Microdevices for Nanomedicine - Molecular Pharmaceutics (ACS

Review pubs.acs.org/molecularpharmaceutics

Microdevices for Nanomedicine Michinao Hashimoto,†,‡ Rong Tong,†,‡ and Daniel S. Kohane*,† †

Laboratory for Biomaterials and Drug Delivery, Department of Anesthesiology, Division of Critical Care Medicine, Children’s Hospital Boston, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 02115, United States ‡ The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, Massachusetts 02142, United States ABSTRACT: This review surveys selected methods of manufacture and applications of microdevicesminiaturized functional devices capable of handling cell and tissue cultures or producing particlesand discusses their potential relevance to nanomedicine. Many characteristics of microdevices such as miniaturization, increased throughput, and the ability to mimic organspecific microenvironments are promising for the rapid, low-cost evaluation of the efficacy and toxicity of nanomaterials. Their potential to accurately reproduce the physiological environments that occur in vivo could reduce dependence on animal models in pharmacological testing. Technologies in microfabrications and microfluidics are widely applicable for nanomaterial synthesis and for the development of diagnostic devices. Although the use of microdevices in nanomedicine is still in its infancy, these technologies show promise for enhancing fundamental and applied research in nanomedicine. KEYWORDS: nanomedicine, microdevice, microfluidics, organ-on-a-chip, high-throughput screening, nanoparticle synthesis, diagnostic chip

1. INTRODUCTION Nanomedicine is the application of nanotechnology to medicine and human health, in particular to the diagnosis and treatment of diseases. A wide range of nanomaterials have been developed in the past few decades, including polymeric nanoparticles, liposomes, quantum dots, carbon nanotubes, and inorganic nanoparticles. The sizes, shapes, and other physicochemical properties of such materials have been tailored in the nanoscale range to determine specific function, resulting in tunable optical, electronic, magnetic, and biological properties.1 These properties aid the development of drug delivery vehicles, contrast agents, and diagnostic devices. Some of them have been approved by the FDA for use in humans or are under clinical investigations. Despite the clinical promise that nanomaterials hold, the translation of nanomedicine from bench to the clinic has been relatively slow.2 There have been many reasons for this slowness, including the difficulty of reproducibly synthesizing nanomaterials in sufficient quantity with identical properties, the lack of understanding of the relationship between in vitro physicochemical properties of nanomaterials and their in vivo fates in animals and humans, and the paucity of methods of screening nanomaterials rapidly in a manner that predicts their in vivo behaviors.2 Microdevices are devices that are fabricated with the aid of technologies such as microfabrication, surface patterning, and microfluidics, and are often integrated with cell and tissue cultures. Advances in methods of microdevice fabrication and application could address challenges faced in nanomedicine. Microdevices are generally economical, reproducible, and readily amenable to modification and redesign. Microdevices © 2013 American Chemical Society

can be readily adapted to nanomedicine: for example, lithographic technologies for micro/nanofabrication and computer-aided design (CAD) have been easily transferred to the fabrication of biological platforms for cell and tissue culture.3,4 Similar techniques have been applied to improving the methods for synthesis of nanoparticles with tunable properties.5 Traditional cell culture studies performed on two-dimensional surfaces are limited in simulating the complex cell microenvironments that exist in vivo and may therefore be inadequate for biological assays.6,7 Microdevices can produce three-dimensional cellular environments that create fluid flows and mechanical forces mimicking physiological environments in vitro. Such devices can rapidly evaluate the toxicity and therapeutic efficacy of nanomaterials while maintaining the simplicity and controllability of in vitro models.8,9 They can capture the complex interplay between cells and tissues that is not as readily studied in two-dimensional plates.10 Microfabrication techniques, combined with robotic control, enable the creation of reproducibly micropatterned cell cultures for screening candidate materials in a high-throughput manner. This is of particular value considering the expanding size of libraries of therapeutic candidates, and the increase in cost and time required to test them. High-throughput approaches also Special Issue: Emerging Technology in Evaluation of Nanomedicine Received: Revised: Accepted: Published: 2127

November 13, 2012 February 4, 2013 March 22, 2013 March 22, 2013 dx.doi.org/10.1021/mp300652m | Mol. Pharmaceutics 2013, 10, 2127−2144

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decrease reliance on in vivo characterization throughout the process of drug development, with all its ethical, scientific, and financial downsides. Here, we review advances in technologies for the fabrication of microdevices, methods to create microfabricated cellular environments, and applications of those cell and tissue cultures in nanomedicine. The purpose of this review is to present a set of technologies with considerable potential usefulness in nanomedicine. Some applications we describe have not yet been used to test nanomaterials, but could be readily adapted for that purpose. The overall structure of the paper is outlined (Figure 1). We will highlight different techniques for fabricating

inside devices, such as migration,13 proliferation, differentiation,14 and apoptosis.15 Patterned cell cultures are used in a broad range of applications: high throughput screening,16 cellular assays,17 drug discovery and development,18,19 and tissue engineering.20−22 2.1. Microcontact Printing (μCP). Microcontact printing (μCP) is a technique of soft lithography to transfer microscaleto-nanoscale patterns of molecules via stamping. In μCP, a premade stamp with raised micropatterns is inked with a solution containing molecules of interest; the patterns of molecules are then transferred to other substrates by “stamping” (Figure 2A). This process offers versatile regulation

Figure 1. The structure of this review. Section 2 introduces several technologies to fabricate microdevices and to pattern and integrate cells and tissues with them. Section 3 overviews methods to evaluate nanomaterials for their therapeutic efficacy and toxicity using such microdevices. Section 4 discusses applications of microdevices in nanomedicine, particularly in relation to the synthesis of nano- and biomaterials and the development of diagnostic devices. μfluidics = microfluidics.

Figure 2. Schematics of techniques for microfabrication and surface patterning. (A) Microcontact printing. [1] A micropatterned stamp of PDMS is fabricated via replica molding, and [2] the patterns are exposed to the ink. [3] Stamping transfers the micropatterns onto the substrate. (B) Microchannel fabrication. [1] A stamp of PDMS is created via replica molding and [2] sealed with a flat substrate to create closed channels. (C) Spotting or printing. A solution or hydrogel containing desired compounds is printed through an electronically controlled nozzle. (D) Dip-pen nanolithography (DPN). Molecules are transferred on to the substrate from the tip of an atomic force microscope (AFM). PDMS = polydimethylsiloxane, Si = silicon, SU-8 = a photocurable epoxy-based material.

microdevices, and discuss methods for the manipulation and patterning of cells at the microscale (section 2). Various application for such platforms in the evaluation of nanomedicine will be discussed (section 3), including their use in high-throughput screening (section 3.1), the mimicry of tumor microenvironments (section 3.2), the introduction of mechanical cues to cells (section 3.3), and chips used as organ surrogates and for biodistribution studies (section 3.4) and as physical models of blood vessels (section 3.5). Section 4 introduces the application of microdevices for the controlled synthesis of nanomaterials and biomaterials (section 4.1) and diagnostics in nanomedicine (section 4.2).

of the surface chemistry on various substrates and with which to study biological processes affected by cell−surface interactions. Most typically, stamps have been prepared using soft lithographic techniques23 where micropatterns on polydimethylsiloxane (PDMS) surfaces were fabricated by replica molding using patterns of SU-8 (a photocurable epoxy-based material) fabricated on silicon wafers. PDMS micropatterns can be fabricated in different conformations for various usages, including stamps,23 channels,24 microwells,25,26 and stencils27 (Figure 2A,B). PDMS stamps fabricated this way were used for the early work in μCP.28−30 Other materials have been used as stamps as well,31,32 since certain material characteristics inherent to PDMS might be disadvantageous in some applications: it is relatively soft, which can cause collapse of micropatterns, and it is hydrophobic, and may have solvent compatibility problems with ink solutions.

