Preparation Of Honokiol-sophorolipid Micelles And Its Efficacy On Staphylococcus Aureus Biofilm Infections

Staphylococcus aureus（S.aureus）is a common conditional pathogen causing pulmonary infections.Biofilms formed by S.aureus at the site of infection are often associated with persistent pulmonary diseases.Bacterial biofilms comprise an extracellular polymeric matrix（extracellular polymeric substance,EPS）and an internally colonized bacterial population.EPS is to protect the organism from the body’s immune system and various antimicrobial agents.Sophorolipids（SL）produced by the pseudo filamentous yeast can scavenge EPS and self-assemble to form micelles,potentially making them good nanocarriers.Honokiol（HK）has significant antibacterial effects and is hydrophobic enough to be encapsulated in the hydrophobic core of sophorolipid micelles（SL Ms）.In this paper,hydrophobic HK was encapsulated into the inner core of SL Ms by the solubilizing ability of SL Ms to form HK-SL Ms.SL was used to enhance the effect of HK on bacterial viability within biofilms by dispersing EPS.The main studies are as follows:（1）Preparation and characterization of HK-SL Ms.The ring-pulling method detected the SL solution’s critical micelle concentration（CMC）.The results showed that the CMC value of the SL solution was 3.586μg/m L.HK-SL Ms were prepared by film hydration method,and the encapsulation rate and drug loading capacity were used as evaluation indexes to determine the best preparation process.The optimal preparation process was obtained:30 mg HK,180 mg SL,10 m L anhydrous ethanol,10 min hydration time,20 m L saline,spin temperature 55°C.The encapsulation rate and drug loading capacities were 97.41%±0.70%and 14.25%±0.71%,respectively.TEM and particle size meters characterized the HK-SL Ms.The results showed that the HK-SL Ms prepared under the optimal process conditions were spherical in shape and uniform,with a particle size of 99.44±1.45 nm and a zeta potential of-65.7±1.68 m V.（2）Study on the inhibition of S.aureus by HK-SL Ms in vitroThe minimum inhibitory concentration（MIC）of HK-SL Ms on S.aureus and the effect on the growth of S.aureus was determined by the microdilution method;the impact of HK-SL Ms on S.aureus was detected by the plate count method.Compared with 16μg/m L SL and 16μg/m L HK,16μg/m L HK-SL Ms reduced the total number of colonies by 6.09log₁₀^CFU/mlunits and 3.83 log₁₀^CFU/mlunits（P<0.05）.Byβ-galactosidase,protein,and DNA extravasation assays,scanning electron microscopy（SEM）,and transmission electron microscopy（TEM）results,the to determine the effect of HK-SL Ms on cell membrane damage.The results showed that HK-SL Ms affected the integrity of the cell membrane of S.aureus.HK-SL Ms resulted in significantly higherβ-galactosidase,protein,and DNA exudation than SL Ms and HK（P<0.05）.The 16μg/m L HK-SL Ms caused severe deformation and roughness of the cell membrane surface;some did not have any intact cell membrane structure,and many bacterial contents leaked out.（3）Inhibition of S.aureus biofilm formation by HK-SL Ms in vitroA crystalline violet staining test was performed to determine the preliminary inhibitory effect of HK-SL on S.aureus biofilm formation.The results showed that HK-SL Ms inhibited bacterial biofilm well.4μg/m L HK-SL Ms inhibited up to 91.10%±0.63%,significantly higher than HK and SL Ms（P<0.05）.The preliminary investigation of the effect of HK-SL Ms on S.aureus biofilm formation was investigated by detecting the extracellular proteins,extracellular polysaccharides,and PIA formation in bacterial biofilms.The results showed that HK-SL Ms had some effects on the inhibition of extracellular protein,extracellular polysaccharide,PAI formation.The ability of HK-SL Ms on the initial adhesion of S.aureus was investigated by plate colony counting and SEM.The results showed that HK-SL Ms had some effect on the initial adhesion of bacteria.4μg/m L of HK-SL Ms inhibited the initial adhesion of bacteria better than that of HK and SL Ms visible to the naked eye.（4）In vitro removal of S.aureus biofilm by HK-SL MsThe crystalline violet staining test initially determined the effect of Ms.HK-SL on the removal of S.aureus biofilm.The results showed that HK-SL Ms could remove mature S.aureus biofilms.16μg/m L and 32μg/m L HK-SL Ms showed significantly higher clearance of S.aureus biofilms（P<0.05）than HK and SL Ms with 71.73%±4.27%and 87.36%±1.39%,respectively.The scavenging effect of HK-SL Ms on S.aureus biofilms was initially investigated by detecting the formation of extracellular proteins and extracellular polysaccharides in bacterial biofilms.The results showed that HK-SL Ms could scavenge extracellular proteins and extracellular polysaccharides.Plate counting and fluorescence staining were used to determine the effect of HK-SL Ms on bacterial viability within S.aureus biofilms.The results showed that HK-SL Ms led to a significant decrease in the number of bacteria within the biofilm of S.aureus.16μg/m L of HK-SL Ms resulted in a loosening of the biofilm structure and an increase in the intensity of red fluorescence,indicating that the bacterial mortality rate was significantly higher than that of the control,SL Ms and HK.（5）HK-SL Ms for the treatment of lung infection in miceThe bacterial fluid causes bacterial infection in the lungs through nasal drops HK-SL Ms at 8μg/m L and 16μg/m L was administered by intraperitoneal injection of 1 ml daily for3 d.The effectiveness of HK-SL Ms in treating lung infections was determined at day 5 by measuring bacterial lung load,expression of inflammatory factors,and observing pathological tissue sections of the lungs.The results showed that 16μg/m L HK-SL Ms effectively reduced the bacterial lung load,decreased the expression of IL-6,TNF-α,and IL-1β,enhanced the expression of SP-A in lung tissues,and reduced the pathological changes in the lung.HK-SL Ms improved the lung infection in mice and inhibited the further development of lung infection.In summary,negatively charged,appropriately sized,and rounded HK-SL Ms were successfully prepared in this experiment.They have good antibacterial,antibacterial biofilm,and therapeutic effects on lung infections.It provides a theoretical basis for clinical control and eradication of Staphylococcus aureus biofilm.