响应面法优化T-2毒素脂质体处方及其抗肿瘤活性
|
摘要
用薄膜超声法制备T-2毒素脂质体(LP-T2),用高效液相色谱(HPLC)法测定包封率,并考察测定方法的线性、加样回收率、精密度和重复性;用单因素法考察有机溶剂、磷脂种类、m(药)∶m(脂)、水合体积、m(磷脂)∶m(胆固醇)对包封率的影响及响应面法优化处方;用高压均质法对脂质体整粒,并测定粒径分布;用噻唑蓝(MTT)法检测T-2毒素和LPT2对5种肿瘤细胞和正常细胞的生长抑制作用.结果表明:LP-T2最佳制备工艺为以蛋黄卵磷脂(EPC)为膜材,薄膜超声法制备,m(药)∶m(脂)=3.5,m(磷脂)∶m(胆固醇)=3.74,4-羟乙基哌嗪乙磺酸(Hepes,20mmol/L,pH=7.4)水合,水合体积11.8mL,超声10min;高压均质整粒后平均粒径为267nm;LP-T2可显著抑制肿瘤细胞的增殖,活性与丝裂霉素相当,对正常细胞的IC_(50)值(1 881.54ng/mL)高于T-2毒素(1 427.83ng/mL),即毒副作用减小;LP-T2的包封率和粒径均符合药剂标准,具有体外抗肿瘤活性.
T-2 toxin liposome(LP-T2)was prepared by thin film ultrasonic method.Entrapment efficiency was determined by high performance liquid chromatography(HPLC)method,and the linearity,additive recovery,precision and repeatability of the method were investigated.Effects of organic solvent,phospholipid species,m(drug)∶m(lipid),hydration volume,m(phospholipids)∶m(cholesterol)on encapsulation efficiency were investigated by single factor method,and the prescription of T-2 toxin liposome was optimized by response surface method.The liposome was granulated by high pressure homogenization and particle size distribution was determined.The inhibitory effects of T-2 toxin and LP-T2 on growth of 5 tumor cells and one normal cell were detected by thiazolyl blue(MTT)method.The results show that the best preparation process of LP-T2 are eggphosphatidylcholine(EPC)as membrance material,thin film ultrasonic method,m(drug)∶m(lipid)=3.5,m(phospholipid)∶m(cholesterol)=3.74,4-hydroxyethyl piperazine ethylsulfonic acid(Hepes,20 mmol/L,pH=7.4)hydration,hydration volume 11.8 mL,ultrasound for 10 min.The average particle size after high pressure homogenization is 267 nm.LP-T2 can significantly inhibit the proliferation of tumor cells,and its activity is similar to that of mitomycin.IC_(50) value of normal cell(1881.54 ng/mL)is higher than that of T-2 toxin(1 427.83 ng/mL).The entrapment efficiency and particle size of LP-T2 conform to the standard of drug,and it has anti-tumor activity in vitro.
T-2 toxin liposome(LP-T2)was prepared by thin film ultrasonic method.Entrapment efficiency was determined by high performance liquid chromatography(HPLC)method,and the linearity,additive recovery,precision and repeatability of the method were investigated.Effects of organic solvent,phospholipid species,m(drug)∶m(lipid),hydration volume,m(phospholipids)∶m(cholesterol)on encapsulation efficiency were investigated by single factor method,and the prescription of T-2 toxin liposome was optimized by response surface method.The liposome was granulated by high pressure homogenization and particle size distribution was determined.The inhibitory effects of T-2 toxin and LP-T2 on growth of 5 tumor cells and one normal cell were detected by thiazolyl blue(MTT)method.The results show that the best preparation process of LP-T2 are eggphosphatidylcholine(EPC)as membrance material,thin film ultrasonic method,m(drug)∶m(lipid)=3.5,m(phospholipid)∶m(cholesterol)=3.74,4-hydroxyethyl piperazine ethylsulfonic acid(Hepes,20 mmol/L,pH=7.4)hydration,hydration volume 11.8 mL,ultrasound for 10 min.The average particle size after high pressure homogenization is 267 nm.LP-T2 can significantly inhibit the proliferation of tumor cells,and its activity is similar to that of mitomycin.IC_(50) value of normal cell(1881.54 ng/mL)is higher than that of T-2 toxin(1 427.83 ng/mL).The entrapment efficiency and particle size of LP-T2 conform to the standard of drug,and it has anti-tumor activity in vitro.
引文
[1]王虎军,薛华丽,赵军,等.T-2毒素的产生、毒性及脱毒研究进展[J].食品安全质量检测学报,2014,5(8):2392-2398.(WANG Hujun,XUE Huali,ZHAO Jun,et al.Advance on Researches of Production,Toxicity and Detoxification of T-2Toxin[J].Journal of Food Safety&Quality,2014,5(8):2392-2398.)
[2]侯毅鞠,袁忠海,国巍,等.镰刀菌T-2毒素对人急性早幼粒白血病细胞HL60增殖与凋亡的影响[J].卫生研究,2013,42(3):381-385.(HOU Yiju,YUAN Zhonghai,GUO Wei,et al.Effects of T-2Toxin of Fusarium on Proliferation and Apoptosis of Human Acute Promyelocytic Leukemia Cellline HL60[J].Journal of Hygiene Research,2013,42(3):381-385.)
[3]晋圣坤.白藜芦醇稳定性及与T-2毒素对HepG2细胞作用的研究[D].重庆:西南大学,2012.(JIN Shengkun.Study on Stability of Resveratrol and Effect of T-2 Toxin and Resveratrol on HepG2Cells[D].Chongqing:Southwest University,2012.)
