LETTER pubs.acs.org/NanoLett
pH Responsive Polymer Cushions for Probing Membrane Environment Interactions Rita J. El-khouri,† Daniel A. Bricarello,‡ Erik B. Watkins,§ Caroline Y. Kim,^ Chad E. Miller,|| Timothy E. Patten,† Atul N. Parikh,*,‡ and Tonya L. Kuhl*,^,# Department of Chemistry, ‡Department of Applied Science, and §Biophysics Graduate Group, University of California, Davis, Davis, California 95616, United States Center for Advanced Molecular Photovoltaics and Stanford Synchrotron Radiation Lightsource, SLAC National Laboratory, Menlo Park, California 94025, United States ^ Department of Chemical Engineering and Materials Science and #Department of Biomedical Engineering, University of California, Davis, Davis, California 95616, United States
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bS Supporting Information ABSTRACT: A robust and straightforward method for the preparation of lipid membranes upon dynamically responsive polymer cushions is reported. Structural characterization demonstrates that complete, well-packed membranes with tunable mobility can be constructed on the polymeric cushion. With this system, membrane conformational changes induced by cellular cytoskeleton interactions can be modeled. The membrane can be tailored to screen the cushion from changes in pH or allow rapid response to the pH environment by incorporation of protein ion channels. This elementary system offers a means to replicate the conformational changes that occur with the cellular cytoskeleton and has great potential for fundamental biophysical studies of membrane properties and membraneprotein interactions decoupled from the underlying solid support. KEYWORDS: Polyelectrolyte, polymer cushion, pH responsive, supported membrane, lipid bilayer, poly(acrylic acid), neutron reflectivity, X-ray reflectometry
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hysicalchemical properties as well as many functions of biological membranes are intimately linked to their aqueous surroundings. Many ions (e.g., Ca2þ, Hþ, etc.), molecules (e.g., enzymes and proteins), and even polymerized macromolecules (e.g., actin) present in the aqueous phase interact with the membrane modulating its structure. Conversely, membranes also play a key role in regulating ion concentrations through means such as selective transport. Among a myriad of membraneenvironment interactions,1,2 two types of mechanisms are particularly important because of their versatility and roles in modulating biological functions. First, a generic mechanism by which membraneenvironment coupling occurs involves the interplay between membrane permeability and ionic concentration gradients between the cytosolic interior and the extracellular environment. Such concentration gradients can be specifically driven by various protein channels and pumps within the membrane. For example, during many cellular processes, e.g., solute transport as well as during oxidative and photosynthetic phosphorylation,35 a hydrogen-ion concentration difference (or pH gradient) is maintained across the membrane by membrane-mediated transport. Second, the asymmetric interactions between cytoskeletal components and the plasma membrane play a large role in membrane processes. These interactions not only provide lateral compartmentalization of the membrane but by polymerizationdepolymerization also induce dynamic r 2011 American Chemical Society
deformations of its physical structure. The latter includes changes in membrane elasticity, curvature, and lateral mobilities as well as spatial distributions of membrane components.6,7 Such interactions are important in many vital cellular processes including motility, mechanotransduction, phagocytosis, and endocytosis.811 Similarly, changes in the membrane environment also reorganize the cytoskeleton.12 The ability to recapitulate these mutual interactions between the membrane and its surrounding aqueous milieu using model membrane configurations are desirable for systematically mapping membrane environment interactions. Here, we describe a class of model membranes that integrate the essential membrane structure, a phospholipid bilayer, with a structurally tunable and environment-sensitive interface, which serves to model and assay membraneenvironment interactions. We demonstrate that a pH responsive, hydrophilic poly(acrylic acid) (PAA) cushion layer serves as a suitable substrate for the formation of supported membranes. The platform enables fundamental biophysical investigations of both classes of membraneenvironment interactions described above. First, using gramicidin-doped bilayers, we demonstrate that the pH dependence of the polymer cushion thickness provides a sensitive and Received: March 14, 2011 Published: April 18, 2011 2169
dx.doi.org/10.1021/nl200832c | Nano Lett. 2011, 11, 2169–2172
Nano Letters
LETTER
Figure 1. Neutron scattering length density profile of a DPPC bilayer on a PAA cushion. Dashed line shows the profile unsmeared by interfacial roughness. NR data are shown in the inset and are divided by the Fresnel reflectivity in order to better visualize features and the quality of fit (χ2 = 3.7).
Figure 2. (a) AFM image of a UVozone patterned PAA cushioned DMPC membrane at pH 9.6, color scale maximum is 40 nm. (b) Measured thickness of a PAA cushioned DMPC membrane at pH 4, 7, and 9.6. (c) FM image of PAA cushioned DMPC membrane doped with 1% Texas Red DHPE.
convenient means to measure hydrogen-ion transport across the membrane. Second, we show that the lateral diffusivity within the membrane can be manipulated by simply adjusting the pH of the aqueous phase, which induces changes in the structural properties of the cushion layer. Together, these initial studies suggest that PAA-supported lipid bilayers represent a promising class of model membrane systems for studies of membrane environment coupling by enabling systematic measurements of (1) interactions between the membrane bilayer and the underlying polymeric anchor and (2) ionic permeation and transport across the bilayer. We start with the preparation of the PAA cushion layer. Briefly, (aminopropyl)triethoxysilane (APTES) was first deposited onto UVozone oxidized silica (e.g., glass, quartz, or silicon) substrates from a 1 mM toluene solution.13 Subsequently, a lightly cross-linked, 450k MW PAA (Aldrich, St. Louis, MO) was deposited by spin coating from a methanol solution (2000 rpm, 30 s, 15 mg/mL) onto APTES coated surfaces.14 In order to improve the surface adhesion of the PAA cushion layer, an additional thermal curing step was employed, which induced amide formation between the PAA carboxylic acid and the amine functionality at the surface APTES layer.15 By simply changing the PAA concentration in the methanol solution during the spincoating step, the thickness of the cushion layer was systematically varied to obtain PAA cushions of ∼1050 nm thickness reproducibly. For thinner PAA cushion layers (
