Surface Functionalization of Living Cells with Multilayer Patches

Nov 5, 2008 - The patch does not completely occlude the cellular surface from the surrounding environment; this approach allows a functional payload t...
0 downloads 11 Views 1MB Size
Download PDF
NANO LETTERS

Surface Functionalization of Living Cells with Multilayer Patches

2008 Vol. 8, No. 12 4446-4453

Albert J. Swiston,† Connie Cheng,‡ Soong Ho Um,†,§ Darrell J. Irvine,†,§ Robert E. Cohen,| and Michael F. Rubner*,† Department of Materials Science and Engineering, Department of Biological Engineering, and Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge Massachusetts 02139 Received August 7, 2008; Revised Manuscript Received October 16, 2008

ABSTRACT We demonstrate that functional polyelectrolyte multilayer (PEM) patches can be attached to a fraction of the surface area of living, individual lymphocytes. Surface-modified cells remain viable at least 48 h following attachment of the functional patch, and patches carrying magnetic nanoparticles allow the cells to be spatially manipulated using a magnetic field. The patch does not completely occlude the cellular surface from the surrounding environment; this approach allows a functional payload to be attached to a cell that is still free to perform its native functions, as suggested by preliminary studies on patch-modified T-cell migration. This approach has potential for broad applications in bioimaging, cellular functionalization, immune system and tissue engineering, and cell-based therapeutics where cell-environment interactions are critical.

A cell’s surface composition determines its interactions with the environment, ability to communicate with other cells, and trafficking to tissues. To date, surface modification of living cells has fallen primary within the purview of molecular biology. Methods such as DNA transfection utilize the cells’ natural machinery to achieve specifically designated outcomes. Though the wealth of knowledge about gene regulation, protein function, and cellular biology is ever increasing, these techniques are fundamentally limited to the natural ensemble of functionalities available to the cell. Some approaches, such as those demonstrated by Yarema1 and Bertozzi,2,3 have begun to utilize the power of synthetic materials synergistically with biological systems, though even these methods are restricted by the existing metabolic pathways that can handle synthetic compounds. Other methods exist to introduce non-native macromolecules, such as the direct reaction of N-hydroxysuccinimide esters with surface amines,4,5 passive adsorption of glycosylphosphatidyinositol-conjugated peptides,6 the reaction between cysteine and maleimide conjugates,7 “click” reactions involving synthetic amino acids,8 and adsorption of novel noncytotoxic polymers onto cell surfaces.9 All of these methods expand * Corresponding author, [email protected]. † Department of Materials Science and Engineering, Massachusetts Institute of Technology. ‡ School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138. § Department of Biological Engineering, Massachusetts Institute of Technology. | Department of Chemical Engineering, Massachusetts Institute of Technology. 10.1021/nl802404h CCC: $40.75 Published on Web 11/05/2008

 2008 American Chemical Society

the repertoire of available functionalities, but uniformly coating the cell membrane could potentially occlude the cell surface and prevent useful cell-environment interactions. In contrast, altering only a small portion of the surface of living cells using synthetic nanomaterials offers the possibility of expanding the range of surface properties that a cell may exhibit, while not interfering with the cell’s ability to communicate and interact with its surroundings. Although much effort has been directed at the application of nanomaterials for intracellular drug delivery and imaging applications, few approaches have addressed the task of systematically modifying the surface of living cells. Recently, polymer multilayer assemblies have been explored for cell surface modification, including the encapsulation of living10-13 and dead fixed cells.14,15 These examples focused on total encapsulation of relatively simple organisms such as bacteria and yeast. Much less work has examined surface modification of mammalian cells. Initial work on polyelectrolyte coatings of mammalian cells combined traditional cell encapsulation16 with sequential multilayer assembly to immunoisolate pancreatic islet cells.17 Other researchers have focused on the sequential layering of biological polyelectrolytes and hepatocytes,18 functionalizing gold nanoparticles with various ligands to induce different cell surface-particle interactions,19 and integrating liposomes into cell membranes to provide mechanochromatic sensors.20,21 Polymer multilayers offer a variety of favorable properties for cell surface modification (tunable structure and composition,22,23 functionalizability,24-29 ease of fabrication.30,31)

