Evaluation of stability and internalization by eukaryotic cells of polymer-lipids complexes formed by lactic acid bacterial lipids.

SZYMANOWSKI, F.; HUGO, A. A. AND PÉREZ, P. F.

Simposio; V Simposio Internacional de Bacterias Lácticas (SIBAL 2016); 2016

Liposomes are attractive vehicles for drug delivery and their lipid composition is crucial for the stabilization of liposome formulations. Bacterial lipids are mainly found in cell membranes and play a main role in stabilizing the membrane structure when cells are exposed to stress. Lipids from lactic acid bacteria (LAB) represent natural formulations that may be potentially useful in drug delivery. The internalization of liposomes by target cells can be favored by the incorporation of pH-sensitive polymers, such as Poly (2-(dimethylamino) ethyl methacrylate) (PDMAEMA). The aim of this work was the study of the mechanisms of cellular uptake of liposomes formed by lipids of Lactobacillus delbrueckii subsp. lactis CIDCA 133 and their serum stability in vitro. Fluorescent liposomes were prepared by reverse-phase evaporation. The dried lipid film was rehydrated with calcein or carboxifluorescein (CF) buffer. To form polymer lipid complexes (PLCs), cholesterol PDMAEMA (CHO-PDMAEMA) was added. Lecithin (LC) liposomes were used as controls. Stability of liposomes was evaluated by release of entrapped CF in the presence of different concentrations of normal fetal bovine serum (FBS). Liposome uptake by eukaryotic cells (HEK 293 and Caco-2/TC7) was evaluated by flow cytometry in the presence of endocytosis inhibitors. Intracellular localization of liposomes was assessed by confocal microscopy. CHO-PDMAEMA containing liposomes showed higher cargo capacity and were more internalized by eukaryotic cells than bare liposomes. Bacterial liposomes were more stable than lecithin (LC) liposomes at all the serum concentrations assayed. Bacterial and LC liposomes suffered a noticeable decrease of internalization by cells at 4°C. The uptake of liposomes showed a significantly decrease with caveolae inhibitors whereas it was similar to control with clarthrin and macropinocytosis inhibitors. Confocal images correlated with those results showing colocalization between liposomes and albumin which is a marker of caveolae pathway. Liposomes did not colocalize with lysotracker indicating that they did not route to lysosomes. The main problems associated to drug delivery systems are the degradation in the bloodstream and/or the intracellular destruction of liposome cargo. In this work we demonstrated that PLCs formed by LAB lipids are very stable in the presence of serum and that they internalize through the caveolae endocytic pathway (a non-degradative pathway). Our results support the potential use of LAB lipids in liposome drug delivery systems.