Article Cite This: Langmuir XXXX, XXX, XXX−XXX
pubs.acs.org/Langmuir
Stressful Surfaces: Cell Metabolism on a Poorly Adhesive Substrate Richard L. Surmaitis, Carlos J. Arias, and Joseph B. Schlenoff* Department of Chemistry & Biochemistry, The Florida State University, Tallahassee, Florida 32306, United States S Supporting Information *
ABSTRACT: The adhesion and proliferation of cells are exquisitely sensitive to the nature of the surface to which they attach. Aside from cell counting, cell “health” on surfaces is typically established by measuring the metabolic rate with dyes that participate in the metabolic pathway or using “live/dead” assays with combinations of membrane permeable/impermeable dyes. The binary information gleaned from these testswhether cells are attached or not, and whether they are living or deadprovides an incomplete picture of cell health. In the present work, proliferation rates and net metabolism of 3T3 fibroblasts seeded on “biocompatible” ultrathin polyelectrolyte multilayer films and on control tissue culture plastic were compared. Cells adhered to, and proliferated on, both surfaces, which were shown to be nontoxic according to live/dead assays. However, adhesion was poorer on the multilayer surface, illustrated by diffuse organization of the actin cytoskeleton and less-developed focal adhesions. Proliferation was also slower on the multilayer. When normalized for the total number of cells, it was shown that cells on multilayers experienced a five-day burst of metabolic stress, after which the metabolic rate approached that of the control surface. This initial state of high stress has not been reported or appreciated in studies of cell growth on multilayers, although the observation period for this system is usually a few days.
■
INTRODUCTION Cellular adhesion plays a vital role in regulating processes such as apoptosis and cell differentiation.1−4 The adhesion of living cells to substrates is a complex topic in surface science. It is widely understood that cells adhere to proteins rather than directly to synthetic surfaces.5 Most surfaces begin to adsorb proteins as soon as they are immersed into biological fluids, forming a corona that isolates the surface from direct contact with cells.6 Because of their high concentration, serum proteins such as serum albumin, SA, adsorb onto the substrate first.7,8 Under an exchange mechanism termed the Vroman effect,9−11 which occurs both on planar and (nano)particle substrates, initially adsorbed proteins are gradually displaced by proteins with lower serum concentration but with higher affinity for the surface, yielding a “hard” corona12−14 forming the actual contact to the cell.8,11,14 Cell attachment is much slower than protein adsorption,15 providing time for the expression of adhesive proteins such as fibronectin and vinculin by the cell itself. Many studies have been undertaken on the adsorption and stability of single-component proteins, mostly SA.16 Yet, a large gap in understanding exists regarding the complex cascade of adsorption and desorption/displacement of proteins, followed by cell recognition then adhesion, mainly via integrins.2,3 Once integrins are firmly engaged with the surface, commonly via the Arg-Gly-Asp (RGD) peptide motif found on fibronectin, subsequent adhesion events are under the control of complex cellular processes.4 The connection between the fundamental physical chemistry of individual protein adsorption and coupled cell biochemistry is often glossed over when the object is merely to find whether a surface is “adhesive” or not. Cell morphology indirectly © XXXX American Chemical Society
reflects the strength of cell−protein and presumably protein− surface interactions: under the conditions of weak adhesion, cells are rounded and motile, whereas when they adhere firmly, they are well-spread and flattened.17,18 Crude binary assessments are common when evaluating cell adhesion, such as whether or not they adhere and if they adhere, whether the surface is toxic. These tests are done with cell counting on a surface and “live/dead” staining kits, respectively.19 With the development of techniques and materials to modify surfaces, the level of control of protein and cell adhesion has evolved. For example, self-assembled monolayers of oligoethylene oxide units or zwitterions20 are effective in preventing protein adsorption.21−23 Polymeric zwitterions, especially those in brush format, perform exceptionally well in this respect.24−26 Thin films of alternately layered polyelectrolytes (polyelectrolyte multilayers, PEMUs)27 offer control over surface charge, hydrophobicity, and also the local elasticity a cell experiences.28−31 A wide spectrum of cell adhesion type is possible on PEMUs, promoting various phenotypes32 or even differentiation of stem cells.33,34 We have recently described a minimally adhesive PEMU surface that induces spherical cell clustering35 where cell−cell interactions (typically regulated by cadherins) are stronger than cell−surface binding via integrins.36 Most cells require adhesion to maintain a healthy state and to proliferate. If they are not able to strongly anchor, cells undergo controlled disassembly (apoptosis) without releasing inflammatory materials (e.g., cytokines) into the extracellular Received: December 7, 2017 Revised: January 10, 2018
A
DOI: 10.1021/acs.langmuir.7b04172 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
