administration,it was found that the fluorescence of AnCar-Exo^LaCTRL and AnCar-Exo^LaIMTS3 was mainly detected in the liver,spleen,and kidneys.Additionally,Hematoxylin and Eosin（H&E）staining showed that AnCar-Exo^LaIMTS3 did not cause pathological damage such as cell death and fibrosis in the heart,liver,spleen,lungs,and kidneys,indicating no apparent toxicity of AnCar-Exo^LaIMTS3to these organs.5.AnCar-Exo LaIMTS3 could specifically target synovial macrophages in mice with inflammatory arthritisThe distribution of AnCar-Exo^LaIMTS3 in the joint after intra-articular injection was observed at different time points（0h,1h,3h,6h,12h,24h,48h,72h）.AnCar-Exo^LaIMTS3,rather than AnCar-Exo^LaCTRL,was significantly enriched in the inflamed joints.The intensity of AnCar-Exo^LaIMTS3 peaked at 1h and almost disappeared at 72h,while the intensity of AnCar-Exo^LaCTRL almost disappeared at 48h.Furthermore,the targeting ability of AnCar-Exo^LaIMTS3 to synovial macrophages in arthritis mice was detected using in vivo imaging.The green fluorescence of AnCar-Exo^LaIMTS3gradually intensified in synovial tissue,primarily localized within the red-labeled synovial macrophages.In contrast,the green fluorescence of AnCar-Exo^LaCTRL in synovial macrophages was significantly weaker than that of AnCar-Exo^LaIMTS3.6.AnCar-Exo LaIMTS3 was mainly enriched in pro-inflammatory macrophages in synovial tissues of inflammatory jointCollagenase-induced inflammatory arthritis led to an increase in synovial membrane thickness and the number of F4/80+cells,iNOS+cells,and F4/80+iNOS+cells（pro-inflammatory macrophages）.Subsequently,mice were intra-articularly injected with AnCar-Exo^LaIMTS3and AnCar-Exo^LaCTRL.The AnCar-Exo^LaIMTS3 group showed enhanced DID fluorescence in the synovium,while the AnCar-Exo^LaCTRL group exhibited no significant changes and coexisted with a large number of F4/80+iNOS+positive cells.Furthermore,systemic administration of AnCar-Exo^LaIMTS3and AnCar-Exo^LaCTRL was performed to observe their distribution in the synovium.It was found that the AnCar-Exo^LaIMTS3group exhibited increased DID fluorescence in the synovium,while the AnCar-Exo^LaCTRLgroup showed no significant changes and coexisted with a large number of F4/80+iNOS+positive cells.7.Targeting HIF-1αin synovial macrophages by AnCar-Exo LaIMTS3ameliorated the severity of inflammatory arthritisCompared to the PBS-treated group,the AnCar-Exo^LaIMTS3-siRNA-HIF-1αgroup showed increased contact area,maximum contact area,aspect ratio,oscillation speed,and maximum fluorescence intensity,while the oscillation time decreased.Additionally,comparedtotheAnCar-Exo^LaCTRL-siRNA-HIF-1αgroup,the AnCar-Exo^LaIMTS3-siRNA-HIF-1αgroup exhibited greater changes in gait-related parameters after treatment.Furthermore,MRI imaging revealed increased synovial fluid,synovial thickening,and increased infrapatellar fat pad area in the OA treatment group compared to the Sham group.HE staining demonstrated that,compared to the AnCar-Exo^LaCTRL-siRNA-HIF-1αgroup,AnCar-Exo^LaIMTS3-siRNA-HIF-1αmore significantly reversed arthritis and reduced the synovitis score in mice.Moreover,we also observed the expression of HIF-1αin OA synovial macrophages.As shown in Figure 3-11,injection of AnCar-Exo^LaIMTS3-siRNA-HIF-1αsignificantly reduced the thickness of inflammatory synovial membrane and the number of F4/80+cells compared to the AnCar-Exo^LaCTRL-siRNA-HIF-1αgroup.8.A survey on joint injuries in populations undergoing exercise training under hypoxic conditionsWhile high-altitude conditions influenced the exercise capacity of the study subjects,it did not alter the relative differences in exercise capacity observed among individuals in plain regions.Individuals with strong endurance fitness in plain areas maintained their high exercise capacity even after moving to high-altitude regions.Additionally,individuals with strong endurance fitness experienced an increase in hemoglobin production upon arrival at high altitudes,facilitating a faster adaptation to the high-altitude environment.The high-altitude environment can cause myocardial damage and weaken immune function in individuals,but these effects are minimal in individuals with strong endurance fitness and more pronounced in those with weaker endurance fitness.Conclusions:we construct new engineered exosomes that can efficiently target pro-inflammatory macrophages to exert the effect of targeted treatment of arthritis in this study.Wherein,the optimal AnCar-Exo^LaIMTS3 can efficiently deliver therapeutic contents to pro-inflammatory macrophages and ameliorate the severity of arthritis without obvious toxicity,which may provide a novel strategy for the treatments of arthritis and other inflammatory diseases in the future.