2. TECHNOLOGIES TO FABRICATE MICRODEVICES INTEGRATED WITH CELLS AND TISSUES This section highlights different techniques to fabricate devices within which to manipulate cells and control their behaviors.11 Controlling cellular microenvironments and cell−biomaterial interfaces (such as surface chemistry, rigidity, and topology) is crucial for patterning cells to design cell cultures.12 Those microenvironments determine the behaviors and fates of cells 2128

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fields releases the trapped cells. Microfabrication can integrate the control devices that apply external fields (such as microelectrodes and magnetic field concentrators) inside miniaturized devices. Electrical forces can be used to manipulate cells via two mechanisms: electrophoresis (based on charge) and dielectrophoresis (based on polarizability).70 Applied electrical fields create heat and voltages across cell membranes that can potentially affect cell physiology adversely or even damage cells.70 However, dielectrophoresis using weak electrical fields (5 V, 30 min) can trap cells on microelectrodes while maintaining their morphology and ability to proliferate.71 Such microelectrodes can be patterned or printed72,73 and integrated into the substrates onto which cells will be trapped and grown. Dielectrophoresis can create cellular microarrays for assays done in perfusion culture (a type of cell culture with controlled flow of media to enhance the exchange of oxygen and nutrients). For example, human breast cancer cells (MCF7 cell line) were seeded and uniformly patterned inside a microchannel via dielectrophoresis.74 Dielectrophoresis concentrated cells with high uniformity for each electrode (92 ± 5 cells per electrode, roughly one-third as many cells compared to a comparable assay using a 96-well plate). The miniaturization of the culture, and increase in the volume density of cells (i.e., the number of cell per unit volume) that resulted from the decrease in the volume of the culture, will reduce the required amount of reagents for the assay. These characteristics are of particular interest if the cost and quantity of the reagents are limiting the research. Integration of cell cultures with microchannels enabled the control of perfusion and the exchange of media containing nutrients and of test materials to be evaluated (e.g., drugs), and made it possible to study drug efficacy and toxicity in situ (see also section 3.1). Magnetic fields have also been utilized for patterning of cells. In this approach, cells were labeled with magnetic components, including nanowires,75 nanoparticles,76,77 and liposomes that contained magnetite nanoparticles in their cores.78,79 The magnetic components were bound to cell surfaces via electrostatic interactions (e.g., cationic magnetic liposomes bound to the negatively charged cell surface78) and ligand recognition (e.g., biotinylated cell membrane proteins bound to streptavidin-modified paramagnetic particles77). These approaches allowed the magnetic force to transfer cells to defined positions via magnetophoresis. Optics offers another way to manipulate cells. Focused laser beams generate radiation pressure and can manipulate biological entities (viruses, bacteria, and cells) at small scales.80,81 These “optical tweezers” have been used in cell culture to achieve label-free transport between microwells82 and microfluidic channels.83,84 Optical tweezers have been used for the fabrication of cellular microarrays with high precision.85 Multiple optical tweezers have been employed to organize different types of cells (e.g., E. coli carrying plasmids coded for three differently colored fluorescent proteins) delivered through laminar fluid flows into three-dimensional configurations with 4.3 μm spacing between adjacent cells. The cells were then encapsulated in poly(ethylene glycol) diacrylate, a photopolymerizable hydrogel mimicking ECM, to maintain their location and their three-dimensional structure. Optical methods can manipulate the locations of individual cells to reconstruct three-dimensional architectures with precision; these “synthetic” tissues might serve as platforms for the development of more complex in vitro assays.

A variety of materials in solution were transferred to a substrate using μCP. The solution containing molecules to be patterned is termed a molecular ink. Initially, alkanethiol was used as the molecule to be patterned via μCP;33,34 a selfassembled monolayer (SAM) of terminally functionalized alkanethiol was readily stamped on gold surfaces to form microscale patterns. Other molecular inks have also been employed, such as silane, 35−37 DNA,38,39 cell-adhesive proteins,40,41 and bioactive polymers (PEG-copolymer42 and polydopamine43,44). Many of these molecules are useful to control cell adhesion, and are therefore useful in cell patterning. μCP has also been used with common cell culture substrates such as glass, silicone rubber, and polystyrene.45 In addition to flat substrates with traditional materials, curved surfaces,46 microspheres,47 and biodegradable polymers48 have also been used as substrates for pattern transfer via μCP, demonstrating the versatility of this technique. μCP can create multiple copies of the same chemical patterns such as microarrays of different materials (e.g., cell adhesive proteins, antibodies, carbohydrates).45,49 These surfaces can control the binding of microorganisms such as bacteria. For example, antibodies that specifically bind to the surfaces of Escherichia coli O157 H:7 (E. coli) and Renibacterium salmoninarum (RS) were patterned via μCP and used to detect those bacteria.50 μCP is also useful for studying fundamental cell biology; the local geometry and size of cell microenvironments can be controlled for studies of cell behaviors such as proliferation and apoptosis.15 2.2. Direct Writing: Spotting, Inkjet Printing, and DipPen Nanolithography. Spotting, or inkjet printing, is widely used for micropatterning (Figure 2C). Printing allows for deposition of minute quantities of materials at precisely defined positions.51 Originally used to transfer electronically designed patterns onto paper using ink, printing technologies are now applied for patterning of biologically relevant materials. Patterning of proteins,52,53 bioactive polymers,54 biodegradable polymers,55 and cells56 has been demonstrated. Microarrays created by inkjet printing are utilized for high-throughput screening of polymeric biomaterials, and for high-throughput studies of cell−material interaction54,57,58 (section 3.1). Dip-pen nanolithography (DPN) is another method for surface patterning (Figure 2D). DPN is based on the transfer of molecules from an atomic force microscope (AFM) tip to substrates;59 a solution containing various molecules (e.g., organic, biological, and ionic) was transferred to the substrate via capillary force when the AFM tip was brought close to the surface of the substrate. The initial work on DPN demonstrated patterning of alkanethiols on gold surfaces with 30 nm line width.59 Subsequently, other processes have been developed for patterning of various inks, including organic molecules,60,61 polymers,62 nanoparticles,63,64 and biological molecules (peptides,65 proteins,66 and DNA67). DPN is useful for creating homogeneously nanopatterned environments in a reproducible manner, and is capable of patterning molecules on both soft and rigid substrates with high registration and sub 50 nm resolution.68 These capabilities have been applied for the productions of nanodots (i.e., patterned clusters of chemical ink with fixed dimensions) with various chemical functional groups in order to study the regulation of cell growth.69 2.3. Active Cell Manipulations with External Fields. Cell patterning at the microscale can also be achieved by applying external fields to directly transport individual cells to precise locations on a substrate in a contact-free manner. These approaches are often reversible; discontinuation of the external 2129



