Chemistries for Making Additive Nanolithography in OrmoComp

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Biological and Medical Applications of Materials and Interfaces

Chemistries for Making Additive Nanolithography in OrmoComp® Permissive for Cell Adhesion and Growth David A. Kidwell, Woo-Kyung Lee, Keith Perkins, Kathleen Gilpin, Thomas J. O'Shaughnessy, Jeremy T Robinson, Paul E. Sheehan, and Shawn Patrick Mulvaney ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b04096 • Publication Date (Web): 02 May 2019 Downloaded from http://pubs.acs.org on May 2, 2019

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ACS Applied Materials & Interfaces

Chemistries for Making Additive Nanolithography in OrmoComp® Permissive for Cell Adhesion and Growth David A. Kidwell,* † Woo-Kyung Lee, † Keith Perkins,§ Kathleen M. Gilpin,∥ Thomas J. O'Shaughnessy,⊥ Jeremy T. Robinson,§ Paul E. Sheehan,† and Shawn P. Mulvaney†

†

Chemistry Division, U.S. Naval Research Laboratory, Washington, DC 20375, United States Electronics Science and Technology Division, U.S. Naval Research Laboratory, Washington, DC 20375, United States ∥American Society for Engineering Education (ASEE) Post-Doctoral Fellow at US Naval Research Laboratory, Washington, DC 20375, United States ⊥Materials Science and Technology Division, U.S. Naval Research Laboratory, Washington, DC 20375, United States §

Abstract Two-photon lithography allows writing of arbitrary nanoarchitectures in photopolymers. This design flexibility opens almost limitless possibilities for biological studies, but the acrylate-based polymers frequently used do not allow for adhesion and growth of some types of cells. Indeed, we found that lithographically defined structures made from OrmoComp® do not support E18 murine cortical neurons. We reacted OrmoComp® structures with several diamines thereby rendering the surfaces directly permissive for neuron attachment and growth by presenting a surface coating similar to the traditional cell biology coating achieved with poly-D-lysine (PDL) and laminin. However, in contrast to PDL-laminin coatings that cover the entire surface, the amine-terminated OrmoComp® structures are orthogonally modified in deference to the surrounding glass or plastic substrate, adding yet another design element for advanced biological studies. Keywords: E18 murine cortices; two-photon lithography; Michael Addition; diamines; biocompatibility; Ormocomp Introduction On-demand printing of 3D structures of arbitrary shape is sparking a design revolution by empowering the researcher with additional degrees of freedom in experimental design. One of the most popular techniques to achieve sub-micron writing is two-photon lithography. In twophoton lithography a laser is focused through optics immersed in a photosensitive polymer bath to write and make layer-by-layer structures with submicron resolution. Indeed, 3D tools have already received attention in the biological community; nanolithographic scaffolding has been used to encapsulate and direct biological growth 1,2,3,4,5 and develop 3D micro-scale implants. 6,7 For example, Spagnolo et al., have used two-photon lithography to build 3D polymer cages to constrain cancer cells and evaluate their metastasis potential in response to the cage structure. 8 In another study, Jeon and coworkers have used nanolithography to fabricate parallel and grid patterns that influence cellular functions and provide contact guidance for fibroblast cells. 9 We remain very interested in biological studies with neuronal cells. 10 For our latest studies, we need to constrain neuronal growth to specific areas of our device while directing the axon and dendrite growth over/near electrodes to perform both stimulation and to make measurements of the action potentials. Additive nanolithography can be performed with a number of polymers; Page 1 ACS Paragon Plus Environment

