Gold nanoparticles for regulation of cell function and behavior

Nano Today 13 (2017) 40–60

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Nano Today journal homepage: www.elsevier.com/locate/nanotoday

Review

Gold nanoparticles for regulation of cell function and behavior Gustavo Bodelón a,∗ , Celina Costas a , Jorge Pérez-Juste a , Isabel Pastoriza-Santos a , Luis M. Liz-Marzán a,b,c,d,∗ a

Departamento de Química Física and CINBIO, Universidade de Vigo, 36310 Vigo, Spain Bionanoplasmonics Laboratory, CIC biomaGUNE, Paseo de Miramón 182, 20014 Donostia-San Sebastián, Spain Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Spain d Biomedical Research Networking Center in Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), 20014 Donostia-San Sebastián, Spain b c

a r t i c l e

i n f o

Article history: Received 2 September 2016 Received in revised form 19 November 2016 Accepted 20 December 2016 Available online 3 January 2017 Keywords: Gold nanoparticles Nanostructured materials Cell behavior Cell function Cellular receptor Cell signaling Plasmonics LSPR Optothermal Nanotechnology

a b s t r a c t The cell possesses the remarkable ability to perceive and process chemical and physical stimuli, which in turn modulate cellular behavior. One of the great wonders about nanotechnology is that it enables the fabrication of tools on the same length scale as biomolecules, thereby providing us with a unique handle for characterizing and controlling basic cellular processes. Owing to their tunable size and shape dependent physical properties, biocompatibility and facile surface modification, gold nanoparticles become potentially powerful tools to probe fundamental aspects of cell biology. Consequently, innovative approaches based on gold nanoparticles are under development toward the manipulation of cell function and improvement of techniques currently used in biomedicine and biotechnology. In this review we provide an overview of recent applications based on gold nanoparticles and nanostructured materials for the modulation of cellular activity and behavior, mediated by their interactions with cell surface receptors. © 2016 Elsevier Ltd. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Why gold nanoparticles? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Cell function and behavior are modulated by receptor-mediated cell signaling processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Colloidal gold nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Modulation of cell surface receptor activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Modulation of angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Opto-thermal modulation of neural activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Nanostructured substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Nanopatterned materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Nanocomposite scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Nanocomposite materials for physical modulation of cell behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Conclusion and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

∗ Corresponding authors at: Departamento de Química Física and CINBIO, Universidade de Vigo, 36310, Vigo, Spain/CIC biomaGUNE, 20014 Donostia-San Sebastián, Spain. E-mail addresses: [email protected] (G. Bodelón), [email protected] (L.M. Liz-Marzán). http://dx.doi.org/10.1016/j.nantod.2016.12.014 1748-0132/© 2016 Elsevier Ltd. All rights reserved.

G. Bodelón et al. / Nano Today 13 (2017) 40–60

Introduction As the basic unit of life, the cell is not only a biochemical factory with the capacity to self-replicate, but also a sophisticated sensor-actuator entity that can perceive a wide range of stimuli (i.e. chemical or physical cues) and translate such information into appropriate cellular responses that contribute to cell homeostasis and regulated cell behavior. In order to precisely orchestrate such multitasking activity, cells have developed via evolution a plethora of molecular sensory mechanisms that are arranged into complex networks capable of detecting incoming signals and transferring the information to cellular effectors that regulate basic cellular processes such as metabolism, proliferation or cell fate [1,2]. The functional properties of tissues arise through interactions of numerous cell types [3]. An orderly exchange of signals allows cells to arrive at collective decisions and organize their collective behavior [4]. Remarkably, it turns out that distinct spatiotemporal activation profiles of the same repertoire of signaling proteins result in different gene activation patterns and diverse physiological responses. One of the most important functions of cell signaling is the control of a normal physiological balance within the body. Activation of different signaling pathways leads to diverse physiological responses, such as cell proliferation, cytoskeletal reorganization, cell cycle checkpoints, apoptosis (active cell death), differentiation, and metabolism. During the last few years, signal transduction therapy has become one of the most important areas of modern drug research. In a healthy organism, the processes of cellular growth and differentiation are tightly controlled, but in the pathological state, they are uncoupled in such a way as to result in further damage-causing signals, or the growth of the malfunctioning cells. Dysregulation or malfunctioning in cellular information processing is responsible for a large number of diseases including cancer, autoimmunity, neural disorders or diabetes. Thus, by understanding of the mechanisms underlying cell function and behavior, diseases may be prevented or treated more effectively. A key aim of cellular biology is the characterization of every cell component and their mutual interactions, in order to determine the function of this extraordinarily complex machinery, both at the single cell and multi-cellular levels [5]. Traditional techniques using biochemical or genetic approaches made important contributions into elucidating the molecular ground, and thereby shaping our understanding of cellular function. Additional approaches, such as advanced imaging tools and “omics”, defined as high-throughput technologies that aim at generating a comprehensive view of molecular cell components and their interactions, have rendered huge progress in the exploration and understanding of the role of molecules and their interactions in the cellular environment at increasingly high spatial and temporal resolution [6,7]. The fast advancement of nanotechnology on the other hand, has enabled the fabrication of materials at nanometer scale resolution, with applications as smart devices for probing cellular function with unprecedented precision and accuracy, thus providing these materials with the potential to effect changes in cellular systems in unforeseen ways [8–10]. The availability of sophisticated new experimental techniques and tools based on nanotechnology can be applied to monitor dynamic complex biological processes in real time, at the single cell level. Over the last two decades, different nano-engineered biomaterials based on gold nanoparticles (AuNPs) have been applied in biological and biomedical research for the modulation of various extracellular and intracellular biochemical and biophysical events at subcellular resolution. The ultimate objective is to gain complete knowledge of how cells work at the molecular level, both in healthy and diseased states. We focus in this review on providing a detailed perspective about the use of plasmonic AuNPs and nanostructures as smart

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devices and interfaces to modulate, and further understand, the activity of cell surface receptors, which play key roles in governing cell function and behavior. We decided to keep cell uptake, toxicity and interactions of AuNPs inside cells out of the scope of this review, since they have been extensively reviewed and discussed in a number of recent publications [11–14].

Why gold nanoparticles? The integration of metal nanoparticles in biological systems has triggered a revolution in biology and medicine. Gold nanoparticles are particularly attractive in this respect because they display unique optical properties, low inherent toxicity, large surface area and easy surface functionalization. Therefore, AuNPs have been used in applications such as biosensing, bioimaging, drug delivery, therapy and tissue engineering [15–20]. Gold nanostructures are versatile platforms for the design of recognition elements toward specific biological targets. Moreover, the enhanced radiative properties responsible for fluorescence quenching or enhancement, surface enhanced Raman scattering, as well as their electrochemical activity, can be harnessed to signal the transduction of binding events. Hence, incorporation of AuNPs can simplify system design whilst enhancing the sensitivity of biosensors. The typical size of AuNPs, between one hundred and ten thousand times smaller than human cells and comparable to large biological molecules such as enzymes and antibodies, leads to interactions with biomolecules, both at the surface and inside cells, which may revolutionize disease diagnosis and treatment. AuNPs can act as either photothermal therapeutic (PTT) agents or probes for in vitro or in vivo bioimaging due to their high absorption and scattering cross sections in the visible-NIR range, on account of the so-called localized surface plasmon resonances, (LSPRs, see below). Additionally, AuNPs are promising scaffolds for gene and drug delivery applications, acting as active delivery platforms (triggering release) or just as passive carriers. The bright future for gold nanoparticles in biology and medicine is closely related with their unique optical properties, which originate from LSPRs. We do not intend to provide here a complete description of LSPRs, since the full theoretical treatment is rather lengthy and beyond the scope of this review; we therefore direct the reader to excellent reviews and feature articles [21–23]. LSPRs can be described as the collective coherent oscillation of conduction electrons with respect to a positive metallic lattice, occurring when a metal nanoparticle is stimulated by the electromagnetic field of an incident light beam. The resonance condition takes place when the frequency of the incoming light matches the frequency of free electrons oscillating against the inherent restoring force. Consequently, an enhanced electric field with respect to that of the incident light is generated at the particle surface. LSPR frequencies of AuNPs typically lie in the visible-NIR range and can be easily modulated by tuning particle size and shape, as well as interparticle distance and the refractive index of the surrounding medium. Surface plasmon oscillations decay via two pathways: (1) radiative decay where photons with the same energy as the incident light are emitted (LSPR light scattering); or (2) nonradiative decay via photon-to-thermal energy conversion [24]. These relaxation processes can produce strong enhancements (sometimes quenching) in the optical properties (fluorescence, Raman scattering, absorption, etc. . .) of molecules nearby/adsorbed on the nanoparticles surface. The optical cross section of AuNPs is orders of magnitude larger than those for dye molecules. Calculations based on Mie theory [24] showed that the absorption cross section of 40 nm Au nanospheres is 5 orders of magnitude higher than that for conventional absorbing dyes, while the light scattering of 80 nm Au nanospheres is 5 orders of magnitude higher than the light emis-

