Hybrid Thin Film Organosilica Sol–Gel Coatings To Support Neuronal

Oct 7, 2016 - Mi-Hee Kim , Ji Hun Park , Sunghoon Joo , Daewha Hong , Matthew Park , Ji Yu Choi , Hye Won Moon , Yang-Gyun Kim , Kyungtae Kang , and ...
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Hybrid Thin Film Organosilica Sol−Gel Coatings To Support Neuronal Growth and Limit Astrocyte Growth Larissa Brentano Capeletti,†,‡,§ Mateus Borba Cardoso,† Joaõ Henrique Zimnoch dos Santos,‡ and Wei He*,§,∥ †

LNLS - Laboratório Nacional de Luz Síncrotron, Caixa Postal 6192, CEP 13083-970 Campinas, SP, Brazil Chemistry Institute, Universidade Federal do Rio Grande do Sul, CEP 91501-970, Porto Alegre, RS, Brazil § Department of Materials Science and Engineering and ∥Department of Mechanical, Aerospace and Biomedical Engineering, University of Tennessee, Knoxville, Tennessee 37996, United States ‡

S Supporting Information *

ABSTRACT: Thin films of silica prepared by a sol−gel process are becoming a feasible coating option for surface modification of implantable neural sensors without imposing adverse effects on the devices’ electrical properties. In order to advance the application of such silica-based coatings in the context of neural interfacing, the characteristics of silica sol−gel are further tailored to gain active control of interactions between cells and the coating materials. By incorporating various readily available organotrialkoxysilanes carrying distinct organic functional groups during the sol−gel process, a library of hybrid organosilica coatings is developed and investigated. In vitro neural cultures using PC12 cells and primary cortical neurons both reveal that, among these different types of hybrid organosilica, the introduction of aminopropyl groups drastically transforms the silica into robust neural permissive substrate, supporting neuron adhesion and neurite outgrowth. Moreover, when this organosilica is cultured with astrocytes, a key type of glial cells responsible for glial scar response toward neural implants, such cell growth promoting effect is not observed. These findings highlight the potential of organo-group-bearing silica sol−gel to function as advanced coating materials to selectively modulate cell response and promote neural integration with implantable sensing devices. KEYWORDS: silica, sol−gel, surface modification, functional coatings, neural interface localized delivery of therapeutics from the coating.20−24 Considering the sensing nature of such neural implants, it is of paramount value to ensure that the implant electrical properties are not adversely affected by the applied coating. A recent work by Pierce et al. has implied thin-film silica sol−gel coatings as an encouraging option for neural implant surface modification.25 The motivation to study silica sol−gel as neural implant coating stems from the high biocompatibility of silicabased materials26 and the opportunity for drug release from the porous structure generated by the sol−gel process.27−29 It was shown that 100 nm thick silica coating adhered to neural electrodes successfully without adversely affecting the electrical properties at physiologically relevant frequency (i.e., 1 kHz).25 This finding has warranted further development of silica sol− gel coatings for neural implants by introducing features to control cell−implant interaction and promote neural integration. One desirable feature to incorporate is the ability to support neuron adhesion and neurite outgrowth, as such close

1. INTRODUCTION The use of artificial materials as implantable neural sensors to tap into the central nervous system has presented very promising opportunities for both advancements of fundamental neuroscience and treatments for patients with debilitating sensory and/or motor diseases.1−3 Over the years significant strides have been made in this field, and remarkable clinical potentials of such neural sensors were exemplified from recent human subject studies where microscale intracortical recording arrays enabled paralyzed humans independently control prosthetic devices through their thoughts.4 However, in order for the successful clinical translation becomes a reality, a number of critical issues have to be addressed, one of which being the longevity and functional reliability of these implantable neural sensors when in service. It has been suggested that the biological host response against the implants is an important factor affecting the chronic performance of the implants.5−7 Thus, many strategies have been explored to mitigate the response and promote neural integration with the implants.8−23 One of such strategies is to apply a coating to the implant surface to either directly modulate cell−substrate interactions12−19 or indirectly control cellular response by © XXXX American Chemical Society