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SU-8, IP-DIP, and OrmoComp® are amongst the most commonly used. SU-8 is an epoxy polymer, but its refractive index is not as well matched to the optics of our Nanoscribe (Nanoscribe GmbH) tool as is the other two polymers, reducing the possible print resolution. IPDIP and OrmoComp® are both acrylate based polymers, with OrmoComp® being a mixed acrylate-siloxane polymer. 11 IP-DIP is a less rigid polymer compared to OrmoComp® and perhaps has a better Young’s modulus for cell compatibility. 12 However, the UV sensitizer that allows photo-crosslinking of IP-DIP fluoresces in the visible spectrum and subsequently interferes with visualization of fluorescently stained cells on the final structures. Therefore, OrmoComp® is preferred. Biological studies using cell culture is a well-established field and it is known that neurons do not adhere well to untreated glass or plastic. Instead, the substrate is typically prepared for seeding with cells by modification with poly-lysine (D or L), to introduce a positive charge and increase the initial cell adhesion, and laminin to influence cell differentiation, migration, and also improve adhesion. The cost of these reagents and time required for their application are not inconsequential, but their application is necessary to make surfaces permissible for cell culture experiments. Importantly, treatment with PDL-laminin is a bulk coating, meaning the whole substrate or device surface is covered with the same chemistry and cells therefore can grow anywhere on the surface. If one desired to control the locations of cells on a PDL-laminin coated surface the architecture of the underlying substrate would need to influence cell adhesion or the surface would need to be patterned such that the PDL-laminin coating was excluded from specific areas. 13 Either solution introduces significant complexity to the experimental design. Here we offer an alternative approach to bulk coatings, rendering the OrmoComp® permissible for neuron adhesion and growth by reacting it with diamines to mimic the PDL-laminin treatment coating by presenting amine groups as the pendent surface chemistry after modification. This manuscript describes the applications of and principles behind this new surface treatment. Results and Discussion Using two-photon 3D lithography we can arbitrarily write architectures that are specifically designed for our experiment. As described earlier, we chose to use the Nanoscribe instrument with structures built from OrmoComp®, however, there was little adhesion of E18 murine cortical neurons to the bare OrmoComp® structures (Figure 1a). Treatment of the OrmoComp® structure with PDL-laminin allowed for neuronal cell adhesion and growth, however growth was not limited to the OrmoComp® structures, but also occurred on the surrounding glass substrate (Figure 1b). However, if we performed a Michael addition with 1,3 propane diamine the OrmoComp® structures were rendered permissive for neuronal cell adhesion and growth with no additional PDL-laminin treatment (Figure 1c). Furthermore, cell adhesion was limited to the OrmoComp® structures with more adherent cells and more robust neurite outgrowth than either PDL-laminin treated OrmoComp® structures or glass substrates (Figure 1b and c). Michael addition of α,β unsaturated carbonyls and esters are common reactions that link a nucleophile to the β carbon (Supporting Information, Figure S1a). Under the appropriate conditions and moderate temperature any primary or secondary amine can undergo Michael addition. 14,15 We reacted diamines (Supporting Information, Figure S1b) with OrmoComp’s® free acrylate groups to provide a surface that would enhance cellular adhesion and growth (Supporting Information, Figure S1c). The reaction is conducted at room temperature to reduce side reactions considered to be detrimental (Supporting Information, Figures S1b and S1c). Page 2 ACS Paragon Plus Environment

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With this chemistry, selective growth of neurons on OrmoComp® occurred (Figure 1c). Importantly, this biocompatibility was achieved without PDL-laminin treatment. It instead mimics that coating by presenting amine groups as the pendent surface chemistry after modification. FTIR spectra of developed OrmoComp® structures (Figure 2) showed bands consistent with free acrylate groups that have not been reacted during the polymerization. We extensively explored both the conditions for the polymer development and post-development washing to remove any unpolymerized monomer, but the presence of free acrylate groups persist. In fact, the persistence of free acrylates appears to be common to this class of photopolymers; FTIR data with IP-DIP also has evidence of free acrylate bands post-development (Supporting Information, Figure S2). b.