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sion from fluorescein, a dye commonly used in biological imaging. Since both phenomena are the basis of numerous bioapplications, the effectiveness of gold nanoparticles is strongly dependent on their scattering and absorption cross sections. For instance, particles with high light scattering intensity are potential agents for bioimaging through dark field microscopy or biosensing based on surface-enhanced Raman scattering (SERS) spectroscopy. On the other hand, particles with high absorption cross sections are particularly interesting for photothermal therapy or for bioimaging based on multiphoton plasmon resonance microscopy and photoacoustic tomography. It is thus crucial to keep in mind that the relative contributions of absorption and scattering can be modulated by means of the size, shape and composition of the nanoparticles. The relative contribution of scattering increases with the particle effective size. Particle morphology is highly relevant, as calculations show that the absorption and scattering coefficients of nanorods are one order of magnitude higher than those for nanoshells and nanospheres [24]. For biological applications, nanoparticles should ideally display an efficient LSPR in the NIR region of the biological transparency window (700–900 nm), where tissues display the highest transmissivity, meaning that nanorods, nanoshells, nanostars or nanocages appear to be the most suitable morphologies [20]. The modulation of cell behavior using AuNPs often requires targeting to a specific receptor for subsequent perturbation of its signaling activity (see next section). This can be achieved through careful biofunctionalization of the nanoparticles surface, which is also related to their biocompatibility because some of the most commonly used capping ligands for the fabrication of Au nanoparticles are toxic to cells. A prominent example is CTAB, an essential component during the preparation of Au nanorods, which displays high toxicity at the micromolar range [25]. The most common strategy for the attachment of biomolecules to Au surfaces involves the use of thiolated molecular linkers, since thiols strongly bind to gold (bond energy ca. 44 kcal mol−1 ), so that they are firmly anchored, even under biological conditions [26]. Other moieties, albeit with lower bonding strength, are also employed as anchoring groups include amines, dithiocarbamates, carboxylates, isothiocyanates, and phosphines. Molecular linkers facilitate conjugation of AuNPs with the desired biofunctional molecules by means of hydrophobic interactions, electrostatic forces, covalent cross coupling (carbodiimide, maleimide, or click chemistry), dative covalent bonding, oligonucleotide hybridization, and photolabile linkages (Fig. 1) [20]. Alternative functionalization strategies involve the encapsulation of AuNPs within a polymeric or dielectric shell, which can be subsequently conjugated. As an example, our group has recently reported a new class of SERS tags based on poly(N-isopropylacrylamide) (pNIPAM) encapsulated gold nanoparticles, which were bioconjugated with antibodies for multiplex immunophenotyping of cellular receptors and imaging of tumor cells [27]. A special mention should be made to silica coated Au nanoparticles (Au@SiO2 ), since silica has been proven not just to facilitate bioconjugation and labeling of Au nanoparticles but also to increase their biocompatibility and stability in biological media, additionally allowing the fabrication of multifunctional systems. Au@SiO2 nanoparticles have been used as biosensors [28], as labels for in vivo and in vitro bioimaging [29–31], drug delivery systems [32], therapy carriers [33], or theranostic transducers [34]. Besides targeting cell surface receptors, the modulation of cellular processes may be also achieved by the interaction with intracellular receptors and organelles, which is not trivial since the bioengineered AuNPs must escape from endosomes/lysosomes after cellular uptake. In vivo applications of AuNPs face additional challenges like accumulation in healthy organs or tissue-specific barriers. Therefore, a number of issues such as in vivo targeting efficiency, and toxicological effects of functionalized AuNPs are

under investigation. The biocompatibility and biodegradation of such nanomaterials need to be thoroughly investigated before they can be safely applied in clinical trials. Targeting nanoparticles to biomolecules (e.g. protein receptors) is usually accomplished by the use of antibodies attached on the Au surface [27]. Another alternative is the conjugation of AuNPs to thiolated lipids, so to facilitate their binding to the cell membrane [35]. If AuNPs are aimed to target the nuclear interior, the size of the transport channel of the nuclear pore complex (NPC) has to be considered. NPCs allow passive diffusion of ions and small molecules through aqueous channels with a diameter of ∼9 nm. Successful transport of AuNPs up to 39 nm in diameter into cell nuclei has been achieved through their bioconjugation with peptides or proteins containing a nuclear localization signal (NLS) [36,37], which were originally discovered to mediate the selective import of nuclear proteins by signal-mediated mechanisms [38]. Mitochondria however do not contain large pores to provide easy access for AuNPs, and therefore the conjugation of particles with mitochondrial targeting sequence (MTS) peptides have so far achieved limited success [39]. Another strategy to achieve mitochondrial targeting makes use of delocalized lipophilic cations (DLCs), small positively charged molecules that favor mitochondrial accumulation in response to the high negative membrane potential. This is the case of triphenylphosphonium (TPP), which has been to induce accumulation in the mitochondria of HeLa cells [40]. As mentioned above, the surfactant CTAB has been reported to be highly toxic because it damages lipid bilayers facilitating permeation and leading to the association of CTAB functionalized AuNPs to the mitochondrial membrane [41,42]. Finally, it has been reported that AuNPs are transported into the peroxisomal matrix via conjugation to proteins bearing the peroxisomal targeting signal [43].

Cell function and behavior are modulated by receptor-mediated cell signaling processes All cells are constantly exposed to a myriad of extracellular cues present in their environment. Their ability to perceive and correctly respond to this biological information is crucial for cell survival, development, tissue repair, or immunity. As a result, the cells have evolved different mechanisms that enable them to “read”, “translate” and “react” to such extracellular signals with appropriate cellular responses, which are collectively termed cell signaling. Cell signaling systems govern basic cellular activities, greatly influencing cell behavior, and thus, their dysregulation or malfunctioning result in diseases such as cancer [44]. At the heart of the cell signaling lie protein receptors that may be selectively activated by an ample variety of extracellular signals, including hormones, growth factors, ions, metabolic products, gases, and various chemical or physical agents. Cellular receptors come in many different varieties, but they can be classified into two main categories: intracellular and cell surface receptors (Fig. 2). Intracellular receptors are soluble cytoplasmic or nuclear proteins that are activated by molecules that can pass through the plasma membrane of the cell. Cell surface receptors bind to molecules and ligands that cannot traverse the plasma membrane. They typically present three regions; an extracellular domain that specifically recognizes a particular ligand, a transmembrane domain that tethers the receptor in the plasma membrane, and an intracellular domain, which interacts with the interior of the cell, relaying the signal. Cells have evolved a variety of signaling mechanisms to accomplish the transmission of environmental information. A general outline is as follows: Upon ligand-binding the cell surface receptor may adopt a new conformation, form aggregates (multimerize), or become chemically modified (e.g. phosphorylated). This change

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Fig. 1. Schematics illustrating various methods by which gold nanoparticles can be conjugated with biofunctional molecules. (A) hydrophobic interactions, (B) electrostatic forces, (C) covalent cross coupling, (D) dative covalent bonding, (E) oligonucleotide hybridization, and (F) photolabile linkages. Adapted from [20] with permission from The Royal Society of Chemistry.

usually results in the activation of the receptor, which may subsequently associate to adapter signaling molecules that transduce and amplify the signal through the cell by activating specific effector molecules. The outcome of the signal transduction cascade is a physiological response, such as secretion, movement, growth, division, or death [45,46]. Broadly, cell surface receptors fall into four main classes; Ligand-gated ion channel receptors, enzyme-coupled receptors, Gprotein-coupled receptors and adhesion receptors. Ligand-gated ion channel receptors are ion channels that are opened or closed (gated) by ligands, which causes a drastic change in the permeability of the channel to a specific ion. Enzyme-coupled receptors act as enzymes or associate with enzymes inside the cell. When stimulated, the enzyme activates intracellular signaling pathways involved in growth, migration, proliferation, differentiation and survival. G-protein-coupled receptors (GPCR’s) activate membrane-bound, trimeric GTP binding proteins, which in turn activate either an enzyme or an ion channel (effector) in the plasma membrane [45,46]. Adhesion receptors (integrins, cadherins, selectins, and immunoglobulin-like cell adhesion molecules) mediate cell–cell and cell-extracellular matrix (ECM) adhesion (Fig. 3) [47]. The ECM typically consists of a viscoelastic network of proteins and polysaccharides that provide biological and chemical moieties, as well as a physical scaffold supporting cell attachment and growth. At the nanoscale, the ECM shows an intricate mesh of protein fibers such as fibrillar collagens and elastins, ranging from 10 to several hundreds of nanometers, and nanoscale adhesive proteins, such as laminin and fibronectin, which provide specific binding sites for adhesion receptors, such as integrins and cadherins. In particular, integrin receptors recognize motifs such as Arg-Gly-Asp (RGD) within proteins of the ECM. Upon ligand interaction, integrins tether the cell cytoskeleton to the fibers of the ECM, forming focal adhesions, which play a major role in adhesion- and mechano-dependent signal transduction [48–50]. Recent studies within materials science have evidenced that the cellular response to environmental signaling goes far beyond the ability of the cell to sense soluble chemical cues (chemical composition, chirality or ligand density), but additionally encompasses a wide range of physical

cues (matrix stiffness, topography and interfacial hydrophobicity) generated at the cell/ECM interface [51–53]. Besides its prominent role in cell adhesion and spreading, cell–cell and cell/ECM interactions regulate major cellular physiological activities such as cell proliferation, motility, shape, differentiation, or pathological processes such as tumorigenesis [54,55]. Collectively, the receptors and intracellular proteins that convey an extracellular stimulus toward its destination in the cell’s interior constitute a signal transduction pathway. Myriad signaling cascades are initiated at the plasma membrane, upon interaction of extracellular signals with cell surface receptors. Such signaling pathways are high-fidelity, robust information-processing machineries that can distinguish weak signals from a noisy environmental background with high precision and selectivity. Depending on the nature of the external stimuli, specific signaling transduction pathways become activated, which very often form part of larger, highly interconnected networks [1]. Intracellular networks of signaling pathways (Fig. 4), allow the cell to respond specifically to a nearly limitless set of external inputs with controlled sensitivity, speed and duration. Proteins that interact with the wrong partners or are at the wrong place within cells cause various diseases. Therefore, the cell has evolved sophisticated regulatory mechanisms to ensure that the signal transduction machinery encounters intracellular substrates at the right place and at the right time [56]. Because of their functional significance in both normal and pathological conditions, cellular signaling provides attractive targets for disease therapy [57]. Colloidal gold nanoparticles Modulation of cell surface receptor activity While the signaling mechanisms and intracellular pathways of cell surface receptors have been the subject of intensive studies for over 50 years, we are only beginning to understand how this vast molecular network is interconnected. A major limitation to our understanding has relied on the experimental approaches being used to investigate cellular processes [58]. Tools capable of perturbing (and imaging) signaling pathways with fine spatiotemporal

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Fig. 2. Five major classes of cellular receptors. (A) Intracellular (cytoplasmic/nuclear) receptors bind to small or lipophilic molecules that can cross the cell membrane. The resulting ligand-receptor complexes may travel to the nucleus and bind to transcription-control regions in DNA thereby affecting expression of specific genes; (B) Ionchannel receptors, which selectivelly allow the passage of specific ions, thereby effecting changes in the membrane potential of a cell. They are involved in the transmission of electrical signals between excitable cells (e.g. neurons and muscle cells); (C) Receptors with an intracellular region containing an enzymatic domain. This group includes receptor tyrosine kinases involved in the response to many growth factors; (D) G-protein-coupled receptors that associate with cytosolic or membrane-bound proteins with enzymatic activity for signaling; (E) Adhesion receptors involved in binding with other cells (not depicted) or with the extracellular matrix (ECM).

