Graphene Oxide Finely Tunes the Bioactivity and Drug-Delivery of

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Biological and Medical Applications of Materials and Interfaces

Graphene Oxide Finely Tunes the Bioactivity and Drug-Delivery of Mesoporous ZnO Scaffolds Marco Laurenti, Andrea Lamberti, Giada Graziana Genchi, Ignazio Roppolo, Giancarlo Canavese, Chiara Vitale Brovarone, Gianni Ciofani, and Valentina Cauda ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20728 • Publication Date (Web): 11 Dec 2018 Downloaded from http://pubs.acs.org on December 14, 2018

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ACS Applied Materials & Interfaces

Graphene Oxide Finely Tunes the Bioactivity and Drug-Delivery of Mesoporous ZnO Scaffolds Marco Laurenti,a Andrea Lamberti,a Giada Graziana Genchi,b Ignazio Roppolo,a Giancarlo Canavese,a Chiara Vitale-Brovarone,a Gianni Ciofani,b,c and Valentina Cauda*,a a

Department of Applied Science and Technology, Politecnico di Torino, C.so Duca degli Abruzzi

24, 10129 Turin, Italy. b

Istituto Italiano di Tecnologia, Smart Bio-Interfaces, Viale Rinaldo Piaggio 34, 56025 Pontedera

(Pisa), Italy. c

Department of Mechanical and Aerospace Engineering, Politecnico di Torino, C.so Duca degli

Abruzzi 24, 10129 Turin, Italy. KEYWORDS. Mesoporous zinc oxide, graphene oxide, bioactivity, drug delivery, biocompatibility, bone tissue engineering.

ABSTRACT. Mesoporous zinc oxide (ZnO) scaffolds coated by drop-casted graphene oxide (GO) flakes are proposed as a novel bilayer system featuring bioactivity, biocompatibility and promising loading/release properties for controlled drug-delivery systems. The high-surface area ZnO scaffolds shows clear apatite deposition but the particular surface chemistry and topography prevent the formation of a continuous coating, resulting in micrometric crystalline apatite aggregates after 28 days in simulated body fluid (SBF). When gentamicin sulfate (GS) is considered as a model molecule, the pure ZnO scaffolds also show functional GS loading efficiency, with fast in vitro release kinetics driven by simple diffusion mechanism. Strikingly, the bioactivity and GS delivery 1 ACS Paragon Plus Environment

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properties of mesoporous ZnO are efficiently triggered by drop-casting GO flakes atop of the mesoporous scaffold surface. The resulting ZnO@GO bilayer scaffolds show the formation of a uniform apatite coating after 28 days in SBF. A biocompatible behavior of the ZnO@GO bilayer scaffolds is also observed, supporting the culture of SaOS-2 osteoblast-like cells. Moreover, the GO coating also leads to a barrier-layer effect, preventing fast GS release especially in the short-time range. This barrier effect, coupled to the existence of nanopores within the GO structure, sieves drug molecules from the mesoporous ZnO matrix and allows for a delayed release of the GS molecule. We thus demonstrated a new-generation ZnO@GO bilayer system as effective multifunctional and biocompatible scaffold for bone tissue engineering.

1. INTRODUCTION New-generation biomedical devices based on zinc oxide (ZnO) nanomaterials are capturing considerable attention from various application fields, including tissue engineering,1 biosensing and bioimaging,2 and drug-delivery systems.3 In vivo studies demonstrated that ZnO nanoflowers actively promoted the formation of new blood vessels and bone tissue.4,5 Likewise, the electromechanical coupling between ZnO nanosheet generators and osteoblast-like cells was successfully demonstrated in vitro.6 In this case, the mechanical interaction between the living cells and the piezoelectric nanosheet array locally induced the arise of a piezoelectric potential, modulating the cells viability, differentiation and proliferation. Biosensing units consisting of enzyme-immobilized ZnO nanowires and exploiting the piezo-enzymatic-reaction mechanism were also integrated into self-powered, artificial-skin systems for perspiration analysis.7 To the same extent, graphene-based materials are receiving increasing consideration in biomedical fields.8 Among them, graphene oxide (GO) flakes are intensively explored thanks to their high surface area and generous surface chemistry.9 Moreover, various physical and chemical stimuli like thermal reduction, ultraviolet light irradiation and pH stimulation may be externally provided to remotely control the GO flakes porosity10,11 and interlayer spacing,12 as well as the surface states of GO 2 ACS Paragon Plus Environment

