Lian T, Ho R J
Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle 98195-7610, USA.
Pharm Res. 1997 Oct;14(10):1309-15. doi: 10.1023/a:1012195932724.
To determine whether the non-toxic pentameric B subunit of Cholera toxin (CTB) binding to ganglioside GM1 on both the lipid vesicles and epithelial cells may provide a means to target lipid vesicles to mucosal cells expressing surface GM1.
Sonicated lipid vesicles containing ganglioside GM1 were prepared. Inter-vesicle cross-linking due to pentameric CTB binding to these GM1 vesicles was determined with a sub-micron particle analyzer. Association of CTB to GM1 vesicles was analyzed with continuous sucrose gradient centrifugation. CTB-mediated binding of GM1 vesicles to human mucosal epithelial cells (Caco-2 and HT-29), mucous membranes of mouse trachea, and nasal tissues were detected with fluorescent labeled vesicles.
An increase in lipid particle size due to binding of CTB to lipid vesicles and inter-vesicles cross-linking was detected. At a 30-to-1 mole ratio of membrane-bound GM1-to-CTB, optimum increase in GM1 vesicle aggregation, was detected. Under such conditions, all the added CTB molecules were associated with GM1 vesicles. Time course analysis showed that inter-vesicles cross linking by CTB was detectable within 10 min. and reached a maximum value at 60 min. CTB associated GM1-vesicles bind to mucosal epithelial cells HT-29 and Caco-2 with similar affinity [Kd = 7.8 x 10(-4) M lipid (Caco-2) and 7.6 x 10(-4) M lipid (HT-29)]. GM1 mediated binding specificity was demonstrated by blocking with anti-GM1 antibody and the insignificant degree of CTB-associated GM1 vesicle binding to GM1 deficient C6 cells.
The CTB-mediated GM1 binding to multiple membrane surfaces provides selective localization of GM1 vesicles to GM1 expressing mucosal cells and tissues. The strategy may be useful in localizing drugs and proteins to gut and respiratory tract mucosa.
确定霍乱毒素（CTB）无毒的五聚体B亚基与脂质囊泡和上皮细胞上的神经节苷脂GM1结合，是否可为将脂质囊泡靶向至表达表面GM1的黏膜细胞提供一种方法。
制备含有神经节苷脂GM1的超声破碎脂质囊泡。使用亚微米颗粒分析仪测定由于五聚体CTB与这些GM1囊泡结合导致的囊泡间交联。通过连续蔗糖梯度离心分析CTB与GM1囊泡的结合。用荧光标记的囊泡检测CTB介导的GM1囊泡与人黏膜上皮细胞（Caco-2和HT-29）、小鼠气管黏膜和鼻组织的结合。
检测到由于CTB与脂质囊泡结合及囊泡间交联导致脂质颗粒大小增加。在膜结合GM1与CTB的摩尔比为30∶1时，检测到GM1囊泡聚集的最佳增加。在这种条件下，所有添加的CTB分子都与GM1囊泡结合。时间进程分析表明，CTB介导的囊泡间交联在10分钟内可检测到，并在60分钟时达到最大值。CTB相关的GM1囊泡以相似的亲和力[解离常数（Kd）=7.8×10⁻⁴M脂质（Caco-2）和7.6×10⁻⁴M脂质（HT-29）]与黏膜上皮细胞HT-29和Caco-2结合。通过用抗GM1抗体阻断以及CTB相关的GM1囊泡与GM1缺陷的C6细胞结合程度不显著，证明了GM1介导的结合特异性。
CTB介导的GM1与多个膜表面的结合，使GM1囊泡选择性定位于表达GM1的黏膜细胞和组织。该策略可能有助于将药物和蛋白质定位于肠道和呼吸道黏膜。