Received: July 29, 2016 Accepted: September 30, 2016

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DOI: 10.1021/acsami.6b09393 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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a given organosilane were mixed with ethanol and water in a molar ratio of 1:0.2:4:2. Silane hydrolysis was promoted by using a catalytic amount of nitric acid at a molar ratio of 0.1:1 with TEOS. After 12 h of stirring at room temperature, the resulting solution was deposited on piranha-treated glass substrates using a spin-coating technique. To ensure uniform coating, the silica sol was first deposited on the clean glass substrates and then subjected to a two-step spin-coating program: 1500 rpm for 27 s followed by 3000 rpm for 3 s to avoid any edge effect. The resulting gel films were dried for 2 h on a hot plate set at 45 °C. For comparison purposes, a bare silica film derived from TEOS alone was also prepared with the same protocol. The film morphology was investigated by atomic force microscopy (AFM). The analyses were carried out at Brazilian Laboratory of Nanosciences (LNNano) using a Digital Instruments Nanoscope III microscope in tapping mode. The images were processed using WSxM software.35 The thickness of each sol−gel film was determined by profilometry, and the methods and corresponding results (Table S1) are shown in the Supporting Information. The surface wettability was characterized by water contact angle (WCA) measurements. The analyses were performed in quintuplicate by sessile-drop contact angle method with 5 μL of deionized water droplet applied on the samples at room temperature using a CAM-Plus contact angle goniometer (Cheminstruments, USA). X-ray photoelectron spectroscopy (XPS) analyses were performed at the Synchrotron Light National Laboratory (LNLS, Campinas, Brazil). A SPECSLAB II (PhoibosHsa 3500 150, 9 channeltrons) SPECS with Al Kα (E = 1486.6 eV) source operating at 15 kV, Epass = 40 eV, pass of 0.6 eV, and acquisition time of 2 s per point. Samples were attached to a steel sample holder using a carbon tape and transferred to prechamber under an inert atmosphere. The initial pressure at the analysis chamber was 1 × 10−9 Torr. Binding energies of C 1s, O 1s, N 1s, and Si 2p were calibrated according to C 1s peak at 285 eV providing accuracy of ±0.2 eV. Data analyses were performed using CasaXPS software. Cell Culture. PC-12 cells (ATCC), a rat adrenal pheochromocytoma cell line, were cultured in F12-K medium supplemented with 15% horse serum (HS, Invitrogen), 2.5% fetal bovine serum (FBS, HyClone), and 1% penicillin/streptomycin (P/S) with partial medium replacement every other day. To subculture, cells were detached from flasks and mechanically dissociated into single cells by passing through a 22 G needle repeatedly. Primary cortical neurons were obtained following procedures modified from a previous report.36 Briefly, forebrains of 7-day-old chicken embryos were removed, stripped off meninges, minced into small pieces, and enzymatically dissociated with 0.25% trypsin in phosphate buffered saline (PBS) at 37 °C. After 20 min incubation, Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS was added to quench trypsin activity. The cell suspension was then centrifuged, and the obtained cell pellet was resuspended and dissociated by mechanical trituration in DMEM supplemented with 10% FBS, 1% P/S, and 1% L-glutamine. The A-172 astrocytic cells (ATCC) were cultured in DMEM medium supplemented with 10% FBS and 1% P/S with total medium replacement every 2 days. All cell cultures were carried out at 37 °C in a 5% CO2 atmosphere. Prior to cell seeding, all samples were sterilized by immersing in 70% ethanol for 30 min and rinsed extensively with sterile water. As a positive control, PLL-coated glass substrates were also prepared fresh by immersing clean glass substrates in a 0.1 mg mL−1 PLL solution for 1 h at 37 °C followed by rinsing with sterile water three times. Fluorescent Staining. To examine cell viability, a live/dead staining assay was performed. Briefly, cell culture samples were gently washed with PBS and then incubated for 20 min at 37 °C in PBS containing 2 μM calcein AM to stain live cells, 4 μM ethidium homodimer 1 (EthD-1, Molecular Probes, USA) to stain dead and dying cells, and 3.2 nM of Hoechest 33342 (Thermo Sci) to stain cell nuclei. Cell morphology was studied at predetermined time points for these various types of cells after fixation in 4% paraformaldehyde for 20 min at room temperature (RT). For a typical immunofluorescent staining, cells were first permeabilized using 0.1% X-100 Triton (Fischer Scientific, USA) for 5 min and blocked by incubation with either 1% bovine serum albumin (BSA, Sigma, USA) or 4% goat serum

