| BackgroundChronic rhinosinusitis (CRS) is one of the common disease in rhinology,the occurrence of CRS is the result of interaction of many factors,bacterial infection and the formation of bacterial biofilm is one of the most important factors.Bacterial biofilm (BBF) is a highly organized structure with a certain spatial configuration formed by bacteria,which is another form of existence that is different from free bacteria to adapt to the natural environment.When the biofilm formed,bacterial biofilms require up to 1000 times greater antibiotic dose for effective treatment compared to their planktonic types,which make the eradication even more difficult.Although some CRS patients receive techinically successful endoscopic sinus surgery (ESS) and standardized comprehensive medical treatment,their clinical efficacy is not significantly improved.Although surgical operation can directly remove the mucous membrane covered with the biofilms and its pathological changes,in the process of operation,we can not determine the location of the existence of biofilm and the involvement of the specific scope of mucosal lesions directly,so if you just rely on surgery to remove biofilm is not enough.In the field of rhinology,it has been proven that Staphylococcus aureus biofilms are associated with the unfavorable evolution after ESS,more severe disease parameters,and intracellular infection,suggesting that S.aureus biofilms may contribute greatly to the recalcitrant nature of CRS.Nitric Oxide (NO),a gas naturally produced in high concentrations in healthy sinuses,is found in significantly lower concentrations in CRS patients.NO has been demonstrated that it has antibacterial and antiviral properties,it plays an important role in innate immunity and mucociliary clearance and is thought to provide a significant contribution to maintaining the balance of normal flora in non-diseased sinuses.Preliminary data showed that NO can effectively eliminate S.aureus biofilms in vitro.And our preliminary experiment has also proved that nitric oxide can effectively kill the S.aureus biofilms.Isosorbide Mononitrate (ISMN) is a kind of NO donor drug,which has been widely used in the clinical practice of Cardiovascular Medicine.At present,the comprehensive treatment to the CRS patients whose nasal mucosa appeared S.aureus biofilms is poor.Hence,the isosorbide mononitrate (ISMN)liposomes is produced which is modified with alpha toxin antibody,and its basic physical and chemical properties are investigated.In this project,experimental methods including Elisa,Realtime polymerase chain reaction (Realtime PCR) and observation with confocal laser scanning microscope (CLSM) will be applied to study the mechanism,efficacy and safety of nanoparticle both in vitro and in vivo.This work will be a prior study in the treatment of recalcitrant CRS.Our study is composed of four parts as following.Part One:The preparation of HLA monoclonal antibodiesObjectiveThe HLA monoclonal antibody was prepared to provide the conditions for the preparation of isosorbide mononitrate immunoliposomeMethodsFirstly,the pET28a-Hla recombinant plasmid was constructed,then HLA protein was expressed as prokaryotic expression.Finally,the purified HLA recombinant protein was obtained.The purified HLA recombinant protein was used as antigen to immunize the BALB/C mice.Freund’s complete adjuvant and Freund’s complete adjuvant were used to emulsify the antigen.The suspension of the spleen cells of the immunized mice was fused with the myeloma cell SP2/0 to screen the hybridoma cell which could stably secrete the antibody.The hybridoma cell was inoculated into the abdomen of mice,after that,the hydroperitoneum was collected and purified with the octanoic acid-ammonium sulfate method to obtain the monoclonal antibodies against HLA.Western blot was used to identify their specificities.Results1.PCR amplification was performed on the genome of staphylococcus aureus strains,we got the target gene fragment and constructed the pET28a-Hla recombinant plasmid.2.The pET28a-Hla recombinant plasmid was successfully transfected into cells