resolution at the plasma membrane may not only contribute to elucidate the molecular mechanisms underpinning cell signaling and behavior, but they may also provide novel strategies for therapeutic interventions. As AuNPs can be engineered within a wide range of sizes, aspect ratios and surface chemistries, they have been applied as controllable spacers to mechanically manipulate the spatial organization and activity of cell receptors at the plasma membrane. The epidermal growth factor receptor (EGFR) is an archetypal cell surface receptor. The initial step in receptor activation involves binding of the EGF peptide to the extracellular domain leading to dimerization or activation of pre-existing dimers. In the canonical pathway, EGFR phosphorylates downstream targets following activation, initiating signaling cascades that drive various cellular responses, including cell proliferation and inhibition of apoptosis. Overexpression of EGFR leads to uncontrolled cell signaling, which is associated with poor prognosis in many tumors including glioblastoma, breast, lung, ovarian, colorectal, renal carcinomas and brain cancer [57]. Studies on the activation of EGFR revealed that the receptor assemble in nanometer-scale clusters, which may enhance inter-receptor communication, intracellular signaling efficiency, and information processing capacity [59]. However, this process is not completely understood, in part, due to the inability to control the spatial distributions of recep-

tors on the biologically relevant scale (10–100 nm). Paviolo and collaborators employed Au nanospheres and nanorods of different dimensions, functionalized with thiolated-EGF as tunable spacers to control the activation of EGFR receptors on living cells and their subsequent effects on cell proliferation [60]. Both EGF conjugated nanorods (10 × 37 nm) and spheres (80 nm) stimulated cell proliferation, though to a lesser extent than soluble EGF, likely due to EFGR activation. Interestingly, larger EGF-nanorods (25 × 94 nm) inhibited cell proliferation to a 43% relative to control cells incubated with non-conjugated nanorods. The authors hypothesized that the complex construct sterically hindered EGFR at different protein densities, since the separation between two EGFs bound to opposite nanorod tips (94 nm) exceeds the length of highly ordered EGFR clusters (32–56 nm) [61]. On the other hand, the coverage of EGF molecules on 80 nm spheres is higher and the dimensions of small EGF-nanorods (10 × 37 nm) match the EGFR cluster length (32–56 nm), so that these nanostructures may promote EGFR activation and induce cell proliferation. Ligand-mediated receptor oligomerization and clustering are also involved in mast cell activation [62]. This results in the active secretion of chemical mediators of inflammation (i.e. ␤hexosaminidase) from intracellular granules (e.g. degranulation), as part of the adaptive immune response, which is also responsible for the symptoms of allergy. The first steps of this process occur

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Fig. 3. The behavior of individual cells and the dynamic state of multicellular tissues are regulated by reciprocal molecular interactions between cells and their surroundings. This extracellular microenvironment is a hydrated protein- and proteoglycan-based gel network comprising soluble and physically bound signals as well as signals arising from cell–cell interactions. The specific binding of these signaling cues with cell surface receptors induces complex intracellular signaling cascades that converge to regulate gene expression, establish cell phenotype and direct tissue formation, homeostasis and regeneration. Ellipsis (. . .) indicates that the lists of signals are not intended to be complete. PLC, phospholipase C; GAGs, glycosaminoglycans; PGs, proteoglycans; CAMs, cell adhesion molecules. Reprinted by permission from Macmillan Publishers Ltd: Nature Biotechnology [47], Copyright 2005.

at the plasma membrane, where antigen-specific immunoglobulin E (IgE) bound to its receptor, Fc␧RI, is cross-linked by a soluble multivalent antigen. Cross-linking of IgE-Fc␧RI complexes triggers a signaling cascade that leads to Ca2+ mobilization and cellular degranulation [63]. Huang and co-workers explored the use of AuNPs as a platform for multivalent binding of IgE-Fc␧RI complexes to either activate or inhibit mast cell activation [64]. This study showed that dinitrophenyl (DNP)-conjugated AuNPs larger than 20 nm demonstrated the greatest effect on ␤-hexosaminidase secretion and were very potent effectors of RBL-2H3 mast cells for inflammatory response. They also found that the degranulation response decreased by lowering the amount of DNP present on the surface of the 20 nm nanoparticles. However, by decreasing the DNP surface coverage to 10 molecules per nanoparticle, effective inhibitors can be generated by competitively inhibiting the degranulation stimulated by a polyvalent antigen consisting of bovine serum albumin (BSA) conjugated with multiple DNP haptens (BSA-DNP). The authors suggested that DNP-AuNPs of different architectures can actively engage and mediate the molecular processes that are essential for regulating cell functions (Fig. 5A). In a recent study, Yang and co-workers screened a peptideAuNP hybrid library to inhibit Toll-like receptor (TLR) signaling, which plays a central role in the pathophysiology of many acute and chronic human inflammatory diseases [65]. The study focused on TLR4, which recognizes the lipopolysaccharide (LPS) component of

the bacterial cell wall and identified a specific nanoparticle hybrid (designated P12) that largely inhibited both arms of the TLR4 signaling pathway by abrogating NF-kB/AP-1 and IRF3 activation, as well as the secretion of a variety of pro-inflammatory cytokines. Significantly, by means of structure activity relationship studies, this study identified key chemical components of the NP hybrids related to the hydrophobicity and aromatic ring structure of the amino acids for tuning TLR4 responses. In addition, the anti-inflammatory activity of P12 in a murine model of acute systemic inflammation triggered by LPS was also evaluated. The authors found that pretreatment with P12 significantly reduced levels of cytokines, IL-6 and TNF-␣ in mouse serum following LPS challenge, whereas a control nanoparticle hybrid (P13), which only differs from P12 by substitution of a phenylalanine to alanine at the C-terminal position, displayed no inhibitory activity [65]. High-density lipoproteins (HDLs) are natural nanoparticles (7–13 nm in diameter) that bind cholesterol in the bloodstream. Cholesterol-rich HDLs are then trafficked to the liver, delivering their cholesterol cargo to hepatocytes through a specific cell surface receptor termed SR-B1 where it is properly processed and ultimately excreted in the feces. Significantly, cholesterol associated with HDLs has been termed “good” cholesterol, and epidemiology studies demonstrated that HDLs directly protect against the development of atherosclerosis and resultant illnesses such as heart disease and stroke [66]. It is now becoming apparent that some

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Fig. 4. Intracellular signaling networks regulate the operations of the cancer cell. An elaborate integrated circuit operates within normal cells and is reprogrammed to regulate hallmark capabilities within cancer cells. Separate subcircuits, depicted here in differently colored fields, are specialized to orchestrate the various capabilities. At one level, this depiction is simplistic, as there is considerable crosstalk between such subcircuits. In addition, because each cancer cell is exposed to a complex mixture of signals from its microenvironment, each of these subcircuits is connected with signals originating from other cells in the tumor microenvironment. Reprinted from [54], Copyright 2009, with permission from Elsevier.

Fig. 5. (A) Schematic illustration of nanoparticle-mediated cellular response. The multivalent AuNPs were generated through covalent attachments of thiolated dinitrophenyl onto AuNPs of various sizes. The attachment of multiple ligands onto the nanoparticle surface allows the formation of DNP molecules with different architectural features based on particle sizes and ligand densities, which, in turn, allows the selective control of specific interactions between DNP and IgE-Fc␧RI complexes in RBL-2H3 mast cells and the attendant alteration of cellular signal transduction [64]. (B1) Illustration of the inhibitory activity on TLR signaling (triggered by the canonical ligand, LPS) by peptideAuNP. (B2) Screening of nanoparticle modulators on THP-1 cell-derived macrophages with NF-kB/AP-1 and IRF reporter systems. Nanoparticle concentration = 100 nM. Adapted with permission from [65]. Copyright 2015 American Chemical Society.

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cancer cells express receptors for HDLs due to their increased need for cholesterol. Thus, research is focused on fabricating synthetic nanoparticles as delivery vehicles that can be loaded with diverse therapeutic cargo (e.g. chemotherapies, nucleic acids, proteins) and specifically targeted to cancer cells [67,68]. Gold nanoparticles are being used as cores to fabricate biomimetic HDL nanostructures (HDL-like AuNPs) within the size range of natural HDL by templating the assembly of a mixed phospholipid layer and apolipoprotein A-I [69–75]. Such HDL-like AuNPs recapitulate the cholesterolbinding properties of natural HDLs, and they specifically bind to the same receptors as natural HDLs [69,70,73]. The Thaxton group showed that HDL-like AuNPs can compete with natural HDLs to bind the high-affinity HDL receptor termed SR-B1. Uniquely, the AuNP at the core of the HDL-like AuNP enables differential modulation of cholesterol metabolism as compared with natural HDL, which leads to selective induction of apoptosis in B lymphoma cells [73]. In a follow-up study, this group demonstrated that HDL-like AuNPs binding to SR-B1 blocks the influx of cholesteryl ester leading to a dramatic reduction of cellular exosome uptake, whereby exosome-based information transfer between cells may be inhibited [74]. It is also noteworthy that HDL-like AuNPs induce the clustering of the SR-B1 receptor expressed as a fusion to green fluorescent protein in A375 melanoma cells, which may modulate the signaling pathway involved in exosome uptake. Since exosomes have been shown to promote melanoma disease progression, the identification of targeted inhibitors of this process may be translationally relevant [74]. In a recent study, Thaxton and collaborators recently showed that rhodamine labeled HDL-like AuNPs bind cell surface SR-B1, resulting in internalization and incorporation into newly formed exosomes. Notably, these nanoparticles can also bind SR-B1 in free exosomes, as demonstrated by flow cytometry, which may provide a rapid analysis for exosome detection [75]. A major limitation to the study of receptor signaling of cell has been related to the experimental approaches used to investigate such cellular processes [58,76]; In the past, crystallography has often been the method of choice for investigating receptor multimerization. However, crystallography requires harsh preparation conditions, a noncellular environment, and the crystallized state might not always represent the biochemically active form [77]. On the other hand, light microscopy studies are limited by the diffraction limit of visible light whereas electron microscopy cannot be applied to study live cells and relies on fixation and staining protocols, which may cause artifacts. Biochemical methods have therefore been the tools of choice to study cell signaling and other cellular processes. Although biochemical approaches have successfully identified and characterized many signaling components of the cell, they are destructive, and therefore lack spatiotemporal details [58]. Because of its high sensitivity and noninvasiveness, fluorescence imaging has been widely used to describe the organization and dynamics of cellular processes in live cells [77]. However, the resolution of a standard fluorescence system is limited by light wavelength to approximately 200 nm [78], whereas an IgG2 antibody molecule (Protein Data Bank accession code, 1IGT) is approximately 10 × 15 × 2.5 nm [76]. Therefore, individual components localized hundreds of nanometers apart will still appear co-localized in conventional fluorescence microscopy. Recent developments in single-molecule imaging involve improvements on two complementary aspects: microscopy techniques and optical probe synthesis, allowing better biocompatibility, brightness (for precise detection), stability (for longer detection), specificity, monovalency, together with a small size as possible, so as to not perturb the molecular function of the tagged moiety. Super-resolution fluorescence microscopy techniques including stochastic optical reconstruction microscopy (STORM), (fluorescent) photoactivatable localization microscopy (fPALM), near-field

