Patterned Cellulose Nanocrystal Aerogel Films with Tunable

Article Cite This: ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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Patterned Cellulose Nanocrystal Aerogel Films with Tunable Dimensions and Morphologies as Ultra-Porous Scaffolds for Cell Culture Tyler Or,† Sokunthearath Saem,† Aurore Esteve,‡ Daniel A. Osorio,§ Kevin J. De France,§ Jaana Vapaavuori,¶ Todd Hoare,§ Aline Cerf,‡ Emily D. Cranston,§,# and Jose M. Moran-Mirabal*,† Downloaded via 81.22.47.25 on July 22, 2019 at 01:23:52 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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Department of Chemistry and Chemical Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada ‡ LAAS-CNRS, Université de Toulouse, CNRS, Toulouse 31400, France § Department of Chemical Engineering, McMaster University, Hamilton, Ontario L8S 4M1, Canada ¶ Départment de Chimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Quebec H3C 3J7E, Canada S Supporting Information *

ABSTRACT: Aerogel films are interesting as coatings due to their unique properties including high surface area, sorption capacity and insulating properties. To date, silica-based aerogel films have been most widely explored due to their ultrahigh surface areas and wellknown chemistry. However, the fragile nature of silica aerogels coupled with the limited control over film thickness and dimensions when using traditional deposition techniques limits their use in applications requiring films with good mechanical stability (e.g., in flexible devices). To address these challenges, we present a pressureaided freeze casting method to pattern, on a variety of substrates (e.g., glass or flexible polyethylene terephthalate), mechanically robust aerogel films composed of covalently cross-linked cellulose nanocrystals (CNCs) with controlled dimensions and internal morphology. To accomplish this, a film of the desired aerogel thickness was deposited on the substrate and a mold with the specific shape for the aerogel was fabricated by xurography (>1 mm lateral dimensions, 7−85 μm thickness) or photolithography (2−500 μm lateral dimensions, 3 μm thickness). An aqueous gel of reactive CNCs or CNCs with poly(oligoethylene-glycol-methacrylate) was drop cast onto the substrate, and pressure was applied so that the gel adopted the mold shape. The gel was subsequently frozen and lyophilized, and the mold was lifted off the substrate, leaving behind patterned porous aerogel films, which were first explored as cell culture scaffolds. Human prostate cancer cells strongly adhered to the aerogels, where individual cells could be isolated on small aerogel arrays while cell clusters were obtained on larger arrays. This system has potential applications in studying single-cell phenotype and developing miniaturized cell-based assays. The simplicity of this freeze casting and lift-off patterning technique makes it attractive for the fabrication of cellulose-nanocrystal-based aerogels with a variety of compositions for applications requiring materials with high surface area, low density, and good mechanical stability. KEYWORDS: micropatterning, freeze casting, microporous materials, hierarchically porous, flexible, CNC, POEGMA, cell scaffold

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INTRODUCTION Aerogels are dry, ultralow-density, and highly porous monolithic materials derived from a gel composed of nanoparticles or polymers that physically or chemically crosslink to form a three-dimensional network. During fabrication, gelation is achieved through reactive surface groups that form covalent, electrostatic, or hydrogen bonds, after which the gel is carefully desolvated using a method that preserves to the greatest extent possible the internal porous morphology (e.g., critical point drying, CPD; lyophilization).1 Their exceptional properties, such as high-surface-area-to-volume ratio, high © XXXX American Chemical Society

thermal insulation, high acoustic dampening, superabsorbent ability, and low dielectric constant make aerogels ideal for a broad range of applications, where high porosity, high surface areas, and low densities are required.1−3 While bulk/macroscopic aerogels with ultrahigh surface area made from different materials have been reported for a variety of applications, there has been considerably less work on Received: April 6, 2019 Accepted: June 13, 2019 Published: June 13, 2019 A

