鱼精蛋白凝聚法测定脂质体和纳米脂质体包封率

Determination of Encapsulation Efficiencies of Liposomes and Nanoliposomes by Protamine Aggregation Method

目的 建立鱼精蛋白凝聚法测定脂质体、纳米脂质体包封率的方法。方法 鱼精蛋白凝聚法分离脂质体、纳米脂质体，以高效液相色谱法为分析手段，测定脂质体药物包封率，并与葡聚糖凝胶柱色谱法比较。考察脂质体粒径、脂质体Zeta电位、鱼精蛋白用量以及药物不同溶解性、荷电性、分子量大小等因素对鱼精蛋白凝聚法测定脂质体包封率的影响。结果 鱼精蛋白凝聚法可以准确测定不同溶解性质药物脂质体的包封率；应用葡聚糖凝胶G-50柱色谱法与鱼精蛋白凝聚法均能很好测定粒径≥220nm碘海醇脂质体的包封率。脂质体粒径减小，葡聚糖凝胶柱色谱法药物回收率下降；碘海醇脂质体粒径小于200nm时，葡聚糖凝胶G-50柱色谱法测得药物回收率较鱼精蛋白凝聚法低。葡聚糖凝胶柱色谱法不适合脂溶性药物脂质体的分离且不能准确测定脂溶性药物托氟啶脂质体的药物包封率；鱼精蛋白凝聚法可准确测定粒径在100—500nm的托氟啶脂质体的药物包封率。脂质体荷电性影响鱼精蛋白凝聚法测定脂质体包封率；调节介质pH值使托氟啶脂质体Zeta电位分别为-44.07，-4．76和14．5mV，鱼精蛋白凝聚法方法回收率分别为98.1％，95.3％和86.6％。不同荷电性小分子药物不影响鱼精蛋白凝聚法测定脂质体包封率，但大分子药物荷电性有影响；解离的奥沙西罗（荷负电）和pH2的胰岛素（荷正电）回收率较好，而pH7的胰岛素（荷负电）的回收率则较低。结论 鱼精蛋白凝聚法可以测定不同溶解性质药物脂质体、纳米脂质体的包封率，此法适合小分子药物及正电荷大分子药物脂质体的测定；适于中性或负电荷脂质体的测定。

OBJECTIVE To establish the protamine aggregation method for the drug encapsulation efficiency determinations of liposomes and nanoliposomes. METHODS Protamine aggregation method was applied to separate the free drug and liposomes. The drug concentration was determined by HPLC. The encapsulation efficiency determined by the sephadex chromatography method and protamine aggregation method was compared. The liposomes sizes and zeta potentials, protamine contents and the dissolubilities, charges and molecular weights of drugs were taken as factors to evaluate the feasibility of protamine aggregation method. RESULTS Liposomes containing drugs of different dissolubilities were well separated by protamine aggregation method, Lohexol liposomes（size≥220 nm）and the free lohexol were well separated by sephadex G- 50 chromatography method and protamine aggregation method. The lohexol recovery in blank liposomes declined when the liposomes size decreased using sephadex G - 50 chromatography method, especially when lohexol liposomes size was below 200 nm. Free TFu and TFu liposomes were not be separated by sephadex G- 50 chromatography method due to the lipophilic characteristics of TFu. Free TFu and TFu liposomes （sizes between 100 to 5013 nm） were separated by protamine aggregation method accurately. The separation efficiencies of protamine aggregation method were affected by liposomes electric charges. When the Zeta potentials of TFu liposomes were changed to - 44.07, - 4.76 and 14.5 mV by adjusting the pH of the liposome suspensions, the drug recoverys of protamine aggregation method were 98.1%, 95.3 % and 86.6%, respectively. Micromolecule drugs with different electric charges didn＇t affect separation efficiency of protamine aggregation method. The drug recovery of the dissociated oxaceprol （negative charge）was good. Macromolecule drugs with different electric charges, however, had different effects on the drug recoverys of protamine aggregation method. Insulin（positive charge,pH 2）had good method recovery, but insulin （negative charge, pH 7）had lower drug recovery. CONCLUSION Protamine aggregation method can be used to determine the drug entrap- ment efficiencies of the liposomes and nanoliposomes which encapsulated drugs with different dissolubilities. It is suitable to separate liposomes from encapluated micromolecule drugs and positive charge macromolecule drugs and to separate liposomes from neutral and negative charge drugs.