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functional groups.10,11 These aspects might be explored for the design of novel drug delivery systems with remotely controlled release properties. The present study demonstrates for the first time that high-surface-area ZnO scaffolds featuring a mesoporous network show in vitro bioactivity and efficient loading of an antibiotic drug (gentamicin sulfate, GS, as a model molecule). Thereafter, the use of GO flakes is proposed as a novel triggering approach to further promote the formation of a continuous and compact apatite layer and efficiently control the release kinetics of GS at the same time. The resulting ZnO@GO bilayer scaffolds display faster apatite-forming ability than pure ZnO, biocompatible properties and, more strikingly, controlled GS delivery properties in the short-time range with respect to the immediate antibiotic desorption recorded from pure ZnO. This behavior is due to a concomitant barrier-effect and the presence of nanopores within the GO structure, allowing for a time-controlled release of the model molecule from the porous ZnO scaffold. 2. EXPERIMENTAL SECTION 2.1 Preparation of ZnO and ZnO@GO scaffolds. Porous zinc layers were grown on pre-cleaned silicon (Si) wafers (area ~ 1 cm2) by radio-frequency magnetron sputtering and finally converted into mesoporous ZnO through thermal oxidation in air, with a muffle furnace operating at 380 °C for 2 h.13,14 Graphene oxide (GO) flakes were obtained by dispersion of commercial GO powders in bi-distilled water (concentration 0.5 mg mL-1).15 ZnO@GO scaffolds (having GO wt% 1.95, see Figure S1 of Supporting Information, SI) were prepared by drop-casting 50 μL of GO solution atop the ZnO surface and air-drying the samples overnight. 2.2 In-vitro bioactivity and drug-delivery assays. Mesoporous ZnO and ZnO@GO scaffolds were soaked in orbital shaking conditions at 37 °C in a Simulated Body Fluid (SBF) solution, prepared according to Kokubo’s protocol.16 The samples were collected from the SBF solution after 5 h, 1 day, 3 days, 7 days, 14 days, and 28 days, rinsed with bi-distilled water and air-dried overnight. Refresh of SBF solution was performed every 48 h. The pH was time-monitored and close to 3 ACS Paragon Plus Environment


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physiological neutrality values during the overall tests. After each soaking time, the SBF solution was stored at 4 °C and then analyzed by ICP-MS to monitor the time evolution of Ca, P and Zn ions concentrations. Gentamicin sulfate (GS) was loaded on ZnO scaffolds by soaking the samples in a drug/SBF solution for 2 h at a starting concentration of 250 µg mL-1. Then GS release was evaluated in SBF for up to 7 days in orbital shaking conditions at 37 °C.17 2.3 In-vitro biocompatibility assay. Biocompatibility of the samples was investigated with the SaOS-2 osteoblast-like cell model (ATCC CRL7939). To the purpose, 20,000 SaOS-2 cells cm-2 were seeded on the substrates and were cultured with high-glucose Dulbecco’s Modified Eagle’s Medium (Sigma D6546) added with 10% fetal bovine serum (Sigma F4135), 2 mM L-glutamine (Sigma G7513) and 100 U penicillin-100 µg mL-1 streptomycin (Sigma P4333) for 24 h in a 5% CO2, saturated humidity incubator set at 37 °C. As an indication of cell viability, the activity of mitochondrial dehydrogenases was investigated with WST-1 assay (BioVision). Briefly, cell culture medium was replaced with 300 µL of WST-1 reagent 1:11 fold-diluted in phenol-red free proliferation medium. The samples were incubated for 1 h at 37 °C, and then supernatant absorbance was read at 440 nm with Perkin Elmer Victor X3 plate reader. Fluorescence microscopy was also performed on the samples upon fixation with 4% paraformaldehyde in Dulbecco’s Phosphate Buffered Saline (Sigma D8662) and following staining with 100 µg mL-1 tetramethylrhodamine B isothiocyanate-phalloidin (Sigma P1951) and 5 µg mL-1 Hoechst dye (Sigma 94403) in 10% goat serum (Gibco 16210072) for 30 min. Ten representative images were acquired with Nikon Eclipse Ti microscope and used for semi-automated analysis with ImageJ software. 2.4 Characterization methods. Morphological and average thickness analyses were performed by Field Emission Scanning Electron Microscopy (FESEM, Zeiss Merlin). Energy-Dispersive Spectroscopy (EDS) maps were obtained by a desktop SEM Phenom XL equipped with EDS analyzer (map resolution 128 pixel, acquisition time per pixel 20 ms). XRD measurements were performed with a Panalytical X’Pert PRO diffractometer. Cu-Kα monochromatic radiation was used 4 ACS Paragon Plus Environment

