Composition-Tailoring of ZnO-Hydroxyapatite Nanocomposite as

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Composition-Tailoring of ZnO-Hydroxyapatite Nanocomposite as Bioactive and Antibacterial Coating Ori Geuli, Israel Lewinstein, and Daniel Mandler ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.9b00369 • Publication Date (Web): 22 Apr 2019 Downloaded from http://pubs.acs.org on April 23, 2019

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ACS Applied Nano Materials

Composition-Tailoring of ZnO-Hydroxyapatite Nanocomposite as Bioactive and Antibacterial Coating Ori Geuli,† Israel Lewinstein, ‡ and Daniel Mandler †*.

†

Institute of Chemistry, the Hebrew University of Jerusalem, Jerusalem 9190401, Israel

‡ The

Maurice and Gabriela Goldschleger School of Dental, Medicine, Department of

Oral Rehabilitation, University of Tel Aviv, Tel Aviv 6997801, Israel

Keywords: hydroxyapatite, ZnO, nanoparticles, electrophoretic deposition, antibacterial

Abstract

Overcoming post-implantation infections is considered as a major challenge in medicine, where continuous efforts have been invested in developing bactericidal functional coatings. The synergic combination of hydroxyapatite (HAp) and ZnO holds beneficial properties, such as excellent bioactivity that is reinforced with antibacterial nature. Here,

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highly-phase tunable pure ZnO-HAp nanocomposite coatings were fabricated via electrophoretic deposition. HAp and ZnO nanoparticles were synthesized separately, dispersed simultaneously and co-electrophoretically deposited onto a titanium substrate. By manipulating the deposition potentials, a single-stage graded-coating of ZnO-HAp was successfully obtained. In addition, by controlling the composition of the nanomaterials in the dispersions, we managed to precisely tailor composite coatings with a dictated phase ratio between ZnO and HAp. In-vitro studies, bioactivity, cytotoxicity and antibacterial tests, showed excellent performance by enhancing the mineralization of the coating and improving cells proliferation while successfully eradicating E.coli bacteria. . Introduction

The prevention of implantation-associated bone infections is still a major challenge for both orthopedic and dental surgeries. The prophylaxis treatment is based on both intravenous and oral antibiotic administration. The main limitations of the conventional therapy is the risk of systemic toxicity (usually renal and liver complications) and insufficient penetration of the drugs into the infected tissues. In addition, once a biofilm is formed, the debridement of the infected tissues is inevitable, which ultimately leads to implantation failure.1 Numerous efforts have been made in order to overcome these limitations, in particular, by using local delivery systems. The concept of delivering high concentrations of drugs to the vicinity of the infected area has been vastly investigated, where commercial products, such as antibiotics-loaded poly(methyl methacrylate) 2 ACS Paragon Plus Environment

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(PMMA) beads have already reached the surgery rooms 2. The main drawback of using non-biodegradable systems, such as PMMA, is the need for additional operation for beads removal. Other biodegradable systems, such as collagen,3 and lactic acid polymers,4 have been fabricated. In spite of the fact that bioresorbable polymers have excellent biocompatibility, their tendency to reduce healing time by enhancing bone ingrowth towards the implant is very limited. Therefore, biomaterials, such as calcium phosphates have been widely used as drug-carriers for local delivery systems. In addition to their high biocompatibility, calcium phosphates have superior ability to induce bone regeneration.5 Hydroxyapatite (HAp, Ca5(PO4)3OH) has gained much recognition as bone filler due to its high structural similarity to the natural bone mineral. HAp has a unique ability to enhance biochemical interactions with biological systems, which promotes the regeneration of the hosted bone towards the grafted material.6 Due to its poor mechanical properties, such as brittleness and low impact resistance, HAp is usually applied as a coating material on metallic supports. Ti and its alloys, especially Ti-6Al-4V have been extensively used for surgical implantation due to its good biocompatibility and high corrosion resistance.7 Ti is biologically inert, therefore, coating Ti supports with HAp can enhance the formation of new bone tissue within a short time period by promoting osteoblasts adhesion and inducing the biomineralization of the implant.8 Incorporation of antibiotics with HAp may hold significant benefits, such as superior osteoconductivity and bactericidal properties. As evidence, HAp has been studied as efficient drug-delivery platform for sustained, local and controlled antibiotics release.6 3 ACS Paragon Plus Environment


Currently, overuse of common antibiotics has led to significant prosperity of antibioticsresistance bacteria, which have become a major concern globally. Therefore, several alternatives have been utilized as efficient antimicrobial agents, such as Ag nanoparticles (NPs), quaternized amines, metal oxides among others, which have wide antibacterial spectrum with barely no bacterial resistance.9 Ag NPs are one of the most powerful antibacterial activity in biomedical applications. The main drawback of Ag NPs is the continuous dissolution and formation of Ag+, which has high toxicity towards all biological systems, even in very low concentrations.10 Metal oxides, such as TiO2, CuO, Fe2O3, ZnO and others, have been extensively studied as efficient bactericidal agents, due to their high stability and relative non-toxicity.11 ZnO has been selected as a preferred antibacterial agent

12

as compared with other metal oxides for its high biocompatibility

and low toxicity. As evidence, ZnO has been listed as generally-recognized as safe (GRAS) by the United States Federal Drug Administration (21CFR182.8991). The antibacterial activity of ZnO is linked with several suggested mechanisms, such as generation of reactive oxygen species (ROS), membrane dysfunction upon direct contact by electrostatic interactions and accumulation of zinc ions, which interfere with respiratory processes inside the bacterial cell.13 The antibacterial activity of ZnO has been suggested to be greatly affected by the morphology, structure and size, where ZnO NPs showed enhanced biocidal effect.14 Therefore, combining HAp NPs as a bioactive component with ZnO NPs as an antimicrobial agent, may provide a superior composite coating for biomedical implants. 4 ACS Paragon Plus Environment

