苦参碱长循环脂质体的制备及药效学研究
摘要
目的：
临床上用来治疗肝纤维化的苦参碱注射液存在体内分布广泛,易被代谢,副作用大等缺陷。为了改善这些不足,本课题制备苦参碱长循环脂质体,将脂质体包封的苦参碱导向肝星状纤维细胞,提高药物在肝星状细胞中的浓度；通过体外观察对肝星状细胞增殖的影响,评价苦参碱长循环脂质体对肝星状细胞抗肝纤维化的作用,为研发苦参碱抗肝纤维化提供药剂学和药效学实验依据。
方法：
制备苦参碱脂质体,观察脂质体的成膜情况；考察工艺因素对脂质体包封率的影响,采用正交实验优化工艺参数；考察超声条件对脂质体稳定性的影响。对脂质体的制备方法(薄膜分散法、逆相蒸发法、乙醇注入法、乙醚注入法)进行了比较。采用逆相蒸发法,以包封率为考察指标优选最佳处方及制备工艺,研制出苦参碱长循环脂质体；通过层析法、微柱离心法、高速离心法和透析法对含药脂质体和游离药物的分离度进行了考察,选用微柱离心法,分段收集离心液进HPLC法考察洗脱液对苦参碱脂质体包封率的影响；用负染色电镜观察脂质体的形态；用动态光散射法测定粒度分布和平均粒径；在室温与3℃条件下比较脂质体的稳定性；用动态透析法考察脂质体的体外释药特性；用10%的DMEM培养液于37℃,5%CO2培养箱中培养肝星状纤维细胞,于酶标仪490nm测定各孔的OD值,用抑制率衡量不同苦参碱制剂对肝星状细胞的抑制效果。
结果：
采用逆相蒸发法制备脂质体成膜效果好,以包封率为指标确定的最佳工艺条件压力为0.055±0.005Mpa、温度为40℃、转速为100r/min旋转蒸发1h得到脂质体,最佳处方条件为卵磷脂：胆固醇脂比为8、卵磷脂：苦参碱比为8、长效剂的量为8%、pH值为6.8,在此条件下制备的长效苦参碱脂质体,用HPLC法测定药物的含量,峰面积定量线性关系良好,药物包封率达80%以上,载药量达7%以上,符合相关制剂规定；0.9%NaCl溶液作为微柱离心的洗脱溶剂,脂质体与游离药物能得到很好的分离；含药脂质体在200W的细胞粉碎仪中超声3min,间隔1s超声1s,在电镜下观察脂质体为圆形、椭圆形粒子,平均粒径在100nm左右。脂质体放在3℃条件下,产品比较稳定。不同苦参碱制剂对肝星状细胞实验比较,长效苦参碱脂质体对肝星状细胞有明显的抑制作用。
结论：
用逆相蒸发法制备苦参碱脂质体的工艺可行,药物的包封率高,体外肝星状细胞试验表明,苦参碱脂质体比常规苦参碱注射液的效果好,达到试验设计的目的。
Objective
Matrine injection used to treat liver fibrosis in vivo have some defects, such as widely distributed, easily metabolism, side effects and so on. For this reason, a long-acting matrine liposome was prepared in this work, which can orient the hepatic stellate cells and increase the concentration of matrine in the hepatic stellate cells. According to the effect of martine liposome on the proliferation of hepatic stellate cells in vitro, the anti-liver fibrosis effect of martine liposome can evaluated. And this work could provide experimental evidences of the pharmacodynamics for the development of novel preparations of matrine anti-liver fibrosis.
Methods
Matrine liposome was prepared and the film of the liposomes was observed. The differences of these preparations, film dispersion method, reverse evaporation method, ethanol injection method, and ethyl ether injection method were investigated. Taking encapsulation efficiency as the marker the best prescription and process was optimized with orthogonal experimental design method. And the long-acting matrine liposome was prepared with reverse evaporation method. The resolution between drug-containing liposome and free drug was explored by the chromatography, microcolumn centrifugation, the high-speed centrifugation and dialysis method; the effect of the eluent on encapsulation rate of matrine liposome was studied with HPLC through microcolumn centrifugation and segmented collection of centrifugal; the appearance of liposome was observed by TEM with negative staining; the particle diameter of liposome was measured by dynamic light scattering method; the in vitro release of the liposome was examined by dynamic dialysis method; stability of Liposome was compared under the conditions of room temperature and3℃. Under the condition:10%DMEM culture liquid was cultured at37℃,5%CO2incubator, the inhibitory effect of the new matrine liposome on hepatic stellate cells was studied by inhibition rate.
Results
The matrine liposome was prepared with reverse evaporation technique. One optimum recipes of long-acting matrine liposome, obtained from orthogonal experimental design, showed that cholesterol/lecithin8:1, cholesterol/matrine8:1, pH6.8under the optimum conditions is0.055±0.005Mpa,40℃,100r/min for1h. The results showed that using the defined method, we prepared the matrine liposome with new dosage form:it has round, oval-shaped particles with average particle size of about100nm through TEM. Research indicated that the measuration of encapsulation efficiency is reliable, and measured the encapsulation efficiency is80%or more. Using HPLC we found that both the peak figure of drug and standard curve were ideal, the liposome was separated well with0.9%NaCl solution as a micro-column centrifugal elution solvent. In vivo experiment showed that long-acting matrin liposome targeting and long-acting matrine liposome can inhibit the grown of hepatic stellate cells.
Conclusion
New dosage form technology for matrine of reverse-phase evaporation method is feasible, the experiment results of hepatic stellate cells show that the effects of new dosage form for matrine was better than that of conventional matrine and the purpose of the trial design had achieved.
