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Nanoparticles Meet Cell Membranes: Probing Nonspecific Interactions using Model Membranes Kai Loon Chen, and Geoffrey D. Bothun Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 16 Dec 2013 Downloaded from http://pubs.acs.org on December 22, 2013

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Environmental Science & Technology

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Nanoparticles Meet Cell Membranes: Probing Nonspecific

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Interactions using Model Membranes

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Feature Article

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Revised on December 10, 2013

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Kai Loon Chen*, † and Geoffrey D. Bothun*, ‡

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University, Baltimore, Maryland 21218-2686

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Department of Geography and Environmental Engineering, Johns Hopkins

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Department of Chemical Engineering, University of Rhode Island, Kingston, Rhode Island 02881-2018

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*Co-corresponding authors: Kai Loon Chen, E-mail: [email protected], Phone: (410) 516-7095;

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Geoffrey D. Bothun, E-mail: [email protected], Phone: (401) 874-9518. 1 ACS Paragon Plus Environment


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Abstract

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Nanotoxicity studies have shown that both carbon-based and inorganic engineered

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nanoparticles can be toxic to microorganisms. Although the pathways for cytotoxicity are

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diverse and dependent upon the nature of the engineered nanoparticle and the chemical

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environment, these studies have provided evidence that direct contact between nanoparticles and

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bacterial cell membranes is necessary for cell inactivation or damage, and may in fact be a

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primary mechanism for cytotoxicity. The propensities for nanoparticles to attach to and disrupt

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cell membranes are still not well understood due to the heterogeneous and dynamic nature of

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biological membranes.

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investigations of nanoparticle–membrane interactions. In this article, current and emerging

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experimental approaches to identify the key parameters that control the attachment of ENPs on

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model membranes and the disruption of membranes by ENPs will be discussed. This critical

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information will help enable the “safe-by-design” production of engineered nanoparticles that are

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non-toxic or biocompatible, and also allow for the design of antimicrobial nanoparticles for

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environmental and biomedical applications.

Model biological membranes can be employed for systematic

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TOC/Abstract Art

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Introduction

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The fast-growing utilization of nanomaterials in diverse applications, including electronic

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devices, drug and gene delivery, consumer products, and environmental remediation, will

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undoubtedly lead to the release of these materials into the environment.1

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nanoparticles (ENPs) that are released into natural and engineered aquatic systems can undergo

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physical and chemical transformation, and the nature and degree of transformation are directly

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dependent on the solution chemistry, environmental conditions (e.g., sunlight and temperature),

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and constituents present in the environment.2 The transformation of ENPs will influence their

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mobility and transport,3 as well as their biological effects on microorganisms and higher

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organisms.4

Engineered

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While evidence from recent studies has shown that both carbon-based and inorganic

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ENPs can be toxic to microorganisms, the mechanisms for their cytotoxicity are varied and not

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always completely elucidated. Carbon nanotubes (CNTs) can cause cellular membrane damage

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upon direct CNT–membrane contact with microorganisms and hence result in their inactivation.5-

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multiwalled CNTs (MWNTs) and the toxicity of the CNTs was attributed to a combination of

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physicochemical interactions with the cell membranes and oxidative stress.5, 6 Metallic SWNTs

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were recently discovered to be more toxic to Escherichia coli than semiconducting SWNTs and

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the adhesion of SWNTs to E. coli cells was necessary before the toxicity pathways occurred.7

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Individually dispersed SWNTs were more toxic to both gram-positive and gram-negative

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bacteria than SWNT aggregates and it was postulated that dispersed SWNTs were more effective

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in piercing cell membranes than SWNT aggregates.8 Graphene oxide nanosheets have also been

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reported to cause membrane and oxidative stress to E. coli upon attachment to the bacterial

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cells.9

Single-walled CNTs (SWNTs) were found to exhibit a higher level of toxicity compared to

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While direct contact between carbon-based ENPs and bacterial cells has been shown to

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result in membrane damage and cell inactivation, similar observations have been made for

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inorganic ENPs. The dissolution of silver nanoparticles (AgNPs) resulting in the release of Ag+

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ions is expected to be a key mechanism for their cytotoxicity.10 The attachment or close

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proximity of AgNPs to cell membranes will thus enhance the exposure of microorganisms to the

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ionic Ag+ species. AgNPs can also accumulate in the membranes of E. coli cells and cause pit

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formation in the cell walls.11

Similarly, ZnO nanoparticles were found to damage the 3 ACS Paragon Plus Environment


