Article pubs.acs.org/Langmuir
Effectiveness of Nanometer-Sized Extracellular Matrix Layer-by-Layer Assembled Films for a Cell Membrane Coating Protecting Cells from Physical Stress Atsushi Matsuzawa,†,‡ Michiya Matsusaki,† and Mitsuru Akashi*,† †
Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Imaging Media Division, Kyoto R&D Laboratory, Mitsubishi Paper Mills Limited, 1-6-6 Kaiden, Nagaokakyoshi, Kyoto 617-8666, Japan
‡
ABSTRACT: In recent approaches to tissue engineering, cells face various stresses from physical, chemical, and environmental stimuli. For example, coating cell membranes with nanofilms using layer-by-layer (LbL) assembly requires many cycles of centrifugation, causing physical (gravity) stress. Damage to cell membranes can cause the leakage of cytosol molecules or sometimes cell death. Accordingly, we evaluated the effectiveness of LbL films prepared on cell membranes in protecting cells from physical stresses. After two steps of LbL assembly using TrisHCl buffer solution without polymers or proteins (four centrifugation cycles including washing), hepatocyte carcinoma (HepG2) cells showed extremely high cell death and the viability was ca. 15%. Their viability ultimately decreased to 6% after 9 steps of LbL assembly (18 cycles of centrifugation), which is the typical number of steps involved in preparing LbL nanofilms. However, significantly higher viability (>85%) of HepG2 cells was obtained after nine steps of LbL assembly employing fibronectin (FN)-gelatin (G) or type IV collagen (Col IV)-laminin (LN) solution combinations, which are typical components of an extracellular matrix (ECM), to fabricate 10-nm-thick LbL films. When LbL films of synthetic polymers created via electrostatic interactions were employed instead of the ECM films described above, the viability of the HepG2 cells after the same nine steps slightly decreased to 61%. The protective effects of LbL films were strongly dependent on their thickness, and the critical thickness was >5 nm. Surprisingly, a high viability of over 85% was achieved even under extreme physical stress conditions (10 000 rpm). We evaluated the leakage of lactate dehydrogenase (LDH) during the LbL assembly processes to clarify the protective effect, and a reduction in LDH leakage was clearly observed when using FN-G nanofilms. Moreover, the LbL films do not inhibit cell growth during cell culturing, suggesting that these coated cells can be useful for other experiments. LbL nanofilm coatings, especially ECM nanofilm coatings, will be important techniques for protecting cell membranes from physical stress during tissue engineering.
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(FN-G) films as a nanoextracellular matrix (nano-ECM) on the cell surfaces.4,6 Because FN is one of the most important celladhesive ECMs,14 nanometer-sized FN-G thin films can promote cell−cell interactions without cytotoxicity.15,16 For example, we have reported the long-term stability and viability of 3D fibroblast tissue,17 3D blood vessel wall models,18−20 and 3D- tumor models containing stromal cells.21 Moreover, approximately 100-μm-thick (∼20 L) microtissues containing endothelial tube networks are obtained using our recently published cell-accumulation technique, which can induce immediate 3D cell−cell interactions and create thick multilayers during just 1 day of incubation.5 Because the coating of LbLassembled nanofilms on the cell membranes requires many
INTRODUCTION Various tissue engineering approaches have been discovered, such as 3D culturing inside biodegradable hydrogels,1,2 a layerby-layer (LbL) assembly technique,3−6 a microfluidic system,7 a cell sheet technique,8 and inkjet printing of cells and polymers.9,10 However, these advances in tissue engineering have created various stresses for cells from physical, chemical, and environmental stimuli. For example, Boland et al. reported the formation of nanometer-sized pores on cell membranes due to shear stress during the inkjet printing process.11 Accordingly, we have to consider cell viability and function carefully during tissue engineering. The LbL assembly of polymers or proteins onto cell membranes has attracted much attention because of the easy functionalization of cell surfaces.3,12,13 We recently discovered a simple and unique bottom-up approach, termed hierarchical cell manipulation, to develop 3D cellular multilayers with the desired layer number and location by the fabrication of nanometer-sized, LbL-assembled fibronectin (FN)-gelatin (G) © 2012 American Chemical Society
