穿膜肽修饰多功能靶向阿霉素脂质体处方优选及理化性质研究

Formulation Optimization and Physicochemical Properties of Multifunctional Targeting Doxorubicin Liposomes Modified by Cell Penetrating Peptides

目的对以PFVYLI(PFVY)修饰的阿霉素与五味子乙素共载脂质体进行处方优化,并建立脂质体中阿霉素和五味子乙素的含量测定方法。对制备得到的脂质体进行物理化学性质及药效学研究。方法利用薄膜-超声分散法与硫酸梯度法结合的方式制备PFVY修饰的阿霉素与五味子乙素脂质体。利用Box-Behnken响应面法对拟定处方中影响最终包封率的3因素(膜材比例、药脂比例、超声功率)进行优化。利用高效液相色谱法(HPLC)对脂质体中五味子乙素和阿霉素进行含量测定。通过透射电子显微镜及马尔文激光粒度仪对已制备的多功能阿霉素靶向脂质体形态、粒径、Zeta电位等理化性质进行研究。以含10%胎牛血清(FBS)的磷酸缓冲盐溶液(PBS)为释放介质,分别测定阿霉素与五味子乙素的体外释放率。利用不同组制剂对A549细胞和小鼠肺癌细胞(LLC)的细胞毒性试验进行药效学评价。结果优选后的处方为磷脂44 mg,胆固醇8 mg,五味子乙素1.8 mg,阿霉素0.49 mg,超声功率500 W,处方量为5 mL。此配方下的包封率为(96.30%±0.98)%(n=3),脂质体中五味子乙素含量为(359.80±1.26)μg/mL(n=3),阿霉素含量为(98.45±0.70)μg/mL(n=3)。制备得到的多功能靶向脂质体形态近球形,粒径适宜均匀,阿霉素和五味子乙素在48 h时的体外释放率分别为(32.91±2.08)%和(32.29±0.89)%(n=3)。体外药效学也在一定程度显示穿膜肽PFVY的作用与五味子乙素对阿霉素细胞毒性的增效作用。结论优化后所得处方稳定,包封率高,适用于PFVY修饰的阿霉素与五味子乙素脂质体的制备。针对脂质体建立的HPLC法准确且重现性好,可应用于五味子乙素和阿霉素的含量测定。制备得到的多功能靶向脂质体具有较好的临床应用潜力。

Objective To optimize the formulation of PFVY modified doxorubicin plus schisandrin B liposomes and to establish a method for content determination of PFVY modified doxorubicin plus schisandrin B liposomes. Methods PFVY modified doxorubicin plus schisandrin B liposomes was prepared by film-ultrasonic diffusion and sulfuric acid gradient method. Three factors,membrane material ratio,drug lipid ratio and ultrasonic power,that affect the encapsulation efficiency were optimized by Box-Behnken design methodology. The content of schisandrin B and doxorubicin in liposomes was determined by HPLC. Physicochemical properties(morphology,particle size,Zeta potential)were studied by transmission electron microscopy and Marvin laser particle size analyzer. The in vitro release rates of doxorubicin and schisandrin B were determined by PBS containing 10% FBS. The pharmacodynamics of varying formulation on A549 cells and Lewis lung cancer cells(LLC)were evaluated. Results The optimal formulation of 5 m L PFVY modified doxorubicin plus schisandrin B liposomes was consist of phospholipid 44 mg,cholesterol 8 mg,schisandrin B 1.8 mg,doxorubicin 0.49 mg,ultrasonic power 500 W. The encapsulation efficiency of liposomes was(96.30±0.98)%(n=3),the content of schisandrin B in liposomes was(359.80±1.26)μg/m L(n=3),and the content of doxorubicin was(98.45±0.70)μg/m L(n=3). The release rates of doxorubicin and schisandrin B were(32.91±2.08)% and(32.29±0.89)%(n=3),respectively. In vitro pharmacodynamics also showed that PFVY and schisandrin B had synergistic effect on doxorubicin cytotoxicity. Conclusion The optimized formulation is stable and suitable for the preparation of PFVY modified doxorubicin plus schisandrin B liposomes. The HPLC method is accurate and reproducible,and can be used for the determination of schisandrin B and doxorubicin.The prepared multifunctional targeting liposomes have potential clinical application.