DOI: 10.1021/acsanm.9b00640 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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ACS Applied Nano Materials realizing the potential of aerogel films.2 This is in part because of the difficulty in applying the methods used to produce bulk aerogels (e.g., casting, followed by CPD or subcritical drying with additives) toward the fabrication of thin films, where the stress during the casting and drying process can lead to cracking of the fragile material. The methods to reported to date have fabricated aerogel films from metal oxides (often silica or titania) and have had limited control over the aerogel dimensions (thickness or lateral size).2,4−6 Silica aerogel films fabricated using dip, spray, spin, and surface tension coating techniques have resulted in crystalline films with nanometersized pores and mostly submicron-scale thickness.2,7−9 The predominant challenge in these fabrication approaches has been ensuring that solvent evaporation is limited throughout deposition and prior to drying. To address this, strategies have been developed to perform the coating step under conditions, where the environment is saturated with solvent vapor or have modified the silica with organosilyl groups to resist capillary tension.10 Nevertheless, the silica aerogel films produced through these methods remain challenging to work with, since the high porosity and crystalline arrangement of the material carries with it the trade-off of increased fragility. To improve the mechanical properties and the interconnectivity between the pores, metal oxide mesoporous films have been produced through sol−gel synthesis approaches, where organic materials (e.g., surfactants, polymer beads) are added to the mixture as sacrificial pore templates.11,12 Using this approach, Zhao et al. fabricated continuous silica mesoporous films by dip-coating, followed by solvent drying and calcination to remove the organic templates.13 This resulted in films with thicknesses of 300−1000 nm, pore sizes of 40−90 Å, and porosities in the 51−75% range. Using similar fabrication approaches, patterned mesoporous silica thin films with high surface areas and nanometer scale pore sizes have also been reported. In one implementation, Fan et al. first created a mesoporous thin film (100−200 nm thickness) through a block-polymer-templated sol−gel process followed by calcination, and then etched part of the material away to produce patterned mesoporous silica strips with ∼8 nm aligned pores.14 Similarly, surfactant-templated silica microdots have been fabricated through electrochemically driven nanolithography15 or inkjet printing,16 where the lateral dimension was controlled to a limited extent by the size of the ultramicroelectrode and duration of the electrochemical deposition step or by the size of the printed droplet and evaporation profile. While these approaches were successful in patterning mesoporous silica thin films, they required complex instrumentation, expensive lithographical techniques or afforded limited control over the patterned thin film dimensions (i.e., lateral size and thickness). Given that high-surface-area low-density films have been identified as attractive materials for integrated circuits,17 sensing and filtration membranes, insulating coatings,2 and tissue engineering scaffolds and in view of the limitations of silica aerogels (i.e., brittleness, fragility), the ability to produce mechanically robust aerogel films with tunable dimensions and properties could significantly increase their range of applications. Cellulose nanocrystals (CNCs) are rigid needle-shaped nanomaterials (with length and diameter for wood-derived materials in the 100−200 nm and 5−20 nm range, respectively) typically produced through the sulfuric acid hydrolysis of cellulose fibers.18 They are attractive nanoscale “building blocks” due to their nontoxicity, biodegradability,

large scale production, low cost, and renewable origin. The breadth of potential applications for CNCs stems from their high aspect ratio, rigidity (elastic modulus of 100−150 GPa),18 and the presence of surface hydroxyl groups that enable the grafting and physical adsorption of functional materials such as fluorophores, sensing molecules, nanoparticles, and conductive polymers.19 Recently, CNCs have been introduced as fillers to reinforce polymeric matrices in aerogels or as the main components that make up the aerogel.20 Like traditional silica aerogels, CNC-based aerogels have superabsorbent properties, ultralow densities, and high specific surface areas. However, they are also elastic and compressible, which is compatible with applications requiring mechanical robustness.21 For instance, contrary to silica alcogel networks that cannot withstand the crystallization of solvent within pores and thus have to be processed using CPD,22 CNC-based aerogels can be readily prepared by lyophilization or even simple solvent drying.23,24 This has opened doors for the use of CNC-based aerogels as scaffolds for metal−organic framework water purification devices,23 3D supercapacitors,25 and tissue engineering,26 where the high porosity and flexibility are critical for functionality. We have previously demonstrated that highly porous, lightweight, and mechanically robust aerogels with tunable morphology can be created from covalently cross-linked CNCs (“all-CNC” aerogels)21,24,25,27 or a composite of CNC and poly(oligoethylene glycol methacrylate) (“CNC-POEGMA” aerogels).28,29 To form covalent cross-links, the constituent species (CNCs with or without POEGMA) were surface modified with reactive aldehyde or hydrazide groups. When brought into close proximity, the reactive groups can form hydrazone bonds, creating a stable 3D structure. The gel thus formed is entirely aqueous, which eliminates concerns over toxicity and rapid solvent volatilization as seen in silica alcogels.2 In addition, the hydrazone chemistry used to crosslink the components can be performed without the need for UV/heat and additional cross-linker or initiator material, which eliminates time-consuming steps, such as solvent exchange to purify the gels.30 The CNC-based gels can then be frozen and lyophilized or simply dried, resulting in ultraporous CNCPOEGMA or all-CNC aerogels.21 These aerogels demonstrate a hierarchical pore structure containing macropores (∼50 nm to tens of micrometers, in diameter) templated from the growth of ice crystals (freeze casting) and mesopores (2−50 nm) formed by the cross-links between individual CNCs or POEGMA chains. Thus, CNC-based aerogels are easy to produce and provide a versatile system for various applications. For instance, the bulk and cross-link density of the aerogel can be easily controlled by varying the concentration of CNC or POEGMA, as well as by changing the aldehyde/hydrazide content. The mechanical strength and porosity of CNCPOEGMA aerogels can be improved by increasing the CNC to POEGMA ratio. However, if a compressible aerogel is desired, the relative amount of POEGMA can be increased. Because of the nanoscopic dimensions of CNCs, all-CNC aerogels have a high specific surface area (190−320 m2/g, dependent on the fabrication method) comparable to that of silica mesoporous aerogels (highest reported values ∼1000 m2/g).21,27 In this work, we developed and optimized a simple benchtop approach to pattern all-CNC and CNC-POEGMA aerogel films with controlled millimeter to micrometer lateral dimensions and thickness in the micrometer range. To our knowledge, these are the first CNC-based aerogel films B


