Letter www.acsami.org
Gradient and Patterned Protein Films Stabilized via Nanoimprint Lithography for Engineered Interactions with Cells Li-Sheng Wang,†,§ Bradley Duncan,†,§ Rui Tang,† Yi-Wei Lee,† Brian Creran,† Sukru Gokhan Elci,† Jiaxin Zhu,‡ Gülen Yesilbag Tonga,† Jesse Doble,† Matthew Fessenden,† Mahin Bayat,† Stephen Nonnenmann,‡ Richard W. Vachet,† and Vincent M. Rotello*,† †
Department of Chemistry, University of MassachusettsAmherst, 710 North Pleasant Street, Amherst, Massachusetts 01003, United States ‡ Department of Mechanical and Industrial Engineering, University of Massachusetts, Amherst, Massachusetts 01003, United States S Supporting Information *
ABSTRACT: Protein-based biomaterials provide versatile scaffolds for generating functional surfaces for biomedical applications. However, tailoring the functional and biological properties of protein films remains a challenge. Here, we describe a high-throughput method to designing stable, functional biomaterials by combining inkjet deposition of protein inks with a nanoimprint lithography based methodology. The translation of the intrinsically charged proteins into functional materials properties was demonstrated through controlled cellular adhesion. This modular strategy offers a rapid method to produce customizable biomaterials.
KEYWORDS: biomaterial, nanoimprint lithography, inkjet printing, cell patterning, thin films
P
printed without vastly altering the intrinsic properties of the protein.19 We hypothesized that the parametric control offered by inkjet printing would allow us to dial in the biological response to combinatorial protein films. As shown in Figure 1a, inkjet deposition of protein-based inks generates patterns whose components can be modularly assembled. Following this directed deposition, the proteins are stabilized into a functional film using an additive-free, NIL-based method that is stable to cell culture conditions. We chose to probe cellular adhesion as a model biological response as the regulation of cellular adhesion/migration has been shown to be a critical factor in a variety of biological processes including cell differentiation,20 tissue development,21 and cancer progression.22 Bovine serum albumin (BSA, MW: 66.3 kDa, pI: 4.8) and lysozyme (Lyso, MW: 14.4 kDa, pI: 11.0) were selected as our model anionic and cationic protein inks, respectively. Films were generated through the deposition of the protein inks in a parametric fashion. Film composition was varied from 100% BSA to 100% Lyso in 20% increments (see Supporting Information for details). Inkjet printing of water-based inks produces coffee rings that result in high roughness of printed films.23 Addition of organic solvents or polymers has been utilized to reduce coffee ring formation;24,25 however, the compatibility of organic solvents and the toxicity of polymers hindered the application of inkjet printing protein films.26 In our approach, we found that the
rotein-derived materials offer an inherently sustainable and structurally diverse platform for the fabrication of functional materials.1−3 Protein films provide particularly attractive scaffolds for biomaterials, combining biodegradability and biocompatibility in versatile materials comprised of natural precursors.4−6 Furthermore, the protein surface creates a molecular template for controlling interaction with biological systems.7−9 These favorable attributes have made these proteinbased materials highly amenable to interface with cells for tissue engineering10,11 and wound healing applications.12,13 Recently, we have developed an additive-free, nanoimprint lithography (NIL) based method for the generation of waterstable protein films.14 Based on previous studies of patterned and multicomponent thin films,15−17 we hypothesized that inkjet printing of proteins would provide a suitable method for the “direct-writing” of two-dimensional biomolecular patterns to complement our NIL protein film fabrication strategy. Herein, we describe a combined inkjet printing based deposition with NIL stabilization methodology that generates materials surfaces with tunable biological interactions. The utility of these films was demonstrated through the controlled adhesion and migration of mammalian fibroblasts. By manipulating the cell behaviors of macrophage (RAW264.7) and human embryonic kidney cells (HEK293) using inkjet-printed protein films, a cell pattern composite of multiple cell types was generated. This versatile nonmanufacturing is a promising system for the rapid fabrication of macrophage coculture platforms. Inkjet printing provides a reproducible method for controlling the mixing and deposition of nanomaterials.18 Previous studies have demonstrated that proteins can readily be inkjet © XXXX American Chemical Society
Received: October 28, 2016 Accepted: December 23, 2016 Published: December 23, 2016 A
DOI: 10.1021/acsami.6b13815 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Figure 1. (a) Schematic of film processing strategy to generate protein films. Inkjet-directed deposition controls both the film composition and spatial presentation of the protein components. (b) Topography of protein films with different ratio of BSA and Lyso before and after NIL processing. The scale bar for z-axis was 10 μm. The lateral areas measured were 0.70 mm × 0.53 mm.
Table 1. Thickness and Roughness of Protein Films with Different Ratio of BSA and Lyso before and after NIL Processinga pristine films composition 100% BSA 20% Lyso 40% Lyso 60% Lyso 80% Lyso 100% Lyso a
thickness (nm) 2168 2255 2036 1742 1235 1626
± ± ± ± ± ±
184 85 177 57 89 54
NIL processed films
roughness (nm) 375 386 558 753 631 789
± ± ± ± ± ±
14 58 81 45 29 51
thickness (nm) 814 1010 843 733 562 665
± ± ± ± ± ±
13 11 26 4 19 41
roughness (nm) 163 192 270 322 324 265
± ± ± ± ± ±
19 33 23 15 27 22
The data were measured using a profilometer (n = 3).