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Figure 3. Three-dimensional cell cultures created by stacking layers. (A) Cell sheet technology (CST). A single-layer cell sheet, released from temperature responsive culture dishes, can be stacked with other homotypic or heterotypic sheets. Cocultured cell sheets can also be prepared. Reproduced with permission from ref 92. Copyright 2005 Elsevier B.V. (B) Paper-supported three-dimensional cell culture. Cells spotted on papers can be stacked to create three-dimensional cell cultures, which can be destacked to study spatial effects of the gradient of nutrients and oxygen in the z-dimension. The impermeable layer is located at the bottom of the stack, and creates the oxygen gradient. Reproduced with permission from ref 95. Copyright 2009 National Academy of Sciences, U.S.A.

dimensional cell cultures with multiple discrete zones.96 Destacking of stacked sheets was shown to be a convenient way to study the spatial considerations of processes affecting cell growth and proliferation (such as migration, and oxygen, and nutrient transfer). This capability enabled the threedimensional study of the growth of a tumor model at different locations in the z-dimension, and of cell migration within those constructs.96 2.5. Microfluidics. There are two distinct types of research in which microfluidic systems are useful: as analytical tools (sections 3 and 4.2) for culturing and analyzing cells for biological, pharmaceutical, and toxicological studies3,97 and as engineering tools (section 4.1) for synthesizing nano/ biomaterials as therapeutic drugs, and making building blocks for larger tissue constructs.98,99 PDMS is one of the most widely used materials to build microfluidic devices for both purposes (Figure 2B). To create closed PDMS-based microfluidic channels, patterned surfaces are created by replica molding, oxidized to render their surfaces adhesive, and sealed with another flat, oxidized substrate (e.g., glass, PDMS, etc).24 PDMS has useful characteristics for cell culture and in situ observation such as high gas permeability, transparency to visible wavelengths, and non-cytotoxicity. The elasticity of PDMS provides the capability to engineer valves for the compartmentalization and regulation of flows.100,101 The gas permeability of PDMS allows the generation of concentration gradients of oxygen102 and carbon dioxide103 inside the channels. Appropriate design of the geometry of channels and obstacles allows for hydrodynamic trapping of cells (for example, with U-shaped geometrical traps perpendicular to flow);104,105 see also section 3.1. Cells cultured in the device

2.4. Three-Dimensional (3D) Printing and Layer-byLayer Stacking. Traditional cell culture on two-dimensional surfaces may not provide ideal simulation of real cell microenvironments. Complex (i.e., three-dimensional and/or heterogeneous) cell cultures may be more appropriate for assays that evaluate the interaction between materials and living tissues. The construction of larger tissue constructs with complex functions also requires methods to engineer heterogeneous (or multi-cell-type, e.g. vascularized) tissues.86 Printing technology can provide three-dimensional substrates for such applications. For example, a cell spotter sequentially deposited cell-laden hydrogels containing ECM in a layer-bylayer approach, creating a three-dimensional construct.87−89 In an alternative approach, two-dimensional sheets of cells prepared via detachment from a cell culture surface functionalized with a thermoresponsive hydrogel (cell sheet technology, CST)90−93 were used to form three-dimensional constructs via shaping, layer-by-layer stacking, and rolling (Figure 3A). CST offers facile ways to create three-dimensional cultures that resembled in vivo system more closely than do traditional culture systems; complex heterogeneous tissues (e.g., blood− brain barrier, small intestinal mucosa, and renal glomerulus) and tissues including capillary-like prevascular networks have been fabricated using CST.94 The hope is that these complex tissue models might serve as real tissue surrogates to assess and evaluate new drug candidates as alternative to reliance on animal models. Recently, papers used in chromatographyporous materials consisting mainly of cellulosehave been used as support materials for ECM hydrogels (Figure 3B).95 Printing barriers composed of hydrophobic ink on such papers, then stacking multiple of them, provided a simple way to create three2130



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can be monitored while perfused with media over extended periods.106−108 Microfluidic channels have also been created inside hydrogels containing ECM (i.e., microfluidic hydrogel). A range of hydrogels, including matrigel, alginate, and collagen,109−111 have been modified in this way. One way to fabricate such structures is to employ sacrificial structures (e.g., gelatin) that can be incorporated into the ECM and later removed by dissolution to create empty channels.109 The microfluidic hydrogels serve as scaffolds for seeded cells, and the embedded channels are used for perfusion with physiological fluids. Due to the porosity of hydrogels, molecules in the perfusate are able to diffuse throughout. Alginate-based microfluidic hydrogels have been used to control the distribution of water-soluble compounds in the scaffolds via diffusion. Calcein-AM was used as a model compound, which was cleaved within the cytosol of living cells to generate fluorescence.110 Collagenbased microfluidic hydrogels have been shown to be mechanically stable over many weeks, and capable of restricting the growth of cells to within designed patterns.111 In a related approach, printing technology was used to pattern and embed rigid fibers composed of a mixture of carbohydrates in a threedimensional lattice structure within ECM. Subsequent dissolution of the fibers generated perfusable vascular networks. The networks were used to perfuse blood to maintain the function of primary rat hepatocytes in engineered tissue constructs, which would have formed necrotic cores in the absence of vascularization.112 These approaches are applicable to a range of cell types and matrices.

Figure 4. Microfabricated devices for cell-based assays. (A) Collagen gel spots (0.6 mm) containing MCF7 cells, visualized with live-cell staining. The inset shows the magnification of the area depicted by the red square. Reproduced with permission from ref 116. Copyright 2008 National Academy of Sciences, U.S.A. (B) Array of single-cell cultures inside a microfluidic device. Cells are trapped and cultured under continuous perfusion of media. The scale bar is 50 μm. Reproduced with permission from ref 129. Copyright 2006 Royal Society of Chemistry. (C) Confocal section of a heterogeneous spheroid after 7 days of culture. PC-3 cancer cells stably transfected with the fluorescent protein DsRed (red) were cocultured with osteoblasts (MC3T3-E1) and endothelial cells (human umbilical vein endothelial cells, HUVEC) in a microfluidic device. Live cells were stained with Calcein-AM (green). Reproduced with permission from ref 132. Copyright 2009 Elsevier B.V. (D) Schematic illustrations and an optical micrograph of a lung-on-a-chip microfluidic device. The device contains two compartments in the middle that reconstitute alveolar− capillary interface, and control side chamber to generate mechanical motion. The scale bar is 200 μm. Reproduced with permission from ref 161. Copyright 2010 American Association for the Advancement of Science.

3. MICRODEVICES INTEGRATED WITH CELL CULTURES TO EVALUATE NANOMATERIALS This section highlights advances in the application of microengineered devices in pharmaceutical research, with a focus on the evaluation of nanomaterials. In particular, microengineered cell cultures can be useful in assessing materials via cell-based screening. 3.1. Platforms for High-Throughput Screening. Cellular microarrays are useful for screening large-scale libraries of materials for drug discovery and toxicity testing113−115 at high throughput, while reducing the time and cost required for the assays and increasing their portability. Many methods (e.g., μCP, DPN, inkjet-printing, and microfluidics) can perform microscale patterning of hundreds of physicochemically distinct environments to be screened by cells in a high-throughput manner. Microarrays on glass,116 PDMS,25,117 and hydrogelbased materials118−121 have been easily prepared and used for the evaluation of materials. For example, cultured cell arrays have been developed for cytotoxicity testing using 1080 individual minute MCF7 cell-laden hydrogels (collagen, 60 nL) patterned on a functionalized glass slide (Figure 4A, DataChip). Each cell culture was reacted with varying doses of nine model compounds to assess their cytotoxicity116 The precision of the assay was not impaired compared to those from 96-well plates. The incubation time for MCF7 cells was shorter for microarrays (doubling time of 14−19 h) than for threedimensional culture in 96-well plates (doubling time of 37 h); the diffusion of the oxygen was less limiting, which facilitated cell proliferation.116 In a related study, cellular microarrays were developed for high-throughput immunofluorescence-based quantification of the level of an intracellular protein that accumulates in response to hypoxia (hypoxia-inducible factor; HIF-1α) when treated with an agent that leads to the