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as the X-ray source with λ=1.54059 Å. Infrared (IR) spectroscopy was performed in transmission mode, by a Nicolet5700 FTIR Spectrometer (ThermoFisher, 2 cm−1 resolution, 64 scans accumulation). The time evolution of Ca, P and Zn ions in SBF was determined with an Inductively Coupled

Plasma

Mass

Spectrometer

analyzer

(ICP-MS,

mod.

7500cc,

AGILENT

TECHNOLOGIES). UV-Vis absorbance spectra of GS solutions (350 μL) were acquired in a quartz cuvette (optical path length of 1 mm) in the range 200–285 nm, with a double-beam Varian Cary5000 UV-vis-NIR spectrophotometer. Thermogravimetric analyses were performed using a NETZSCH TG 209F1 Libra instrument between 25 and 800 °C (heating rate 10 °C min−1). 3. RESULTS & DISCUSSION The bioactivity of mesoporous ZnO and ZnO@GO scaffolds was investigated by soaking the samples in simulated body fluid (SBF) for up to 28 days16 and evaluating their ability to induce the formation of an apatite-like coating. The surface morphologies of ZnO and ZnO@GO scaffolds before interacting with SBF are shown in Figure 1a and 1b. The ZnO structure (average thickness 7 μm, surface area: 14 m2g−1, pore volume: 0.095 cm3g−1, Figure S2 of SI)13,18 is featured by interconnected ZnO nanocrystals (< 50 nm) forming mesopores of 12−27 nm. After drop-casting GO flakes atop the pure ZnO scaffold, a very thin layer uniformly covering the whole surface is observed (Figure S3 of SI). The underlying ZnO framework is observable as well, due to the reduced GO thickness. After soaking in SBF for up to 28 days, both the pure ZnO and ZnO@GO scaffolds showed clear bioactive responses. However, the final apatite coating featured different morphologies. For pure ZnO, apatite dots suddenly formed after 5 h in SBF, then evolving into more-defined globular-shaped structures after 24 h (Figure S4). By increasing the soaking time up to 28 days, the apatite globules coalesced together, leading to micrometric apatite aggregates both in size and thickness, spotted on the ZnO surface (Figure 1c and 1e, Figure S4 and S5 of SI). In contrast, the deposition of GO flakes on the ZnO surface promoted the rapid development of a compact and uniform apatite layer, which is highly desired for efficient osseointegration of implants 5 ACS Paragon Plus Environment


