Visualizing Cell Extracellular Matrix (ECM) Deposited by Cells

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Visualizing Cell Extracellular Matrix (ECM) Deposited by Cells Cultured on Aligned Bacteriophage M13 Thin Films Laying Wu,†,‡ L. Andrew Lee,† Zhongwei Niu,§ Soumitra Ghoshroy,‡ and Qian Wang*,† †

Department of Chemistry and Biochemistry and Nanocenter, University of South Carolina, Columbia, South Carolina 29208, United States ‡ Electron Microscopy Center, University of South Carolina, Columbia, South Carolina 29208, United States § Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China ABSTRACT: Topographical features ranging from micro- to nanometers can affect cell orientation and migratory pathways, which are important factors in tissue engineering and tumor migration. In our previous study, a convective assembly of bacteriophage M13 resulted in thin films which could be used to control the alignment of cells. However, several questions regarding its underlying reasons to dictate cell alignment remained unanswered. Here, we further study the nanometer topographical features generated by the bacteriophage M13 crystalline film, which results in the alignment of the cells and extracellular matrix (ECM) proteins. Sequential imaging analyses at micro- and nanoscale levels of aligned cells and fibrillar matrix proteins were documented using scanning electron microscopy and immunofluorescence microscopy. As a result, we observed baby hamster kidney cells with higher degree of alignment on the ordered M13 substrates than NIH-3T3 fibroblasts, a difference which could be attributed to the intrinsic nature of the cells’ production of ECM proteins. The results from this study provide a crucial insight into the topographical features of a biological thin film, which can be utilized to control the orientation of cells and surrounding ECM proteins.

’ INTRODUCTION Virus and viral-like particles (VLPs) have been used as building blocks for a broad range of applications in chemistry and material science.1 4 The advantages of viral systems include simple production, monodisperse size and well-known structures, and the potential to be assembled into hierarchical structures.5 7 The advances in structural biology and molecular biology make it possible to display a variety of functional groups at high densities with submolecular precision via molecular cloning and protein bioconjugation strategies.8,9 Recently, an emerging interest in virus-related research is to employ plant viruses and bacteriophages as drug carriers or probes for cell imaging10 16 and as substrates for cell growth and tissue engineering applications.17 21 Bacteriophage M13 is a filamentous virus that measures 880 nm in length and 6.6 nm in diameter. A single-stranded DNA genome is encased by five different proteins (P8, P9, P7, P6, and P3). Each virus consists of ∼2700 identical copies of the major coat protein P8 and five copies each of four minor coat proteins (P9, P7, P6, and P3) which are located at the two apical ends. M13 phage can be functionalized by chemical modification as well as genetic engineering on its exterior surface to express peptides that can specifically bind to a target (termed as “phage display”).8,22 M13 has been applied as both structural component and carrier of functionalities for tissue engineering. For example, at high concentration M13 has the tendency to assembly and form various smectic liquid crystal phases,23,24 and therefore, a convective assembly can be used to generate a thin film of aligned r 2011 American Chemical Society

bacteriophage M13 that controls the directionality of cells.25 The thin film substrate has been shown to support different types of mammalian cell lines without any cytotoxic effect and guide cell spreading along the same direction of the virus film orientation.25 Using this method, Lee and co-workers have reported that genetically modified RGD-M13 can also form thin films, which provide directional guidance of neural cells and fibroblasts.18,19,26 These studies recapitulated our previous studies with chemically modified M13, which can also form thin films by convective assembly and guide the various mammalian cells.25 Furthermore, the use of bacteriophage in recent human clinical trials suggest that the phage dose not exhibit acute toxicity or immune response within the human host.27 On the basis of these studies, M13 bacteriophage can be envisioned as a powerful biological nanomaterial, which can be modified with biologically relevant functional groups at high ligand densities and assembled into thin films to guide cells and facilitate our attempts to control the ever-complicated cell behavior. Previous studies have demonstrated that cellular morphology and migration behaviors can be governed by physical cues provided by nanometer scale topographies.28 30 Sufficient ridge width and depth appear to be important factors for cell elongation along the nanopatterns, where a critical depth of 80 nm has been Received: April 28, 2011 Revised: June 8, 2011 Published: June 16, 2011 9490

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Langmuir observed for endothelial cell types.28 In our studies, the intrinsic ability of M13 phage to form well-organized thin films for controlling cell orientation and outgrowth provided a simple method of regulating cellular response including migration, proliferation, and differentiation. Therefore, in this paper, we examined the nanoscale topographical features of the virus film generated from the convective assembly process and visualized the extracellular matrices deposited by the cells cultured on this film. These results altogether provide insight into how the virus film can dictate the orientation of cell outgrowth.