containing only culture medium. Relative fluorescent units (RFUs) for each day were compared and later used with cell counts per well per day to determine the metabolic activity. Cell Counting. After extraction of alamarBlue-infused media from each well following the 4 h incubation period, 200 μL of 10% trypsin/ TE solution was added to each well.43 The plates were then incubated for 10 min at 37 °C to allow the trypsin to remove all of the cells. The cells were then extracted using a transfer pipette into 1 mL microcentrifuge tubes and cooled to −20 °C to preserve the DNA. After day 6, all cells from each well had been extracted into microcentrifuge tubes and frozen. All trials were then thawed, and 50 μL of Proteinase K (Sigma-Aldrich) was added to each trial. Each trial was then placed in a water bath at 50 °C for 24 h to allow the release of DNA. Each trial sample (25 μL) was added to wells in a 96-well opaque plate in triplicate and cooled to rt. This yielded 9 wells per condition per day. AccuClear Ultra High Sensitivity dsDNA Quantitation (Biotium) was used to correlate the number of cells per trial. The AccuClear dye was diluted at a ratio of 1:100 in an AccuClear buffer, and 200 μL of this working solution was added to each well. The fluorescent dye binds only to dsDNA.44 The fluorescence was measured in a plate reader using λEX = 468 nm and λEM = 507 nm maxima. This was outside the range of the alamarBlue, so no overlapping signal was acquired. A set of DNA standards of known concentrations was generated by diluting AccuClear DNA Standard 25 ng μL−1 calf thymus DNA in TE buffer which is a mixture of 10 mM Tris and 1 mM ethylenediaminetetraacetic acid. Each DNA standard (25 μL) and working solution (200 μL) were added to individual wells in a 96-well opaque plate in triplicate. The standard curve (see Figure S1 Supporting Information) was used to translate RFUs observed from each trial to number of cells, assuming 6.6 pg of DNA per single diploid cell.45 Live/Dead Cell Double Staining. Fibroblast viability under all conditions was determined using a double staining kit (Calbiochem, EMD Chemicals). Fibroblasts were seeded at 10 000 cells per well and cultured for 3 days. Media were aspirated on the third day, and the wells were rinsed with phosphate-buffered saline solution which was also aspirated. Cells remaining in the wells were exposed to the double staining kit following the manufacturer protocol. The Live/Dead kit consists of two fluorescent dyes: CytoGreen dye, λEX = 488 nm and λEM = 518 nm, and propidium iodide, λEX = 488 nm and λEM = 615 nm. CytoGreen dye is cell membrane permeable, so both live and dead cells will fluoresce green. Propidium iodide, impermeable to live cell membranes, only stains dead cells red.46 A Nikon Eclipse TS100 microscope with FITC and Texas Red filters was used to capture images of the cells. Cell counting was performed using ImageJ software.
medium.37 Adherent cells are typically counted, after release, using flow cytometry or hemocytometry. Alternatively, the number of cells is deduced from the net metabolism in the culture using a metabolic dye that is converted by cell mitochondria.19 The latter approach assumes that each cell produces the same metabolic turnover of dye. There are many sources of cell stress,38 such as an imbalance of chemical species needed for signaling or proper function,39 for example, hypoxia, or a change in the physical environment, such as temperature40 or cell compression.41 Metabolic stress is indicated by accelerated activity in the mitochondria. We have discovered that fibroblasts adhering poorly to PEMU surfaces generate much higher metabolic activity. This feature of cell adhesion is rarely reported, as it requires simultaneous and precise determination of cell number and metabolism. However, it is an important consideration for synthetic surfaces for implants that have been rendered “biocompatible” with an appropriate coating: a burst of high metabolic rate on contact with the in vivo environment may be an undesired property of such a coating. In the example shown below, the metabolic activity gradually recovers.
■
MATERIALS AND METHODS
Materials. Poly(4-styrenesulfonic acid) (PSS; 19 wt % in water, ∼70 000 g mol −1 from Scientific Polymer Products), poly(diallyldimethylammonium chloride) (PDADMAC; 21.3 wt % in water, 400 000−500 000 g mol−1 Sigma-Aldrich), and sodium chloride (99.5%) were used as received. All solutions were prepared with 18 MΩ deionized water (Barnstead, E-Pure). PSS and PDADMAC were 10 mM (based on the monomer repeat unit) polymer solutions with a NaCl concentration of 1.0 M. The pH of the solutions was adjusted to 7.0 with 1.0 M NaOH. Flat bottom, polystyrene 24-well plates (JET BIOFIL, Tissue Culture Products) with a growth area of 1.9 cm2 were substrates for PEMU buildup used in the cell experiments. Polyelectrolyte Multilayer Buildup. 24-Well plates were removed from sterile packaging immediately before buildup. [PDADMA/PSS, X]n multilayers, where “n” indicates the number of bilayers and X represents the NaCl concentration used for the buildup, were built manually at room temperature (rt) layer by layer. For example, [PDADMA/PSS, 1.0]10 indicates 20 alternating layers of PDADMA and PSS, starting with PDADMA on the 24-well plate, using 1.0 M NaCl in both polyelectrolyte solutions. The dipping time in each polyelectrolyte solution was 5 min followed by three consecutive water rinses for 1 min, after which the PEMU was allowed to dry in a clean room. The PEMU-coated culture plates were then exposed to UV light for 5 min in a laminar flow hood to sterilize them. Cell Culture. 3T3-Swiss albino fibroblasts (initially purchased from American Type Culture Collection as ATCC CCL-92 cells and maintained in the lab for numerous generations) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% Cosmic Calf Serum (Thermo Scientific), 1 g L−1 L-glutamine, 1.2 g L−1 sodium bicarbonate (Sigma-Aldrich), 0.25 μg mL−1 amphotericin B, 10 μg mL−1 gentamicin, 100 U mL−1 penicillin G, and 100 μg mL−1 streptomycin (Invitrogen). Cells were incubated at 37 °C with 5% CO2 (Nu-4750, NuAire incubator). Uncoated 24-well plates were used as controls. Cellular Metabolism. alamarBlue (Thermo Scientific) was used to observe the metabolism of the fibroblasts. alamarBlue contains resazurin, a nontoxic, cell-permeable dye that is reduced to fluorescent resorufin (λEX = 570 nm and λEM = 600 nm).36 alamarBlue (100 μL) was added to each well for each trial day in triplicate and incubated for 4 h at 37 °C to allow the metabolic-based reduction via reactions of the respiratory chain.42 Media from each well were then extracted with a pipette into a new 12-well plate, so fluorescence could be measured using a SpectraMax M5 Multi-Mode Microplate Reader. Background fluorescence measurements were determined by placing dye in wells