However, while complete coating of the entire apical face of an adherent cell may work well in applications where modification of a sessile structure is the goal, in other situations global modification of the cell surface may be undesirable. We became interested in creating a functional patch that could contain environment-responsive materials, release drugs, or change the mechanics of the cell surface without blocking active functions of the cell. Such patchmodified cells could be used to combine favorable innate properties of a cell with synthetic materials endowing new therapeutic or sensing capabilities. Of particular interest is the functionalization of immune cells with cargo-carrying multilayers, in order to combine targeted tissue-homing properties of these cells with synthetic material payloads. For example, T-cells home to lymphoid organs, sites of infection, and tumors, and patch-modified cells could be used to deliver synthetic sensory, vaccine, or chemotherapeutic payloads. Other work has demonstrated that synthetic drugcarrying or therapeutic nanoparticles can be fed to phagocytic macrophages, which subsequently carry these particles to lymphoid organs32 or tumors.33 However, this strategy is confined to treatments that can operate from inside a cell. Clearly, a broad range of sensing and therapeutic strategies could be enabled by functionalizing the external surface of immune cells while allowing the cell, and possibly the synthetic material, to communicate with the surrounding environment. Here, we show how to use existing photolithographicpatterning techniques34,35 to engineer novel patch heterostructures containing both a payload component (superparamagnetic nanoparticles in the present study) and a cell-adhesive face that partially comprises hyaluronic acid. When living lymphocytes are exposed to the patches, individual cells attach to a surface-bound patch. A specific temperature stimulus triggers release of the patch from the surface without sacrificing the cell-patch attachment, thus yielding a synthetically functionalized and living cell. These cells respond to an externally applied magnetic field and continue to function metabolically as a native lymphocyte as evidenced by patch-modified T-cell migration. While other work has focused on the lithographic patterning of polymer films, uniform functionalization of cell surfaces, and interaction of living systems with nanomaterials, we believe our approach is the first biologically friendly method to functionalize only a part of the surface of a living cell which is then free to perform its native functions. We used the patterning method of McShane and coworkers34,35 to create patterned multilayer heterostructure assemblies. Briefly, this process uses a traditional lift-off photolithographic approach to pattern ultrathin polymer films (see Supporting Information for a more detailed discussion). A positive photoresist is deposited onto a substrate, which is then exposed with a 365 nm UV light source through a chromium on glass mask patterned with 10 or 15 µm diameter holes. After development, the features in the photoresist extend down to the substrate. A heterostructured polymer multilayer film (details below) is deposited conformally on both the patterned photoresist and the exposed Nano Lett., Vol. 8, No. 12, 2008

Figure 1. Overview of our cell functionalization scheme, with CLSM images demonstrating each step. The film system considered here is (PMAA3.0/PNIPAAm3.0)80.5(FITC-PAH3.0/Fe3O4 NP4.0)10 (CHI3.0/HA3.0)3. (a) A regular array of surface-bound patches spaced 50 µm apart. The green fluorescence is from the FITCPAH used to fabricate the payload region. After CH27 B-cell incubation and attachment (b), a majority (85 ( 3%) of the surfacebound patches are occupied. The red fluorescence is from CellTracker Red CMPTX, which nonselectively tags the interior of living cells. (c) After the temperature is reduced to 4 °C for 30 min, the patches are released from the surface while remaining attached to the cell membrane. All scale bars are 25 µm.

substrate. In the final step, the sample is sonicated in acetone to dissolve the photoresist and release the polymer film deposited directly onto the photoresist. This procedure leaves only the polymer that had been deposited within the features and attached directly to the substrate. Using this approach, we are able to make uniformly patterned, heterostructured, surface-bound patches (see Figure 1 and Supporting Information) over areas as large as 1 in.2, although we expect that this fabrication method may be scaled to much larger areas. We used an aqueous-based layer-by-layer technique to deposit the functional, heterostructured polymer film. This technique has been used extensively for both polyelectrolyte and hydrogen-bonded films, and details can be found elsewhere.22,30 Each cycle of complementary polymer deposition produces a complexed, interpenetrated structure referred 4447

to as a bilayer and the following notation is commonly used: (Poly1X/Poly2Y)n. Here, Poly1 and Poly2 refer to the abbreviation for the specific polymers or nanoparticles used in a selected assembly process, X and Y refer to the pH of the solution during multilayer deposition, and n is the number of bilayers that have been deposited. Further details for the heterostructured film deposition method may be found in the Supporting Information. The general process for creating living cells with functional multilayer patches is illustrated in Figure 1. First, a patterned array of heterostructured patches is created using a photolithographic lift-off process combined with multilayer assembly. Since no living cells are involved at this point, a wide range of assembly conditions and materials can be utilized without concern for cytotoxic effects. Second, live cells are sedimented onto the patterned array and selectively bind to the surface of each patch to produce a large-area array of immobilized individual cells. Finally, a triggering mechanism is used to release the cell-patch complexes from the substrate resulting in free-floating cells fitted with a functional multilayer patch. Specific details about all key elements of this process follow. To create a releasable functional patch system suitable for attachment to living cells, three functional regions are essential. The order of deposition to the surface is as follows: (1) a releasable region that deconstructs in noncytotoxic conditions, (2) a payload region that holds the functional cargo that will be exposed to the medium, and (3) a celladhesive region that anchors the payload to the cell membrane. In this study, each surface-bound patch was a heterostructure consisting of three identifiable lamellar regions or strata, each of which consists of several bilayers of electrostatically complexed or hydrogen-bonded materials (Figure 1a). For each region, see the Supporting Information for details on solution concentrations and other deposition conditions. The first region of the patch heterostructure was designed to deconstruct readily upon exposure to specific noncytotoxic conditions. Although there are a number of different triggering mechanisms that can be used to deconstruct suitably designed polyelectrolyte multilayers,36 we focused on the controlled dissolution of two different hydrogen-bonding systems based on poly(methylacrylic acid) (PMAA), poly(vinylpyrolidone) (PVPON), and poly(N-isopropylacrylamide) (PNIPAAm), specifically (PMAA2.0/PVPON2.0) and (PMAA3.0/PNIPAAm3.0). It has previously been shown that hydrogen-bonded multilayer systems containing carboxylic acid groups can be readily assembled at low pH but will dissolve quickly when exposed to a pH sufficiently high to ionize the hydrogen-bonded acid groups.37 In the case of the PMAA/PVPON multilayer system, the critical pH for dissolution is ∼6.4. Since the PMAA/PNIPAAm multilayer system contains a thermally responsive polymer (PNIPAAm), release only occurs both above the critical solution pH (∼6.2) and below a specific triggering temperature (∼32 °C, to be discussed). All multilayer depositions during heterostructured patch assembly must be carried out below the critical dissolution pH. 4448

