| Salmonella is a kind of facultative intracellular zoonotic parasitic of gram negative bacilli.It lives and spread in cells,and can evade the host of phagocytosis and killing effect through a variety of mechanisms,which resulting in persistent and recurrence infection.Most antimicrobial agents are difficult to enter the cells.There intracellular concentration cannot reach required effective concentration to clear the bacteria,and fails to realize effective therapy for intracellular salmonella infection.Enrofloxacin has a strong antibacterial activity against Salmonella and owns an effective transcellular diffusion ability,which is widely used for the prevention and treatment of intracellular salmonella infection.However,it is always characterized by having scarce accumulation performance in host cells,and cannot maintain the effective concentration in cells for a long time,resulting in a poor treatment effect for intracellular salmonella infection.Studies have shown that nanoparticles can significantly improve the intracellular concentration and retention time of antibacterial drugs,providing a new strategy to solve the difficulties of enrofloxacin in treating Salmonella intracellular infection.This study used different fatty acids as lipid matrix to prepare enrofloxacin-loaded SLN.By comparing the effects of fatty acids,particle size and charge on the intracellular uptake and intracellular accumulation of enrofloxacin-loaded SLN,SLN which can significantly improve the intracellular concentration and residence time of enrofloxacin has been screened out.To provide a new breakthrough for the treatment of intracellular bacterial infections,the antibacterial activity on intracellular salmonella and pharmacokinetics of different administration ways in pigs of the optimizational enrofloxacin-loaded SLN were studied.1.Study on the preparation and intracellular delivery performance of enrofloxacin-loaded fatty acid SLNThis study used tetradecanoic acid,palmitic acid,stearic acid and docosanoic acid as lipid matrix and polyvinyl alcohol as the active agent,respectively.Enrofloxacin-loaded fatty acid SLN were prepared by hot homogenization and ultrasonication method.The encapsulation efficiency and loading capacity of nanoparticles were determined by high performance liquid chromatography(HPLC).The shape,size,polydispersity index(PDI)and zeta potential of SLN were measured by photon correlation spectroscopy(PCS)by using atomic force microscopy(AFM)and malvin laser nano particle size analyzer.The optimal drug lipid ratio of nanoparticles was determined by single factor control experiment.Selecting SLN which can efficiently transmit enrofloxacin into targeted cells through investigating the effects of SLN on RAW264.7 cellular uptake and cellular accumulation by comparing different fatty acids.AFM showed that all the four fatty acid SLNs were well dispersed with uniform quasi-spherical shape.The encapsulation efficiency of enrofloxacin-loaded fatty acid(tetradecanoic acid,palmitic acid,stearic acid and docosanoic acid)SLN was 65.26,67.53,72.57 and 86.56%,respectively.The loading capacity was 6.44,6.65,7.12 and 8.37%,respectively.Particle size was 341.4,348.8,408.5 and 414.5 nm,respectively.The PDI was 0.241,0.264,0.352 and 0.265,respectively.And the zeta potential was-19.9,-20.6,-21.3 and-22.1 mV,respectively.The saturated solubility of enrofloxacin in melting fatty acid was 1:9,so the optimal ratio of drug to lipid was 1:9.Intracellular uptake indicated that fatty acid SLN encapsulated enrofloxacin accumulating in RAW.264.7 cells was higher up to 4.34 to 37.71-fold than that of free drug at the same extracellular concentration and the same incubation time.Among them,the intracellular concentrations of docosanoic acid SLNs encapsulated enrofloxacin reaching a maximum intracellular concentration of 0.8861 μg/mg protein at 0.5h.The study found that the intracellular residence time of docosanoic acid SLN entrapped enrofloxacin was also the longest.The docosanoic acid SLNs payload drug remained 0.4031 μg/mg after removal of the extracellular drug with continuing incubation for 2 h,while the other three fatty acid encapsulated enrofloxacin and free drug were not detectable in cells at the time.Thus,docosanoic acid SLN entrapped