Special Issue: Interfacial Nanoarchitectonics Received: August 28, 2012 Revised: October 22, 2012 Published: October 23, 2012 7362
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rinsing with pure water and drying with N2 gas in order to clean the surface. The cleaned QCM chip was immersed in a 50 mM Tris-HCl buffer solution (pH 7.4) containing another protein or polymer. Deposition was repeated 10 times. A QCM can be used to monitor the amount of assembly deposition (Δm) based on its frequency shift (ΔF). This relationship for a 9 MHz QCM with an electrode diameter of 4.5 mm can be expressed as follows according to Sauerbrey’s equation23 when ΔF is measured in air: −ΔF/Hz = 1.15Δm/ng. LbL Assembly on Cell Membranes and Cell Viability after LbL Assembly. LbL assembly on cell membranes was performed on the basis of our previous report.5 Briefly, 1 × 107 cells mL−1 of HepG2 or other cells collected by centrifugation after trypsinization were alternately incubated with 0.2 mg mL−1 FN and G in 50 mM Tris-HCl (pH 7.4) for 1 min at 37 °C. After each procedure, the cells were washed with 50 mM Tris-HCl (pH 7.4) using centrifugation at 2500 rpm (419g) for 1 min in order to remove unabsorbed polymers. After nine steps of immersion, FN-G multilayer nanofilms were coated onto single cell surfaces. The other kinds of LbL films were also prepared in the same way. The viability of HepG2 after LbL assembly was measured by trypan blue staining. Lactate Dehydrogenase (LDH) Assay. To evaluate the leakage of cytosol proteins during the centrifugation steps of LbL assembly, the amount of released LDH was detected using an LDH cytotoxicity assay kit (Cayman Chemical Company, MI). Briefly, 100 μL of supernatant was collected after each centrifugation cycle in the LbL assembly process, and the amount of LDH that was released in each step was assayed. To determine the amount of LDH released, the production of formazan was measured spectrophotometrically at 490 nm according to the manufacturer’s protocol. Formazan production is proportional to the amount of LDH released. Proliferation of HepG2 Cells Coated with Nanofilms. The effect of LbL nanofilms prepared on cell membranes with respect to proliferation and morphology was evaluated for 8 days of culturing in DMEM containing 10% FBS. HepG2 cells (1 × 105) coated with nanofilms were seeded in 6-well cell culture plates. Cell numbers were measured using a cytometer after trypsinization. The cell morphology on the culture plate was observed using a phase-contrast microscope.
cycles of centrifugation (physical stress), the physical stress may damage the cell membranes, causing the leakage of cytosol molecules or ultimately cell death. However, there are no reports on physical stress during LbL assembly processes. In this article, we investigated for the first time the effect of LbL assembly centrifugation processes (gravity stress) on cell viability and the leakage of cytosol enzymes. After 18 cycles of centrifugation without polymers or proteins (only buffer), which represents the number of centrifugation cycles needed for nine steps of LbL assembly (including rinsing steps), over 90% cell death of human hepatocyte carcinoma (HepG2) cells was clearly observed. However, a viability of >85% was obtained even after the same 18 cycles of centrifugation related to the 9-step LbL assembly of FN-G-coated cells (in which the coating thickness was approximately 7 nm). The protective effect of LbL films was strongly dependent on their composition and thickness. Moreover, the leakage of cytosol enzyme lactate dehydrogenase (LDH) during the centrifugation cycles was clearly decreased by the fabrication of the FN-G nanofilms. Because there was no inhibition of cell growth profiles, the FN-G nanofilm coating can be useful not only for preventing physical stress but also for other experiments. The results of this study demonstrate the novel possibility of using LbL-assembled film coatings on cell membranes to protect against physical stress.
