Studies On Multivesicular Liposome Of Tyroserleutide

Tyroserleutide(YSL)is a type of active,low molecular weight polypeptide,comprised of three amino acids,L-tyrosine,L-serine and L-leucine with a corresponding chemical structure C₁₈H₂₇N₃O₆. Pharmacodynimic studies have proved that YSL may directly affect the signal transduction and significantly inhibit human hepatocarcinoma Be17402 growth in vitro and in vivo resulting necrosis and apoptosis of the tumor cell by adjusting the concentration of Ca+ with no obvious adverse effects.YSL discompose rapidly in plasma,resulting in a short half-life,and needs to be injected once a day and 30 days for one treatment.Sustained and controlled release delivery systems could be applied to lessen the dose times and extend the pharmaceutical effects of the drug in vivo.In this article,pharmaceutical research was carried out in order to prepare Tyroserlenutide-Multivesicular liposome(YSL-MVL) with sustained release property and the physiochemical properties of the YSL-MVL were also examined.This study includes main contents as below:First,preformulation studies were carried out.The isoelectrical point of YSL was determined to be 4.8 by IEF-PAGE method.The solubility of YSL in different solvent was tested and it indicated that YSL was easily dissolved in acetic acid,dissolved in water,slightly dissolved in alcohol, hardly dissolved in tetrahydrofuran and don't dissolve in diethy ether and chloroform.The saturated solubility of YSL in 50mmol/L acetate buffer of pH4.0 was 21.670mg/mL and was 17.625mg/mL in the range of pH5.0～7.5.The apparent octanol-water partition coefficient was determined to be 0.035,indicating that YSL was strongly hydrophilic.HPLC methods for assaying YSL was established,limit of detection was 2ng and limit of quantitation was 6ng and it showed fine lineal relationship in the range of 2.00～50.00μg/mL. Factorial experiment design was used to scrutinize the factors which influence on the stability of YSL in solutions.The degradation rate constant and degradation half-life in different conditions were measured and the results showed that the pH value of the solution,the type of buffer,the ionic concentration and the temperature exerted obvious impacts on the stability of YSL in the solutions.Raising temperature accelerated the degradation of YSL.YSL showed a good stability in ABS and less stability in PBS and was most instable in CBS.The best pH value for YSL in ABS,PBS and CBS were 4.08,4.02 and 8.08 respectively.The existing of oxygen may affect the stability of YSL in coordination with the varying pH value and the degradation of YSL confirmed to the first order kinetic equation.Second,the formulations and pharmaceutical procedures were examined based on the results of preformulaiton studies.SPC,CH,OA and TO were chosen as main lipid materials for preparation of YSL-MVL and the concentration of SPC in the oil phase was 16.5mg/mL,CH 8.0mg/mL,YO8.0mg/mL,OA1.0mg/mL by single factor study and orthogonal experimental designs.The inner aqueous phase was chosen as 50mmol/LABS with pH value of 4.0 and the concentration of YSL was 10mg/mL,sucrose 7.5%;The outer aqueous phase was chosen as 40mmol/L L-lysine with 5%sucrose and pH value of 9.27;The suspension media was 50mmol/L ABS with 5%sucrose and pH value of 7.00.Single factor screening was carried out to determine the oil and water phase ratio to be 1:1,the temperature for preparing primary emulsion to be 10～25℃,the shear rate to be 9000rpm for 10min,the primary emulsion and outer aqueous phase ration to be 1:4,the shear rate for preparing double emulsion to be 3000rpm for 10 seconds,the nitrogen to be the eliminate gas,the centrifuge rate for depositing MVL to be 2500rpm for 10min.Finally,the basic physiochemical properties of YSL-MVL were studied.The average particle size of optimum preparation was 17.23μm and 88.57%was in the range of 10～30μm.The entrapment efficiency was up to 90.8%.The surface of the particles was negative charged with the zeta potential of(-44.53±4.10)mv.The release profile and its relevant influencing factors were examined to draw a conclusion that the release of YSL from YSL-MVL included eroding and diffusing processes.The stability of YSL and YSL-MVL in plasma were tested and the results showed that YSL-MVL may protect the YSL from degradation and could provide a sustained release property confirming to the first order kinetic equation.