【作者】 杨亚军；

【导师】 李引乾；

【作者基本信息】 西北农林科技大学 ， 基础兽医学， 2007， 硕士

【摘要】 本研究旨在研制出包封率高、稳定性好、安全、高效的甲砜霉素脂质体(TAPL)。1. TAPL包封率测定方法的建立透析法分离脂质体和游离药物,紫外-可见分光光度法在225 nm波长处测定药物含量。透析法分离脂质体的平均回收率为96.1%;在6μg/mL～16μg/mL浓度范围内,浓度与吸光度的线性关系良好,平均回收率为99.22%。样品的平均包封率为37.02%(n=3),甲砜霉素含量为10.21 mg/mL(n=3)。该方法可用于TAPL的分离及包封率和含量的测定。2. TAPL制备方法筛选以外观性状、包封率为指标,从薄膜分散法、改良薄膜分散法、逆相蒸发法和改良逆相蒸发法中筛选TAPL的最佳制备方法。改良逆相蒸发法制备的脂质体为小单室囊泡,包封率为37.02%(n=3),与改良薄膜分散法和逆相蒸发法差异显著(P<0.05)。改良逆相蒸发法是制备TAPL的最佳方法。3. TAPL的制备与质量控制以包封率为指标,采用正交试验优化TAPL的配方和改良逆相蒸发法的工艺条件。优化后的配方和工艺条件分别为:药物与磷脂比例为1︰5(W︰W),磷脂与胆固醇比例为4︰1(W︰W),PBS液pH 7.4;超声7 min(连续超声裂解10 s,间隔1 s),蒸发温度为40℃,冻融3次。制备的TAPL为淡黄色均一乳液,显微镜观察(10×100)为小单室囊泡,视野中没有药物结晶,滤膜滤过后透射电镜观察为类球形囊泡,粒径在180 nm～300 nm之间,包封率为62.37%(n=3),甲砜霉素含量为8.78 mg/mL(n=3),无热原。4. TAPL稳定性与安全性在4℃、室温、40℃条件下对TAPL进行热稳定性实验,低温光照条件下进行光稳定性实验,低温离心考察TAPL悬浮稳定性;以肌肉刺激实验、溶血性实验、毒性试验对TAPL进行安全性评价。试验结果表明,TAPL对热、光不稳定,悬浮稳定性好;TAPL无刺激性、无溶血反应,小鼠、家兔、罗曼公雏大剂量注射后未出现明显的毒性反应。TAPL应低温避光保存。5. TAPL体外抑菌实验选取临床常见致病菌,以试管二倍稀释法对TAPL进行体外抑菌实验。结果表明,TAPL对致病性大肠杆菌、金黄色葡萄球菌、无乳链球菌的MIC分别为游离甲砜霉素的1/4、1/4和1/8;实验浓度下两者的MBC没有差异。更多还原

【Abstract】 Thiamphenicol liposome (TAPL) which was high entrapment efficiency, well stability, safety and more efficient was studied in this paper.1. Determination of entrapment efficiency of TAPL. The liposome and free thiamphenicol were separated by dialysis. Total and free thiamphenicol were measured by ultraviolet and visual light spectrophotometry at 225 nm. The drug recovery of dialysis was 96.1%. Calibration curve was linear in the range of 6μg/mL～16μg/mL, and mean recovery was 99.22%. Mean entrapment efficiency and content of three samples were 37.02% and 10.21 mg/mL respective. The method of determining was convenient, sensitive, accurate and suitable for determining the separation, entrapment efficiency and content of the liposome.2. To select the best method for TAPL preparation. The best method of TAPL preparation was selected from thin-film dispersion method, modified thin-film dispersion method, reverse phase evaporation method and modified reverse phase evaporation method by means of TAPL character and entrapment efficiency. TAPL which prepared by modified reverse phase evaporation method was small unilamellar vesicles, and 37.02% of total thiamphenicol was entraped. The difference of modified reverse phase evaporation method from modified thin-film dispersion method and reverse phase evaporation method of entrapment efficiency was significant (P<0.05). The results showed modified reverse phase evaporation method was the best method to prepare TAPL.3. TAPL preparation and quality control. To optimize the prescription and technology of TAPL, orthogonal design was made by means of entrapment efficiency. Orthogonal design results showed that prescription factors were: ratio of thiamphenicol to egg phosphatidyl choline was 1:5, ratio of egg phosphatidyl choline to cholesterol was 4:1, pH of PBS was 7.4. Technology factors were: 7 minutes treated with ultrasonic, evaporation at 40℃, freezing and thawing 3 times. The prepared TAPL was yellowish homogeneous emulsion, and samll unilamellar vesicles, no thiamphenicol crystal observed under microscope (10×100). TAPL which was filtered in turn by microporous membrane of 0.45 and 0.22 micrometer was spheroid vesicles under transmission electron microscope (×20 000), and the range of particle size of was 180 to 300 nanometer. Entrapment efficiency and content of the liposome were 62.37% (n=3) and 8.78 mg/mL (n=3) respective, and there was no pyrogen reaction. 4. Stability and safety tests of TAPL. Heat stability of TAPL was studied at 4℃, room temperature and 40℃. Photo-stability and suspension stability of liposome were studied by low temperature with light exposure and centrifugation respectively. Safety of liposome were assessed by muscle stimulation test, haemolysis experiment and toxicity test. The tests indicated TAPL wasn’t stable to heat and light but stable to centrifugation. Liposome was not irritative to muscle and also no hemolytic reaction. It was no toxicity reaction to mice, rabbits and chickens when the liposome was injected with large dosage. Those results suggested that TAPL should be stored at low temperature and away from light.5. Antibacterial effect in vitro of TAPL. Antibacterial activity in vitro of the liposome to 3 common strains in veterinarian clinical practice was tested by tube double dilution method. Minimal inhibitory concentration of the liposome was 1/4, 1/4 and 1/8 to that of free thiamphenicol for Escherichia coli, Staphylococcus aureus and Streptococcus agalactiae in vitro. However, minimal bactericidal concentration of the liposome and free thiamphenicol were no difference to those at experimental concentration in vitro.更多还原