proximity with neurons will benefit the sensing function of the implant. Toward this goal, researchers have added poly-L-lysine (PLL), an amino acid-based nonspecific adhesive polymer, to the silane precursor sol mixture to obtain PLL-doped silica sol− gel thin films.30 Although the doped substrate was neural permissive, the stability of entrapped PLL needs to be considered. Since the presence of PLL is made possible through the physical trapping mechanism, care must be taken to prevent leaching of PLL. Loss of PLL is undesirable, as it will adversely affect cell adhesivity of the silica substrate. More importantly, the leached PLL causes cytotoxicity concern due to its cationic nature.31 To overcome these drawbacks, we propose to capitalize on the versatility offered by the sol−gel method to transform plain silica into organosilica where functional moieties are permanently displayed via stable chemical bonding to the silica network. Such a direct functionalization of silica is commonly accomplished in a one-pot manner through co-condensation of tetraalkoxysilane and organo-trialkoxysilane. 32 So far a wide variety of applications have been explored for organosilica thin films, with examples ranging from optical sensors, electrochemical sensors, and catalysis to molecular valves or gates for controlled drug release.33 Yet, little attention has been given to the potential of organosilica to serve as neuro-integrative coating for neural implants. In this study, we developed a library of hybrid organosilica coatings simply using a direct one-step sol−gel route on a number of readily available organo-trialkoxysilanes carrying distinct organic functional groups. It is known that surfaces containing amine groups are an already established protocol.34 However, different organo-alkoxysilanes containing distinct organic moieties and presenting different molecular volumes were employed in order to look for nontrivial results. Thus, the ability of these coatings to support neuron adhesion and differentiation was evaluated with in vitro PC12 cell culture. The results indicate that different organic groups confer varying levels of neural support. The neural permissive nature of the best performing hybrid organosilica was demonstrated by primary cortical neuron study. An unexpected but promising finding is that such support of cell growth by the hybrid organosilica was not applicable for astrocytes, a highly desirable effect considering the adverse astroglial scarring commonly encountered by neural implants. It implies that hybrid organosilica sol−gel coating could provide a simple yet versatile option to selectively modulate cellular interactions with neural implants.

2. EXPERIMENTAL SECTION Materials. Tetraethyl orthosilicate (TEOS, C0, 99%), triethoxymethylsilane (C1, 99%), octadecyltrimethoxysilane (C18, 90%), (3aminopropyl)triethoxysilane (NH2, 98%), (3-mercaptopropyl)trimethoxysilane (SH, 95%), (3-isocyanatopropyl)triethoxysilane (NCO, 95%), (3-iodopropyl)trimethoxysilane (Ip, 95%), and (3glycidoxypropyl)trimethoxysilane (Gp, 98%) were purchased from Sigma-Aldrich. Octyltriethoxysilane (C8, 90%), vinyltrimethoxysilane (Vy, 90%) and phenyltrimethoxysilane (Ph, 90%) were obtained from Dow Corning, and (3-chloropropyl)trimethoxysilane (Cl, 99%) was from Wacker. Ethanol (99.5%) and nitric acid (65%) were purchased from Synth. Poly-L-lysine hydrobromide (mol wt 70 000−150 000 Da) was obtained from Sigma-Aldrich. All chemicals and reagents were used as received without further purification. Hybrid-Silica Film Preparation and Characterization. Hybridsilica-based films were prepared by a sol−gel process from TEOS and the 11 organosilanes specified above. In a typical synthesis, TEOS and B

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Figure 1. Surface characterization of the various silica sol−gel films. (A) AFM images for the plain silica C0 and the hybrid organosilica with specified functional groups. The AFM scan was 5 μm × 5 μm. (B) Average surface roughness (RMS value) obtained from the corresponding AFM images [glass AFM image is provided as Supporting Information] and (C) WCA results for glass substrate and the prepared silica films. for 30 min at RT to minimize nonspecific binding. Then cells were incubated in primary antibody solutions for 1 h at RT. After rinsing with wash buffer (0.05% Tween-20 in PBS), fluorescently conjugated secondary antibodies were used to treat the samples for 1 h at RT to enable visualization. Finally, samples were rinsed, counterstained for nuclei using Hoechest 33342, and mounted on coverslips with Fluoromount-G antifading solution (Southern Biotechnology Associates, Inc.). Primary antibodies used in this study included mouse monoclonal anti-α-tubulin (Sigma, 1:200 dilution) to label microtubules and vinculin monoclonal primary antibody (Santa Cruz Biotechnology, 1:500 dilution). Goat anti-mouse IgG1 conjugated with Alexa Fluor 488 (Molecular Probes, 1:100 dilution) was used as a secondary antibody. Actin filaments were stained using either Alexa Fluor 488-labeled or Alexa Fluor 594-labeled phalloidin (Molecular Probes, 1:50 dilution). Fluorescent images were captured using a Zeiss Axio Observer A1 inverted fluorescent microscope (Zeiss, Germany). PC12 Cell Behaviors. PC12 cell interactions with the different hybrid silica substrates were studied in terms of cell adhesion, differentiation, and morphology. Briefly, cells were seeded on the samples at a density of 1 × 104 cells cm−2. After overnight incubation at 37 °C in a 5% CO2 atmosphere, the number of viable cells attached was quantified based on the live/dead assay. To study cell differentiation, culture medium was replaced with differentiation

