微柱离心-HPLC测定胰岛素脂质体的包封率

目的使用微柱凝胶离心分离结合高效液相色谱法(HPLC)定量的方法,建立胰岛素脂质体包封率的测定方法。方法采用了改良的薄膜分散法来制备胰岛素脂质体。使用Sephadex G-50填充凝胶离心微柱,分别使用去离子水及磷酸缓冲液作为洗脱液,分离载药脂质体和游离胰岛素后,经HPLC测定胰岛素浓度并计算包封率。结果通过洗脱曲线确定了洗脱方式,上样体积为600μL,使用去离子水洗脱5次(每次600μL)可将载药脂质体完全洗脱,再使用pH 7.4磷酸盐缓冲液洗脱6次可将其中的游离药物完全洗脱。实现了完全分离载药脂质体与外水相游离药物,经HPLC测定并计算得出3批胰岛素脂质体的平均包封率为36.03%。结论对于亲水性多肽药物脂质体,微柱离心可以实现有效分离脂质体与游离药物的目的,结合HPLC测定其中胰岛素的含量,可以作为测定胰岛素脂质体包封率的有效方法。

Determination of Encapsulation Efficiency of Insulin Nanoparticles by Minicolumn Centrifugation-HPLC Method

OBJECTIVE To establish a minicolumn cetrifugation-HPLC method for the determination of entrapment efficiency for insulin liposomes. METHODS Insulin liposomes were prepared by modified membrane dispersion method. Firstly, the centrifuge minicolumn was filled with Sephadex G-50 gel. Then, deionized water and phosphate buffer were used as eluents to separate the entrapped and free insulin in liposome solution. Eventually, insulin concentration was measured through HPLC method and entrapment efficiency was calculated. RESULTS The optimum parameters of minicolumn centrifugation was defined by the elution curve. The volume of centrifugal was 600 μL with deionized water elution for 5 times(600 μL per time) firstly and pH 7.4 phosphate buffer elution for 6 times afterwards to completely separate free drugs in the outer water phase. The average entrapment efficiency of insulin liposomes(3 batches) was 36.03% through HPLC determination. CONCLUSION For hydrophilic polypeptide drug liposomes, minicolumn centrifugation method could effectively separate free drugs in liposome solution. Combined with HPLC method for insulin concentration determination, minicolumn centrifugation method is applicable for the measurement of entrapment efficiency of insulin liposomes.