Preparation Of Nano-iron Sulfide Hydrogel And Its Application In The Treatment Of Suppurative Otitis Media

BackgroundSuppurative otitis media(Suppurative otitis media,SOM)is a common middle ear disease in otorhinolaryngology,and bacterial infection is the main pathogenic factor.According to the course of disease,SOM can be further divided into acute suppurative otitis media(Acute suppurative otitis media,ASOM)and chronic suppurative otitis media(Chronic suppurative otitis media,CSOM).If it can not be treated in time and effectively,it can lead to symptoms such as tinnitus,hearing loss and even intracranial infection,seriously threatening the life and health of patients.At present,there are many problems in the clinical treatment of suppurative otitis media,such as the low targeting rate of systemic drug administration and the abuse of antibiotics will lead to the increase of bacterial drug resistance.There are some problems in conventional ear drops,such as easy loss of drugs,frequent use of drugs,and other diseases related to the inner ear through the round window membrane.It is difficult for drugs to kill the bacteria wrapped in the biofilm by extracellular matrix.Once the bacterial biofilm is formed,it becomes extremely difficult to completely remove and kill the related bacteria.Therefore,there is an urgent need to find new ideas and methods to replace traditional antibiotic therapy.In the field of inorganic nanomaterials,nanozymes have attracted extensive attention because of their enzyme-like catalytic activities.Different nanozymes have different antibacterial mechanisms,but the main mechanism is that they regulate the level of reactive oxygen species(ROS)produced by cells.Too much ROS can destroy cell membrane,protein,nucleic acid and kill pathogenic microorganisms including bacteria,fungi and viruses.Nanozyme is essentially a nano-material,which greatly reduces the possibility of drug resistance of bacteria,and it can effectively prevent the attachment of bacteria and kill bacteria through physical and mechanical interaction with bacteria.In this thesis,Cys-nFeS was synthesized with L-cysteine as sulfur source,and PLGA-PEG-PLGA(PPP)temperature-sensitive hydrogel was used as delivery carrier to assemble Cys-nFeS/PPP.Then,the killing effects of Cys-nFeS and Cys-nFeS/PPP on bacteria and bacterial biofilms in vitro were evaluated,and a mouse model of suppurative otitis media caused by Staphylococcus aureus infection was further constructed to explore the therapeutic effect of Cys-nFeS/PPP in vivo.It provides reference for the clinical application of nanomedical biomaterials in the future.Methods(1)Cys-nFeS was synthesized by solvothermal method,and PPP was synthesized by ring-opening polymerization.Both of them were characterized and analyzed,and Cys-nFeS/PPP was assembled and characterized.(2)The antibacterial effects of Cys-nFeS and Cys-nFeS/PPP were tested respectively,and the morphological changes of bacteria and bacterial biofilm were observed by scanning electron microscope and fluorescence microscope,and the level of ROS in bacteria was further detected to explore the mechanism of bacterial death.(3)Staphylococcus aureus was inoculated into the middle ear cavity of mice through tympanic membrane puncture to establish a mouse model of suppurative otitis media.The pathological changes in the middle ear cavity of mice were determined by the determination of inflammatory factors in middle ear lavage fluid,auditory brainstem response(ABR)detection and HE staining of middle ear tissue sections,and the formation of bacterial biofilm on middle ear mucosa was observed by scanning electron microscope and confocal microscope.(4)Cys-nFeS/PPP was injected into the tympanic cavity along the external auditory canal for treatment,and the ofloxacin control group was set up to observe the pathological changes of the middle ear cavity and the destruction of bacterial biofilm before and after treatment.The biosafety of using Cys-nFeS/PPP was evaluated by HE staining of middle ear mucosa and fluorescence staining of hair cells.Results(1)Under electron microscope,Cys-nFeS presented polygonal sheet and spheroid structure,mainly composed of sulfur and iron elements,and had remarkable peroxidase activity.However,Cys-nFeS were easily affected by oxidation in non-vacuum storage environment,resulting in changes in morphology,function and other properties.Therefore,we used PPP as the carrier to deliver Cys-nFeS and prepared Cys-nFeS/PPP.The results showed that both Cys-nFeS and Cys-nFeS/PPP showed excellent antibacterial ability.Moreover,Cys-nFeS/PPP could release Cys-nFeS slowly and continuously inhibit the growth of bacteria and bacterial biofilms because of the drug delivery characteristics of PPP.(2)The mice inoculated with S.aureus in the middle ear showed some symptoms,such as weight loss,arched back,mucous membrane redness and middle ear effusion.HE staining results showed a large number of necrosis and loss of epithelial cells in the middle ear mucosa,mucosal hyperplasia and thickening,inflammatory cell infiltration and bone destruction,and we found that the hearing threshold of mice decreased at different frequencies,which may be related to the inflammatory changes in the middle ear.The formation of bacterial biofilms on the mucous membrane of the middle ear was observed by scanning electron microscopy and confocal microscopy.The results indicated that the mouse model of suppurative otitis media was successfully constructed.(3)Cys-nFeS/PPP is liquid when stored at low temperature,which is convenient for injection and easy to go deep into the lumen.When Cys-nFeS/PPP was injected in the tympanic cavity in the form of liquid,it can rapidly undergo phase transformation in the middle ear cavity of mice,and gradually solidify and precipitate.Then,it can form semi-solid gel and become a drug reservoir,in which Cys-nFeS is slowly released with the dissolution and degradation of the gel.Finally,it plays a role as an antibacterial agent.After treatment,the inflammation of the middle ear was improved and hearing was restored.However,Cys-nFeS/PPP showed stronger killing effect than ofloxacin in destroying bacterial biofilms.The safety evaluation results showed that Cys-nFeS/PPP only acted locally and did not affect other normal tissue functions,and had good biological safety.ConclusionCys-nFeS/PPP has the ability to release Cys-nFeS slowly,has a good lasting antibacterial effect,and can destroy the bacterial biofilm for the treatment of suppurative otitis media without irritating and damaging normal tissues.These results indicate that Cys-nFeS/PPP is a promising new antibacterial agent.