纳豆激酶微胶囊制备及其释放与Caco-2细胞模型吸收的评价

为防止纳豆激酶在胃中失活,提高其在小肠内的吸收能力,本研究以聚乙二醇-聚乳酸-羟基乙酸共聚物(PEG-PLGA)为壁材,以包封率为指标,通过正交实验探究双重乳液蒸发法制备纳豆激酶微胶囊的最适条件,接着采用上述条件分别以PEG-PLGA和叶酸-聚乙二醇-聚乳酸-羟基乙酸共聚物(FA-PEG-PLGA)为壁材制备纳豆激酶微胶囊,最后对两种微胶囊体外缓释效果、细胞毒性及在Caco-2单层细胞模型中的吸收效果进行评价。结果表明,PEG-PLGA纳豆激酶微胶囊最适制备条件为壁材浓度5 mg/mL、聚乙烯醇(PVA)浓度1%、二级均质转速17500 r/min、二级均质时间7.5 min;PEG-PLGA和FA-PEG-PLGA纳豆激酶微胶囊的平均粒径分别为271.33和255.75 nm,包封率分别为61.54%±2.36%和58.76%±2.54%,ζ电位分别为(-20.17±1.42)和(-24.73±2.36) mV。体外模拟缓释结果表明,经胃环境(pH2.0)2 h后,两种微胶囊中超60%纳豆激酶被保留,经肠环境(pH7.0)22 h缓释效果良好。两种微胶囊对Caco-2细胞均无明显毒性且细胞吸收良好,顶侧表观渗透系数分别高达2.367×10-6和3.497×10-6 cm/s,激光共聚焦显微镜观察结果同样表明两种微胶囊在Caco-2细胞中均有很好的吸收效果,且FA-PEG-PLGA微胶囊比PEG-PLGA微胶囊吸收效果更佳。

Preparation and Release of Nattokinase Microcapsules and Evaluation of Absorption in Caco-2 Cells Model

To protect nattokinase from inactivation and improve its absorption in gastrointestinal tract,the optimum preparation condition of nattokinase microcapsules formed by double emulsion evaporation method was obtained by orthogonal experiment,with poly( ethylene glycol-b-( DL-lactic acid-co-glycolic acid)-b-ethylene glycol)( PEG-PLGA) as the wall material,and entrapment efficiency as the index.Then nattokinase microcapsules were respectively prepared with PEG-PLGA and folate-poly( ethylene glycol-b-( DL-lacticacid-co-glycolic acid)-b-ethylene glycol)( FA-PEG-PLGA) as the wall material,under the optimum condition.Finally,the gastrointestinal release in vitro and cytotoxicity and absorption in Caco-2 monolayer model of the two microcapsules were evaluated. Results showed that,the optimum preparation condition of PEG-PLGA nattokinase microcapsules were listed as follows: Wall material concentration of 5 mg/m L,PVA concentration of 1%,homogenization speed of 17500 r/min and homogenization time of 7.5 min for double emulsion. For PEG-PLGA and FA-PEG-PLGA nattokinase microcapsules,their average particle sizes were 271.33 and 255.75 nm,the encapsulation efficiency was 61.54%± 2.36% and58.76%± 2.54%,the Zeta potential was(-20.17 ± 1.42) and(-24.73 ± 2.36) m V,respectively. Results of gastrointestinal release in vitro showed that more than 60% nattokinase was retained in the two microcapsules after 2 h in gastric pH,and the sustained release effect was good in the intestinal environment( pH7.0) within 22 h.Both microcapsules had almost no toxicity to Caco-2 cells at a concentration of 500 μg/m L,and could be well absorbed,the apparent permeation coefficients of the two microcapsules were as high as 2.367 × 10^-6 and 3.497 × 10^-6 cm/s,respectively. Results of laser confocal microscopy also showed that both microcapsules were well absorbed in Caco-2 cells,and FA-PEG-PLGA microcapsules were absorbed better than PEG-PLGA microcapsules.