enrofloxacin had the strongest capability in intracellular delivery.As a result,it can be used as the candidate nanoparticles for further optimization.2.The effect of particle size and charge on intracellular delivery property of enrofloxacin-loaded docosanoic acid SLNAfter screening out the optimal fatty acid,the effects of particle size and charge of docosanoic acid SLN on the RAW264.7 cells uptake was investigated further.Different volume(10,20 or 30 mL)of 1,2 or 4%PVA solution alone or combined with a certain concentrations(0.5,2,3 and 4%)of the cationic surfactant dimethyldioctadecyl ammonium chloride(DDAC)solution,sonicated for 8 min using the 3,6 or 30 mm microprobes with 60%or 80%amplitude(VCX 130Vibra-CellTM,Sonics&Materials,Inc.,Newtown,CT,USA)to form docosanoic acid SLN encapsulated enrofloxacin with different size(605,415 and 150 nm etc.)and different charge(-24.9,-17.5,-8.1,7.1 and 18.8 mV etc.).Intracellular uptake showed that the intracellular enrofloxacin concentration was enhanced with the increase of particle size.The intracellular concentration of 605,415,and 150 nm enrofloxacin encapsulated docosanoic acid nanoparticles were 1.1474,0.8343 and 0.3356 μg/mg,respectively.Study results showed that the cellular uptake efficiency is independent of the positive or negative charge value,but it is positively correlated with the absolute value of charge.When the charge of docosanoic acid SLN was-8.1,-17.5 and-24.9 mV,the corresponding cellular uptake amount was 0.9597,1.0778 and 1.1474 μg/mg,respectively.In addition,research showed docosanoic acid SLN with a charge and particle size of 7.1 mV and 501 nm had a higher intracellular concentration than the SLN with a charge and particle size of 18.8 mV and 345nm in RAW264.7 cells.It indicated that the particle size of enrofloxacin-loaded docosanoic acid SLN can significantly affect the efficiency of cellular uptake more than its charge.In summary,the optimal SLN possessed a particle size,charge,PDI,encapsulation efficiency and drug loading was 605 nm,-24.9 mV,0.241,95.9%and 8.9%,respectively.Its corresponding prescription was 0.2 g of enrofloxacin,1.8 g of docosanoic acid.The concentration and volume of PVA was 1%and 10mL.3.Quality evaluation of enrofloxacin-loaded docosanoic acid SLNDocosanoic acid SLN suspension was be evaluated in accordance with the corresponding guiding principle.The results showed that the appearance of enrofloxacin-loaded docosanoic acid SLNs was milky white.The redispersed time of docosanoic acid SLN suspension in a of bottom sediment was only 1 min under a magnetic shaker rotating at 20 r/min.Enrofloxacin-loaded docosanoic acid SLN suspension go through the 9-gage needle with the withdrawal volume of 8.9 mL/min after shaking,which indicated that docosanoic acid SLN suspension can go through the 9-gage needle smoothly.The sedimentation rate and pH value of enrofloxacin-loaded docosanoic SLN suspension were 1 and 6,respectively.Influencing factor test showed the appearance,redispersibility,syringeability,sedimentation rate and pH value of the suspension has no obvious change except a little increase of particle size and polydispersity index of enrofloxacin-loaded docosanoic SLN suspension under strong light conditions.Its long-term stability is good.The injection site of docosanoic acid SLN entrapped enrofloxacin in swine without apparent pathological changes.And there was no adverse reaction such as redness,congestion,exudation,degeneration or necrosis.Hemolytic test showed that docosanoic acid SLN suspension encapsulated enrofloxacin without hemolysis or coagulation within 3 h.4.Study on intracellular pharmacodynamics of enrofloxacin-loaded docosanoic acid SLN in vitroAfter a successful construction of salmonella infected cell system,enrofloxacin-loaded docosanoic acid SLN with 1,4 and 10MIC were added respectively and setting enrofloxacin as the control group.Cells were lysed after 4,14,24 and 48 h,respectively.Through coating plate,the intracellular colony count was conducted.The test determined the antibacterial activity of enrofloxacin-loaded docosanoic acid SLN on RAW264.7 intracellular salmonella bacteria CVCC541,The results showed that enrofloxacin-loaded docosanoic acid SLN for intracellular salmonella CVCC541 had