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Figure 1 – Fluorescent images of neuronal cells on two-photon 3D written ring structures. These fluorescent images are falsely colored to represent the green emission of the calcein-AM dye. (a) Neuronal cell culture showing poor cell adhesion and almost no neurite extension to the OrmoComp® structure written with the Nanoscribe. (b) Neuronal cell culture with PDL-laminin coating both the OrmoComp® structure and surrounding glass substrate showing cell adhesion and neurite extension equally across both surfaces. (c) Neuronal cell culture on an OrmoComp® structure reacted with 1,3-propane diamine showing improved cell adhesion, in the form of higher cell density, and increased neurite extension and complexity relative to the PDL-laminin treated structure. There is little cell adhesion on the surrounding glass substrate in this case even though the entire surface was exposed to the same chemistry. 100

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Figure 2 – FTIR of OrmoComp structures reacted with 1,2 ethane diamine (EDA). OrmoComp® was spin coated on glass coverslips and cross-linked under UV illumination. The surface was reacted with varying amounts of EDA and FTIR spectra collected. The blank shows the expected stretching of an acrylate functionality (1560 cm-1) that decreases upon reaction with increasing amounts of EDA. The inset shows the shift of the ester bands from 1725 cm-1 to 1731 cm-1 after the reaction. The cause ®

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of this reproducible shift in the peak maximum is unknown. This peak is characteristic of ester stretching vibrations. However, the ester groups on the rest of the polymer chain should not be affected by the residual acrylate groups. Other functionalities are identified in the Supporting Information, Figure S3.

To better understand this chemical modification and its influence on cell culture experiments, it became necessary to make many more OrmoComp® structures. Unfortunately two-photon nanolithography has throughput limitations, so we employed more conventional photolithography with OrmoComp® spin coated on top of glass coverslips. Figure 2 shows the reaction of an OrmoComp® film with different amounts of 1,2 ethane diamine showing the loss of the acrylate group and the formation of other functionalities, including amine and amide bands. The IR assignments are more fully discussed in Supporting Information, Figure S3. After reaction with diamine, the acrylate groups are removed and the surface becomes positively charged; this mimics a surface coated with poly-lysine, presenting a pendent amine group, which enhances cell adhesion. The diamine treatment works with a number of different molecules spanning from ethane diamine to hexane diamine (Supporting Information, Figures S4-S7). Although there appeared to be preferable concentrations for neuron growth for each diamine (more experiments are need to determine an optimal concentration), there appeared to be little difference between modified surfaces in terms of overall neuron growth and axon or dendrite extension. As ethane and propane diamine are liquids and easy to handle and purify, we have concentrated our efforts on surface treatments using these reagents. The most important aspects of this approach are that the researcher has design control over the number of amines present on the final structure, the lengths of the attached diamine backbone, and the final surface charges because the acrylate groups will react first before amide formation with the esters (Supporting Information, Figure S1). We also note that the treatment is a covalent modification and robust as evidenced by strong cell growth for up to two weeks as tested in the cell culture experiments (Supporting Information, Figure S8). That the diamine modification treatment renders OrmoComp® permissive for cell growth is but one advantage offered for designing advanced biological experiments. Equally important, the chemical modification of OrmoComp® occurs in deference to the glass substrate upon which the arbitrary architecture is written. Figure 3 demonstrates the directed growth of the neuronal cells on OrmoComp® structures and not the surrounding glass substrate. A shadow mask with varying pad dimensions was used to define squares of three different dimensions. The neurons prefer the chemically modified OrmoComp® surface showing strong growth on all structures. Additionally we see evidence that diamine treatment may help direct growth and extension along lithographically defined structures. Figure 4 shows a close up view of an OrmoComp® surface with 5 µm lines connecting the individual pads. In the fluorescence image, neurons are clearly extending along the connecting lines. The limits of this guidance is a matter of further study, but the directional growth has been seen on many surfaces and across wide areas (Supporting Information, Figure S12 is a larger scale image of the same surface as Figure 4). Lastly, we note that the orthogonal derivatization of the substrate can be extended to chemistries attached specifically to the glass substrate. Starting with a modified OrmoComp® substrate, we have subsequently treated the surface with a fluorinated silane, thereby coating the glass areas. XPS data of that surface evidenced fluorine on both the glass areas and modified OrmoComp® pads (Supporting Information, Figure S9b) but this did not appear to inhibit neuronal cell growth. Alternatively, the surface can be treated with a fluorinated anhydride and react only with the polymer and not the glass surface (Supporting Information, Figure S9a), which blocks the amines but not the glass surface and stops neuronal attachment.