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EXPERIMENTAL SECTION
Materials. Fibronectin (FN) from bovine plasma, gelatin (G), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), sulfuric acid (H2SO4), anhydrous calcium chloride (CaCl2), and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Type IV collagen (Col IV) from human placenta, poly(styrene sulfonate sodium salt) (PSS, Mw = 2.0 × 105), and Roswell park memorial institute (RPMI)-1640 medium were obtained from Sigma-Aldrich (St. Louis, MO). Natural mouse laminin (LN) was purchased from Life Technologies (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from Biowest (Miami, FL). Poly(diallyldimethylammonium chloride) (PDDA, Mw = 2.4 × 105) was purchased from Polyscience (Warrington, PA). All chemicals were used without further purification. Cell Culture. Human hepatocyte carcinoma cells (HepG2, Hep3B, PLC/PRF/5, and Huh-7), normal human dermal fibroblasts (NHDF), mouse 10T1/2 cells (10T1/2), and one type of human pancreatic cancer cells (MIA PaCa-2) were cultured in DMEM containing 10% FBS and GA-1000. Human pancreatic cancer cells (BxPC-3) and human colon cancer cells (HT-29) were cultured in RPMI-1640 medium containing 10% FBS and GA-1000. Human umbilical vein endothelial cells (HUVEC) were cultured in endothelial basal medium-2 (EBM-2) containing hFGF-B, vascular endothelial growth factor (VEGF), R3-IGF-1 (IGF = insulin-like growth factor 1), ascorbic acid, FBS, human epidermal growth factor (hEGF), and GA-1000. Keratinocytes (KC) were cultured in MCDB153 medium as described previously.22 All cell cultures were maintained in 5% CO2 at 37 °C. Physical Stress Study during the LbL Centrifugation Process. HepG2 cells (1 × 107 cells mL−1) and other cells in 50 mM Tris-HCl (pH 7.4) were prepared after trypsinization. Then, physical stress was applied repetitively by centrifugation at 2500 rpm (419g) for 1 min using a MiniSpin plus centrifuge (Eppendorf, Germany). At each step, 50 mM Tris-HCl (pH 7.4) buffer was replaced. Cell viability was measured by trypan blue staining. Quantitative Analysis of LbL Assembly. The quantitative analysis of LbL assembly using a quartz crystal microbalance (QCM) was performed essentially as reported in our previous studies.4,15 An AT-cut quartz crystal (9 mm diameter) with a parent frequency of 9 MHz and a frequency counter (model 53131 A) were purchased from USI (Fukuoka, Japan). Before the assembly measurements, the QCM electrodes were treated three times with a piranha solution (H2SO4/ 40% H2O2 aqueous solution = 3:1 by volume) for 1 min, followed by
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RESULTS AND DISCUSSION HepG2 is one of the candidate cell lines for the study of liver tissue engineering because of its reproducible, inexpensive, and polarized properties, but it displays much lower liver-specific functions at abnormal levels.24 The 2D or 3D cocultures using tissue engineering approaches have been widely employed to enhance liver function.3,25 Thus, we focused on HepG2 cells to understand the effect of LbL assembly as one of the tissue engineering approaches. Figure 1 shows the viability changes in
Figure 1. Cell viability of HepG2 with or without FN-G films for ninestep LbL assembly. Cell viability of HepG2 (○) with or (Δ) without FN-G films at each centrifugation was plotted (n = 3). Cell viability was measured by trypan blue staining and was calculated by the ratio of the number of living cells to the total number of cells. Error bars denote standard deviations. 7363
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Figure 2. Effect of multiple types of LbL films. Frequency shift of the QCM for the stepwise assembly of (a) FN(○)/G(●), (b) Col IV(○)/LN(●), and (c) PDDA(○)/PSS(●) on a gold-based surface at 37 °C in 50 mM Tris buffer (pH 7.4) (n = 3). (d) Viability and (e) total yield of cells with FN-G, Col IV-LN, and PDDA-PSS nanofilms by nine-step LbL assembly or uncoated cells that underwent the same centrifugation regime (n = 3). In part e, the total cell yield indicates the ratio of the number of remaining to initial cells. White and black columns indicate the proportion of living cells and dead cells, respectively, and the values shown on the top of the column indicate the cell viability after nine-step LbL assembly. The asterisks (*) denote a statistically significant difference between the samples or versus all samples calculated by a two-sample t test (p < 0.01). N.S. means no significant difference. Error bars denote standard deviations.