accumulation of HIF-1α (MG-132) and an agent that prevents that accumulation (2ME2).122 Three-dimensional cellular microarrays consisting of 560 spots of 60 nL alginate hydrogels encapsulating human pancreatic tumor cells were created via robotic spotting. The results from the microarray-based study agreed closely with those from the conventional Western immunoblotting, but the microarrays facilitated washing and removal of reagents more efficiently than conventional multiwell plates did.122 These results suggested that scaledown of cell cultures by 2000-fold (by volume) did not adversely affect the capability to perform cell-based assays, while offering benefits such as reduction of the amount of reagents, reduction of the time to complete the assay, and facilitation of experimental processes such as washing. These approaches can be extended to the evaluation of nanomaterials, which could enhance studies that are currently done with more conventional methods, such as the cell-based toxicity screening of nanoparticles of Ag, Au, Pt, Al2O3, etc. performed with 384well plates.123 The appropriate design of microfluidic systems can enable the combination of cell culture and in situ high-throughput evaluation of materials. In one example, channels with hydrodynamic traps offered a simple way to perform cell2131



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such as differentiation, proliferation, migration, and apoptosis.144 In vivo systems have abundant mechanical stimuli from multiple sources such as blood flow, muscular contraction, breathing, etc. The ability to study cellular responses to mechanical stimuli in vitro is therefore important not only for fundamental cellular and molecular biology145,146 but also for applications such as the discovery and development of therapeutic materials.147 Microdevices have been developed to provide experimental in vitro models that have moveable components.148 The elastomeric nature of PDMS is beneficial for creating mobile modules.149,150 The biomechanics of the cardiovascular systems have been simulated using cell-cultured, elastomeric membranes: a stretchable cell culture was integrated with a fluidic system, so that fluid shear stress and cyclic stretch, two major types of mechanical stimulation in the cardiovascular systems, could be simulated.151 The vascular endothelium is a potential target of a wide range of therapeutic drugs. Shear stress influences the uptake of therapeutic nanoparticles by endothelial cells.152,153 Microfluidic systems allow the facile control of shear stress on the surfaces of cultured cell,154 facilitating the study of the relationship between shear stress and nanoparticle uptake. For example, controlled flow rates through microchannels have been used to perform quantitative investigations of CdTe and SiO2 nanoparticle uptake by cultured endothelial cells.155 3.4. Organs-on-Chips and Microdevices for Biodistribution Studies. In vitro cell assays have notoriously poor predictive value for the in vivo efficacy and toxicity of drugs; this poor correlation may impede the translation of drug candidates from bench to clinic.156 Recently, three-dimensional models of tissue cultures that better mimic the mechanical and physiological microenvironments of living organs (organs-onchip) have been developed.157,158 These engineered organspecific structures consisting of structural materials (PDMS, glass, hydrogels) with defined mechanical structures (channels, pores, thick walls, and thin membranes) and three-dimensional cell cultures have been developed with the hope that they have the potential to replace animal models in pharmaceutical research. One example of such a microdevice is a heart-on-achip consisting of contractile thin films of cardiac myocytes,159 with which the mechanical effects of myocyte contraction and the pharmacological effects of epinephrine could be studied. Lung-on-a-chip devices have been fabricated to mimic airway epithelia160 and alveolar−capillary interfaces161 in the human lung (Figure 4D). The latter biomimetic alveolar−capillary interface device consists of a main channel divided into two compartments by a porous, flexible membrane, with vacuum chambers on each side of the main channel. Human alveolar epithelial cells and human pulmonary microvascular cells were cultured on different sides of the porous membrane (Figure 4D). Application of cyclic suction by the vacuum chambers generated mechanical forces that stretched the cell-cultured membrane, simulating the mechanical strain caused by breathing. A nanotoxicology study using this device suggested that mechanical stimulation enhanced the uptake of nanoparticles by epithelial and endothelial cells, and also enhanced the inflammatory response of the lung to nanoparticles.161 Organ-on-a-chip devices have been created for liver,162 kidney,163 gut,164 breast,165 and other tissues. These devices show promise for studying human physiology in organ-specific environments.158 The hope is that they eventually serve as inexpensive and scalable in vivo platforms for drug development.

based cytotoxicity tests for cadmium (Cd) nanoparticles. Microfluidic channels with cell culture chambers (C-shaped traps with the open side facing the flow) were fabricated, in which human embryonic kidney cells (HEK293) were seeded and cultured. Subsequently, different types of Cd nanoparticles were introduced to the channel to study the cellular events they induced.124 Similar methods have been developed for in vitro screening of nanoparticles for cancer treatment125−127 using tumor spheroids as the cell-based component. Tumor spheroids are three-dimensional multicellular constructs that mimic cell microenvironments relevant to in vivo tumors.128 Microfluidic methods enable the coculture of heterogeneous cells into three-dimensional tumor spheroids. Various methods including hydrodynamic trapping and self-assembly,129,130 culturing in arrays of microcompartments,131−133 and encapsulation in hydrogels134 have been developed for the controlled formation of tumor spheroids and assays based on them (Figure 4B,C). A high density of tumor spheroids (7500 spheroids/cm2) can be grown on a chip.130 These formed spheroids were used for screening of anticancer drugs in situ without the need to transfer them to multiwell plates or other containers.134 Microfluidic traps or compartments offered precise control over the location of spheroid formation. Placing multiple spheroids on the same chip separately helps conduct multiple assays simultaneously. In combination with image analysis, these methods provide the potential to perform highthroughput testing for therapeutic nanoparticles. 3.2. Generating Heterogeneous Cellular Constructs Mimicking Tumor Microenvironments with Microfluidics. Microfabrication and microfluidics can create, in a controlled manner, cellular constructs that are heterogeneous in both cell type and spatial organization. Such constructs may then be relevant models of conditions within tumors, and may be useful for cell-based screening of materials intended for imaging or treating tumors. For example, the abnormal tumor vasculature in tumors causes nutrient gradients such that cells in different parts of a tumor can be necrotic, quiescent, or rapidly proliferating.135 Most chemotherapeutics, including nanoparticles, are only effective against cancer cells in the tumor periphery that are proliferating, and cannot penetrate the poorly perfused areas in sufficient concentrations to be effective.136−138 Recently, a microfluidic device consisting of PDMS and glass that mimicked the nutrient gradients in tumors was engineered to form micrometer-scale tumor cell masses inside the fluidic chamber for long-term growth and study; the chamber was designed to expose the cells to constant flow of medium that controlled nutrient gradient.139 The tumor masses showed viable and necrotic microregions, with acidic pH gradients similar to those found in in vivo tumors. The optical transparency of the PDMS and glass device enabled quantitative transmission and fluorescence microscopy of all regions of the cell masses. The diffusion of therapeutic molecules and nanoparticles (e.g., liposomes containing doxorubicin) was directly measured by fluorescence microscopy.139 Microfluidics have been used to better model the tumor microenvironment in a variety of other ways, including tuning the growth-factor gradient to affect tumor cell migration,140 or culturing tumor cells with tumor-associated cells to study tumor−stromal cell interactions,141,142 and to mimic tumor−endothelial cell interactions.143 3.3. Applying Mechanical Stimuli to Cells. Mechanical stimuli are essential factors that influence the behaviors of cells 2132