■
RESULTS AND DISCUSSION Fibroblasts are appropriate for studying adhesion to a substrate because they efficiently produce collagen, fibronectin, and other extracellular matrix (ECM) components.46 3T3 fibroblasts were deposited on the PEMU-coated polystyrene 24-well plates in groups of three wells and cultured for 1−6 days. Two multilayers were tested for cellular adhesion, [PDADMA/ PSS, 1.0]10 and [PDADMA/PSS, 1.0]10[PDADMA, 1.0]. These PEMUs were about 300 nm thick and differed in the terminating or top layer: PDADMA is positively charged and PSS negatively charged. In addition, the morphology of the film depends on the terminating layer.47 PEMUs ending with PSS are more glassy, whereas PEMUs capped with PDADMA are softer.28,48 Over the time period observed, control cells on polystyrene adhered and proliferated to 100% confluency, whereas cells grown on [PDADMA/PSS, 1.0]10 adhered and proliferated more slowly, as seen in Figure 1. Cells on [PDADMA/PSS, 1.0]10[PDADMA, 1.0] died, undergoing what appeared to be a necrotic mechanism (Figure 1). B
DOI: 10.1021/acs.langmuir.7b04172 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 2. Cell viability of live fibroblasts seeded at 10 000 cells cm−2 onto three surfaces at day 3 and day 6. ( ) TCP, control surface, ( ) [PDADMA/PSS, 1.0]10 surface, and (purple square) [PDADMA/PSS, 1.0]10[PDADMA, 1.0] surface. Cells remain viable on both the control surface and the [PDADMA/PSS, 1.0]10 multilayer, although cell count is reduced on the latter. Figure 1. Brightfield microscope images at 10× objective lens on day 3 of fibroblasts seeded at 10 000 cells cm−2 onto three surface conditions. (A) Tissue culture plastic (TCP), control surface, (B) [PDADMA/PSS, 1.0]10 surface, (C) [PDADMA/PSS, 1.0]10[PDADMA, 1.0] surface, and (D) structures of PSS and PDADMA. The scale bar is 100 μm.
film51 which can complex bovine serum albumin (BSA). In contrast, the PSS-capped multilayers were shown to have nearly stoichiometric amounts of positive and negative polyelectrolyte on their surface51 and adsorb monolayer quantities of BSA.53 The number of fibroblasts on the PSS-terminated surface increased but at a much slower rate than the control cells. Live cell imaging comparing the [PDADMA/PSS, 1.0]10 surface to the polystyrene surface shows the morphology of cells on [PDADMA/PSS, 1.0]10 to be more rounded and less spread than those adhered to the control surface. Figure 3 compares
Unlike many surfaces, the [PDADMA/PSS, 1.0]10 and [PDADMA/PSS, 1.0]10[PDADMA, 1.0] multilayers are wellcharacterized, according to our previous work, including thickness, roughness, modulus, charge density, and charge type. A summary of these characteristics is given in Table 1. Table 1. Physical and Chemical Characteristics of [PDADMA/PSS, 1.0]10 and [PDADMA/PSS, 1.0]10[PDADMA, 1.0] PEMU Films and Surfaces property a
PSS terminated
PDADMA terminated
thickness, wet thickness, dryb RMS roughness, weta RMS roughness, drya static water contact anglec surface chemistry
340 nm 260 nm 30 nm 25 nm 18°
360 nm 280 nm 80 nm 6 nm 68°
aromatic sulfonate
surface S/N ratiod,e surface charge surface charge densityb adsorbed BSAb surface modulus wetc
1.1 negative −0.44 μmol m−2 1.3 mg m−2 24 MPa
aliphatic tetraalkylammonium 0.6 positive n/d 170 mg m−2 7 MPa
a
b
c
d
Figure 3. Fluorescence of cells immunofluorescence stained for vinculin (with antibody hVIN-1) in the top row, panels A and B, and of cells stained for F-actin in the bottom row, panels C and D, after 3 days of incubation. (A,C) [PDADMA/PSS, 1.0]10 surface and (B,D) TCP, control surface. The scale bar is 10 μm.
e
From ref 49. From ref 35. From ref 50. From ref 51. Upper 10 nm.
[PDADMA/PSS, 1.0]10[PDADMA, 1.0] differs in several respects (Table 1), but the difference in viability is attributed to the positive charge of the surface. The cytotoxicity of the increased positive surface charge density from PDADMA has been observed previously.46,48,52 Viability studies using a common live/dead counting combination of stains (see Supporting Information Figure S2 for examples of micrographs) revealed the difference in toxicity between the PSS- and PDADMA-capped multilayers (Figure 2). Table 1 indicates that PDADMA-capped PEMUs take up large amounts of protein. This “spongelike” effect was described previously53 and is due to the large amount of excess PDADMA throughout such a
the organization of vinculin, localized at focal adhesions, and actin, comprising a major part of the cytoskeleton, of cells on control and [PDADMA/PSS, 1.0]10. On the control surface, vinculin is clearly localized in punctate regions on the cell periphery consistent with well-developed focal adhesions, whereas it is more uniformly and diffusely distributed around the periphery of cells on the PEMU. Also, consistent with good adhesion, actin is organized into stress fibers for cells on the control surface, whereas in the poorly adhered cells on PEMU, it is again diffusely distributed. Actin staining also reveals C
DOI: 10.1021/acs.langmuir.7b04172 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 4. Brightfield images of cells on control, TCP surface and [PDADMA/PSS, 1.0]10 surface taken each day (day number in boxes, inset) for the 6 days of the experiment. For each day, the control image is on the left and the [PDADMA/PSS, 1.0]10 surface image is on the right. The scale bar is 100 μm.