To evaluate the protective contribution of the nanofilm component, type IV collagen (Col IV)-laminin (LN) nanofilms as another nano-ECM film (basement membrane component) and poly(diallyldimethylammonium) chloride (PDDA)-poly(styrene sulfonate sodium salt) (PSS) as a typical LbL nanofilm of synthetic polymers were employed. The synthetic PDDAPSS films are polyelectrolyte LbL films, and their driving force is electrostatic interaction. However, the driving force of the Col IV-LN nanofilms is biological recognition, the same as for the FN-G nanofilms.26 The estimated thicknesses of natural Col IV-LN and synthetic PDDA-PSS films after nine-step LbL assembly calculated from the frequency shifts of QCM were 9.4 ± 0.8 and 14.0 ± 0.7 nm, respectively (Figure 2b,c). They were in the range of thickness of natural FN-G films (approximately 10−20 nm). When HepG2 cells were coated with natural Col IV-LN nanofilms, the viability of the HepG2 cells was 89%, similar to that achieved with natural FN-G nanofilms (Figure 2d). Meanwhile, the HepG2 viability after coating with synthetic PDDA-PSS films decreased to about 60% even with the thickest films. The difference is likely related to cationic polymers. We recently reported the cytotoxicity of polyelectrolyte LbL
HepG2 cells with or without coating FN or G in Tris-HCl buffer (pH 7.4) during nine-step LbL assembly including the washing step. Nine steps of LbL assembly include a total of 18 cycles of centrifugation, each at 2500 rpm (419g). For LbL assembly, an odd number means centrifugation after incubation in FN or G solution, and an even number means the washing step. The viability of HepG2 cells without LbL-assembled nanofilms drastically decreased to less than half even after the second centrifugation and finally declined to 6% after 18 cycles of centrifugation. However, when FN and G solutions were employed to fabricate ECM nanofilms on cell membranes, HepG2 cells maintained a viability of >86% even after the final centrifugation. The HepG2 cells should be influenced by the cumulative gravity (physical stress) during centrifugation. In particular, cell membrane structures were probably deformed. The thickness of the nine-step LbL assembly of FN-G nanofilms was investigated by the frequency shift of the quartz crystal microbalance (QCM), and the estimated thickness was 6.9 ± 0.2 nm (Figure 2a). Interestingly, FN-G films prepared on micrometer-sized cell surfaces as thin as 10 nm had a sufficient protective effect against harsh physical stress. 7364
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films prepared on cell surfaces5,15 due to the unspecific adsorption of cationic polymers to anionic cell membranes. However, nano-ECM films were prepared by biological recognition through anionic proteins only.6,20 Accordingly, the synthetic PDDA-PSS films achieved lower viability compared to that of the nano-ECM films probably because of the unspecific adsorption of cationic PDDA on the surfaces of HepG2 cells. However, because the synthetic PDDA-PSS nanofilms also showed a certain protective effect compared to that of the uncoated cells, it is clear that coating cell membranes with LbL nanofilms is essential for creating a protection from physical stress from centrifugation. Additionally, biocompatible LbL nanofilms such as nano-ECM films should have better viability outcomes against physical stress. The total yield of HepG2 cells after 18 cycles of centrifugation was high at approximately 70− 90% in all cases, even for uncoated cells (Figure 2e). To evaluate the protective effect of the FN-G nanofilms against physical stresses in more detail, the rotational speed was increased from 2500 to 10 000 rpm for 1 min for the nine-step LbL assembly with or without FN or G in Tris-HCl buffer (Figure 3a). In the case of uncoated cells, obvious cell death was observed at 2500 rpm as described before, and low viabilities (less than 10%) were also observed at higher rotational speeds. In contrast, a high viability of >85% was achieved even under extreme physical stress conditions (10 000 rpm). We were surprised by this result because 10 000 rpm is 10-fold higher than the typical speed for collecting cells (1000 rpm). Moreover, because 18 cycles of centrifugation were carried out at the same speed, the cumulative physical stress would theoretically be 180-fold higher. To understand the main factor responsible for this interesting protective effect, the nanofilm thickness was varied from 0 to 7 nm. The mean thickness of the FN-G nanofilms was estimated under dry conditions by the Sauerbrey equation,23 and thus these thicknesses were presumed values. To create identical physical stress conditions, LbL assemblies with varying nanofilm thicknesses were subjected to additional centrifugation using a washing buffer until 18 centrifugations were performed in