and the purified Hla protein was obtained.3.The purified HLA recombinant protein was used as antigen to immunize the BALB/C mice,eventually,the hybridoma cells that secreted Hla protein monoclonal antibodies were obtained.4.In the end,the antibodies was successfully prepared,and the Western blot method was used to verify the specificity of the antibodies we prepared.ConclusionThe HLA monoclonal antibody is successfully produced,it can be linked to liposomes as targeted groups due to its specificity.Part Two:The preparation of the targeted nanoparticles of isosorbide mononitrateObjectiveTo prepare the ISMN loaded nanoparticles conjugated with anti-Staphylococcus alpha-toxin.MethodsA single factor was used to optimize the preparation process of ISMN liposomes to screen out the best phospholipid concentration,the ratio of phospholipid to cholesterol,the ratio of phospholipid to drug.The optimal prescription was selected by orthogonal test,then,the basic physicochemical properties of liposomes were investigated.The method of glutaraldehyde crosslinking was used to connect the Hla monoclonal antibody with liposome,the targeted nanoparticles of isosorbide mononitrate was produced.The targeted nanoparticles of isosorbide mononitrate was investigated for its particle size,encapsulation rate,drug loading efficiency and the linkage rate of antibody.ResultsThe basic physicochemical properties of ISMN liposomes were studied,the particle size of ISMN liposomes was 136.8±7.414nm,the encapsulation rate was 19.03±1.595%,the Zeta potential was -1.145±0.095,the ISMN liposomes was stored at 4℃,on the 15th day,the particle size of ISMN liposomes was 137.9±7.432nm,the encapsulation rate was 18.53±0.838 %,the Zeta potential was -1.09±0.042.On the 30th day,the particle size of ISMN liposomes was 147.7±7.325nm,the encapsulation rate was 17.10±1.435 %,the Zeta potential was-1.05±0.061。 The particle size,encapsulation rate and potential of three different time points were analyzed by statistical methods,there were no statistically significant differences between any two groups at three different time points(P<0.05).When the ISMN liposomes was stored at 4℃ for a month,The particle size,the encapsulation rate and potential basically remained unchanged.The particle size of ISMN immunoliposomes was 169.3±6.780nm,the encapsulation rate was 16.9±1.52%,the drug loading efficiency was 2.36±0.19%,the linkage rate of antibody was 30.68±2.39%.ConclusionThe targeted nanoparticles of isosorbide mononitrate is successfully produced,its particle size is suitable for the experiment,the physicochemical properties of the targeted nanoparticles of isosorbide mononitrate are stable after the modification and antibody coupling.It can be used in the future experiment.Part Three:In vitro the effect of targeted nanoparticles of isosorbide mononitrate on Staphylococcus aureus biofilmsObjectiveTo investigate the in vitro effect of targeted nanoparticles of isosorbide mononitrate on S.aureus biofilms.MethodsSpecific S.aureus biofilms model was established by using reference strains(ATCC25923).In vitro,Staphylococcus aureus biofilms model was established by 96-well culture plate and 8-well culture slides.After challenged by targeted nanoparticles of isosorbide mononitrate in various concentrations (the drug concentration was 45mg/ml,23mg/ml,11mg/ml) and at different biofilm forming stages,biofilm models were investigated by using the crystal violet assay,Alamar Blue assay and CLSM observation.ResultsThe crystal violet assay was used to evaluate the effect of Immunoliposome to the forming biofilm.The results were as follows,after the intervention of ISMN immunoliposomes (the drug concentration was 45mg/ml,23mg/ml,llmg/ml),the formation of biofilm was reduced to 2.16±0.22%,38.42±4.44%,62.38±5.01 %,after the intervention of ISMN liposomes (the drug concentration was 45mg/ml,23mg/ml,11mg/ml),the formation of biofilm was reduced to 18.26±5.21 %,58.31±3.15%,82.50±3.10%, after the intervention of ISMN (the drug concentration was 45mg/ml,23mg/ml,11mg/ml),the formation of biofilm was reduced to 35.7±2.87 % ,76.37±5.65%,82.61±5.68%,When the drug concentration was 45mg/ml,23mg/ml and 11 mg/ml,the inhibitory effect of ISMN immunoliposomes on biofilm formation was stronger than that of ISMN liposomes and ISMN(P<0.05),When the drug concentration was 45mg/ml,23mg/ml,the inhibitory effect of ISMN liposomes on biofilm formation was stronger than ISMN(P<0.05),When the drug concentration was 11mg/ml,the inhibitory effect of ISMN liposomes on biofilm formation was the same as ISMN (P>0.05).Alamar Blue assay was used to evaluate the effect of Immunoliposomes to the formed biofilm.The results were as follows,after the intervention of ISMN immunoliposomes (the drug concentration was 45mg/ml,23mg/ml,11mg/ml),the inhibition rate was 96.67±2.08%,50.33±5.86%,21.33±3.06%,after the intervention of ISMN liposomes (the drug concentration was 45mg/ml,23mg/ml,11mg/ml),the inhibition rate was 77.33±3.06 %,36.671±3.51 %,19.00±5.29%, after the intervention of ISMN (the drug concentration was 45mg/ml,23mg/ml,11mg/ml),the inhibition rate was 62.67±4.04%,21.33±1.53%,16.33±2.08%.When the drug concentration was 45mg/ml,23mg/ml,the inhibitory effect of ISMN immunoliposomes on the formed biofilm was stronger than that of ISMN liposome and ISMN(P<0.05),the inhibitory effect of ISMN liposomes on the formed biofilm was stronger than ISMN(P<0.05),When the drug concentration was 11mg/ml,ISMN immunoliposomes,ISMN liposomes and ISMN had the same effect on the removal of the formd biofilms(P>0.05).The quantitative analysis of green fluorescence intensity obtained from CLSM were consistent with the results of Alamar Blue assay.ConclusionCompared with the ISMN liposomes and ISMN,the ISMN immunoliposomes have the most obvious anti-biofilm effects at the some drug concentration,the concentration of the 45mg/ml immunoliposomes own the most obvious anti-biofilm effects.The result of CLSM indicates that the 45mg/ml immunoliposomes can completely destroy the structure of biofilm.ISMN immunoliposomes can be widely applied in the treatment of infectious diseases caused by Staphylococcus aureus biofilms in the future.Part Four:The effects of targeted nanoparticles of isosorbide mononitrate on Staphylococcus aureus biofilms in chronic rhinosinusitisObjectiveTo investigate the effect of ISMN immunoliposomes on staphylococcus aureus biofilms in chronic rhinosinusitis by establishing a model of chronic rhinosinusitis.MethodsS.aureus biofilm-related sinusitis animal mode was established,CT was used to confirm the mode was successful.In this study,45mg/ml ISMN immunoliposomes,45mg/ml ISMN liposomes and 45mg/ml ISMN were used to douche the maxillary sinus respectively,in the process of modeling,the blood sample was obtained from the rabbits in the different time points,ELISA experiment was used to observe the level of inflammatory factors in vivo during the process of modeling and after the drug action.After the drug intervention,Realtime PCR was applied to detect the mRNA change of inflammatory factor,in order to explore the effect of ISMN immunoliposomes on Staphylococcus aureus biofilms in chronic rhinosinusitis.ResultsS.aureus biofilm-related sinusitis animal mode was successfully established and the method was simple and easy to operate.Elisa showed that after the action of 45mg/ml ISMN immunoliposomes,the levels of IL-4,IL-8,IL-17A and IFN-γ in the blood were lower compared with the 35th day,the difference was statistically significant (P <0.05),Compared with the ISMN liposomes and ISMN,the ISMN immunoliposomes owned the most obvious ability to reduce the levels of IL-4,IL-8,IL-17A and IFN-γ,the differences were statistically significant (P <0.05).Realtime PCR showed that after the action of 45mg/ml ISMN immunoliposomes,the IL-4,IL-8,IL-17A and IFN-γ in mRNA levels were lowest compared with the model group,ISMN liposomes and ISMN,the differences were statistically significant (P <0.05).ConclusionISMN immunoliposomes can remove bacterial biofilms and reduce inflammation in vivo,ISMN immunoliposomes will provide a new way for the treatment of RCRS in the clinical. |
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