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Yet, the number of studies that describe the formation of mixed coatings that contain both HAp and ZnO and their antibacterial application, is limited. Several approaches have been applied for the fabrication of such coatings. One technique is based on doping HAp with Zn ions to produce Zn-HAp NPs.15 However, this technique has significant limitations. For instance, too high concentration of Zn2+ could result in the precipitation of Zn-Ca-P species, such as Scholzite (CaZn2(PO4)2∙2H2O) and other zinc/calcium phosphates. Therefore, the loading concentration of Zn is very limited. In addition to releasing of antibacterial Zn2+, the antimicrobial of ZnO is also associated with ROS production and bacterial membrane destabilization by a direct contact. Therefore, doping HAp with Zn2+ will not necessarily provide the same benefits provided by ZnO. Another approach involves the fabrication of ZnO-HAp coating by different techniques, such as spincoating,16 spraying,17 electrohydrodynamic atomization (EDHA),18 and sputtering.19 The main disadvantages of these techniques are expensive equipment and insufficient control on the coating process. For instance, application of spin-coating to complex geometries, such as dental implants is inapplicable. On the other hand, electroplating is a non-line of sight technique, inexpensive and simple for coating substrates with complex geometries,20 which has been vastly applied for both HAp

21-22

and ZnO

23-24

coatings.

Interestingly, only few studies have been applied for the electroplating of HAp-ZnO. Gopi

et al electrochemically deposited HAp-ZnO-Sr composite coating from ionic species to fabricate both bioactive and corrosion protective coatings.25 Boccaccini et al



electrophoretically deposited ZnO-bioactive glass-alginate composite bactericidal coating for orthopedic implants.26 Here, we report on the electrophoretic deposition (EPD) of pure HAp and ZnO NPs for the fabrication of both bioactive and antibacterial composite coating for biomedical applications (Scheme 1). This techniques serves as fundamental platform for producing highly-controllable composite coatings, such as nanocomposite graded coatings. Welldefined NPs were synthesized by different methods and dispersed in 2-propanol (2PrOH) by adding triethanolamine (TEOA). EPD was carried out under different potentials and in dispersions containing various NP concentrations of ZnO and HAp. We managed to control the composition of the coatings by tuning the applied potential and by alternating the ratio between HAp to ZnO in the dispersion. Hence, utilizing EPD as the driving force for the fabrication of nanocomposites enables the precise control of the physical properties as well as the chemical composition of the coating. Scheme 1.

Experimental Section

HAp NPs Synthesis: 4.722 g of Ca(NO3)2 (ACS EMSURE®, Merck) was dissolved in 18 mL of deionized water (18.3 M cm, EasyPure UV, Barnstead) using a magnetic stirrer. The solution was


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adjusted to pH 12 by adding 0.6 mL ammonium hydroxide (25%, Baker Analyzed®, J. T. Baker) and 17.4 mL of water. 1.584 g of (NH4)2HPO4 (BioUltra≥99.0%, Sigma) was dissolved in 30 mL water while stirring. The solution was adjusted to pH 12 by adding 15 mL of concentrated ammonium hydroxide and 19 mL of deionized water. The diammonium phosphate solution was dropwise added to the calcium nitrate solution using a separatory funnel while vigorously stirring. The slow addition resulted in a turbid suspension. The latter was boiled for 1 h. Then, the suspension was cooled to room temperature and aged for 3 days. The precipitated NPs were washed with water and centrifuged at 10,000 rpm for 5 min. This was repeated three times. The HAp NPs precipitate was collected and freeze-dried. The HAp NPs were characterized by X-ray diffraction (XRD, Bruker, D9 Advance), X-ray photoelectron spectroscopy (XPS, Axis Ultra), high-resolution scanning electron microscopy (XHR-SEM, FEI Magellan 400L), and high resolution transmission scanning electron microscopy (HR-TEM, Tecnai F20 G2). All XRD results were compared to the ICSD (Inorganic Crystal Structure Data) files. Solvothermal Synthesis of ZnO NPs ZnO NPs were synthesized by a solvothermal synthesis. 15.36 gr of zinc acetate dehydrate (≥99%, Sigma) was suspended in 50 mL ethanol (ACS, Merck) at 60 °C. The solution was vigorously stirred for 15 min, then 5.22 gr of triethanolamine (TEOA, BAKER ANALYZEDTM, J. T. Baker®) dissolved in 20 mL ethanol was rapidly added, and the solution was stirred for additional time at 60 °C. After approximately 1 hour, clear solution 7 ACS Paragon Plus Environment


was obtained, then the solution was aged for additional 1 hour at room temperature. Then, the solution was transferred into 100 mL PPL (para-polyphenylene) lined vessel, which was placed inside a stainless steel autoclave. The autoclave was placed inside an oven for 18 hours at 150 °C. Then, the sample was allowed to cool down at room temperature, the white precipitate was washed in deionized water and centrifuged for 5 min at 6,000 rpm. The washing process was repeated 3 times. Then, the ZnO precipitates were collected and dried in an oven at 70 °C overnight. The formed clusters were milled by a mortar and pestle to form a powder. The characterization of ZnO NPs was similar to that of HAp NPs. Titanium Surface Pretreatment Ti (Grade 4) plates and Ti–6Al–4V rods were purchased from Barmil Ltd. The surface area of the Ti plate was 1.08 cm2, and that of the Ti-6Al-4V rod was 2.68 cm2. The Ti plates were manually ground on grit 600 grinding paper (Microcut®, Buehler), rinsed in acetone, ethanol and water in ultrasonic bath (Elmasonic P, Elma) for 10 min, and etched in (30%)HF/(65%)HNO3 (2/20 %v, respectively) for 1.5 min. Commercial dental implants made of Ti–6Al–4V from SGS Dental Implants (Schaan, Liechtenstein) were also used. ZnO-HAp NPs dispersions Pure ZnO/HAp dispersion was prepared by adding 50 mg of NPs and 20 mg of TEOA to 20 mL of 2-PrOH. For ZnO-HAp dispersions certain amount of both NPs were added.


