| Staphylococcus aureus is a facultative intracellular bacteria that can survive and multiply in host cells,which also exist in different subcellular organelles or cytosols,thus causing persistent or periodic infections.However,more than two-thirds of existing antibiotics are inefficient to kill intracellular pathogens both in vitro and in vivo.All three antibiotics,vancomycin,linezolid and daptomycin,currently used as the standard of care for invasive MRSA infections against extracellular planktonic bacteria,failed to kill a MRSA strain sequestered inside macrophages exposed to clinically achievable concentrations of the antibiotics.Additionally,the antibiotic concentration in the intracellular compartment is often sub-therapeutic,resulting in a drastic reduction in their effectiveness against intracellular pathogens,but also in promotion of antibiotic resistance.Therefore,the treatment of intracellular bacterial infections remains a major pharmaceutical challenge and there is a tremendous interest in improving antibiotics' entry within cells,in order to treat these intracellular infectious diseases.Exosomes have recently emerged as a promising drug delivery system with high biocompatibility and high delivery efficacy.Exosomes have already been used to deliver drugs including chemotherapy drugs,nucleic acid drugs and proteins to target tissues/cells,leading to enhanced anti-tumor or anti-inflammatory activities.Based on these observation,we proposed to use a biocompatible exosomes as drug carriers to prepare exosomes-antibacterial drug conjuctions for the treatment of intracellular MRSA infection.The main research contents are listed as follows:(1)Preparation,purification,and characterization of exosomes from the culture supernatants of RAW264.7 cellsExosomes were isolated from the culture supernatants of RAW264.7 cells by polymers precipitation.The morphologies of exosomes were characterizated by Dynamic Light Scattering(DLS)and Transmission Electron Microscopy(TEM),and the results showed that exosomes possess spherical morphology with sizes about 70 nm and zeta potential was-7.0 mV.As revealed by western blotting,the exosomes were more enriched with CD63 and flotillin 1,two exosomal markers,than the cell lysate.By concentrating the cell supernatant and extracting with the kit,the obtained exosomes are high in content and structurally intact,which lays a good foundation for drug loading in our following experiments.(2)Exosome-encapsulated linezolid against intracellular MRSA infectionsA nanoformulation of the antibiotic linezolid(ExoLZD)was prepared using exosomes harvested from mouse RAW264.7 macrophages.TEM and DLS studies revealed that the loading procedures did not significantly alter the morphology of nanocarriers.And linezolid could be effectively released in two media with pH 7.4 or pH 4.5.The internalization results of ExoLZD indicated that the drug payload did not affect the ability to deliver exosomes to macrophages,and no signs of cytotoxicity was observed in macrophages,which suggested that exosomes had good biocompatibility and might be a preeminently potential drug carrier.Moreover,the exosomal formulation of linezolid was more effective against intracellular MRSA infections in vitro and in vivo,than the free linezolid.In addition,the effect mechanism of ExoLZD were further analyzed.A plausible mechanism of action of this drug is as follows after being released into the acidic environment of the lysosomes,(1)a portion of the LZD kills bacteria in the lysosomes,and(2)some LZD crosses the intracellular endolysosomal membranes into the cytoplasm to therein exert a bactericidal effect.Thus,the strategy is an effective alternative method for intracellular antibiotic delivery of antibiotic.(3)Exosome-encapsulated vancomycin against intracellular MRSA infectionsVancomycin was incorporated into exosomes using sonication.The TEM images revealed that sonication caused a reformation/deformation for exosomes,the average particle size increased from 70 to 130 nm,but it does not affect the ration of uptake.Moreover,the exosomal formulation of vancomycin induced an improved intracellular antibacterial activity on the facultative intracellular pathogen MRSA as compared with free vancomycin.These results demonstrated again that exosomes could delivery antibiotic into cells and possess intracellular bactericidal effect.(4)Exosome-encapsulated Top I inhinitor calothrixin A against intracellular MRSA infectionsCalothrixin A,a Top I inhinitor,has shown strong antibacterial activity,but it displayed cytotoxicity at nanomolar concentrations which limits its development as an antibacterial agent.In this part,calothrixin A was incorporated into exosomes by incubation at 37 °C.The obtained exosomal formulation of calothrixin A led to decreased intracellular MRSA CFU,similar as exosome-encapsulated vancomycin.Furthermore,its cytotoxicity was also significantly reduced than the free calothrixin A.The reduced cytotoxicity could be explained by the fact that exosomes deliver calothrixin A into the lysosome rather than cell nucleus to inhibit Topoisomerase Iactivity.This strategy provides the possibility for calothrixin A exerting intracellular antibacterial activity,and also a new option for other similar broad-spectrum antibiotic to play the role of intracellular antibacterial agents.(5)Preparation of surface functionalized exosomesThe metabolized exosomes could be obtained by co-culturing L-azidohomoalanine(AHA)with exosome-secreting RAW264.7 cells for three days and then isolated the exosomes by using differential ultracentrifugation.The diameters of the AHA-metabolized exosomes are approximately 90 nm measured by DLS and TEM.To confirm the successful incorporation of AHA into exosomes during the cell co-culture process and the efficient click conjugation of these engineered exosomes,AHA-metabolized exosomes were incubated with fluorescent DBCO-RohB and then RohB conjugation efficiency was quantified after removing the excess DBCO-RohB from the reaction.The preliminary click conjugation of AHA-metabolized exosomes with DBCO-RohB confirmed the efficient integration of azides into these exosomes.Furthermore,the intracellular delivery of DBCO-RohB conjugated exosomes indicated that the engineered exosomes could click cargos and transport them into cells.Our strategy combines the metabolic labeling of newly synthesized proteins of exosome-secreting cells with active azides and bioorthogonal click conjugation to modify and functionalize the exosomes.The incorporation of active target ligands and therapeutics into exosomes will enable the use of exosomes as novel nanocarriers for targeted drug delivery.(6)Mannose-exosome encapsulated antibiotics against intracellular MRSA infectionsMannose-DBCO was synthesized and then were incubated with AHA-metabolized exosomes for the efficient click conjugation.The obtained AHA-mannose-clicked exosomes(MExos)were efficiently taken up by RAW264.7 macrophages and exhibited higher uptake than that of the control exosomes.Vancomycin or lysostaphin was incorporated into MExos or the control exosomes using sonication.The vancomycin loaded MExos(MExoVan)exhibited an improved inhibitory capability against intracellular bacteria compared to the exosome encapsulated vancomycin and free vancomycin.Moreover,MExoVan in combination with the lysostaphin loaded MExos(MExoLy)had superior inhibition of intracellular MRSA than that alone.In summary,three antibacterial drugs(linezolid,vancomycin and lysostaphin)with weak intracellular antibacterial effect,and a Top I inhibitor calothrixin A with highcytotoxicity were incorporated into exosomes or mannose modified exosomes that targeted macrophages.Their intracellular antibacterial effect was evaluated on MRSA infected macrophages in vitro and MRSA peritonitis model in vivo.Their in vivo distribution and cytotoxicity were also determined.Additionally,their intracellular effect mechanism inside macrophages were analyzed based on the result of intracellular distribution.These studies provide a theoretical basis and a new research platform for the application of exosomes as antimicrobial drug delivery systems for intracellular infections. |
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