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Figure 3 – Orthogonal neuronal cell culture on EDA modified OrmoComp® pads. Fluorescent image of cells grown on modified EDA OrmoComp® pads. The substrate was photolithographically patterned with 2800 µm, 200 µm, and 100 µm squares. Note the differential growth of neurons on the treated polymer pads compared to the surrounding glass areas. Because PDL-Laminin is not needed to grow cells on these surfaces, the glass being untreated, has no cell adherence or growth. The slightly varying background is an artifact of the stitching process used in gathering the high-resolution images. A further example of orthogonal growth is shown in the Supporting Information, Figure S11.

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Figure 4 – Directed growth of neuronal cells along pad interconnections. (a) Fluorescence image of neuronal cells grown on photolithographically defined pads, with 5 µm interconnecting lines between pads. In this example, there is little growth on the glass surface and the neurons appear to develop axons and dendrites along some of the polymer traces between pads. An expanded image of this surface is shown in the Supporting Information, Figure S12. It is not known if this directed growth is the result of the chemistry or the topography. (b) The portion of the photomask used in patterning the polymer pads corresponding to the area in Figure4a.



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Orthogonal neurite growth can be achieved using microprinting for selective modification but that is difficult to achieve on the micron scale. 16 Our technique, of making OrmoComp® cell adhesion permissive, will allow selective patterning during the fabrication process. Additionally, coupled with selective photo patterning through photo deprotection 17,18,19 after fabrication and derivatization should allow even more flexibility of directing neuronal growth on the micron scale as protecting groups may be removed selectively. This aspect will be explored in a subsequent paper. Conclusion We have developed a new surface modification that renders photopolymerized acrylate polymers permissive for neuronal cell adhesion and growth. In particular this treatment can be applied to OrmoComp®, a polymer commonly employed to print arbitrary shapes with twophoton 3D nanolithography. Our modification results in pendent amine groups, mimicking classical cell culture surface preparations. Importantly, our treatment is flexible allowing the researcher the ability to control the number and type of diamine attached to the 3D polymer structure. Equally important the treatment is an orthogonal modification meaning the polymer surface and surround glass substrate can be modified in deference to each other. In total, our approach empowers the modern biological research with several new tools for advanced study in cell culture experiments. Materials and Methods OrmoComp® ring structures written with Nanoscibe Small volumes of OrmoComp photoresist were dropcast onto 170 µm thick glass coverlips that had been previously coated with roughly 15 nm Al2O3 by means of atomic layer deposition. This was necessary to create a refractive index difference between the polymer and the substrate, a requirement for the Nanoscribe tool in order to locate the interface along the z optical axis. The tilt of the substrate relative to the plane of stage motion was calculated over the patterned area from measurements of the relative displacement of the surface, and this was used for dynamic a priori correction in z on a frame-by-frame basis. The typical planar skew was such that the displacement across a mm structure was considerably greater than the structure thickness, and measurements at each frame would have considerably extended the write time. Structures were written layer by layer, comprised of adjacent 140 µm square frames. Several variations of scaffold patterns were investigated for the highest yield neuron growth, and the most successful was found to be three layers of crossed wires, the uppermost being ½ micron wires on a 2 micron pitch. The overall layer thickness was about two µm. After pattern writing, the cover slip was removed and developed in multiple sequential baths of MIBK:IPA in a 1:1 ratio followed by IPA. Longer soak time of at least two weeks appeared to allow less toxicity. Substrates were then transferred wet into a CO2/IPA critical point drier apparatus to minimize the effects of surface tension, which would otherwise collapse the fine structures. Following drying, the samples were flood-exposed to 4.5 J/cm2 of 360 nm wavelength light, and baked on a 150 °C hotplate in air for final hardening and drying.


