Characterization of CD44-Mediated Cancer Cell Uptake and Intracellular Distribution of Hyaluronan-Grafted Liposomes

Hyaluronan (HA) is a biocompatible and biodegradable linear polysaccharide which is of interest for tumor targeting through cell surface CD44 receptors. HA binds with high affinity to CD44 receptors, which are overexpressed in many tumors and involved in cancer metastasis. In the present study, we investigated the impact of HA molecular weight (MW), grafting density, and CD44 receptor density on endocytosis of HA-grafted liposomes (HA liposomes) by cancer cells. Additionally, the intracellular localization of the HA liposomes was determined. HAs of different MWs (5-8, 10-12, 175-350, and 1600 kDa) were conjugated to liposomes with varying degrees of grafting density. HA surface density was quantified using the hexadecyltrimethylammonium bromide turbidimetric method. Cellular uptake and subcellular localization of HA liposomes were evaluated by flow cytometry and fluorescence microscopy. Mean particle sizes of HA liposomes ranged from 120 to 180 nm and increased with increasing size of HA. HA liposome uptake correlated with HA MW (5-8 < 10-12 < 175-350 kDa), grafting density, and CD44 receptor density and exceeded that obtained with unconjugated plain liposomes. HA liposomes were taken up into cells via lipid raft-mediated endocytosis, which is both energy- and cholesterol-dependent. Once within cells, HA liposomes localized primarily to endosomes and lysosomes. The results demonstrate that cellular targeting efficiency of HA liposomes depends strongly upon HA MW, grafting density, and cell surface receptor CD44 density. The results support a role of HA liposomes for targeted drug delivery.