extensive filopodia for cells on the multilayer, which is typical of motile cells searching for anchorage. The modulus of [PDADMA/PSS, 1.0]10 is much higher than the minimum needed for efficient cell adhesion and spreading.54,55 The difference in adhesion is attributed to weaker protein attachment on [PDADMA/PSS, 1.0]10. Because cells attach to the proteins already adhered to the surface, they have difficulty forming focal adhesions to a surface with poor protein adhesion regardless of the stiffness of the film.8,31,46 Surfaces with adsorbed fibronectin should promote cell adhesion and spreading,56 as demonstrated for certain multilayer surfaces.57 However, [PDADMA/PSS, 1.0]10[PDADMA, 1.0] coated with fibronectin still yielded a toxic surface (see Supporting Information Figure S3). Apparently, a coating of fibronectin is not sufficient to insulate the cells from the toxic properties of this cationic surface. Adhesion to [PDADMA/ PSS, 1.0]10 precoated with fibronectin was not noticeably improved, the cells remaining more rounded than control (Figure S3). Figure 4 shows a gradual increase in the fibroblast density on [PDADMA/PSS, 1.0]10 surfaces as the cells adjust to the poorly adhesive substrate. Because the coverage of cells on the PSSterminated PEMU was much lower than that on the control surface, some methods for cell counting were unsuitable. Flow cytometry required more cells than were available on a sparsely populated surface and hemocytometry produced too much variability (again due to low cell count). To provide a reliable indication of the total number of cells on a surface, AccuClear Ultra High Sensitivity dsDNA Quantitation (Biotium) was used, which binds to the DNA of the cells.44,58,59 This method had a lower limit of detection of about 2 cells per cm2. Calibration curves were consistently linear and reproducible over the range of 3 to 80 000 cells per cm2 (see Supporting Information Figure S1 for an example of such a calibration
curve). Cell counts as a function of time for [PDADMA/PSS, 1.0]10 and control are presented in Figure 5.
Figure 5. Total adhered cells (from the DNA assay), upper panel and relative fluorescence units (from metabolic tests), lower panel; for sixday trials on TCP, control surface (blue circle) and [PDADMA/PSS, 1.0]10 (maroon triangle). Cell proliferation is encouraged by better adhesion as observed on TCP.
Cellular metabolism was assessed on the same population using the alamarBlue assay. alamarBlue (resazurin) is reduced by mitochondria to resorufin (see Figure S4, Supporting Information, for the structures). alamarBlue has been cited as a highly reproducible method for the detection of 3T3 fibroblast proliferation.42 The indicator dye becomes an intermediate in the electron transport chain that is reduced to a fluorescent D
DOI: 10.1021/acs.langmuir.7b04172 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir compound.60,61 The changes in relative fluorescence are normally understood to correlate with the number of cells exposed to the alamarBlue dye.42 As expected, fibroblast populations on the control surface showed increasing total metabolism as they proliferated (Figure 5). The metabolic output of cells on the [PDADMA/PSS, 1.0]10 surface also increased. Cells on [PDADMA/PSS, 1.0]10[PDADMA, 1.0] showed no significant transformation of resazurin into resorufin because they were dead (Figure 2).42 The use of the two accurate and precise methods for cell counting and cell metabolism allowed us to determine the relative metabolic rate per cell (Figure 6). This revealed a
integrins to the ECM and acts at the site of cell−ECM interactions.70 When FAK is blocked by an inhibitor, cell proliferation and viability are reduced.71 For integrins, the receptor phosphorylation reacting to collagen is a slow process.72 Because cell proliferation and migration are dependent on the actin cytoskeleton and focal adhesion contacts, this would explain why the cell counts are low for the initial days of the experiment for the [PDADMA/PSS, 1.0]10 surface.72
■
CONCLUSIONS Fibroblast adhesion, survival, and proliferation depend strongly on many surface variables. Although nontoxic surfaces are usually labeled “biocompatible,” the true effect of the surface on cell response may be unappreciated with the use of live/dead staining kits or metabolism dyes. When the surface exhibits poor protein adhesion but is not actually cytotoxic, a stressful environment for the cells is created. To adhere to such a surface, these stressed cells must increase the expression of genes to produce components necessary for cytoskeletal development and focal adhesion complexes. Generating this additional ECM causes the cells to increase their metabolic rate. Such an outcome may be harmful for proper integration of implants into living tissue. Other undesired outcomes, such as the release of (inflammatory) cytokines, may accompany an enhanced metabolic rate. It would be interesting to explore a more expanded panel of stress biomarkers and to determine which specific genes are being upregulated in response to a poorly adhesive surface. The question as to why different surfaces, all hydrophilic, lead to different strengths of protein adsorption is currently under investigation.
Figure 6. RFU per cell, representing normalized metabolism rates per cell, as a function of time for six-day trials on TCP, control surface (blue circle) and [PDADMA/PSS, 1.0]10 (maroon triangle). Metabolism per cell is initially much higher on [PDADMA/PSS, 1.0]10 compared to TCP but falls to levels close to the control.
