| 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. |
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