Ultrananocrystalline Diamond Thin Films Functionalized with

Feb 18, 2009 - Houjin Huang,†,‡ Mark Chen,§ Paola Bruno,| Robert Lam,‡ Erik Robinson ... National Laboratory, Argonne, Illinois 60439, and Robe...
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2009, 113, 2966–2971 Published on Web 02/18/2009

Ultrananocrystalline Diamond Thin Films Functionalized with Therapeutically Active Collagen Networks Houjin Huang,†,‡ Mark Chen,§ Paola Bruno,| Robert Lam,‡ Erik Robinson,†,‡ Dieter Gruen,| and Dean Ho*,†,‡,⊥ Department of Biomedical Engineering, Northwestern UniVersity, EVanston, Illinois 60208, Department of Mechanical Engineering, Northwestern UniVersity, EVanston, Illinois 60208, Departments of Chemistry and Biological Sciences, Northwestern UniVersity, EVanston, Illinois 60208, Materials Science DiVision, Argonne National Laboratory, Argonne, Illinois 60439, and Robert H. Lurie ComprehensiVe Cancer Center, Northwestern UniVersity, Chicago, Illinois 60611 ReceiVed: January 14, 2009; ReVised Manuscript ReceiVed: February 4, 2009

The fabrication of biologically amenable interfaces in medicine bridges translational technologies with their surrounding biological environment. Functionalized nanomaterials catalyze this coalescence through the creation of biomimetic and active substrates upon which a spectrum of therapeutic elements can be delivered to adherent cells to address biomolecular processes in cancer, inflammation, etc. Here, we demonstrate the robust functionalization of ultrananocrystalline diamond (UNCD) with type I collagen and dexamethasone (Dex), an anti-inflammatory drug, to fabricate a hybrid therapeutically active substrate for localized drug delivery. UNCD oxidation coupled with a pH-mediated collagen adsorption process generated a comprehensive interface between the two materials, and subsequent Dex integration, activity, and elution were confirmed through inflammatory gene expression assays. These studies confer a translational relevance to the biofunctionalized UNCD in its role as an active therapeutic network for potent regulation of cellular activity toward applications in nanomedicine. Introduction The emergence of implantable medical devices has prompted the development of a spectrum of materials that are capable of suppressing inflammatory responses and other processes that can preclude long-term implant activity. For example, polymeric, lipid-based, and metallic nanomaterials have been explored as drug delivery agents for applications in inflammatory suppression and chemotherapeutic release, among others.1-6 Among these classes of therapeutic delivery platforms, ultrananocrystalline diamond (UNCD)-based substrates have been characterized as a promising technology for biological applications owing to their low cytotoxicity, scalable fabrication parameters, chemical reactivity, and biocompatible physical properties.7 Utilizing these favorable properties as a foundation for biointerface applications, the addition of strategies to attenuate surfacemediated inflammatory cytokine expression and release will further enhance the medical significance of UNCD. The fusion of biology and nanotechnology in the form of nanodiamondbased materials has created novel strategies for the fabrication of medically relevant nanodiamond hybrids.8-16 The facile nature of UNCD deposition on silicon surfaces, and the straightforward * Corresponding author. Address: 2145 Sheridan Road, Evanston, IL 60208. Phone: (847) 467-0548. Fax: (847) 491-3915. E-mail: d-ho@ northwestern.edu. † Department of Biomedical Engineering, Northwestern University. ‡ Department of Mechanical Engineering, Northwestern University. § Departments of Chemistry and Biological Sciences, Northwestern University. | Argonne National Laboratory. ⊥ Robert H. Lurie Comprehensive Cancer Center, Northwestern University.