■
ASSOCIATED CONTENT
S Supporting Information *
strong difference in the normalized metabolism between the adhesive (control) and the poorly adhesive ([PDADMA/PSS, 1.0]10) surfaces. For example, 24 h after seeding, the metabolism of cells on the PEMU surfaces was about eight times that of the cells on the control surface. While the metabolic rate on the control surface dropped slightly over the next few days, 3T3 metabolism on the PEMU decreased significantly until it was close to the rate on polystyrene. The results in Figure 6 clearly show an unrecognized feature of cell adhesion on different surfaces: the poorly adhesive surface causes a high level of metabolic stress. The stress level decreases with time, presumably as a result of decreased production of adhesive ECM proteins and other ECM components such as a collagen network by the fibroblasts.62 Stress is believed to be a result of poor mechanical adhesion of ECM proteins required for integrin-based cell anchoring on [PDADMA/PSS, 1.0]10. Compared to control, cells expend more energy attempting to form permanently adhesive contacts with the surface. Fibroblasts in stressed conditions are known to express genes that produce higher levels of ECM molecules and to produce protease inhibitors that cause accumulation of ECM by decreasing ECM degradation.63,64 Studies have shown upregulation of cytoskeleton and focal adhesion components for stressed fibroblasts.65 Mechanical stimuli cause the cell to respond by changing gene expression through signal transduction.66 To bind to fibronectin and connect to the actin cytoskeleton, fibroblasts increase the production of integrin receptors to facilitate the formation of focal adhesion complexes.64,67,68 The focal adhesion complex formation relies upon tyrosine phosphorylation, so focal adhesion kinase, FAK, is upregulated.69 FAK is activated following the binding of
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b04172. DNA calibration curve, examples of live/dead images for counts, and images of cells on fibronectin-coated surfaces (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Joseph B. Schlenoff: 0000-0001-5588-1253 Notes
The authors declare no competing financial interest.
■ ■
ACKNOWLEDGMENTS This work was supported by grant DMR1506824 from the National Science Foundation. REFERENCES
(1) Juliano, R. L.; Haskill, S. Signal Transduction from the Extracellular Matrix. J. Cell Biol. 1993, 120, 577−585. (2) Hynes, R. O. Integrins: Versatility, Modulation, and Signaling in Cell Adhesion. Cell 1992, 69, 11−25. (3) Ruoslahti, E.; Ö brink, B. Common Principles in Cell Adhesion. Exp. Cell Res. 1996, 227, 1−11. (4) Buckley, C. D.; Rainger, G. E.; Bradfield, P. F.; Nash, G. B.; Simmons, D. L. Cell Adhesion: More Than Just Glue (Review). Mol. Membr. Biol. 1998, 15, 167−176.
E
DOI: 10.1021/acs.langmuir.7b04172 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir (5) Ruoslahti, E.; Ö brink, B. Common Principles in Cell Adhesion. Exp. Cell Res. 1996, 227, 1−11. (6) Walczyk, D.; Bombelli, F. B.; Monopoli, M. P.; Lynch, I.; Dawson, K. A. What the Cell “Sees” in Bionanoscience. J. Am. Chem. Soc. 2010, 132, 5761−5768. (7) Andrade, J. D.; Hlady, V. Protein Adsorption and Materials Biocompatibility: a Tutorial Review and Suggested Hypotheses. Adv. Polym. Sci. 1986, 79, 1−63. (8) Wassel, E.; Jiang, S.; Song, Q.; Vogt, S.; Nöll, G.; Druzhinin, S. I.; Schönherr, H. Thickness Dependence of Bovine Serum Albumin Adsorption on Thin Thermoresponsive Poly(Diethylene Glycol) Methyl Ether Methacrylate Brushes by Surface Plasmon Resonance Measurements. Langmuir 2016, 32, 9360−9370. (9) Vroman, L.; Adams, A. L. Findings with the Recording Ellipsometer Suggesting Rapid Exchange of Specific Plasma Proteins at Liquid/Solid Interfaces. Surf. Sci. 1969, 16, 438−446. (10) Vroman, L.; Adams, A. L.; Fischer, G. C.; Munoz, P. C. Interaction of High Molecular Weight Kininogen, Factor Xii, and Fibrinogen in Plasma at Interfaces. Blood 1980, 55, 156−159. (11) Jung, S.-Y.; Lim, S.