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scanning optical microscopy (NSOM), stimulated emission depletion (STED) microscopy and fluorescence correlation spectroscopy (FCS) have broken the diffraction limit and can now be applied to image the local structure of living cells from the micro to the nanoscale [58,79,80]. However, phototoxicity, low signal intensities and photophysical stability of organic dyes, together with photobleaching effects, fundamentally limit the ability of fluorescence microscopy to continuously monitor cellular dynamics over extended periods of time with high temporal resolution [81]. To date, fluorescence-resonance energy transfer (FRET) has been the fundamental tool in imaging and understanding interand intramolecular interactions. However, the technique is sensitive only to molecular interactions that occur at short distances (typically 5 nm), which requires protocols to precisely position the donor and acceptor fluorophores within the molecules of interest. Alternative methods to fluorescence microscopy involve labeling with nonfluorescent nanoparticles made of latex, silica, polystyrene, or metal and detection methods based on Rayleigh scattering using standard microscopy techniques. This approach, however is limited to particles bigger than 40 nm [82] Plasmonic nanoparticles have the extraordinary ability to control light beyond the diffraction limit through LSPRs. They display optical cross-sections that are many times larger than those of fluorescent dyes, proteins or even quantum dots, they are chemically inert and have stable signal intensity because they do not suffer from photobleaching or blinking effects. Colloidal gold labeling of cellular proteins were first introduced by Faulk and Taylor in 1972, when they absorbed anti-salmonella rabbit gamma globulins to gold particles for one-step identification and localization of Salmonella antigens [83]. Since then, immunogold labeling in combination with scanning electron microscopy (SEM) and transmission electron microscopy (TEM) have been successfully applied to identify and localize cell receptors in fixed cells and tissues. Since then, AuNPs have been implemented as labels in a variety of optical techniques for cell bioimaging studies, including dark-field microscopy, reflectance confocal microscopy, photoacoustic and photothermal imaging, one- and two-photon fluorescence imaging, SERS imaging, plasmon-resonance energy transfer (PRET), plasmon-modulated fluorescence and plasmon coupling microscopy (PCM) [79,84–86]. In particular, PCM has been applied as a non-fluorescent, all-optical approach to study the dynamics of cellular receptors and their spatial organization into clusters, employing AuNPs as labels [81]. Individual plasmonic NPs can strongly interact with each other producing distancedependent electromagnetic interactions that lead to collective spectral responses (e.g. plasmon coupling effects), detectable in the far-field [87]. The advantages of plasmon coupling include a dramatic nonlinear increase in scattering cross-section per interacting particle, alterations in LSPR frequency and depolarization of linearly polarized light. Since the distances over which coupling is significant can be as much as three times the particle radius, the range of detectable protein–protein interaction distance is extended by more than an order of magnitude relative to FRET [88]. By plasmon coupling microscopy (PCM), the spectral shifts in the scattering response of clustered NPs facilitate the detection and sizing of individual NPs and NP assemblies. The advantages of PCM include the ability to image sub-diffraction limit interactions between NP tagged species, high temporal resolution, and extraordinary photophysical stability of the probes [81]. The dramatic changes in optical properties correlate with nanometer-scale alterations in the organization of labeled biomolecules, which facilitates statistical analysis of the processes under observation. Thus, AuNPs have been applied in PCM studies as plasmonic probes for labeling specific cell surface receptors, including EGFR, Her-2, Erb1, Erb2, CD24 or CD44. This has enabled imaging the spatial distribution of receptors at nanometer length scales in live cells [89–94].

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A nanotechnology tool has been recently reported, which is based on monovalent targeted magnetoplasmonic nanoparticles (MPNs) composed of a magnetic Zn-doped ferrite core, a dielectric silica layer and a plasmonic gold shell, capable of targeting, visualizing and mechanically activating cell surface receptors (Notch and E-cadherin), at the single-cell or single-molecule level, with high spatiotemporal resolution in vivo [95]. Spatial control is achieved through application of a focused magnetic field gradient to a particular cellular location and subsequent nanoparticle re-localization. This study extends the use of magnetic nanoparticles for controlled activation of cell behavior [96], and adds the imaging functionality provided by the plasmonic shell. As mentioned in previous sections, the activation of mechanosensitive protein receptors is regulated by ligand binding, oligomerization, spatial organization, mechanical loading, and processing by a number of downstream proteins. The MPNs with the ability to bind monovalently, provide precise chemical and physical control of ligand-receptor binding [95]. The superior properties of MPNs as a perturbation tool over traditional microbeads are shown in Fig. 6. Microbeads (M280 Dynabeads, ThermoFisher) and MPNs showed dramatic differences in all aspects tested. Not only microbead attachment was sparse (Fig. 6A), but a significant perturbation of the cell-surface structure could be detected upon microbead labeling, as shown by SEM (Fig. 6C). Importantly, microbead labelling led to immobilization of Notch receptors (Fig. 6E), E-cadherin clustering and F-actin polymerization (Fig. 6F) without the application of an external magnetic field. On the other hand, monovalent MPNs yielded dense Notch receptor labelling (Fig. 6B) and minimal perturbation of cell-surface structure in the absence of an applied magnetic field (Fig. 6D). The authors stated that they could not detect alterations in Notch receptor diffusion (Fig. 6E) or E-cadherin signal activation (Fig. 6G) by MPN labelling. In this study, MPNs enabled to decouple spatial from mechanical cues in activation of E-cadherin junction formation (Fig. 7). Spatial localization of the nanoprobes—and, hence, E-cadherin— was induced by placing the magnetic tweezer 2 mm above a target subcellular location (Fig. 7A). E-cadherin clustering induces recruitment of F-actin at or adjacent to E-cadherin clusters. Under the weak-force regime (1 pN), F-actin assemblies showed a circular shape rather than defined filamentous actin network structure (Fig. 7B, top). In contrast, under a strongforce mode (9 pN), where E-cadherin receptors were both spatially localized and mechanically activated, the recruited F-actin assemblies exhibited diverse structural patterns and a filamentous network (Fig. 7B, bottom). Significant differences in actin fluorescence intensity (Fig. 7D and E) and fluorescence area (Fig. 7F and G) were observed when E-cadherin was clustered with and without mechanical loading. Also recruitment of vinculin occurred (Fig. 7H). These observations support the notion that a force-mediated conformational change stabilizes E-cadherin-cytoskeletal complexes, illuminating how living cells respond to force at the nanoscale.

Modulation of angiogenesis Angiogenesis is a process in which new blood vessels originate from existing ones, generating a new vascular network to support a local metabolic demand for oxygen. It initiates upon activation of specific cellular receptors located in the plasmamembrane of endothelial cells, an event that initiates a signaling cascade that results in cellular proliferation to form the new vascular structures. This is a tightly regulated process that is influenced by the microenvironment and modulated by numerous pro-angiogenic and anti-angiogenic factors. When dysregulated, angiogenesis contributes to numerous malignant, ischemic, inflammatory, infectious and immune disorders [97–99]. Since Folkman

first proposed the strategy of starving tumor cells of its blood supply more than 40 years ago, angiogenesis has become a major therapeutic target. In particular, the vascular endothelial growth factor (VEGF) and its receptor (VEGFR) have been shown to play major roles in physiological as well as in most pathological angiogenesis. Thus, numerous chemotherapeutic agents such as antibodies, aptamers and small-molecule inhibitors have been developed to inhibit VEGF signaling [100]. Despite some preclinical success, tumor resistance mechanisms limit the long-term benefit of current anti-VEGF signaling therapies [101]. Therefore, novel angiogenic modulators for the treatment of cancer are urgently needed. With this aim, several nanoparticle-based systems are being developed as an alternative strategy for anti-angiogenic therapies [102]. Several studies reported the capacity of AuNPs for the inhibition of VEGF-stimulated angiogenesis both in vitro and in vivo [103–107]. These approaches are based on the intrinsic capacity of citrate-capped AuNPs to bind to the heparin-binding domain found in certain proangiogenic growth factors like VEGF165 or bFGF and, as a result, inhibit their biological signaling activity. Notably, circular dichroism experiments showed the structural conformation of the heparin-binding growth factors VEGF165 or bFGF is significantly altered upon preincubation with the AuNPs, whereas non heparin-binding growth factors remain unaffected [105]. As an example of the anti-angiogenic effect of unmodified AuNPs, Mukherjee and co-workers showed that citrate-capped AuNPs significantly inhibited signaling events induced by VEGF165, such as VEGF receptor-2 phosphorylation, intracellular calcium release, cellular proliferation, migration and RhoA activation in vitro, without eliciting apparent toxicity. Importantly, AuNPs inhibited VEGF-induced angiogenesis in vivo employing a nude mouse ear model [108]. In a follow up study, this group showed that the capacity of the nanoparticles to inhibit VEGF165 signaling is abrogated upon surface modification of the AuNPs with different ligands [105]. An independent study confirmed the aforementioned results, additionally showing that AuNPs reduced the levels of VEGFR2 and AKT phosphorylation induced by VEGF165, as well as associated nanodomain/microdomain formation of the VEGFR2 receptor. The authors hypothesized that AuNPs bind to the heparin binding site of VEGF165, thereby inhibiting VEGF165-mediated signaling and subsequent cell proliferation and angiogenesis [106]. More recently, the same group reported the AuNP-mediated inhibition of VEGF165-induced migration and tube formation of endothelial HUVEC cells via the Akt pathway [107]. Since heparin-binding growth factors are involved in tumor development and metastasis, the group of Mukherjee evaluated citrate-capped AuNPs as a therapeutic agent in two separate orthotopic models of ovarian cancer. Strikingly, in this study the nanoparticles inhibited both tumor growth and metastasis, most likely due to abrogating MAPK signaling and preventing epithelial to mesenchymal transition of tumor cells [109]. Therapeutic angiogenesis for treatment of ischemia and vascular diseases relies on the delivery of exogenous growth factors, such as VEGF, to stimulate neovasculature formation. This can be achieved by either direct injection into the ischemic region or by systemic administration. Owing to the short half-life of VEGF in the blood circulation, lack of specific targeting, and the requirement for the growth factor to be present for relatively long periods of time, the clinical translation of this approach proved to be challenging. To solve these limitations, methods employing targeted delivery of biomaterials and nanoscale devices are being explored toward the controlled release of therapeutic agents in a time- and space-controlled manner [110–112]. Different investigations have shown that sustained and localized release of VEGF encapsulated within biodegradable hydrogels leads to significant recovery of ischemic regions in animal models [113,114]. Unfortunately, most of these methods require invasive implantation into the tissue. On