’ MATERIALS AND METHODS Bacteriophage M13 Assembly and AFM Analysis. Bacteriophage M13 was harvested from infected E. coli according to previously reported method.31 M13 film was assembled by a convective method reported previously.25 In short, the film was generated by dragging 40 μL of virus solution (20 mg/mL in deionized nanopure water) along 3-aminopropyltriethoxysilane (3-APTES)-coated glass slides. The film was then freeze-dried. Before seeding cells, M13 thin films were crosslinked with 0.1% glutaraldehyde (Electron Microscopy Sciences) solution for 30 min and then reduced with 1% NaBH4 (Acros Organics) for 15 min. After multiple washing steps with phosphate buffered saline (PBS) and serum-free media, the substrates were used for cell seeding. The roughness and the three-dimensional structure of the film substrates were characterized by using a NanoScope IIIA MultiMode atomic force microscope (AFM, Veeco). A dried substrate was mounted on a tin plate and loaded onto the sample stage. The height profile images were obtained at ambient conditions in tapping mode. A silicon cantilever (spring constant 25 75 N/m and resonant frequency 200 400 kHz, NanoScience) was used for scanning. Cell Culture. NIH-3T3 fibroblasts were seeded on bacteriophage M13 substrates in a 12-well plate (Corning) at a density of 5  104 cells mL 1. Cells were cultured in high glucose Dulbecco’s modified eagle’s medium (DMEM) (HyClone) supplemented with 10% neonatal calf serum (NCS, HyClone), 4 mM L-glutamine, 1% penicillin, and streptomycin (HyClone) in a humidified incubator at 37 °C supplemented with 5% CO2. Cells were passaged at 80 90% confluency. Baby hamster kidney (BHK) cells were maintained in high glucose DMEM with 10% fetal bovine serum (FBS, Hyclone), 4 mM L-glutamine, 1% penicillin, and streptomycin and passaged every 2 3 days. Decellularization. Postconfluent cells were washed with PBS and then soaked in PBS containing 0.3% Triton X-100 and 5 mM NH4OH. Monitored by light microscopy, when cell removal was almost complete, the solution was removed using a pipet, leaving the remaining solution to decellularize the rest of cells for 1 2 min. And afterward, the cell debris was washed once with PBS containing 0.5% glutaraldehyde for 5 min and with PBS for three times immediately. Then the samples were fixed with 2.5% glutaraldehyde solution for 30 min. This procedure removed most or the entire cell layer and left much of the extracellular matrix (ECM) firmly attached to the substrates. Immunostaining. To ascertain that the cell body removal was complete, the decellularized substrates were stained with DAPI to monitor residual nucleic acids. For visualizing the ECM structures, the substrates were stained with anticollagen (Sigma) or antifibronectin (Santa Cruz Biotechnology) antibodies for indirect immunofluorescence imaging. Confluent samples of the cell growing on both coverslips and M13 patterned substrates were washed three times with PBS. Cells were then fixed with 2.5% GTA in PBS (Electron Microscopy Sciences) for 2 h. After three washes with PBS, the samples were permeabilized for 15 min in PBS with 0.1% Triton X-100 and then blocked by soaking cells in 1% bovine serum albumin (BSA)/PBS for 60 min. For decellularized samples, the substrates were fixed with 2.5% glutaraldehyde solution for 60 min, then washed three times, and blocked with 1% BSA/PBS