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ACS Applied Nano Materials

Figure 1. Aerogel fabrication. (A) Schematic of the pressure-aided freeze casting procedure. Red arrows represent steps only necessary when a Parylene mold is used. Aerogel films were fabricated using sol−gel concentrations ranging from 0.3 to 3.5 wt %. An excess amount of gel was drop cast to fill the mold. Pressure was applied using a 100 g weight (Figure S4) to fill the mold and expel excess gel. (B) Schematic of the photolithographic patterning protocol. (i) Parylene is deposited on the substrate. (ii) Photoresist layer spin-coated. (iii) Patterns on photoresist created using a UV mask. (iv) Oxygen plasma etches both Parylene to create patterns. Excess photoresist on Parylene is removed with acetone and isopropanol rinses. (C) Aerogel films fabricated into various patterns on a glass substrate using a 7 μm-thick Parylene mold.

a 1:1 mass ratio of hPOEGMA:aCNC or hCNC:aCNC, unless otherwise specified. Aerogel film fabrication was performed on various substrates ranging in wettability. Figure 1C shows a particular case of CNC-POEGMA and all-CNC aerogel film patterns on glass substrates. Successful fabrication of the patterned aerogels required extensive optimization. Substrate preparation prior to casting was key to the reproducible patterning of the films. Glass substrates were cleaned using Piranha solution to remove organic residues and any other contaminants that interfered with casting (Figure S1A). An additional advantage of the Piranha treatment was that it strongly oxidized the surface, creating silanol and siloxide groups that increased the surface hydrophilicity and allowed the gel to uniformly wet the substrate. This treatment improved the uniformity of the films and reproducibility of the results. For substrates incompatible with Piranha treatment (i.e., plastics and metals), mild oxidation using air plasma or UV−ozone achieved a similar effect. The casting reproducibility was enhanced by coating a thin layer of poly(allylamine hydrochloride) (PAH) onto the substrate, which promoted electrostatic interactions with the sulfate half-ester groups on CNCs and served as an anchoring layer for the aerogel.31 This was especially useful in substrates where the aerogels did not adhere well, such as fluorine-doped tin oxide (FTO)-coated glass, flexible indium-doped tin oxide (ITO)-coated poly(ethylene) terephthalate (PET), and Ptcoated PET (Figure S2). The aerogel dimensions were tailored by fabricating a mold that matched the desired aerogel thickness and patterned lateral dimensions. Adhesive tape molds are accessible and straightforward to work with. However, their thickness is limited based on manufacturer availability. Adhesive molds may also leave residue on the substrate after lift-off, which can be mitigated by using specialty tapes, such as UV-release tape. Parylene molds produced through chemical vapor deposition

demonstrated to date. The morphology and mechanical strength of the aerogel can be tailored by altering the sol− gel concentration, composition, and freezing kinetics. As scaffolding for cell culture and tissue engineering is a particularly promising application of CNC-based aerogels, in this work we demonstrate a proof-of-concept with CNCPOEGMA aerogel films and human prostate cancer epithelial cells (PC3). The aerogel surface porosity and nanoscale roughness promoted strong cell adhesion. With this new patterning approach, we have also fabricated aerogel microarrays (5−500 μm in lateral dimensions), which may find use in miniaturized cell-based assays. Because of the simplicity and versatility of this strategy, we anticipate that it can be adapted to various aerogel compositions and to novel applications in addition to biomedical devices; for example, we envision aerogel films as highly efficient catalyst scaffolds, where the large pore volume can promote facile diffusion and reaction kinetics throughout the network.

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RESULTS AND DISCUSSION Aerogel Film Fabrication. Aerogel films were fabricated by a pressure-aided freeze casting and stencil lift-off patterning approach (Figure 1). Briefly, a Parylene or adhesive film of the desired aerogel thickness was adhered on the substrate and a mold with the specific shape for the aerogel was patterned onto the film through xurography or photolithography. The allCNC or CNC-POEGMA suspension, containing components with reactive aldehyde and hydrazide functionality (i.e., hCNC-aCNC or hPOEGMA-aCNC), was premixed and drop cast onto the exposed substrate. Pressure was applied to the gel such that it adopted the shape of the mold. The confined gel was then frozen and lyophilized, creating aerogels with a continuous pore morphology. The mold was subsequently lifted off the substrate to leave behind the patterned aerogel films. All aerogels were prepared using either C


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°C, with the aerogels prepared at the lower temperature being more homogeneous (−20 °C aerogels appear similar to Figure S1B). Gels frozen at −30 °C were also more opaque, which was attributed to the formation of smaller ice crystals at the faster freezing rates.32 Finally, it was important to ensure that the CNC and POEGMA suspensions were well dispersed. This was done by probe sonicating both suspensions prior to mixing and casting. Note that these optimization steps were unnecessary in the formation of all-CNC aerogels, as the sol suspension did not immediately form covalent cross-links upon mixing. This is because the individual CNCs repel each other due to electrostatics from the anionic sulfate groups on their surface and lack the flexibility to entangle, which limited their ability to cross-link in suspension. During the freezing step, the CNCs were excluded from the growing ice crystals and thus pushed together, promoting covalent cross-linking through hydrazone bond formation.21 Figure 1A summarizes the final optimized protocol for fabricating thin patterned aerogels using Parylene or adhesive tape molds. Aerogel Film Characterization. The thickness of the aerogel films made from various compositions and concentrations was characterized using a stylus profilometer and compared to the mold thickness used for casting (Figure 2).

