Study On The Controlled Release System And Antibacterial Activity Of Florfenicol With Poly (Lactic-co-glycolic Acid) As Carrier

Florfenicol（FF）,a fluoride analogue of thiophenol,is a broad-spectrum veterinary antibiotic effective against both Gram-positive and Gram-negative bacteria,with no potential aplastic anemia effect and other advantages,and is widely used in veterinary animal husbandry.However,FF has poor water solubility,poor bioavailability and shortened plasma half-life.These disadvantages have many inconveniences for clinical applications.In this paper,a simple single emulsification solvent volatilization method with poly（lactic acid hydroxyacetic acid）（PLGA）as the carrier material was used to investigate the florfenicol nanoparticle formulations prepared with different coating materials to improve the water solubility of florfenicol and control the drug release to achieve better therapeutic effects,combined with freeze-drying technology to improve the stability of nanoparticle formulations.Finally,the in vitro antibacterial activity of florfenicol nanoparticle formulations was investigated.The main research contents and results of the article are as follows.1.In this paper,the FF high performance liquid chromatography（HPLC）analysis method was established.G50 dextran gel column chromatography was used to effectively separate nanoparticles from free drug in order to determine the encapsulation rate and drug loading of nanoparticles.The equilibrium solubility of FF in different solvents were determined to provide basis for the later physicochemical property investigation and in vitro release evaluation.2.The nano-controlled release drug delivery system is a promising new drug delivery system with the advantages of improving the long-term controllability of the effective therapeutic dose of drugs,including reduce the side effects,decrease the number of dosing times and reduce the toxic reactions of drugs.In this article,PLGA nanoparticles containing florfenicol were prepared by O/W emulsion solvent volatilization method.The preparation process（sonication time and sonication power）and preparation prescription（emulsifier types,emulsifier concentration,drug-PLGA ratio,oil-water phase volume ratio,and drug concentration）were varied to provide data basis for orthogonal test optimization with PVA concentration（A）,drug-lipid ratio（B）,oil-water phase volume ratio（C）and drug concentration（D）.The encapsulation rate was used as the evaluation index to finally obtain FF-PLGA nanoparticles（FF-PLGA NPs）with high encapsulation rate.The optimal prescription was verified and the physicochemical properties（FTIR,DSC,XRD,TEM）were investigated.The results showed that the optimal prescription of FF-PLGA NPs was obtained with a PVA concentration of 4%,a drug-lipid ratio（FF:PLGA）of 1:15,an oil-water phase volume ratio of 1:8,and an FF concentration of 1.5 mg·mL^-1.The encapsulation rate and drug loading prepared by the optimal prescription were 80.99%±0.87%and1.65%±0.01%,respectively.The particle size,PDI and Zeta potential were201.40±3.50 nm,0.08±0.02 and 0.95±0.04 m V,respectively,which met the injection requirements.The saturation solubility in water was 1.92±0.04mg mL^-1,which was significantly higher than that of the FF（P<0.05）.The in vitro release results showed that the FF-PLGA NPs started to release a large amount of drug with some sudden release,and followed by slow release,maintaining a high blood concentration level until the drug was completely released.The results of the mathematical model fitting showed that the in vitro release behavior of FF was in accordance with the primary pharmacokinetics with the fitted equation ln（1-Q/100）=-0.6251t-0.2335,R²=0.9963.The FF-PLGA NPs were in accordance with the Ritger-peppas equation with the fitted equation lnQ=0.451lnt+2.974,R²=0.9609,n<0.5,which was consistent with the Fick diffusion.3.The stability and physicochemical properties of the nanosystems were investigated using hydrophilic 2-HP-β-cyclodextrin（2-HP-β-CD）as the coating material.The FF-PLGA NPs encapsulated by 2-HP-β-CD（FF-2-HP-β-CD-PLGA NPs）with high encapsulation rate and stability were prepared by single-factor investigation and orthogonal test optimization.The emulsifier concentration,drug-lipid ratio,oil-water phase volumetric drug concentration and 2-HP-β-CD concentration were studied based on the previous experiments.The results showed that the concentration of PVA,2-HP-β-CD and FF,drug-lipid ratio（FF:PLGA）and oil-water phase volume ratio were 2%,1.5%,2 mg·mL^-1,1:15 and 1:8,respectively.The encapsulation rate,drug loading,particle size,PDI value and Zeta potential of FF-2-HP-β-CD-PLGA NPs were 82.02±0.82%,2.03±0.02%,185.90±2.80 nm,0.10±0.05 and-5.92±1.80 m V,respectively.The saturation solubility in water was 5.60±0.32 mg mL^-1（about 5 times that of the FF and3 times that of FF-PLGA NPs）,indicating that the presence of 2-HP-β-CD effectively improved the dispersion of FF in water.In vitro release results showed that FF-PLGA NPs released approximately 44.96%in the first 4hours,whereas FF-2-HP-β-CD-PLGA NPs released approximately 37.85%in the first 4 hours with a significantly weaker sudden release（P<0.05）,and then maintained high blood concentrations for slow release,which indicated that 2-HP-β-CD encapsulation contributes to the slow release of the drug and prevents the sudden release of FF nanoparticles.The mathematical model fitting results showed that FF-2-HP-β-CD-PLGA NPs fit the Ritger-peppas equation with the fitted equation