[4]赵颖,张琳,王澈.抗肿瘤脂质体的制备及其靶向性研究进展[J].中国生化药物杂志,2017,37(1):311-314.(ZHAO Ying,ZHANG Lin,WANG Che.Advances in Studies of Anti-cancer Liposomes with Its Preparation and Targeting[J].Chinese Journal of Biochemical Pharmaceutics,2017,37(1):311-314.)
[5]何文,胡翠萍,郭咸希.水飞蓟宾脂质体制备方法研究[J].中国药业,2016,25(1):49-52.(HE Wen,HU Cuiping,GUO Xianxi.Studies on the Preparation Methods of Silybin Liposomes[J].China Pharmaceuticals,2016,25(1):49-52.)
[6]王宝宝,武元,付强,等.高效液相色谱法测定血清碘海醇浓度方法的建立和应用[J].基础医学与临床,2015,35(7):963-967.(WANG Baobao,WU Yuan,FU Qiang,et al.Determination of Iohexol Concentration in Serum and Its Clinical Application by HPLC in Children[J].Basic&Clinical Medicine,2015,35(7):963-967.)
[7]杨雪薇,杨爽,董冰雪,等.用统计学方法优化硝苯地平脂质体处方[J].吉林大学学报(理学版),2015,53(3):572-576.(YANG Xuewei,YANG Shuang,DONG Bingxue,et al.Optimize the Preparation Formulation of Nifedipine Liposome by Statistical Methods[J].Journal of Jilin University(Science Edition),2015,53(3):572-576.)
[8]施政,王建平,章建军,等.响应面法优化穿山龙总皂苷提取工艺[J].中国现代应用药学,2017,34(1):68-71.(SHI Zheng,WANG Jianping,ZHANG Jianjun,et al.Optimizing the Extraction Process of the Total Saponin of Dioscoreae Nipponicae Rhizoma by Response Surface Methodology[J].Chinese Journal of Modern Applied Pharmacy,2017,34(1):68-71.)
[9]李向春,邸瑞玲,董立新.MTT法检测化疗药物对口腔癌细胞体外抑制率的结果分析[J].中国煤炭工业医学杂志,2017,20(4):402-405.(LI Xiangchun,DI Ruiling,DONG Lixin.The Inhibitory Rate of Chemotherapeutic Drugs on Oral Carcinoma Cells in vitro by MTT Method[J].Chinese Journal of Coal Industry Medicine,2017,20(4):402-405.)
[2]侯毅鞠,袁忠海,国巍,等.镰刀菌T-2毒素对人急性早幼粒白血病细胞HL60增殖与凋亡的影响[J].卫生研究,2013,42(3):381-385.(HOU Yiju,YUAN Zhonghai,GUO Wei,et al.Effects of T-2Toxin of Fusarium on Proliferation and Apoptosis of Human Acute Promyelocytic Leukemia Cellline HL60[J].Journal of Hygiene Research,2013,42(3):381-385.)
[3]晋圣坤.白藜芦醇稳定性及与T-2毒素对HepG2细胞作用的研究[D].重庆:西南大学,2012.(JIN Shengkun.Study on Stability of Resveratrol and Effect of T-2 Toxin and Resveratrol on HepG2Cells[D].Chongqing:Southwest University,2012.)
[4]赵颖,张琳,王澈.抗肿瘤脂质体的制备及其靶向性研究进展[J].中国生化药物杂志,2017,37(1):311-314.(ZHAO Ying,ZHANG Lin,WANG Che.Advances in Studies of Anti-cancer Liposomes with Its Preparation and Targeting[J].Chinese Journal of Biochemical Pharmaceutics,2017,37(1):311-314.)
[5]何文,胡翠萍,郭咸希.水飞蓟宾脂质体制备方法研究[J].中国药业,2016,25(1):49-52.(HE Wen,HU Cuiping,GUO Xianxi.Studies on the Preparation Methods of Silybin Liposomes[J].China Pharmaceuticals,2016,25(1):49-52.)
[6]王宝宝,武元,付强,等.高效液相色谱法测定血清碘海醇浓度方法的建立和应用[J].基础医学与临床,2015,35(7):963-967.(WANG Baobao,WU Yuan,FU Qiang,et al.Determination of Iohexol Concentration in Serum and Its Clinical Application by HPLC in Children[J].Basic&Clinical Medicine,2015,35(7):963-967.)
[7]杨雪薇,杨爽,董冰雪,等.用统计学方法优化硝苯地平脂质体处方[J].吉林大学学报(理学版),2015,53(3):572-576.(YANG Xuewei,YANG Shuang,DONG Bingxue,et al.Optimize the Preparation Formulation of Nifedipine Liposome by Statistical Methods[J].Journal of Jilin University(Science Edition),2015,53(3):572-576.)
[8]施政,王建平,章建军,等.响应面法优化穿山龙总皂苷提取工艺[J].中国现代应用药学,2017,34(1):68-71.(SHI Zheng,WANG Jianping,ZHANG Jianjun,et al.Optimizing the Extraction Process of the Total Saponin of Dioscoreae Nipponicae Rhizoma by Response Surface Methodology[J].Chinese Journal of Modern Applied Pharmacy,2017,34(1):68-71.)
[9]李向春,邸瑞玲,董立新.MTT法检测化疗药物对口腔癌细胞体外抑制率的结果分析[J].中国煤炭工业医学杂志,2017,20(4):402-405.(LI Xiangchun,DI Ruiling,DONG Lixin.The Inhibitory Rate of Chemotherapeutic Drugs on Oral Carcinoma Cells in vitro by MTT Method[J].Chinese Journal of Coal Industry Medicine,2017,20(4):402-405.)