medium containing 1% HS, 0.2% FBS, 1% P/S, and 50 ng/mL of nerve growth factor (NGF, Roche) at 24 h postseeding. Cells were then allowed to grow for 48 h before analysis. For neurite outgrowth evaluation, phase contrast images were collected. At least 12 images were collected at random locations for each sample and analyzed using ImageJ software. Quantitative neurite analysis was performed following criteria reported by Leach and co-workers.37 The parameters of interest include average neurite length, number of neurites per cell, branching, and percent of cells expressing neurites. During quantification, only neurites or branches longer than 5 μm were considered. Also, only cells bearing at least one neurite were included in the determination of number of neurites per cell. Branching was considered when one neurite splits into two branches. The branching value represents the number of branches counted for each sample divided by their total neurite length. Cell morphology was studied via actin filament staining. Cortical Neuron Behaviors. Cortical neurons were seeded at a density of 5 × 104 cells cm−2. Similar to PC12 cells, neuron attachment was quantified based on the live/dead assay. Neuron morphology was studied after 72 h of culture. Besides immunofluorescent staining of microtubules and actin filaments, the cultures were also examined using scanning electronic microscopy (SEM). SEM samples were prepared following an established protocol.38 Briefly, cells were fixed in C

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Figure 2. PC12 cell adhesion study for the various silica sol−gel films. (A) Adherent cell density quantified following overnight culture. (B−D) Fluorescent images showing viability of cells attached on (B) NH2 silica, (C) SH silica, and (D) the PLL control. Live cells were stained green with calcein AM, and dead cells were stained red with ethidium homodimer-1. Scale bar = 100 μm. 3% glutaraldehyde solution and dehydrated using ascending grades of ethanol (25%, 50%, 75%, and 100%) followed by ethanol and hexamethyldisilazane (HMDS) mixtures with descending ethanol:HMDS ratios (2:1, 1:1, and 1:2), vacuum-dried, mounted on aluminum stubs, and finally gold coated by SPI sputtering device for 20 s at 20 mA. Samples were then examined using a LEO 1525 scanning electron microscope operated at an accelerating voltage of 5 keV. Astrocyte Cell Behaviors. A172 astrocytes were seeded at a density of 1 × 104 cells cm−2. Cell number was quantified at both 4 and 48 h postseeding. Cell morphology was examined at 48 h via vinculin and actin filament staining. Statistical Analysis. Characterization data were acquired in triplicate for AFM measurements and quintuplicate for WCA measurements and triplicate for AFM as well as cell experiments. For all cases average values were reported. The numbers of cells and neurite outgrowth evaluation were quantified from 12 images collected at random locations for each of the three replicates, totalizing 36 images per group. All the images were considered for both cell counting and neurite outgrowth evaluation, and the average values were reported. Statistical analysis was performed by using Student’s t test, and statistical significance was defined at p < 0.05.

using the sol−gel process were characterized with atomic force microscopy and water contact angle. AFM images representing the surface morphology of the 12 different sol−gel samples are shown in Figure 1A along with the measured root-mean-square (RMS) surface roughness summarized in Figure 1B. RMS measurements presented standard deviation values ranging from ±0.01 nm for the less rough films to ±2.8 nm for those films with higher roughness. The plain silica sol−gel film C0 has a smooth surface. When organo-functional groups such as octyl (C8), phenyl (Ph), aminopropyl (NH2), or glycidyl (Gp) are incorporated using the corresponding organo-trialkoxysilanes, the resulting hybrid film surface morphology and roughness are comparable to the plain silica film. When groups such as methyl (C1) and vinyl (Vy) are introduced, the surfaces appear to be more granulated than the plain silica film, and a slight increase in RMS roughness is noted. A more drastic change in surface morphology is observed for organo-functional groups such as octadecyl (C18), thiol (SH), isocyanate (NCO), chlorine (Cl), or iodine (Ip), where large islands could be identified in the AFM images and the calculated RMS roughness being at least 1 order of magnitude higher than that of the plain silica. Such a wide range of surface morphological characteristics can be explained by the different hydrolysis and condensation reaction rates of these organo-trialkoxysilanes with varying functional groups during the sol−gel process.27 Specifically, the rates can differ because of the organo-functional group being electron donating or withdrawing or due to the size-induced steric

3. RESULTS AND DISCUSSION Hybrid Organosilica Film Characterization. Surface properties of biomaterials such as surface roughness, morphology, and wettability all play important roles in affecting cell adhesion, proliferation and differentiation behaviors. Therefore, these key surface characteristics of our hybrid films obtained D