-M.; Albertorio, F.; Kim, G.; Gurau, M. C.; Yang, R. D.; Holden, M. A.; Cremer, P. S. The Vroman Effect: A Molecular Level Description of Fibrinogen Displacement. J. Am. Chem. Soc. 2003, 125, 12782−12786. (12) Lundqvist, M.; Stigler, J.; Cedervall, T.; Berggård, T.; Flanagan, M. B.; Lynch, I.; Elia, G.; Dawson, K. The Evolution of the Protein Corona around Nanoparticles: A Test Study. ACS Nano 2011, 5, 7503−7509. (13) Fleischer, C. C.; Payne, C. K. Nanoparticle−Cell Interactions: Molecular Structure of the Protein Corona and Cellular Outcomes. Acc. Chem. Res. 2014, 47, 2651−2659. (14) Ritz, S.; Schöttler, S.; Kotman, N.; Baier, G.; Musyanovych, A.; Kuharev, J.; Landfester, K.; Schild, H.; Jahn, O.; Tenzer, S.; Mailänder, V. Protein Corona of Nanoparticles: Distinct Proteins Regulate the Cellular Uptake. Biomacromolecules 2015, 16, 1311−1321. (15) Arima, Y.; Iwata, H. Effects of Surface Functional Groups on Protein Adsorption and Subsequent Cell Adhesion Using SelfAssembled Monolayers. J. Mater. Chem. 2007, 17, 4079−4087. (16) Vasina, E. N.; Paszek, E.; Nicolau, D. V.; Nicolau, D. V. The Bad Project: Data Mining, Database and Prediction of Protein Adsorption on Surfaces. Lab Chip 2009, 9, 891−900. (17) von der Mark, K.; Gauss, V.; von der Mark, H.; Müller, P. Relationship between Cell Shape and Type of Collagen Synthesised as Chondrocytes Lose Their Cartilage Phenotype in Culture. Nature 1977, 267, 531−532. (18) Villar-Suárez, V.; Calles-Venal, I.; Bravo, I. G.; FernándezÁ lvarez, J. G.; Fernández-Caso, M.; Villar-Lacilla, J. M. Differential Behavior between Isolated and Aggregated Rabbit Auricular Chondrocytes on Plastic Surfaces. J. Biomed. Biotechnol. 2004, 2004, 86−92. (19) Berridge, M. V.; Herst, P. M.; Tan, A. S. Tetrazolium Dyes as Tools in Cell Biology: New Insights into Their Cellular Reduction. Biotechnol. Annu. Rev. 2005, 11, 127−152. (20) Fabianowski, W.; Coyle, L. C.; Weber, B. A.; Granata, R. D.; Castner, D. G.; Sadownik, A.; Regen, S. L. Spontaneous Assembly of Phosphatidylcholine Monolayers Via Chemisorption onto Gold. Langmuir 1989, 5, 35−41. (21) Tegoulia, V. A.; Rao, W.; Kalambur, A. T.; Rabolt, J. F.; Cooper, S. L. Surface Properties, Fibrinogen Adsorption, and Cellular Interactions of a Novel Phosphorylcholine-Containing Self-Assembled Monolayer on Gold. Langmuir 2001, 17, 4396−4404. (22) Holmlin, R. E.; Chen, X.; Chapman, R. G.; Takayama, S.; Whitesides, G. M. Zwitterionic SAMS That Resist Nonspecific Adsorption of Protein from Aqueous Buffer. Langmuir 2001, 17, 2841−2850. (23) Chen, S.; Zheng, J.; Li, L.; Jiang, S. Strong Resistance of Phosphorylcholine Self-Assembled Monolayers to Protein Adsorption: Insights into Nonfouling Properties of Zwitterionic Materials. J. Am. Chem. Soc. 2005, 127, 14473−14478.
(24) Feng, W.; Zhu, S.; Ishihara, K.; Brash, J. L. Adsorption of Fibrinogen and Lysozyme on Silicon Grafted with Poly(2Methacryloyloxyethyl Phosphorylcholine) Via Surface-Initiated Atom Transfer Radical Polymerization. Langmuir 2005, 21, 5980−5987. (25) Jiang, S.; Cao, Z. Ultralow-Fouling, Functionalizable, and Hydrolyzable Zwitterionic Materials and Their Derivatives for Biological Applications. Adv. Mater. 2010, 22, 920−932. (26) Nguyen, A. T.; Baggerman, J.; Paulusse, J. M. J.; van Rijn, C. J. M.; Zuilhof, H. Stable Protein-Repellent Zwitterionic Polymer Brushes Grafted from Silicon Nitride. Langmuir 2011, 27, 2587−2594. (27) Decher, G. Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites. Science 1997, 277, 1232−1237. (28) Richert, L.; Lavalle, P.; Vautier, P.; Senger, B.; Stoltz, J.-F.; Schaaf, P.; Voegel, J.-C.; Picart, C. Cell Interactions with Polyelectrolyte Multilayer Films. Biomacromolecules 2002, 3, 1170− 1178. (29) Boura, C.; Menu, P.; Payan, E.; Picart, C.; Voegel, J. C.; Muller, S.; Stoltz, J. F. Endothelial Cells Grown on Thin Polyelectrolyte Mutlilayered Films: An Evaluation of a New Versatile Surface Modification. Biomaterials 2003, 24, 3521−3530. (30) Richert, L.; Boulmedais, F.; Lavalle, P.; Ferreux, E.; Decher, G.; Schaaf, P.; Voegel, J.-C.; Picart, C. Improvement of Stability and Cell Adhesion Properties of Polyelectrolyte Multilayer Films by Chemical Cross-Linking. Biomacromolecules 2004, 5, 284−294. (31) Ren, K.; Crouzier, T.; Roy, C.; Picart, C. Polyelectrolyte Multilayer Films of Controlled Stiffness Modulate Myoblast Cell Differentiation. Adv. Funct. Mater. 