不同粒径聚乙二醇化维生素K_(1)脂质体的制备及体内外评价

Preparation and in vitro and in vivo evaluation of PEGylated vitamin K_(1)liposomes with different particle sizes

目的制备不同粒径聚乙二醇化维生素K_(1)(VK_(1))脂质体,并对其进行制剂学表征,考察体内药动学和促凝药效。方法采用薄膜分散法制备不同粒径的VK_(1)脂质体,采用马尔文粒径仪测定粒径、聚合物分散性指数(PDI)、Zeta电位;透射电子显微镜观察形态;以粒径为指标,考察脂质体在4°C下储存1个月、在磷酸缓冲液(PBS,pH 6.8)及pH 1.2水溶液中48 h的稳定性;采用超滤离心法测定包封率与载药量;采用大鼠在体肠吸收模型考察肠吸收特性;ig给药考察脂质体在大鼠体内药动学行为;以华法林钠诱导大鼠低凝血酶原血症,采用ELISA试剂盒检测血浆中凝血因子Ⅱ、Ⅴ、Ⅶ和Ⅸ含量,检测凝血酶原时间(PT),评价不同粒径VK_(1)脂质体(2 mg·kg^(−1))促凝效果。结果制备了3种VK_(1)脂质体(Lip-180、Lip-120、Lip-60),粒径分别为(182.40±2.17)nm、(114.38±0.60)nm和(68.42±0.73)nm,PDI分别为0.21±0.01、0.12±0.00和0.17±0.01,电位分别为(−27.67±1.58)、(−22.93±1.81)、(−26.63±1.37)mV,脂质体均呈球形,分布均匀,包封率均>90%,载药量均>2.90%,稳定性良好。与Lip-180相比,Lip-120和Lip-60表现出更缓慢的释放性能和更好的跨膜吸收速率。Lip-120及Lip-60的药时曲线下面积(AUC_(0-∞))分别是Lip-180的1.52和1.80倍,并且Lip-120与Lip-60的AUC_(0-∞)分别是维生素K_(1)注射剂(市售对照)的1.24与1.46倍。与低凝血酶原血症模型大鼠比较,经ig给药Lip-180、Lip-120、Lip-60及维生素K_(1)注射剂后,PT显著降低(P<0.001),凝血因子Ⅱ、Ⅶ、Ⅸ、Ⅹ水平均升高,其中,Lip-60作用最显著,除给药后6 h的凝血因子Ⅹ外,均差异显著(P<0.05、0.01、0.001)。结论聚乙二醇化VK_(1)脂质体分布均匀,稳定性高,释放缓慢,口服生物利用度高,体内促凝效果较好,优选粒径最小的Lip-60。

Objective To formulate and characterize the PEGylated Vitamin K_(1)(VK_(1))liposomes with different particle sizes,and investigate the corresponding pharmacokinetics and coagulation-promoting activity.Methods VK_(1)liposomes with different particle sizes were prepared by thin film dispersion method.Particle size,polymer dispersion index(PDI)and Zeta potential were measured by Malvin particle size analyzer.The morphology was observed by transmission electron microscope.The stability of liposomes at 4℃ for one month and in phosphoric acid buffer solution(PBS,pH 6.8)and pH 1.2 aqueous solution within 48 h were investigated using particle size as index.The encapsulation rate and drug load were determined by ultrafiltration centrifugation.In vivo intestinal absorption model of rats was used to investigate intestinal absorption characteristics.Pharmacokinetic behavior of liposome in rats was investigated by ig administration.The contents of plasma coagulation factors Ⅱ,Ⅴ,Ⅶ and Ⅸ were detected by ELISA kit,and the prothrombin time(PT)was detected in rats with hypothrombinemia induced by warfarin sodium,to evaluat the effect of VK_(1)liposomes(2 mg·kg^(−1))with different particle sizes on promoting coagulation.Results Three kinds of VK_(1)liposomes were prepared,termed as Lip-180,Lip-120 and Lip-60,with a uniform particle size distribution of(182.40±2.17),(114.38±0.60)and(68.42±0.73)nm,and Zeta potentials of(−27.67±1.58),(−22.93±1.81)and(−26.63±1.37)mV,PDI were 0.21±0.01,0.12±0.00 and 0.17±0.01,respectively.Meanwhile,these as-formulated liposomes were uniformly spherical shape of good stability with a satisfied encapsulation efficiency of 90%and a drug loading capability of 2.90%of the drug load.Compared with Lip-180,the in vitro release and in situ intestinal perfusion results demonstrated that Lip-120 and Lip-60 achieved a superior release performance and transmembrane absorption rate.In consistence with the in vitro results,the area under the drug time curve(AUC_(0-∞))of Lip-120 and Lip-60 were 1.52 and 1.80 times of Lip-180,respectively.Lip-120 and Lip-60 generated 1.24-and 1.46-fold higher bioavailability than commercial VK_(1)solution.After oral administration,all of these liposomes and commercial solution were able to decrease plasma PT,and enhance the produce of coagulation factors Ⅱ,Ⅴ,Ⅶ and Ⅸ in warfarin-triggered anticoagulant rat models,where Lip-60 exerted the best procoagulant efficiency.Conclusion Pegylated VK_(1)liposomes have uniform distribution,high stability,slow release,high oral bioavailability,and wonderful in vivo coagulation effect,and Lip-60 with the smallest particle size is preferred.