Tools for studying cellular uptake and trafficking of archaeosomes: A first approach

DEFAIN TESORIERO, M.V; PETER GAUNA, R.; PEREZ AP; MORILLA MJ; ROMERO EL

Congreso; Clinical Nanomedicine & Targeted Medicine-CLINAM; 2013

INTRODUCTION Nanotechnology has shown new and promising strategies for the prevention, diagnosis and treatment of human diseases. One of the major challenges is understanding the interaction that occurs between nano-objects and living cells and in which way they are internalized. The archaeolipids (lipids extracted from Archaebacteria) are non saponificable molecules that form self sealed mono or bilayer vesicles named archaeosomes (ARC). Unlike liposomes (L) made from conventional glycerophospholipids, the ARC possess a higher structural resistance to physicochemical and enzymatic degradation and surface hydrophobicity. [1] In this research, techniques for studying the endocytic uptake pathway(s) and the intracellular trafficking were described. In particular, inhibitors of clathrin and caveolae mediated endocytosis, macropinocytosis and phagocytosis were used to determine the mechanism(s) of ARC uptake by J774 macrophage cell line. MATERIALS AND METHODS Materials: Hydrogenated phosphatidylcholine from soybean (HSPC) was obtained from Northern Lipids (Vancouver, Canada), Modified Eagle?s medium with non-essential aminoacids (MEM-NEAA), was purchased from Gibco (Buenos Aires, Argentina). Heat-inactivated fetal bovine serum (FBS), glutamine (200 mM), antibiotic-antimycotic solution (10,000 units/ml penicillin G sodium, 10,000 mg/ml streptomycin sulphate and 25 mg/ml amphotericin B), pyruvate, and trypsin/ ethylenediamine tetra-acetic acid were purchased from PAA Laboratories GmbH (Pasching, Austria). Phosphatidylethanolamine-C14-Lissamine? rhodamine B (RhPE), 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS), Chloroquine diphosphate salt (CQ), cytochalasin D (Cyt D) from Zygosporium mansonii, genistein (Genis), 5-(NEthyl-N-isopropyl)amiloride (Amil), wortmannin (Wort) and 3-(4,5-dimethythiazole-2-yl)-2,5- diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Buenos Aires, Argentina). Nocodazole (Noc) and methyl-β-cyclodextrin (MβCD) were acquired from Fluka (Buenos Aires, Argentina) and chlorpromazine (CPZ) was a gift from Laboratorios Ceballos, Argentina. Transferrin-Alexa Fluor 633 (TFR); BODIPY-LacCer (LacCer) and Dextran-Alexa Fluor 647(DEX) were purchased from Life Technologies, (Buenos Aires, Argentina). Culture growth and characterization: Halorubrum tebenquichense archaeabacteria, were isolated from soil samples from Salina Chica, Península de Valdés, Chubut, Argentina, as described by Gonzalez et al. [1] ARC and L preparation and physicochemical characterization: ARC were prepared according to the thin film hydration method. TPL (20 mg) were mixed in chloroform:methanol (1:1; v/v) and dried with a rotary evaporator at 40°C in a round bottom flask until organic solvent elimination. The thin lipid film was flushed with N2 and hydrated at 40°C with 1 ml of 10 mM Tris-HCl buffer plus 0.9% w/v NaCl, pH 7.4 (Tris/NaCl buffer). The resulting suspensions were sonicated (1 h) in a bath sonicator at 80 W. Multilamellar archaeosomes (ARC M), so formed, were forced through polycarbonate membrane of a 100 nm defined pore size using a Miniextruder ® (Avanti Polar Lipids, Alabaster, Alabama, U.S.A.). L made of HSPC were prepared in the same way. Phospholipids were quantified by Bötcher method [2]. The Z-average size and zeta potential of ARC and L were determined using a Zetasizer NanoZS instrument (Malvern) at 25 ◦C in Tris/NaCl buffer. For flow citometry studies, Rh-PE (lipids/Rh-PE ratio 200:1 w/w) was added to prepare the films and ARC-RhPE or L-RhPE were obtained. For fluorescence microscopy studies, HPTS (35 mM) was added to the buffer and then the free fraction HPTS was separated with molecular exclusion column method (Sephadex G-50), and ARC- HPTS or L-HPTS were obtained. Kinetics of cellular uptake: Cell uptake was further investigated using flow cell cytometry (Becton Dickinson FACSCalibur, San Jose, CA). J774 cells were seeded at a density of 2 × 105 cells/well, in six-well plates and allowed to attach 24 h. Subsequently, the medium was replaced with fresh supplemented MEM-NEAA without FBS containing ARCRhPE (0,5 mM, final concentrations) and the cells were incubated for 0,5; 1; 3 and 5 hours at 37°C in 5% CO2. After each incubation period, the medium was removed, the cells were washed three times with phosphate-buffered saline (PBS, pH 7.4), and harvested by trypsinization. After trypsin inactivation and washing with PBS, the cells were fixed with 1% formaldehyde in PBS and stored at 4ºC until analysis. A total of 10,000 cells were analyzed by flow cytometry. In all cases fluorescence was quantified by the number of cells that emitted above the level of autofluorescence in control cells. Cell debris was excluded based on the forward-and side-scatter characteristics of the cell populations Data were analyzed using WinMDI 2.9 software. L-RhPE were used as control. Toxicity of endocytosis inhibitor treatments: To evaluate the effect of inhibitor treatments on ARC uptake by cells, the nontoxic concentration of inhibitors was chosen. For this, different inhibitor doses were pre-incubated during 1 h and then ARC (0,5 mM, final concentration) were added and incubation was extended 3 h. Citoxicity was evaluated with MTT assay and LDH leakage. Non-toxic concentrations are shown in Table 1. Uptake in presence of endocytosis inhibitors: J774 were seeded in six-well plates as stated previously. The medium was then replaced with fresh supplemented MEM-NEAA without FBS containing endocytic inhibitors at a nontoxic