lnQ=0.417lnt+2.794,R²=0.9655,n<0.5,which was consistent with the Fick diffusion.4.The amphiphilic copolymer of polyethylene glycol(PEG₄₀₀₀)modified PLGA,PEG₄₀₀₀-PLGA was used as the carrier material to optimize the preparation of FF nanoparticle formulations(FF-PEG₄₀₀₀-PLGA NPs)with high encapsulation rate using single-factor examination and orthogonal tests.The focus was on investigating the effects of several major factors in the preparation process of FF-PEG₄₀₀₀-PLGA NPs by emulsification solvent volatilization method:emulsifier concentration,drug-lipid ratio,oil-water phase volume ratio,and drug concentration on nanoparticle preparation.The results showed that the PVA and FF concentration,drug-lipid ratio(FF:PEG₄₀₀₀-PLGA)and oil-water phase volume ratio were 1%,0.8 mg·mL^-1,1:15 and 1:6.The encapsulation rate,drug loading,particle size,PDI value and Zeta potential of FF-PEG₄₀₀₀-PLGA NPs were 81.04%±1.04%,2.63±0.03%,129.10±3.90 nm,0.11±0.03 and-4.53±0.37 m V,respectively.The saturation solubility in water was 4.64±0.56 mg mL^-1（about 4 times that of the FF and 2.5 times that of the FF-PLGA NPs）,which fully indicates that the modification of the nanoparticles by the hydrophilic segment PEG₄₀₀₀improved the hydration performance of the carrier material.The in vitro release results showed that FF-PLGA NPs released approximately 44.96%and FF-2-HP-β-CD-PLGA NPs released approximately 37.85%in the first 4h,while FF-PEG₄₀₀₀-PLGA NPs released approximately 33.79%in the first 4h.The release rate of FF-PEG₄₀₀₀-PLGA NPs was significantly slower and smoother than FF and the above two nanoparticle formulations（P<0.05）,which indicated that the modification of PLGA by PEG₄₀₀₀would affect the degradation process and drug release behavior of polymeric nanoparticles and help to maintain drug activity.The mathematical model fitting results FF-PEG₄₀₀₀-PLGA NPs showed compliance with the Ritger-peppas equation with the fitted equation lnQ=0.611lnt+2.208,R²=0.9701,n<0.5,which was consistent with the Fick diffusion.5.Freeze-drying prescriptions of three nanoparticle formulations were prepared using freeze-drying technology to optimize the freeze-drying process of nanoparticles using appearance,color and redispersibility as indicators.The optimal freeze-drying prescription was prepared using a pre-freezing temperature of-50°C,a pre-freezing time of 6 h,a drying time of 32 h and the optimal lyophilization prescriptions were prepared with 10%mannitol as lyophilization protectant for FF-PLGA NPs and 5%mannitol as lyophilization protectant for FF-2-HP-β-CD-PLGA NPs and FF-PEG₄₀₀₀-PLGA NPs.The properties of freeze-dried samples such as particle size,Zeta potential,encapsulation rate and redispersibility were investigated separately.The results showed that the changes of encapsulation rate,particle size and Zeta potential before and after freeze-drying were small and the redispersibility was good.The preliminary stability experiments showed that the lyophilized powder was placed at 4℃for 90 days,and the particle size,Zeta potential and encapsulation rate did not change significantly with good stability.The bulk release showed similar to that of nanoparticles before lyophilization,which was consistent with the Ritger-peppas equation.6.The results of the inhibition zone test showed that the inhibition zone diameters of FF and FF nanoparticles against the Pseudomonas aeruginosa,Staphylococcus aureus,Klebsiella pneumoniae and Escherichia coli were22.0,20.0,22.5 and 19.0 mm,respectively;the inhibition zone diameters of FF-PLGA NPs against the four strains were 23.0,23.0,26.0and 21.0 mm,respectively;the inhibition zone diameters of FF-2-HP-β-CD-PLGA for the four strains were 23.0,22.5,25.5 and 21.0 mm,respectively;the inhibition zone diameters of FF-PEG₄₀₀₀-PLGA NPs for the four isolates were 22.5,22.0,24.5 and 20.0 mm,respectively,indicating that all three nanoparticle formulations were significantly more effective than the FF against the common isolates.The MIC of the nanoparticle formulations against P.aeruginosa all were 1/2 that of FF.The MIC of FF-PLGA NPs against Klebsiella pneumoniae was 1/2 that of FF,while the MIC of FF-2-HP-β-CD-PLGA NPs and FF-PEG₄₀₀₀-PLGA NPs against Klebsiella pneumoniae were not significantly different from FF.The MIC of the three preparations to Escherichia coli were 1/4,1/2,1/2 that of FF,respectively（P<0.05）.The MBC of FF-PLGA NPs and FF-2-HP-β-CD-PLGA NPs against Klebsiella pneumoniae were 1/2 that of FF（P<0.05）,and no significant difference between the MBC of Klebsiella pneumoniae and FF of FF-PEG₄₀₀₀-PLGA NPs;the MBC of the three preparations to Escherichia coli were 1/4,1/2 and 1/2 that of FF,respectively（P<0.05）.The results of hemolysis test showed that the three FF nanoparticle formulations had no significant hemolytic effect on erythrocytes in the concentration range of100-1000μg·mL^-1,and were safe for intravenous injection.In this paper,we initially discussed the controlled release system of florfenicol drug with PLGA as the carrier,which improved the solubility of FF in the formulation,uniform and stable particle size,good biocompatibility,high safety of the formulation for intravenous drug delivery,and improved the antibacterial activity of the drug to some extent.The nanoparticles modified by 2-HP-β-CD or PEG₄₀₀₀could significantly reduce the sudden release of nanoparticles,sustain the slow release at high concentration and improve the drug stability,which proving that the FF nanoparticle drug delivery system with PLGA is a very promising new drug formulation.