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containing 0.1% Triton X-100. Afterward, both intact cells and decellularized samples were treated with the same procedures. First, the samples were incubated for 60 min at ambient temperature with primary antibody of collagen type I or fibronectin in PBS with 1% BSA. Subsequently, the samples were washed three times and incubated with 5 μg/mL DAPI and secondary antibody conjugated with fluorescein isothiocyanate (FITC) for 60 min with gentle agitation in dark at room temperature. The samples were then washed with PBS, mounted on microscope slides, sealed with nail polish, and viewed under an Olympus IX81 fluorescence microscope. Scanning Electron Microscopy (SEM). For SEM, the cells were washed three times in PBS and fixed with cacodylate-buffered 2.5% glutaraldehyde for 2 h at room temperature. After washing three times with 0.1 M cacodylate buffer (pH 7.2), cells were postfixed with 1% OsO4 buffered with 0.2 M cacodylate for 60 min at 4 °C, and washed three times with 0.1 M cacodylate buffer. The cells were then dehydrated in a series of ethanol washes (30%, 50%, 70%, 80%, 95%, and 2  100%) for 10 min each. In order to avoid the distortions caused by changing surface tension, the specimens were processed though critical point drying apparatus (Ladd Research Industries, Inc.). A thin layer of gold (around 20 nm) was sputter-coated onto the samples, after which the samples were viewed with FEI Quanta 200 ESEM.

’ RESULTS AND DISCUSSION M13 Assembly and Substrate Stabilization. The thin films were fabricated from a solution of bacteriophage M13 by convective assembly at room temperature in a humidity-controlled chamber and of M13 aligned parallel to the direction of assembly. One of the major issues for these fabricated films had been that these virus films would dissolve over time when used for cell cultures. This issue was resolved by lyophilization and mild cross-linking with glutaraldehyde. The virus films, once freeze-dried, remained stable over much longer period of time in cell culture media (days to weeks), whereas the untreated films would slowly dissolve into the medium within 24 h and some portions of the film would begin to peel away from the underlying silane-coated glass substrate. The reason would be that the removal of water in the process of lyophilization allows more crystalline M13 viruses interaction with the positive charged silane surface. The substrate was further treated with glutaraldehyde solution to stabilize the viral film to prevent the film from peeling off during cell culturing. Glutaraldehyde has been used broadly as an effective cross-linking agent for proteins and cells. At either acidic or basic condition, glutaraldehyde can react with multiple amino acid residues, among which the primary amino group of lysyl residues is the most reactive one.32,33 In general, the degree of cross-linking varies in pH, temperature, time, and glutaraldehyde concentration.34,35 In our experiments M13 film can be effectively cross-linked upon treating with a 0.1% (w/w) solution of glutaraldehyde for 30 min. To reduce the reversible Schiff’s bases and the cytotoxicity of the leached glutaraldehyde,36,37 the crosslinked M13 film was soaked in NaBH4 aqueous solution (1% w/w) for 15 min. As observed under the optical microscope (data not shown), the cross-linking of viral after the lyotropic liquid crystal transition upon lyophilization exhibited clearer pattern than untreated viral film. AFM Analysis of Virus Film. Prior to seeding the M13-coated substrates with cells, the cross-linked and freeze-dried virus films were analyzed by AFM. The convective assembly had generated a patterned surface that was solely attributed to the M13 bacteriophage assemblies. To determine the influence of nanopattern dimensions on cells adhesion and spreading, several 20  20 μm areas were scanned for each substrate, and multiple M13 thin 9491

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Figure 1. (a) AFM height image of a smooth glass surface. (b) AFM height image of the aligned crystal M13 film sheet with alternate ridges and grooves. (c) 3D topographical image of the M13 film. (d) Representative section analysis to show the surface roughness of the virus film along the X direction in image c, distance between ridge positions 1 and 2, and a relative height value of position 3 in reference to position 4. (e) Representative plot of the height distribution of virus films. Along the X direction (the direction of viruses alignment), the height ranges from 40 to 80 nm (orange). Along the Y direction, it ranges from 60 to 110 nm (black). The reference point is the lowest position of the entire film. Scale bars for AFM images are 5 μm.