When utilizing hydrogen-bonded multilayers to release a heterostructured thin film, it is essential to determine how subsequently assembled layers influence the release behavior. Decher reported that an electrostatically bonded region built on top of a hydrogen-bonded region requires a critical thickness of the hydrogen-bonded region for successful dissolution and release.38 Caruso also noted that deposition of polyelectrolytes onto hydrogen-bonded films seems to stabilize these films at high pH.39 We observed similar behavior in a number of hydrogen-bonded polymer systems. In all cases, despite variations in polymer systems, molecular weights, and subsequent electrostatic layer depositions, a release region thickness of at least ∼250-300 nm was required to achieve successful patch lift-off. When pH is used as the only release mechanism, the narrow pH range suitable for cell survivability coupled with the very rapid release that occurs above the critical release pH resulted in a difficult to control release process (cell binding and patch release are occurring simultaneously). To address the need for better control over patch release with an on-demand release mechanism, we developed a thermally responsive multilayer system based on PMAA/PNIPAAm. PMAA/PNIPAAm multilayers, when built into a patterned heterostructure, dissociate in water by a combined mechanism that is controlled by both pH and temperature. The pH mechanism depends on the ionization level of PMAA’s acid groups incorporated in the film. Below the critical pH, PMAA and PNIPAAm will form hydrogen-bonded multilayers that are stable at all biologically useful temperatures. The temperature mechanism relies on the interaction between water and PNIPAAm. Above the lower critical solution temperature (LCST, 32 °C for PNIPAAm in pure water), polymer-polymer interactions are favoredover polymer-water interactions, leading to insoluble PNIPAAm. Below the LCST, PNIPAAm prefers to hydrogen bond with water, leading to a homogeneous, single phase polymer-water solution. When PNIPAAm is incorporated into a patterned multilayer heterostructure, the solubility of PNIPAAm determines the dissolution behavior of the entire film. We find that PMAA/PNIPAAm films deconstruct in physiological pH conditions (∼7.4, above the critical pH) at 4 °C (below the LCST, PNIPAAm is soluble) but not at 37 °C (above the LCST, PNIPAAm is insoluble). Thus, binding cells to the surface-confined patches can be carried out at 37 °C for as long as needed, followed by controllable release by simply lowering the temperature to 4 °C. We believe that this is the first demonstration of a thermally responsive thin film based on a hydrogen-bonded multilayer that can be controllably erased (rather than simply swollen)40,41 using a temperature trigger. It should be noted that nonpatterned PMAA/PNIPAAm multilayers without the capping payload layers are not stable in pH 7.4 phosphate-buffered saline at 37 °C and that the thermal control described here is only observed in the patterned heterostructure. The origin of this behavior is the subject of ongoing research. As previously mentioned, we have attached patches to cell surfaces using two different mechanisms (temperature and pH or just pH) for release region dissolution. The first method Nano Lett., Vol. 8, No. 12, 2008

Figure 2. CLSM images of two patch functionalized lymphocytes: (a) a CH27 B-lymphocyte and (b) a HuT 78 T-cell. The green fluorescence is from the FITC-PAH used in the payload region, and both scale bars are 10 µm. Both patches had the following composition: (PMAA3.0/PNIPAAm3.0)80.5(FITC-PAH3.0/ Fe3O4 NP4.0)10(CHI3.0/HA3.0)3.

is depicted in Figure 1, which presents a single cell docking with a single patch followed by thermally triggered release, yielding an individual cell with a single patch. Figure 2 shows confocal laser scanning microscopy (CLSM) optical brightfield and fluorescence images of a patch on the surface of two different types of lymphocytes suspended in media immediately after patch attachment (using the thermally controlled PMAA/PNIPAAm release system). Figure 2a is a CH27 B-lymphocyte with a patch oriented in profile to the microscope objective; Figure 2b is a HuT78 human T-lymphocyte with a patch that has partially folded upon itself. In both of these images, the cell does not attach to the patch’s entire surface area despite initially having CD44 receptors available everywhere on the cell surface, an issue discussed in greater detail later. With this approach, patch-functionalized cells could be preprogrammed to assemble into sheet, ring, or cluster structures by judicious placement of adhesive regions as shown with colloidal bodies in the work of Glotzer et al.42,43 Patches that are cell-adhesive on both faces may even allow the polymerization of cellular species, with individual cells being the monomers and the patches serving as bifunctional linkages. The second method we used to attach cells to patches was to release patches with a pH-only controlled (PMAA/ PVPON) release region into a small (∼300 µL) volume of cell solution. Here, release is determined solely by pH conditions (above or below the critical pH), and since the cells are introduced at pH 7.4 (which is significantly above the critical pH for this hydrogen-bonded system), numerous Nano Lett., Vol. 8, No. 12, 2008