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OrmoComp® test structures fabricated with photolithography Borosilicate, square 18 mm glass coverslips were plasma cleaned in oxygen plasma for 10 min (YES G500 plasma generator, RF 135 set point and 10 SCC/min O2, Yield Engineering System, Inc.). Neat OrmoComp® was spin coated at: RPM Rate Time (RPM/Sec) (sec) 1000 20 80 5000 100 60 A film thickness of about 14.5 µm was calculated from etalons in the transmittance FTIR spectrum measured by coating on undoped silicon (used to transmit IR light). The thickness was confirmed by profilometry on other coupons. For thinner coatings, the OrmoComp® may be diluted 1:1 in acetone. A higher boiling solvent, such as anisole may provide a more uniform coating. As uncured OrmoComp® is sticky, contact printing must be avoided, which limits the resolution of each feature. For easy mask exposures, the spin coated coverslips were placed on a 2x4” glass slide and two sets of coverslips, to act as spacers for the mask, were placed on each end. As, we were more interested in large patterns, a standard chrome photomask on soda-lime glass was designed in EAGLE layout editor and commercially produced at a 5 µm resolution. The slides were exposed in a Karl Suss MJB3 at 15 mW/cm2 lithography system for 3-6 min with 4 min producing an optimal pattern. They were developed in 1:1 IPA:MIBK at room temperature for 5 min. Longer development times did not produce better patterns or less cellular toxicity (by extracting potentially toxic, non-polymerized materials). Procedure for Reaction of Polymers with Diamines The OrmoComp® samples were placed in 29 x 65 mm dram vials and 6 mL of isopropanol (IPA) added. Varying amounts of diamine were added as indicated in the figures. For example, EDA 50 is prepared by adding 50 µL of 1,2-ethane diamine to 6 mL of IPA forming a 0.12 M solution of EDA in IPA. The diamine abbreviations are: EDA  1,2-ethane diamine PDA  1,3-propane diamine BDA  1,4-butane diamine HDA  1,6-hexane diamine Butane diamine and hexane diamine are solids at RT and are melted on a hot plate before dissolving in IPA. EDA was distilled before use to ensure purity. The other diamines were used as received. The vial is sealed with a plastic cap and placed on a rocker. The reaction was allowed to proceed at room temperature typically 16-24 hr. Next, the coupon was rinsed 2x with IPA, 2x with dH2O, and stored in IPA before cell growth. Cell growth did not appear to depend upon how long the samples are stored in IPA. OrmoComp® biocompatibility Although OrmoComp®, IP-DIP, and related acrylate polymers are reported to be biologically compatible, 20,21,22,23,24,25,26 our initial attempts to grow primary neuronal cells on OrmoComp® with or without PDL-laminin treatment showed clear evidence of toxicity. Ultimately, this toxicity was eliminated by careful preparation of the OrmoComp® surfaces, whether they were produced with the Nanoscribe or through spin coating. Preparation included long exposure to UV light to maximize polymerization within the OrmoComp®, followed by soaking in isopropyl alcohol for 37 days to leak out any left-over solvents or unpolymerized monomer that might be responsible for the toxicity to the cells. The combination of these two steps reduced or eliminated the toxicity initially noted in the OrmoComp® surfaces. Page 7 ACS Paragon Plus Environment