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Fig. 6. Effect of MPNs and microbeads on cell architecture and signaling after probe labeling. Optical imaging of (A) microbead- or (B) MPN-labeled U2OS cells expressing SNAP-hN1-mCherry receptors. MPN probes show dense labeling. Scanning electron microscopy images of (C) microbead- or (D) MPN-labeled cells expressing Notch. Inset image show a 10 x magnification on a single MPN. The cell edge is outlined in white. (E) Diffusion of Notch receptors labeled with magnetic microbeads or MPN probes. (F) Confocal microscopy of U2OS cells expressing E-cadherin, after labeling with (F) a microbead or (G) MPNs in the absence of any magnetic perturbation. Error bars indicate SE. ****p < 0.0001. NS, not significant. Reprinted from [95], Copyright 2009, with permission from Elsevier.

the other hand, systemic delivery of growth factor-loaded nanoparticles with subsequent homing to the ischemic region due to the so-called enhanced permeability and retention (EPR) effect is far less invasive and represents an attractive solution [115]. The use of AuNPs as a carrier of vascular endothelial growth factor (VEGF) for the treatment of ischemic lesions was reported by Kim and coworkers [116]. In this work, VEGF165-conjugated AuNPs bearing an hydrodynamic size of 124 nm (less than the 200 nm diameter required for effective EPR effect), preferentially accumulated in the ischemic region of mouse model of hind-limb ischemia, and stimulated growth of approximately twice as many blood vessels as free VEGF or unconjugated nanoparticles. Intriguingly, covalent conjugation of VEGF to the nanoparticles did not affect the activity of the angiogenic factor and the biomolecule maintained its ability

to stimulate endothelial cell proliferation [116]. This discrepancy with previously mentioned studies was not discussed. In a different approach, AuNPs were functionalized with peptides for modulating the expression of genes involved in angiogenesis by activating specific cell surface receptors [117]. Oligo-ethylene glycol (OEG)-capped gold nanospheres were conjugated with different peptides: (1) P1 (KPQPRPLS) which binds to the vascular endothelial growth factor receptor-1 (VEGFR-1) and promotes signal cascade activation of angiogenic genes, (2) P2 (KPRQPSLP) which does not interact with any receptor and is taken up by the cells (scrambled) and (3) P3 (KATWLPPR) which predominantly binds to neuropilin-1 receptor (NRP-1), which promotes receptor internalization and subsequent signaling. Significantly, competition experiments with free peptides demonstrated that the

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Fig. 7. Response of E-Cadherin to Spatial and Mechanical Perturbation with MPNs. (A) Subcellular localization of E-cadherin receptors (green) in U2OS cells by MPNs induces F-actin (red) recruitment. Scale bars, 5 mm and 2 mm (inset). (B–H) Force response of E-cadherin domain formation at the cell membrane. (B) Time series of F-actin fluorescence images before and after force application by MPNs: (top) weak force (1 pN at d = 2.0 ␮m) or (bottom) strong force (9 pN at d = 0.7 ␮m). Scale bars, 2␮m. (C) Outline of thresholding and segmentation algorithm used for analysis of actin recruitment and residual area. (D) Representative intensity trajectories of F-actin fluorescence within a 2-mmcircle of tweezing area after removal of the weak- or strong-force mode tweezer. (E) Statistical residual intensity analysis of multiple replicates. (F) Representative time trajectories of F-actin area upon tweezer removal after either weak- or strong-force modes. (G) Statistical residual area analysis ofmultiple replicates. (H) Immunofluorescence staining for vinculin recruitment after weak- or strong-force application. Scale bar, 2 mm **p < 0.01; ****p < 0.0001. Error bars indicate SE. Reprinted from [95], Copyright 2009, with permission from Elsevier.

P1- and P3-AuNPs bind to their receptors specifically. Gene expression analysis of in vitro cultured cells grown under serum starvation and hypoxic conditions showed that P1-AuNPs induced a 30-fold expression of angiogenic Vegf-A gene, as well as upregulation of Hif1␣, and c-Myc genes. On the other hand P3-AuNPs inhibited their expression, and no significant gene response was observed for the P2-AuNPs as the P2 peptide did not interact with the chosen receptors. Thus, gene stimulation or inhibition can be manipulated at will with specific nanovectorization systems. The same group investigated the capacity of Au nanospheres conjugated with P1, P2 or P3 peptides to manipulate the formation of capillaries by endothelial cells in vitro and in vivo using the chick embryo chorioallantoic membrane (CAM) assay. Both studies showed that depending on the peptide function, activating or inhibiting P-OEG-NPs can stimulate or block capillary formation, without causing toxicity to the cells, presenting experimental evidence for controlled blood vessel growth [118,119]. Pro-angiogenic gold nanoparticles bio-synthesized from chloroauric acid through a green chemistry approach employing

a proangiogenic extract obtained from Hamelia patens leaves (b-Au-HP), exhibited blood vessel formation activity in vitro and in a chick embryo model [120]. The treatment of endothelial (HUVEC) cells with the nanoparticles induced Akt phosphorylation (p-Akt) and generation of reactive oxygen species (ROS), which suggested the involvement of redox signaling for b-Au-HP induced angiogenesis. Interestingly, as the authors indicated, even though highly concentrated ROS are detrimental for most tissues, low levels of ROS can activate signaling pathways that eventually promote regeneration and vessel growth. The biocompatibility of b-Au-HPs was demonstrated by cell proliferation assays in vitro and by the lack of changes in NF-kB expression upon incubation with nanoparticles [120]. The ability of plasmonic AuNPs to absorb specific wavelengths of light and efficiently generate heat through the LSPR effect has been exploited for thermal modulation of cellular responses (i.e. membrane permeability and gene expression) in endothelial cells and inhibition of tumor angiogenesis in vitro [117,121]. In one study, different types of OEG-coated AuNPs (nanorods, hollow spheres,

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and nanoshells) functionalized with a KPQPRPLS peptide that preferentially binds to VEGFR-1 receptors, were used for assessing NIR laser-induced treatment of HUVEC cells at low power densities (10–24 W/cm2 ), below the threshold of most destructive studies [117]. This study shows that targeting nanoparticles to the cellular receptor induces transient changes in cell permeability upon laser treatment, whereas internalized nonfunctional particles (OEG-NPs) in endosomes cause cell death. Notably, gene expression profiles obtained from cells treated with either peptide-OEG-NPs or OEGcoated NPs after irradiation (10–24 W/cm2 ) for 10 min, showed that the expression levels of ELAM-1 and ICAM-1 genes, which are indicators of endothelial activation, were significantly downregulated, depending on laser power. The authors reported that this effect was more pronounced with hollow spheres and nanorods as compared to nanoshells. Control experiments without nanoparticles did not show any significant change in gene expression profile, indicating an absence of stress response at the laser intensities used. In a follow up study, OEG-AuNRs conjugated with the anti-angiogenesis peptide P3 (KATWLPPR) that selectively binds to neuropilin-1 (NRP-1) [118], were applied to inhibit capillary formation mediated by tumor cells in a synergistic manner, employing NIR laser intensities up to 24 W/cm2 . By tuning the nanoparticle dose and the laser irradiation the authors identified optimum conditions for the efficient inhibition of in vitro vascular growth [121].

Opto-thermal modulation of neural activity Neuromodulation, the stimulation or inhibition of an electrical change (e.g. action potential) through the nervous system, represents a powerful approach in neuroscience for the understanding of neural function and for clinical applications treating neurological diseases and restoring lost neural functions. Since Galvani first demonstrated in the 18th century that neurons could be electrically stimulated, voltage and current controlled electrical neural stimulation (ENS) has historically been the main technique for controlling neuronal activity and played a crucial role in neuroscience ever [122]. Despite the success of this approach, there has been interest in developing alternative techniques that avoid the disadvantages and limitations of electrical stimulation. For instance, although stimulating and recording electrodes offer unparalleled sensitivity and unparalleled temporal resolution, they generally offer poor spatial resolution and inability to selectively activate different neuronal subtypes [123,124]. To address this issue, alternative techniques employing light, magnetic fields or ultrasound, combined with engineered nanomaterials, have been proposed to stimulate neurons with high spatial resolution and cell-type specificity in a noninvasive or minimally invasive fashion [125,126]. In particular, infrared neural stimulation (INS) has been demonstrated as an effective technique for photo-thermal stimulation of neural tissue [123,124,127]. INS uses short pulses of infrared light to generate a local transient rise in temperature that is mediated through absorption of IR radiation by water. Such a local heating reversibly alters the electrical capacitance of the plasma membrane, resulting in cell depolarization and action potential propagation [128–130]. Complementary to the capacitative theory of INS action, it has been proposed that changes in temperature also can affect the conductance of temperature-sensitive ion channels embedded in the cellular membrane, such as members of the transient receptor potential vanilloid channel (TRPV) family, which upon thermal stimulation cause an influx of calcium ions, thereby depolarizing the membrane to fire action potentials [131]. Although the efficacy of INS has been demonstrated, direct heating of the bulk solution may cause off-target effects or cellular damage. Thus, an important aspect toward improving INS is to avoid excessive thermal heating that may cause tissue damage. Reducing the radiant