have advantages over adhesive tape molds, as mold thickness can be precisely controlled based on the mass of dimer precursor added to the deposition unit (Figure S3). Additionally, their thickness can span a range below conventional adhesive tape thicknesses (2 μm) would allow seeded cells to penetrate the structure, while mesopores would facilitate the diffusion of nutrients and metabolic waste products. Furthermore, the filamentous 3D structure of these aerogels would more uniformly expose the cultured cells to environmental factors, such as nutrients, growth factors, cytokines, oxygen, and pH, F


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Figure 6. Confocal imaging of PC3 cells cultured on CNC-POEGMA aerogel films. Confocal image stacks were obtained from cells cultured on aerogels made using (A) 7 μm-thick Parylene molds patterned using xurography (only top edge of a mm-long pattern is shown) and (B−C) 3 μmthick Parylene molds patterned using photolithography (aerogel in panel C was doped with fluorescein). Images are average intensity projections of the z-stacks. Cells constitutively expressed a GFP (green fluorescent protein) reporter and were tagged with DAPI and Cy5-phalloidin to visualize the nucleus and actin, respectively. The first three columns panels show gray scale images for the blue, green, and red channels, respectively, with an inverted grayscale look-up table to facilitate visualization of dim features, while the fourth column shows an overlay for the three channels presented in conventional fashion but with enhanced brightness and contrast to enable the visualization of all channels.

lines.36 Confocal images showed that the cells on the scaffolds had diverse morphologies, spanning from round cells to highly elongated ones, indicating adhesion and contact guidance by the underlying scaffold (Figure 6). The PC3 cells adhered strongly on the scaffolds, without requiring the conjugation of extracellular matrix ligands to the support, which is significant since it has been reported that native cellulose does not promote the adhesion of most mammalian cell types, including PC3.37−39 The lack of adhesion of cells to cellulose has been attributed to a number of factors, including the lack of natural integrin-binding sites (e.g., arginine-glycine-aspartic acid or RGD motif), their hydrophilic nature, which limits nonspecific binding of integrin-binding proteins in the cell media, and the possibility of anionic groups on the cellulose surface that can repel phosphate groups on the cell membrane. In our case, the enhanced adhesion to the CNC-containing aerogels can be attributed to the nanoscale features and porosity of the aerogel, which enhance the roughness and surface area for binding. Confocal images also showed that PC3 cells were able to penetrate into the aerogels through filopodia extensions or embed into them when large macropores were available (Figure S6). This is consistent with prior studies that have shown that human breast cancer cells adhered and proliferated

which cannot be done when culturing on traditional 2D surfaces, where only a portion of the membrane is directly exposed to the culture media.34 In this sense, CNC-POEGMA scaffolds could be interesting surrogates for natural cell microenvironments, as their mechanical properties can also be tailored (by varying the CNC:POEGMA ratio) to match those of various tissues. To test their suitability as 3D scaffolds for cell culturing, CNC-POEGMA macro- and micropatterned aerogel films with different thicknesses were seeded with human prostate cancer epithelial (PC3) cells at a density of 64,000 cells/cm2 for 24 h and the cells were monitored through confocal microscopy (Figures 6 and S7). The strain of PC3 cells used constitutively expressed a GFP reporter, which allowed monitoring cell adhesion over time. After 24 h culture, the cells were fixed and stained for actin with Cy5-phalloidin and for nucleic acids using DAPI (4′,6-diamidino-2-phenylindole dihydrochloride). The fact that the cells remained firmly attached after the multiple washing steps involved in the fixation and staining protocol indicated that there was strong adhesion to the aerogel scaffold, which could only be ascribed to the presence of CNCs and a fibrous nanostructure, since bulk POEGMA is known to strongly repel protein and inhibit cell adhesion,35 but electrospun POEGMA can support the adhesion of some cell G


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ligands, human prostate cancer cells adhered to the aerogel due to its high porosity, nanotopography, and surface area. This showcased the suitability of CNC-based aerogel films for cancer cell culture and suggests the possibility of using such hierarchically structured substrates to study cancer cell metastasis and extravasation processes.