all cases. Interestingly, high viability was clearly observed when the thickness was >5 nm (Figure 3b). Our previous report on the tissue engineering suggested that FN-G films needed to be at least 6 nm thick to induce stable cell adhesion for a second layer to develop 3D cellular multilayers.4 Although the exact reason is unclear, this phenomenon may be related to our previous finding. A certain thickness might be necessary to initiate the protective effects of FN-G nanofilms prepared on the cell surface. To understand the protective effects of the nanofilms better, we evaluated the viability of HepG2 cells before and after additional centrifugation (Figure 3c). In the case of uncoated cells, the cell viability decreased to less than 20% after four cycles of centrifugation as described before. However, the nanofilm coatings on the HepG2 cells induced high resistance to physical stress, and it took 10 cycles of centrifugation to decrease the cell viability to 20%, even though the cells had already undergone 18 cycles of centrifugation to create the 7 nm films on the cell membrane. These results clearly suggest the strong protective property of FN-G nanofilms against physical stress through centrifugation. To understand how physical stress induces cellular damage, we hypothesized that the main pathway responsible for damage correlates with the leakage of cytosol molecules or ions outside the cell. We developed this hypothesis because some previous
Figure 3. Effects of FN-G nanofilms on cell viability. (a) The viability of HepG2 with FN-G nanofilms after nine-step LbL assembly and uncoated cells that underwent the same centrifugation regime was measured at different rotational speeds, as described in the Experimental Section (n = 3). (b) The relationship between cell viability and FN-G nanofilm thickness (n = 3). The nanofilm thickness was calculated on the basis of the frequency shift of the QCM during stepwise assembly. To apply the same amount of stress in all cases, additional cycles of centrifugation were administered so that all cells received a total of 18 cycles. (c) Physical stress study of uncoated HepG2 and HepG2 with nanofilms by nine-step LbL assembly. The viability of uncoated cells after trypsinization and FN-G coated cells after nine-step LbL assembly was determined under physical stress conditions created by centrifugation.
reports demonstrated pore formation on the cell membrane after shear stress.11 To detect the leakage of a cytosol molecule, a lactate dehydrogenase (LDH) assay was performed. Because LDH is one of the major proteins located in cytosol and LDH release is known to be a good indicator of cellular damage during physical stresses such as shear stress or sonication,27 LDH activity is widely employed to check cell/tissue damage. The leakage of LDH is estimated on the basis of an LDH activity assay after the centrifugation process. If the supernatant created by centrifugation shows high LDH activity, then LDH is being released from the cytosol. Figure 4a shows a calibration curve for LDH activity, and Figure 4b shows the LDH activity for each LbL assembly step. The HepG2 cells without FN or G in Tris-HCl buffer showed a linear increase in LDH activity depending on the number of LbL steps; however, cells with FN or G in the buffer continued to show low activity even after 5 steps of LbL assembly (a total of 10 cycles of centrifugation). 7365
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Figure 6. Schematic illustration of the effect of physical stress on (a) uncoated cells or (b) cells with films by LbL assembly.
as an example of cytosol molecules and cell damage, and thus the leakage of cytosol proteins might be one of the reasons for cell damage by physical stress. Although the protective property of the nano-ECM films prepared on cell membranes was clarified, the biocompatibility of the nano-ECM films, including the Col IV-LN films, was not evaluated during the cell culture period. We previously reported the biocompatibility of FN-G nanofilms on L929 mouse fibroblast cells15 and on normal human dermal fibroblast cells.5 However, we have not yet evaluated their biocompatibility with hepatocyte cells. In particular, Col IV-LN nanofilms appear to be novel LbL films, and their biocompatibility should be carefully evaluated. FN-G or Col IV-LN films that were 7 or 9 nm thick were prepared on the surfaces of HepG2 cells, and the growth curve of the coated cells was compared against that of uncoated cells as a control (Figure 5a). The coated cells
Figure 4. (a) Standard curve for the LDH test. The standard curve is prepared in each experiment. (b) LDH activity of uncoated cells or cells with FN-G and Col IV-LN nanofilms (n = 3). The LDH activity of the supernatant was measured after each centrifugation cycle. Error bars denote standard deviations.
