HepG2.2.15细胞对同时包载丁香苦苷和羟基酪醇纳米粒的摄取机制研究(1)
摘要：目的 考察HepG2.2.15细胞对同时包载丁香苦苷和羟基酪醇纳米粒(nanoparticles co-loaded with syringopicroside and hydroxytyrosol，SH-NPs)的摄取机制。方法 采用沉淀法制备SH-NPs，以异硫氰酸荧光素为荧光标记物，采用流式细胞仪研究HepG2.2.15细胞对SH-NPs的摄取机制。结果 秋水仙素为抑制剂，孵育时间在0.5～24 h范围内，阳性细胞百分数由1.9%增加到56.4%；药物浓度为125、250、500 ?g/mL时，阳性细胞百分数分别为4.9%、3.4%、3.9%。氯喹为抑制剂，孵育时间在0.5～24 h范围内，阳性细胞百分数由7.4%增加到55.4%；药物浓度为125、250、500 ?g/mL时，阳性细胞百分数分别为19.5%、22.5%、27.6%。结论 秋水仙素与氯喹对HepG2.2.15细胞摄取有抑制作用，且HepG2.2.15细胞对SH-NPs的摄取与药物浓度、孵育时间呈正相关，推断HepG2.2.15细胞对SH-NPs细胞的摄取机制为非特异性吸附内吞。
关键词：丁香苦苷；羟基酪醇；聚乙二醇-聚乳酸乙醇酸共聚物纳米粒；HepG2.2.15细胞；摄取机制
DOI：10.3969/j.issn.1005-5304.2018.03.018
中图分类号：R285.5 文献标识码：A 文章编号：1005-5304(2018)03-0081-05
Abstract： Objective To investigate the uptake mechanism of HepG2.2.15 cells to the nanoparticles co-loaded with syringopicroside and hydroxytyrosol (SH-NPs). Methods The nanoparticles were prepared by using a nanoprecipitation method with mPEG-PLGA as nano-carrier co-loaded with syringopicroside and hydroxytyrosol. The uptake mechanism of HepG2.2.15 cells to SH-NPs was studied by fluorescence microscopy and flow cytometry using fluoresceineisothiocyanate (FITC) as a fluorescent marker. Results With colchicine as the inhibitor， the incubation time ranged from 0.5 to 24 h， the percentage of positive cells increased from 1.9% to 56.4%； When the drug concentration was 125， 250 ?g/mL and 500 ?g/mL， the positive cell percentages were 4.9%， 3.4% and 3.9%. With chloroquine as the inhibitor； the incubation time ranged from 0.5 to 24 h， the percentage of positive cells increased from 7.4% to 55.4%； When the drug concentration was 125， 250 and 500 ?g/mL， the percentage of positive cells was 19.5%， 22.5% and 27.6%. Conclusion Colchicine and chloroquine have an inhibitory effect on HepG2.2.15 cells uptake， and the uptake of SH-NPs in HepG2.2.15 cells was positively correlated with drug concentration and incubation time. It can be concluded that the uptake mechanism of HepG2.2.15 cells to SH-NPs was nonspecific adsorption endocytosis.
Keywords： syringopicroside； hydroxytyrosol； mPEG-PLGA nanoparticles； HepG2.2.15 cells； uptake mechanism
丁香苦苷(syringopicroside)和羟基酪醇(hydroxytyrosol)是木犀科植物洋丁香、朝鲜丁香或紫丁香干燥叶中的活性物质，二者体内代谢迅速，稳定性及肝靶向性差，常规给药途径不利于药效的发挥。近年来，由于肝癌发病率逐渐增高，因此，开发提高丁香苦苷和羟基酪醇靶向于肝细胞的新型递药系统是目前亟需解决的问题。
中药纳米递药系统不仅具有较强的靶向性，而且在生物利用度和缓释功能方面也具有较大优势。研究发现，納米粒(NPs)的表面用聚乙二醇(PEG)等亲水性的高分子材料修饰后，能够形成一层水化保护膜，减少巨噬细胞对纳米粒的识别和吞噬，同时能延长纳米粒血液循环时间，从而产生缓释作用，提高生物利用度，降低毒副作用，为药物输送提供了新途径[1-2]。本课题组将二者制备成聚乙二醇-聚乳酸乙醇酸共聚物(monomethoxy polyethylene glycol-poly lactide- co-glycolide，mPEG-PLGA)纳米递药系统，并且于前期进行了关于同时包载丁香苦苷和羟基酪醇纳米粒(nanoparticles co-loaded with syringopicroside and hydroxytyrosol，SH-NPs)的制剂性状研究，结果表明，SH-NPs总包封率为(32.38±2.76)%，总药物负荷为(12.01±0.42)%，粒径为(91.70±2.11)nm，多分散指数为0.22±0.01，通过透射电子显微镜观察， SH-NPs颗粒形态圆整，均匀分布，且对肝脏具有较强的靶向性[3]，同时，经过HepG2.2.15细胞对SH-NPs摄取研究，发现SH-NPs能够被HepG2.2.15细胞摄取，在此基础上进一步进行细胞摄取机制的研究，选择秋水仙素和氯喹为内吞抑制剂，以异硫氰酸荧光素(fluoresceine isothiocyanate，FITC)为荧光标记物，用纳米沉淀法制备SH-NPs，以HepG2.2.15细胞为模型细胞，借助流式细胞仪研究HepG2.2.15细胞对SH-NPs的细胞摄取机制，为存在肝靶向性差、稳定性差、半衰期短等缺陷的药物的纳米化提供新思路。, 百拇医药(管庆霞 李云行 吕邵娃 孙佳琳 张亮 封文静 王利萍 李永吉)