10.1021/jp9004086 CCC: $40.75

approach toward functionalizing the UNCD films with therapeutically active collagen networks enables UNCD interfacing with a broad array of emerging implant technologies, particularly microfabricated devices on silicon substrates. Owing to the scalability and broad relevance of these devices toward the treatment of a spectrum of physiological disorders, they are thus finding applicability in the neurological, cardiovascular, orthopedic, and real-time health monitoring domains, among others. As such, UNCD coatings may play an important role toward enhancing the bioamenability of an important class of advanced implantation technologies. Here, we demonstrate the ability to generate a biologically relevant interface between soft and hard materials, by depositing and interfacing collagen fibrils integrated with the anti-inflammatory element dexamethasone (Dex) atop UNCD thin films, resulting in a therapeutically relevant active substrate. This novel hybrid technology is based upon a facile strategy for nanodiamond film biofunctionalization that can provide potent and requisite inflammatory attenuation toward translational applications. The UNCD film exhibits favorable biocompatibility and has previously been functionalized with DNA oligonucleotides for immobilizing biomolecules and proteins for biosensors.17-19 Through the particular process presented, collagen fibrils were adsorbed to the UNCD film surface, and cross-linkages between collagen fibrils were observed to form via atomic force microscopy (AFM). In addition, biomolecular studies were performed to confirm the successful conjugation and elution of dexamethasone from the collagen matrix via suppression of  2009 American Chemical Society

Letters inflammatory gene expression shown through significant reduction in interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-R). Collagen is a major protein within the extracellular matrix that provides a good foundation for tissue growth on implants and other devices for cellular interrogation studies. The collagen molecule has a length of 3000 Å, a width of 15 Å, and a molecular weight of 300 kDa and is composed of three polypeptide chains of a common repeating amino acid sequence intertwined in a triple helix structure.20,21 The amino acids glycine, proline, and hydroxyproline are held together by covalent bonding and hydrogen bonding between -CO and -NH groups.20 Due to the great tensile strength achieved through bundling, collagen is a major component in muscles and bones.21,22 As such, collagen is an ideal material for biomedical applications with its innate biocompatibility, strength through cross-linking, self-assembly, and aggregation, as well as easily modifiable characteristics.21 This work merges the benefits afforded from both the innately medically relevant UNCD properties23,24 and collagen to generate a resultant hybrid film with Dex-based therapeutic activity. The soft interface serves as a versatile platform that can be functionalized with virtually any drug or effector molecule for translational applications that merge technology with biology and medicine. Experimental Methods UNCD Film Synthesis and Deposition. The UNCD thin films under study were grown using a commercially available IPlas 2.45 GHz microwave plasma enhanced chemical vapor deposition system. A nominal gas composition of 1% CH4 and 99% Ar with 100 sccm total flow rate made up the synthesis gas. Silicon wafer substrates were mechanically polished with fine diamond powder, which yielded a surface roughness of 5-10 nm in order to provide a high density of nucleation sites for UNCD film growth.25 During the deposition process, the substrate temperature was independently set at 800 °C, while the total ambient pressure and input power were kept at 150 mbar and 1200 W, respectively. UNCD films prepared in this way have been extensively characterized by a variety of techniques.23,24 Their microstructure consists of randomly oriented 3-5 nm diamond crystallites strongly held together by largely sp2 bonded carbons at 0.2-0.3 nm wide grain boundaries. UNCD films are distinct from nanocrystalline diamond (ND) films in that the latter are composed of diamond crystallites that are about an order of magnitude or more larger. The unique microstructure of UNCD films is responsible for their being smooth on the nanometer scale. Such films are therefore exceptionally well suited for the production of conformal coatings of biomaterials that can display nanoscale roughness properties. Surface Characterization and Collagen Adhesion. The surfaces of UNCD films were oxidized in concentrated HNO3 at 60-70 °C for 24 h. This oxidation reaction transformed the face of the film from a hydrophobic surface to a hydrophilic surface by adding carboxylate groups to the film. Subsequently, the surfaces were washed with nanopure water, and 50 µL of aqueous type I rabbit collagen (pH 8, Sigma-Aldrich, St. Louis, MO) adjusted with glacial acetic acid, sodium hydroxide, and nanopure water was pipetted onto a 2 × 3 cm2 oxidized UNCD film and allowed to spread across the surface of the film by means of diffusion. The coated UNCD film was then dehydrated in an oven at 40 °C for 30 min under a vacuum to promote solvent evaporation. Following dehydration, the samples were cooled and washed with nanopure water and dried with air to