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Specifically, for ZnO-HAp (7:3), ZnO-HAp (1:1) and ZnO-HAp (3:7), 49 mg of ZnO and 21 mg of HAp, 25 mg of ZnO and 25 mg of HAp, 21 mg of ZnO and 49 mg of HAp, respectively, were added to 20 mL 2-ProH containing 20 mg of TEOA. Stable dispersions were obtained followed by 25 min sonication by using tip-sonicator (Sonics, Vibra cell) with pulse rate of 1 sec on, 1 sec off at 70% amplitude. The suspension stability was examined by measuring the (zeta) potential and particle size distribution (Zetasizer, Malvern ZS). Electrophoretic Deposition Electrophoretic coatings were obtained by using a DC power supply (Major Science, Mini 300) in a conventional two-electrode cell. Pretreated Ti (Grade 4) was used as cathode, while stainless steel 316L with the same dimensions was used as anode. The two electrodes were placed in parallel, and the distance between them was approximately 1 cm. For each experiment, a fresh 15 mL dispersion was used. Deposition was carried out either by applying different potentials (10-60 V) for 2 min, or under a constant potential of 60 V for 5 min with different ZnO/HAp ratio. After deposition, the samples were left to dry at ambient. Commercial dental implants were used also as substrates. Coating Characterization The coated substrates were analyzed by XRD (2θ = 20–70° at step size 0.02 °s–1 at). Quantitative phase content of the coatings was obtained by Rietveld analysis. High



magnification images of the coatings were acquired by XHR-SEM and HR-TEM. Crosssection images and thickness measurements were obtained by SEM equipped with focused ion beam (FIB). Element analysis was performed by XHR-SEM and HR-TEM (FEI Titan Themis 60-300 kV) equipped with energy-dispersive X-ray spectroscopy (EDS). Fourier transform infrared (FTIR) spectra were recorded with a Bruker Vertex 70v spectrometer in the reflection mode (80°) using a liquid-nitrogen-cooled mercury cadmium telluride (MCT) detector. The samples were scanned 1023 times with 4 cm‒1 resolution. The spectra were recorded between 400 and 4500 cm‒1. The strength of adhesion of ZnO-HAp (3:7) coating to the Ti substrate was tested by a standard shear test. Prior to the adhesion test, the coatings were annealed at 700 °C for 60 min, at a heating rate of 2 °C/min. Each test specimen was composed of a coated and uncoated substrate with the same dimensions and surface pretreatment. The two substrates were glued together by a thin layer of epoxy resin (Poxipol®) followed by 24 hrs curing at room temperature. The experiments were performed with a mechanical testing machine (Instron 4502) at a cross-head velocity of 10 mm min-1. Bioactivity Test Simulated body fluid (SBF) was prepared according to a previous procedure.27 The coated implant was soaked in SBF solution at 37±1 °C for 4 weeks in a humidity chamber (Memmert, HCP 108). Every 5 days the SBF solution was replaced by a fresh one. The morphology of the implant was examined by SEM, XRD and EDS. 10 ACS Paragon Plus Environment

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Cytotoxicity Test In-vitro Cytotoxicity test was conducted on Jurkat (human lymphoma, purchased from ATCC Inc) and HaCaT (human keratinocytes, purchased from CLS, Cell lines Services). Briefly, Jurkat cells were incubated at 37 °C with ZnO-HAp (3:7) scraped coating (100 g/mL) for 3 days. The cell viability was examined by two cytotoxicity assays, MTT and resazurin. Both assays indicate of metabolic activity, which correlates with the number of viable cells. The results were normalized to the untreated samples defined as 100%. In MTT assay,28 the cells were incubated with 0.5 mg/mL (3–4,5-dimethylthiazol-2–yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma Aldrich) for 1-2 hrs at 37 °C. The resulting precipitated stain was extracted in DMSO with shaking for 15-30 min at room temperature. The cell viability was determined spectrophotometrically by subtracting the OD at 570 nm by the OD 690 nm. By the resazurin assay,29 the cells were treated with a fresh growth medium containing the resazurin dye (Sigma-Aldrich) (0.01 mg/mL) for 1-2 hrs at 37 °C. The dye penetrated inside viable cells, and reduced by the cellular mitochondrial activity into a fluorescent product, which was detected in the culture supernatant. The cell viability was determined by measuring the fluorescence at the excitation and the emission wavelengths (544 and 590 nm, respectively). Dose response experiments were carried out by incubating HaCaT cell lines with different concentrations of ZnO-HAp (3:7) for 3 days at 37 °C. The cells viability was determined by the MTT assay. Antibacterial Activity

Escherichia Coli (purchased form ATCC, USA) was used as a model bacteria for this study. A colony of E.coli was cultivated at 37 °C with shaking (120 rpm) in 15 mL of LB (Lennox, DifcoTM, Fisher Scientific) overnight. The cell density of the bacterial suspension was adjusted to OD600= 0.3 by adding fresh LB to the overnight cultivated suspension. A 10 L aliquot of bacterial suspension was dropped on top of each coated Ti and allowed to incubate for 4 hours in a 6 well plate under agitation (120 rpm). This was essential to



ensure that all the bacteria settled on the coated substrate. After 4 hours, the substrates were washed by adding 5 mL of LB and incubated with shaking (120 rpm) for additional 18 hours. After incubation, 1.5 mL aliquot was analyzed spectrophotometrically (at 600 nm) in order to verify bacterial proliferation. The antibacterial tests were performed in triplicates and repeated three times. Results and Discussion

