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Effect of supporting polyelectrolyte multilayers and deposition conditions on the formation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine lipid bilayers Magdalena Wlodek, Michal Szuwarzynski, and Marta Kolasi#ska-Sojka Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b02560 • Publication Date (Web): 03 Sep 2015 Downloaded from http://pubs.acs.org on September 8, 2015
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Effect of supporting polyelectrolyte multilayers and deposition conditions on the formation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/-palmitoyl-2oleoyl-sn-glycero-3-phosphoethanolamine lipid bilayers Magdalena Wlodek†*, Michal Szuwarzynski‡, Marta Kolasinska-Sojka† †
Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, 30-239 Cracow,
Poland ‡
Jagiellonian University, 30-060 Cracow, Poland
*Correspondence: Magdalena Wlodek Jerzy Haber Institute of Catalysis and Surface Chemistry, Niezapominajek 8, 30-239 Cracow, Poland Tel.: +48 126395212 Fax: +48 124251923 E-mail:
[email protected] Abstract: The formation of complete supported lipid bilayers by vesicle adsorption and rupture was studied in relation to deposition conditions of vesicles and underlying cushion formed from various polyelectrolytes. Lipid vesicles were formed from zwitterionic 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine (POPE) in phosphate buffer of various pH with or without NaCl addition. Polyelectrolyte multilayer films (PEM) were constructed by sequential adsorption of alternately charged polyelectrolytes from their solutions - layer-by-layer deposition (LBL). The mechanism of the formation of supported lipid bilayer on polyelectrolyte films was studied by quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). QCM-D allowed following the adsorption kinetics while AFM measurements verified the morphology of lipid vesicles and isolated bilayer patches on the PEM cushions providing local topological images in terms of lateral organization. Additionally, polyelectrolyte cushions were characterized with ellipsometry to find thickness and swelling properties and their roughness was determined using AFM.
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It has been demonstrated that, the pH value and an addition of NaCl in the buffer solution as well as the type of the polyelectrolyte cushion influence the kinetics of bilayer formation and the quality of formed bilayer patches. Keywords: supported lipid bilayer, polyelectrolyte multilayer cushion, atomic force microscopy, quartz crystal microbalance and swelling of PEM.
INTRODUCTION Formation of supported lipid bilayers on soft polymer cushions has been extensively studied in recent years due to their numerous potential applications for biotechnology as cellmembrane models, for biosensors, drug delivery systems or as platforms for cells.1,2 However, the supported lipid bilayers (SLB) formation on cushion material is not fully understood. The combination of assemblies of amphiphilic molecules bearing specific features and polyelectrolytes with their great potential of effectively decoupling lipids from solid support due to electrostatic interactions and their fluidity is a general principle for the fabrication of new materials.3 Supported lipid bilayers were introduced by Brian and McConnell and they can be easily formed by vesicle fusion method.4 There exist three typical pathways for formation of supported lipid bilayers by the vesicle rupture and fusion. The first one is that a single, isolated vesicle ruptures and transforms into a SLB patch. Another is that vesicles adsorb until threshold coverage and the rupture of a vesicle causes the SLB growth by stimulating the neighboring vesicles. The third pathway is a formation of a stable layer of adhesive vesicles, which does not transform into a planar lipid bilayer.5 Adsorption kinetics of SLB formation strongly depends on liposome composition, charge, size, solution pH, ionic strength and buffer composition6-8, surface charge and surface roughness. Development of sensitive techniques for surface analysis, such as dissipationenhanced quartz crystal microbalance (QCM-D), atomic force microscopy (AFM) and surface plasmon resonance (SPR) enabled in situ observations of the SLB formation, necessary to understand the process of vesicle adsorption/fusion and the formation of complete lipid bilayer.9,10 Several studies have shown that stable lipid bilayer can be formed on polyelectrolyte multilayers
(PEM)
composed
of
poly(sodium
4-styrenesulfonate)
(PSS)
and
11
poly(diallyldimethylammonium) chloride (PDADMAC) , chitosan (CHI) and hyaluronic acid (HA)12 or polyethyleneimine (PEI) and polystyrene sulfonate (PSS)13. Formation of lipid