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membranes of E. coli cells and internalization of the nanoparticles was observed through

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transmission electron microscopy (TEM).12

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Based on existing nanotoxicity studies, it is apparent that the attachment of ENPs to the

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membranes of microorganisms is the critical initial process that precedes the toxicity pathways.13

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For example, greater nanoparticle adhesion has been observed to correlate with greater cellular

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internalization.14-17 Because cell membranes are complex and dynamic and contain multiple

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components both within the membranes and on the membrane surface (such as phospholipid

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bilayers, proteins, extracellular polymeric substances, and lipopolysaccharides),18 it is

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advantageous to employ model biological membranes of known compositions to systematically

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investigate the key parameters that control the attachment of ENP attachment. Likewise, the role

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of the biophysical and chemical properties of cell membranes, as well as the physicochemical

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properties of ENPs, on the propensity for physical disruption or penetration of cell membranes is

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still not well understood. Model biological membranes have the potential to be used to elucidate

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the mechanisms for the disruption of membranes by ENPs.

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In this article, we present commonly used models for biological membranes and highlight

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several techniques that can be employed to investigate the nonspecific interactions (or

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interactions that do not involve specific cell receptors) between ENPs and model cell membranes.

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We focus on experimental approaches that detect the attachment of ENPs on model membranes,

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as well as the physical disruption of model membranes by ENPs. Since the area of nanoparticle–

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membrane interactions is still relatively new, challenges related to these types of measurements

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and opportunities to further this field of study are also discussed.

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Models for Biological Membranes

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In addition to providing mechanical structure and separating intracellular and

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extracellular environments, the main role of the membrane is to provide an anisotropic fluid

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phase for supporting proteins and to regulate molecular transport into and out of the cell (e.g.,

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resisting water and controlling ion permeation). Figure 1 shows the main outer-most structural

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components, excluding integral and peripheral proteins, of bacterial, plant, and mammalian cell

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membranes. In general, all cell membranes exhibit negative charge and contain structural

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carbohydrate-rich layers anchored to a lipid bilayer membrane (intracellular cytoskeletons are

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not shown in Figure 1). They are not equilibrium structures, but rather dynamic structures that 4 ACS Paragon Plus Environment

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undergo transient chemical and physical changes depending on the environment the cells are

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exposed to and the stage the cells are in within their lifecycle. Given the inherent complexity of

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cell membranes that spans multiple length scales, model cell membranes composed of natural or

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synthetic lipid bilayers are typically used to gain fundamental insight into membrane

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organization and structure. In addition to reducing complexity, using model membranes

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eliminates the effects of cell metabolism and growth. FIGURE 1

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Lipid bilayers have been used as model membranes to examine the effects of pollutants,

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such as surfactants, organics, and metal ions on membrane-related cytotoxicity.19 Model

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membranes can be formed with biologically-relevant lipids and used to analyze mechanical,

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thermodynamic, and kinetic information relating to how compounds partition into membranes

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and cause disruption. In bilayers, partitioning and disruption are often assessed by measuring

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changes in lipid phase behavior (i.e., thermal transitions between ordered and disordered lipid

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phases), curvature, elasticity, and permeability, which are tied to membrane structure, domain

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formation, and pore formation. Systematically varying membrane composition provides a

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hierarchical approach where interaction mechanisms can be determined in ‘simple’ models and

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then extended to increasingly ‘lifelike’ membranes. Model membrane techniques can be

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extended to ENP–membrane systems to determine how nanoparticle size, shape, and surface

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chemistry influence their interactions.

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ENPs have been shown to damage bacterial and mammalian membranes through

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membrane disruption and to internalize cells through passive or active membrane transport.20

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Cytotoxic events associated with nanoparticle–membrane interactions involve, for example,

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destabilization of membrane proteins and membrane leakage,15, 16 which, among other things,

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affects transmembrane pH and ion gradients. These properties depend critically on membrane

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order and structure, as well as the membrane’s resistance to disordering or restructuring. There is

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precedence to using model membranes to examine membrane disordering or restructuring, and

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this precedence has been extended to nanoparticle–membrane interactions.