HAp NPs were synthesized by a wet chemical precipitation reaction between calcium and phosphate precursors and characterized by various techniques. Figure 1SAI shows a SEM image of the HAp synthesized NPs. It can be seen, that the NPs have an oval shape, similarly to previous reports.30 The average particles size was 30±12 nm based on TEM (Figure 1SBI). Figure 1SCI shows the selected area electron diffraction (SAED) pattern of the HAp NPs. The spotty diffraction speckles in the diffuse rings confirm the polynanocrystalline structure of HAp NPs.31 Table 1S shows the XPS element analysis of the HAp NPs. The obtained powder is composed of Ca, P, O and C, where the latter is associated with carbon impurities. The atomic Ca/P ratio is 1.73, which is slightly higher than the expected stoichiometric Ca/P (1.67) in HAp. Therefore, we conducted an XRD analysis to further clarify the phase content. Figure 1SDI shows the diffraction peaks of the HAp NPs and the corresponding indexes of pure HAp (ICSD-01-084-1998). It is evident that no other peaks than HAp are detected. The calculated degree of crystallinity


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(based on XRD) is 74%.32 The average hexagonal crystalline size (based on the Scherrer equation) is 17 nm. The cell parameters are a=9.41 and c=6.88 Å, which are in agreement with the unit cell of stoichiometric HAp. ZnO NPs were prepared by solvothermal synthesis, were zinc acetate was utilized as the zinc donor, TEOA used as a capping agent and ethanol as an oxygen donor. The elaborated mechanism of the reaction can be found elsewhere.33 Figure 1SAII shows a SEM image, which reveals that ZnO NPs have hexagonal structure with elongated shape. Figure 1SBII is a TEM image of the ZnO NPs, which shows a similar structure compared with the SEM. SAED (Figure 1SCII) confirms the single crystal hexagonal structure of well-crystallized ZnO NPs.34-35 The average particles size was 141±59 nm, indicating a certain polydispersity in size. The XRD pattern of ZnO (Figure 1SDII) shows the typical peaks of highly crystallized well-defined ZnO (ICSD-01-089-7109). The strong orientation towards (101) index supports the elongated morphology of ZnO. No other impurities were detected. The average crystalline size (based on Scherrer equation) was 69.7 nm. The cell parameters (a=3.24, c=5.20 Å) were identical to the theoretical lattice parameters of ZnO and the average crystalline size (based on Scherrer equation) is 68 nm. XPS elemental analysis (Table 1S) shows that the obtained ZnO powder is composed of Zn and O. Any traces of nitrogen as a result of TEOA adsorption during the synthesis have not been found. The detection of carbon might be related to carbonaceous contaminations, as mentioned above.



Stable ZnO-HAp dispersions were prepared by adding certain amounts of NPs to 2-PrOH. 2-PrOH was selected as solvent due to the high dispersibility of both ZnO and HAp. TEOA, a well-known biocompatible36 dispersant, was added in order to increase to the stability of the NPs in 2-PrOH. TEOA forms strong complexation with Zn2+ and therefore can increase the stability of ZnO by increasing the -potential.37 Specifically, TEOA as a strong organic base can induce the ionization of alcohols to form alkoxide ions and positively charged ammonium, which can be adsorbed on the surface of NPs and increase the -potential.38 Table 1 summarizes the dispersions parameters of ZnO and HAp as a function of TEOA addition. The incorporation of TEOA on the NPs surfaces has a significant impact on the surface charge of the NPs, where the -potential is considerably increased. The high stability of the dispersions of both NPs can be also manifested by measuring the particles size distribution (by dynamic light scattering), where the tendency of the NPs to aggregate is dramatically diminished. As evidence, the particles size distribution curves of both nanomaterials (Figure S2) were shifted towards smaller diameters. These results are in agreement with Figure S1, where the native structure is maintained in the dispersion after TEOA was added. The formation of stable dispersions made it possible to continue to the next phase, which aimed at the formation of composite coatings based on the EPD of both components, i.e. HAp and ZnO NPs. Table 1.


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ZnO-HAp coatings were obtained by EPD, where charged NPs migrate under electric field towards the electrode surface possessing the opposite charge. Both ZnO and HAp NPs acquire positive charge in alcoholic solvents, particularly, with the addition of TEOA, which enabled their co-deposition on the cathode. In these set of experiments Ti (Grade 4) was used as cathode. Firstly, we investigated the impact of the applied potential on the coating composition, taking into account that both of the NPs have similar -potentials. All the experiments in this section were carried out in equal concentration of ZnO and HAp, namely 0.125% wt/v of each component in the dispersion. Interestingly, we were witnessed to an unexpected result, where at relatively low potentials (10 V) the deposition of ZnO favored that of HAp. This uneven deposition gradually decreased under higher potentials. Figure 1A shows the cross-section of STEM images of a few nanometer thick ZnO-HAp lamellas, obtained by FIB cutting. It can be seen that applying higher potentials resulted in thicker layers of ZnO-HAp. The brighter NPs are made of ZnO while the darker are HAp (due to charging effects). Closer inspection of the cross section reveals that at higher potentials, the amount of HAp NPs in the coatings increased. Figure 1B shows the corresponding element analysis by EDS of ZnO-HAp lamellas (shown in Figure 1A). It can be seen, that the at% of Zn decreased by more than a factor of two as the potential was increased from 10 to 50 V, whereas the at% of HAp (Ca and P) increased as a function of applied potential. Figure 1C shows the phase composition 15 ACS Paragon Plus Environment


of the nanocomposite coatings based on XRD measurements. Clearly, the ratio between the ZnO and HAp reaches that of the deposition solution, i.e., 1:1, at high potentials. It is worth mentioning, that due to the low signal of HAp at potentials of 10 and 20 V, the XRD results are based on semi-quantitative Rietveld analysis. Since, the thickness of the film increases almost linearly with potential (Figure 1C) we attribute the change in the ZnO:HAp ratio to the change in the kinetics of HAp deposition as a function of potential. This, might further be associated with the difference in the -potential. According to Smoluchowski equation (Equation 1) the electrophoretic mobility (e) is linearly dependent on the -potential.39 (1)

