36 ENDOCYTOSIS UPTAKE OF LIPOSOMES IN UROTHELIUM CELLS DETECTED BY TRANSMISSION ELECTRON MICROSCOPY

Abstract

INTRODUCTION AND OBJECTIVES: Intravesical liposomes (phospholipid membrane vesicles) have been used as a drug delivery platform and more recently, empty liposomes has shown promise as a treatment of interstitial cystitis/painful bladder syndrome. The exact mechanism for liposome uptake in bladder cells remains to be investigated and in the present study, we investigated the role of endocytosis in uptake of liposomes by urothelium cells. METHODS: Urothelial cell line (UROtsa) were left to adhere and grow for 48h in 100mm petridishes using Dulbecco modified Eagle medium (DMEM) containing 5% v/v fetal bovine serum with incubation at 37°C in a 5% CO2:95% air atmosphere. Liposomes encapsulating colloidal gold particles size 10nm were prepared by thin film hydration method and were incubated with serum free UROtsa cells for 2h either at 37°C or at 4°C. The morphology of liposome–cell interactions at two temperatures was assessed by transmission electron microscopy. RESULTS: Transmission Electron Microscopy (TEM) images located endocytic vesicles containing liposomes inside the UROtsa cells incubated at 37°C [panel B of attached picture] as indicated by dark stain of colloidal gold particles 10nm. In contrast, only extracellular binding was noticed in cells incubated at 4°C[panel A of attached picture]. Thin section electron micrographs show gold–containing liposomes trapped in intracellular vesicles, and in secondary lysosomes incubated at 37°C with colloidal gold liposomes. Absence of liposome internalization at 4°C indicate the need of energy dependent endocytosis as the primary mechanism of entry of liposomes into urothelium. CONCLUSIONS: Based on these observations, we propose that the main route of entry of liposomes into urothelium cells is by endocytosis and ultrastructural study provide evidence for a possible mechanism by which liposomes can act as drug delivery platform. These findings support the usefulness of liposomes in intravesical drug delivery. Source of Funding: NIDDK R01DK083323