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exposure level required for INS would increase the safety margin between threshold and tissue damage [127]. An alternative strategy has been proposed to enhance INS, based on the use of plasmonic gold nanoparticles as photothermal nanotransducers to enable photothermal stimulation of neural activity [132–140]. Upon irradiation at their resonant wavelengths, plasmonic nanoparticles can produce rapid heating due to photon-to-heat energy conversion, which can be transferred to the immediate environment surrounding the nanoparticle. Targeting the plasmonic nanoparticles to the cell plasma membrane allows a more precise and localized effect, while avoiding tissue damage by excessive heating [141] Opto-thermal modulation of neuronal activity by means of plasmonic gold colloids as extrinsic absorbers was demonstrated by Yong and collaborators using silica-coated nanorods and a pulsed laser at the LSPR wavelength. Irradiation of neuronal cells with membrane-bound plasmonic nanoparticles induced a significant increase in their electrical activity compared with neurons that were incubated with non-absorbing gold nanospheres or cells with no AuNPs [136]. Yoo and collaborators, employed nanorods to inhibit action potentials of cultured hippocampal neurons using membrane-bound NIR-activateable nanotransducers. Binding to the cellular plasma membrane was facilitated by functionalizing the particles with positively charged amineterminated polyethylene glycol (NH2 -PEG). The authors also showed a possible involvement of the TREK-1 thermosensitive potassium channel in the photothermal inhibition of action potentials through membrane-localized photothermal effects [137]. Recently, Carvalho-de-Souza used AuNPs conjugated with celltargeting entities (scorpion toxin Ts1, which targets voltage-gated sodium channels, and antibodies against TRPV1 and P2 × 3 ion channels) to stimulate specific neuronal cells upon excitation with a visible continuous wave laser [138]. Interestingly, this study showed that laser-induced heating of planar lipid bilayers produced changes in bilayer capacitance. Since this process occurred in the absence of any membrane proteins, the authors suggested that ion channels and other membrane proteins are not necessary for the mechanism of neuronal stimulation via AuNPs [138]. A recent study employing nanorods functionalized with a cationic form of a high-density lipoprotein (catHDL) for targeting the cell plasma membrane, demonstrated that photo-stimulation of cultured DRG neurons occurred by activation of the thermosensitive ion channel TRPV1, which achieved Ca2+ influx and membrane depolarization [135]. Lavoie-Cardinal and co-workers followed a cell-targeting strategy to carry out photo-stimulation of subcellular regions on cultured hippocampal neurons, termed NALOS (NPAssisted Localized Optical Stimulation). The authors demonstrated that this approach triggered action potentials and activation of local Ca2+ transients and Ca2+ signaling via CaMKII in dendritic domains (Fig. 8) [139]. Finally, Eom and collaborators showed opto-thermal stimulation of neural cells both in vitro and in vivo [134,140]. Notably, the demonstration of enhanced stimulation in vivo suggests that this approach could be used to remotely manipulate and stimulate diverse excitable tissues [134]. Based on these studies, and while the underlying mechanism of optothermal stimulation remains to be fully understood, the use of plasmonic nanoparticles as nano sources of heat has several advantages over conventional INS approaches in terms of faster cell response, higher stimulation efficiency, and stronger behavior change. Significantly, the energy required to achieve stimulation is reduced in approximately two orders of magnitude [124], and the cooling kinetics observed after a laser pulse are faster than conventional INS by an order of magnitude or more [138]. Despite some potential limitations, such as the inability to independently excite multiple cell types using different wavelengths due to broad and multiple peaks of plasmon absorption, limited lifetime of cell photosensitivity due to clearance and/or degradation of deliv-

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Fig. 8. NALOS with functionalized AuNPs. (a,b) Representative neurons expressing (a)HA-GluA1and GCaMP6 s (n = 5) or (b) only GCaMP6 s (n = 5) and incubated 90 min with fAuNPs. The right panels in (a) and (b) show the same neuron as on their left, following fixation and immunostaining for the Ha-tag (revealed with Alexa 546). Without the presence of the HA-GluA1, the fAuNPs (functionalized with monoclonal anti-HA antibodies) did not bind on the neurons. (c–e) NALOS with fAuNPs triggered localized and repeatable Ca2+ transients (n = 10). (c) Other representative neuron transfected and imaged as in (a). (d) Magnification of the region marked in (c) for three consecutive time points (marked with 1, 2 and 3 on the graph in (e)); NALOS was applied on the region marked with a white circle. (e) Ca2+ signal (F/F) of the corresponding colored oval regions marked in (c), before and after NALOS at the time points marked by dotted lines. Scale bars (a,b) 10 ␮m and (c,d) 5 ␮m. Reprinted from [139] with permission.

ered plasmonic conjugates, as well as nanoparticle internalization, which may cause variability and short-term cytotoxicity, the aforementioned studies show that plasmonic AuNPs serve as a powerful complement to other light-dependent methods (e.g. optogenetics, caged molecule approaches) aiming to modulate the activity of excitable cells (e.g. neurons, muscle cells, etc). Finally, since membrane potential is believed to play important functional roles in non-excitable cells, -it has been involved in the proliferation, migration, and differentiation of cancer cells [142]-, this strategy could also be applied to finely tune cellular processes controlled by membrane potential in non-excitable cells. Nanostructured substrates Nanopatterned materials Engineered nanostructures with designed geometry and chemical functionality have been implemented in cellular studies to provide effective mimics of the ECM, in order to understand the regulation of these signaling processes at the nanoscale [143,144]. Such materials have found application in differentiating stem cells into neurons, repairing bone and inducing angiogenesis [47,48,143]. In particular, nanopatterned materials have emerged as a valuable tool for revealing instructive roles of individual biophysical cues of the ECM, such as cell shape and geometry, ECM topography, mechanical forces, and spatial organization of cell-adhesive pro-

teins, in regulating major cellular behaviors such as cell migration, proliferation or differentiation [48,145,146]. In particular, biofunctionalization of surfaces with patterning techniques allows the precise spacing of adhesion proteins and peptide sequences such as RGD recognized by integrin receptors, and regulating cell adhesion and mechano-transduction. Thanks to these techniques it has been possible to understand at the molecular level some of the parameters controlling the very initial integrin recruitment steps in the formation of focal adhesions [48]. ‘Bottom-up’ methods based on self-assembly of nanoscale components offer an inexpensive and convenient route for creating in-plane nanoscale features over large areas [147]. In particular, colloidal lithography is a method for fabricating nanostructured biomaterial interfaces [148], that has been applied for gaining understanding in the molecular mechanisms used by the cells to sense their physical environment. One particularly successful approach is block copolymer micelle lithography (BCML) developed by Spatz and co-workers [149]. The basis of this approach is the self-assembly of polystyrene-b-poly[2-vinylpyridine (HAuCl4 )] diblock copolymer micelles into uniform monomicellar films on solid supports such as glass. The reduction of the precursor generates AuNPs, which are embedded in the centre of the copolymer micelles. Subsequent plasma treatment of the dry film leads to uniformly patterned sub-10 nm gold nanoclusters, with inter-spot distances varying from 20 to 100 nm (depending on the micelle size prior to plasma treatment). Spatz applied such nanoparticle arrays as anchor points to pattern thiolated proteins with single molecule

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Fig. 9. (A) Alexa Fluor 555 conjugated mouse IgG coupled to a gold nanoparticle array through the site-directed immobilization of histidine-tagged protein A on nitrilotriacetic acid (NTA). The spacing of gold nanoparticles within the 5 ␮m circles is 70 nm, and the hydrogel consists of PEG-700-DA. (B) Immunofluorescence micrograph of a REF52YFP-paxillin fibroblast on a PEG-10000-DA hydrogel. The microstructure consists of 5 ␮m circles separated by 5 ␮m. Cells are stained for the nucleus (blue) and the actin cytoskeleton (green), and stably expressed YFP-paxillin localized at focal adhesions is shown in red. Adapted with permission from [164]. Copyright 2010 American Chemical Society.

resolution for investigating the function behind the molecular arrangements of specific cellular receptors. In an early work, quasihexagonally close-packed rigid templates of cell-adhesive gold nanodots coated with thiolated cyclic RGDfK (c-RGDfK-thiol) were used to evaluate the nanoscale spacing required for effective adhesion mediated by integrins. It is well known that RGD ligand clustering of integrin receptors is a prerequisite to trigger intracellular signaling pathways involved in cell adhesion, motility and spreading. The size of the NPs (ca. 8 nm) is small enough to allow the binding of a single integrin receptor molecule, whereas the area between these adhesive sites was passivated with PEG to prevent cell and protein adhesion, thereby ensuring that membrane receptor binding is specific. By precisely positioning RGD-functionalized gold nanodots at 28, 58, 73, and 85 nm spacing the authors showed that when the spacing exceeded approximately 70 nm, cell adhesion and spreading, focal adhesion, and actin stress fiber formations were significantly impaired, likely owing to the restricted clustering of integrin molecules imposed by the distance between adjacent gold nanoarrays [150]. In subsequent studies, the same group applied this method for the study and control of integrinmediated cell adhesion, and downstream signaling processes in various cell types, by fine tuning the distribution of RGD-binding motifs at the molecular level in nanopatterns, revealing some of the parameters controlling the initial steps of integrin recruitment during the formation of focal adhesion complexes [151–156]. These studies have highlighted the important role of ligand density and nanoscale presentation in cell spreading and motiliy. For example, in a recent report, the role of ␣5␤1 and ␣v␤3 integrins on fibroblast adhesion and migration was characterized on BCML gold nanopatterned substrates coated with the extracellular matrix glycoproteins fibronectin and vitronectin. They found that fibroblasts manifested high directional motility during migration on fibronectin-, but not vitronectin-coated substrates. Also, directional migration on fibronectin seems to require the engagement of both ␣v␤3 and ␣5␤1 integrins to the substrate, and does not support the general view that ␤3 integrins promote directional migration, while ␤1 integrins favor random migration [156].

Amschler and collaborators used a similar BCML-based approach to investigate the integrin-mediated adhesion of melanoma cells as a function of the density of nanopatterned RGD ligand sites. They showed that melanoma cells spread optimally at a ligand site density of 349 NPs/␮2 (lateral spacing of 60 nm). Either increasing or decreasing the ligand site density from this optimum value abrogated full cellular spreading [157]. Spatz and co-workers studied T cell activation on BCML gold nanopatterned substrates functionalized with poly-histidine tagged ligands [158,159]. In one study the major histocompatibility complex class II (pMHC) proteins to investigate the influence of ligand density in T cell activation. They found that local pMHC clustering is not important for T cell activation and identified a threshold ligand density of approximately 112 molecules per ␮m2 [158]. In a different study, immune cell stimulating nanoarrays bearing T cell or NK cell stimulatory ligands attached to an array of evenly spaced gold nanoparticles generated by BCML were used to investigate how spacing affects signaling from two paradigmatic immune receptors: the T cell antigen receptor (TCR) and the activating NK cell receptor CD16. In both cases, the strength of the response decreased with increasing nanoparticle spacing, falling to background levels by 69 nm in the T cell/anti-CD3 system and 104 nm for the NK cell/anti-CD16 system. These results again demonstrate that immune receptor activation can be influenced by the nanoscale spatial organization of receptor/ligand interactions [160]. ECM stiffness is recognized for playing an important role in regulating cell fate and tissue development [161,162]. To study the effects of varying matrix elasticity on cell behavior, PEG hydrogels have been widely used to create a biomimetic artificial substrate with tunable stiffness, which can be easily tuned by varying the molecular weight or concentration of the polymer. However, despite its biocompatibility, pure PEG based hydrogels do not support cell adhesion and proliferation, unless cell-adhesion ligands (e.g. RGD peptides, collagen, etc) are incorporated into the PEG scaffold. Unfortunately, this hydrogel system does not allow to decouple mechanical properties (e.g. stiffness) from ligand presentation [163]. This issue was addressed by Spatz and co-workers, who developed a method for