on bacterial cellulose scaffolds containing large macropores (>100 μm), whereas uniform bacterial cellulose films resulted in poor adherence and proliferation. 25 Similarly, bulk POEGMA repels cells, but electrospun POEGMA nanofibers can support some cell adhesion.36 Our findings suggest that CNC-POEGMA aerogels could be interesting scaffolds for the study of extravasation, since PC3 cells have high metastatic potential, with the bone being the most common site of prostate cancer metastases. Furthermore, the adhesion and mechanical properties of the CNC-POEGMA scaffolds can be altered by changing the relative amount of CNCs in the starting suspension, which will modulate the nanotopography and the rigidity of the matrix to which the cells are exposed. An increase in CNC content would result in a stiffer scaffold that further promotes PC3 cell adhesion and spreading due to mechanotransduction feedback that cells receive as they form focal adhesions to anchor them to their surroundings.40 The microfabricated arrays showed multiple cells attached onto the micropatterned aerogels with large-dimensions (≥50 μm, Figure 6B), while single-cell adhesion and isolation was possible with smaller micropatterns. Cell isolation was limited by the size of the pattern and the probability for a cell to land on the micropatterned aerogel, such as in Figure 6C, where individual cells have only adhered to two of the four available 20 × 20 μm squares. Since the micropatterned aerogels were thin (fabricated using ∼3 μm Parylene mold) and the morphology was densely fibrillar near the substrate (cf., Figure 4), cell penetration into the aerogel was limited. However, because of the nanoscale features of the aerogel, the cells strongly adhered on the scaffold. Single-cell micropatterning is a powerful tool that can reveal heterogeneity within cell populations.41 Traditional cell-based assays only reflect the dominant phenotype within a population, which can mask critical information from rare cell types. This has important clinical relevance for disease detection and formulating therapeutic strategies based on patient biopsies. For instance, recent findings indicate that a small number of cells within a tumor population transiently assume a drug-resistant phenotype to protect the population from eradication.42 This micropatterned aerogel system could be an attractive avenue to isolate and study individual PC3 and other cancerous cells, and trace their response to specific treatments. Along these lines, future studies could involve conjugating binding ligands that target specific cancer cells to arrays of aerogel scaffolds, which would represent a better biomimetic environment than traditional 2D surfaces functionalized with binding proteins.

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EXPERIMENTAL SECTION

Materials. All-CNC suspensions used in this study were produced in-lab from ashless cotton filter aid (Whatman, GE Healthcare Life Sciences, CAT No. 1703-050, Mississauga, Canada) and stored in acid form at 4 °C. Whatman glass microfiber filter paper (1 μm pore size, CAT No. 1821-070) was used for filtering CNC suspensions. Di(ethylene glycol) methyl ether methacrylate (M(EO)2MA, 2 EO repeat units, Sigma-Aldrich, Oakville, Canada) and oligo(ethylene glycol) methyl ether methacrylate (OEGMA500, number-average MW = 500 g/mol, 8−9 EO repeat units, Sigma-Aldrich) were purified using a basic alumina column (CG-20, Sigma-Aldrich) to remove inhibitors. Acrylic acid (AA, Sigma-Aldrich), adipic acid dihydrazide (ADH, ≥ 98%, Sigma-Aldrich), 2,2-azobisisobutyric acid dimethyl ester (AIBMe, Wako Chemicals, Richmond, VA, USA), ethanol (Caledon Laboratories, Georgetown, Canada), dioxane (Caledon Laboratories), N′-ethyl-N-(3-(dimethylamino)propyl)-carbodiimide (EDC, Sigma-Aldrich), ethylene glycol (≥99.5%, Caledon Laboratories), 0.1 M hydrochloric acid (HCl, LabChem, Zelienople, PA, USA), N-hydroxysuccinimide (NHS, 98%, Sigma-Aldrich), silver(I) oxide (Ag2O, ≥99.99%, Sigma-Aldrich), 0.1 M sodium hydroxide (NaOH, LabChem), sodium hypochlorite solution (NaOCl, SigmaAldrich), sodium periodate (NaIO4, Sigma-Aldrich), sulfuric acid (H2SO4, Caledon Laboratories), and 2,2,6,6-(tetramethylpiperidin-1yl)oxyl (TEMPO, 99%, Sigma-Aldrich) were used as received without further purification. Reactions and dilutions were performed in purified water (18.2 MΩ·cm resistivity at 25 °C from a Milli-Q A10 Purification System, Millipore, Etobicoke, Canada) unless stated otherwise. Piranha solution consisting of 5:1 (v:v) concentrated H2SO4 to hydrogen peroxide (30%, Caledon Laboratories) was used to clean glass (GoldLine, VWR International, Mississauga, Canada) and silicon (University Wafer, Boston, MA, USA) substrates. Aluminum weighing dishes (CAT No. 25433−008, VWR International) were used for pressure casting. Poly(allylamine hydrochloride) (PAH, MW = 120 000−200 000 g/mol, Sigma-Aldrich) was dissolved at 0.1 wt %. Preparation of Sulfated CNC Suspensions. Ashless cotton filter aid was blended into a fine pulp and heated at 80 °C for at least 1 h to remove residual moisture. Cotton pulp (40 g) was hydrolyzed in 700 mL of 64 wt % H2SO4 and mechanically mixed for 45 min at 45 °C. The hydrolysis was quenched by a 10x dilution with water chilled to 4 °C. The CNC aggregates were centrifuged at 5500g for 10 min, decanted, and redispersed in water. This was repeated until the suspension appeared murky, indicating sufficient removal of acid which led to colloidal stability. The CNC suspension was placed in cellulose dialysis tubes (14 kDa MWCO, Sigma-Aldrich) and placed in dialysis columns (∼15 L). The purified water in the dialysis columns was replaced daily for at least 10 days until the pH stabilized at ∼5.5. CNC aggregates were dispersed with a point probe sonicator (Sonifier 450, Branson Ultrasonics, Danbury, CT, USA) for 30 min at 60% amplitude and vacuum filtered through a glass microfiber filter. The suspension was concentrated to 2 wt % CNC by evaporation at 50 °C while stirring to prevent aggregation. Synthesis of Hydrazide-Functionalized POEGMA. Hydrazidefunctionalized POEGMA (hPOEGMA, branched polymer containing 10 mol % OEGMA500, 90 mol % M(EO)2MA, and 30 mol % hydrazide functionality) was synthesized as previously described.18,33 Briefly, AIBMe (74 mg), M(EO)2MA (6.2 g), OEGMA500 (1.8 g), AA (1050 μL, corresponding to 30 mol % of M(EO)2MA + OEGMA500), and TGA (150 μL, 10 wt % in dioxane) were mixed in dioxane (40 mL) and the reaction mixture was bubbled with nitrogen for ≥20 min. The mixture was stirred for 4 h at 75 °C and subsequently cooled. After evaporation of dioxane, the resulting copolymer was grafted with

