CHOLERA-TOXIN B-MEDIATED TARGETING OF LIPID VESICLES CONTAINING GANGLIOSIDE GM1 TO MUCOSAL EPITHELIAL-CELLS

Purpose. To determine whether the non-toxic pentameric B subunit of Ch olera toxin (CTB) binding to ganglioside GM1 on both the lipid vesicle s and epithelial cells may provide a means to target lipid vesicles to mucosal cells expressing surface GM1. Methods. Sonicated lipid vesicl es containing ganglioside GM1 were prepared. Inter-vesicle cross-linki ng due to pentameric CTB binding to these GM1 vesicles was determined with a sub-micron particle analyzer. Association of CTB to GM1 vesicle s was analyzed with continuous sucrose gradient centrifugation. CTB-me diated binding of GM1 vesicles to human mucosal epithelial cells (Caco -2 and HT-29), mucous membranes of mouse trachea, and nasal tissues we re detected with fluorescent labeled vesicles. Results. 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 membr ane-bound GM1-to-CTB, optimum increase in GM1 vesicle aggregation, was detected. Under such conditions, all the added CTB molecules were ass ociated with GM1 vesicles. Time course analysis showed that inter-vesi cles cross linking by CTB was detectable within 10 min. and reached a maximum value at 60 min. CTB associated GM1-vesicles bind to mucosal e pithelial cells HT-29 and Caco-2 with similar affinity [K-d = 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 antibod y and the insignificant degree of CTB-associated GM1 vesicle binding t o GM1 deficient C6 cells. Conclusions. The CTB-mediated GM1 binding to multiple membrane surfaces provides selective localization of GM1 ves icles to GM1 expressing mucosal cells and tissues. The strategy may be useful in localizing drugs and proteins to gut and respiratory tract mucosa.