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Devices with multiple, distinct and separate cell cultures in microfluidic networks have been developed to reflect the fact that in vivo systems consist of multiple organs with organ− organ interactions.166−169 Physiologically relevant designs were used to create in vitro models of mass transfer between organs (i.e., to study biodistribution and clearance). Microscale cell cultures in microfluidic devices, where multiple cell culture chambers are connected by fluidic channels to mimic multiorgan interactions and test drug toxicity and pharmacokinetics, may be useful as preliminary in vitro pharmacokinetic models.169 For example, three cell lines representing the liver, tumor and marrow were initially cultured separately in three chambers. Later they were assembled into a network of interconnected microfluidic channels and used to test the toxicity and distribution of anticancer drugs.169 Although preliminary and simple, such designs may evolve into platforms with improved predictability, that can help researchers gain better insight into a given drug’s distribution and elimination.170 3.5. Mimicking Microvascular Microenvironments. Microfluidic systems inherently deal with microscale flows and therefore lend themselves naturally to the development of physical models stimulating the fluidic aspects of in vivo microenvironments. Recently, three-dimensional microvascular networks mimicking the typical functions of blood vessels were developed (Figure 5A,B).171 Using lithographic techniques, endothelialized microfluidic vessels have been fabricated in a native collagen matrix. The engineered vessels could recruit preseeded vascular pericytes or arterial smooth muscle cells in the collagen matrix and produce new branches by processes that resembled angiogenesis. Such simplified systems may be useful for the study of angiogenesis and the evaluation of drugs affecting vascular functionality.172 Microfluidic devices have been used to mimic more complicated situations. For example, microfluidic cell cultures have been successfully employed to study cancer metastasis. A three-dimensional microfluidic model of the tumor−vascular interface was designed to provide precise control of the concentration of biochemical factors such as epidermal growth factors (EGF) and tumor necrosis factor alpha (TNF-α) that can potentially stimulate tumor cells to cross endothelial barriers and enter the bloodstream.173 The device consisted of two independent parallel microchannels seeded separately with tumor or endothelial cells, separated by a 1500 μm wide middle channel filled with an ECM hydrogel. The process of tumor intravasation across the endothelial barriera continuous endothelial monolayer at the interface between ECM and the endothelial channelcould be visualized by fluorescence imaging through the transparent devices (Figure 5C,D). The tumor cells could be seen cross into the ECM hydrogels into the endothelial channel in three dimensions, in response to EGF added to the ECM hydrogel, or to TNF-α added to the endothelial channel. Such three-dimensional microfluidic devices allow for both paracrine signaling (the EGF in the ECM hydrogel affecting the tumor cells) and juxtacrine signaling (the TNF-α in endothelium channel affecting the tumor cells) to stimulate tumor cells’ intravasation. Twodimensional models might have difficulty capturing this type of event. When macrophages that excrete TNF-α and other cytokines were seeded in the endothelial channel and ECM hydrogel, the endothelial monolayer at the interface of the ECM and endothelium channel became more permeable, as assessed by fluorescence imaging, which potentially facilitated

Figure 5. Microfluidic vessel and tumor models. (A) Schematics of microfluidic vessel networks (μVNs). (B) Confocal images of μVNs consisting of collagen scaffolds, seeded with HUVEC and cultured for two weeks (red, CD31, denoting maintenance of the endothelial phenotype; blue, nuclei). The scale bar is 100 μm. Reproduced with permission from ref 171. Copyright 2012 National Academy of Sciences, U.S.A. (C) Schematic illustration of microfluidic channels to culture the tumor−vascular interface. Channels are for endothelial cells (green), tumor cells (red), and extracellular matrix (dark gray). (D) Three-dimensional rendering of a confocal image of a model tumor− vascular interface cultured in a microfluidic channel. Endothelial cells formed junctions in the z-direction (green: VE-cadherin), and tumor cells (red: HT1080-mCherry) are seen invading in the y-direction (blue: DAPI; nuclei). The scale bar is 30 μm. Reproduced with permission from ref 173. Copyright 2012 National Academy of Sciences, U.S.A.

tumor cell intravasation across the barrier. These findings in the microfluidic model agreed well with in vivo murine studies174−176 and findings in clinical specimens.177 Microfluidic models of in vitro tumor microenvironments may also allow the study of drugs or nanomaterials that can inhibit the tumor metastasis process.

4. APPLICATIONS OF MICRODEVICES FOR MATERIALS SYNTHESIS AND DIAGNOSIS Micro/nanofabrication technology and microdevices are useful in other aspects of nanomedicine, such as the synthesis of nanomaterials and the development of diagnostics based on nanotechnology. 4.1. Synthesis of Nanomaterials and Other Biomaterials. Lithographic technologies (section 2.1) are used for controlled synthesis of nanomaterials. For example, an approach has been recently developed to imprint nanoparticles using low surface-energy micromolds consisting of perfluoropolyether, a fluorocarbon elastomer. The mold surfaces were nonwetting and nonswelling to both organic and inorganic materials. Consequently, it was possible to fabricate isolated nanostructures having well-defined shapes and sizes with sub2133



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Figure 6. Lithographically prepared materials as drug delivery carriers. (A) Nanoparticles encapsulating siRNA replicated by photolithography. The scale bar is 2 μm. Reproduced with permission from ref 180. Copyright 2012 American Chemical Society. (B) Microparticles composed of distinct regions with different degradation rates can provide sequential release of compounds over time. Reproduced with permission from ref 190. Copyright 2009 American Chemical Society. (C) Schematic diagram of the synthesis of coded particles using flow lithography and (D) an optical image of the synthesized particles. The scale bar is 50 μm. Reproduced with permission from ref 191. Copyright 2007 American Association for the Advancement of Science.

50 nm resolution without residual material between nanostructures. Such techniques can produce various nanostructures composed of or containing different materials, including synthetic polymers, natural biomacromolecules such as proteins178,179 and siRNAs (Figure 6A),180 which can be used for delivery of small molecule drugs or large macromolecules.181 Particles of this type were used to investigate the influence of nanostructure size, shape, and aspect ratio on cell internalization kinetics.182 This technique can be used to tune a variety of key physical properties of particles, such as drug loading, elasticity, and surface poly(ethylene glycol) (PEG) density. A series of particles with different shapes, sizes, and aspect ratio were prepared by lithography to investigate the effect of particle size and shape on internalization into HeLa cells.183 Cylindrical particles with a diameter of 150 nm and an aspect ratio of 3 exhibited a higher rate of internalization than particles with a diameter of 100 nm and the same aspect ratio. The elasticity of particles can also potentially affect circulation.184,185 Red blood cell shaped microparticles were fabricated with different concentrations of acrylate crosslinker inside particles to tune their elasticity. Those particles were applied into narrow microfluidic channels to test their deformability. For example, 1% cross-linked particles with a diameter of 6 μm could flow through 3.5 μm channels by elongating elastically, and the particles readily recovered their original shapes when removed from the confining channels. In vivo experiments showed that the elasticity of particles dramatically affected their circulation times and biodistribution. An 8-fold decrease in elasticity increased the half-life of elimination by a factor of 30. These examples demonstrate that such lithographic technologies can be useful in elucidating the relationship among physiochemical properties of nanostructures and their in vivo behavior.