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CONCLUSIONS In this work, a simple benchtop technique was developed and optimized to fabricate cross-linked CNC-based aerogel films with tunable morphology and dimensions. Various aerogel morphologies were produced by varying the sol−gel concentration and their physical dimensions were controlled using a mold. The mold was patterned by xurography to fabricate various aerogel film shapes with macroscopic lateral dimensions, while aerogel arrays with micrometer-scale dimensions were fabricated using photolithography. This technique invokes many potential applications for aerogel films that require precise optimization, such as the incorporation of interlayer dielectrics and functional nanoparticle supports for use in electronic devices. As proof-of-concept, we explored the application of the thin aerogel films as scaffolds for cell culture. Despite the lack of natural ECM binding H


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content of 0.50 ± 0.07 mmol/g. The DLS z-average and zeta potential were 108 ± 1 nm and −36 ± 5 mV, respectively. Pressure-Aided Freeze-Casting. Glass substrates were cleaned using Piranha solution for 15 min at 110 °C. NOTE: Piranha solution should be handled with care, since it can be explosive when in contact with organic solvents. Plastic and metal substrates were cleaned using UV-Ozone (Novascan, Boone, IA, USA) for 2 h. UV-release tape (85 μm-thick, DU 300, Semiconductor Equipment Corp., Worcester, MA, USA), 3 M Scotch tape (50 μm-thick), and Parylene C (3−7 μmthick, Specialty Coating Systems, Indianapolis, IN, USA) were used for the fabrication of stencils used as casting molds. Large scale mold patterns were created by xurography using a blade cutter (ROBOPro CE5000-40-CRP, Graphtec America Inc., Irvine, CA, USA). For adhesive films (UV-release tape and Scotch tape), the patterned adhesive molds were transferred to a clean substrate and served as stencils during aerogel casting. On the other hand, for the fabrication of Parylene stencils, the substrates (i.e., glass, ITO, FTO) were pretreated by spin-coating (4000 rpm, 30 s, Spin-coat G3P-12, Specialty Coating Systems) a Micro-90 surfactant solution (1% v/v, International Products Corp., Burlington, NJ, USA) to form a thin film that reduced the Parylene-substrate adhesion and ensured that the Parylene could be peeled off without leaving residues. The Parylene films were then deposited to the desired thickness using a PDS-2010 Labcoater (Specialty Coating Systems). Large-scale molds were cut into 7 μm-thick Parylene films via xurography and the cutouts were manually lifted off to expose the underlying substrate. Similarly, micrometer-scale mold patterns were created on 3 μm-thick Parylene films using standard photolithography procedures (described below). To ensure no interference with gel casting, the Micro-90 film remaining on the exposed glass areas was removed by bath sonication (Branson 2510, Danbury, CT, USA) in water for 15 min. To enhance cellulose adhesion, PAH was then applied to the exposed substrate by submerging it in a 0.1 wt % PAH solution for 15 min and subsequently rinsing it in water for 5 min to remove excess polymer. The substrates were then dried in an oven at 60 °C. CNC-POEGMA and all-CNC aerogels were formed by mixing aCNCs with either hPOEGMA or hCNCs respectively and vortexing the sol suspension for 10 s. The gel was subsequently drop cast onto the exposed glass substrate and immediately frozen at −30 °C under pressure with an aluminum weighing dish (VWR International, Cat No. 2433-008) in between a 100 g calibration weight and the gel/substrate. The weight and aluminum dish were removed, and the frozen gel was lyophilized (Labconco Benchtop Freeze-Dryer, Kansas City, MO, USA) to form an aerogel. The molds were then lifted off, revealing the aerogel of desired thickness and pattern. N.B.: The average density measured for an aerogel produced using a 1 wt % sol−gel concentration was ∼0.01 g/cm3. Photolithographic Patterning of Parylene Films. The micropatterned Parylene films were prepared using standard photolithography techniques. Briefly, a solution of SC-1827 positive photoresist (Dow MICROPOSIT, Austin, TX, USA) was spin-coated onto the Parylene coated glass at 2000 rpm for 30 s to yield a film with a nominal thickness of 3.8 μm. The photoresist was baked on the substrates at 90 °C for 1 min, then exposed through a chrome photomask using a Karl Suss MJB3 mask aligner (SUSS MicroTec, Garching, Germany) at 5.7 mW for 30 s. The exposed resist was developed to reveal circular and square micropatterns using MF-319 (Dow MICROPOSIT) developer for 30 s, followed by extensive rinsing in Milli-Q water, and drying under a stream of nitrogen gas. The exposed Parylene areas were etched away using a Technics Micro-RIE Series 800 Plasma System (Arlington, TX, USA) at an oxygen flow rate of 28 sccm and 100 W power for 35 min, resulting in an average Parylene etch rate of ∼100 nm/min. To remove residual photoresist, the substrates were washed sequentially in acetone and isopropanol baths, followed by drying under a nitrogen stream and exposed to a 15 min UV−ozone treatment (Novascan). Aerogel Characterization. The morphology of the aerogel surface and cross-section was assessed with SEM (JEOL JSM-7000F, Tokyo, Japan) using a working distance between 6 and 8 mm and an acceleration voltage of 2 kV. Prior to imaging, the aerogels were