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Geometrical and experimental considerations. As in cellular membranes, model

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membranes are composed of lipid bilayers that self-assemble via hydrophobic forces. Model

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membranes are generally prepared in spherical or planar geometries (Figure 2). Spherical

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geometries consist of freestanding (unsupported) lipid bilayer vesicles,21 supported vesicles that 5 ACS Paragon Plus Environment


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are deposited intact on a substrate,22 or bilayers supported on spherical nanoparticles or

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microparticles that are dispersed in an aqueous phase.23, 24 Planar geometries include bilayers

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supported on planar substrates,25,

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deposited at air/water or solvent/water interfaces.28 The geometry employed depends on the

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types of measurements to be conducted (Table 1). Spherical vesicles provide high lipid

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concentrations to examine nanoparticle binding, lipid phase behavior, and membrane

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permeabilization using, for example, calorimetric or spectroscopic techniques. Varying vesicle

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size allows one to determine the effects of membrane curvature, which are related to membrane

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compressibility and elasticity,29 on modulating nanoparticle interactions. Spherical vesicles are

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also amenable to cryogenic transmission electron microscopy (cryo-TEM) techniques. In planar

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geometries, the lipid concentration is dictated by the size of the support, interface, or orifice. A

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distinct advantage of planar geometries is that they are amenable to a range of microscopy and

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spectroscopy techniques commonly employed in surface science.

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bilayers suspended across orifices,27 and monolayers

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FIGURE 2

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TABLE 1

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Once formed, intermolecular forces between lipid molecules govern the organization and

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phase behavior of a membrane, as well as the membrane’s integrity or stability against physical

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deformation. Membrane-active molecules can bind to the surface or partition into membranes

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and disrupt these forces. ENPs can also bind to membrane surfaces, but in this case the particle

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acts more like a solid surface and interacts locally with groups of lipid molecules or lipid

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domains. ENP–membrane interaction schemes are depicted in Figure 3. Once a nanoparticle

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adheres to a membrane, driven by intermolecular and surface forces, it can lead to lipid

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restructuring, domain formation, and local deformation (Figure 3A, B).30, 31 These processes can

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lead to membrane leakage due to transient voids or pore formation (Figure 3B),21, 32 passive

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membrane translocation of the ENP due to poration or invagination (Figure 3C),12, 20 or lipid

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extraction from the membrane (Figure 3D).28, 31 Experimentally, ENP binding can be examined

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using any model membrane configuration, while membrane leakage studies are limited to

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configurations that yield freestanding membranes (i.e., where aqueous reservoirs are in contact

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with both sides of the membrane, analogous to cellular membranes separating intracellular and

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extracellular fluids). However, unsupported freestanding membrane vesicles are the best

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candidates to examine simultaneous binding and leakage processes as solid supports or confined 6 ACS Paragon Plus Environment

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geometries influence lipid organization and phase behavior, and restrict elastic membrane

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deformation. To further add to the complexity, these same properties that can be influenced by

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ENP binding (lipid organization, phase behavior, and elasticity) can also affect the nature and

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extent of ENP binding. While there are preferred model membrane geometries for determining

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ENP binding and membrane disruption, there is no single experimental approach that provides a

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complete picture of the processes. FIGURE 3

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Probing Interactions between Nanoparticles and Model Membranes:

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Attachment and Disruption

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Several recent nanotoxicity studies have shown that ENPs can adsorb and penetrate (or

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disrupt) bacterial and mammalian cell membranes, both processes likely to play important roles

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in NP toxicity.11, 33 The approach of ENPs towards cell membranes resulting in either their

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attachment or close proximity to the membrane surface is expected to be the critical step

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preceding the toxicity pathways that result in cell inactivation or damage (including nanoparticle

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uptake or penetration). However, beyond the obvious electrostatic interactions, very little is

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currently known about the factors that control the interactions between ENPs and cell

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membranes. This includes how these interactions act to resist membrane disruption by ENPs;

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how ENP binding influences intermolecular lipid interactions and membrane organization; and

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how the shape and orientation of isotropic ENPs with respect to cell membranes, as well as the

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nanoparticle surface chemistry, affect ENP–membrane interactions.

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Since biological membranes, as well as most ENPs, carry surface charges, Derjaguin–

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Landau–Verwey–Overbeek (DLVO) interactions,34 namely, electric-double layer and van der

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Waals interactions, likely play important roles in controlling the propensity of the ENPs to attach

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on cell membranes.35 A recent study has reported a correlation between electrostatic attraction

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and bacterial minimal inhibitory concentrations for ZnO.36 Furthermore, because phospholipid

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bilayers, a key component of the cell membranes, are extremely hydrophilic and undergo

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dynamic fluctuations,34, 37 repulsive hydration and undulation forces are expected to contribute to

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ENP–membrane interactions. A detailed discussion on these ENP–membrane interfacial forces

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is presented in Nel et al.’s article,18 while Negoda et al.38 have recently reviewed current

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experimental and theoretical methods to characterize ENP–membrane interactions. 7 ACS Paragon Plus Environment

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feature article, we focus on complimentary techniques focused more at the biophysical level that

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can experimentally assess the mechanisms by which ENPs attach to and disrupt (or rupture)

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membranes.