NIL process can be applied for flattening the inkjet-printed films (Figure 1b). The thickness and roughness of pristine films were found decreased twice after NIL (Table 1). Previously, we have shown that NIL can be used to generate water-stable protein films while maintaining their inherent surface properties. These results were reproduced on inkjetprinted protein films as well. The stability of inkjet-printed protein films was demonstrated by measuring the thickness change of films after washing by water (Figure 2a). No significant loss of films was observed after 3 days incubation. Kelvin probe force microscopy (KPFM) was employed to demonstrate the control of surface potentials on inkjet-printed protein films. The surface potentials of mixed films were gradually increased with the increase of lysozyme component (Figure 2b).
Figure 2. (a) Thickness changes of protein films after immersing in water. (b) Surface potential determined by KPFM. Protein films were generated by varying the BSA:Lyso ratio of the film in 20% increments. B
DOI: 10.1021/acsami.6b13815 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces We determined whether this physiochemical property was translatable to biological systems. We quantified the adhesion of mammalian fibroblast cells using films generated from increasing ratios of BSA:Lyso. As shown in Figure 3, cells adhere to films
generated with greater percentages of Lyso with a drastic increase observed with films comprised of 80% or more of Lyso. Films fabricated with higher BSA amounts demonstrated minimal adhesion, confirming the incorporation of protein charge into the overall materials properties of the film. We generated a gradient across which the BSA:Lyso ratio was varied to further probe the adhesion based on protein component. As shown in Figure 4a and Figure 4b, the cells preferentially adhere to the Lyso-containing portion of the film. Notably, the results on gradient pattern demonstrate the ability to fine-tune cell adhesion using different compositions of protein inks. Inkjet printing advantageously affords spatial control over the deposition of film components. To probe the control over cell growth as a function of protein component, we deposited a rectangle of Lyso surrounding by a circle of BSA. As shown in Figure 4c and Figure 4d, the cells preferentially adhere to the Lyso pattern and can be easily washed away from the BSAcoated surface. This dynamic process of cellular attachment was also observed at the boundary of BSA and Lyso patterns as shown in Video S1. Cellular coculture systems have been applied for inflammatory studies by mixing macrophages with other cells.27 Side-byside coculture facilitated the studying of cell−cell interaction at the interface; however, attaching different cells on specific area has been challenging.28 Taking advantage of different cellular interactions with protein films, we were able to demonstrate a patterned side-by-side coculture of RAW 264.7 and deGFPexpressed HEK293 cells. Similar to fibroblast cells, HEK293 only adhered on the Lyso area (Figure S3). On the other hand, RAW 264.3 were able to adhere on both BSA and LYSO regions (Figure S4). After 24 h incubation of HEK 293 cells on patterned protein film, a patch of HEK293 cells was formed, and RAW 264.3 was then introduced. Since the HEK293 has preoccupied the Lyso area, RAW 264.3 was forced to adhere on
Figure 3. (a) Adhesion of mammalian fibroblasts on films with varying ratios of protein components. Cells were stained with Hoescht 33343 and Calcein AM to label the cell nuclei and cytosol, respectively. Scale bars are 200 μm. (b) Number of cells with respect to different ratios of protein components (see Figure S1 for raw data).
Figure 4. Adhesion of mammalian cells on micropatterned films. (a) Fibroblast adhesion to protein film generated with a gradient pattern. Cells were stained with Hoescht 33342 and Calcein AM to label cell nuclei and cytosol, respectively. (b) Number of cells with respect to position along gradient (see Figure S2 for raw data). (c) Fibroblast adhesion to patterned film with discrete Lyso and BSA domains. The solid line (Lyso) and dotted line (BSA) were drawn to aid the eye. (d) Fluorescence micrograph of cells adhered to Lyso pattern surrounded by BSA. (e) Optical and (f) fluorescence micrograph of the coculture pattern (green: GFP-expressed HEK293; red: DiD lipophilic tracer labeled RAW264.7). Scale bars were 100 μm for (e) and (f). C
DOI: 10.1021/acsami.6b13815 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
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only the BSA region, resulting in a side-by-side cell culture (Figure 4e and Figure 4f). In conclusion, we have developed a highly modular method to generate stable protein films in a rapid fashion with diverse components. The environmentally friendly processing taken with the parametric control over the surface chemistry provides a multidimensional platform for understanding and controlling biological interactions with protein-coated surfaces. NIL not only provides stabilization of protein films but also smoothens the coffee rings resulting from inkjet deposition. Due to the spatial and compositional control of inkjet deposition, various functional films can be generated using the combination of different protein precursors to generate functional biomaterials.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b13815. Experimental section; image analysis of cell counting for protein films with different ratios and gradient components; optical image of RAW 264.7 and HEK293 cellular adhesion (PDF) Live video of cell migration on protein film (AVI)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Richard W. Vachet: 0000-0003-4514-0210 Vincent M. Rotello: 0000-0002-5184-5439 Author Contributions §
L.-S. W. and B. D. contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Professor Kenneth R. Carter, from the Department of Polymer Science and Engineering at University of Massachusetts Amherst, for the use of the nanoimprinter. This research was supported by the NSF (CHE-1307021 and CMMI-1025020).
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REFERENCES
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DOI: 10.1021/acsami.6b13815 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