𝜇𝑒 =

𝜀𝑟 ∗ 𝜀0 ∗ 𝜉 𝜂

Where, r is the dielectric constant of the solvent, 0 is the permittivity of vacuum, and  is the viscosity of the medium. All the parameters besides the -potential were constant, where the latter was similar for both ZnO and HAp, namely 46.2 and 42.1 mV. In addition, the -potential of both of the two nanomaterials was measured separately. We assume that the minor difference in potential (≈4 mV) could not exclusively clarify the difference in the deposition kinetics. Therefore, in order to study the deposition kinetics of ZnO and HAp, we electrophoretically deposited ZnO and HAp separately and measured the thickness of the coatings as a function of the applied potential (Figure 3S). Figure 4SA-B shows the SEM images of the films obtained by the EPD of HAp and ZnO NPs, respectively. It can be seen that the HAp 16 ACS Paragon Plus Environment

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coatings were substantially thicker than the coatings of ZnO NPs, which can indicate on higher electrophoretic mobility. The deviation in the electrophoretic mobility from Smoluchowski equation, which showed similar -potential, may be associated with the substantial difference in the particles size. This variation in the NPs size may affect the electrophoretic velocity () according to the following Equation 240: (2)

𝜈=

𝑄 4𝜋𝑟𝜂

Where, Q and r are the charge and radius of the particle, respectively, and  is as above. Since, the surface charge or the -potential, is similar for both of the NPs, the difference in size apparently governs the electrophoretic mobility. Interestingly and as shown above, when ZnO and HAp were mixed together in a dispersion, followed by their EPD, ZnO exhibited higher electrophoretic mobility than HAp. Since, the two nanomaterials showed different electrophoretic mobility separately, we speculate that there are certain interactions between ZnO and HAp NPs that affect their joint migration under electric field. Since, ZnO is more soluble than HAp (≈7 mg/L 41 and 43 g/L 42, respectively), we assume that partial dissolution of ZnO to generate Zn2+ may interfere with HAp deposition by surface adsorption as shown in previous studies.43 In order to assess our assumption, we measured the -potential of HAp NPs in different concentration of Zn ions (Figure 5S). It is important to mention that the dispersion of HAp was prepared exactly as described in the experimental section. It can be seen that the Zn ions have a considerable impact on the -potential of HAp NPs, where even at low 17 ACS Paragon Plus Environment


concentration (1 M) the surface charge of HAp decreased from 42.1 mV to 50 V, the thickening rate for ZnO was substantially increased to 1.3 m/min, where for HAp it was increased to 2.6 m/min. The significant increase in the thickening rate of the deposited films manifests the impact of the high electric field, which can compensate the difference in the -potential between ZnO and HAp in the mixed dispersion.

Figure 1.

Taking advantage of the effect of potential on the kinetics of deposition mechanism, was further applied for the formation of a graded coating of ZnO-HAp by stepwise increasing the applied potential from 10 to 30 to 50 V. Each potential was applied for 2 min. Figure 2A shows a HR-TEM cross-section image of the ZnO-HAp coating lamella. Elemental analysis by EDS and mapping are also provided (Figure 2B, C, respectively). The graded coatings are divided into three areas (based on the deposition rate shown in Figure 2B) which correspond to the applied potential. Namely, the bottom area was deposited at 10


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V, whereas the upper area was deposited at 50 V. We assume that the middle area was formed at 30 V. It can be seen that the bottom layer is enriched with ZnO, which gradually decreased up to the top layer. The reversed behavior is shown for HAp, i.e., the content increases with the development of the film. EDS and mapping clearly suggest a similar trend, where the concentrations (% at) of Ca and P increase from area 1 to 3 and vice

versa for Zn. These results definitely prove that electrophoretically deposited graded coating can be formed by alternating the potential in addition to changing the concentrations of each material in the dispersion as described in other systems.44-45

Figure 2.

Controlling of the coating composition was also accomplished by varying the ZnO-HAp concentration ratio in the deposition dispersions as can be seen in Figure 6S. Figure 3A shows top-view SEM images of HAp, ZnO and ZnO-HAp composite coatings deposited from solutions with different ZnO-HAp ratios. Specifically, ratios of 7:3, 1:1 and 3:7 w/w of ZnO-HAp were prepared and electrophoretically deposited. Images are shown in two magnifications. It should be noted that the EPD was performed at 60 V, where the 19 ACS Paragon Plus Environment


coatings are expected to have the same composition as in their dispersion. Since the size of the ZnO NPs is larger than that of the HAp, the pristine ZnO coating has more porous structure as compared with the close-packed structure of pure HAp coating. The structural morphology of ZnO-HAp 7:3 and 1:1 looks similar, where the ZnO NPs are surrounded with HAp NPs.

On other hand, ZnO-HAp (3:7) had a different morphology, where the ZnO NPs were sputtered across the HAp matrix more distantly. Further characterization of the coatings composition was carried out by element analysis and mapping (EDS). Figure 7S shows the elemental mapping and analysis of ZnO, HAp and ZnO-HAp composite coatings. Obviously, pure HAp coating is composed of Ca, O and P, where ZnO is composed of Zn and O. The small content of carbon in both coatings is probably attributed to TEOA adsorption. The element analyses of ZnO-HAp composite coatings are in agreement with the ZnO and HAp content in the dispersions, where the %at of Zn decreases from ZnOHAp (7:3) to ZnO-HAp (3:7), and vice versa for Ca and P. Qualitative perspective is provided by elemental mapping of the coatings (Figure 7S insets).