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Attachment of ENPs on Model Membranes. Atomic force microscopy (AFM) has

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been used to examine the adsorption of ENPs on synthetic membranes.

Currently, most

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published work on ENP–model membrane interactions has reported the use of AFM to obtain a

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top image of supported lipid bilayers (SLBs; Figure 2B) that have been exposed to ENPs.33, 39, 40

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AFM can also be used to obtain a cross-sectional analysis of the SLBs to determine the degree of

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penetration of ENPs into the model membranes.39, 40

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Other than the AFM, optical tweezers have been employed to study ENP–membrane

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interactions. Rusciano et al.41 employed optical tweezers to trap phospholipid vesicles that were

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exposed to carbon ENPs. By performing Raman spectroscopy analysis, the authors were able to

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conclude from their measurements that the carbon ENPs can either partition within or penetrate

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the bilayers (they were unable to distinguish the two processes).41 However, the optical tweezer

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setup is relatively sophisticated and is still not commercially available. Moreover, it does not

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allow for the rapid and convenient analysis of the propensity of an ENP to adsorb or penetrate a

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model membrane.

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Since a tremendous amount of research on lipid bilayers has already been conducted with

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the quartz crystal microbalance with dissipation monitoring (QCM-D),25, 42 this technique holds a

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great promise in the measurements of the interactions of ENPs and model cell membranes.13, 22, 43

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The QCM-D is commercially available and it allows for the monitoring of the frequency and

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dissipation responses of a quartz crystal as adsorption of polymers, polyelectrolytes, or

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nanoparticles takes place on the crystal surface. By monitoring both signals, the mass and

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viscoelastic properties of the adsorbed layer can be determined real time. Hence, the QCM-D

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technique allows for the in situ detection of ENP adsorption on model membranes. Other than

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the ability for real-time measurements, the QCM-D technique is highly sensitive and can detect a

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mass change on the crystal of as low as tens of nanograms. Thus, this technique will be sensitive

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enough for ENP suspensions of low concentrations, as well as ENPs with low propensities to

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adsorb on cell membranes. Furthermore, only a small volume of ENP suspension (about a few

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milliliters) is required for this assay because the volume of the flow chamber of the QCM-D is

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only about 0.05 cm3. Also, because of the flow-through design of the QCM-D, this technique 8 ACS Paragon Plus Environment

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potentially can be automated and included as one of the components in a production/process train

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in a nanomaterial production plant to assess the nanomaterial’s propensity to adsorb on cell

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membranes.

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Using the approach of Richter et al.,25,

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Yi and Chen22 recently assembled a SLB

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composed of zwitterionic 1,2-dioleoyl-sn-glyero-3-phosphocholine (DOPC) in a QCM-D to

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investigate the effects of solution chemistry on the adsorption of oxidized MWNTs on the model

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biological membranes. They demonstrated that the presence of Ca2+ cations, as well as low pH

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conditions, will allow for favorable deposition of MWNTs on DOPC membranes through

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electrostatic attraction. These findings are consistent with observations made by other groups

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that electric-double layer interactions play an important role in controlling the attachment of

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ENPs on membranes.23, 24 While the QCM-D can measure the adsorption of ENPs to a SLB, it

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does not provide information about the location of the adsorbed ENPs with respect to the bilayer

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(e.g., on the outer leaflet or within the hydrophobic core of the bilayer). If such information is

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needed, AFM imaging39, 40 or cryo-TEM (discussed in later section) can be used to locate the

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ENPs on or in the bilayer.

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Disruption of Model Membranes by ENPs. Since the conditions which favor ENP

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adsorption on model membranes may not necessarily favor ENP penetration, experiments should,

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ideally, be designed to study both processes independently. One of the most common methods

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currently employed to test the propensity for ENPs to disrupt model membranes is the dye-

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leakage assay.21, 44 This method involves the preparation of an aqueous mixture comprising lipid

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vesicles (or liposomes) that encapsulate a fluorescent dye and ENPs that are being evaluated

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(Figure 2A). The fluorescence intensity is monitored and an increase in the measurements would

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indicate a leakage of dyes from within the vesicles due to the disruption of the bilayers by the

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ENPs. Moghadam et al.21 employed this technique to demonstrate that positively charged ENPs

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have a higher propensity than negatively charged ENPs to disrupt DOPC vesicles. The same

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technique was also employed by Shi et al.44 to show that SWNTs stabilized with sodium

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dodecylbenzenesulfonate (SDBS) do not disrupt egg L-α-phosphatidylcholine (egg-PC) vesicles.