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ACS Applied Materials & Interfaces hindrance effect.39 When using an organosilane exhibiting a higher hydrolysis rate, it will stimulate the cross-linking of the sol fraction, resulting in a relatively smooth surface. On the other hand, if the condensation reaction rate is increased, the formation of particles is promoted in the sol, and their deposition on the film surface leads to a higher roughness of the final sol−gel silica. In addition, the films roghness can be also affected by the drying process after film deposition by spincoating technique. The solvent evaporation rate can differ according to hydrophilicity of each hybrid system resulting in different film internal stress that can affect the surface characteristics.40 All the hybrid organosilica films except the glycidyl (Gp) group are more hydrophobic than the plain silica film (C0), as shown by the increase in WCA (Figure 1C). However, no direct correlation can be made between the extent of increase in surface wettability and the type of functional group introduced. This is due to the known fact that surface wettability can be influenced by both surface chemistry and surface roughness,39 and the AFM results clearly show that some functional groups significantly change surface roughness. Although surface chemistry was not examined in the present work, the successful incorporation of the various functional groups to these hybrid organosilica materials has been shown in our previous study, where infrared and Raman spectroscopies were applied on the bulk materials prepared under the same composition and characteristic bands associated with these functional groups were observed.41 Neuronal Cell Behavior on the Hybrid Silica Films. PC12 Cell Adhesion and Viability. Support of neural adhesion and viability of PC12 rat adrenal pheochromocytoma cells by the silica hybrid materials was investigated using Live/Dead staining (Figure 2). All 12 silica films were seeded with PC12 cells, and the number of viable cells attached after 24 h was quantified. Bare glass and PLL-coated glass were included as negative and positive control, respectively. PLL is a well-known coating for neuron culture. However, different types of cells can develop on this surface since PLL does not exhibit selectiveness. As summarized in Figure 2A, plain silica C0 and hybrid silica C1, Ph and Gp behaved similarly to the bare glass negative control. Slight improvement in PC12 cell adhesion was observed for the C8, C18, Vy, SH, NCO, Cl, and Ip hybrid silica samples. Drastic increase in cell adhesion was noted for the NH2 hybrid silica sample, where the number of attached cells was comparable to the PLL-coated glass control. None of the hybrid silica films exhibited any toxic effect toward the attached PC12 cells, as evident by the lack of red fluorescent staining on the cultured samples (Figure 2B,C and Figure S1). Clearly, the adhesion results show that introduction of amine groups to the silica sol−gel film can effectively enhance neural cell attachment. Although surface hydrophilicity is known to improve protein adsorption to the surface and consequently improve protein-mediated cell adhesion,34,42 such a wettability effect is less likely to be the key reason underlying the observed improvement in neural adhesion to the silica substrates herein. As shown from the water contact angle data (Figure 1C), hybrid silica such as C8, C18, Vy, Ph, SH, Cl, and Ip were comparable to or even more hydrophilic than the NH2 sample. Yet, only marginal or moderate improvement in cell adhesion was observed for these groups. It suggests that the chemical nature of amine groups plays a more significant role in promoting neural adhesion. Such an effect can be inferred from the prevalent practical use of amine-bearing polycations, such as

polyethylenimine (PEI), PLL, or poly-L-ornithine, to coat tissue cultureware to facilitate neuron attachment.34 The underlying principle has been shown to be the electrostatic interaction between the negatively charged cell membrane due to the presence of the glycocalyx layer and the positively charged culture surface endowed by the polycationic coating.43,44 Therefore, we postulate that for the NH2 hybrid silica sample amine groups are presented on the surface to confer electrostatically based adhesiveness toward neurons. X-ray photoelectron spectroscopy characterization of the NH2 hybrid silica sample directly validated our postulation. In contrast to the lack of any nitrogen signal in the range of 396−406 eV for the plain silica sample (Figure 3A), a new N 1s peak was observed for the NH2 hybrid silica sample (Figure 3A). Further peak deconvolution revealed that two types of amine groups are present on the hybrid silica surface (Figure 3B), with one type

Figure 3. (A) High-resolution XPS spectra for N 1s of plain silica C0, the hybrid organosilica with NH2 groups, and the PLL control. Curve fitting was applied to (B) the NH2 organosilica and (C) the PLL control. Fitted spectra are shown in red and superimposed with the original data. E

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Figure 4. Actin filament staining of PC12 cell differentiation on hybrid silica substrates with (A) NH2 groups, (B) SH groups, and (C) the PLL control substrate. Quantitative neurite outgrowth analyses (D−G) were also performed for the NH2 hybrid silica and the PLL control. No statistical difference (p > 0.05) was found in terms of average neurite length (D), average number of neurites per cell (E), percentage of cells expressing neurites (F), and neurite branching (G). Scale bar = 100 μm.

Figure 5. Primary cortical neuron adhesion study. (A) Adherent cell density quantified following 24 h culture. Statistical difference was observed between the NH2 hybrid silica and the PLL control (*p < 0.05). Phase-contrast micrographs (B, C) and fluorescent images of live/dead staining (D, E) present neuron morphology and viability on the NH2 hybrid silica (B, D) and the PLL control (C, E). Live cells were stained green and dead cells were stained red. Scale bar = 100 μm.