J. Phys. Chem. B, Vol. 113, No. 10, 2009 2967 remove any weakly bound collagen fibrils on the surface. The collagen-Dex samples were composed of a solution of 45 µL of aqueous type I rabbit collagen (pH 8) and 5 µL of Dex that was coated on the UNCD film based on the procedure detailed above. The final concentration of Dex in cell culture solution was 1.67 µg/mL. Atomic Force Microscopy. An Asylum MFP3D atomic force microscope (Santa Barbara, CA) was used to image the surface of the UNCD thin film deposited on silicon, a UNCD film deposited in silicon and oxidized in concentrated HNO3, as well as collagen-functionalized UNCD films. Imaging parameters included the application of tapping mode atomic force microscopy at a scan rate of 1 Hz. Image sizes included 1 µm × 1 µm and 5 µm × 5 µm. XPS Analysis. XPS analysis was performed on UNCD, oxidized UNCD, collagen, and UNCD-collagen using an Omicron ESCA probe equipped with an EA125 energy analyzer. Spectra were obtained using monochromatized Al K a1 radiation (1486.6 eV) at 10 kV with a current of 7.74 mA under UHV. EIS software (Omicron,Taunusstein, Germany) was used to collect and evaluate survey and high resolution scans. The electron gun was operated at 8 eV and 0.005 mA to compensate for the low electrical conductivity of both collagen samples. Cell Culture. RAW 264.7 (ATCC, Manassas, VA) murine macrophage cells were cultured in 1× DMEM (Cellgro, Herndon, VA) containing 10% FBS (ATCC) and 1% penicillin/ streptomycin (Cambrex, East Rutherford, NJ) at 37 °C. After the cell culture reached 70-80% confluence, the macrophage cultures were split and plated on the four substrates: glass, UNCD film, UNCD film with collagen, and UNCD film with collagen-Dex. After 20 h of growth, lipopolysaccharide (LPS) was added to a concentration of 5 ng/mL in each cell/substrate culture. LPS stimulation lasted for 4 h. The cells were allowed to grow on the substrates for a total of 24 h at 37 °C and then harvested for gene expression studies. Quantitative Real-Time Polymerase Chain Reaction (RTPCR). RNA was purified via cell lysis with 1 mL of TRIzol (Invitrogen, Carlsbad, CA) followed by extraction with 200 µL of chloroform. The samples were then centrifuged for 15 min at 14 000 RPM and 4 °C, and the supernatant was transferred into isopropyl alcohol for RNA isolation and overnight storage at -80 °C to promote nucleic acid precipitation. Following RNA purification, the samples were thawed and centrifuged for 30 min at 14 000 RPM and 4 °C to form an RNA pellet. The supernatant was removed and the pellets were washed with 70% ethanol to remove isopropyl alcohol and salts, and then centrifuged for 5 min at 14 000 RPM and 4 °C. After centrifugation, the supernatant was removed and the pellets were dried in air for 5 min. Nucleic acid analysis with a UV-vis spectrophotometer (Beckman Coulter, Fullerton, CA) was then performed on the RNA samples to determine the amounts of RNA present in each sample, and to normalize RNA concentrations for cDNA synthesis. Using the appropriate RNA volumes based off of A260 values, cDNA was synthesized using an iScript Select cDNA Synthesis Kit (Bio-Rad, Hercules, CA) with reverse transcriptase. The samples were incubated in a water bath at 37 °C for 90 min to complete cDNA synthesis. RT-PCR was run with the iCycler thermocycler (Bio-Rad) on 25 µL samples comprised of DEPC water, SYBR Green Supermix (Bio-Rad), the forward and reverse primers for the respective cytokines, and the cDNA sample dissolved in DEPC water. The amplification conditions were the following0: 95 °C (3 min), 45 cycles of 95 °C (20 s), 55 °C (30 s). The β-actin, mTNF-R, and mIL-6 primer sets were purchased from Integrated

