Eugenol Nanoliposomes:Preparation And Antibacterial Activity Against Escherichia Coli

Eugenol(Eug)is a light yellow phenolic compound with special aroma,and it is also the main active ingredient of clove oil.Many studies have shown that Eug has a variety of pharmacological effects such as antioxidant,anti-inflammatory,antibacterial,immunomodulatory,analgesic and anticancer,and has a good safety profile,and the U.S.Food and Drug Administration(FDA)has included it in the food additive list.However,Eug has defects such as high volatility,poor water solubility,low stability and bioavailability,which limit its efficacy,so there is an urgent need to develop new dosage forms to improve its application.Liposomes are a bilayer vesicle carrier system that can self-assemble in aqueous media through amphiphilic groups.Encapsulation of drugs in liposomal vesicles enhances their solubility and stability,and reduces drug degradation.In addition,the controlled release properties of liposomes can prolong the circulation time of drugs in the blood,while reducing side effects and target toxicity of various drugs.In this study,eugenol nano-liposome(ENL)was prepared by loading Eug into liposome using thin film hydration ultrasonication method,and its preparation process was optimized by Box-behnken design(BBD)response surface method to characterize the morphological structure and release and other properties of ENL.Then,its antibacterial effect on Escherichia coli(E.coli)in vitro and in vivo was evaluated,which provided technical support for the development of new preparations of Eug.1.Preparation of ENL and optimization by BBD response surface methodology.To solve the problems of Eug’s volatility,insolubility in water and instability,it was prepared into ENL by thin film hydration sonication method,and the actual encapsulation rate of ENL was calculated by organic solvent extraction method and emulsion breaking method.To determine the preparation conditions,a five-factor,five-level single-factor analysis of ENL was performed for the mass ratio of soy lecithin to cholesterol(W/W),Eug concentration(%),Tween-80 mass(mg),hydration temperature(℃),and sonication time(min).The first three factors,which had the greatest influence on the encapsulation rate,were selected to optimize the preparation process by BBD response surface method.The results showed that the optimal preparation process was a soy lecithin to cholesterol mass ratio of 5.4:1,an Eug concentration of 1.5%,and an ultrasonic time of 10 min.The mean value of the encapsulation rate obtained accordingly was 72.42 ± 0.22%,which was less different from the predicted encapsulation rate of 72.51%.The results indicate that single factor analysis combined with BBD response surface method optimization can obtain ENL with higher encapsulation rate.2.Characterization of the properties of ENLTo investigate the preparation of ENL,its morphology,particle size,polydispersity index(PDI),zeta potential,Fourier infrared spectroscopy(FTIR),in vitro release,and stability were characterized.The ultrastructure of ENL was observed by transmission electron microscopy(TEM);the particle size and zeta potential were determined by nano particle size analyzer;the infrared spectrum was scanned by potassium bromide(KBr)compression;the in vitro release of Eug from ENL was determined by dialysis and UV spectrophotometry in a simulated in vivo gastrointestinal environment;the ENL was placed at 4℃ and 25℃ for 45 d.The particle size and PDI of ENL were determined at 4℃ and 25℃,respectively.The storage stability of ENL was determined by measuring the particle size and PDI changes at 4℃ and 25℃,respectively.The results showed that:the morphology of ENL was nearly spherical with uniform size;the average particle size was 203.97±8.66 nm,the PDI was 0.23±0.03,and the zeta potential was-2.18±0.18 mV;the characteristic peaks of Eug were red-shifted and blueshifted in ENL,probably due to hydrogen bonding interactions;ENL was slowly released in the gastrointestinal mimetic solution;and it could be stored at 4℃ and 25℃ for 45 d under two conditions.The above results indicate that the ENL prepared by thin film hydration sonication has good stability and slow release.3.Study on antibacterial effect of ENL in vitro.In order to investigate the inhibition of E.coli by ENL in vitro,the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of ENL were determined by micro broth dilution and agar diffusion method;the inhibition of E.coli was examined by