Subscriber access provided by Virgina Commonwealth University Libraries & VIVA (Virtual Library of Virginia)
Letter
Formation of supported lipid bilayers by vesicle fusion – effect of deposition temperature Tania Kjellerup Lind, Marité Cárdenas, and Hanna Pauliina Wacklin Langmuir, Just Accepted Manuscript • Publication Date (Web): 16 Jun 2014 Downloaded from http://pubs.acs.org on June 17, 2014
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 17
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
Formation of supported lipid bilayers by vesicle fusion – effect of deposition temperature Tania Kjellerup Lind1,2*, Marité Cárdenas1,3 and Hanna Pauliina Wacklin1,2
1Nano-Science 2European
Center and Institute of Chemistry, Copenhagen University, Denmark.
Spallation Source ESS AB, Lund, Sweden. 3 Malmoe University, Health & Society, 20506 Malmoe, Sweden
*Corresponding author:
[email protected] Abstract
We have investigated the effect of deposition temperature on supported lipid bilayer formation via vesicle fusion. By using several complementary surface sensitive techniques, we demonstrate that despite contradicting literature on the subject, high-quality bilayers can be formed below the main phase transition temperature of the lipid. We have carefully studied the formation mechanism of supported DPPC bilayers below and above the lipid melting temperature (Tm) by quartz crystal microbalance and atomic force microscopy under continuous flow conditions. We also measured the structure of lipid bilayers formed below or above Tm by neutron reflection and investigated the effect of subsequent cooling to below the Tm. Our results clearly show that a continuous supported bilayer can be formed with high surface coverage below the lipid Tm. We also demonstrate that the high dissipation responses observed during the deposition process by QCM-D correspond to vesicles absorbed on top of a continuous bilayer and not to a surface-supported vesicular layer as previously reported.
ACS Paragon Plus Environment
Langmuir
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Introduction Supported lipid bilayers (SLBs) are commonly used as simple model systems for cell membranes1, for instance to study enzymatic lipolysis2, or to understand the binding mechanisms of antibacterial drugs.3, 4 They can be formed by several methods, including vesicle fusion5, the lipid-detergent method6, 7 and Langmuir-Blodgett deposition8. The physico-chemical properties in terms of lipid composition, leaflet asymmetry, in-plane structure, mobility and fluidity have been studied extensively by a range of techniques including atomic force microscopy (AFM)9, 10, 11, dissipation-enhanced quarts crystal microbalance (QCM-D)9, 10, 11, 12, ellipsometry9, neutron reflection11, 12, 13, fluorescence microscopy11, 14 and interferometric scattering microscopy15. A key parameter for SLB formation by vesicle fusion is thought to be the deposition temperature, especially for lipids that are in the gel phase at room temperature.16 In many cases contradictory information about SLB formation (for example of E. coli bacterial lipids) exists in the literature. This seems to be coupled to the type of method used for evaluation of the SLB formation.17, 18, 19, 20 We present a detailed study of the effect of deposition temperature on the quality of SLBs formed by DPPC. We have used three different surface sensitive techniques: QCM-D, neutron reflection and AFM under continuous flow conditions.4 QCM-D gives an estimate of the wet adsorbed mass and it is thus particularly sensitive to the presence of water-filled vesicles, whether they are attached to the sensor surface as a supported vesicle layer, co-adsorbed in defects in a bilayer or on top of it. This technique is commonly used to asses the quality of SLBs and the success of various protocols for SLB formation21. Neutron reflection on the other hand gives detailed information of the structure and composition of the SLB in the direction perpendicular to the interface, but it is not particularly sensitive to the presence of a small number of vesicles, as they have a low scattering contrast to the surrounding solution. AFM gives complementary information about the lateral organization of the lipid bilayers and allows for real-time imaging of bilayer formation under continuous flow conditions4. In this letter, we show
ACS Paragon Plus Environment
Page 2 of 17
Page 3 of 17
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
that a combination of these three techniques is essential for a complete understanding of the structure and formation mechanism of SLBs. Experimental Materials. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). D2O was provided by the Institut LaueLangevin, Grenoble, France, or purchased from Sigma-Aldrich Inc. Ultrapure Milli-Q water with a resistivity of 18.2 MΩ⋅cm (Thermo Scientific, Branstead Nanopure 7145) was used for all cleaning procedures and sample preparation. Hellmanex 2 % (Hellma GmbH & Co, Germany) and absolute ethanol were used for cleaning QCM-D sensor crystals (purchased from QSense, Biolin Scientific, Stockholm, Sweden). Chloroform was purchased from Sigma Aldrich Inc. Small Unilamellar Vesicles (SUVs) were prepared by tip sonication of lipid films hydrated in ultrapure water above the lipid Tm (42 °C) and then kept in the fluid phase or at room temperature, as described earlier.4, 11 Quartz crystal microbalance with dissipation was performed with the Q-SENSE E4 system (QSense, Biolin Scientific, Stockholm, Sweden). Silicon oxide sensors, 50 nm, were purchased from QSense and cleaning was performed as earlier described.4, 11 The fundamental frequency and six overtones were recorded during lipid bilayer formation at either 50 °C or at 25 °C. SUVs in ultrapure water, at a concentration of 250 μg/ml, were injected into the cells using a peristaltic pump (Ismatec IPC-N 4) at 100 μl/min. After successful bilayer formation, the membranes were rinsed with ultrapure water followed by phosphate buffered saline (PBS; 10mM, NaCl 100mM, pH 7.4) before lowering the temperature to 25 °C (in cases of deposition at 50°C). Bilayer formation above the Tm was very reproducible in the QCM. We found a standard deviation of 2.5 % in mass adsorption between different depositions (19 depositions in 11 different experiments). Deposition at 25 °C was less reproducible as the adsorbed mass depends on the number and size of the attached vesicles, which is highly influenced by factors such as the equili-
ACS Paragon Plus Environment
Langmuir
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
bration time, the size distribution, the buffer conditions etc. 13 depositions (in four different experiments) of DPPC at RT in pure water were performed and we found a standard deviation between depositions of 14.5 %. Atomic force microscopy measurements were carried out on a Nanoscope IV multimode AFM (Veeco Instruments Inc.). Images were generated in the PeakForce QNM® (Quantitative Nanomechanical Property Mapping) mode with a silicon oxide tip (Olympus micro cantilever OTR8 PS-W) having a spring constant of 0.15 N/m and a radius of curvature of
