| Staphylococcus aureus(S.aureus)is a familiar zoonotic pathogen,which can cause many diseases such as skin infections,mastitis and bacteremia in livestock.It can not only cause huge economic losses in animal husbandry,but also spread to human beings through the food chain,which seriously threaten human health.At present,bacterial drug resistance is the major challenge in the prevention and treatment of clinical S.aureus.Methicillin-resistant S.aureus(MRSA),most of which are multidrug-resistant stains,have become the main pathogen that causes livestock infections.More seriously,as a facultative intracellular bacteria,MRSA can survive in phagocytes to escape the killing of antibacterial agents and immune system,and leading to persistent infections.However,conventional antibacterial drugs are diffcuclt to reach effective therapeutic concentration in cells due to poor cell membrane permeability,weak intracellular accumulation ability and the reduction or loss of drug activity,it is difficult for conventional antibacterial drugs to reach effective therapeutic concentration in cells.The long-term treatment with sub-inhibitory concentration would accelerate the development of drug resistance,which challenges intracellular MRSA treatment.In addition,the livestock breeding in our country has entered the era of "reduction and restriction use of antibiotics",therefore,it is a great significance to develop new antibacterial drugs and strategies that are safe,efficient and not prone to drug resistance in the clinical treatment of intracellular infections.As a valuable natural medicinal resource,traditional Chinese medicine are diffcuclt for bacteria to develop resistance that have been widely used for the prevention and treatment of bacterial infections in livestock.The active ingredients of traditional Chinese medicine are also potent source in the development of new drugs for antibiotic alternatives.Glabridin(GLA)is the main active ingredient in Glycyrrhiza glabra,previous studies have shown that GLA has excellent anti-MRSA activity,but it has certain toxicity.And,poor solubility and low bioavailability of GLA limit its clinical application and development.Nano-preoparation based nano drug delivery system can improve drug solubility and bioavailability,enhance drug accumulation in infected sites,and can further increase the active uptake of host cells through targeted group modification,provideing a new way to solve the problem of intracellular bacterial infection treatment in livestock.Therefore,in this study,the biodegradable natural essential oil,cinnamaldehyde was conjugated to dextran,which can target MRSA and macrophage lectin receptors,by pH-responsive acetal bond.Then,the prepared cinnamaldehyde-dextran conjugate(CA-Dex)was further used as a nanocarrier materials to prepare GLA loaded nanoparticles(GLA@CA-Dex)for the treatment of intracellular bacteria infection.The antibacterial effect of this system was evaluated through an in vitro and in vivo antibacterial tests.The specific results are as follows:(1)CA-Dex conjugate was synthesized by aldol condensation reaction,and the grafting rate of cinnamaldehyde in this conjugate was 7%.The critical micelle concentration of CA-Dex nanoparticles was 42 μg/m L,and the morphology of this nanoparticles were spherical with an average particle size of 80.65±3.25 nm.The functionality of CA-Dex nano drug delivery system was verified by laser confocal microscope,flow cytometry and DLS.It was found that CA-Dex nano-drug delivery system can actively target bacteria and macrophages,and can dissociate in the acidic environment.(2)GLA@CA-Dex nanoparticles were prepared by dialysis.With the input mass ratio of drug and carrier as a variable and drug loading as an index,the optimal preparation process was determined as the input mass ratio of drug and carrier was 1:12.The results of transmission electron microscope showed that the GLA@CA-Dex nanoparticles were spherical and evenly distributed,with an average particle size of 123.5±4.4 nm.In addition,the nanoparticles released GLA in weakly acidicenvironment.In addition,the particle size distribution remained stable within 30 d at 4℃ and room temperature,demonstrating the stability of storage.(3)Sheep blood cells were selected to investigate the biocompatibility of nano-drug delivery system and CCK-8 method was used to investigate the cytotoxicity of GLA@CA-Dex nanoparticles.The results showed that CA-Dex nanocarriers had good biocompatibility with no obvious cytotoxicity.In addition,GLA and GLA@CA-Dex nanoparticles had no obvious toxicity to RAW264.7 cells at treatment concentration,and the cell survival rates were both higher than90%,Moreover,the toxicity of GLA was reduced after encapsulatin.(4)GLA@CA-Dex nanoparticles can actively target MRSA and increase the drug accumulation in bacteria.And the bactericidal performance of GLA@CA-Dex nanoparticles on extracellular MRSA is better than GLA monomer and vancomycin.In addition,GLA@CA-Dex nanoparticles can target macrophages directly and uptake by macrophages through clathrin-mediated endocytosis.Compared with GLA monomer,GLA@CA-Dex nanoparticles can significantly enhance the bactericidal effect of GLA on intracellular MRSA by increasing the accumulation of drugs in macrophages/intracellular bacteria.Most importantly,GLA@CA-Dex nanoparticles do not readily develop resistance of MRSA and are beneficial for long-term clinical application.(5)The MRSA-induced mouse peritonitis model was established and treated with GLA@CA-Dex nanoparticles.The results showed that GLA@CA-Dex nanoparticles had a good bactericidal effect on both intracellular MRSA and extracellular MRSA,and the survival rate of MRSA was significantly reduced after treatment.In addition,GLA@CA-Dex nanoparticles can also alleviate the inflammatory reaction and reduce tissue damage by down-regulating the expression of IL-6 and TNF-α.To sum up,the intelligent GLA@CA-Dex nanoparticles prepared in this study can increase the active uptake of MRSA and MRSA-infected macrophages through lectin receptors and release drugs in response to the pH of the target site.This nanoparticles can improve the solubility of GLA and reduce its toxicity,decrease the occurrence of bacterial drug resistance,and further treat intracellular MRSA infections safely and efficiently.This study not only brings new ideas to the application of the active components of insoluble traditional Chinese medicine,but also provides a feasible way for the treatment of intracellular MRSA infections in the era of "reduction and restriction use of antibiotics". |
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