films were analyzed to profile the topographical features of the film. In Figure 1a, the bare glass substrates had an average height of 15 nm. In contrast, the M13 thin film exhibited topography with patterned ridges and grooves (Figure 1b). The anisotropic surface formed by the M13 bacteriophage could be better observed when the height image was converted to a three-dimensional (3D) image (Figure 1c). The ridges and grooves alternate with prominent width and height differences when measured the same direction of the virus film alignment (X direction, Figure 1c), and these differences are far more pronounced in the Y-axis, which is perpendicular to the alignment of the film. Figure 1d displays a section analysis represents a roughness curve along the white line in the Y direction. The distance between ridges (1 and 2) or grooves was measured. The average width is several hundred nanometers (∼600 nm) in the X direction but more than 1 μm (∼1.32 μm) in the Y direction (data not shown). Likewise, a relative height of position 3 was also measured in reference to the position 4 (pointed by the red arrow). Several of these cross sectional lines were drawn across the entire film to determine the height distribution profile, which was plotted as a black curve in Figure 1e. Similarly, a graph of the height distribution along the X direction was also drawn (the orange line). The reference point is the lowest position of the whole M13 film. Clearly, the average surface roughness of the film ranges in 40 80 nm in the direction of M13 assembly. Nevertheless, when measuring the height perpendicular to the viral assembly direction, the majority of the film is filled with alternate ridges and grooves at a height range of 60 110 nm. This surface feature is significant to provide contact cues for controlling cell directionality. As reported by the Gensen group, a higher depth of ridge/groove and a larger groove width have profound effects on osteoblast cells alignment.28 In

the case of the convectively assembled M13 film, the surface exhibits a larger groove/ridge depth and a broader groove/ridge distance in the Y than those in X direction, which provides potential guidance on cells aligned along the X direction—the viral assembly direction. Cell Alignment on Virus Film. After profiling the topographical feature of M13 substrates, these patterned M13 film serving as scaffolds were incubated with cells. Similar to our previous report,25 when 3T3 and BHK cells were cultured for 24 h on the aligned M13 films, the majority of the cells displayed an elongated, spindle-like morphologies. The cells were aligned parallel to the convective assembly direction of M13 viruses rather than traversing the ridge and groove, as seen in Figure 2b,e. In contrast, cells grown on coverslips were randomized with stellate morphologies (Figure 2a,d). This tendency of the cell orientation suggests that the presence of contact cues between cells and the patterned M13 scaffolds plays an important role in cell morphology and spreading. Such observations have also been documented for cells cultured on nanopatterned substrates with varying heights and ridge widths.38 41 When the cells were incubated for longer time period (72 h), 3T3 fibroblasts appeared less elongated and lost their overall orientation (Figure 2c). More cells began to exhibit stellate morphologies rather than a spindle-like morphology. On the contrary, BHK cells remained to their original orientation and appeared to be more significantly elongated as seen in Figure 2f. The cell alignment was measured using previously reported method to compare elongation factor and degree of orientation for the two cell lines.42 As shown in Figure 2g, the degree of orientation refers to the angle (θ) between cell long axis of the cell body (X-axis) and the general direction of the virus film. An angle of 0° refers to perfect alignment of cells along the 9492

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Figure 2. Orientation angle and elongation factor of mammalian cells on patterned M13 films. (a f) Optical image of NIH-3T3 cells (incubated for 24 h) on coverslip (a) and on M13 films for 24 h (b) and for 72 h (c). Optical image of BHK cells grown on coverslip for 24 h (d) and on M13 films for 24 h (e) and for 72 h (f). (g) Schematic illustration of orientation angle θ and elongation factor X/Y. (h) Summary of the elongation factors and orientation angles of 3T3 and BHK cells incubated on the M13 film, with mean ( SD from three separate experiments performed in duplicates. Orientation of (i) NIH-3T3 cells and (j) BHK cells on M13 films when cultured for 24 and 72 h, respectively (0° degree represents perfect alignment and >45° represents random orientation). Scale bars are 20 μm for (a f).

direction of the virus film and 45° would note random orientation. The ratio of X/Y represents the elongation factor, where the higher values indicate longer and thinner morphologies, which are typically observed with spindle-shaped cells that align along a surface topography. Based on these conditions, the elongation factor and orientation angle of 3T3 and BHK cells were calculated (Figure 2h). As the incubation time increased from 24 to 72 h, the average elongation factor for 3T3 fibroblasts decreased from 3.20 to 2.42, and the average orientation angle increased from 11.2° to 25°. These changes indicate that 3T3 fibroblasts deviate from their original uniform directionality, which would be dictated by the M13 phage film. The cells over time orient in a random tendency as growth is prolonged on the M13 substrates. This shift in orientation can be observed by light microscope (Figure 2b,c), and this observation is represented as a graph (Figure 2i). Similar elongation and orientation measurements with BHK cells were