cells are introduced to rapidly releasing patches. In addition to single cell-single patch complexes, large multicellmultipatch aggregates (visible to the naked eye as particles in solution) were made. These aggregates form because patches do not conformally attach to the cell surface (see Figure 2) allowing more than one cell to be associated with one patch, and when a cell has two or more patches attached to its surface, large aggregates are possible. Cells also seem to weakly associate with the outer face of the patch, which further contributes to cell aggregation. A sample of several CH27 B-cell aggregates can be found in Figure 3. We feel that control of this aggregation phenomenon will allow opportunities for bottom-up tissue engineering. This approach, for example, could yield macroscopic cell and tissue constructs in which cells, rather than porous scaffolds, serve as the basis for synthetic tissue structures. Control over this phenomenon, such as by adjusting the ratio of cells to released patches, is the subject of ongoing work. The second important region of the heterostructured patch is the payload region. Upon dissolution of the release region, the payload region of the PEM patch is presented to the extracellular environment and is anchored to the surface of the cell via the cell-adhesive region (Figure 1b). Examples of possible cargoes that may be incorporated into this region include drugs,25 proteins,26,27 or nanoparticles.28,29 In this study, anionic, superparamagnetic nanoparticles (Fe3O4 NP) were alternately deposited with fluorescein-labeled poly(allylamine hydrochloride) (FITC-PAH) to create a fluorescent labeled and magnetically responsive PEM patch. Ten bilayers of magnetic nanoparticles and FITC-PAH yield a ∼130 nm thick payload region. To test that we have conferred magnetic properties to the cell via the attached patch, B-lymphocytes were exposed to superparamagnetic patches containing a PMAA/PVPONrelease region. The free-floating lympocytes were washed off the previously patch-laden surface into a LabTek chambered coverslip and imaged using an inverted microscope. After cells were allowed to settle, a rare earth magnet was placed close to the imaged region but outside of the chamber. Figure 4 shows how a single B-cell-single patch complex responds to the applied magnetic field. This cell moved ∼200 µm in 11 s, much faster than a freely suspended cell would normally move. Further, cells without patches, as seen in Figure 4, do not respond to the applied magnetic field. Since the PMAA/PVPON system was used, large cell-patch aggregates (such as seen in Figure 3) also move in the direction of the magnet. A video of this may be found in the Supporting Information. The adhesion between the patch and cell is strong enough that the cell is pulled along with, rather than releasing, the patch. A first-order analysis of the drag force exerted on the cell in Figure 4 is ∼3 pN.44 In general, cells do not adhere to the entire surface area of the patch (see Figure 2), which means that the cell-patch adhesion may be quite high. The final region of the assembled heterostructure is celladhesive, anchoring the underlying payload region to the cell membrane. The cell-adhesive region must be chosen with consideration of the cells to be functionalized. We chose a 4449

Figure 3. Overlayed fluorescent microscopy images of different cell-patch complexes created using the following patch system: (PMAA2.0/ PVPON2.0)40.5(FITC-PAH3.0/Fe3O4 NP4.0)10(CHI3.0/HA3.0)3. The red signal comes from CellTracker Red CMPTX, and the green from the fluorescein-containing payload region. The scale bar in each image is 10 µm.

Figure 4. A patch functionalized B-cell responds to an applied magnetic field. The patch system used was (PMAA2.0/ PVPON2.0)40.5(FITC-PAH3.0/Fe3O4 NP4.0)10(CHI3.0/HA3.0)3. The cell moved ∼200 µm in 11s toward a rare earth magnet placed near the imaging chamber. Because the cell is free-floating in solution, it moves out of the field of focus during the course of imaging. Note that cells without patches do not respond to the applied field.

hyaluronic acid/chitosan (HA/CHI) multilayer, since lymphocytes contain CD44 cell-surface receptors whose natural ligand is a three-structural unit repeat of the polysaccharide hyaluronic acid.45 HA thus forms the outermost layer of the cell-adhesive region in each heterostructured surface-bound 4450

