An evaluation of different cationic lipids on uptake of liposomes by endothelial cells and influence of the lipid type on binding to albumin plasma protein: A mechanistic approach in vitro.

摘要

In order to meet the increasing demands of a developing tumor mass, tumor endothelial cells actively participate in the formation of new blood vessels through a process known as neovascularization. Since tumor cells rely on the function of endothelial cells for a steady supply of oxygen and essential nutrients, any significant damage caused to tumor endothelial cells would ultimately result in tumor death. Unlike tumor cells which are located some distance away from the vascular wall, tumor endothelial cells are easily accessible to systemically applied therapeutics. Apart from that, tumor endothelial cells exhibit characteristic differences in membrane surface charge characteristics compared to endothelial cells lining vessels in quiescent tissues. Tumor endothelial cells have abundant anionic groups (i.e., glycosaminoglycans and phosphatidyl-serine) exposed on the outer leaflet of the plasma membrane which contribute to the negatively charged surface potential. This characteristic of the tumor vasculature can be exploited for therapy by using positively charged therapeutic drug carrier molecules. The positive surface charge potential of the drug carrier, combined with the net negatively charged tumor vascular surface, will facilitate the uptake of therapies by tumor endothelial cells resulting in the destruction of the vascular supply.;In this area, cationic liposomes have shown an ability to preferentially target tumor endothelial cells. We have used pegylated cationic liposomes (PCLs) in the present study. We have evaluated the ability of PCL preparations to interact with various endothelial cells; each PCL variety consisted of a different cationic lipid type, used at a fixed molar concentration and ratio to other liposome components, for the sake of comparison. Four endothelial cell lines representing different organ systems were used along with one mouse fibroblast cell line. Three endothelial cell lines HMEC-I, MS-1 VEGF and bEnd.3 are transformed cell lines, whereas HUVEC is a primary (non-transformed) endothelial cell line. All of the cell lines were carefully selected on the basis of organ specificity; the endothelial cell lines thus represent an in vitro model of the respective tissue environment. In order to assess cell-PCL interactions, cell association studies and FACS analyses were performed under appropriate experimental conditions. We also characterized PCL formulations based on their physicochemical properties such as net surface charge potential and their size. To compare the relative toxicity level of the PCLs as a function of the specific cationic lipid employed, cell viability studies were performed. Liposomes are known to destabilize and rapidly clear from the circulation when administered intravenously.;Several studies suggest that plasma protein-liposome interaction is responsible for liposome's untimely and undesirable fate. In order to mimic the in vivo environment, we evaluated plasma protein binding to PCLs; an ELISA kit was used to determine plasma protein's relative affinity for binding PCLs. Albumin was the model protein used for this study owing to its abundant levels in blood, and since similar studies involving the use of PCLs have not been published to date. Moreover, the experimental conditions including concentration of plasma proteins used resemble the concentrations in blood for extrapolation purposes.;Studies have revealed the presence of anionic phospholipids on the external leaflet of plasma membrane of tumor endothelial cells. Phosphatidylserine (PS) is a highly abundant anionic phospholipid present in mammalian cells. The positively charged cationic liposomes undergo electrostatic interactions with negatively charged anionic phospholipids on tumor vasculature, thereby increasing the uptake of PCLs by tumor endothelial cells. Hence, we decided to measure the extent of interaction of our PCL preparations with PS. For this study, we used 96-well plate pre-coated with PS. ^ These studies revealed that the in vivo fate of liposomes may not only depend on the overall physicochemical properties (i.e., particle size and surface charge potential) of the PCLs, but on the specific chemistry of the cationic lipid used to achieve the desired physicochemical properties. Moreover, the specific lipid head group and acyl chain composition may influence the overall extent to which the different PCLs bind to plasma proteins.;The use of seven different PCL formulations on the basis of size, cationic charge potential and chemical composition offered a unique opportunity to investigate advantages and disadvantages of using the different PCL varieties.