assessed (Figure 2h). In comparison to 3T3 fibroblasts, BHK cells exhibited smaller orientation angles and a higher elongation factor, which corroborates the images obtained with a light microscope (Figure 2e,f). The average elongation factor of BHK increased from 6.82 to 8.79, and the average orientation angle decreased from 8.86° to 5.63° as cell incubation was increased from 24 to 72 h. The majority of BHK cells remain significantly aligned along on the M13 substrate as the incubation time increased (Figure 2j). These observations suggest that in the presence of the patterned M13 film both 3T3 and BHK cells were elongated and aligned along with the virus assembly direction at an early time point (24 h); however, with cell incubation prolonged to 72 h, BHK cells showed a more uniform directionality than 3T3 cells. ∼90% of the BHK cells show an orientation angle within (10°. ECM Structure. ECM fiber architecture has been reported to determine the directionality of breast carcinoma cell migration.43 9493

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Langmuir The parallel ECM fiber arrangement promotes the carcinoma cells migrating with directionality. With the knowledge of cell ECM substrate interactions, to account for the above observation on cell behaviors, we propose that the physical cue from the nanopatterned M13 topographical surface can guide cell ECM architectures, which in turn mediates cell spreading. In the early stage, proteins such as fibronectin, vitronectin, and collagen-like protein from serum can adsorb on the substrate to facilitate cell substrate interactions and promote cell adhesion.44,45 In this stage cells accommodate themselves to adhere to and spread on the substrate (with possible adsorption of ECM proteins from media) and barely produced their own ECM. Influenced by physically topographical feature of the M13 film, cells show directionality during the initial spreading. As cell incubation extends, they start to secret ECM. Different types of cells produce different amount of ECM even at the similar environmental condition. At this stage, the cytoskeleton extension and migration of cells could be regulated by both the Scheme 1. Schematic Illustration of M13 Substrates Preparation and Cell Secreted ECM Productiona

A thin film of bacteriophage M13 is aligned on the surface of a glass slide. The substrate, once dried and stabilized, is seeded with cells. After cells are removed, the remaining ECM structures are analyzed. a

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topography of the substrate as well as the microenvironment formed by the ECM proteins secreted by cells.46 The 3T3 fibroblast cells can produce more ECM proteins; therefore, their morphology will be significantly affected by their secreted ECM, revealing more tendencies to deviate from the original orientation and elongated cell morphologies. On the contrary, the BHK cells generally secrete less ECM proteins,47 resulting in more tendency to response of directionality of the original nanopatterned M13 film. To confirm aforementioned explanation for the cell alignment and elongation on patterned M13 substrates, we tried to decellularize cell bodies and uncover the underneath ECM structure. Scheme 1 illustrates the general procedures including the preparation of the virus film and the additional steps for analyzing the ECM proteins embedded on top of the virus film. The first step involved the preparation of bacteriophage films on microscope slides. Cells were seeded on the M13 thin film and cultured to confluency, and afterward these cells were removed with minimal damage to the surrounding extracellular matrices. In a typical experiment, the denuded cell ECM structure was achieved by removing postconfluent cell bodies from their ECM matrix using a solution of Triton X-100 and ammonium hydroxide salt. In this solution, Triton X-100 was used to disrupt lipid lipid and lipid protein interactions while leaving protein protein interactions intact; ammonium hydroxide salt was used to disrupt nucleic acids.48 Figure 3 illustrates cell secreted ECM structures by decellularizing cells growing both on coverslips and on the patterned M13 substrates. To ensure the ECM was secreted from cells themselves instead of the growth media, some controlled experiments were carried out by soaking the M13 substrates in 10% serum-supplemented DMEM for 4 days. The film was still stable after the four-day incubation (Figure 3a), and fibronectin were observed to adsorb on the M13 film with a pale green color under immunofluorescence microscopy (Figure 3e). Apparently, the absorbed fibronectin was from the serum which contains a