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Figure 3B shows the FTIR spectra of ZnO, HAp and ZnO-HAp composite coatings. The characteristic peaks of ZnO are shown in the region of 400 to 600 cm−1, where the three intense peaks at 475, 522 and 600 cm−1 are associated with metal-oxide [M-O] stretching.46 The peak at 1130 cm−2 is attributed to C-N vibrations due to TEOA adsorption.47-48 Further indications of TEOA ubiquity in the coatings can be found at 1400, 2850 and 2930 cm−1 bands, which are attributed to methylene stretching vibrations33 the peak at 1553 cm−1, associated with N-H vibrations, and the peaks at 1583 and 1610 cm−1, which might be assigned to C-O vibrations.33, 48 The typical peaks of HAp are shown at 472, 560, 588, 605, 961, 1015, 1086 and 1165 cm−1, which are due to PO4−3 vibrations.49 The peak at 875 cm−1 is associated with HPO4−2 vibrations. The peaks at 631 and 3570 cm−1 are attributed to the OH group.50 The two intense peaks at 1420 and 1460 cm−1 are associated with CO3−2 impurities. It can be seen, that the peaks of HAp are intensified as the HAp content in the dispersion increases, where the strong peak of ZnO at 470 cm−1 can be observed in all the composite coatings.



Figure 3C shows the XRD patterns of ZnO, HAp and ZnO-HAp composite coatings. The XRD pattern of pure ZnO shows the typical peaks of pure crystalline ZnO with the same strong (101) orientation, indicating that the electrophoretically deposited ZnO NPs maintained their native structure. The peaks of pure HAp coatings are less visible compared to ZnO, which is in agreement with the XRD pattern of HAp NPs powder (see Figure 1S).

It can be seen, that the peaks of ZnO gradually decrease as its concentration in the dispersion diminishes. On other hand, the peak of HAp at (112) index continuously increases with HAp concentration. Aside from, the highest peak of HAp at (211) index is probably masked by (100) index of ZnO. The peaks at (002), (300), (213) and (004) indexes of HAp are clearly shown in the ZnO-HAp (3:7) spectrum.

Figure 3.

Further investigation of the coatings was conducted in order to determine the ratio between each phase, ZnO and HAp, in the composite coatings. Good correlation between the nanomaterials ratio in the dispersions and the coatings, enables us to tailor 22 ACS Paragon Plus Environment

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nanocomposite coatings based on their initial composition in the suspensions. Therefore, we compared the ratio of ZnO and HAp in the dispersions with their ratio in the coatings. The phase composition of the coatings was determined by Rietveled quantitative phase analysis based on XRD. Table 2 shows the weight ratio between ZnO and HAp in the dispersions and the phase ratio of ZnO-HAp composite coatings based on XRD. Rietveld quantitative phase analysis is a powerful method for determining the quantities of crystalline components in multiphase mixture.51 As evidence, the ratio between the two components in the composite coatings is similar to their ratio in the dispersions, enabling excellent control on the composition of the film.

Table 2.

The adhesion of the nanoparticulate coating to the metallic substrate has a crucial role in determining its application in different biomedical fields, such as orthopedic or dental implantations. The as-prepared electrophoretically deposited ZnO-HAp (3:7) had poor adhesion to the Ti with approximately a maximum shear stress of 5 MPa. In order to improve the bonding, the samples were annealed at 600 °C in air in order to densify the



coating and to form oxide layer on the titanium surface, which is likely to provide stronger interactions with the inorganic film.52-53 Indeed, the maximum shear stress of the ZnOHAp (3:7) coating after the thermal treatment was significantly increased to 14.3±2.0 MPa. Visual inspection of the detached coating reveals that the locus of failure was adhesive rather than cohesive, since the coating was completely detached from metallic substrate, where the adhesive epoxy glue remained intact. The decomposition temperatures of HAp and ZnO are >1000 ˚C.54-55 Hence, the thermal treatment should not deteriorate or alternate the ratio between the two nanomaterials. As evidence, Figure S8A,B shows a SEM image and XRD pattern of ZnO-HAp (3:7) coating before and after annealing, respectively. It can be seen that the coating was densified as a result of the thermal treatment. Specifically, the crystallite size of HAp was slightly increased from 17 to 23 nm, where the crystalline size of ZnO remained the same. The XRD pattern of the coating indicates that none of the nanomaterials was decomposed.

The main advantage of electro-assisted deposition is the ability to fabricate a continuous film on complex structures, as compared with other line-of-sight coating techniques, such


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as spraying and spin coating. Therefore, we electrophoretically coated a commercial screw-like dental implant made of Ti-6Al-4V. Figure 4 shows the SEM images of ZnOHAp (3:7) coated dental implant at different magnifications. The low-magnification reveals that the substrate was entirely covered. The homogeneity and morphology of the implant coating were similar to the coatings obtained on a flat surface.

Figure 4.

Preliminary evaluation of the bioactive nature of ZnO-HAp composite coating was conducted by in-vitro bioactivity test. The ability of the coated titanium to enhance the precipitation of bone-like apatite, which mainly composed of calcium and phosphate with other bone minerals, such as magnesium, sodium and potassium, implies on the in-vivo activity with bone tissues.27 Therefore, coating titanium implants with HAp holds superior benefits compared to bare Ti by initiating better and fast interactions with biological systems, which ultimately yields enhanced bone regeneration.56-57 In addition, Zn has been also reported, both in-vivo and in-vitro, for its fundamental ability to induce stimulatory effects of bone integration by promoting osteoblasts proliferation.58-59 Figure



5A,B shows SEM images of ZnO-HAp (3:7) coated titanium after four weeks immersion in SBF at 37±1 °C. It can be seen that agglomerated pellet-like clusters are formed on the surface of ZnO-HAp (3:7) coating. A comparison between pure-HAp (Figure S9A) and ZnO-HAp (3:7) coating reveals interesting findings. The morphology of pure-HAp coating is different, where needle-like mineralized apatite nanocrystals are formed, which is in agreement with previous reports.60-61 The difference in the precipitate morphology, which is formed as a result of immersing in SBF solution, is related to the presence of ZnO in the coating, where zinc ions have significant impact on the crystallization of HAp. It was suggested58, 62 that partially dissolved ZnO generated Zn2+ ions, which are adsorbed on the active sites of HAp, inhibit the precipitation of bone-like apatite from the SBF solution. Therefore, the fewer nucleation sites on the surface of ZnO-HAp (3:7) cause the formation of larger bone-like apatite crystallites (Figure 5A) after prolong immersion in SBF solution. Figure S9B-C shows SEM images of pristine ZnO coating and bare Ti after immersion in SBF for 4 weeks, respectively. Obviously, the precipitation of minerals did not occur on the surface of bare titanium substrate indicating the poor biomineralization activity of Ti.


