Evaluation of stability and internalization by eucariotic cells of polymer lipids complexes formed by lactic acid bacterial lipids

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

San Miguel de Tucumán

Simposio; V Simposio Internacional de Bacterias Lácticas; 2016

Centro de Referencia para Lactobacilos (CERELA)

Liposomes are attractive materials for drug delivery and the lipid composition is responsible for the stabilization of liposome formulations. Bacterial lipids are mainly found in cell membranes and have a crucial role in stabilizing the membrane structure when cells are exposed to stress processes. Thus, lipids from lactic acid bacteria (LAB) represent natural formulations that may be potentially useful in drug delivery. The internalization of liposomes by target can be favoured by the incorporation of pH-sensitive polymer, such as Poly (2-(dimethylamino) ethyl methacrylate) (PDMAEMA). The aim of this work was the study of the mechanisms of cellular uptaking of liposomes formed by lipids of Lactobacillus delbrueckii subsp. lactis CIDCA 133 and their serum stability in vitro. Fluorescent liposomes were prepared by the reverse-phase evaporation procedure. The dried lipid film was rehydrated with calcein or carboxiflurescein (CF) buffer. To form polymer lipid complexes (PLCs), cholesterol PDMAEMA (CHO-PDEMAEMA) was added. Lecithin (LC) liposomes were used as controls. Stability of liposomes was evaluated by the leakage of entrapped CF in the presence of different concentration of non-inactivated fetal bovine serum (FBS). Liposome uptake by eukaryotic cells (HEK 297 and Caco-2/TC7) was evaluated by flow cytometry in the presence of endocytosis inhibitors. Intracellular localization of liposomes was assessed by confocal microscopy. PLCs formed by the addition of CHO-PDMAEMA exhibited 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 suggesting an endocytic uptake mechanism. The uptaking of both liposomes showed a significantly decrease with caveolae inhibitors whereas it was similar to control with clarthrin and macropinocytosis inhibitors. Confocal images were in agreement with those results showing colocalization between liposomes and albumin which is a marker of caveolae pathway. Liposomes exhibited no co-localization with lysotracker indicating that they avoid acid compartments as lysosomes. The main problems of liposomes as drug delivery systems is the bloodstream instability and the intracellular destruction of liposome cargo. In this work we demonstrated that PLCs formed by LAB lipids were extremely stable in the presence of serum and employed the caveolae endocytic pathway, which typically avoid intralcellular acidification. The results supports the potential use of LAB lipids in liposome drug delivery systems.