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Phospholipid vesicles can be used with the QCM-D to evaluate the propensity for an ENP

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to disrupt model membranes. Yi and Chen22 developed an assay that involved the assembly of a

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supported vesicular layer (SVL, Figure 2D) on a QCM-D crystal surface and the subsequent

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exposure of the SVL to the ENPs of interest under flow-through conditions. If the vesicles were 9 ACS Paragon Plus Environment


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to be disrupted by the ENPs, the solution that was initially encapsulated in the vesicles will be

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released into the bulk solution, hence resulting in a decrease in the deposited mass on the crystal

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surface (which will be reflected by an increase in the crystal frequency response). Similar to the

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findings of Shi et al.,44 Yi and Chen22 showed that MWNTs do not cause any noticeable damage

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to DOPC vesicles. One advantage of the QCM-D assay is that it does not require the use of a

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fluorescent dye. Furthermore, it enables the monitoring of changes in the viscoelastic properties

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of the SVL upon contact with the ENPs. The viscoelastic parameters, namely, viscosity and

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shear modulus, can be derived by using the Voigt-based model45 to fit the frequency and

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dissipation responses collected by the QCM-D.

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Electrophysiological measurement is an emerging approach to detect the disruption of

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model membranes by ENPs. This technique involves the measurements of the electrical/ionic

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conductance across an unsupported, planar lipid bilayer that is formed across an orifice (Figure

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2E).38 The disruption or penetration of a lipid bilayer by ENPs can result in the formation of

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pores and hence, lead to an increase in the electrical conductance across the bilayer. The

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advantage of electrophysiological measurements is that the measurements are highly sensitive to

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changes in electrical conductance and thus, can detect minute perturbations of the membrane.

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The measurements, however, require an elaborate experimental setup, which is not yet

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commercially available. Corredor et al.27 conducted electrophysiological measurements which

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showed that MWNTs have the ability to disrupt a planar DOPC bilayer resulting in an increase in

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the current through the bilayer. The differing results regarding the propensity of CNTs to disrupt

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model cell membranes22, 27, 44 are not surprising considering the preparation methods for the

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CNTs were different in the experiments performed by the various groups. Instead, they highlight

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the pressing need for more in-depth studies of the role of surface chemistry of CNTs (and ENPs

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in general) on the propensity of the nanomaterials to disrupt cell membranes. Furthermore, the

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use of a combination of the techniques discussed above to investigate a single ENP–membrane

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system will allow for the validation of experimental data and also the elucidation of the

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mechanisms for membrane disruption by ENPs.

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Visualization of ENP–Membrane Interactions.

Direct visualization of ENP–

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membrane binding and membrane disruption can be achieved through cryo-TEM. An advantage

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of this technique is that it can be performed on unsupported spherical vesicles (Figure 2A),

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which can exhibit elastic deformations and rupture. Chen and Bothun31 have recently 10 ACS Paragon Plus Environment

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demonstrated the effect of ENP size (anionic iron oxide nanoparticles) on the binding and

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disruption of oppositely charged membranes. Small ENPs (16 nm hydrodynamic diameter) were

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shown to bind to membrane vesicles without causing the rupture of vesicles. In contrast, larger

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ENPs (30 nm hydrodynamic diameter) led to local changes in membrane curvature and

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significant vesicle rupture. Differences in the nature of the membrane interaction for the two

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ENPs were based upon the balance between membrane bending elasticity (2kb), which reflects

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how the membrane resists deformation, and the adhesion energy (Eadh, per area). This balance

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can be used to determine the critical nanoparticle radius, R2 = 2kb/Eadh, that causes deformation.46

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The total adhesion energy provided by larger ENPs (i.e. > 20 nm diameter), EadhR2, was enough

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to overcome the energy penalty for bending the membrane around the nanoparticle, which

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effectively led to the extraction of lipids from the membranes. Le Bihan et al.20 utilized this same

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concept of critical nanoparticle radius to analyze ENP invagination into membrane vesicles

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driven solely by membrane adhesion. Finally, cryo-TEM has also been used to examine the

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interactions between CNTs and vesicles, where it was shown that zwitterionic vesicles bound

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readily to MWNTs.22, 44 In this case the vesicles remained intact, indicating that there was

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limited contact area and/or weak ENP-membrane adhesion. An important point to note is that

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since the vitrified film has to be sufficiently thin (ca. 200–500 nm) in order for electrons to pass

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through, the size of the vesicles that can be observed is limited by the sample thickness.