2008, 18, 1378−1389. (32) Salloum, D. S.; Olenych, S. G.; Keller, T. C. S.; Schlenoff, J. B. Vascular Smooth Muscle Cells on Polyelectrolyte Multilayers: Hydrophobicity-Directed Adhesion and Growth. Biomacromolecules 2005, 6, 161−167. (33) Moby, V.; Labrude, P.; Kadi, A.; Bordenave, L.; Stoltz, J. F.; Menu, P. Polyelectrolyte Multilayer Film and Human Mesenchymal Stem Cells: An Attractive Alternative in Vascular Engineering Applications. J. Biomed. Mater. Res., Part A 2011, 96, 313−319. (34) Gribova, V.; Auzely-Velty, R.; Picart, C. Polyelectrolyte Multilayer Assemblies on Materials Surfaces: From Cell Adhesion to Tissue Engineering. Chem. Mater. 2012, 24, 854−869. (35) Arias, C. J.; Keller, T. C. S.; Schlenoff, J. B. Quasi-Spherical Cell Clusters Induced by a Polyelectrolyte Multilayer. Langmuir 2015, 31, 6436−6446. (36) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Investigation of the Alamar Blue (Resazurin) Fluorescent Dye for the Assessment of Mammalian Cell Cytotoxicity. Eur. J. Biochem. 2000, 267, 5421−5426. (37) Elmore, S. Apoptosis: A Review of Programmed Cell Death. Toxicol. Pathol. 2007, 35, 495−516. (38) Thomas, M. P.; Lieberman, J. Live or Let Die: Posttranscriptional Gene Regulation in Cell Stress and Cell Death. Immunol. Rev. 2013, 253, 237−252. (39) Jacobs, A. T.; Marnett, L. J. Systems Analysis of Protein Modification and Cellular Responses Induced by Electrophile Stress. Acc. Chem. Res. 2010, 43, 673−683. (40) Wallace, E. W. J.; Kear-Scott, J. L.; Pilipenko, E. V.; Schwartz, M. H.; Laskowski, P. R.; Rojek, A. E.; Katanski, C. D.; Riback, J. A.; Dion, M. F.; Franks, A. M.; Airoldi, E. M.; Pan, T.; Budnik, B. A.; Drummond, D. A. Reversible, Specific, Active Aggregates of Endogenous Proteins Assemble Upon Heat Stress. Cell 2015, 162, 1286−1298. (41) Tse, J. M.; Cheng, G.; Tyrrell, J. A.; Wilcox-Adelman, S. A.; Boucher, Y.; Jain, R. K.; Munn, L. L. Mechanical Compression Drives Cancer Cells toward Invasive Phenotype. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 911−916. (42) Voytik-Harbin, S. L.; Brightman, A. O.; Waisner, B.; Lamar, C. H.; Badylak, S. F. Application and Evaluation of the Alamar Blue Assay for Cell Growth and Survival of Fibroblasts. In Vitro Cell. Dev. Biol.: Anim. 1998, 34, 239−246. (43) Al-Nasiry, S.; Hanssens, M.; Luyten, C.; Pijnenborg, R. The Use of Alamar Blue Assay for Quantitative Analysis of Viability, Migration F
DOI: 10.1021/acs.langmuir.7b04172 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
on Fibronectin and Enhanced by Integrins α11β1 and α2β1. J. Biol. Chem. 2002, 277, 37377−37381. (65) Klein, C. E.; Dressel, D.; Steinmayer, T.; Mauch, C.; Eckes, B.; Krieg, T.; Bankert, R. B.; Weber, L. Integrin α2β1 Is Upregulated in Fibroblasts and Highly Aggressive Melanoma Cells in ThreeDimensional Collagen Lattices and Mediates the Reorganization Of collagen I Fibrils. J. Cell Biol. 1991, 115, 1427−1436. (66) MacKenna, D.; Summerour, S. R.; Villarreal, F. J. Role of Mechanical Factors in Modulating Cardiac Fibroblast Function and Extracellular Matrix Synthesis. Cardiovasc. Res. 2000, 46, 257−263. (67) Jokinen, J.; Dadu, E.; Nykvist, P.; Käpylä, J.; White, D. J.; Ivaska, J.; Vehviläinen, P.; Reunanen, H.; Larjava, H.; Häkkinen, L.; Heino, J. Integrin-Mediated Cell Adhesion to Type I Collagen Fibrils. J. Biol. Chem. 2004, 279, 31956−31963. (68) Zhang, X.; Ibrahimi, O. A.; Olsen, S. K.; Umemori, H.; Mohammadi, M.; Ornitz, D. M. Receptor Specificity of the Fibroblast Growth Factor Family: The Complete Mammalian FGF Family. J. Biol. Chem. 2006, 281, 15694−15700. (69) Kinch, M. S.; Clark, G. J.; Der, C. J.; Burridge, K. Tyrosine Phosphorylation Regulates the Adhesions of Ras-Transformed Breast Epithelia. J. Cell Biol. 1995, 130, 461−471. (70) Abedi, H.; Zachary, I. Vascular Endothelial Growth Factor Stimulates Tyrosine Phosphorylation and Recruitment to New Focal Adhesions of Focal Adhesion Kinase and Paxillin in Endothelial Cells. J. Biol. Chem. 1997, 272, 15442−15451. (71) Cabrita, M. A.; Jones, L. M.; Quizi, J. L.; Sabourin, L. A.; McKay, B. C.; Addison, C. L. Focal Adhesion Kinase Inhibitors Are Potent Anti-Angiogenic Agents. Mol. Oncol. 2011, 5, 517−526. (72) Olaso, E.; Labrador, J.-P.; Wang, L.; Ikeda, K.; Eng, F. J.; Klein, R.; Lovett, D. H.; Lin, H. C.; Friedman, S. L. Discoidin Domain Receptor 2 Regulates Fibroblast Proliferation and Migration through the Extracellular Matrix in Association with Transcriptional Activation of Matrix Metalloproteinase-2. J. Biol. Chem. 2002, 277, 3606−3613.