关键词：丁香苦苷；羟基酪醇；聚乙二醇-聚乳酸乙醇酸共聚物纳米粒；HepG2.2.15细胞；摄取机制
DOI：10.3969/j.issn.1005-5304.2018.03.018
中图分类号：R285.5 文献标识码：A 文章编号：1005-5304(2018)03-0081-05
Abstract： Objective To investigate the uptake mechanism of HepG2.2.15 cells to the nanoparticles co-loaded with syringopicroside and hydroxytyrosol (SH-NPs). Methods The nanoparticles were prepared by using a nanoprecipitation method with mPEG-PLGA as nano-carrier co-loaded with syringopicroside and hydroxytyrosol. The uptake mechanism of HepG2.2.15 cells to SH-NPs was studied by fluorescence microscopy and flow cytometry using fluoresceineisothiocyanate (FITC) as a fluorescent marker. Results With colchicine as the inhibitor， the incubation time ranged from 0.5 to 24 h， the percentage of positive cells increased from 1.9% to 56.4%； When the drug concentration was 125， 250 ?g/mL and 500 ?g/mL， the positive cell percentages were 4.9%， 3.4% and 3.9%. With chloroquine as the inhibitor； the incubation time ranged from 0.5 to 24 h， the percentage of positive cells increased from 7.4% to 55.4%； When the drug concentration was 125， 250 and 500 ?g/mL， the percentage of positive cells was 19.5%， 22.5% and 27.6%. Conclusion Colchicine and chloroquine have an inhibitory effect on HepG2.2.15 cells uptake， and the uptake of SH-NPs in HepG2.2.15 cells was positively correlated with drug concentration and incubation time. It can be concluded that the uptake mechanism of HepG2.2.15 cells to SH-NPs was nonspecific adsorption endocytosis.
Keywords： syringopicroside； hydroxytyrosol； mPEG-PLGA nanoparticles； HepG2.2.15 cells； uptake mechanism
丁香苦苷(syringopicroside)和羟基酪醇(hydroxytyrosol)是木犀科植物洋丁香、朝鲜丁香或紫丁香干燥叶中的活性物质，二者体内代谢迅速，稳定性及肝靶向性差，常规给药途径不利于药效的发挥。近年来，由于肝癌发病率逐渐增高，因此，开发提高丁香苦苷和羟基酪醇靶向于肝细胞的新型递药系统是目前亟需解决的问题。
中药纳米递药系统不仅具有较强的靶向性，而且在生物利用度和缓释功能方面也具有较大优势。研究发现，納米粒(NPs)的表面用聚乙二醇(PEG)等亲水性的高分子材料修饰后，能够形成一层水化保护膜，减少巨噬细胞对纳米粒的识别和吞噬，同时能延长纳米粒血液循环时间，从而产生缓释作用，提高生物利用度，降低毒副作用，为药物输送提供了新途径[1-2]。本课题组将二者制备成聚乙二醇-聚乳酸乙醇酸共聚物(monomethoxy polyethylene glycol-poly lactide- co-glycolide，mPEG-PLGA)纳米递药系统，并且于前期进行了关于同时包载丁香苦苷和羟基酪醇纳米粒(nanoparticles co-loaded with syringopicroside and hydroxytyrosol，SH-NPs)的制剂性状研究，结果表明，SH-NPs总包封率为(32.38±2.76)%，总药物负荷为(12.01±0.42)%，粒径为(91.70±2.11)nm，多分散指数为0.22±0.01，通过透射电子显微镜观察， SH-NPs颗粒形态圆整，均匀分布，且对肝脏具有较强的靶向性[3]，同时，经过HepG2.2.15细胞对SH-NPs摄取研究，发现SH-NPs能够被HepG2.2.15细胞摄取，在此基础上进一步进行细胞摄取机制的研究，选择秋水仙素和氯喹为内吞抑制剂，以异硫氰酸荧光素(fluoresceine isothiocyanate，FITC)为荧光标记物，用纳米沉淀法制备SH-NPs，以HepG2.2.15细胞为模型细胞，借助流式细胞仪研究HepG2.2.15细胞对SH-NPs的细胞摄取机制，为存在肝靶向性差、稳定性差、半衰期短等缺陷的药物的纳米化提供新思路。, 百拇医药(管庆霞 李云行 吕邵娃 孙佳琳 张亮 封文静 王利萍 李永吉)