placing ENL at 4℃ and 25℃ for 0 d,7 d,15 d,30 d and 45 d,respectively.The OD600 of E.coli at different time periods after treatment with different concentrations of drugs was determined by enzyme standard and the growth curves were plotted;2MIC and 4MIC concentrations of ENL were used to treat E.coli at different time points,and the colony numbers were determined and the time-fungicidal curves were plotted;SEM and TEM were used to observe the morphological and structural changes of E.coli after treatment with different concentrations of drugs.The morphological and structural changes of E.coli after different concentrations of drug treatment were observed by SEM and TEM;the changes of E.coli extracellular inner membrane were observed by fluorescence microscopy after cotreatment of E.coli with fluorescent probes 1-N-phenylnaphthylamine(NPN)and propidium iodide(PI)at different concentrations of drug treatment.The results showed that the MIC of ENL against E.coli strain was 0.31 mg/mL and MBC was 0.63 mg/mL,and the inhibition effect was enhanced 2-4 times compared with Eug;ENL had good inhibition stability within 45 d,and the MIC and MBC of ENL against E.coli under two storage conditions were almost unchanged;2MIC of ENL treatment for 2 h could kill the standard The results of SEM and TEM showed that the treatment with ENL caused severe wrinkling,cell membrane breakage,and cell wall damage.The results of SEM and TEM showed that ENL treatment caused severe wrinkling,cell membrane breakage,cell wall and hair loss,etc.The results of fluorescence microscopy showed that ENL could destroy the outer and inner cell membranes of E.coli,increase its permeability,and show a certain drug concentration dependence.The above results indicated that ENL had obvious inhibitory and killing effects on E.coli,mainly caused by disrupting its cell membrane.4.Study on the therapeutic effect of ENL on E.coli enteritis.To investigate the in vivo therapeutic effect of ENL on E.coli enteritis,this experiment firstly established an enteritis model by instilling clinical isolate E.coli(AHM5C23)in BALB/c mice,and then treated them with different concentrations of ENL(5,10 and 20 mg/kg)after modeling.4 d later,the mice were executed and pathologically dissected and sampled,and then mortality,body weight and organ index,intestinal histopathological changes and small intestinal absorptive capacity,intestinal tissue inflammatory cytokines and intestinal tissue inflammation-related protein expression to determine the therapeutic effect of ENL on E.coli enteritis.The results showed that compared with the blank group,the survival rate of mice in the model group was 80%,body weight decreased significantly,liver and spleen indices increased significantly,and HE staining of the jejunum and colon showed obvious pathological changes such as swelling,intestinal wall thinning,villi atrophy,hemorrhage,inflammatory cell infiltration,and a significant decrease in the villi crypt ratio(V/C);mice in the ENL-treated group did not show mortality,and the above symptoms improved significantly.RT-qPCR assay revealed that ENL treatment significantly inhibited the mRNA expression of intestinal tissue-associated inflammatory cytokines NLRP3,IL-1β,IL-6,TNF-α,and western blot assay demonstrated that ENL treatment significantly inhibited the expression of intestinal tissue inflammation-associated proteins TLR4,MyD88,p-P65,and p-IκB.The above results indicate that ENL has obvious anti-inflammatory effects and can significantly inhibit the overactivation of NF-κB signaling pathway caused by bacterial enteritis,while exerting good therapeutic effects.In summary,after optimizing the preparation conditions by BBD response surface method,we can obtain ENL with high encapsulation rate,which is homogeneous spherical in size with small particle size,slow release and storage stability.Meanwhile,ENL showed significant inhibition of multiple drug-resistant E.coli strains and killed the bacteria within minutes;this was related to the destruction of cell membrane and the shrinkage and damage of bacteria.In addition,ENL inhibited the activation of TLR4/MyD88/NF-κB signaling pathway induced by E.coli infection and down-regulated the mRNA expression of inflammatory cytokines NLRP3,IL-1β,IL-6,and TNF-α to have therapeutic effects on E.coliinfected enteritis.The above results indicate that ENL has good antibacterial and antiinflammatory effects in vitro and in vivo,and is expected to be a new alternative anti-drug.