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the preparation of soft micro-nanopatterned PEG hydrogels based on block copolymer nanolithography of AuNPs, as anchoring points for biomolecules, with tunable stiffness and independent control over biomolecule arrangement at the nanometer scale (Fig. 9) [164]. This method was employed by Trappman and collaborators to show that the tethering of keratinocytes to collagen, and not the stiffness of the gel, influences cell-fate decisions [165]. Recently, Ding and collaborators also employed nanopatterns/micropatterns of gold nanoarrays on non-fouling PEG hydrogels fabricated by block copolymer micelle nanolithography and transferring technologies to decouple the stiffness effect from protein tethering in the differentiation of mesenchymal stem cells (MSCs) [166], and in the dedifferentiation of chondrocytes [167]. Recently, the group of Basabe-Desmonts reported a Printing and Vacuum lithography (PnV lithography) combined technique employing micro-contact printing, directed self-assembly of AuNPs and microfluidics for the production of a chip based platform in which mammalian cells can assemble into predefined patterns. The plasmonic AuNPs function as sensing and actuating elements, which can be introduced in the cells’ microenviroment, providing a new, powerful substrate for cell studies [168].

Nanocomposite scaffolds Artificial biomaterial scaffolds designed to support the growth of cells and tissues have traditionally designed at a macroscopic scale, so as to reproduce the biological properties of the tissues they are to replace, without recreating the nanoscale features found on real organs [50]. Notably, studies of engineered cell–biomaterial interactions at the subcellular level are providing evidence regarding the potential importance of submicrometer cues for cell signaling, adhesion, growth, and differentiation [143]. The rationale behind incorporating nanostructures is to compensate for scaffold limitations such as weak mechanical properties, the absence of adhesive and microenvironment-defining moieties, and the inability of cells to organize into tissues [169]. Thus, different inorganic nanostructures such as carbon nanotubes, silicon nanowires, and metallic nanoparticles, including AuNPs, have been incorporated into tissue engineering scaffolds as a means for improving the mechanical and physical properties of the material for directing cell growth and tissue morphogenesis [169]. Collagen is the major component of the ECM and therefore it has been proposed as a natural choice for an engineered scaffold biomaterial as it has the advantage of providing both structural and microenviromental support. However, collagen matrix lacks the mechanical properties desired for vascular grafts. In an attempt to improve the biocompatibility of this material, Hung and co-workers showed that composites containing AuNPs enhance the properties of collagen biomaterials. In this study, a nanogold-collagen composite was obtained by mixing a solution of collagen type I with AuNPs (5 nm). Nanocomposites (containing 17.4, 43.5 and 174 ppm of AuNPs) favored the proliferation and migration of mesenchymal stem cells, reduced activation of monocytes and platelets, as well as the attenuation of ROS production. The authors indicated that the cell behavioral changes take place through regulation of the avb3 integrin/CXCR4 receptor, FAK, MMP-2, and Akt/eNOS molecular signaling [170]. In another study, Xing and collaborators employed a one-step strategy to synthesize stable colloidal gold-collagen core-shell nanoconjugates in aqueous solution, without the addition of any reducing and stabilizing agents. The film formed by layer-by-layer (LbL) assembly of such colloidal nanoconjugates supported the adhesion and growth of NIH-3T3 cells [171]. A silk nanofiber matrix was fabricated by electrospinning, incorporating AuNPs and subsequent chemical modification of the AuNPs to immobilize the integrin-binding RGD peptide (Fig. 10) [172]. Silk

was used as a model material because of its favorable mechanical properties, biocompatibility and potential uses as a scaffold in engineered tissue. The authors found that the spreading and adhesion of MSCs were enhanced by the biofunctionalized nanostructure as compared with bare silk fibers [172]. Gold nanoparticles were also employed for improving the functional properties of a gelatin-based fibrous scaffold for cardiac tissue engineering [173]. In this study, a polycaprolactone–gelatin mixture was electrospun to obtain fibrous scaffolds with an average fiber diameter of 250 nm. Subsequently, AuNPs were evaporated on the surface of the fibers, thereby creating nanocomposites with a nominal gold thickness up to 14 nm. Compared to pristine scaffolds, cardiac cells assembled on the nanocomposite into more elongated and aligned tissues, promoted massive cardiac sarcomeric actinin expression and exhibited anisotropic transfer of electrical signals, leading to higher contraction rates and stronger contraction forces. The authors hypothesized that the cell–cell interactions promoted by AuNPs are likely responsible for the improved properties of the engineered tissue. In a recent study, inspired by the coiled structure of perimysial fibers present in the native heart matrix, a coiled electrospun fiber substrate doped with AuNPs was fabricated in order to improve the mechanical properties of the nanocomposite [174]. A key challenge in cardiac tissue engineering is the fabrication of non-immunogenic scaffolds that would not induce adverse immune response upon transplantation. To address this issue, Shevach and collaborators employed omental tissue devoid of cells to obtain a non-immunogenic scaffold [175]. Since naturally derived matrices have impaired or low electrical conductivity, the decellularized scaffold was subsequently functionalized with 4 or 10 nm AuNPs as in previous studies. To investigate the effect of the AuNPs within the scaffolds on tissue organization and function, cardiac patches obtained from neonatal rat hearts were grown on the platform. The assembly of the engineered tissue was assessed by immunostaining for sarcomeric actinin, a protein associated with cell contraction, and connexin 43, associated with electrical coupling between adjacent cells. The hybrid nanocomposite exhibited improved properties toward enhanced electrical conductivity, such as elongated and aligned cell morphology, massive striation, and organized distribution of connexin 43, which led to fast, anisotropic propagation of the electrical signal (Fig. 11). The authors suggested that besides engineering non-immunogenic cardiac patches to improve heart function after myocardial infarction, this strategy could be also beneficial for other electrogenic cells such as neurons and may assist to engineer other tissues such as the injured spinal cord. Gold nanocomposites have been also used for wound healing applications. A nanocomposite mixture of AuNPs, epigallocatechin gallate (EGCG), and ␣-lipoic acid (ALA) was used to stimulate Hs68 and HaCaT proliferation and migration, as well as to accelerate the wound healing of mouse skin through anti-inflammatory and antioxidant effects [176]. Chitosan/AuNPs nanocomposites, as well as a gold nanocomposite comprising phytochemically stabilized gold nanoparticles coated on a hydrocolloid membrane (HCM) used for dermatological care, enhanced the rate of wound healing, the increase of angiogenesis, and the formation of connective tissue in rat models [177]. Nanocomposite materials for physical modulation of cell behavior Modulating cell behavior using controllable, on-demand nonbiochemical methods, such as electrical stimulation is an attractive area of research. It is widely accepted that electrical currents modify the cell membrane potential and subsequent ion flux across the membrane. The physiological effects of electrical stimulation have been shown to be due to the redistribution of cell surface receptors and cytoskeleton reorganization in response to applied electrical

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Fig. 10. Synthesis and fabrication of AuNPs-doped SNF (A-C) and SNF cell interactions (D, E). (A) The building blocks: silk fibroin and gold seed nanoparticles. (B) A SNF doped with AuNPs (SNFAu) on the surface and throughout the fiber cross section. (C) An illustration of surface modi cation of the SNFAu. In this case the surface of the AuNPs was chemically modified with RGD motif peptide. (D) Confocal imaging of (I) SNF, (II) SNFAu, (III) SNF + RGD, and (IV) SNFAu + RGD (II). Stains: nucleus (blue), actin laments (red), and vinculin (green). Scale bar is 25 ␮m (E) I. Morphology of hMSC cultured on SNFAu + RGD. Scale bar is 20 ␮m. II. Expanded view of the white dashed box, white arrow mark adhesion points. Scale bar is 400 nm.

fields [178]. Nanostructured AuNPs have been investigated as conductive materials to electrically stimulate cells or tissues, thereby influencing their behavior. For example, AuNPs deposited onto poly(ethyleneimine) (PEI)-coated surfaces have been shown to promote neurite outgrowth in PC12 nerve cells, upon application of pulsed electrical stimulation [179]. In another study, AuNPs modified with fibronectin and PEI were exploited to induce stem cell differentiation of human embryonic stem cells (hESCs), resulting in a loss of stem cell marker OCT-4 expression and enhanced expression of the osteogenic markers collagen type I and Cbfa1, in contrast to non-stimulated cells [180]. Orza and collaborators fabricated gold-coated collagen nanofibers (GCNFs) and used them as electrically conductive substrates for human placental-derived stem-cell differentiation. The spatial arrangement of collagen, along with the

potential of GCNFs to deliver electrical stimulation, induce faster neuronal and cardiac differentiation of the stem cells, as compared to control substrates, when the metal-collagen substrate is exposed to electrical stimulation [181]. Modulation of cardiac cell function was assessed with thiol-HEMA/HEMA scaffold containing AuNPs prepared by a polymer templating technique, which leads to a homogeneous synthesis of Au nanoparticles throughout the gel. The authors show that conductive and mechanical properties of the nanocomposite can be controlled via scaffold formulation. Although the conductivity observed in thiol-HEMA/HEMA scaffolds was high with respect to physiological values, they exhibited values comparable to typical conductive hydrogels (between 0.1 and 30 S/m). Notably, neonatal rat cardiomyocytes exhibited increased expression of the protein connexin 43 (Cx43), which forms gap

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Fig. 11. (A). Schematic overview of the concept. Omental tissue is isolated from the patient and undergoes a quick decellularization process to produce the 3D scaffold biomaterial. The decellularized scaffold is decorated with conductive motifs using an e-beam evaporator to produce a hybrid scaffold. Cells are isolated from the same patient, cultured in vitro, and seeded on the hybrid scaffold to produce a personalized cardiac patch. The engineered patch is transplanted on the infarcted heart. (B-G) Cardiac cell organization and engineered tissue function on day 5. (B-D) Immunostaining of cardiac sarcomeric actinin (pink), connexin 43 (green), and nuclei (blue) of cardiac cells within the pristine (B), 4 nm (C), and 10 nm (D) scaffolds. Lower panels are higher magnifications. White arrows indicate the location of connexin 43 molecules. (E) Spontaneous contraction amplitude. (F) Excitation threshold. (G) Velocity of calcium transients during spontaneous contractions. The velocity is normalized to tissues engineered in pristine scaffolds. Bar = 20 ␮m.