Although the other nano-ECM films, Col IV and LN, also showed lower LDH activity than did uncoated cells, the activity level was slightly higher than for the FN-G nanofilms, suggesting a small release of LDH from the cytosol. These results clearly showed the correlation between the loss of LDH
Figure 5. (a) Proliferation profile of uncoated HepG2 or HepG2 with FN-G and Col IV-LN nanofilms prepared on the cell surface by nine-step LbL assembly (n = 3). (b−d) Phase-contrast images of HepG2 with (b) FN-G and (c) Col IV-LN nanofilms or (d) uncoated cells after incubation for 8 days. Uncoated cells were cultured under standard conditions without any treatment. The scale bars are 100 μm. 7366
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recently reported improvements in the insertion efficiency of a nanoneedle into cells by preparing nano-ECM films on the surface of the cells.29 This improvement might be related to changes in the mechanical properties of the cell membrane. Because of these physicochemical protective properties, the coated cells would show high viability against the physical stress induced by centrifugation. To understand the universality of this protective effect for other kinds of cells, we evaluated the physical stress damage to normal cells (Figure 7a,b) and other types of hepatocyte carcinoma cells (Figure 7c,d). Interestingly, human keratinocyte (KC) and Huh-7 cells behaved like HepG2 cells during centrifugation without LbL components, and their viabilities dropped to 77 and 41%, respectively; other cells did not exhibit cell death. This result suggests that the toughness of cells against physical stress depends on the cell type. Cytosol molecules might also be released from these tough cells even when cell death is not observed. When FN and G were employed in LbL films for KC and Huh-7 cells, their viability increased to 85 and 97%, respectively, after nine steps of the LbL process (data not shown). This result was identical to that observed for HepG2 cells, suggesting a universal protective effect of the LbL nanofilms, especially the nano-ECM films, against physical stress created by centrifugation.
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CONCLUSIONS We successfully demonstrated the protective effect of the LbL nanofilms prepared on cell membranes against physical stress derived from centrifugation. In particular, the LbL films consisting of ECM proteins revealed higher protective properties than synthetic polyelectrolyte LbL films, probably because of the cytotoxicity of the cationic components. The protective property of the LbL films was strongly dependent on their thickness, with a critical thickness of >5 nm. One hypothesis of the protective effect is the inhibition of cytosol molecule leakage during centrifugation. Furthermore, the nano-ECM films do not inhibit cell growth during cell culturing, suggesting the usefulness of the coated cells for further experiments. Because the protective effect of the nano-ECM films may be applicable to the other types of cells, this technique can be important to protecting cells from physical stress during tissue engineering.
Figure 7. Change in viability of (a) multiple types of normal cells and (c) other types of human carcinoma cells with stress administered by centrifugation in Tris-HCl buffer (pH 7.4) (n = 3). Total cell yields of (b) multiple types of cells and (d) other types of human carcinoma cells after 18 cycles of centrifugation in Tris-HCl buffer (pH 7.4) (n = 3). White and black columns indicate the proportion of living cells and dead cells, respectively, and the values shown at the top of the column indicate the cell viability. All values after 18 cycles are shown in parentheses. The upward arrow in part c shows the viability shift for Huh-7 by FN-G nanofilm preparation. The asterisks (*) denote a statistically significant difference between samples calculated from a two-sample t test (p < 0.01). Error bars denote standard deviations.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +81-6-6879-7356. Fax +81-6-6879-7359. E-mail: akashi@ chem.eng.osaka-u.ac.jp.
showed good growth profiles that were similar to that of uncoated cells. Furthermore, the cellular morphology of the coated cells demonstrated the extended cobblestone shape noted for control cells similar to that shown in Figure 5b−d. These results clearly suggested the high biocompatibility of both FN-G and Col IV-LN nanofilms with the hepatocyte cells. Finally, the protective effect of the LbL films with respect to physical stress is summarized visually in Figure 6. The polymers or proteins, which are components of the LbL nanofilms, adsorb onto cell membranes during the LbL process and the LbL films inhibit the leakage of cytosol molecules during additional centrifugation by physical or chemical entrapment. Another possible explanation of the protective effect is mechanical protection from gravity. Svaldo-Lanero et al. have reported an increase in the mechanical properties of cell membranes through the preparation of the LbL films.28 We
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was mainly supported by the Funding Program for Next Generation World-Leading Researchers (NEXT Program: LR026) and partially by a Grant-in-Aid for Scientific Research (S) (A232250040) from the Japan Society for the Promotion of Science (JSPS).
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dx.doi.org/10.1021/la303459v | Langmuir 2013, 29, 7362−7368