Imprinting lithography can produce drug carriers with various shapes. However, the technique is limited in its ability to incorporate multiple components and achieve sub-micrometer scale morphologies. Recently, flow lithography has enabled the customization of the shape and composition of particles consisting of cross-linkable polymers by controlling UV exposure in microfluidic channel with tunable flow rates.186−188 Such flow lithography can spatially control hydrophobic compartments in hydrophilic matrices, allowing encapsulation and release of multiple compounds from the resulting particles.189 The composition of matter can be varied by controlling the duration of the UV exposure so that different regions will have distinctive degradation rates, allowing sequential release of multiple drugs (Figure 6B).190 Flow lithography has also enabled the synthesis of microparticles with graphic barcodes encoding information placed on a specific compartment of each particle (Figure 6C). Such particles have one or multiple areas encapsulating drug and the other area bearing a unique barcode for the identification of drug formulation via imaging. These types of particles are potentially useful for generating libraries for drug screening (Figure 6D).191 Microfluidic methods have also been used for the preparation of micro- to nano- polymeric particle encapsulating small molecule drugs. Microfluidic hydrodynamic focusing has been used to produce emulsions with various configurations (e.g., single, double, and triple emulsions) and compositions (e.g., polymer, hydrogel and lipid bilayer).192 Representative geometries of microchannels, and configurations of the fluids, for particle syntheses using microfluidic hydrodynamic focusing are illustrated in Figure 7. For example, nanoparticles composed of diblock amphiphilic copolymers for applications in drug delivery have been synthesized by hydrodynamic flow-focusing in microfluidic channels that enabled controlled mixing of 2134



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of flow rates in microfluidic channels, and changing of the concentration and compositions of polymers, enable tuning of the size, polydispersity, and drug loading of the nanoparticles. The use of microfluidic device for nanoprecipitation can reduce the size distribution of resulting nanoparticles compared with bulk nanoprecipitation.193 Poly(lactide-co-glycolide)-b-poly(ethylene glycol) nanoparticles of about 50−100 nm size prepared by such microfluidic devices have been used to deliver cisplatin and docetaxel for targeted cancer therapy.193,195 However, polymeric nanoparticles with narrowly distributed sizes below 20−30 nm are still difficult to prepare using microfluidic methods. Making smaller particles may be important given accumulating evidence that nanoparticles with sizes around 12−20 nm have superior tumor penetration compared with larger particles.196 Monodisperse vesicles have been synthesized using droplets generated by microfluidic hydrodynamic focusing (Figure 8B). Conventional approaches to preparing vesicular structures, such as electroformation or bulk hydration of dried amphiphiles, have achieved only limited control over the size and structure of synthesized materials. Furthermore, compounds dissolved in a continuous phase are randomly encapsulated during vesicle formation, leading to a low efficiency of encapsulation. Using microfluidic devices, the size of vesicles can be controlled by changing the diameters of the double emulsion droplets that were generated.197,198 Hydrodynamic focusing can generate single emulsions (Figure 7A) and double emulsions (Figure 7C). Double emulsions were used for the synthesis of vesicles. The outer phase of the double emulsions was an organic phase containing amphiphiles (i.e., poly(ethylene glycol)-b-polylactic acid, PEG-b-PLA); the inner phase of the double emulsions and the continuous (i.e., bulk) phase were aqueous. Evaporating the organic solvent from the outer phase of the double emulsions made micrometer-sized PEG-b-PLA solidify and assemble into the membranes of vesicles for drug encapsulation and delivery.197 In another microfluidic approach for the synthesis of vesicles, the aqueous droplet was first created in an oil phase containing lipids. Downstream, the oil phase was extracted and the lipid assembled to form unilamellar vesicles in the external aqueous phase from an additional channel; this process does not require postsolvent evaporation of oil.198 Nanoscale vesicles can also be produced with sizes around 50−150 nm using flowfocusing methods to control solvent extraction between two miscible phases, which is the same approach as that described for the synthesis of polymeric nanoparticles (Figure 7B). In this method, a phospholipid-containing alcohol solution and an aqueous solution formed colaminar flow in the microfluidic channel; once the alcohol to water ratio reached a critical concentration, the lipids could spontaneously self-assemble into liposomes.199−201 The capability to synthesize particles with different physicochemical characteristics (e.g., dimension, morphology, polymer matrix, and drug molecules) rapidly and reproducibly could accelerate the formulation, development, and discovery of drugs and their drug delivery systems. A microfluidic chip-based method has been used to prepare large-scale libraries of siRNAcontaining lipid nanoparticles (LNPs). Each nanoparticle consisted of different synthetic lipid-like materials that could affect the efficacy of siRNA delivery, and a total of 70 LNPs (each consisting of a unique lipid-like material that contained siRNA) were formulated.202 The speed and reproducibility of this microfluidic approach made it possible to synthesize a large number of high-quality siRNA-containing LNPs to be tested.

Figure 7. Schematic illustrations of microfluidic hydrodynamic focusing for the synthesis of micro- to nanomaterials. In most cases, the flow rates of 1, 2, and 3 are controlled electronically, tailoring the sizes of particles to be synthesized. (A) Solutions 1 and 2 are immiscible (e.g., water and fluorocarbon): droplets of solution 1 form in the flow-focusing orifice (green arrow). Formed droplets can be solidified to obtain microspheres. (B) Solutions 1 and 2 are miscible (e.g., water and acetonitrile): solution 1 is mixed with solution 2, and nanoprecipitation of 1 forms. (C) Solutions 1 and 3 are immiscible with solution 2. Double emulsions form where solution 1 is encapsulated by solution 2.

solvents so as to cause nanoprecipitation (Figure 8A). In nanoprecipitation, amphiphilic copolymers dissolved in a watermiscible organic solvent will precipitate and drive the formation of micelles upon mixing with water.193,194 The stable, controlled mixing of polymer solutions and water and variation

Figure 8. Polymeric and inorganic particles synthesized using microfluidic reactors. (A) Nanoparticles synthesized by nanoprecipitation of PLGA-PEG using hydrodynamic focusing in a microfluidic channel. The scale bar is 100 nm. Reproduced with permission from ref 193. Copyright 2008 American Chemical Society. (B) Polymersomes consisting of PEG-b-PLA synthesized from double emulsion droplets generated via microfluidic hydrodynamic focusing. The scale bar is 50 μm. The arrows indicate aggregated excess PEG-bPLA. Reproduced with permission from ref 197. Copyright 2008 American Chemical Society. PLGA = poly(lactic-co-glycolic acid), PEG = poly(ethylene glycol), PLA = poly(lactic acid). 2135



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Figure 9. Microfluidic reactors for the synthesis of inorganic nanoparticles. (A) Continuous flow reactor. Mixing of two solutions (shown in red and blue) is facilitated by diffusion only. (B, C) Segmented flow reactors. The reaction can be carried out in droplets (segments of the dispersed fluid separated by the immiscible carrier fluid; B) or slugs (segments of the carrier fluid separated by the immiscible dispersed fluid or a gas; C), where mixing is facilitated via recirculation within each segment. The droplet or slug reactors are separated by immiscible spacers, which can be gas (bubbles) or liquid. (D) A schematic diagram and an optical micrograph of a droplet flow microreactor for the synthesis of CdS nanoparticles. Reproduced with permission from ref 206. Copyright 2004 Royal Society of Chemistry. (E) TEM (transmission electron microscopy) image of gold nanorods synthesized using droplet microfluidic reactors. Reproduced with permission from ref 208. Copyright 2009 John Wiley & Sons, Inc.