patch. Chitosan was chosen as a complementary polycation for its biocompatibility when complexed with HA in multilayer films.12,46 Three bilayers of HA and CHI yield a ∼20 nm thick cell-adhesive region. To test if the CD44-HA interaction is in fact mediating attachment of cells to patches, we incubated cells in HA-containing media before introducing them to HA-terminated patches and measured the relative number of cells associated with patches. We saw an 83% reduction in the number of patches occupied with a cell for the aliquot incubated in HA-containing media, which suggests that the CD44-HA interaction is the mechanism by which these cells attach to the patch. Ideally, cells settle to the patch-laden surface and associate at a ratio of one cell to one patch, though several more situations are possible. First, there may be more than one cell associated with a patch. Previous studies on colloidal particles adsorbing on patterned surfaces have shown that the ratio between the diameter of the particle and the diameter of a circular feature will determine the cluster size.47 During the lithography step, the diameter of the patch can be easily controlled; in fact, when we used 15 µm diameter patches, we noticed many dimers (two cells per patch, Figure 5a), whereas 10 µm patches almost exclusively displayed single cells (Figure 5b), consistent with previous results. Next, there are some cells that do not associate with a patch, because either the cell remains in solution or settles onto an interstitial area between patches. Increasing the density of surface patches (using a photomask with greater feature density) and decreasing the number of cells in solution can control this effect. Last, patches may not attach to a cell but will still release in the neutral 4 °C media. We investigated the relative frequency of these scenarios by fabricating patches without a releasable region and surveying the cell attachment behavior to the patches and surface interstitial areas. With the patch system (Fe3O4 NP4.0/FITC-PAH3.0)10.5(CHI3.0/HA3.0)3, 53 ( 5% of patches are occupied after one agitation/incubation cycle and 85 ( 3% are occupied after two cycles (see Figure 5b). These Nano Lett., Vol. 8, No. 12, 2008

Figure 5. Representative optical brightfield images showing cell attachment to patches fabricated without a release region: (a) dimers (two cells per patch) on 15 µm diameter patches after two agitation/incubation cycles; (b) 10 µm diameter patches after two agitation/incubation cycles, with 84.5% of the patches occupied. The scale bar is 100 µm in each image, and the inset of (a) is 200 µm × 200 µm.

results are characteristic of any (CHI3.0/HA3.0)3 terminating patch. Thus, we offer a straightforward method for nonadherent cell patterning based solely upon the natural interaction between CD44 and HA with an efficacy that rivals previous methods.48,49 A critical design parameter in our system is the balance between cell adhesion and release region dissolution. The strength of the interaction between the cell and the celladhesive region must be greater than that between the functional and release regions. While the present CD44-HA binding strategy does provide enough interaction, several other options exist to increase this interaction, such as nonselective biotin/streptavidin48,49 or RGD-integrin24 strategies. Temperature also plays a key role in mediating the cell-patch and release region dissolution. We find that CH27 B-lymphocytes attach to HA-containing surfaces most efficiently at 37 °C. Using an LCST-based release region allows us to attach the cells at a temperature optimal for encouraging attachment and thermodynamically preventing release. We may then very briefly lower the temperature below the LCST to encourage patch release and then return the cells to physiological temperature. We have shown how to fabricate a heterostructured patch and how various functionalities embedded in the patch (fluorescence and magnetic responsiveness) may be used. A fundamental question that arises is how do the cells respond to the attached patch and specifically whether the patch negatively impacts native cell behavior. Possible cytotoxicity caused by the attached patch is of paramount concern for meaningful application of this technique. B-cells incubated on top of (PMAA3.0/ PNIPAAm3.0)80.5(FITC-PAH3.0/Fe3O4 NP4.0)10(CHI3.0/ HA3.0)3 patches that were not thermally released show nearly 100% viability up to 48 h following attachment, suggesting no acute toxicity attributable to the PEM patch. Furthermore, we see greater than 85% viability for B-cells thermally released, collected, and cultured up to 72 h after patch attachment (see Supporting Information for details on both cytotoxicity assays). As a test of whether patch attachment would negatively affect intrinsic cell functions, we assessed the ability of patchmodified T-cells to migrate. Hut 78 T-cells spontaneously Nano Lett., Vol. 8, No. 12, 2008

Figure 6. Migration of a HuT 78 model T-cell on an ICAM1coated coverslip. The cell was observed to travel at ∼0.5 µm/ min for at least 6 h, at which point the patch adsorbed to the coverslip preventing the cell from migrating further. The scale bar is 25 µm.

migrate on substrates coated with intercellular adhesion molecule-1 (ICAM-1), an adhesion ligand present in tissue and on endothelial cells that binds to the T-cell integrin lymphocyte function-associated molecule-1. We attached fluorescent, superparamagnetic nanoparticle-containing patches to the surfaces of T-cells and tracked their migration over time by videomicroscopy. We found that this type of T-cell attached to patches with less efficiency than CH27 B-cells, likely due to their lower expression of CD44 cell surface receptors. While several T-cells decorated with patches were found to migrate on ICAM-1-coated surfaces, we chose to closely monitor one, and this cell is shown in the time-lapse sequence in Figure 6 at three different time points for the same field of view. This cell polarized, developed a characteristic lamellipodium-extending leading edge and trailing uropod, and migrated continuously for over 6 h (video available in Supporting Information). The patch was not conformally attached to the cell membrane, which seems to suggest that the cell has chosen to locally cluster some of the available surface receptors responsible for cell-patch 4451