hydrazide groups by reacting the AA residue carboxyl groups with ADH using EDC as an activation agent. Water (200 mL) and ADH (8.66 g) were added to the polymer mixture, and the pH was adjusted to 4.8 ± 0.1 using HCl, after which EDC (3.87 g) was added. The pH was maintained at 4.8 ± 0.1 using HCl for 4 h and the solution was then left stirring overnight. To isolate hPOEGMA, the mixture was dialyzed (3.5 kDa MWCO) in water with six daily rinses and subsequently lyophilized. The polymer was stored as a 10 wt % aqueous suspension at 4 °C prior to use. Characterization of CNCs. The mean particle size of the dispersed CNCs (0.025 wt %, with 10 mM NaCl added) was measured by dynamic light scattering (DLS, Malvern Zetasizer 3000, Malvern, UK). The resulting z-average is the average diameter of a sphere with equivalent Brownian motion to the needle-shaped CNC. Zeta potential of the CNC suspension (0.25 wt %, with 10 mM NaCl added) was determined using a ZetaPlus Analyzer (Brookhaven Instruments, Holtsville, NY, USA). The number of functional groups on CNCs were quantified through conductometric titration.43 In all titrations, the conductivity of the CNC suspension was first adjusted to ∼200 μS/cm using NaCl. For unmodified CNCs, the sulfate halfester content by conductometric titration was 193 ± 4 μmol/g. The DLS z-average and zeta potential were 88 ± 5 nm and −27 ± 4 mV, respectively. Functionalization of CNCs with Aldehyde Groups. Mass ratios (X:Y) represent the mass of a liquid/solid reagent (X) relative to the dry mass of CNCs (Y). CNCs were mixed with NaIO4 at a mass ratio of 4:1. The pH was adjusted to 3.5 ± 0.2 with HCl or NaOH. The mixture was stirred in the dark while submerged in an oil bath at 45 °C for 4 h. Ethylene glycol was then added at a 2:1 mass ratio to quench the reaction. The suspension was dialyzed for at least 10 days until the pH of the water bath stabilized at ∼5.5. The degree of aldehyde functionalization on the CNCs was quantified by conductometric titration. In this process, an aliquot of aldehydefunctionalized CNCs (aCNCs) was mixed with Ag2O and pure (solid form) NaOH, at respective 3.86:1 and 0.54:1 mass ratios, and stirred overnight to oxidize all aldehyde groups present. The resulting carboxylated CNCs (COOH−CNCs) were protonated with HCl (pH of suspension ∼3) and subsequently titrated with 10 mM standardized NaOH while monitored with a conductometric probe (VWR sympHony). The aldehyde content corresponds to the region of the titration curve where conductivity is stable. Conductometric titration yielded an aldehyde content of 1.56 ± 0.09 mmol/g. The DLS zaverage and zeta potential were 92 ± 8 nm and −16 ± 2 mV, respectively. Functionalization of CNCs with Hydrazide Groups. Mass ratios (X:Y) represent the mass of a liquid/solid reagent (X) relative to the dry mass of CNCs (Y). CNCs were first grafted with carboxylic acid groups through a TEMPO oxidation. A 200 mL aqueous solution of TEMPO and NaBr prepared at respective 0.03:1 and 0.32:1 mass ratios was added dropwise to a suspension of CNCs at ambient conditions with stirring. Immediately after, 12 wt % NaOCl was added dropwise to the suspension at a 6:1 mass ratio. The reaction was stirred for 3 h with the pH maintained at 10.0 ± 0.2. Afterward, ethanol was added at a 3.6:1 mass ratio to quench the reaction. The suspension was centrifuged at 5500g and the supernatant was decanted. The CNC pellet was then resuspended in water and dialyzed as described in the previous section. The resulting COOH− CNC suspension was then grafted with hydrazide (NH2NH−) groups by mixing it with ADH at a mass ratio of 0.3:1. NHS (0.007:1) and EDC (0.03:1) were suspended in separate 1 mL 1:1 (volume) DMSO:water solutions and added dropwise to the suspension. The pH was adjusted to 6.8 with HCl or NaOH until it stabilized, and the suspension was subsequently dialyzed. The degree of hydrazide functionalization on the CNCs (hCNCs) was quantified indirectly by conductometric titration. This was done by titrating the COOH− groups after the TEMPO oxidation step with standardized NaOH as described in the prior section. After the hydrazide functionalization, the remaining COOH− groups were quantified again by titration. The difference between these two measurements yielded a hydrazide I