临床上用来治疗肝纤维化的苦参碱注射液存在体内分布广泛,易被代谢,副作用大等缺陷。为了改善这些不足,本课题制备苦参碱长循环脂质体,将脂质体包封的苦参碱导向肝星状纤维细胞,提高药物在肝星状细胞中的浓度；通过体外观察对肝星状细胞增殖的影响,评价苦参碱长循环脂质体对肝星状细胞抗肝纤维化的作用,为研发苦参碱抗肝纤维化提供药剂学和药效学实验依据。
方法：
制备苦参碱脂质体,观察脂质体的成膜情况；考察工艺因素对脂质体包封率的影响,采用正交实验优化工艺参数；考察超声条件对脂质体稳定性的影响。对脂质体的制备方法(薄膜分散法、逆相蒸发法、乙醇注入法、乙醚注入法)进行了比较。采用逆相蒸发法,以包封率为考察指标优选最佳处方及制备工艺,研制出苦参碱长循环脂质体；通过层析法、微柱离心法、高速离心法和透析法对含药脂质体和游离药物的分离度进行了考察,选用微柱离心法,分段收集离心液进HPLC法考察洗脱液对苦参碱脂质体包封率的影响；用负染色电镜观察脂质体的形态；用动态光散射法测定粒度分布和平均粒径；在室温与3℃条件下比较脂质体的稳定性；用动态透析法考察脂质体的体外释药特性；用10%的DMEM培养液于37℃,5%CO2培养箱中培养肝星状纤维细胞,于酶标仪490nm测定各孔的OD值,用抑制率衡量不同苦参碱制剂对肝星状细胞的抑制效果。
结果：
采用逆相蒸发法制备脂质体成膜效果好,以包封率为指标确定的最佳工艺条件压力为0.055±0.005Mpa、温度为40℃、转速为100r/min旋转蒸发1h得到脂质体,最佳处方条件为卵磷脂：胆固醇脂比为8、卵磷脂：苦参碱比为8、长效剂的量为8%、pH值为6.8,在此条件下制备的长效苦参碱脂质体,用HPLC法测定药物的含量,峰面积定量线性关系良好,药物包封率达80%以上,载药量达7%以上,符合相关制剂规定；0.9%NaCl溶液作为微柱离心的洗脱溶剂,脂质体与游离药物能得到很好的分离；含药脂质体在200W的细胞粉碎仪中超声3min,间隔1s超声1s,在电镜下观察脂质体为圆形、椭圆形粒子,平均粒径在100nm左右。脂质体放在3℃条件下,产品比较稳定。不同苦参碱制剂对肝星状细胞实验比较,长效苦参碱脂质体对肝星状细胞有明显的抑制作用。
结论：
用逆相蒸发法制备苦参碱脂质体的工艺可行,药物的包封率高,体外肝星状细胞试验表明,苦参碱脂质体比常规苦参碱注射液的效果好,达到试验设计的目的。
Objective
Matrine injection used to treat liver fibrosis in vivo have some defects, such as widely distributed, easily metabolism, side effects and so on. For this reason, a long-acting matrine liposome was prepared in this work, which can orient the hepatic stellate cells and increase the concentration of matrine in the hepatic stellate cells. According to the effect of martine liposome on the proliferation of hepatic stellate cells in vitro, the anti-liver fibrosis effect of martine liposome can evaluated. And this work could provide experimental evidences of the pharmacodynamics for the development of novel preparations of matrine anti-liver fibrosis.
Methods
Matrine liposome was prepared and the film of the liposomes was observed. The differences of these preparations, film dispersion method, reverse evaporation method, ethanol injection method, and ethyl ether injection method were investigated. Taking encapsulation efficiency as the marker the best prescription and process was optimized with orthogonal experimental design method. And the long-acting matrine liposome was prepared with reverse evaporation method. The resolution between drug-containing liposome and free drug was explored by the chromatography, microcolumn centrifugation, the high-speed centrifugation and dialysis method; the effect of the eluent on encapsulation rate of matrine liposome was studied with HPLC through microcolumn centrifugation and segmented collection of centrifugal; the appearance of liposome was observed by TEM with negative staining; the particle diameter of liposome was measured by dynamic light scattering method; the in vitro release of the liposome was examined by dynamic dialysis method; stability of Liposome was compared under the conditions of room temperature and3℃. Under the condition:10%DMEM culture liquid was cultured at37℃,5%CO2incubator, the inhibitory effect of the new matrine liposome on hepatic stellate cells was studied by inhibition rate.
Results
The matrine liposome was prepared with reverse evaporation technique. One optimum recipes of long-acting matrine liposome, obtained from orthogonal experimental design, showed that cholesterol/lecithin8:1, cholesterol/matrine8:1, pH6.8under the optimum conditions is0.055±0.005Mpa,40℃,100r/min for1h. The results showed that using the defined method, we prepared the matrine liposome with new dosage form:it has round, oval-shaped particles with average particle size of about100nm through TEM. Research indicated that the measuration of encapsulation efficiency is reliable, and measured the encapsulation efficiency is80%or more. Using HPLC we found that both the peak figure of drug and standard curve were ideal, the liposome was separated well with0.9%NaCl solution as a micro-column centrifugal elution solvent. In vivo experiment showed that long-acting matrin liposome targeting and long-acting matrine liposome can inhibit the grown of hepatic stellate cells.
Conclusion
New dosage form technology for matrine of reverse-phase evaporation method is feasible, the experiment results of hepatic stellate cells show that the effects of new dosage form for matrine was better than that of conventional matrine and the purpose of the trial design had achieved.
引文
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