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Challenges, opportunities, and recommendations

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Establishing links between model and ‘real’ membranes. The goal of any model

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membrane study is to mimic cellular membrane behavior and to provide fundamental biophysical

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insight into membrane-related phenomena that can be used to explain physiological responses. In

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nanotechnology environmental health and safety (EHS) research, such information would help

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identify the specific role of nanoparticle–membrane interactions in cytotoxicity, and would

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provide criteria for designing or selecting nanomaterials with that exhibit minimal (or no)

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membrane disruption. A first step towards improving our understanding of nanoparticle–

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membrane interactions requires transitioning from simple homogenous model membranes to

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more life-like (and more complex) heterogeneous membranes. This includes (i) using

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multicomponent membranes composed of biologically-relevant lipids, (ii) using membranes

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reconstituted from cell membrane extracts, and, specific to bacterial membranes, (iii) using 11 ACS Paragon Plus Environment


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membranes that incorporate polysaccharide or peptidoglycan surface coatings and bacterial lipids

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(Figure 1A, B). Multicomponent membranes could include mixtures of zwitterionic and anionic

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lipids, which would provide variable surface charge density, or lipids with different tail lengths

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or degree of saturation, which would provide membranes that contain co-existing phases or

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domains (e.g., sterol-rich domains). These approaches would allow one to determine if

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nanoparticles preferentially interact with specific lipids or lipid structures. Reconstituted

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membranes could be an ideal platform to study such interactions; however, the studies would

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need to include analyses of lipid composition and lipid organization to be complete.

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Accounting for environmental transformations. The second transition needed is from

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nanoparticles dispersed in “clean” media to nanoparticles dispersed in environmentally relevant

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media containing proteins, ions, and/or organic molecules (e.g., natural organic matter).2 The

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presence of these components is known to lead to the formation of a nanoparticle “corona” that

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alters nanoparticle surface chemistry and aggregation state (Figure 4). Recent research indicates

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that protein adsorption onto nanoparticles (carboxylated polystyrene NPs in serum) reduces cell

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adhesion and subsequent nanoparticle uptake.13 Adding to the complexity is the observation that

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ENPs with mixed hydrophilic/hydrophobic surface coatings can rearrange these coatings to

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maximize hydrophobic matching with the membrane and achieve penetration.47 Therefore, it is

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not just the composition of the coating in biological or natural environments, but the dynamic

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nature of this coating to restructure and adapt. FIGURE 4

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Complexities associated with predicting ENP–membrane interactions. Over the last

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decade, there have been many insightful studies conducted to determine the effects of ENP–

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membrane binding on membrane disruption. Fundamental information on ENP–membrane

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interactions has been gained primarily using model membranes. However, common platforms or

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techniques to determine ENP–membrane interactions have not been developed, and predictive

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frameworks for determining the membrane activity of an ENP are lacking. Addressing these

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issues is no small task given the inherent complexity and diversity of both biological membranes

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and ENPs.

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Attachment (adhesion) represents the first step for nanoparticle–membrane interactions.

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Electrostatic interactions have proven to be critical for ENP adhesion to and disruption of

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model35 and intact12, 48 cellular membranes. Classic approaches using DLVO or extended DLVO 12 ACS Paragon Plus Environment

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theories seem to capture the attachment process and can be adjusted based on nanoparticle size

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and surface functionality. However, these theories do not take into account membrane surface

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tension or membrane bending rigidity, which influence the adhesion process and the extent of

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membrane deformation (after adhesion).49 These factors could be accounted for by varying

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membrane lipid composition, which would change the mechanical properties of the membrane.

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Adhesion and membrane deformation will drive lipid reorganization and govern the capacity for

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transient void or pore formation. Lipid reorganization is also dependent upon lipid composition,

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namely, the presence of charged lipids, sterols, and lipids with varying degrees of tail saturation.

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Collectively, these processes occur due to changes in intermolecular interactions between

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membrane components that arise due to nanoparticle adhesion. High adhesion energies due to

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strong ENP–membrane surface interactions alter these intermolecular interactions and can lead

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to membrane disruption. Connecting ENP adhesion to changes in intermolecular membrane

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interactions, which may be assessed through calorimetry, spectroscopy, or AFM, is another

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critical step in predicting ENP membrane activity.