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Neuronal cell culture Glass coverslips, with or without diamine modified OrmoComp®, were ethanol sterilized by briefly rinsing in 70% ethanol bath, soaking in 2 mL of 70% ethanol for 15 min., removing the ethanol, and letting the coverslips air dry. For experiments where the growth surfaces were prepared with PDL and laminin, the procedure was as follows. Sterilized coverslips were pretreated with 50 µg/mL PDL (Sigma-Aldrich, St. Louis, MO) by placing 100 µL drops in the center of the coverslips for 1 hour, rinsing once with water, and allowing to air dry. Next, 100 µL drops of 20 µg/mL laminin (Sigma-Aldrich) in Dulbecco’s PBS (without Ca2+/Mg2+; Thermo Fisher Scientific Inc., Waltham, MA) were placed at the center of the coverslips for 1 hour, then removed and the coverslips air dried. Coverslips were prepared on the same day as the cell culture and placed in 35 mm dishes. Microsurgically dissected E18 murine cortices were obtained commercially (C57/BL6 mouse; BrainBits LLC, Springfield, IL) and stored at 4°C until cultured. Cortices were mechanically dissociated, centrifuged at low speed, and resuspended in serum-free Neurobasal media supplemented with B27 and GlutaMAX (NB/B27; all components Thermo Fisher Scientific Inc.). Cells were counted and seeded at 1x105 cells/coverslip (100 µL of 1x106 cells/mL) onto the prepared coverslips. The dishes with the coverslips were then placed in a 37°C/5% CO2 incubator for 1 hour prior to flooding with 2 mL of NB/B27 media. Cultures were maintained at 37°C/5% CO2 for 4-14 days before imaging. Microscopy The neurons were stained with calcein-AM (Life Technologies, Grand Island, NY), a cell permeant dye that stains live cells after the acetoxymethyl ester is cleaved by intracellular esterases. Media was removed from the dishes and replaced with Dulbecco’s 1X PBS (with Ca2+/Mg2+; Thermo Fisher Scientific Inc.). Calcein-AM was added to the PBS for a final concentration of 5 µM. The dishes were returned to the 37°C/5% CO2 incubator for 30 min. prior to imaging. Cells were imaged using an EVOS FL Auto microscope (Thermo Fisher Scientific Inc.) with either a 10X or 20X objective. For imaging of large areas of the coverslips, multiple images were taken of the region of interest in a grid pattern using the EVOS software’s built in routines. The resulting image sets were processed in Fiji 27 by applying a logarithmic map and gamma adjustments to enhance the fine details of the neurites as well as background subtraction to clean up the images. Multiple images were stitched together using the Grid/Collection Stitching plugin 28 in Fiji. Given this processing, staining intensity should not be interpreted; staining was done solely to visualize the cells on the substrates. Supporting Information Potential reactions of diamines with acrylate groups FTIR characterization of residual reactive acrylates in various polymers Toxicity of OrmoComp® films after reaction with differing amounts of EDA, PDA, BDA, and HDA XPS data showing reactivity of functionalized polymer Close-up and areas images of neuronal cell culture on lithographically defined OrmoComp® treated with EDA-50 OrmoComp® on borosilicate coverslips Conflict of Interest Disclosure DAK and TJO have filed a Provisional Patent on the use of diamines to derivatize acrylate polymers. The additional authors declare no competing financial interest. Page 8 ACS Paragon Plus Environment

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Funding This work was supported by the Office of Naval Research under work unit 61-1E23. AUTHOR INFORMATION Corresponding Author *Email: [email protected] ORCID David Kidwell: 0000-0002-2690-971X Woo-Kyung Lee: 0000-0002-8235-5336 Jeremy Robinson: 0000-0001-8702-2680 Keith Perkins: 0000-0003-1404-7590 Paul Sheehan: 0000-0003-2668-4124 Shawn Mulvaney: 0000-0003-2931-6079 References 1