obvious inhibition effect after adding the drug 4 h.With the incubation time and extracellular add drug concentration increasing,the inhibitory effect was more significant than the control group.When the incubation time increased from 24 h to 48 h,the colony logarithmic value of enrofloxacin-loaded docosanoic acid SLN group with 1,4 and 10 MIC changed from 3.60,3.00 and 1.48 CFU/mL to 3.80,3.30 and 1.50 CFU/mL,respectively,with no obvious growth.While the colony logarithmic value of enrofloxacin group with 1,4 and 10 MIC increased from 5.04,3.95,3.04 CFU/mL to 6.91,5.65,4.15 CFU/mL,respectively,with significance growth.Thus,the inhibitory effect of enrofloxacin-loaded docosanoic acid SLN on intracellular salmonella was more significant,which maintained stronger inhibition ability constantly.5.Pharmacokinetic studies of enrofloxacin-loaded docosanoic SLN in pigsHealthy pigs(n=24)for test were randomly divided into 4 groups.The experimental group and the control group were 6 head,respectively.Intramuscular injection enrofloxacin-loaded docosanoic acid SLN and Baytril at the dose of 2.5 mg/kg body weight respectively.And intragastric administration of enrofloxacin-loaded docosanoic acid SLN and enrofloxacin soluble powder at the dose of 5 mg/kg body weight respectively.Blank plasma collected before administration.And blood was collected at different time points after administration.Then plasma was separated.HPLC was used to detect the concentration of enrofloxacin in plasma.Pharmacokinetic parameters were fitted by Winnonlin software.The blood concentration of baytril group increased rapidly after intramuscular administration,reaching a peak concentration of 1.572+0.222 μg/mL at 1 h.Then the blood drug concentration began to decline,it was close to the detection limit at 48 h,which was 0.024±0.011 μg/mL.The absorption of the enrofloxacin-loaded docosanoic acid SLN group was slow after administration,reaching a peak concentration of 0.746±0.074 μg/mL at 4 h.Then the blood drug concentration falling slowly,dropping to 0.034±0.01 μg/mL at 120 h which was higher than the concentration of baytril group at 48 h.The results showed that the docosanoic acid SLN entrapped enrofloxacin had a significant sustained-release effect.The Cmax of the enrofloxacin-loaded docosanoic acid SLN and baytril was 0.685±0.059 μg/mL and 1.363±0.235 μg/mL,respectively.T1/2ke was 20.053±5.203 h and 6.326±0.889 h,respectively.The AUC was 22.257±6.261 μg/mL and 13.603±2.106 h·μg/mL,respectively.The MRT was 37.761±4.460 h and 11.272±0.756 h,respectively.The relative bioavailability of enrofloxacin loaded docosanoic acid SLN increased to 163.62%compared with the free drug.The blood concentration of the enrofloxacin soluble powder group increased rapidly after intragastric administration,reaching a peak concentration of 1.166±0.079 μg/mL at 1 h.Then the blood drug concentration began to decline,dropping to 0.150±0.029 μg/mL at 24 h.And it was lower than the detection limit after 48 h.While the absorption of the enrofloxacin-loaded docosanoic acid SLN group was slow after intragastric administration,reaching a peak concentration of 0.924±0.194 μg/mL at 4 h.Then the blood drug concentration fell slowly,continue until 120h after administration.The relevant pharmacokinetic parameters of enrofloxacin-loaded docosanoic acid SLN and enrofloxacin soluble powder in plasam were as follows:Cmax was 0.824±0.152 μg/mL and 1.027±0.059μg/mL,respectively;T1/2ke was 12.058±5.470 h and 5.582±1.381 h,respectively;AUC was 23.559±3.669 h·μg/mL and 9.886±1.996 h·μg/mL,respectively;MRT was 35.153±0.537 h and 12.330±1.558 h,respectively.The relative bioavailability of enrofloxacin-loaded docosanoic acid SLN group is 238.31%.In summary,this study successfully developed the enrofloxacin-loaded docosanoic acid SLN,which could significantly improve the intracellular concentration,residence time and the antibacterial activity of intracellular salmonella CVCC541 infections of enrofloxacin.It realized sustained release and significantly improved the bioavailability of enrofloxacin in pigs,which provides a new possibility to solve the problem in treating bacterial infection of Salmonella and other intracellular bacteria. |
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