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At this point, studies are still underway to determine the possible (and dominant)

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mechanisms of ENP–membrane interactions and the extent to which they contribute to

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nanotoxicology. Model membrane studies have provided critical insight into these mechanisms

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and have demonstrated the importance of adhesion. Relatively few studies, however, have

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connected adhesion with changes in membrane function. This connection and the transition to

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heterogeneous membranes and environmentally-transformed ENPs should represent the next

375

stage in ENP–membrane interactions studies.

376 377 378

Biography Kai Loon Chen is an Assistant Professor in the Department of Geography and

379

Environmental Engineering at the Johns Hopkins University.

His research focuses on

380

environmental applications and implications of nanotechnology, interactions between engineered

381

nanomaterials and biological membranes, and membrane filtration processes for water treatment

382

and purification.

383

Geoffrey D. Bothun is an Associate Professor in the Department of Chemical

384

Engineering at the University of Rhode Island. His research focuses on the role of nanoparticle-

385

membrane interactions in nanotoxicology and nanomedicine, biological membrane adaptation to 13 ACS Paragon Plus Environment


386

membrane-active solutes and surfaces, and nanoparticle-based environmental remediation

387

technologies.

388 389

Acknowledgments

390

This material is based upon work supported by Semiconductor Research Corporation

391

(award 425-MC-2001, project 425.041) and the National Science Foundation under Grant No.

392

CBET-1055652.

393

14 ACS Paragon Plus Environment

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394

References

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35. Xiao, X. Y.; Montano, G. A.; Edwards, T. L.; Allen, A.; Achyuthan, K. E.; Polsky, R.; Wheeler, D. R.; Brozik, S. M., Surface Charge Dependent Nanoparticle Disruption and Deposition of Lipid Bilayer Assemblies. Langmuir 2012, 28, (50), 17396-17403. 36. Feris, K.; Otto, C.; Tinker, J.; Wingett, D.; Punnoose, A.; Thurber, A.; Kongara, M.; Sabetian, M.; Quinn, B.; Hanna, C.; Pink, D., Electrostatic Interactions Affect NanoparticleMediated Toxicity to Gram-Negative Bacterium Pseudomonas aeruginosa PAO1. Langmuir 2010, 26, (6), 4429-4436. 37. Marra, J.; Israelachvili, J., Direct Measurements of Forces between Phosphatidylcholine and Phosphatidylethanolamine Bilayers in Aqueous-Electrolyte Solutions. Biochemistry-Us 1985, 24, (17), 4608-4618. 38. Negoda, A.; Liu, Y.; Hou, W.-C.; Corredor, C.; Moghadam, B. Y.; Musolff, C.; Li, L.; Walker, W.; Westerhoff, P.; Mason, A. J.; Duxbury, P.; Posner, J. D.; Worden, R. M., Engineered nanomaterial interactions with bilayer lipid membranes: screening platforms to assess nanoparticle toxicity. Int. J. Biomedical Nanoscience and Nanotechnology 2013, 3, 52-83. 39. Roiter, Y.; Ornatska, M.; Rammohan, A. R.; Balakrishnan, J.; Heine, D. R.; Minko, S., Interaction of nanoparticles with lipid membrane. Nano Letters 2008, 8, (3), 941-944. 40. Spurlin, T. A.; Gewirth, A. A., Effect of C-60 on solid supported lipid bilayers. Nano Lett 2007, 7, (2), 531-535. 41. Rusciano, G.; De Luca, A. C.; Pesce, G.; Sasso, A., On the interaction of nano-sized organic carbon particles with model lipid membranes. Carbon 2009, 47, (13), 2950-2957. 42. Keller, C. A.; Kasemo, B., Surface specific kinetics of lipid vesicle adsorption measured with a quartz crystal microbalance. Biophys J 1998, 75, (3), 1397-1402. 43. Zhang, X. F.; Yang, S. H., Nonspecific Adsorption of Charged Quantum Dots on Supported Zwitterionic Lipid Bilayers: Real-Time Monitoring by Quartz Crystal Microbalance with Dissipation. Langmuir 2011, 27, (6), 2528-2535. 44. Shi, L.; Shi, D. C.; Nollert, M. U.; Resasco, D. E.; Striolo, A., Single-Walled Carbon Nanotubes Do Not Pierce Aqueous Phospholipid Bilayers at Low Salt Concentration. J Phys Chem B 2013, 117, (22), 6749-6758. 45. Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B., Viscoelastic acoustic response of layered polymer films at fluid-solid interfaces: Continuum mechanics approach. Phys Scripta 1999, 59, (5), 391-396. 46. Lipowsky, R.; Dobereiner, H. G., Vesicles in contact with nanoparticles and colloids. Europhys Lett 1998, 43, (2), 219-225. 47. Van Lehn, R. C.; Alexander-Katz, A., Penetration of lipid bilayers by nanoparticles with environmentally-responsive surfaces: simulations and theory. Soft Matter 2011, 7, (24), 1139211404. 48. Zhang, L. L.; Jiang, Y. H.; Ding, Y. L.; Povey, M.; York, D., Investigation into the antibacterial behaviour of suspensions of ZnO nanoparticles (ZnO nanofluids). J Nanopart Res 2007, 9, (3), 479-489. 49. Yi, X.; Shi, X. H.; Gao, H. J., Cellular Uptake of Elastic Nanoparticles. Phys Rev Lett 2011, 107, (9).