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Obi, S. Replicated Optical Microstructures in Hybrid Polymers: Process Technology and Applications. Doctor of Science Dissertation, Institute of Microtechnology, University of Neuchâtel, Neuchâtel, Switzerland 2006. 12 V.; Brandalise, R.N.; Savans, M. Engineering of Biomaterials, Topics in Mining, Metallurgy and Materials Engineering, Springer, 2017, pp. 5-15. DOI 10.1007/978-3-319-58607-6 13 Chen, H.; Yuan, L.; Song, W.; Wu, Z.; Li, D. Biocompatible Polymer Materials: Role of Protein–Surface Interactions. Prog. Polym. Sci. 2008, 33(11),1059–1087. 14 Escalante, J.; Carrillo-Morales, M.; Linzaga,I. Michael Additions of Amines to Methyl Acrylates Promoted by Microwave Irradiation. Molecules 2008, 13, 340-347. 15 Hallden-Abberton, M.P. The Preparation of Methyl Methacrylate/Methacrylic Anhydride Copolymers from PMMA and Dialkyl Amines via Reactive Extrusion, in G. Swift, C.E. Carraher,Jr., and C.N. Bowman (eds.), Polymer Modification, Springer Science+Business Media New York 1997, pp. 3-9. 16 Hamid, Q.; Wang, C.; Snyder, J.; Williams,S.; Liu, Y.; Sun, W. Maskless Fabrication of CellLaden Microfluidic Chips with Localized Surface Functionalization for the Co-Culture of Cancer Cells. Biofabrication 2015, 7(1), (015012). doi: 10.1088/1758-5090/7/1/015012 17 Cheng, N.; Cao, X. Photosensitive Chitosan to Control Cell Attachment. J. Colloid Interface Sci. 2011, 361(1), 71-78. https://doi.org/10.1016/j.jcis.2011.05.045 18 Wang, B.; Zheng, A. A Photo-Sensitive Protecting Group for Amines Based on Coumarin Chemistry. Chem. Pharm. Bull. 1997, 45(4), 715-718. doi: 10.1248/cpb.45.715 19 Bhatnagar, P. Malliaras, G.G.; Kim, I.; Batt, C.A. Multiplexed Protein Patterns on a Photosensitive Hydrophilic Polymer Matrix. Adv. Mater. 2010, 22(11),1242–12. DOI: 10.1002/adma.200903255 20 Qiu, F.; Zhang, L.; Peyer, K.E.; Casarosa, M.; Franco-Obregón, A.; Choie, H.; Nelson, B.J. Noncytotoxic Artificial Bacterial Flagella Fabricated from Biocompatible ORMOCOMP and Iron Coating. J. Mater. Chem. B 2014, 2, 357-362. DOI: 10.1039/C3TB20840K 21 Nemani, K.V.; Moodie, K.L.; Brennick, J.B.; Su, A.; Gimi, B. In vitro and In Vivo Evaluation of SU-8 Biocompatibility. Mater Sci Eng C Mater Biol Appl. 2013, 33(7), 4453-4459. doi: 10.1016/j.msec.2013.07.001 22 Vernekar, V.N.; Cullen, D.K.; Fogleman, N.; Choi, Y.; García, A.J.; Allen, M.G.; Brewer, G.J.; LaPlaca, M.C. SU-8 2000 Rendered Cytocompatible for Neuronal BioMEMS Applications. J. Biomed Mater Res A. 2009, 89(1) 138-151. doi: 10.1002/jbm.a.31839 23 Hamid, Q.; Wang, C.; Snyder, J.; Sun, W. Surface Modification of SU-8 for Enhanced Cell Attachment and Proliferation within Microfluidic Chips. J. Biomed. Mater. Res. B Appl. Biomater. 2015, 103(2), 473-484. doi: 10.1002/jbm.b.33223. 24 Doraiswamy, A.; Patz, T.; Narayan, R.; Chichkov, B.; Ovsianikov, A.; Houbertz, R.; Modi, R.; Auyeung, R.; Chrisey, D.B. Biocompatibility of CAD/CAM ORMOCER Polymer Scaffold Structures. In Materials Research Society Symposium Proceedings, Vol. 845, Symposium AA – Nanoscale Materials Science in Biology and Medicine, Boston, MA, United States. Warrendale, PA: Materials Research Society 2005, pp. 51–56. doi: 10.1557/PROC-845-AA2.4 25 Käpylä, E.; Sorkio, A.; Teymouri, A.; Lahtonen, K.; Vuori, L.; Valden, M.; Skottman, H.; Kellomäki, M.; Juuti-Uusitalo, K. Ormocomp-Modified Glass Increases Collagen Binding and Promotes the Adherence and Maturation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells. Langmuir 2014, 30(48), 14555−14565. doi.org/10.1021/la5023642 26 Schlie, S.; Ngezahayo, A.; Ovsianikov, A.; Fabian, T.; Kolb, H.A.; Haferkamp, H.; Chichkov, B.N. Three-Dimensional Cell Growth on Structures Fabricated From ORMOCER® by TwoPhoton Polymerization Technique. J. Biomater. Appl. 2007, 22(3), 275–287. 27 Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig,V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; Tinevez, J-Y.; White, D.J.; Hartenstein, V.; Eliceiri, K.; Tomancak, P.; Cardona, A. Fiji An Open-Source Platform for Biological-Image Analysis. Nat. Methods 2012, 9(7), 676-682. Page 10 ACS Paragon Plus Environment