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in bone tissue. In this case, the formation of apatite globules was achieved after already 5 h (Figure S4 of SI). The formation of a compact apatite coating was observed after 14 days, which further evolved into a continuous micrometric-thick layer after 28 days (Figure 1d and 1f, Figure S5 of SI). The different morphologies of the so-formed apatite coatings are further highlighted by considering the mapping of the corresponding chemical composition. Figure 2a shows the surface distribution of Ca and P elements for ZnO and ZnO@GO scaffolds soaked in SBF for 28 days. Maps for lower soaking times are shown in Figure S4 of SI. For pure ZnO, nucleation of apatite structures occurred at specific sites, with P being uniformly distributed while Ca localized only close to apatite structures (Figure 1e). On the contrary, a uniform surface distribution of Ca and P is observed for the ZnO@GO scaffold. In both the cases, the Ca/P ratio differs from stoichiometric hydroxyapatite value (1.67) and suggests the existence of a phosphorous-rich phase in the observed apatite layers. This is also represented by IR results and ICP-MS release profiles, and is due to a strong chemical interaction occurring between Zn and P elements for ZnO, as well as between the GO surface and phosphate groups.19,20

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Figure 1. FESEM images of mesoporous ZnO and ZnO@GO scaffolds: (a, b) as-prepared and (c, d) after soaking in SBF for 28 days. The squared regions are shown in panel (e) and (f) at higher magnification.

After soaking ZnO and ZnO@GO scaffolds in SBF for 5 h, an intense phosphate band at 1100-1050 cm-1 of the υ3(PO43-) vibration is observed, unveiling a fast reaction mechanism in both cases (Figure S6 and S7 of SI). This is combined with the broad amorphous band of υ4(PO43-) in the range of 600-560 cm-1, along with the appearance of characteristics OH bands at 1652 cm-1 and in the 7 ACS Paragon Plus Environment


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range of 3600-3500 cm-1. By increasing the soaking time, IR spectra featured similar absorption contributions. Strikingly, remarkable differences among the pure ZnO and ZnO@GO scaffolds are observed after 28 days (Figure 2b). In pure ZnO scaffold crystalline phosphate modes appear, with υ3(PO43-) and υ4(PO43-) bands showing doublet formation and witnessing the presence of nanocrystalline apatite.19 This is combined with the rise of additional absorption bands at 960 cm-1 and 624 cm-1 and due to υ1(PO43-) and υL(OH-) vibrational modes, respectively. Absorption contributions of amorphous phosphate vibrational modes are instead obtained for ZnO@GO scaffolds. It is thus suggested a delayed crystallization process of apatite with respect to pure ZnO. Such delay is further underlined by X-Ray Diffraction (XRD) patterns (Figure 2c). Beyond the typical [100], [002] and [101] contributions of hexagonal wurtzite ZnO, additional peaks at 21.9° and 45.3° are detected in the pure ZnO scaffold after 28 days in SBF, and assigned to [200] and [203] directions of hexagonal hydroxyapatite crystal lattice (JCPDS card n. 090432). For the same soaking time, the [203] peak is only slightly visible in ZnO@GO scaffolds, while the [200] peak is almost negligible. For both scaffolds, no additional peaks are detected for shorter soaking times (see Figure S8 and S9 of SI). Moreover, by considering the most intense diffraction peak and according to Debye-Scherrer equation, a similar average particle size of 41.40 nm and 41.60 nm could be estimated for pure ZnO and ZnO@GO scaffolds, respectively. Figure 2d shows the ICP-MS release profiles for Zn, Ca and P ions monitored during the overall soaking time. For pure ZnO, fast Zn release is observed during the first hours. Meanwhile, fast precipitation of P and Ca is observed in the first 5 h, then slowly decreasing and approaching a plateau for the rest of the soaking time. On the contrary, a slower release of Zn ions is noticed for ZnO@GO samples, while both Ca and P ionic concentrations globally decrease during the overall time monitoring, with a slower kinetic during the first days than for pure ZnO. The observed bioactive response for ZnO and ZnO@GO scaffolds, leading to apatite coatings with different features, is due to the combination of multiple effects, involving surface chemistry and topography, as well as the crystal structure of the scaffold materials. For pure ZnO, ICP-MS profiles highlight the partial ZnO dissolution and quick 8 ACS Paragon Plus Environment

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absorption of Ca and P elements for short soaking times (5 h). This led to ZnO hydration and the formation of a negatively-charged surface exposing Zn‒OH groups,14 which is favorable for attracting positively-charged Ca2+ ions and further attraction of negatively-charged PO43- groups, thus inducing the formation of calcium phosphate compounds.