526 527

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Table 1. General Properties of Model Membrane Configurations. Membrane

Sample

Lipid

configuration

Common experimental techniques

concentration

Spherical vesicles

Dispersed in aqueous

High and

Calorimetry; spectroscopy; electronb

(Figure 2A, D)

solutions or

variable or low

and optical microscopy;

deposited on supports

and defined by

microbalanced

support Supported planar

Deposited at defined

Low and

Spectroscopy; atomic force, electron,c

bilayers (Figure 2B)

interfaces

defined by

and optical microscopy; microbalance

Interfacial

support or

Spectroscopy; atomic force,d Brewster

monolayers (Figure

interfacial areaa

angle, electron,d and optical

2C)

microscopy; microbalance

Unsupported planar

Deposited across

Low and

bilayers (Figure 2E)

orifices

defined by orifice area

530

a

531

manipulated by compression.

532

b

533

c

534

d

Spectroscopy; atomic force microscopy; electrophysiology a

Low relative to dispersed vesicles. Monolayer concentrations determined by interfacial area and Requires cryogenic sample preparation.

Limited to scanning electron microscopy. Requires deposition onto a planar solid substrate.

535


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536 (A) Gram-positive bacteria

(B) Gram-negative bacteria outer lipid membrane containing anionic lipopolysaccharides

peptidoglycan layer anchored by anionic lipoteichoic acids

peptidoglycan layer anchored by lipoproteins

lipid membrane inner lipid membrane

(C) Plant cell (D) Mammalian cell

pectin cellulose microfibrils cross-linked with hemicellulose

carbohydrates

lipid membrane

lipid membrane

537 538 539

FIGURE 1. Membrane structure, excluding integral and peripheral proteins, of (A) gram-

540

positive bacteria, (B) gram-negative bacteria, (C) plant cells, and (D) mammalian cells.

541



Page 20 of 22

542 (A) Spherical vesicles

(B) Supported planar bilayers water

(D) Vesicles on planar support water water water

solid support water

543

water

(C) Interfacial monolayers air or solvent

water water

solid support (E) Unsupported planar bilayers water

water

water

544 545

FIGURE 2. Model membrane configurations: (A) spherical vesicles (dispersed), (B) supported

546

planar bilayers, (C) interfacial monolayers, (D) vesicles on planar supports, and (E) unsupported

547

planar bilayers (i.e., spanning an orifice). Spherical vesicles (A) can consist of freestanding

548

bilayers, bilayers supported on microparticles or nanoparticles, or intact vesicles deposited on

549

supports (D).

550


Page 21 of 22


551 (A) Nanoparticle adhesion (binding) at membrane/water interface

-

+

-

+

-

+

+

(C) Passive (adhesive) membrane translocation

+ +

- - -

domain formation

(D) Adhesive lipid extraction

(B) Membrane restructuring and leakage

domains

pores

552 553

FIGURE 3. Effect of ENP–membrane interactions on membrane organization and translocation.

554

The red regions denote the membrane segments that are locally affected by nanoparticle binding.

555



Page 22 of 22

556

+

+ Corona formation

-

-

+

Aggregation

+ +

+

proteins

- + 557

ions organic molecules

+

+

558

FIGURE 4. Environmental transformation leads to the formation of an adsorbed nanoparticle

559

corona, which alters the colloidal stability of the nanoparticles. The composition of the corona

560

depends upon the ‘native’ nanoparticle surface coating and the water chemistry.


Nanoparticles Meet Cell Membranes - American Chemical Society

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