binding. Interestingly, while the cell changes its migration direction several times, including reversing the leading and trailing ends by changing the migration direction nearly 180°, the cell-patch attachment point is always found at the trailing end of the cell. This may reflect the fact that CD44, like a number of other adhesion molecules on T-cells, preferentially accumulates in the uropod at the rear of the cell during migration.50 After 6 h, the patch stuck to the coverslip surface, but the cell continued polarizing. However, the cell-patch association was strong enough to frustrate actual migration, indicating that the strength of binding between the patch and cell surface was greater than the traction force exerted by the cell migrating on ICAM-1. The preliminary, proof-of-concept results presented here suggest that T-cells have the capability to migrate normally while bearing a patch. A statistical understanding of how patches affect cell migration is currently being pursued. We used CD44-hyaluronic acid interactions to anchor patches to leukocytes via a natural cell surface receptor-ligand interaction. CD44 is thought to mediate adhesion of T-cells to hyaluronan-expressing endothelial and stromal cells during homing to inflammatory sites51,52 and may play a role in regulating T-cell activation.53,54 Thus, a complete blockade of CD44 might be expected to interfere with lymphocyte functions. However, we expect that engagment of CD44 with the patch likely does not sequester all of the receptors on the cell due to the low affinity of individual CD44-HA interactions.55 In fact, patched CH27 B-lymphocytes exposed to a fluorescein-tagged anti-CD44 antibody show uniform fluorescence over the entire membrane, indicating little or no receptor sequestering due to the attached patch (see Supporting Information for fluorescence and brightfield micrographs of a CD44-stained cell). Therefore, we do not expect the attached patch to impair other CD44-dependent functions. In addition, the strategy described here is a general one, and the use of other receptor-ligand interactions or covalent conjugation strategies to link patches to lymphocytes is possible and an area of ongoing studies. A great number of strategies have been published enabling the controlled adhesion of anchorage-dependent cells to defined sites on substrates by patterning adhesion molecules on surfaces. Applying the present “cell patch” strategy to nonadherent lymphocytes posed advantages and challenges relative to adherent cell experiments: Lymphocytes exhibit very weak nonspecific adhesion to the background of patcharrayed substrates, compared to adherent cells like fibroblasts. However, these cells also anchor to the specific binding sites much more weakly than adherent cells, perhaps due to their lack of strong adhesion to secreted ECM molecules adsorbed on the substrate and adhesion-mediating structures such as focal adhesions. The approach of using CD44-HA interactions to anchor lymphocytes enabled us to overcome these difficulties. How the patch associates with the cell surface may provide fundamental insights into membrane dynamics and cellular behavior. As seen in Figures 2 and 6, patches tend not to attach conformally to the cell surface despite the mobility of CD44 receptors in the fluid plasma membrane of the cell. 4452

This may reflect an energetic penalty for deforming the cell membrane to achieve conformal contact with the flat patch surface that outweighs the energy of receptor-ligand binding and also suggests that the patch itself is stiff enough to resist deformation by the cell. Further studies on how the cell responds to and interacts with the patch will elucidate fundamental cell processes and lend insight into how mechanical and chemical cell surface stimuli affect cellular behavior. Membrane mechanical properties have been connected with certain types of diseases such as cancer and malaria,56,57 and cell patches may offer a valuable tool to systematically probe physical property-disease state correlations. Patch modification of lymphocytes could be of interest to combine their native homing to lymphoid tissues, tumors, or sites of infection with non-native functions imparted by nanomaterials. The homing properties of leukocytes can be tuned by activation/cytokine treatment, providing further control: B cells might carry antigens into the lymphoid organs to promote antibody responses, while T-cells could carry antitumor drugs, sensing materials, or adjuvant molecules to tumor or infection sites. In the case of living lymphocytes, these cells need to enter and exit the vasculature by diapedesis, a process that requires a dynamic reorganization of the cell membrane. The ability to modify only a limited portion of the cell surface is thus an advantage, to avoid blocking intrinsic cell functions such as tissue homing or cell division. The synthetic material-biological system symbiosis presents a host of fundamental questions. What are the unintended consequences of introducing synthetic materials for both the cell and the material? Are there any cytotoxic effects? Does the presence of synthetic materials alter the native functions of a cell? How does the synthesis or structure of the synthetic material depend on the cooperation or assistance from the biological system? These questions have only begun to be addressed for the host of exciting new synthetic nanostructures now being developed. For instance, we envision magnetic-field-heated nanoparticles embedded in a thermally responsive polymer, such as poly(N-isopropyl acrylamide) which could be attached to a homing immune system cell. This cell would be free to travel to a site of interest (such as a tumor or trauma), either because it is intrinsically programmed for tissue surveillance (e.g., effector memory T-cells) or because it specifically recognizes antigen within a given target tissue/disease site, then precisely activated to release a cargo molecule. A second example lies in using magnetic fields to spatially manipulate magnetically functionalized cells for cell-patterning and homing.58 These cells could simultaneously be functionalized with an IR fluorescent species, such as quantum dots, that would be capable of in vivo tracking. These examples are just a few potential applications of patch-modified cells we envision. We have demonstrated the functionalization of living cells with PEM patches, thereby conferring new properties to the cell without preventing interaction with its environment. Fabrication of these patches requires the construction of multiregion, multilayered heterostructures, which comprise Nano Lett., Vol. 8, No. 12, 2008