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DNA Technologies (Coralville, IA). The primer sequences used are as follows: TNF-R, 5′-GGTGCCTATGTCTCAGCCTCTT3′ and 5′-CGATCACCCCGAAGTTCAGTA-3′; IL-6, 5′-CACAGAGGATAC-CACTCCCAACA-3′ and 5′ TCCACGATTTCCCAGAGAACA-3′;β-actin,5′-TGGAATCCTGTGGCATCCATGAAAC3′ and 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′. Results and Discussion Formation of a uniform fibrous collagen layer on an UNCD surface is not a trivial issue. The surface properties of UNCD, the complicated conformation and aggregation states of collagen in solution, as well as the delicate interaction between UNCD and collagen are all needed to be considered. The functionalization of UNCD films can be facilitated by the functional groups present on the UNCD film surface. Successful biotic-abiotic interfaces such as the UNCD-collagen integrated film are based on interactions that can take advantage of surface properties from both components of the interface. In this case, enhanced wetting on the UNCD surface can be coupled with the soft/ therapeutically active soft surface properties presented by the collagen. Experiments conducted using NDs as catalysts for oxidation reactions have shown that UNCD surfaces are successfully saturated in atomic oxygen.26 In addition, the oxidation of UNCD surfaces has produced hydroxyl and carboxylic acid groups that can enhance surface wettability to improve collagen protein adhesion.16,27 The hydroxide ions in the aqueous collagen (pH 8) act as the deprotonating agent for the carboxyl group when the aqueous collagen is added to the surface of the oxidized UNCD film. This reaction generates carboxylate anions on the surface of the UNCD that can enhance charged-based interactions with the collagen protein. For example, maximum coating and retention of collagen on the surface of the film was observed at pH 8. Dehydration in an oven was required for successful UNCD-collagen integration to aid in removal of water during formation of the collagen-UNCD interface. Therefore, promoting the rate by driving the mechanism of dehydration in an oven near physiological temperatures was crucial to interface formation. It has previously been suggested that an entropy increase due to liberation of water molecules drives mechanism of adsorption, and not collagen conformational changes.28 In addition, increased temperature in the preparation of collagen has been shown to produce longer, thinner, and more flexible collagen fibers than at lower temperatures. The collagen/surface dehydration treatment at 40 °C for 30 min under a vacuum resulted in the potent adsorption of collagen fibril networks. Figure 2 shows the topographical modifications associated with UNCD oxidation that facilitated robust collagen-UNCD integration. Figure 1A represents a UNCD film deposited on a silicon wafer prior to surface oxidation. Surface features are more pronounced and larger in dimension (e.g., diameter) compared to the oxidized UNCD features shown in Figure 1B. Collagen layers adsorbed on hydrophilic surfaces are more stable, possibly due to a lower affinity caused by increased surface hydration and/or electrostatic repulsion or deprotonated carboxyl functions on the surface.29 Hydrophilic surfaces with a lower rate of adsorption cause a conformal dense layer of collagen to form, whereas, in a hydrophobic surface, a high rate of adsorption/desorption causes loose segments to form and aggregate, albeit through multiple sources of individual fibril linkage29 (Figure 2A). We found that the optimal collagen adherence occurred at a slightly basic pH (Figure 2B). Decreasing the pH of the solution produced little to no noticeable collagen attachment and has been implicated in the formation

Figure 1. (A) Atomic force microscope image of an ultrananocrystalline diamond (UNCD) thin film deposited on silicon. (B) Following surface oxidation in concentrated HNO3 at 60-70 °C for 24 h, the feature sizes were visibly altered to possess reduced diameters and a significantly rougher appearance. The oxidation-mediated conferring of surface-bound carboxyl groups served as a precursor to collagen type I functionalization of the UNCD films.

