| Cefquinome sulphate is the fourth-generation cephalosporins animal drug, playing a strong role on the killing most gram-negative bacteria and gram-positive bacteria. Thus it is widely used for the therapy of respiratory infections of pigs, cattle and mastitis cow diseasebecause of its lower drug-resistance.The study has found that S.aureus is easy to form a biofilmand this is one of thereason why it becomes drug-resistant. So it is meaningful and necessary to research an efficacious pharmaceutic preparation that resistant the forming of S.aureus biofilm to control this kind bacterium.This study is mainly about developing a new cefquinome sulfate cationic liposome formulation and exploring its effects on bacterial biofilms to get an efficacious pharmaceutic preparation that resistant S.aureus. Through screening the preparation technologyof cefquinome sulphate cationic liposomes and investigating the in vitro release, physical-chemical properties and preliminary efficacy against S.aureus biofilm of the new formulation, we reached the expected research objectives. The main findings were as follows:1. It had chosen and established an exclusive High Performance Liquid Chromatography (RP-HPLC) to assay in vitro content of cefquinome sulfate. The UV was detected at 270 nm. Linear regression equation was A=51313C-22439, R2=0.9997, the linear relationship was well in the range of 1.25?160 ?g/ml, and the average recoveries were between 99.30%?101.60%, the RSD values of intra-day and inter-day were less than 5%. Therefore, this methoddeterminated the drug content effectively and accurately,eliminated the interference of impurities and excipients.At the same time, it were investigated the demulsifier of liposomes and the separate effect of free drug from liposome by refrigerated high-speed centrifugation, the results showed that adding an appropriate amount of tetrahydrofuran could destroy the phospholipid liposomes bilayer structure well; at 4?,15000r/min for 1 h could separate the two parts completely, the recovery of free drug reached 99.72%?101.17%. These results were a basis of the design and optimization of the formulation process.2. Using the solid dispersion combined with the effervescent hydration method prepared the cefquinome sulphate precursor cationic liposome.Entrapment efficiency was considered as the main parameter combined with its exterior shape to screen the most suitable method. Single-factor test and orthogonal design duplication test were used to optimize the cefquinome sulphate precursor cationic liposome preparation process and the optimal formulation process as follows:the rotary evaporation temperature was 54?, the amount of CCl4was 20 ml, the amount of Tween80 was 75 ?l, the mass ratio of soybean phosphatidylcholine(SPC) with cholesterol(CH) was 2:1, drug and SPC-CH mass ratio was 1:10, Citric acid and NaHCO3 mass ratio was 5:1, drug and stearylamine (SA) mass ratio was 1:5. The liposome mean entrapment efficiency and loading with this process were 63.21% and 4.04%, respectively.3. According to the preferred preparation process recipe, the cefquinome sulphate precursor cationic liposome was dispersed as a white uniform suspension. The optical microscope shape was circular, average particle size was 201.5 nm, Zeta electric potential was 65.29 mV. The in vitro cumulative release tests showed that API cumulative release reached 97.82% of the total drug at 8 h, but the cationicliposome was only 73.94%, this result indicates that the cationic liposome had a degree of sustained release.This formulation safety for intravenous injection without hemolytic and non-irritatingmad. Drug stability tests showed that cationic liposome was unstable at high humidity and light irradiation condition, and should be stored dry and dark.4. This study had measured the MIC of cefquinome sulphate solution, the cefquinome sulphate nomal liposomes and the cefquinome sulphate cationic liposomes against the S.aureus standard strain and 10 procine clinical strainswith micro dilution method. The results showed that three drugs MIC values against S.aureus standard strain were 1,1.34,0.48 ?g/ml, successively, all the MIC values against clinical strains had risen of different degree, which indicated that there was some resistance of clinical strains and the MIC value of cefquinome sulphate cationic liposomes was still less than the solution. The growth inhibition curve of cefquinome sulphate solution and cefquinome sulphate cationic liposomes against S.aureus standard strain was detected by spectrophotometric method. The results was that after adding drug 2 h, the bacterial growth curve began to appear gap and at the same drug concentration of 0.5MIC, the inhibition effect of cationic liposomes was more obvious. The growth of S.aureus got into the decline phase quickly when the drug concentration great than or equal to MIC. The higher of the concentration, the faster of the decline phase time, even skipped log phase and stationary phase of bacterial growth.5. In this study, using the Congo red, crystal violet, silver nitrate and other methods to identify the forming biofilms ability of S.aureus standard strain and 10 procineclinical strains. The results showed that all of the selected 11 strains of S.aureus could form a stable biofilm structure; four has a strong ability. MTT assayed drug against S.aureus biofilms clearance rate and drug treating timeagainst S.aureus biofilm removal efficiency. Under the same respective multiples of MIC of drug concentrations, cationic liposomes to remove bacterial biofilms was more effective at 24 h when the drug concentration was 2.5MIC biofilm clearance rate had reached 81.30%. Drug concentration was positively correlated with bacterial biofilms clearance rate. The ability of clearance showed some time-dependent. ESEM images results showed that after the treatment, both of the biofilm bacteria amount and biofilm matrix had been reduced. These results were confirmed the cefquinome sulphate cationic liposome formulation had certain advantages on anti-S.aureus biofilm.This research results showed that the solid dispersion combined with the effervescent hydration method prepared the cefquinome sulphate precursor cationic liposome controllable quality, good reproducibility, the physical and chemical and other indexes were in line with the requirements of a liposomal formulation. The formulation could effectively inhibit the growth of S.aureus and clear its biofilm in vitro. It has advantage of S.aureus biofilm even the bacterial biofilm treatment and control, this consequence has achieved the ultimate goal of this subject research. |
PDF Full Text Request