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bilayers by vesicle fusion is very sensitive to the surface properties.5 Some aspects have been already discussed.14 The hydrophilicity of the substrate is one of the crucial factors determining the SLB-substrate interactions. In addition, Duarte et al. demonstrated that surface roughness strongly influences liposomes’ adsorption.8 Polyelectrolyte multilayers provide an interesting alternative to other type of polymer cushions. PEM films are created by sequential adsorption of oppositely charged polyelectrolytes from their aqueous solutions.12 It is noteworthy, that their properties (thickness, roughness, surface charge, wettability) can be controlled by varying the number of adsorption cycles, charge density, ionic strength and pH of their solutions.15-20 The aim of presented studies was to understand the mechanism of the lipid bilayer formation on the top of polyelectrolyte multilayers brought into contact with lipid vesicles in solution. We studied the behavior of the negatively charged unsaturated phosphoethanolamine (1palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine,
POPE)
and
the
zwitterionic
unsaturated phosphatidylocholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC) when interacting with PEM modified solid surface. Modification of the support was done by polyelectrolyte multilayers’ formation on their surface. Five types of polyelectrolytes were used: branched poly(ethyleneimine) - (PEI), poly(diallyldimethylammonium)chloride – (PDADMAC), polysodium 4-styrenesulfonate – (PSS), poly–L–glutamic acid sodium (PGA) in their natural pH and poly–L–lysine hydrobromide – (PLL) with pH adjusted to 10 during film formation to decrease its ionization degree. Such the choice of polyions was made to study interactions of resulting PEM with lipid vesicles with respect to various ionization degrees (for details see Materials and Methods) and various chemical composition of PEM. The cushion’s thickness was regulated by the number of polyelectrolyte layers used. The formation of lipid structures on the top of positively charged, polycation-terminated PEMs were studied in situ using QCM-D measurements and the topography of resulting probes was measured by AFM. We believe that the obtained results may have a great significance for the scientific community dealing with supported lipid bilayers since we point out key factors responsible for the quality of the formed SLB.
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MATERIALS AND METHODS
Materials. The polyelectrolytes (PE) used in our studies (shown in Figure 1) were: branched poly(ethyleneimine)
(PEI)
of
molecular
weight
c.a.
750kDa,
poly(diallyldimethylammonium)chloride (PDADMAC) of molecular weight in the range of 100-200kDa, poly–L–lysine hydrobromide (PLL) of molecular weight about 30kDa as polycations and polysodium 4-styrenesulfonate (PSS) of 70kDa, poly–L–glutamic acid sodium salt (PGA) of molecular weight about 50kDa as polyanions. All polyelectrolytes were purchased from Sigma-Aldrich. Lipids used were: zwitterionic 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) and positively charged 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoethanolamine (POPE) both from Avanti Polar Lipids (shown also in Figure 1). NaCl (99,5%) was obtained from Fluka, Na2HPO4, NaH2PO4, chloroform and HCl – from P.O.Ch Gliwice and NaOH –from Aldrich. Phosphate buffer (PBS) was made of Na2HPO4 and NaH2PO4, with pH adjusted by NaOH to the following values: pH=9.5; pH=8 and pH=7.5. All studied buffers were used without or with addition of NaCl, keeping ionic strength of both types of phosphate buffers constant and equal to 0.2M. Ultrapure, Millipore water with resistivity >18MΩ/cm was used for all prepared solutions. As support materials for PEM deposition silicon wafers (On Semiconductor, Czech Republic) and standard gold/quartz sensors QSX 301 (Q-Sense, Sweden) were used. They were cleaned by washing for 30 min. in piranha solution, which is a mixture of equivalent volumes of concentrated sulfuric acid and perhydrol (Precaution! This solution is a very strong oxidizing agent and should be handled carefully). Rinsing with water and then incubated for 30 min. in hot water (70oC) were the following cleaning steps. Deposition of polyelectrolytes onto silicon wafers or QCM sensors was performed from 0.15M NaCl solution. PDADMAC and PSS were used in their natural pH, since they are strong electrolytes, which means that they are completely charged over the whole pH range. PLL solution was adjusted to pH=10 to partially withdraw its dissociation. Zeta potential of PLL at pH=10 was 12mV. As a reference value, zeta potential of fully charged PLL in acidic condition was c.a. 23mV. PGA was used at pH=6, being fully charged with zeta potential equal to -30mV. PEI was used in its natural pH equal to 10.5 at which it is partially dissociated, similarly to PLL. PEI was always used as the first, anchoring layer for the build-up of cushion films.21 Each polyion adsorption step took 10 min and rinsing in between was three times for 1 min. in water. The process was continued up to 3 bilayers, since we know from our previous studies on polyelectrolyte multilayers that for
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similar conditions 3 bilayers are enough to become independent of the solid substrate.21,22
NHCH2CH2
N
Cl
CH2CH2
x
+
-
y
CH2CH2CH3
+
N
PEI
n
n
H3C
CH3
SO3
PDADMAC
PSS O
H2NCH2CH2CH2CH2 +
NH2
-O C
H O C
H O
CH2CH2 NH
C
C
C
n
n
PGA
PLL
O
O O
O O
H
+
P
O
_
N
O
O
POPC O
O O
O O
H
P
O
_
O
Na
+
NH3
+
O
POPE Figure 1. Structural formulas of polyions and lipids applied in the described studies.