PC12 Cell Differentiation and Morphology. Among the various hybrid silica substrates prepared, the NH2 and SH samples were selected for further investigation of neuronal cell growth because of their demonstrated support of PC12 cell adhesion. Of particular interest to us is whether such hybrid silica samples support neurite development, which holds a very important role for neuronal functions. Fluorescent staining revealed different PC12 cell morphologies on NH2 and SH hybrid silica samples as well as the PLL control following 48 h differentiation induced by nerve growth factor (NGF) (Figure 4A−C). The NH2 silica sample supported PC12 cell differentiation as evident from the presence of long and well-defined

being free amine groups (−NH2) as indicated by the peak at 399.8 eV and the other type being protonated amine groups (−NH3+) shown by the peak at 401.9 eV.45 For comparison purposes, XPS analysis was also performed on the PLL-coated glass control. Similarly, a nitrogen peak was detected (Figure 3A), and curve fitting of the N 1s spectrum showed two peaks (Figure 3C). The peak at the higher binding energy of 402.4 eV corresponds to protonated primary amines in the lysine repeat unit, whereas the peak at 400.3 eV can be accounted for by both the uncharged primary amines of the lysine repeat unit and the polymer backbone amides.46 F

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Figure 6. Fluorescent images of cortical neurons cultured on the NH2 hybrid silica (A−D) and the PLL control (E−H) for 72 h. Cells were stained for microtubules (A, E), actin filaments (B, F), and nuclei (C, G). Merged images are also presented (D, H). Scale bar = 100 μm.

Figure 7. (A−D) SEM images of cortical neurons cultured on the NH2 hybrid silica (A, B) and the PLL control (C, D) for 72 h. S: soma; L: lamellipodium. Scale bar = 5 μm. (E) Neuron maturation stage quantification comparing the number of neurons in each stage on the NH2 hybrid silica and the PLL control after 72 h culture. Neurons were classified into respective stages based on their morphological characteristics. Models of stage 0−4 neurons are shown schematically. The number of neurons analyzed for each group is indicated on top of the stacked bar.

NH2 hybrid silica sample, ∼60% cells expressed at least one neurite, slightly higher than the PLL control that supported ∼52% cells to bear neurite (Figure 4F). Finally, the branching index was slightly higher for the PLL control (ca. 3.2 branches per mm neurite) than for the NH2 hybrid silica sample (ca. 2.5 branches per mm neurite) (Figure 4G). These promising results indicate that the surface of the NH2 hybrid silica sample is chemically analogous to the PLL coating in supporting PC12 cell differentiation toward neuronal cells. Cortical Neuron Adhesion and Differentiation. The use of PC12 cell line offered us a cost-effective and simple means leading to the identification of the NH2-containing sample from the 11 different types of hybrid organosilica films to be most worthy of exploration as neuro-integrative coating for implantable neural sensors. However, because PC12 cells are immortalized and require growth factor to exhibit neuron-like behavior, it is important to further gauge the neuro-integrative potential of the NH2 hybrid silica film using primary neurons. Considering that cortex region of the brain is one of the targeted locations for implantable neural sensors, we collected chicken cortical neurons and studied their response to the NH2

neurites extending from the cell body (Figure 4A) in a manner similar to cells cultured on the PLL control surface (Figure 4C), although neurite branching on the NH2 sample was not as pronounced and numerous as the PLL control. In contrast, cells on the SH silica sample showed only short neurite-like processes (Figure 4B), and also the number of attached cells decreased during the incubation period, indicating a poor support for cell differentiation. A similar behavior was reported by Ren et al. using neural stem cells.47 It was found in that study that surfaces functionalized with NH2 or SH groups were both able to support neural stem cell adhesion, but only cells on the NH2-bearing surface exhibited process-extending and growth cone-like structure.47 To better evaluate the cell behavior on the NH2 hybrid silica sample, a quantitative analysis of neurite outgrowth was carried out with reference to the PLL control (Figure 4D−G). Overall, no statistically significant difference (p > 0.05) was observed between the two groups in terms of the various neurite parameters analyzed. For both growth substrates, the average neurite length was about 12 μm (Figure 4D), and the average number of neurites per cell was around 2.4 (Figure 4E). On the G

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Figure 8. Astrocyte cell behavior on plain silica C0 (A, E, I), the NH2 hybrid organosilica (B, F, J), the bare glass control (C, G, K), and the PLL control (D, H, L). (A−H) Phase-contrast micrographs showing cell adhesion after 4 h (A−D) and cell proliferation after 48 h (E−H). (I−L) Fluorescent images of astrocytes stained for vinculin (green), actin filament (red), and nucleus (blue) after 48 h of culture. Scale bar = 100 μm.