That capability enabled the comparison of in vitro and in vivo effectiveness, which revealed that the in vitro studies had a false negative rate of over 90% (i.e., rejection of LNPs that would in fact exhibit in vivo efficacy). Moreover, the use of this approach led to the discovery of seven novel LNPs with in vivo efficacy whereas only one LNP had been discovered in a previous study using conventional method of formulation.202 In addition to polymeric nanoparticles for drug encapsulation and delivery, microfluidic methods have been explored for the synthesis of inorganic (metallic and silica) nanomaterials. The features of the synthesized materials such as size, size distribution, and crystal structure are determined by the types and designs of the microfluidic reactors.203,204 One simple reactor design employs continuous flow; a continuous flow reactor usually consists of a channel with multiple inlets for introduction of reagents (Figure 9A). The synthesis of nanoparticles using such reactors has been demonstrated.205 Although the implementation is simple for this type of reactor, synthesized nanomaterials may exhibit high polydispersity in their properties because the reaction is facilitated only by the diffusion of reagent molecules between laminar streams. Alternatively, approaches that use droplets206−208 and slugs209,210 as microreactors have been developed (Figure 9B,C). In these designs, the reagents are actively mixed within segments (the droplets or slugs) separated by immiscible

phases (gas or liquid). This type of reactor usually consists of a generator of droplets (which may be reactors or inert liquid spacers that separate slugs) or bubbles (gaseous spacers that separate slugs) followed by a long channel to allow sufficient time for the reaction to reach completion (Figure 9D). Using these techniques, synthesis of a wide range of metallic nanoparticles211−213 and quantum dots207,214 has been demonstrated (Figure 9E), and they exhibit narrow size distributions. These nanomaterials are potentially useful for biomedical applications such as in vivo imaging,215 photothermally triggered drug delivery,216 and tissue engineering.217,218 Finally, microfluidic hydrodynamic focusing allows the handling of multiple liquid phases containing cells, enabling the synthesis of cell-laden structures with unique morphologies and constituents such as fibers,219,220 particles,221−223 and capsules.224 For example, hollow microfibers219 and sandwichtype (solid−soft−solid) heterogeneous microfibers220 were fabricated using different configurations of colaminar flows in microchannels. The gelation of alginate with calcium ions in situ formed microfibers. Microcomponents of this kind have been used as building blocks for generating larger hierarchical tissue constructs through bottom up tissue engineering,225 To this end, monodisperse, cell-laden microspheres of PEG-hydrogel have been prepared by microfluidic hydrodynamic focusing and 2136



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Figure 10. (A) Scheme of circulating tumor cell (CTC) dissemination from primary tumors to distant organs to form micrometastases via blood vessels. The detection of CTC from cancer patients’ blood is performed on microfluidic chips with antibody-conjugated microposts to capture CTCs. (B, C) Illustrations of cell interactions with chip surfaces with (B) herringbone micropattern or (C) flat-walled surfaces. The chaotic mixing generated with the herringbone micropattern can enhance CTC capture. Reproduced with permission from ref 230. Copyright 2010 National Academy of Sciences, U.S.A.

used for the fabrication of heterogeneous, three-dimensional constructs.223 As discussed earlier (section 2.4), synthetic threedimensional tissue models might be useful to assess drug candidates as alternative to animal models; microfluidics offers a versatile toolbox for the preparation of such building blocks. 4.2. Microdevices for Diagnostics. Nanoparticles have been used as labels to detect and/or measure molecules or cells in microfluidic devices for in vitro diagnosis. Such devices are often fabricated with colored or fluorescent nanoparticles that allow for optical visualization or even quantification of the targeted analyte once fluidic samples are applied. For example, the urine pregnancy test used anti-hCG antibody-coated gold nanoparticles to detect hCG in urine samples using microfluidic devices.226 Microfluidic devices have been used in conjunction with antibody-coated nanoparticles to detect circulating tumor cells (CTC), providing early diagnosis of cancer mutation, metastasis, and relapse.227 The detection of CTCs provides an instructive illustration of the application of micro- or nanofabrication and microfluidics in diagnostics (Figure 10A). It has been estimated that, on average, there is 1 CTC for every one billion peripheral blood cells. This small number highlights how challenging their isolation and analysis is. Microfluidic devices have been used for efficient and selective separation of CTCs from peripheral whole blood samples from patients without prelabeling procedures. The detection of CTCs was performed by using hexagonal arrays of anti-epithelial-cell-adhesionmolecule (anti-EpCAM) antibody-coated 50 μm microposts that enhanced mixing and capturing CTCs with precisely controlled flow conditions.228 Mixing could also be facilitated by herringbone micropatterns on the wall of the microchannel that generate microvortices229 to increase the chance of collisions between CTCs and antibody-coated surfaces (Figure 10B,C).230 The chip showed a sensitivity of 93% in patients with metastatic prostate cancer, and could detect 63 CTCs/mL blood. This sensitivity is much higher than that of a current FDA-approved procedure (1 CTC per 7.5 mL of blood by magnetic-bead-based CellSearch assay using semiautomated purification174). The enhanced mixing with microvortices230

facilitated the detection of CTC clusters (4−12 cell aggregates) that could not be identified by previous CTC microchips with hexagonal arrays of microposts.228 This improvement is important considering that multicellular aggregates have the potential to establish metastatic lesions. Other polymers (e.g., poly(methyl methacrylate)) have been used to create fluidic devices for high recovery and efficient counting of CTCs from peripheral blood using immobilized anti-EpCAM antibody or anti-prostate-specific membrane-antigen aptamers as recognition elements.231 The integration of electrokinetic manipulation units consisting of linear channels running between two platinum electrodes allowed the direction of CTCs by modulation of an electric field and hydrodynamic flow. CTCs captured by the antibodies on the channel surface were subsequently released into a reservoir for counting and analysis.232 Other efforts to improve the sensitivity of detection included modifying the topography of microfluidic device surfaces with a microfabricated silicon-nanopillar array coated with anti-EpCAM, which enhanced local interactions between the surface receptors of CTCs and antibodies on the nanostructure surface.233 The addition of chaotic mixing units229 further promoted cell−substrate contact.234 Recently, magnetic nanoparticles coated with antibodies targeting CTCs were used to label those cells in patient blood samples, which were subsequently applied to a microfluidic chip. The magnetic moments of CTCs labeled with magnetic particles could be sensitively measured by a detector alongside the channels.235 The device could count CTCs from cancer patients’ blood samples with a sensitivity of 96%, compared to 15% for the CellSearch assay. Such devices coupled with commercially available immunomagnetic nanoparticles can also sensitively detect a broad range of clinically relevant cellular markers.235 They can be used to simultaneously detect multiple biomarkers on individual cells, which can be distinguished by the magnetization properties of different types of magnetic particles. With the beneficial features of sensitivity and multiplex detection, an analytical strategy based on magnetic nanoparticles and microfluidics can potentially provide a 2137