Preparation of small unilamellar vesicles. Lipid mixtures were obtained by mixing of 5mg POPC and 5mg POPE in 1ml chloroform. The solution was evaporated to a thin film on the walls of a glass using vacuum pump overnight. The film was then hydrated in 2ml of selected PBS buffer solution with vortex stirring to obtain monolamellar vesicles. Three various pH values of PBS buffer were studied: pH=7.5, pH=8 and pH=9.5, having ionic strength constant and equal to 0.2M. The resulting suspension was then extruded 15 times through polycarbonate membranes with nominally 100nm pores using mini-extruder (Avanti Polar Lipids) and diluted in phosphate buffer to the final concentration 0.4mg/ml. The hydrodynamic diameter of lipid vesicles in solution after extrusion was determined by dynamic light scattering (DLS) using the Zetasizer Nano Series, Malvern Instruments at scattering angle 90o. All measurements were performed at 25oC.
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The obtained hydrodynamic diameter for vesicle solutions was approximately 90.2±6.0nm for all studied systems. The zeta potential of lipid vesicle suspension measured by electrophoretic mobility depended on the pH value of buffer solution and it was (-3.4±0.6mV for pH=7.5); (-4.1±0.7mV for pH=8) and (-18.8±0.9mV for pH=9.5).
Experimental techniques. Ellipsometry: Thickness of “dry” and “wet” PEM films deposited by the LBL method on silicon wafers was determined using the EP3 imaging spectroscopic ellipsometer (Accurion, Germany - formerly Nanofilm). The same procedures and adsorption conditions as for formation of PEMs at QCM crystals were applied. The optical parameters for polymer films were determined by the multiangle analysis at wavelength λ=658nm. Two layer constant n, k model23 was used with the appropriate refractive index depending on the conditions of experiment and extinction coefficient k=0 for the PEM layer. The refractive index of dried and wet polyelectrolyte films was equal to 1.55 and 1.47, respectively. Thickness of the silica layer on silicon wafer used as substrate was determined separately by the multiangle analysis and it was equal to 3±1nm. The ellipsometric thickness of multilayers was measured at 75o angle of incidence, near the Brewster angle for the support material, in order to obtain results with the highest possible sensitivity.24 Atomic Force Microscopy. The surface morphology of “dry” and “wet” polyelectrolyte films was investigated using AFM. Measurements were taken by tapping mode using Asylum Research Cypher at room condition. Standard silicon cantilevers AC160TS and TR400PB (Olympus, Tokyo, Japan) were used. AFM images with scan areas of 2x2µm2 were obtained at a scan rate of 0.5Hz with 256x256 pixels. The surface roughness has been calculated as the root mean square (RMS) value, R of the distribution of height in the AFM topography images using Gwyddion software. AFM images for PEMs with supported lipid bilayer were obtained with Dimension Icon atomic force microscope (Bruker, Santa Barbara, CA) working in the fluid in the Peak Force Tapping™ (PFT) mode. Standard silicon cantilevers for PFT in fluids (Bruker) with nominal spring constant of 0.7N/m and tip radius