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On the other hand, small amounts of pellet-like clusters were formed on the ZnO coated Ti, implying that ZnO is a better biomineralization agent than Ti.63-64

The XRD pattern of HAp (Figure S10A) shows intensified peaks of HAp at (002), (102), (211), (112), (300), (310), (222) and (213) planes after the SBF experiment. In the case of ZnO-HAp (3:7) (Figure 5C), the diffraction peaks of HAp have more amorphous nature, which can be explained by the presence of Zn2+ due to ZnO dissolution. As mentioned previously, Zn ions have significant impact on the crystallization of HAp, where Zn+2 ions are adsorbed on HAp surface and interfere with further crystallization of the lattice.65-66 Indeed, the peaks of ZnO are significantly lower compared to ZnO-HAp (3:7) coating prior to the exposure to SBF. In addition, Rieteveld quantitative phase analysis shows increase in HAp concentration (89.2 %wt) and decrease in ZnO concentration (10.8 %wt) as compared with Table 2. The XRD pattern of ZnO (Figure S10B) shows only the diffraction peaks of ZnO without any traces of HAp or any other crystalized inorganic species. One possible explanation is that insufficient amounts of HAp clusters were formed and therefore could not be detected by XRD. The other feasible explanation is that the



precipitated HAp has amorphous nature, which cannot be precisely discern by the diffraction pattern. Figure S10C shows the XRD of pristine Ti after the bioactivity test. In agreement with the SEM images, only the typical peaks of Ti are detected.

EDS elemental analysis was conducted in order to study the composition of the coatings before and after the prolonged exposure to SBF. Figure S11A-B shows the EDS spectra of ZnO-HAp (3:7) and HAp coatings, respectively. Both spectra indicate the biomineralization of the coatings, where bone minerals, such as Mg2+, Na+ and Cl− were adsorbed on the negatively charged coated Ti surface to form poorly crystalized nonstoichiometric minerals-containing bone-like apatite precipitate, according to previous reports.67-68 The Ca/P ratio of pure HAp coating did not change as a result of immersing in SBF solution. In contrast, the Ca/P ratio of ZnO-HAp (3:7) coating decreased to approximately unity, suggesting the enrichment of the coating by phosphate. Moreover, the at% of Zn decrease by two after four weeks immersion in SBF, which can be attributed to ZnO dissolution and reprecipitation of Zn-substituted calcium phosphate.69 The XRD pattern of ZnO-HAp (3:7) after SBF experiment shows only HAp and ZnO without any


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other traces. The EDS spectrum and element analysis of pristine ZnO (Figure S10C) clearly shows that the coating was enriched with Ca, P, Mg and Cl, similarly to ZnO-HAp (3:7) coating. The Ca/P ratio was approximately 0.5, which implies that the formed CaP phase was mainly Zn-substituted Ca-poor amorphous calcium phosphate. The EDS spectrum and element analysis of bare Ti (Figure S11D) shows the presence of pure Ti with low concentrations of carbonaceous species. The different mechanisms of the bonelike apatite formation on HAp and ZnO-HAp coating is illustrated in Scheme 2.

Scheme 2.

Figure 5B shows a SEM cross-section image of an electrophoretically deposited ZnO-HAp (3:7) coating and the corresponding elemental mapping. The average thickness of the coating is 7.54±0.31 m, where the average thickness of the agglomerated clusters is 1.81±1.05 m, indicating the irregularity and non-uniformity of the precipitated pellets. The elemental mapping of the cross section shows that both of the electrophoretically deposited coating and the precipitated clusters from the SBF are composed of Ca, P, O



and Zn. The presence of Pt in the upper layers is due to the sputtering of the protective layer before FIB cutting.

Figure 5.

Assessing the biocompatibility of the nanomaterials has a crucial impact on the applicability of the coating. Therefore, we evaluated the cytotoxicity test on two human cell lines, Jurkat T-cells and HaCaT skin cells. The cytotoxicity test was conducted on scrapped ZnO-HAp (3:7) coating. The ZnO-HAp (3:7) powder was dispersed and incubated with different human cells for 3 days. After the incubation period, the cell viability was tested by two different techniques, MTT and resazurin. Figure 6A shows the cell viability (%) of Jurkat cells after 3 days of incubation with ZnO-HAp powder (100 µg/mL). Any evidence of a toxigenic effect on the cells was absent, where by the MTT assay, improved proliferation of cells was observed with increase of ≈20% in the cell viability compared with untreated cells. These considerable results could be associated with the interesting phenomenon, hormesis, where increased biological response can be induced by exposure to low concentrations of toxins and stressors. Further investigation


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on the cytotoxicity of ZnO-HAp powder was conducted on HaCaT skin cells. Figure 6B shows a dose response curve of HaCaT cells in the presence of increasing concentrations of ZnO-HAp. Similar to the Jurkat cells, a positive feedback in the proliferation of the cells was observed with no sign of toxicity (based on the MTT assay).

The antibacterial activity of ZnO-HAp coatings was evaluated against E.coli bacteria. ZnO is well-known for its antimicrobial activity, which is driven by bacteria contact and the release of Zn ions.70 The experiments were carried by dropping a certain volume of known bacteria concentration on the various surfaces. The samples were left for four hours under incubation. Then, the bacterial growth medium was added and left for additional 18 hrs under incubation to reach the stationary state of the bacterial growth. The antibacterial activity was determined by measuring the optical density of the solutions. Pure ZnO and HAp coatings were used as negative and positive controls, respectively. Bare Ti was also used as a positive control.

Figure 7 shows the results. It is evident that in all cases where ZnO was used, the OD600 was 0.00 as compared with sterilized growth medium. In other words, in all ZnO-HAp



coatings, no bacterial proliferation was observed in the growth medium, indicating the superior antibacterial effect of ZnO-HAp. For the HAp coating and bare Ti, the growth media were very opaque after 18 hrs, which clearly indicates on bacterial thrive. These results clearly show that the incorporation of ZnO into HAp layers provides excellent bactericidal activity. Further elucidation of the antibacterial mechanism of ZnO, i.e., to reveal whether the inhibition of bacterial growth is attributed to the release of Zn2+ or by direct contact with ZnO, was conducted using an agar-diffusion test (not reported here). Our results clearly show that an inhibition zone was not formed after 18 hrs, yet, the bacteria were eradicated during 4 hrs by the direct contact with ZnO.

Figure 7.

4. Conclusions

A new approach for the formation of a composite coating in a single step, made of HAp and ZnO nanoparticles (NPs) by electrophoretic deposition (EPD) is demonstrated. Taking advantage of the different EPD rate of both components enabled to control the composition of the deposited layers to form a graded coating in one step. Alternatively, 32 ACS Paragon Plus Environment

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the composition of the coatings could be highly controlled by changing the concentration of each component in the dispersion. The good adhesion of the nanocomposite coating and the ability to coat metallic supports with different geometries suggests the applicability of the system for medical devices. In-vitro studies, antibacterial, cytotoxicity and bioactivity, exhibit notable performance, such as excellent bacterial eradication, superior biocompatibility and efficient mineralization. This simple and economic approach provides a pivotal strategy for tailoring the composition of multi-component nanomaterial based coatings. Therefore, we foresee a wide variety of applications in fields such as medical implants, optical devices and sensing, following this approach.



A

B

C

Figure 1. A,B. Cross-section STEM images and their corresponding EDS element analysis of ZnO-HAp (1:1) lamellas. C. Phase composition (calculated by XRD) of ZnOHAp coatings as a function of applied potential


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A

B

C

Figure 2. HR-TEM cross section of a few nanometers lamella of ZnO-HAp coating on Ti at different applied potentials (10, 30 and 50 V) for 2 min at each potential. A: HR-TEM image and B. the corresponding EDS analysis, and C. HR-TEM elemental mapping.



Figure 3. A. SEM, B. FTIR and C. XRD of ZnO, HAp and ZnO-HAp composite coatings.


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Figure 4.

SEM images (with different magnifications) of ZnO-HAp (3:7 w/w in the

deposition solution) composite coating on commercial dental implant.

Figure 5. A-B. SEM images (top view and cross-section with its corresponding elemental, respectively), and C. XRD of ZnO:HAp (3:7) electrophoretically coated on a Ti surface after immersion in a SBF solution for four weeks.




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Figure 6. A. Cell viability of Jurkat cells after 3 days incubation with ZnO-HAp (3:7) scraped coating (100 g/mL). B. Dose-response curve of HaCaT cells (determined by

MTT assay) after 3 days incubation with different concentration of ZnO-HAp (3:7) scarped coating.

Figure 7. Absorbance measurement (at 600 nm) of bacterial growth medium after 4 hrs contact of bacterial culture with different coatings, followed by 18 hrs incubation in growth medium at 37±1 ° C. Inset shows a photograph of the different coatings and bare Ti in bacteria growth medium after 18 hrs incubation.



Scheme 1. Schematic illustration of the synthesis and EPD of ZnO-HAp composite.

Scheme 2. Schematic illustration of the in-vitro biomineralization process of HAp and ZnO-HAp coatings.


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Dispersion

Size (nm)

-potential (mV)

ZnO

607±120

35±5

ZnO-TEOA

151±61

46±2

HAp

81±35

32±3

HAp-TEOA

36±11

42±2

Table 1. Stability parameters of ZnO and HAp NPs with and without TEOA.

Dispersion composition

Coatings composition

(%wt)

(%wt by XRD)

Sample ZnO

HAp

ZnO

HAp

ZnO-HAp (7:3)

70

30

68±2

31±2

ZnO-HAp (1:1)

50

50

52±1

47±1

ZnO-HAp (3:7)

30

70

34±1

66±1

Table 2. Comparison between ZnO-HAp content in dispersions and in the composite coatings. 41 ACS Paragon Plus Environment


ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS website. Elaborated characterization of ZnO and HAp NPs, such as SEM, TEM, SAED, XRD and XPS. Particles size distribution curves of ZnO and HAp NPs dispersions. Photographs of different ZnO, HAp and ZnO-HAp dispersions. Thickness measurements and crosssection SEM images of ZnO and HAp NPs coatings. -potential graph of HAp NPs as function of Zn2+ addition. SEM and XRD of ZnO-HAp after thermal treatment. EDS and element analysis of ZnO, HAp and ZnO-HAp coatings. XRD, EDS and SEM images of HAp, ZnO, ZnO-HAp and bare Ti after bioactivity test. AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]

Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT This research is supported by the Israeli Ministry of Science and Technology (contract 86569). The Harvey M. Krueger Family Center for Nanoscience and Nanotechnology of the Hebrew University is acknowledged. We acknowledge Dr. Yoram Soroka, Dr. Marina Frušić‐Zlotkin and Prof. Ron Kohen (the Institute for Drug Research, School of Pharmacy, Faculty of Medicine, the Hebrew University of Jerusalem, Israel) for their kind assistance with the cytotoxicity tests.

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Graphical Abstract




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Composition-Tailoring of ZnO-Hydroxyapatite Nanocomposite as

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