Article

ACS Applied Nano Materials coated with 5 nm Pt and nickel paint (Ted Pella, Inc., Redding, CA, USA) was applied to the sample edges to establish conductivity with the aluminum stub. Aerogel cross sections were obtained by preparing frozen gel samples on silicon substrates. The substrate was then fractured at the center of the frozen sample, lyophilized, and the mold was lifted off. Aerogel thicknesses were measured using a stylus profilometer (DEKTAK XT, Bruker, Billerica, MA, USA). Cell Seeding on Aerogel Scaffolds and Imaging. Human prostate cancer cells (PC3) transformed to constitutively express green fluorescent protein (PC3-GFP) were kindly provided by the Cuvillier laboratory at the Institute of Pharmacology and Structural Biology (IPBS, Toulouse, France). Cells were cultured in RPMI-1640 culture medium (Gibco GlutaMAX medium, ThermoFisher, Illkrich Cedex, France) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin−streptomycin (Gibco, Thermo Fisher), and 1% geneticin (G418 Geneticin, Thermo Fisher). All cell cultures were incubated in a humidified atmosphere containing 5% CO2 at 37 °C. Prior to seeding on the aerogels, cells were detached from culture flasks by trypsination (Trypsin-EDTA Gibco, 3−5 min incubation), and the cell concentration determined with a counting chamber (Fast read 102, BioSigma, Cona, Italy). Cells were seeded on the glass substrate containing the patterned aerogels at a surface density of 64 000 cells/cm2. After 24 h incubation, the cells were rinsed with phosphate buffered saline (Gibco PBS 1×, ThermoFisher) and the Parylene stencil was lifted off. Then, the cells were fixed using 4% formaldehyde solution (Formalin solution, neutral buffered, 10%, Sigma-Aldrich, Saint-Quentin-Fallavier, France) for 20 min at room temperature. The formaldehyde solution was subsequently removed, and the cells were rinsed 3× for 5 min in PBS. To fluorescently stain the cells, the membranes were permeabilized with 0.1% triton X-100 for 10 min at room temperature and incubated with Cy5-phalloidin (F-Actin stain) for 30 min (1:200 dilution, ThermoFisher), followed by DAPI solution at 20 mM for 5 min. After the staining, samples were stored in PBS at 4 °C until imaging. Fluorescently labeled cells were imaged on the patterned aerogel films using a Leica SP8 confocal microscope equipped with a 63×/1.4NA PLAPO CS2 oil immersion objective and Leica Application Software. Postacquisition, the images were processed using ImageJ software.

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and Department of Chemical and Biological Engineering, The University of British Columbia, 2360 East Mall, Vancouver, BC V6T 1Z3, Canada. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

J.M.M.-M.: Natural Sciences and Engineering Research Council Discovery Grant RGPIN 418326, Ontario Ministry of Research and Innovation Early Researcher Award, Canada Research Chairs, France Canada Research FundNew Collaborations Program. E.D.C.: Natural Sciences and Engineering Research Council Discovery Grant RGPIN 402329, Ontario Ministry of Research and Innovation Early Researcher Award, Canada Research Chairs. A.C.: French National Research Agency Grant ANR-15-CE19-0020. T.H.: Natural Sciences and Engineering Research Council Discovery Grant RGPIN 2017-06455, Canada Research Chairs. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Profs. R. Wylie and R. Pelton for access to equipment and X. Ding for SEM help. T.O. was partially supported through an NSERC USRA, and S.S. was supported through an OGS award. J.M.M. and E.D.C. are recipients of Early Researcher awards from the Ontario Ministry of Research and Innovation. J.M.M. holds the Tier 2 Canada Research Chair in Micro- and Nanostructured Materials; E.D.C. holds the Tier 2 Canada Research Chair in Bio-Based Nanomaterials; and T.H. holds the Tier 2 Canada Research Chair in Engineered Smart Materials. Funding from the Natural Sciences and Engineering Research Council of Canada through Discovery Grants to J.M.M., E.D.C., and T.H. is gratefully acknowledged. The authors acknowledge the French RENATECH network for technological support. This research made use of instrumentation from the Canadian Centre for Electron Microscopy and Biointerfaces Institute at McMaster University.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.9b00640. Optimization of aerogel casting method, CNC-POEGMA aerogel film casting on various substrates, Parylene coating thickness as a function of dimer mass, pressureapplying setup, surface characteristics for Al dishes used to pressure-cast aerogels, storage and loss moduli for CNC-POEGMS suspensions, and orthogonal projections of z-stack images of PC3 cells grown on CNCPOEGMA aerogels (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Sokunthearath Saem: 0000-0002-9106-8160 Kevin J. De France: 0000-0002-5545-4793 Jaana Vapaavuori: 0000-0002-5923-0789 Todd Hoare: 0000-0002-5698-8463 Emily D. Cranston: 0000-0003-4210-9787 Jose M. Moran-Mirabal: 0000-0002-4811-3085 Present Address

# E.D.C.: Department of Wood Science, University of British Columbia, 2424 Main Mall, Vancouver, BC, V6T 1Z4, Canada,

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Patterned Cellulose Nanocrystal Aerogel Films with Tunable

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