T Lian1, R J Ho. 1. Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle 98195-7610, USA.
Abstract
PURPOSE: 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. METHODS: 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. 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 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. CONCLUSIONS: 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.
PURPOSE: To determine whether the non-toxic pentameric B subunit of Cholera toxin (CTB) binding to gangliosideGM1 on both the lipid vesicles and epithelial cells may provide a means to target lipid vesicles to mucosal cells expressing surface GM1. METHODS: Sonicated lipid vesicles containing gangliosideGM1 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. 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 membrane-bound GM1-to-CTB, optimum increase in GM1vesicle 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. CONCLUSIONS: 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.
Authors: Ilya Sotnikov; Tatyana Veremeyko; Sarah C Starossom; Natalia Barteneva; Howard L Weiner; Eugene D Ponomarev Journal: PLoS One Date: 2013-03-26 Impact factor: 3.240