(at a minimum) a labile release region, a functional PEM region, and a cell-adhesive face. Dissolution of the release region depends strongly on thickness and temperatureinduced hydrophilicity; knowledge of these variables allows us to precisely control patch release. We were able to attach a superparamagnetic PEM patch to the membrane of T- and B-lymphocytes using CD44-HA interactions. B-cells responded to an applied magnetic field, and T-cells continued to chemokinetically migrate on ICAM-coated surfaces following patch attachment. The ability of antigen-specific lymphocytes to home to and accumulate in antigen-bearing tissues suggests potential applications for this technique in immune system engineering, such as bioimaging or lymphocyte-directed drug delivery vehicles. Cell-adhesive patches could lead to novel cell agglomerates that self-assemble into unique and useful cell-tissue constructs, a process we call bottom-up tissue engineering. Acknowledgment. We wish to acknowledge support from NSF MRSEC Award DMR-02-13282. This material is based upon work supported under a National Science Foundation Graduate Research Fellowship. We wish to acknowledge Y. Wang and M. Natter for generous help with cell migration and G. Nogueira for assistance with film preparation. Supporting Information Available: Details on film deposition conditions, photolithography, cell culture, patch attachment, videomicroscopy, patch release efficiency, CD44stained B-cells, and videos of B-lymphocytes traveling in response to a magnetic field and a T-lymphocyte migrating on an ICAM surface. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Sampathkumar, S.; Li, A.; Jones, M.; Sun, Z.; Yarema, K. Nat. Chem. Biol. 2006, 2, 149. (2) Prescher, J. A.; Dube, D. H.; Bertozzi, C. R. Nature 2004, 430, 873. (3) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13. (4) Singh, N.; Yolcu, E.; Taylor, D.; Gercel-Taylor, C.; Metzinger, D.; Dreisbach, S.; Shirwan, H. Cancer Res. 2003, 63, 4067. (5) Singh, N.; Yolcu, E.; Askenasy, N.; Shirwan, H. Ann. N.Y. Acad. Sci. 2005, 1056, 344. (6) Medof, M. E.; Nagarajan, S.; Tykocinski, M. L. FASEB J. 1996, 10, 574. (7) Shen, C.; Lin, M.; Yaradanakul, A.; Lariccia, V.; Hill, J. J. Physiol. 2007, 582, 1011. (8) Link, A. J.; Tirrell, D. A. J. Am. Chem. Soc. 2003, 125, 11164. (9) Wilson, J.; Cui, W.; Chaikof, E. Nano Lett. 2008, 8, 1940. (10) Diaspro, A.; Silvano, D.; Krol, S.; Cavalleri, O.; Gliozzi, A. Langmuir 2002, 18, 5047. (11) Germain, M.; Balaguer, P.; Nicolas, J.-C.; Lopez, F.; Esteve, J.-P.; Sukhorukov, G. B.; Winterhalter, M.; Richard-Foy, H.; Fournier, D. Biosens. Bioelectron. 2006, 21, 1566. (12) Hillberg, A. L.; Tabrizian, M. Biomacromolecules 2006, 7, 2742. (13) Krol, S.; Nolte, M.; Diaspro, A.; Mazza, D.; Magrassi, R.; Gliozzi, A.; Fery, A. Langmuir 2005, 21, 705. (14) Moya, S.; Dahne, L.; Voigt, A.; Leporatti, S.; Donath, E.; Mohwald, H. Colloid Surf., A 2001, 183-185, 27. (15) Georgieva, R.; Moya, S.; Donath, E.; Baumler, H. Langmuir 2005, 20, 1895. (16) Chang, T. M. S. Science 1964, 146, 524–525. (17) Schneider, S.; Feilen, P.; Slotty, V.; Kampfner, D.; Preuss, S.; Berger, S.; Beyer, J.; Pommersheim, R. Biomaterials 2001, 22, 1961.