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(2) Valencia, P. M.; Farokhzad, O. C.; Karnik, R.; Langer, R. Microfluidic technologies for accelerating the clinical translation of nanoparticles. Nat. Nanotechnol. 2012, 7, 623−629. (3) Meyvantsson, I.; Beebe, D. J. Cell Culture Models in Microfluidic Systems. Annu. Rev. Anal. Chem. 2008, 1, 423−449. (4) Huh, D.; Hamilton, G. A.; Ingber, D. E. From 3D cell culture to organs-on-chips. Trends Cell Biol. 2011, 21, 745−754. (5) Petros, R. A.; DeSimone, J. M. Strategies in the design of nanoparticles for therapeutic applications. Nat. Rev. Drug Discovery 2010, 9, 615−627. (6) Lee, J.; Cuddihy, M. J.; Kotov, N. A. Three-Dimensional Cell Culture Matrices: State of the Art. Tissue Eng., Part B 2008, 14, 61−86. (7) Fernandes, T. G.; Kwon, S.-J.; Bale, S. S.; Lee, M.-Y.; Diogo, M. M.; Clark, D. S.; Cabral, J. M. S.; Dordick, J. S. Three-dimensional cell culture microarray for high-throughput studies of stem cell fate. Biotechnol. Bioeng. 2010, 106 (1), 106−118. (8) Marquis, B. J.; Love, S. A.; Braun, K. L.; Haynes, C. L. Analytical methods to assess nanoparticle toxicity. Analyst 2009, 134, 425. (9) Takhar, P.; Mahant, S. In vitro methods for nanotoxicity assessment: advantages and applications. Arch. Appl. Sci. Res 2011, 3, 389−403. (10) Sharma, S. V.; Haber, D. A.; Settleman, J. Cell line-based platforms to evaluate the therapeutic efficacy of candidate anticancer agents. Nat. Rev. Cancer 2010, 10, 241−253. (11) Falconnet, D.; Csucs, G.; Michelle Grandin, H.; Textor, M. Surface engineering approaches to micropattern surfaces for cell-based assays. Biomaterials 2006, 27, 3044−3063. (12) Kim, H. N.; Kang, D.-H.; Kim, M. S.; Jiao, A.; Kim, D.-H.; Suh, K.-Y. Patterning Methods for Polymers in Cell and Tissue Engineering. Ann. Biomed. Eng. 2012, 40, 1339−1355. (13) Mahmud, G.; Campbell, C. J.; Bishop, K. J. M.; Komarova, Y. A.; Chaga, O.; Soh, S.; Huda, S.; Kandere-Grzybowska, K.; Grzybowski, B. A. Directing cell motions on micropatterned ratchets. Nat. Phys. 2009, 5, 606−612. (14) Rosenthal, A.; Macdonald, A.; Voldman, J. Cell patterning chip for controlling the stem cell microenvironment. Biomaterials 2007, 28, 3208−3216. (15) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Geometric control of cell life and death. Science 1997, 276, 1425−1428. (16) Rodríguez-Dévora, J. I.; Shi, Z.-D.; Xu, T. Direct assembling methodologies for high-throughput bioscreening. Biotechnol. J. 2011, 6, 1454−1465. (17) Chin, V. I.; Taupin, P.; Sanga, S.; Scheel, J.; Gage, F. H.; Bhatia, S. N. Microfabricated platform for studying stem cell fates. Biotechnol. Bioeng. 2004, 88, 399−415. (18) Bailey, S. N.; Wu, R. Z.; Sabatini, D. M. Applications of transfected cell microarrays in high-throughput drug discovery. Drug Discovery Today 2002, 7, S113−S118. (19) Fernandes, T. G.; Diogo, M. M.; Clark, D. S.; Dordick, J. S.; Cabral, J. M. S. High-throughput cellular microarray platforms: applications in drug discovery, toxicology and stem cell research. Trends Biotechnol. 2009, 27, 342−349. (20) Borenstein, J. T.; Terai, H.; King, K. R.; Weinberg, E. J.; Kaazempur-Mofrad, M. R.; Vacanti, J. P. Microfabrication technology for vascularized tissue engineering. Biomed. Microdevices 2002, 4, 167− 175. (21) Park, H.; Cannizzaro, C.; Vunjak-Novakovic, G.; Langer, R.; Vacanti, C. A.; Farokhzad, O. C. Nanofabrication and Microfabrication of Functional Materials for Tissue Engineering. Tissue Eng. 2007, 13, 1867−1877. (22) Kim, H. N.; Jiao, A.; Hwang, N. S.; Kim, M. S.; Kang, D. H.; Kim, D.-H.; Suh, K.-Y. Nanotopography-guided tissue engineering and regenerative medicine. Adv. Drug Delivery Rev. 2012, 1−23. (23) Xia, Y.; Whitesides, G. M. Soft lithography. Annu. Rev. Mater. Sci. 1998, 28, 153−184. (24) McDonald, J. C.; Duffy, D. C.; Anderson, J. R.; Chiu, D. T.; Wu, H.; Schueller, O. J.; Whitesides, G. M. Fabrication of microfluidic systems in poly(dimethylsiloxane). Electrophoresis 2000, 21, 27−40.

versatile and modular diagnostic platform for rare cells in bodily fluids.

5. OUTLOOK AND CONCLUSIONS Technologies for the fabrication and application of microdevices have considerable potential usefulness for the evaluation and synthesis of nanomaterials and the diagnosis of disease. These technologies provide many practical benefits such as miniaturization, increased throughput (i.e., speed), lower cost, the capability for dynamic movements and stimuli, the creation of three-dimensional cocultures, and the enhanced mimicry of organ-specific microenvironments. These characteristics could eventually reduce the need for animal experimentation in drug development and nanotoxicology. Despite their great promise, microdevices are yet to have a major impact in nanomedicine, and conversely, nanotechnology is only beginning to make inroads into microfluidics. Broad dissemination of these techniques is perhaps hindered by a lack of standardization of device design, and by the fact that the techniques for fabrication are not available to all researchers. Some of these technologies are relatively new. Further improvements may lead to more sophisticated biological models and a better understanding of the efficacy and toxicity of nanomaterials. The application of microdevices in nanomedicine (and vice versa) is still in its infancy. As in often the case, the convergence of other areas of research with microfabrication technology may lead to synergistic progress.236 For example, the application of nanoelectronics to microfluidic cell cultures in a manner analogous to the way in which they have recently been employed in tissue engineering could allow for real-time highresolution monitoring of local tissue conditions (e.g., pH), cellular electrical activity, and other parameters.218 Such nanoscale systems could also be used to stimulate the tissues, either by an operator or by a computerized closed-loop feedback system. That stimulation either could be direct or could trigger an event by another implanted nanoelectronic device, such as an electrically triggered drug delivery system.237 Technologies of this kind, along with the gamut of approachesmany of them nanoparticle-basedthat provide triggered on-demand drug delivery (in response to light, ultrasound, magnetic fields, and other stimuli)238 will introduce the element of spatiotemporal control to this field. Advances in microfluidics itself will be important. The development of smart systems that can modulate flow autonomously or algorithmically239 could improve the mimicry of complex issue interactions by chip-based cell cultures. Modulation of the nanotopography of the surfaces of the microdevices can affect many performance parameters, and could have a marked effect on many aspects of cell behavior.240 The development of easily made channels that are resistant to solvents commonly used in making drug delivery nanoparticles241 will facilitate formulation and promote dissemination of these techniques.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

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REFERENCES

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