of micropores, increasing disentanglement due to decreased interfibril forces while forming a diffuse layer of globular subunits.30-32 Moreover, collagen layers are more likely to form meshes,31 increase in density through loss of water from collagen fibers, and have higher stability due to increased intermolecular forces caused by the deprotonation of histidine and ionization of other primary acids at higher pH levels.33-36 In addition to the clearly visible collagen fibrils shown in Figure 2, X-ray photoelectron spectroscopy (XPS) was performed to further analyze UNCD film oxidation as well as collagen presence on the UNCD film. Oxygen content on the UNCD surface increased >500% after oxidation in nitric acid. This may have facilitated the presentation of active functional groups for highly efficient adsorption between collagen and UNCD. Furthermore, free collagen contains sodium ions which were subsequently not observed in the collagen that was potently adsorbed onto the UNCD surface. These ions were likely rinsed away by water, while collagen still remained on the surface (Figure 3A). This provides good evidence of a strong physical interface between UNCD and collagen.Duetoimprovedchargedissipationinthecollagen-UNCD system, downshifts (4-6 eV) in the binding energy of all of the chemical elements in collagen-UNCD (with chemical linkage) were observed as compared to those in free collagen physically coated on UNCD, providing further evidence of robust linkage between the collagen and UNCD (Figure 3B). Figure 4A further confirmed that the fibril structures are indeed representative of collagen due to the visible striations characteristic of collagen structure. Furthermore, based upon

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Figure 3. (A) XPS surveys for UNCD (a), oxidized UNCD (b), free collagen (c), and chemically linked on UNCD (d). (B) XPS of carbons (C1s) in free collagen and collagen-UNCD. Besides the shift in binding energy, the shoulder peak is more evident in free collagen. Figure 2. (A) Without dehydration at 40 °C, the collagen adsorption was easily disrupted and the fibrils could be washed off through rinsing with nanopure water. (B) Optimized bonding and retention of collagen on the surface of the film was observed at pH 8. Dehydration in an oven was required for successful UNCD-collagen integration to facilitate the removal of water during the adhesion process, further promoting dense collagen network adsorption. Both images represent scan dimensions of 5 µm × 5 µm.

the analyzed sectional component denoted by the red line in Figure 4A, the diameter of the surface-bound collagen segment was within the range 20-60 nm (tens of nanometers) depending on fibril stacking which was evident as shown by the sectional graph in Figure 4B. A collagen fibril diameter of approximately 20 nm was previously shown to be representative of collagen type I.37 With regards to collagen-induced cellular activity, type I collagen has previously been reported to induce a localized and transient inflammatory response in cultured cells, with no effect on viability.35 While RAW 264.7 macrophages normally adhere poorly to nondenatured fibrillar layers, heat denatured collagen exposes macrophage binding sites through macrophage scavenger receptors.31 As such, increased temperature in the collagen both helped in the adherence of macrophages and resulted in increased levels of inflammation in some of the tests. Increased gene expression from incubation with collagen was expected due to the interfacing of rabbit collagen with murine macrophages (mouse). This phenomenon additionally served as a positive indicator of the efficacy of the assay. However, due to the potent efficacy of the release of admixed Dex molecules, the hybrid drug-collagen interface served as a favorable active substrate for the suppression of inflammatory cytokine gene expression. Real-time quantitative polymerase chain reaction (RT-PCR) bioassays with RAW 264.7 macrophages were performed in

order to quantify the increase in biocompatibility via a reduction in inflammation. Expression of genes involved in inflammation, namely, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-R), was evaluated after 24 h of growth upon various substrates. LPS was added 4 h prior to cell harvest in order to induce inflammation. Both genes are related to inflammation through the NF-κB pathway.38-40 The NF-κB pathway is a highly conserved immune pathway that mediates several genes critical in regulation of inflammation, responses toward infection, and other stressful cellular stimuli. Among these are cytokine encoders which include TNF-R and Il-6, among others.39,40 Upregulation of both genes has been implicated in an inflammatory response toward foreign materials in ViVo and in Vitro.41,42 In addition, the aforementioned cytokines and enzymes have been implicated with inflammatory diseases such as asthma, rheumatoid arthritis, and joint inflammation.38 The proinflammatory IL-6 cytokine has been linked to inflammation and tumorigensis.43 Upregulated expression of the IL-6 protein and its receptor has been found in cancerous cells.44 TNF-R has been proven to be a key contributor in articulating intracellular endotoxicity.45 The absence of implant biocompatibility has also often led to upregulation in expression of IL-6 and TNF-R, which eventually results in chronic inflammation. In addition, extended cyclical chronic inflammation can lead to cardiovascular disease.46 Dexamethasone (Dex), a type of glucocorticoid, has been shown to attenuate inflammatory gene expression through a variety of mechanisms.38,47-50 Incorporation of Dex and halting inflammation at the interface between artificial and biological materials can prevent potentially damaging effects on an implanted device. As an integral defense mechanism against foreign materials, cells associated with the inflammatory