Nano Lett., Vol. 8, No. 12, 2008

(18) Rajagopalan, P.; Shen, C.; Berthiaume, F.; Tilles, A.; Toner, M.; Yarmush, M. Tissue Eng. 2006, 12, 1553. (19) de la Fuente, J. M.; Berry, C. C.; Riehle, M. O.; Curtis, A. S. G. Langmuir 2006, 22, 3286. (20) Orynbayeva, Z.; Kolusheva, S.; Livneh, E.; Lichtenshtein, A.; Nathan, I.; Jelinek, R. Angew Chem., Int. Ed. 2005, 44, 1092. (21) Orynbayeva, Z.; Kolusheva, S.; Groysman, N.; Gavrielov, N.; Lobel, L.; Jelinek, R. J. Virol. 2007, 81, 1140. (22) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 4213. (23) Thompson, M.; Berg, M.; Tobias, I.; Rubner, M.; Van Vliet, K. Biomaterials 2005, 26, 6836. (24) Berg, M. C.; Yang, S. Y.; Hammond, P. T.; Rubner, M. F. Langmuir 2004, 20, 1362. (25) Berg, M. C.; Zhai, L.; Cohen, R. E.; Rubner, M. F. Biomacromolecules 2006, 7, 357. (26) Lvov, Y.; Ariga, K.; Kunitake, T. Chem. Lett. 1994, 23, 2323. (27) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. Thin Solid Films 1996, 285, 797. (28) Wu, Z. Z.; Walish, J.; Nolte, A.; Zhai, L.; Cohen, R. E.; Rubner, M. F. AdV. Mater. 2006, 18, 2699. (29) Li, Z.; Lee, D.; Sheng, X. X.; Cohen, R. E.; Rubner, M. F. Langmuir 2006, 22, 9820. (30) Choi, J.; Rubner, M. F. Macromolecules 2005, 38, 116. (31) Decher, G. Science 1997, 277, 1232. (32) Dou, H.; Destache, C. J.; Morehead, J. R.; Mosley, R. L.; Boska, M. D.; Kingsley, J.; Gorantla, S.; Poluektova, L.; Nelson, J. A.; Chaubal, M.; Werling, J.; Kipp, J.; Rabinow, B. E.; Gendelman, H. E. Blood 2006, 108, 2827. (33) Choi, M. R.; Stanton-Maxey, K. J.; Stanley, J. K.; Levin, C. S.; Bardhan, R.; Akin, D.; Badve, S.; Sturgis, J.; Robinson, J. P.; Bashir, R.; Halas, N. J.; Clare, S. E. Nano Lett. 2007, 7, 3759. (34) ShaikhMohammed, J.; DeCoster, M. A.; McShane, M. J. Langmuir 2006, 22, 2738. (35) ShaikhMohammed, J.; DeCoster, M. A.; McShane, M. J. Biomacromolecules 2004, 5, 1745. (36) Lynn, D. M. AdV. Mater. 2007, 19, 4118. (37) Kharlampieva, E.; Sukhishvili, S. A. Polym. ReV. 2006, 46, 377. (38) Ono, S. S.; Decher, G. Nano Lett. 2006, 6, 592. (39) Cho, J.; Caruso, F. Macromolecules 2003, 36, 2845. (40) Serpe, M. J.; Jones, C. D.; Lyon, L. A. Langmuir 2003, 19, 8759. (41) Jones, C. D.; Lyon, L. A. Macromolecules 2000, 33, 8301. (42) Glotzer, S. C. Science 2004, 306, 419. (43) Zhang, Z. L.; Glotzer, S. C. Nano Lett. 2004, 4, 1407. (44) The Stokes equation for drag (F ) 6πηVr) was used for this calculation. Values used were as follows: η ) 10-3 Pa·s, V ) 20 µm/s, and r ) 8 µm. (45) Underhill, C. J Cell Sci. 1992, 103, 293. (46) Serizawa, T.; Yamaguchi, M.; Matsuyama, T.; Akashi, M. Biomacromolecules 2000, 1, 306. (47) Lee, I.; Zheng, H.; Rubner, M.; Hammond, P. AdV. Mater. 2002, 14, 572. (48) Kim, H.; Doh, J.; Irvine, D. J.; Cohen, R. E.; Hammond, P. T. Biomacromolecules 2004, 5, 822. (49) Kim, H.; Cohen, R. E.; Hammond, P. T.; Irvine, D. J. AdV. Funct. Mater. 2006, 16, 1313. (50) Gomez-Mouton, C.; Abad, J.; Mira, E.; Lacalle, R.; Gallardo, E.; Jimenez-Baranda, S.; Illa, I.; Bernad, A.; Manes, S.; Martinez-A, C. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9642. (51) DeGrendele, H.; Estess, P.; Picker, L.; Siegelman, M. J. Exp. Med. 1996, 183, 1119. (52) Clark, R.; Alon, R.; Springer, T. J. Cell Biol. 1996, 134, 1075. (53) Fo¨ger, N.; Marhaba, R.; Zo¨ller, M. Eur. J. Immunol. 2000, 30, 2888. (54) Hegde, V.; Singh, N.; Nagarkatti, P.; Nagarkatti, M. J. Leukocyte Biol. 2008, 84, 134. (55) Lesley, J.; English, N.; Ga´l, I.; Mikecz, K.; Day, A. J. Biol. Chem. 2002, 277, 26600. (56) Cross, S. E.; Jin, Y.; Rao, J.; Gimzewski, J. Nat. Nanotechnol. 2007, 2, 780. (57) Suresh, S.; Spatz, J.; Mills, J. P.; Micoulet, A.; Dao, M.; Lim, C. T.; Beil, M.; Seufferlein, T. Acta Biomater. 2005, 1, 15. (58) Yoon, T.; Kim, J.; Kim, B.; Yu, K.; Cho, M.; Lee, J. Angew. Chem., Int. Ed. 2005, 44, 1068, 10.1002/anie.200461910.

NL802404H

4453