and Invasion of Choriocarcinoma Cells. Hum. Reprod. 2007, 22, 1304−1309. (44) Bruijns, B.; Tiggelaar, R.; Gardeniers, H. Dataset of the Absorption, Emission and Excitation Spectra and Fluorescence Intensity Graphs of Fluorescent Cyanine Dyes for the Quantification of Low Amounts of dsDNA. Data Brief 2017, 10, 132−143. (45) Lui, Y. Y.; Chik, K. W.; Chiu, R. W.; Ho, C. Y.; Lam, C. W.; Lo, Y. M. Predominant Hematopoietic Origin of Cell-Free DNA in Plasma and Serum after Sex-Mismatched Bone Marrow Transplantation. Clin. Chem. 2002, 48, 421−427. (46) Arias, C. J.; Surmaitis, R. L.; Schlenoff, J. B. Cell Adhesion and Proliferation on the “Living” Surface of a Polyelectrolyte Multilayer. Langmuir 2016, 32, 5412−5421. (47) Richert, L.; Engler, A. J.; Discher, D. E.; Picart, C. Elasticity of Native and Cross-Linked Polyelectrolyte Multilayer Films. Biomacromolecules 2004, 5, 1908−1916. (48) Martinez, J. S.; Keller, T. C. S.; Schlenoff, J. B. Cytotoxicity of Free Versus Multilayered Polyelectrolytes. Biomacromolecules 2011, 12, 4063−4070. (49) Ghostine, R. A.; Jisr, R. M.; Lehaf, A.; Schlenoff, J. B. Roughness and Salt Annealing in a Polyelectrolyte Multilayer. Langmuir 2013, 29, 11742−11750. (50) Lehaf, A. M.; Hariri, H. H.; Schlenoff, J. B. Homogeneity, Modulus, and Viscoelasticity of Polyelectrolyte Multilayers by Nanoindentation: Refining the Buildup Mechanism. Langmuir 2012, 28, 6348−6355. (51) Ghostine, R. A.; Markarian, M. Z.; Schlenoff, J. B. Asymmetric Growth in Polyelectrolyte Multilayers. J. Am. Chem. Soc. 2013, 135, 7636−7646. (52) Zhang, W.; Wang, S.; Lin, M.; Han, Y.; Zhao, G.; Lu, T. J.; Xu, F. Advances in Experimental Approaches for Investigating Cell Aggregate Mechanics. Acta Mech. Solida Sin. 2012, 25, 473−482. (53) Salloum, D. S.; Schlenoff, J. B. Protein Adsorption Modalities on Polyelectrolyte Multilayers. Biomacromolecules 2004, 5, 1089−1096. (54) Richert, L.; Boulmedais, F.; Lavalle, P.; Mutterer, J.; Ferreux, E.; Decher, G.; Schaaf, P.; Voegel, J.-C.; Picart, C. Improvement of Stability and Cell Adhesion Properties of Polyelectrolyte Multilayer Films by Chemical Cross-Linking. Biomacromolecules 2004, 5, 284− 294. (55) Ren, K.; Crouzier, T.; Roy, C.; Picart, C. Polyelectrolyte Multilayer Films of Controlled Stiffness Modulate Myoblast Cell Differentiation. Adv. Funct. Mater. 2008, 18, 1378−1389. (56) Cukierman, E.; Pankov, R.; Stevens, D. R.; Yamada, K. M. Taking Cell-Matrix Adhesions to the Third Dimension. Science 2001, 294, 1708−1712. (57) Wittmer, C.; Phelps, J.; Saltzman, W.; Van Tassel, P. Fibronectin Terminated Multilayer Films: Protein Adsorption and Cell Attachment Studies. Biomaterials 2007, 28, 851−860. (58) Singer, V. L.; Jones, L. J.; Yue, S. T.; Haugland, R. P. Characterization of Picogreen Reagent and Development of a Fluorescence-Based Solution Assay for Double-Stranded DNA Quantitation. Anal. Biochem. 1997, 249, 228−238. (59) Bruijns, B. B.; Tiggelaar, R. M.; Gardeniers, J. G. E. Fluorescent Cyanine Dyes for the Quantification of Low Amounts of dsDNA. Anal. Biochem. 2016, 511, 74−79. (60) Page, B.; Page, M.; Noel, C. A New Fluorometric Assay for Cytotoxicity Measurements in-Vitro. Int. J. Oncol. 1993, 3, 473−476. (61) Rampersad, S. N. Multiple Applications of Alamar Blue as an Indicator of Metabolic Function and Cellular Health in Cell Viability Bioassays. Sensors 2012, 12, 12347−12360. (62) Trappmann, B.; Gautrot, J. E.; Connelly, J. T.; Strange, D. G. T.; Li, Y.; Oyen, M. L.; Cohen Stuart, M. A.; Boehm, H.; Li, B.; Vogel, V.; Spatz, J. P.; Watt, F. M.; Huck, W. T. S. Extracellular-Matrix Tethering Regulates Stem-Cell Fate. Nat. Mater. 2012, 11, 642−649. (63) Strehlow, D.; Jelaska, A.; Strehlow, K.; Korn, J. H. A Potential Role for Protease Nexin 1 Overexpression in the Pathogenesis of Scleroderma. J. Clin. Invest. 1999, 103, 1179−1190. (64) Velling, T.; Risteli, J.; Wennerberg, K.; Mosher, D. F.; Johansson, S. Polymerization of Type I and Iii Collagens Is Dependent G
DOI: 10.1021/acs.langmuir.7b04172 Langmuir XXXX, XXX, XXX−XXX