Figure 2. (a) EDS spectra and elemental mapping for ZnO and ZnO@GO scaffolds soaked in SBF for 28 days. Scale bar is 80 μm. (b) IR spectra and (c) XRD patterns of ZnO and ZnO@GO scaffolds soaked in SBF for 28 days. (d) ICP-MS release profiles for P, Ca and Zn ions in SBF. If the soaking time is further increased, Ca2+ and PO43- ions are continuously absorbed from SBF towards the ZnO surface, even though with a slower kinetics. On the other side, the hexagonal wurtzite crystalline structure of pure ZnO acts as a crystalline seed for the nucleation and growth of 9 ACS Paragon Plus Environment


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crystalline apatite.21 This seeding effect, combined with the slow kinetics of absorption, finally result into the formation of nanocrystalline apatite after 28 days. However, both the marked surface roughness of the porous ZnO scaffold and the sudden release of Zn2+ ions observed during the first hours hinder the formation of a continuous apatite layer. In this range, the combination between PO43- and Zn2+ ions and the formation of zinc phosphate compounds is highly favored because of the lower solubility product constant of zinc phosphate than of calcium phosphate.19 Hence, PO43and Ca2+ ions more difficultly combine together and calcium phosphate formation is partially prevented. This also explains the formation of a phosphorous-rich apatite compound, as evident from chemical composition analyses (Ca/P ~ 0.7), and which has been reported to be helpful in promoting in vivo osseointegration.22-24 On the other hand, the negatively-charged ZnO@GO surface is due to the intrinsic abundance of hydroxyl groups of GO and further promotes absorption of Ca2+ ions from SBF solution. PO43- are then attracted and finally calcium phosphate compounds are formed. The release of Zn2+ ions is more efficiently slowed down due to the presence of GO flakes, hence limiting the formation of zinc phosphates to the benefit of calcium phosphate compounds (Ca/P ~ 1). Moreover, the GO flakes highly smoothened the ZnO scaffold surface, allowing for a continuous apatite coating obtained after 28 days. However, IR and XRD results also show that GO flakes partially limited the seeding effect of the hexagonal wurtzite ZnO structure and delayed the apatite crystallization process. This is confirmed by ICP-MS profiles as well,25 which highlighted an overall faster interaction kinetics between Ca ions, P ions and the ZnO@GO surface than for pure ZnO. In view of the application of the proposed materials for bone tissue regeneration, the biocompatibility properties of ZnO, GO and ZnO@GO scaffolds were also preliminarily examined by studying their interaction with SaOS-2 osteoblast-like cells. Figure 3a reports the results of WST-1 assay performed after 24 h from cell seeding, showing an almost comparable mitochondrial activity for the ZnO, GO and ZnO@GO scaffolds, although significantly lower than that on the routine control substrates. Figures 3b and 3c show representative fluorescence microscopy images 10 ACS Paragon Plus Environment

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of SaOS-2 cells cultured on glass (as control) and on the ZnO@GO scaffolds after 24 h from seeding. In particular, Figure 3c qualitatively demonstrates good SaOS-2 cell adhesion on the ZnO@GO scaffold surface, although the nuclei number on ZnO@GO is lower than that on the control substrates, likely denoting a slower cell adhesion on this sample typology. The obtained results suggest that both cell adhesion and viability are encouraging for the ZnO@GO bilayer structure. As a further consideration, it is clear that the biological results could be influenced by multiple factors typical of the ZnO@GO scaffold architecture, mainly including surface chemistry and roughness, which will be the object of future investigations.

Figure 3. WST-1 assay results, *p

Graphene Oxide Finely Tunes the Bioactivity and Drug-Delivery of

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