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Figure 4. (A) AFM imagery revealed the presence of fibril striations that are characteristic of collagen properties. (B) Section analysis revealed a fibril diameter of approximately 20 nm which has previously been shown to be characteristic of type I collagen.

response have been found to surround foreign materials, forming a fibrous capsule that dissociates the device with the body, a phenomenon known as biofouling.41 With the proper dosage, Dex has been used to suppress IL-6 through inhibition of mRNA expression and secretion of IL-6 from cells in prostate cancer patients with little or no adverse health effects.49,50 The biocompatibility of nanodiamond films were confirmed through TNF-R and IL-6 mRNA expression data based on basal studies, which is on the same order as glass. Gene expression data on macrophages stimulated with LPS show an even more profound difference between the levels of cytokines released by macrophages grown on the UNCD film functionalized with collagen and Dex, and the other nonfunctionalized substrates. The elution of Dex was confirmed through the RT-PCR gene expression data, which shows the cytokine expression response of murine macrophage growth on rabbit type I collagen. In Figure 5, the functionalized UNCD film effectively decreased expression of the inflammatory cytokines we investigated by a significant amount. TNF-R expression was decreased over 5 times, and IL-6 expression was abated nearly 30 times compared to our control culture on glass. As such, we observed confirmation of the integration and elution of Dex from the gene expression results shown in Figure 5 where hybrid UNCD samples interfaced with collagen and Dex generated significantly attenuatedexpressionlevelsofinflammatorycytokines(UNCD+C+D), demonstrating the efficacy of the hybrid collagen-UNCD materials and their enormous potential for clinical translation. This work has demonstrated the robust functionalization of UNCD platforms with bioamenable collagen networks embedded with Dex, a clinically relevant anti-inflammatory therapeutic. The fabrication of interfaces that bridge translational technologies (e.g., UNCD coatings) with their surrounding biological environment that preserve favorable biotic-abiotic interactions

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Figure 5. RT-PCR analysis of the collagen-Dex-mediated suppression of inflammatory gene expression. Gene expression levels for TNF-R and IL-6 were significantly reduced due to the elution of Dex from the collagen networks. (Glass ) glass-only samples, UNCD ) ultrananocrystalline diamond-only samples, UNCD+C ) ultrananocrystalline diamond+collagen samples, UNCD+C+D ) ultrananocrystalline diamond+collagen+dexamethasone samples.) Three separate quantitative RT-PCR trials, with each trial run in triplicate, were performed to assess the gene expression activity of the functionalized UNCD surfaces.

are paramount for sustained applications in cellular interrogation and device functionality. Atomic force microscopy and quantitative real-time polymerase chain reaction were used to examine fabrication of UNCD-collagen hybrids following diamond film oxidation coupled with a pH-mediated collagen functionalization process and the suppression of inflammatory gene expression, respectively. Comprehensive film formation and the appearance of collagen fibril networks were confirmed, and potent suppressive behavior of the films toward inflammatory gene expression was confirmed for multiple cytokines. This study demonstrates the relevance of the UNCD-collagen hybrid as an active substrate for the regulation of cellular activity toward applications in nanomedicine. Acknowledgment. D.H. gratefully acknowledges support from a National Science Foundation CAREER Award, V Foundation for Cancer Research V Scholars Award, National Science Foundation Center for Scalable and Integrated NanoManufacturing (SINAM) Grant DMI-0327077, Wallace H. Coulter Foundation Early Career Award in Translational Research, and National Institutes of Health grant U54 A1065359. Work on UNCD film synthesis was performed under the auspices of the U.S. DOE, BES/Materials Sciences under Contract No. DE-AC02-06CH11357. References and Notes (1) Kim, Y.; Dalhaimer, P.; Christian, D. A.; Discher, D. Nanotechnology 2005, 16, S484–S491.

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