hybrid silica film. Our results show that cortical neurons attached readily to the hybrid silica with high viability, and the adherent cell density was even statistically higher (p < 0.05) than that on the PLL control (Figure 5). In addition, 3-day in vitro culture was carried out to determine whether the NH2 hybrid silica film was supportive of cortical neuron differentiation. Since a hallmark indicator of neuron differentiation is neuronal polarization known to be driven by the cytoskeleton,48 we immunofluorescently stained cytoskeletal components such as actin and microtubules to gain detailed view of neuronal morphology. As shown in Figure 6A− D, cortical neurons were differentiating on the NH2 hybrid silica film as evident from the development of neurites. However, a qualitative comparison with the PLL control (Figure 6E−H) revealed distinct differences in the morphology of differentiating neurons. Based on the tubulin staining, individual cells were growing on the PLL control substrate with well-developed long neurites (Figure 6E), and occasionally cell clusters were observed. On the other hand, for the NH2 hybrid silica substrate, differentiating neurons appeared to be bearing several shorter neurites (Figure 6A), and more cell aggregates were noted than the PLL control. Additionally, the flattened, veil-like membrane extensions around the cell body, commonly known as lamellipodia, were more prominent for cells on the NH2 hybrid silica substrate (Figure 6A) than those on the PLL control (Figure 6E). Such lamellipodia structure was barely observed especially for those long neurite-bearing cells on the PLL control (Figure 6E). Staining with Alexa Fluor 594phalloidin revealed the presence of bundles of filamentous (F) actin at the tip of some of the short processes (Figure 6B). The F-actin-rich domains are potential sites of neurite outgrowth.49 It is also interesting to note that cell nuclei on the NH2 hybrid silica substrate were larger than those on the PLL control

surface (Figure 6C,G), implying the different extent of cell body spreading. To further examine neuron morphology and neuron interaction with the growth substrates, the differentiated cultures were fixed and processed for scanning electronic microscopy (SEM). The SEM micrographs also showed the widespread lamellipodia structure around the soma of neurons on the NH2 hybrid silica substrate (Figure 7A,B), consistent with the findings revealed by immunocytochemical staining. For neurons on the PLL control, the soma was instead surrounded by numerous neurite extensions (Figure 7C,D). Collectively, the morphological difference observed from the differentiation study reflects that the culture substrate is affecting the in vitro development of primary cortical neurons. It appears that major neurite extension from neurons on the NH2 hybrid silica substrate is lagging behind neurons cultured on the PLL control substrate. Early studies by Banker and colleagues with the use of dissociated in vitro hippocampal culture have classically demonstrated that neurons derived from the central nervous system transition through several morphological stages to establish neuronal polarity.50 Adopting their criteria, we classified the cultured cortical neurons into five developmental stages in order to further quantitatively examine the impact of the NH2 hybrid silica substrate on the morphological maturation of neurons. First, neurons attach to the culture substrate showing a round cell body (stage 0). Next, attached neurons exhibit intense lamellipodia structure around the cell body (stage 1). At stage 2, neurons consolidate their lamellae and sprout minor processes, also referred to as immature neurites, as well as transitional processes that bear appearance in between lamellipodia and minor processes. Stage 3 neuron features minor and transitional processes, and more importantly, a major neurite that has grown significantly in length to acquire the characteristics of an axon. Stage 4 neuron H

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growth exhibited by neurons and astrocytes inspired them to engineer arrays of submicrometer sized poly(ethylene glycol) hydrogel nanopillars onto laminin-treated silicon surface to differentially support neurite outgrowth and inhibit astrocyte adhesion.53 The advantage of our approach lies in its simplicity offered by the sol−gel process to obtain similar cellular modulation without the use of advanced instrumentations. The ceramic nature of silica is also worth noting, which can be beneficial to address the potential long-term stability issue faced by glial adhesion-inhibiting strategies based on coating neural implants with nonfouling molecules such as hyaluronic acid.54