junction channels involved in regulating how current spreads between cardiomyocytes. However, Cx43 was also upregulated on conductive scaffolds in the absence of electrical stimulation. The authors concluded that conductive scaffolds may facilitate cardiomyocyte function, though electrical stimulation may not be necessary to regulate cell function [182]. On a further development, Yoo and co-workers fabricated an electro-optical neural platform integrated with a monolayer of randomly oriented gold nanorods (GNRs) (optimal density of 150 GNRs/␮m2 ) for simultaneous electrical excitation and readout, achieving photothermal inhibition of neural activity upon irradiation with a NIR laser [137]. The nanostructured substrate was generated by electrostatic binding between amine-terminated PEGylated GNRs and a glass substrate bearing hydroxyl moieties

(OH−). Photothermal conversion through the monolayer has several advantages over colloidal actuation, as it allows uniform heat generation without aggregation or nonuniform distribution of the nanoparticles. In addition, the nanoplasmonic interface enabled thermal stimulation of target neurons, while rapidly collecting in situ information from adjacent electrodes. The ability to modulate cell adhesion is of high relevance for biomedical research, regenerative medicine, and tissue engineering. In particular, controlled and non-invasive detachment from responsive surfaces devoted to cell culture is of significant interest for many research and commercial applications. The standard procedure for dissociating cells cultured in vitro is an enzymatic method based on proteolytic enzymes that “digest” the proteins that facilitate adhesion to the substrate and between cells. How-

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Fig. 12. (A) Schematic representation of the fabrication of plasmonic substrates for cell culture and harvesting. (B) Vis-NIR spectra of plasmonic substrates before (green line) and after (dark-blue line) the chemical growth of adsorbed Au nanoparticle seeds. (C) SEM image of the plasmonic surface. (D) Photograph of a plasmonic substrate on glass. (E-F) Fluorescence microscopy images of cells (HeLa (E) and HUVEC (F)) as grown on bare plasmonic substrates (Au, pictures on the left) and c- RGD-coated plasmonic substrates (RGD, pictures on the right). Staining was as follows: nuclei (blue), vinculin (green), actin (red). Scale bar is 50 ␮m. Aspect ratio of the cells defined as the ratio between the longest and shortest dimensions. Only HUVEC cells show significant differences by Student’s t-test, *p < 0.05 [183].

ever, such an enzymatic disaggregation may damage the cells, is almost ineffective for certain tissues, and lacks selectivity. Recently, the plasmonic response of AuNPs has been applied to modulate adhesion of cells to nanoestructured materials, by remote irradiation with laser light. Visible green laser light was used to induce the selective detachment of NIH3T3 fibroblasts cultured on a glass substrate covered with AuNPs bearing strong photo-absorption in the green spectral range. Activation of cell detachment took place upon irradiation of the gold surface with a laser beam at 532 nm. The authors proposed a nonthermal photochemical effect due to the generation of ROS, as the plausible mechanism for cell detachment. In addition, as the irradiated surface was recolonized by cells, the authors suggested that this approach could be applied to spatially pattern cells, and create co-culture cell sheets by seeding different cell types on irradiated regions. Recently, Liz-Marzán and collaborators fabricated a plasmonic substrate by BCML based on anisotropic AuNPs for the growth and NIR light induced harvesting of cells upon irradiation with a 980 nm laser

(Fig. 12) [183]. Notably, NIR laser irradiation induced the detachment of different cell lines with nearly complete viability of the lifted cells. Since the viability of the cells was not affected, the authors proposed that a photothermal effect, rather the generation of ROS, is the main cause of cell detachment. The authors also showed that complete cell sheets of 3T3 fibroblasts could be detached, thereby broadening the scope of this procedure not only to different cell lines but also to different forms of cellular organization. In another study, a nanostructured substrate consisting on assembled multilayer films of photosensitizer-coupled polypeptides and collagen-stabilized AuNPs was also employed for targeted detachment of cells upon laser illumination. The negatively charged gold-collagen nanoconjugates serve as building blocks for preparation of LbL assembled multilayers based on the electrostatic force between collagen-conjugated AuNPs, photosensitizer-coupled polypeptides (TPPAc-PLL) and glass pretreated with PEI. The nanostructured substrate allowed cell growth and promoted cell detachment upon irradiation the photosensi-

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tizer film with a laser light at 559 nm. The authors demonstrate that this treatment leads to ROS, which are responsible for causing cell death by apoptosis and subsequent detachment. Importantly, local illumination selectively induces the death of those cells that are located in the beam of light, whereas other cells remain intact. Conclusion and future directions The ability to investigate and manipulate cellular signaling processes provides fundamental information about cellular behavior at the single cell level, and as such facilitates our understanding of their group behavior in tissues and organs in physiological and pathological processes. Precise control of cell function employing nanoscale materials will likely allow better understanding of how the cell senses its microenvironment and inspire novel strategies to manipulate cell function and behavior. This review introduces a new approach: engineered AuNPs and gold nanostructured substrates with designed size, geometry, chemical functionality for the modulation of cell function. The unique properties of AuNPs such as rich surface chemistry, low toxicity, high electron density, and strong optical absorption offer the possibility to physically perturb cell signaling processes and cellular activities at the nanoscale in unprecedented ways. In particular, the capacity to easily functionalize the nanoparticles with proteins, peptides and other biomolecules allow to obtain nanodevices at the same scale of cellular receptors and their assemblies. The plasmonic properties of AuNPs provide additional levels of functionalities, enabling the physical modulation (e.g. opto-thermal) of cell function, as well as imaging applications. In summary, we have provided a review of new advancements of the application of biofunctionalized colloidal AuNPs and nanostructured substrates for modulation of cell behavior. Better understanding of receptor-mediated signal transduction through the nanoscale control of cellular receptors and cell-ECM interactions could open up novel strategies to manipulate cell function in biomedical and tissue engineering applications. An exciting challenge would be to modulate intracellular functions. However, despite their potential, nanomaterials have the ability to induce oxidative stress dependent mechanisms, and they could also impact on signaling pathways resulting in adverse/unanticipated outcomes. Thus, a detailed analysis of toxic events triggered would strongly benefit the safety evaluation of nanomaterials as well as for the development of safer and consumer friendly nanotechnology. We anticipate that in the near future, micro and nanoengineered functional biomaterials based on AuNPs and their integration with plasmonic sensing will provide new exciting opportunities for addressing challenges in fundamental cell biology studies and biomedical applications. Acknowledgement Financial support is acknowledged from the European Research Council through ERC Advanced Grant Plasmaquo. References [1] J.D. Jordan, E.M. Landau, R. Iyengar, Cell 103 (2000) 193. [2] J.A. Papin, T. Hunter, B.O. Palsson, S. Subramaniam, Nat. Rev. Mol. Cell Biol. 6 (2005) 99. [3] Y. Sasai, Nature 493 (2013) 318. [4] C.M. Nelson, M.J. Bissell, Annu. Rev. Cell Dev. Biol. 22 (2006) 287. [5] A.L. Barabasi, Z.N. Oltvai, Nat. Rev. Genet. 5 (2004) 101. [6] S.W. Crowder, V. Leonardo, T. Whittaker, P. Papathanasiou, M.M. Stevens, Cell Stem Cell 18 (2016) 39. [7] Z. Yao, J. Petschnigg, R. Ketteler, I. Stagljar, Nat. Chem. Biol. 11 (2015) 387. [8] N. Huebsch, D.J. Mooney, Nature 462 (2009) 426. [9] S. Mitragotri, J. Lahann, Nat. Mater. 8 (2009) 15. [10] P.M. Mendes, Chem. Soc. Rev. 42 (2013) 9207. [11] L.A. Dykman, N.G. Khlebtsov, Chem. Rev. 114 (2014) 1258. [12] S.J. Soenen, W.J. Parak, J. Rejman, B. Manshian, Chem. Rev. 115 (2015) 2109.

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Jorge Pérez-Juste obtained his chemistry degree from the University of Santiago de Compostela in 1995, and his PhD degree in chemistry from the University of Vigo in 1999. He worked as a postdoctoral fellow at UC Santa Cruz in 2000 and at University of Melbourne (Australia) in 2002 and 2003. Since 2009 he is Associate Professor in Physical Chemistry at the University of Vigo. His current research interests involve the synthesis and characterization of metal nanoparticles and their applications in catalysis and sensing.

Gustavo Bodelón is a postdoctoral research fellow currently working in the Colloid Chemistry group at the Department of Chemistry of the University of Vigo. He received his PhD in Biochemistry and Molecular Biology from the University of Santiago de Compostela in 2003. After postdoctoral training in the Ludwig Institute for Cancer Research (London, UK), he was awarded with a Juan de la Cierva fellowship from the Spanish Ministry of Science to join the National Centre for Biotechnology (CNB) in Madrid. His research interests currently focus on the development of bio-applications for plasmonic nanoparticles.

Isabel Pastoriza-Santos is Associate Professor in the Department of Physical Chemistry of the University of Vigo, at which she also received her BSc (1997) and PhD (2001) degrees. She worked as a postdoc in the Chemistry School at the University of Melbourne with Prof. Paul Mulvaney during 2002–2003. Her current interests involve the synthesis, surface modification, and assembly of metal nanoparticles and their application in bioimaging and biosensing.

Luis Liz-Marzán is an Ikerbasque Professor and Scientific Director of CIC biomaGUNE, in Donostia – San Sebastián, since September 2012. He graduated in chemistry from the University of Santiago de Compostela, was postdoc at Utrecht University (The Netherlands) and Professor at the University of Vigo (1995–2012). He has been Invited Professor at several universities and research centers worldwide and received numerous research awards. His major research activity is devoted to understand the growth mechanisms of metal nanocrystals, to tailor their surface chemistry and direct self-assembly. He also works on the design of biomedical applications based on the plasmonic properties of well-defined metal nanoparticles and

Celina Costas obtained her MS degree in Biochemistry from the University of Oklahoma and her PhD in Biochemistry and Molecular Biology from the University of Santiago de Compostela in 2004. After postdoctoral training in the Barts Institute of Cancer (Queen Mary University, UK) and the Severo-Ochoa Centre for Molecular Biology (CBMSO) in Madrid, she joined the Colloid Chemistry group at University of Vigo to develop applications of plasmonic nanoparticles for diagnosis and bio-detection.

nanostructures.