Figure 3. Denuded ECM structure from the four-day cultured mammalian cells on M13 films and coverslips under fluorescence microscope. SEM image of (a) M13 film after soaking in media for 4 days and without cell seeding, (b) ECM structure after removal of 3T3 cells cultured on a coverslip, (c) ECM structure after removal of 3T3 cell cultured on M13 film, and (d) ECM after removal of BHK cells on M13 film. (e h) Immunofluorescence images of fibronectin (Fn): (e) serum adsorbed on M13 film prior to seeding with cells, (f) ECM after removal of 3T3 cells on a coverslip, (g) ECM from 3T3 fibroblasts on the M13 film, and (h) ECM from BHK cells on M13 film. (i l) Immunofluorescence images of collagen type 1 (Col) of (i) serum on M13 which was soaked in media for 4 days, (j) ECM of 3T3 fibroblasts on a coverslip, (k) ECM of 3T3 on M13 film, and (l) ECM of BHK cells on M13 film (orientation of film indicated by white arrow). Scale bars are 5 μm for (a d) SEM images and 100 μm for (e l). 9494

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Langmuir limited amount of fibronectin (∼25 μg/mL).49 However, no collagen (type I, Col) could be detected on the M13 film (Figure 3i). On the basis of these controls, we characterize the decellularized ECM structures of 3T3 and BHK cells growing on coverslips and the M13 films, respectively. When cultured on bared coverslips, ECM structures secreted by 3T3 cells are randomly organized (Figure 3b). However, when cultured on patterned M13 films, being regulated by the topological cue, the ECM proteins secreted by 3T3 and BHK cells both exhibit wellaligned features under SEM (Figure 3c,d). These results were consistent with fluorescent images of fibronectin and collagen that were immuno-labeled by FITC-conjugated antimouse 2° antibody (Figure 3f h,j l). Additionally, much more fibronectin (Figure 3f, g) and collagen (Figure 3j,k) secreted from 3T3 cells can be observed than from BHK cells (Figure 3h,l). This result implies that the better directionality of BHK cells along the M13 substrate, as opposed to 3T3 cells, results from the poorer capability in ECM secretion of BHK cells.

’ CONCLUSIONS In this study, we have analyzed the topographical features of M13 films. In the direction of M13 assembly, the depth of ridge/ groove of the film surface is around 40 80 nm, while perpendicular to the virus assembly direction, the average height ranges is much larger, i.e., from 60 to 110 nm. This surface feature provides contact cues to promote cells to spread in nice alignment with the viral assembly direction. To calculate cells orientation, a statistical analysis was carried out by measuring the cell elongation factor and the orientation angle. As a result, the majority of BHK cells show a narrower orientation angle (less than (10°) than 3T3 upon long-term cell incubation, implying a better response to the surface topography. The studies on cell ECM imaging by SEM and immunofluorescent microscopy help us understand cell substrate interactions. Directed by physical cues, cells accommodate themselves in an alignment with M13 assembly and secrete oriented fibronectin and collagen I on the M13 film. During this process, the high amount of ECM proteins produced by 3T3 cells causes the deviation of the cells from their original directionality as cell incubated for a longer time. On the other hand, BHK cells, which secrete much less ECM proteins than 3T3 cells, maintain very good directionality along with the M13 film. Our detailed analysis of ECM structure will open the way to understand the interaction of cell and substrate. ’ ACKNOWLEDGMENT This work was partially supported by the National Science Foundation (CHE-0748690 and DMR-0706431), the Alfred P. Sloan Scholarship, the Camille Dreyfus Teacher Scholar Award, DoD-W911NF-09-1-0236, and the W. M. Keck Foundation. ’ REFERENCES (1) Dujardin, E.; Peet, C.; Stubbs, G.; Culver, J. N.; Mann, S. Nano Lett. 2003, 3, 413–417. (2) Lee, L. A.; Wang, Q. Nanomedicine 2006, 2, 137–149. (3) Young, M.; Willits, D.; Uchida, M.; Douglas, T. Annu. Rev. Phytopathol. 2008, 46, 361–384. (4) Lewis, J. D.; Destito, G.; Zijlstra, A.; Gonzalez, M. J.; Quigley, J. P.; Manchester, M.; Stuhlmann, H. Nature Med. 2006, 12, 354–360. (5) Lin, Y.; Su, Z.; Balizan, E.; Niu, Z.; Wang, Q. Langmuir 2010, 26, 12803–12809.

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dx.doi.org/10.1021/la201580v |Langmuir 2011, 27, 9490–9496