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Preibisch, S.; Saalfeld, S.; Tomancak, P. Globally Optimal Stitching Of Tiled 3D Microscopic Image Acquisitions. Bioinformatics 2009 25(11), 1463-1465.



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TOC Graphic

Pendant Acrylate

O

O + H N ( )n 2

NH2

Cell N ( )n NH2 Permissive H


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Figure 1 – Fluorescent images of neuronal cells on two-photon 3D written ring structures. These fluorescent images are falsely colored to represent the green emission of the calcein-AM dye. (a) Neuronal cell culture showing poor cell adhesion and almost no neurite extension to the OrmoComp® structure written with the Nanoscribe. (b) Neuronal cell culture with PDL-laminin coating both the OrmoComp® structure and surrounding glass substrate showing cell adhesion and neurite extension equally across both surfaces. (c) Neuronal cell culture on an OrmoComp® structure reacted with 1,3-propane diamine showing improved cell adhesion, in the form of higher cell density, and increased neurite extension and complexity relative to the PDL-laminin treated structure. There is little cell adhesion on the surrounding glass substrate in this case even though the entire surface was exposed to the same chemistry. 135x44mm (150 x 150 DPI)

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Figure 2 – FTIR of OrmoComp® structures reacted with 1,2 ethane diamine (EDA). OrmoComp® was spin coated on glass coverslips and cross-linked under UV illumination. The surface was reacted with varying amounts of EDA and FTIR spectra collected. The blank shows the expected stretching of an acrylate functionality (1560 cm-1) that decreases upon reaction with increasing amounts of EDA. The inset shows the shift of the ester bands from 1725 cm-1 to 1731 cm-1 after the reaction. The cause of this reproducible shift in the peak maximum is unknown. This peak is characteristic of ester stretching vibrations. However, the ester groups on the rest of the polymer chain should not be affected by the residual acrylate groups. Other functionalities are identified in the Supporting Information, Figure S3. 235x116mm (150 x 150 DPI)


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Figure 3 – Orthogonal neuronal cell culture on EDA modified OrmoComp® pads. Fluorescent image of cells grown on modified EDA OrmoComp® pads. The substrate was photolithographically patterned with 2800 µm, 200 µm, and 100 µm squares. Note the differential growth of neurons on the treated polymer pads compared to the surrounding glass areas. Because PDL-Laminin is not needed to grow cells on these surfaces, the glass being untreated, has no cell adherence or growth. The slightly varying background is an artifact of the stitching process used in gathering the high-resolution images. A further example of orthogonal growth is shown in the Supporting Information, Figure S11. 134x99mm (150 x 150 DPI)



Figure 4 – Directed growth of neuronal cells along pad interconnections. (a) Fluorescence image of neuronal cells grown on photolithographically defined pads, with 5 µm interconnecting lines between pads. In this example, there is little growth on the glass surface and the neurons appear to develop axons and dendrites along some of the polymer traces between pads. An expanded image of this surface is shown in the Supporting Information, Figure S12. It is not known if this directed growth is the result of the chemistry or the topography. (b) The portion of the photomask used in patterning the polymer pads corresponding to the area in Figure4a. 210x100mm (150 x 150 DPI)


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Chemistries for Making Additive Nanolithography in OrmoComp

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