differs from stage 3 neuron in that all lamellipodia-like features are lost. A schematic representation of various stages cortical neuron morphologies and percentage of neurons in each stage are provided in Figure 7E. It clearly shows that although the population of neurons advancing beyond stage 2 is much higher on the NH2 hybrid silica substrate, its support of neuron maturation into stage 4 morphology is slightly less than the PLL control. It may be reasoned that such an effect could be attributed to the different substrate adhesivity. In the study reported by Lochter et al., neuronal morphology was found to be affected when substrate adhesivity was varied by changing the concentration of cationic polyornithine coating.51 Specifically, neurite outgrowth was favored highly on substrate showing intermediate level of adhesivity.51 Astrocyte Cell Behavior on the Hybrid Silica Films. Among the different types of glial cells present in the central nervous system, astrocytes assume multiple roles with relevance ranging from proper neuronal functioning to neuropathology.52 Furthermore, in the context of implantable neural sensors, astrocytes are known to be responsible for the formation of glial scar encasing the implant and compromising its chronic performance.7 Thus, when developing neuro-integrative coatings for implantable neural sensors, it is also important to gauge whether such coatings support undesirable astrocyte growth. We seeded astrocytes on the NH2 hybrid silica substrate due to its favorable interaction with neurons, and characterized astrocyte adhesion and proliferation. For comparison, plain silica C0, bare glass, and PLL coated glass were investigated in parallel. Early adhesion was analyzed 4 h postseeding, and the phase contrast images showed different levels of astrocyte attachment to the culture substrates (Figure 8A−D). On the plain silica C0 (Figure 8A) and NH2 hybrid silica (Figure 8B) substrates, a relatively low density of adherent cells was observed with majority of the attached cells being rounded. Cell attachment on the bare glass was also low but with few cells exhibiting round morphology (Figure 8C). In contrast, the PLL control was more favorable for astrocyte attachment, shown both by the higher density of adherent cells and by their spread morphology (Figure 8D). After 48 h in culture, the number of attached cells showed little increase on the plain silica C0 (Figure 8E) and NH2 hybrid silica substrates (Figure 8F), whereas cell proliferation was clearly evident on both the bare glass (Figure 8G) and the PLL control (Figure 8H). Fluorescent staining of the 48 h culture samples further revealed that morphological development of astrocytes on the silica substrates (Figure 8I,J) was significantly lagging behind that on the glass (Figure 8K) and PLL control (Figure 8L), shown by the qualitatively lower level of cell polarization. Together, these results signify that unlike the PLL control that was not selective and could promote both neuron and astrocyte cells favoring glial scar formation, the presence of amine groups in the hybrid silica did not encourage astrocyte cell adhesion and growth. We hypothesize that the sol−gel silica nature of the hybrid silica, with highly hydroxylated surface originated by the acidic catalyzed hydrolysis step, is contributing to the unfavorable glial cell behavior, judging by the observation that the plain silica sample is the least supportive of astrocyte growth. Essentially, the characteristics of the NH2 hybrid silica can be summarized as cell-adhesive amine moieties decorating the surface of an otherwise nonadhesive silica substrate. Such selective cell−substrate interaction is analogous to the effect achieved in the study reported by Krsko and co-workers, where the distinct cellular length-scale requirements for adhesion and

4. CONCLUSION Our study demonstrates that among the 11 different hybrid organosilica thin films prepared to improve neural cell response to silica via the simple sol−gel process, the one derived with the addition of (3-aminopropyl)triethoxysilane shows the most support of neuron adhesion and neurite development. The presence of amino groups on the surface of the resultant hybrid organosilica thin film, as evident from the XPS data, is found to be a major contributing factor to the observed neural promoting effect. Interestingly, the amino-modified silica was resistant toward astrocyte cell growth, which is in stark contrast to the findings of regular astrocyte growth on the aminopresenting PLL control. These results imply a promising potential of such an amino-bearing hybrid organosilica thin film to be further explored as a bioactive, cell selective coating for neural interfacing implants.

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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.6b09393. Phase contrast and live/dead assay fluorescence images, film thickness measured by profilometry for the samples C0, C1, C8, C18, Vy, Ph, NCO, Cl, Ip, and Gp, and bare glass atomic force microscopy (AFM) image (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (W.H.). Funding

This study was supported in part by a National Science Foundation (NSF) award DMR-1055208 to W.H. L.B.C. is grateful to the fellowship provided by Brazilian agencies Capes (PDSE, Proc. 5380-13-8) and CNPq (Proc. 151626/2010-3). M.B.C. acknowledges the productivity research fellowship granted by CNPq (Grant No. 309107/2014-8) as well as the financial Fapesp support (Grant No. 2014/22322-2). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge LNLS (CNPEM−Brazil) for XPS measurements. LCS-LNNano (CNPEM−Brazil) is acknowledged for AFM measurements (proposal AFM-NSIIIa-15619). LMFLNNano (CNPEM−Brazil) is acknowledged for the spincoating usage and Maria Helena de Oliveira Piazzetta for all support during experiments. I

DOI: 10.1021/acsami.6b09393 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ABBREVIATIONS PLL, poly-L-lysine; TEOS, tetraethyl orthosilicate; WCA, water contact angle; AFM, atomic force microscopy; XPS, X-ray photoelectron spectroscopy; HS, horse serum; FBS, fetal bovine serum; P/S, penicillin/streptomycin; PBS, phosphate buffered saline; DMEM, Dulbecco’s modified Eagle’s medium; EthD-1, ethidium homodimer 1; RT, room temperature; BSA, bovine serum albumin; NGF, nerve growth factor; SEM, scanning electronic microscopy; HMDS, hexamethyldisilazane; RMS, root-mean-square; PEI, polyethylenimine.

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DOI: 10.1021/acsami.6b09393 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX