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Time-dependent growth of TiO2 nanotubes from a magnetron sputtered Ti thin film
TSF-31860; No of Pages 7 Thin Solid Films xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Thin Solid Films journal homepage: www.elsevier.com/locate/tsf
Time-dependent growth of TiO2 nanotubes from a magnetron sputtered Ti thin film Soo-Hyuk Uhm a, b, Doo-Hoon Song b, Jae-Sung Kwon b, Su-Yeon Im a, b, Jeon-Geon Han c, Kyoung-Nam Kim a, b,⁎ a b c
Department and Research Institute of Dental Biomaterials and Bioengineering, College of Dentistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, South Korea Research Center for Orofacial Hard Tissue Regeneration, College of Dentistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, South Korea Center for Advanced Plasma Surface Technology, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, South Korea
a r t i c l e Available online xxxx Keywords: TiO2 nanotube Ti thin film Sputtering Drug loading Gentamicin
i n f o
a b s t r a c t The purpose of this study was to apply a Ti film to various substrates for use in biomaterials. By forming TiO2 nanotubes on a film, the biocompatibility of the TiO2 nanotube and its ability as a carrier can be applied to a variety of materials for use within diverse applications. In this work, we present the fabrication of a self-organized TiO2 nanotube layer that was grown from flat and thin sputter-deposited titanium films on a Si (100) substrate by plasma electrolytic oxidation, and evaluated the use of this material for the release of gentamicin. This resulted in TiO2 nanotubes with lengths ranging from about 200 to 400 nm, as confirmed in a cross-sectional view. Operation at a low temperature was a key to achieving self-organized, timedependent growth of the TiO2 nanotube layer from the thin film on a Si substrate. The drugs were then pipetted on the formed nanotubes and their release was evaluated. The results showed that there was adequate drug release from the samples which suggests that they have the potential to be a drug carrier. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The application of nanoscience and nanotechnology to medicine has led to the achievement of several drug delivery materials by enabling control of the materials' size, porosity, and morphology [1–3]. A range of nanoscale materials have been investigated for this purpose, including nanofibers, nanorods, nanoparticles, nanotubes, and nanogels [4–7]. Among these materials, the vertically-aligned, highly-ordered TiO2 nanotube layer on Ti and Ti-based alloys prepared by plasma electrolytic oxidation (PEO) is widely known as one of the most promising methods for advancing local drug delivery [8–10]. Usually TiO2 nanotubes from the Ti substrates are used not only as the surface of orthopedic and dental implants but also as ideal platforms for antibacterial and antibiotic drug delivery and in medical devices such as implants, stents, and immunoisolation capsules [11–14]. These uses are possible due to the nanotubes' excellent biocompatibility, corrosion resistance, high volume ratio, and controllable pore size and length [13,15–20]. For a drug delivery system composed of TiO2 nanotubes, it is essential that the drug carrier system has a sustained and controlled release pattern with uniform drug elution [21,22], which can be controlled by varying the diameter and length of the TiO2 nanotubes through the PEO process [23–25].
⁎ Corresponding author at: Department and Research Institute of Dental Biomaterials and Bioengineering, College of Dentistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, South Korea. Tel.: +82 2 2228 3081; fax: +82 2 364 9961. E-mail address: [email protected] (K.-N. Kim).
Various techniques of thin film coating on the other substrates have been reported [26–28]. Especially, magnetron sputtering was widely used as an alternative method to coat and synthesize the thin films on a variety of materials [29–32]. In order to fabricate TiO2 nanotubes from the Ti layer on the other substrate (Si substrate), magnetron sputtering was adapted to coat the Ti thin films. In this work, we present two approaches to investigate both the morphological time-dependent growth of a TiO2 nanotube layer on a Si substrate with the possibility of controlling tube's length and diameter through the PEO process of sputtered Ti thin films and the release performance of a drug placed in the tubes used as a drug carrier. A schematic of this drug placement procedure is presented in Fig. 1. Hence, we suggest that TiO2 nanotubes can be applied to a variety of biomaterials that are not Ti-based and which otherwise will be electrochemically impossible to fabricate nanotubes as a drug carrier. 2. Materials and methods 2.1. Deposition of a Ti thin film on a Si substrate A Ti bulk disk (99.9% purity, 2 in. diameter) was adapted for the sputtering target. Ti thin films were deposited on the Si substrate (2.5 × 5 cm 2) using a DC magnetron sputtering system. In order to remove the native oxide layer of Si, the Si substrates were dipped in hydrofluoric acid (HF) solution for 30 s, and then they were ultrasonically rinsed in acetone, ethanol, and deionized water (DI) and dried at room temperature before being introduced into the vacuum chamber. Next, the Si substrate was placed in the magnetron sputtering
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vacuum chamber. In order to eliminate the contamination layer on the target surface, the Ti target was cleaned by pre-sputtering for 3 min. The argon (Ar) gas pressure and the power input to the Ti target during the deposition of Ti thin films on the Si substrate were 0.93 Pa and, 10 W per cm2 respectively. Under these conditions, the deposition rate of the Ti thin films was 35 nm per min, and a 500 nm thick Ti thin film was formed after the sputtering. 2.2. Fabrication of TiO2 nanotubes Prior to PEO, the Ti thin film-coated Si substrates were respectively cleaned with acetone, ethanol, and DI water, and then dried in a vacuum dry chamber for 1 day. Following the drying process, specimens were covered with an insulative polymer on their back side to prevent an interruption of the current between the Ti coating layer and the Si substrate as it undergoes PEO. PEO was performed by a two-electrode system in 0.5 wt.% HF and an ethylene glycol mixture solution at room temperature. The PEO system setup was completed with a platinum (Pt) plate used as a cathode electrode that was maintained at a distance of 10 mm from the specimen used as an anode. The PEO experiments consisted of applying a potential between 5 and 20 V over time intervals between 60 and 1200 s using a DC power supply (Genesys, 600-2.6 Densei-Lambda, Tokyo, Japan). Only current was passed to the Ti thin film on the specimen through alligator clips from the power supply. The PEO specimens were washed gently with DI water and dried in the vacuum dry chamber at room temperature. Afterward, specimen was annealed at 450 °C. 2.3. Characterization of TiO2 nanotubes on Ti thin film The surface morphology and chemical composition of the TiO2 nanotube layer on the Si substrate were qualitatively characterized by field emission scanning electron microscopy (FE-SEM, JSM-6700 F, JEOL, Japan), transmission electron microscopy (TEM, JEM-3010, JEOL, Japan) and energy dispersive spectrometer (EDS, X-MAX, Oxford, UK). FE-SEM at 15.0 kV condition was also carried out on the specimen cross-sectional plane prepared by mechanical fracture. The microstructure of the prepared specimens was identified by high resolution X-ray diffraction (HR-XRD, Rigaku, Kuraray, Japan) with a Cu-Kα radiation under 40 kV and 40 mA from 20° to 80° of 2θ value. 2.4. Corrosion resistance
Fig. 1. Procedure for a drug loaded TiO2 nanotube layer on a Ti thin film.
In order to investigate the corrosion resistance, a potentiodynamic test was conducted in phosphate buffered saline (PBS) at 37 °C. The
Fig. 2. Top and cross-sectional view of the Ti thin film on a Si substrate and its chemical composition.
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Fig. 3. FE-SEM images (×50,000) of fabricated TiO2 nanotubes on a Si substrate coated with a Ti thin film with a fixed PEO time of 1200 s: (a) 5 V, (b) 10 V, (c) 15 V, and (d) 20 V.
specimens were situated in an assembled corrosion cell containing the PBS solution, and which was then connected to the electrochemical interface (VersaSTAT3, Princeton Applied Research, USA). 2.5. Drug loading to the TiO2 nanotubes on a Ti thin film To fill the TiO2 nanotubes on Ti thin films, freeze drying was performed with the lyophilization method [33], and filling was adapted from the modified method in Popat et al. [34]. In brief, a solution of 1 mg/ml gentamicin was prepared in a PBS solution, and then the specimens (1.0 × 1.0 cm 2) were cleaned with distilled water before filling the PEO-fabricated TiO2 nanotubes with the drug. Before filling with the drug, the PBS solution was used to gently clean the specimen. To load the drug, 10 μl gentamicin solution was simultaneously spread around the surface and dried in air at room temperature for 2 h. The drying and pipetting steps were repeated until a sufficient amount of gentamicin was filled in the TiO2nanotube. The specimens were quickly rinsed with 500 μl of PBS over the specimens in order to remove excess gentamicin that covered the surface in the final drying step.
3. Results and discussion The purpose of this study was to determine the optimal conditions for fabricating a TiO2 nanotube layer as a drug carrier on a different substrate and to evaluate the drug release. A magnetron sputtering system was used to coat the substrate with an appropriate density and uniformity of Ti thin film. Fig. 2 shows the top and crosssectional view of the coated Ti thin film. The cross-sectional view in Fig. 2b of the specimen reveals the dense Ti thin film by magnetron sputtering. The thickness of the Ti layer was 500 nm ± 10 nm. The chemical composition investigation was performed using EDS within FE-SEM, and indicated a nearly pure Ti thin film.
2.6. Release from TiO2 nanotubes on Ti thin film In order to investigate the release kinetic of the drug from the TiO2 nanotube layer on a Ti thin film, the specimen was immersed in 800 μl of PBS in a 4-well plate at room temperature in a shaking incubator at 100 rpm. After a specified time, 500 μl of the sample was collected to evaluate the amount of drug released. The collected solution was changed with 500 μl of fresh solution every time to calculate the accumulated sample concentration. The collected samples were analyzed for drug concentration using the UV–VIS spectrophotometer (UV–VIS, UVD-3200, Labomed, USA).
Fig. 4. Current time plot for 1200 s of PEO treatment at 20 V in 0.5 wt.% HF electrolyte.
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Fig. 5. FE-SEM image (×100,000) of PEO time-dependent growth of TiO2 nanotubes with a fixed applying voltage. The potential was kept at 20 V at different oxidation times; code: (a) 60 s; TDG01, (b) 240 s; TDG04, (c) 480 s; TDG08, (d) 720 s; TDG12, (e) 960 s; TDG16, and (f) 1200 s; TDG20 (the tube lengths are indicated in the cross-sectional views).
Fig. 3 shows the results of the PEO treatment with a variable voltage (5, 10, 15, 20 V) applied for 20 min. When the applied potential was 5, 10, and 15 V (Fig. 2a, b, and c, respectively), nano-porous shapes appeared on the surface. This indicated that these shapes have improper morphologies to be drug carriers since they were irregular. However, it can be clearly seen that the experimental condition of 20 V (Fig. 2d) produced the most obvious shape for a highly ordered tube that is effective as a drug carrier [35]. Therefore, we conducted the experiment with a 20 V potential. Fig. 4 shows the current reaction of the bath as function of the PEO time. The growth mechanism of the TiO2 nanotubes was revealed by previous studies [36–38]. After applying the potential to the substrate, the current rapidly decreased in the initial stage within the first seconds. Subsequently, a dense Ti oxide layer is fabricated. This was due to the pores soaking through the barrier oxide film, exposing the Ti thin film to the PEO electrolyte. The current density then decreased with time as a result of the increase in diffusion length for an ionic species in the electrolyte [38–40]. Fig. 5 shows the sequence of the morphology of the PEO treated specimen as the evolution of surface topography under different PEO treatment times at 24 °C. In the initial stage of oxidation, some cracks appeared on the surface because the oxide layer was chemically dissolved by field assistance with a voltage on the Ti thin films. Evidently, a nano-porous layer formed on the specimen at the early stage of ~ 240 s (Fig. 5a and b). With an extended PEO treatment time the current densities decreased and gradually the oxide layer dissolved again (Fig. 5c). Subsequently, after 720 s, the initiation of the tube-shape formations on the surface began (Fig. 5d). At this stage the nano-pores were irregularly formed on the specimen and competed for workable space to combine with another pore and potential [36]. Fig. 5e shows that the porous oxide structure was more uniform and had a more regular tube shape than the nano-porous shape (Fig. 5b, c and d). After 1200 s of the PEO treatment (Fig. 5f), the formed nano-tubular self-ordered TiO2 layer clearly showed homogeneous and uniform nanotubes. These results demonstrate that the present approach can capitalize on the fabrication of TiO2 nanotubes that have a morphology similar to a polycrystalline titanium foil anodized using the procedure demonstrated by Popat et al. [5,34].
The influence of the applied PEO time on the resulting diameter and length of the TiO2 nanotubes is summarized in Fig. 6. At 4 min of PEO treatment, a nano-porous oxide layer with approximately 30 nm diameter and 150 nm thickness was fabricated. After 1200 s of PEO treatment there was an array of TiO2 nanotubes with diameters of 100 nm and lengths of 400 nm. Between 240 and 1200 s, the dimensional tube diameters and lengths showed increasing and nearly linear dependence on the PEO treatment time. Figs. 7 and 8 show the EDS, TEM and XRD analyses resulting from the PEO treatment at a potential of 20 V for 1200 s and then, heat treated. TiO2 consisting of Ti and O ions was detected through the EDS analysis. Also, the XRD results show that it is possible to transition from an amorphous phase to a polycrystalline phase TiO2 [16,41]; furthermore, they show the removal of residual debris such as HF acid and C, F ions due to annealing of specimen [42,43]. However, nanotube with the incomplete form has a lower intensity of anatase phase diffraction pattern [44]. The corrosion resistance of biomaterials influences their functionality and stability and is the key factor in their biocompatibility
Fig. 6. The correlation between PEO time and growth of the TiO2 nanotubes on the Ti thin film.
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Fig. 7. EDS analysis after the formation of heat-treated TiO2 nanotubes by the PEO treatment under an applied voltage of 20 V for 1200 s (TDG20).
Fig. 8. (a) TEM image and (b) XRD patterns after the formation of heat-treated TiO2 nanotubes (TDG20).
[45–47]. The corrosion resistance of biomaterials and their functional performance is important in the in vivo environment if nanotubes are to be used as drug carriers [48]. Fig. 9 shows the results of a potentiodynamic test of the TiO2 nanotubes and the sputtered Ti thin film. These results revealed that the corrosion resistance of the TiO2 nanotubes was better than that of the Ti thin film because the nanotubes showed a lower passive current density than the Ti thin film in the PBS solution. We demonstrated the possibility of fabricating self-ordered TiO2 nanotubes in sputtered Ti thin films on a Si substrate. It is also notable that the procedure for fabricating TiO2 nanotubes on sputtered Ti thin
films (Fig. 10) described here exhibits further advantages for drug carrier functionality in biomedical applications compared to other studies [37,49]. To confirm the potential of using TiO2 nanotubes on the Ti thin film as a drug carrier, 1 mg/ml gentamicin was loaded in the TiO2 nanotube layer. Fig. 11 shows the release data for gentamicin. Generally, the drug had a slower release from the TiO2 nanotubes (TDG16 and TDG20) compared to other test groups. Especially, the drug had a higher release efficacy from the TDG20 group where the adequate concentration of drug release was observed even after 12 h. However, other groups showed lower drug-releasing efficacy because of their thin and incomplete shape of oxide as drug carrier. These results suggested that it is feasible for TiO2 nanotubes on a Ti thin film to act as a drug carrier. Hence, the use of a drugreleasing TiO2 nanotube layer as a coating on other implantable biomaterials which are not Ti-based has a variety of applications. 4. Conclusion
Fig. 9. Graph of the PEO treatment current density vs. potential for Ti thin films and TiO2 nanotubes on a Si substrate, from the potentiodynamic test.
We demonstrated two approaches for fabricating TiO2 nanotubes on sputtered Ti thin films on a Si substrate. Titanium thin films coated by magnetron sputtering onto the Si substrate were applied for PEO in 0.5 wt.% HF solution at room temperature. In this study, it was shown that morphological changes of nanotubes were achieved according to the PEO potential (~ 20 V) and control of the length and diameter of nanotube was dependent on PEO time (~ 1200 s). It was morphologically obvious from the analysis performed using FE-SEM that a self-ordered TiO2 nanotube layer with an average diameter about 100 nm and length about 400 nm could be fabricated using the PEO treatment. Also, we showed that TiO2 nanotubes with a PEO treatment time of 1200 s formed on sputtered Ti thin films are shown to release drug sustainably from nanotubes. It was concluded
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Fig. 10. The final shape of TiO2 nanotube which was fabricated on sputtered Ti thin film used as drug carrier.
Fig. 11. Gentamicin calibration curve and released gentamicin cumulative concentration as a function of immersion time.
that TDG20 will be applicable to biomaterials other than Ti substrates for the application as a drug carrier. Conflict of interest The authors declare no conflicts of interest. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2012R1A1A2008659) and SRC program of National Research Foundation (NRF) of Korea, funded by the Ministry of Education, Science and Technology (20100029418).
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Contents lists available at SciVerse ScienceDirect
Thin Solid Films journal homepage: www.elsevier.com/locate/tsf
Time-dependent growth of TiO2 nanotubes from a magnetron sputtered Ti thin film Soo-Hyuk Uhm a, b, Doo-Hoon Song b, Jae-Sung Kwon b, Su-Yeon Im a, b, Jeon-Geon Han c, Kyoung-Nam Kim a, b,⁎ a b c
Department and Research Institute of Dental Biomaterials and Bioengineering, College of Dentistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, South Korea Research Center for Orofacial Hard Tissue Regeneration, College of Dentistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, South Korea Center for Advanced Plasma Surface Technology, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, South Korea
a r t i c l e Available online xxxx Keywords: TiO2 nanotube Ti thin film Sputtering Drug loading Gentamicin
i n f o
a b s t r a c t The purpose of this study was to apply a Ti film to various substrates for use in biomaterials. By forming TiO2 nanotubes on a film, the biocompatibility of the TiO2 nanotube and its ability as a carrier can be applied to a variety of materials for use within diverse applications. In this work, we present the fabrication of a self-organized TiO2 nanotube layer that was grown from flat and thin sputter-deposited titanium films on a Si (100) substrate by plasma electrolytic oxidation, and evaluated the use of this material for the release of gentamicin. This resulted in TiO2 nanotubes with lengths ranging from about 200 to 400 nm, as confirmed in a cross-sectional view. Operation at a low temperature was a key to achieving self-organized, timedependent growth of the TiO2 nanotube layer from the thin film on a Si substrate. The drugs were then pipetted on the formed nanotubes and their release was evaluated. The results showed that there was adequate drug release from the samples which suggests that they have the potential to be a drug carrier. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The application of nanoscience and nanotechnology to medicine has led to the achievement of several drug delivery materials by enabling control of the materials' size, porosity, and morphology [1–3]. A range of nanoscale materials have been investigated for this purpose, including nanofibers, nanorods, nanoparticles, nanotubes, and nanogels [4–7]. Among these materials, the vertically-aligned, highly-ordered TiO2 nanotube layer on Ti and Ti-based alloys prepared by plasma electrolytic oxidation (PEO) is widely known as one of the most promising methods for advancing local drug delivery [8–10]. Usually TiO2 nanotubes from the Ti substrates are used not only as the surface of orthopedic and dental implants but also as ideal platforms for antibacterial and antibiotic drug delivery and in medical devices such as implants, stents, and immunoisolation capsules [11–14]. These uses are possible due to the nanotubes' excellent biocompatibility, corrosion resistance, high volume ratio, and controllable pore size and length [13,15–20]. For a drug delivery system composed of TiO2 nanotubes, it is essential that the drug carrier system has a sustained and controlled release pattern with uniform drug elution [21,22], which can be controlled by varying the diameter and length of the TiO2 nanotubes through the PEO process [23–25].
⁎ Corresponding author at: Department and Research Institute of Dental Biomaterials and Bioengineering, College of Dentistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, South Korea. Tel.: +82 2 2228 3081; fax: +82 2 364 9961. E-mail address: [email protected] (K.-N. Kim).
Various techniques of thin film coating on the other substrates have been reported [26–28]. Especially, magnetron sputtering was widely used as an alternative method to coat and synthesize the thin films on a variety of materials [29–32]. In order to fabricate TiO2 nanotubes from the Ti layer on the other substrate (Si substrate), magnetron sputtering was adapted to coat the Ti thin films. In this work, we present two approaches to investigate both the morphological time-dependent growth of a TiO2 nanotube layer on a Si substrate with the possibility of controlling tube's length and diameter through the PEO process of sputtered Ti thin films and the release performance of a drug placed in the tubes used as a drug carrier. A schematic of this drug placement procedure is presented in Fig. 1. Hence, we suggest that TiO2 nanotubes can be applied to a variety of biomaterials that are not Ti-based and which otherwise will be electrochemically impossible to fabricate nanotubes as a drug carrier. 2. Materials and methods 2.1. Deposition of a Ti thin film on a Si substrate A Ti bulk disk (99.9% purity, 2 in. diameter) was adapted for the sputtering target. Ti thin films were deposited on the Si substrate (2.5 × 5 cm 2) using a DC magnetron sputtering system. In order to remove the native oxide layer of Si, the Si substrates were dipped in hydrofluoric acid (HF) solution for 30 s, and then they were ultrasonically rinsed in acetone, ethanol, and deionized water (DI) and dried at room temperature before being introduced into the vacuum chamber. Next, the Si substrate was placed in the magnetron sputtering
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vacuum chamber. In order to eliminate the contamination layer on the target surface, the Ti target was cleaned by pre-sputtering for 3 min. The argon (Ar) gas pressure and the power input to the Ti target during the deposition of Ti thin films on the Si substrate were 0.93 Pa and, 10 W per cm2 respectively. Under these conditions, the deposition rate of the Ti thin films was 35 nm per min, and a 500 nm thick Ti thin film was formed after the sputtering. 2.2. Fabrication of TiO2 nanotubes Prior to PEO, the Ti thin film-coated Si substrates were respectively cleaned with acetone, ethanol, and DI water, and then dried in a vacuum dry chamber for 1 day. Following the drying process, specimens were covered with an insulative polymer on their back side to prevent an interruption of the current between the Ti coating layer and the Si substrate as it undergoes PEO. PEO was performed by a two-electrode system in 0.5 wt.% HF and an ethylene glycol mixture solution at room temperature. The PEO system setup was completed with a platinum (Pt) plate used as a cathode electrode that was maintained at a distance of 10 mm from the specimen used as an anode. The PEO experiments consisted of applying a potential between 5 and 20 V over time intervals between 60 and 1200 s using a DC power supply (Genesys, 600-2.6 Densei-Lambda, Tokyo, Japan). Only current was passed to the Ti thin film on the specimen through alligator clips from the power supply. The PEO specimens were washed gently with DI water and dried in the vacuum dry chamber at room temperature. Afterward, specimen was annealed at 450 °C. 2.3. Characterization of TiO2 nanotubes on Ti thin film The surface morphology and chemical composition of the TiO2 nanotube layer on the Si substrate were qualitatively characterized by field emission scanning electron microscopy (FE-SEM, JSM-6700 F, JEOL, Japan), transmission electron microscopy (TEM, JEM-3010, JEOL, Japan) and energy dispersive spectrometer (EDS, X-MAX, Oxford, UK). FE-SEM at 15.0 kV condition was also carried out on the specimen cross-sectional plane prepared by mechanical fracture. The microstructure of the prepared specimens was identified by high resolution X-ray diffraction (HR-XRD, Rigaku, Kuraray, Japan) with a Cu-Kα radiation under 40 kV and 40 mA from 20° to 80° of 2θ value. 2.4. Corrosion resistance
Fig. 1. Procedure for a drug loaded TiO2 nanotube layer on a Ti thin film.
In order to investigate the corrosion resistance, a potentiodynamic test was conducted in phosphate buffered saline (PBS) at 37 °C. The
Fig. 2. Top and cross-sectional view of the Ti thin film on a Si substrate and its chemical composition.
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Fig. 3. FE-SEM images (×50,000) of fabricated TiO2 nanotubes on a Si substrate coated with a Ti thin film with a fixed PEO time of 1200 s: (a) 5 V, (b) 10 V, (c) 15 V, and (d) 20 V.
specimens were situated in an assembled corrosion cell containing the PBS solution, and which was then connected to the electrochemical interface (VersaSTAT3, Princeton Applied Research, USA). 2.5. Drug loading to the TiO2 nanotubes on a Ti thin film To fill the TiO2 nanotubes on Ti thin films, freeze drying was performed with the lyophilization method [33], and filling was adapted from the modified method in Popat et al. [34]. In brief, a solution of 1 mg/ml gentamicin was prepared in a PBS solution, and then the specimens (1.0 × 1.0 cm 2) were cleaned with distilled water before filling the PEO-fabricated TiO2 nanotubes with the drug. Before filling with the drug, the PBS solution was used to gently clean the specimen. To load the drug, 10 μl gentamicin solution was simultaneously spread around the surface and dried in air at room temperature for 2 h. The drying and pipetting steps were repeated until a sufficient amount of gentamicin was filled in the TiO2nanotube. The specimens were quickly rinsed with 500 μl of PBS over the specimens in order to remove excess gentamicin that covered the surface in the final drying step.
3. Results and discussion The purpose of this study was to determine the optimal conditions for fabricating a TiO2 nanotube layer as a drug carrier on a different substrate and to evaluate the drug release. A magnetron sputtering system was used to coat the substrate with an appropriate density and uniformity of Ti thin film. Fig. 2 shows the top and crosssectional view of the coated Ti thin film. The cross-sectional view in Fig. 2b of the specimen reveals the dense Ti thin film by magnetron sputtering. The thickness of the Ti layer was 500 nm ± 10 nm. The chemical composition investigation was performed using EDS within FE-SEM, and indicated a nearly pure Ti thin film.
2.6. Release from TiO2 nanotubes on Ti thin film In order to investigate the release kinetic of the drug from the TiO2 nanotube layer on a Ti thin film, the specimen was immersed in 800 μl of PBS in a 4-well plate at room temperature in a shaking incubator at 100 rpm. After a specified time, 500 μl of the sample was collected to evaluate the amount of drug released. The collected solution was changed with 500 μl of fresh solution every time to calculate the accumulated sample concentration. The collected samples were analyzed for drug concentration using the UV–VIS spectrophotometer (UV–VIS, UVD-3200, Labomed, USA).
Fig. 4. Current time plot for 1200 s of PEO treatment at 20 V in 0.5 wt.% HF electrolyte.
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Fig. 5. FE-SEM image (×100,000) of PEO time-dependent growth of TiO2 nanotubes with a fixed applying voltage. The potential was kept at 20 V at different oxidation times; code: (a) 60 s; TDG01, (b) 240 s; TDG04, (c) 480 s; TDG08, (d) 720 s; TDG12, (e) 960 s; TDG16, and (f) 1200 s; TDG20 (the tube lengths are indicated in the cross-sectional views).
Fig. 3 shows the results of the PEO treatment with a variable voltage (5, 10, 15, 20 V) applied for 20 min. When the applied potential was 5, 10, and 15 V (Fig. 2a, b, and c, respectively), nano-porous shapes appeared on the surface. This indicated that these shapes have improper morphologies to be drug carriers since they were irregular. However, it can be clearly seen that the experimental condition of 20 V (Fig. 2d) produced the most obvious shape for a highly ordered tube that is effective as a drug carrier [35]. Therefore, we conducted the experiment with a 20 V potential. Fig. 4 shows the current reaction of the bath as function of the PEO time. The growth mechanism of the TiO2 nanotubes was revealed by previous studies [36–38]. After applying the potential to the substrate, the current rapidly decreased in the initial stage within the first seconds. Subsequently, a dense Ti oxide layer is fabricated. This was due to the pores soaking through the barrier oxide film, exposing the Ti thin film to the PEO electrolyte. The current density then decreased with time as a result of the increase in diffusion length for an ionic species in the electrolyte [38–40]. Fig. 5 shows the sequence of the morphology of the PEO treated specimen as the evolution of surface topography under different PEO treatment times at 24 °C. In the initial stage of oxidation, some cracks appeared on the surface because the oxide layer was chemically dissolved by field assistance with a voltage on the Ti thin films. Evidently, a nano-porous layer formed on the specimen at the early stage of ~ 240 s (Fig. 5a and b). With an extended PEO treatment time the current densities decreased and gradually the oxide layer dissolved again (Fig. 5c). Subsequently, after 720 s, the initiation of the tube-shape formations on the surface began (Fig. 5d). At this stage the nano-pores were irregularly formed on the specimen and competed for workable space to combine with another pore and potential [36]. Fig. 5e shows that the porous oxide structure was more uniform and had a more regular tube shape than the nano-porous shape (Fig. 5b, c and d). After 1200 s of the PEO treatment (Fig. 5f), the formed nano-tubular self-ordered TiO2 layer clearly showed homogeneous and uniform nanotubes. These results demonstrate that the present approach can capitalize on the fabrication of TiO2 nanotubes that have a morphology similar to a polycrystalline titanium foil anodized using the procedure demonstrated by Popat et al. [5,34].
The influence of the applied PEO time on the resulting diameter and length of the TiO2 nanotubes is summarized in Fig. 6. At 4 min of PEO treatment, a nano-porous oxide layer with approximately 30 nm diameter and 150 nm thickness was fabricated. After 1200 s of PEO treatment there was an array of TiO2 nanotubes with diameters of 100 nm and lengths of 400 nm. Between 240 and 1200 s, the dimensional tube diameters and lengths showed increasing and nearly linear dependence on the PEO treatment time. Figs. 7 and 8 show the EDS, TEM and XRD analyses resulting from the PEO treatment at a potential of 20 V for 1200 s and then, heat treated. TiO2 consisting of Ti and O ions was detected through the EDS analysis. Also, the XRD results show that it is possible to transition from an amorphous phase to a polycrystalline phase TiO2 [16,41]; furthermore, they show the removal of residual debris such as HF acid and C, F ions due to annealing of specimen [42,43]. However, nanotube with the incomplete form has a lower intensity of anatase phase diffraction pattern [44]. The corrosion resistance of biomaterials influences their functionality and stability and is the key factor in their biocompatibility
Fig. 6. The correlation between PEO time and growth of the TiO2 nanotubes on the Ti thin film.
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Fig. 7. EDS analysis after the formation of heat-treated TiO2 nanotubes by the PEO treatment under an applied voltage of 20 V for 1200 s (TDG20).
Fig. 8. (a) TEM image and (b) XRD patterns after the formation of heat-treated TiO2 nanotubes (TDG20).
[45–47]. The corrosion resistance of biomaterials and their functional performance is important in the in vivo environment if nanotubes are to be used as drug carriers [48]. Fig. 9 shows the results of a potentiodynamic test of the TiO2 nanotubes and the sputtered Ti thin film. These results revealed that the corrosion resistance of the TiO2 nanotubes was better than that of the Ti thin film because the nanotubes showed a lower passive current density than the Ti thin film in the PBS solution. We demonstrated the possibility of fabricating self-ordered TiO2 nanotubes in sputtered Ti thin films on a Si substrate. It is also notable that the procedure for fabricating TiO2 nanotubes on sputtered Ti thin
films (Fig. 10) described here exhibits further advantages for drug carrier functionality in biomedical applications compared to other studies [37,49]. To confirm the potential of using TiO2 nanotubes on the Ti thin film as a drug carrier, 1 mg/ml gentamicin was loaded in the TiO2 nanotube layer. Fig. 11 shows the release data for gentamicin. Generally, the drug had a slower release from the TiO2 nanotubes (TDG16 and TDG20) compared to other test groups. Especially, the drug had a higher release efficacy from the TDG20 group where the adequate concentration of drug release was observed even after 12 h. However, other groups showed lower drug-releasing efficacy because of their thin and incomplete shape of oxide as drug carrier. These results suggested that it is feasible for TiO2 nanotubes on a Ti thin film to act as a drug carrier. Hence, the use of a drugreleasing TiO2 nanotube layer as a coating on other implantable biomaterials which are not Ti-based has a variety of applications. 4. Conclusion
Fig. 9. Graph of the PEO treatment current density vs. potential for Ti thin films and TiO2 nanotubes on a Si substrate, from the potentiodynamic test.
We demonstrated two approaches for fabricating TiO2 nanotubes on sputtered Ti thin films on a Si substrate. Titanium thin films coated by magnetron sputtering onto the Si substrate were applied for PEO in 0.5 wt.% HF solution at room temperature. In this study, it was shown that morphological changes of nanotubes were achieved according to the PEO potential (~ 20 V) and control of the length and diameter of nanotube was dependent on PEO time (~ 1200 s). It was morphologically obvious from the analysis performed using FE-SEM that a self-ordered TiO2 nanotube layer with an average diameter about 100 nm and length about 400 nm could be fabricated using the PEO treatment. Also, we showed that TiO2 nanotubes with a PEO treatment time of 1200 s formed on sputtered Ti thin films are shown to release drug sustainably from nanotubes. It was concluded
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Fig. 10. The final shape of TiO2 nanotube which was fabricated on sputtered Ti thin film used as drug carrier.
Fig. 11. Gentamicin calibration curve and released gentamicin cumulative concentration as a function of immersion time.
that TDG20 will be applicable to biomaterials other than Ti substrates for the application as a drug carrier. Conflict of interest The authors declare no conflicts of interest. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2012R1A1A2008659) and SRC program of National Research Foundation (NRF) of Korea, funded by the Ministry of Education, Science and Technology (20100029418).
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