Development Of Azithromycin-rhamnolipid (AZI-RHL) Micelles And Its Efficacy On Staphylococcus Aureus Biofilm

Biofilms are surface-attached bacterial communities that are embedded within extracellular polymeric substances(EPS).Biofilms have complex three-dimensional structures,and EPS can protect bacteria from antibiotics to induce high drug resistance,thus new therapeutic strategies are needed to address biofilm drug resistance.As nanocarriers loaded with antibiotics,micelles can protect them from inactivation,and also have the advantages of reducing drug toxicity,delaying drug release,and easy surface modification.The biosurfactant rhamnolipid(RHL)can self-assemble into micelles to be used as drug carriers.Meanwhile,RHL can play an anti-biofilm effect by affecting the solubility of hydrophobic hydrocarbons and regulating the quorum sensing system.Therefore,RHL micelles can be used as nanocarriers to load antibiotics and prepared as nanoformulations with anti-biofilm potential.Azithromycin(AZI)has a broad antibacterial spectrum,strong hydrophobicity and a large number of hydroxyl groups in its structure,which can be embedded in the hydrophobic core of RHL micelles,and then used to control Staphyloccocus aureus(S.aureus)biofilm infection.Therefore,in this paper,RHL micelles were used as nanocarriers to encapsulate AZI to prepare azithromycin-rhamnolipid(AZI-RHL)micelles.The preparation is designed to increase the sensitivity of S.aureus biofilm to AZI,not only acting on bacteria,but also scavenging EPS,thus exerting a good anti-biofilm effect.The main research results are as follows:1.The critical micelle concentration(CMC)of the RHL aqueous solution was determined by surface tension method,and the results showed that the CMC value of the RHL aqueous solution was 0.25 mg/m L.The AZI-RHL micelles were prepared by thin film hydration method.Taking encapsulation efficiency and drug loading as evaluation indicators,the optimal preparation technology was obtained through single factor test and orthogonal test.The four influencing factors of RHL dosage,aqueous dosage,methanol dosage,and stirring time were investigated.The effect of each factor on the encapsulation efficiency was as follows: RHL dosage> stirring time>methanol dosage,and the aqueous dosage had no effect.Orthogonal test were carried out according to these three influencing factors,and the optimized preparation technology conditions were as follows: RHL dosage was 100 mg,methanol dosage was 12 m L,and the stirring time was 20 min.The AZI-RHL micelles prepared under the optimized preparation technology conditions were spherical when observed under transmission electron microscope,with a hydrodynamic diameter of136.3±68.5 nm,a Zeta potential of-23.1±6.8 m V,an encapsulation efficiency of80.34±0.60%,and a drug loading of 19.42±0.48%.The in vitro cumulative release test showed that AZI-RHL micelles had a certain sustained release effect.The cumulative release curve of AZI-RHL micelles in vitro conforms to the Ritger-Peppas equation,and the release of drugs is dominated by Fick diffusion.AZI-RHL micelles are unstable under continuous strong light exposure.The results of the hemolysis test showed that 32 μg/m L AZI-RHL micelles started to cause severe hemolysis.2.The bacteriostatic ability of AZI-RHL micelles was determined by broth dilution method.The results showed that the bacteriostatic rate of 16 μg/m L AZI-RHL micelles against S.aureus was over 90%.The results of crystal violet staining showed that AZI-RHL micelles could inhibit the formation of S.aureus biofilm,and the inhibition rate of 8 μg/m L AZI-RHL micelles was as high as 92.13%±6.30%,which was significantly higher than that of free AZI and blank RHL micelles(P<0.01).The mechanism of AZI-RHL micelles inhibiting the formation of S.aureus biofilm was investigated by plate colony counting method,plate method,sedimentation determination and ultra-micro spectrophotometry.It was found that AZI-RHL micelles had certain inhibitory effects on the initial adhesion ability,motility ability,self-aggregation ability and e DNA release of S.aureus.Crystal violet staining showed that AZI-RHL micelles could remove mature S.aureus biofilm,and the clearance rate of 64 μg/ m L AZI-RHL micelles against S.aureus biofilm was up to 80.02%±1.32%.Fluorescence staining was used to observe the scavenging effect of AZI-RHL micelles against S.aureus biofilm.The results showed that compared with the untreated control group,after AZI-RHL micelles treated with S.aureus biofilm,the biomass and thickness of biofilm were significantly reduced,and a large number of dead bacteria were increased.The scavenging effect of AZI-RHL micelles on S.aureus biofilm was quantitatively analyzed.The test results showed that compared with the positive control group,after 16 μg/m L,64 μg/m L AZI-RHL micelles acted on S.aureus biofilm,polysaccharide clearance were 46.79%±9.42%,82.99%±14.07%,protein clearance were 36.85%±10.26%,78.63%±7.46%,and live bacteria reduction were47.22%±13.15%,72.19%± 7.96%,respectively.Compare with AZI solution and blank RHL micelles,AZI-RHL micelles had higher clearance rates of polysaccharide,protein and live bacteria.The results of this research show that the preparation process of AZI-RHL micelles is stable and reliable,with small particle size,high encapsulation efficiency and high drug loading capacity.Compared with AZI solution,it has acertain sustained release ability.AZI-RHL micelles can effectively inhibit the formation of S.aureus biofilm and destory mature S.aureus biofilm,and have good anti-biofilm potential.AZI-RHL micelles achieve co-delivery of RHL and AZI by means of preparations,play a good role in destroying S.aureus biofilm,provide a new idea for the prevention and control of biofilm,and at the same time provide a certain reference for the preparation of subsequent surfactant-loaded micelles.