Bioinspired Surface Functionalization of Titanium for Enhanced

Apr 29, 2019 - Titanium and its alloys have long been used as implantable biomaterials in orthopedics; however, to the best of our knowledge, few stud...
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Bioinspired Surface Functionalization of Titanium for Enhanced Lubrication and Sustained Drug Release Ying Han, Sizhe Liu, Yulong Sun, Yanhong Gu, and Hongyu Zhang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00338 • Publication Date (Web): 29 Apr 2019 Downloaded from http://pubs.acs.org on May 5, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioinspired Surface Functionalization of Titanium for Enhanced Lubrication and Sustained Drug Release Ying Han1, Sizhe Liu1, Yulong Sun1, Yanhong Gu2, Hongyu Zhang1,*

1State

Key Laboratory of Tribology, Department of Mechanical Engineering, Tsinghua

University, Beijing 100084, China 2Beijing

Key Laboratory of Pipeline Critical Technology and Equipment for Deepwater

Oil & Gas Development, Beijing Institute of Petrochemical Technology, Beijing 102617, China

*Correspondence

to Prof. Hongyu Zhang

State Key Laboratory of Tribology, Department of Mechanical Engineering, Tsinghua University, Beijing 100084, China Email: [email protected]

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ABSTRACT Titanium and its alloys have long been used as implantable biomaterials in orthopedics, however, to the best of our knowledge, few studies are reported to investigate surface functionalization of titanium for enhanced lubrication and sustained drug release. In the present study, titania nanotube arrays (TNTs) were prepared by anodization as effective drug nanocarriers, using titanium as the substrate. Meanwhile, motivated by articular cartilage-inspired superlubricity and mussel-inspired adhesion, a copolymer containing both dopamine methacrylamide and 2-methacryloyloxyethyl phosphorylcholine was synthesized (DMA-MPC) and spontaneously grafted onto the TNTs surface, which was validated by the characterizations of scanning electron microscopy, water contact angle, and X-ray photoelectron spectroscopy. Additionally, the lubrication test showed that the copolymer-grafted TNTs remarkably reduced friction coefficient compared with the bare TNTs. Furthermore, the drug release test demonstrated that the copolymer-grafted TNTs inhibited burst drug release and achieved sustained drug release in comparison with the bare TNTs. In conclusion, the bioinspired surface functionalization strategy developed here, namely DMA-MPC copolymer-grafted TNTs, can be applied to modify orthopedic biomaterials (such as titanium) for enhanced lubrication and sustained drug release.

Keywords: TNTs; articular cartilage; mussel; hydration lubrication; drug delivery

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INTRODUCTION Osteoarthritis is a degenerative joint disease caused by wear of the articular cartilage.1,2 Artificial joint replacement is the most common surgery for clinical treatment of end stage osteoarthritis.3 Titanium and its alloys have been widely used as implantable biomaterials due to their outstanding properties, including excellent biocompatibility, good mechanical stability and corrosion resistance.4-6 However, titanium and its alloys experience wear in vivo, and the wear debris-induced inflammation response occurs following orthopedic surgery, which can result in serious complications and greatly affect the lifetime of implants.7 Basically, it is difficult for the anti-inflammatory drugs taken via oral administration to reach the joints, partly due to the restrictions including poor targeting, poor distribution and uncontrolled pharmacokinetics.8,9 Consequently, local drug delivery may represent a proper solution to solve this problem because this technique, with lower dosage and extended release time, can deliver drugs directly to the required location.10 Titania nanotube arrays (TNTs) prepared with titanium as the substrate are reported to be promising nanocarriers for drug delivery systems, which have attracted considerable attention due to their excellent biocompatibility, high surface area, thermal stability, and the potential for further surface modification.11-13 TNTs are tubular and selforganized nanostructures that enable them to be loaded with therapeutic agents such as antibiotics, anti-inflammatory agent, and growth factors using physical adsorption and deposition method.14-17 Meanwhile, TNTs can be simply prepared in large quantities via various techniques such as hydrothermal treatment of sol-gel template and anodic 3 ACS Paragon Plus Environment

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oxidation method. Generally, anodization is an effective method that can synthesize TNTs with controlled nanostructure and morphology, being aligned perpendicularly to the titanium substrate.18,19 Nevertheless, TNTs have relatively lower wear and abrasion resistance, and as a consequence TNTs are easy to be removed away under shear force. Therefore, a suitable surface modification is desirable to enhance the lubrication property of TNTs, that is to say, TNTs should be modified with effective lubricating materials to achieve better lubrication. In nature, articular cartilage can form excellent superlubricity under high loading based on hydration lubrication mechanism, which is attributed to the supramolecular synergy of the brush-like biomacromolecules.20-22 Specifically, the charged zwitterionic groups (N+(CH3)3 and PO4–) in the phosphocholine lipids attract a tenacious hydration layer surrounding them, and the hydration layer responds in a fluid-like manner under shear, significantly enhancing lubrication property between two sliding surfaces. Inspired by this, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) brush, with the same zwitterionic charges as phosphocholine lipids, has been paid much attention recently. Typically, Klein et al. modified mica with PMPC brush and obtained an extremely low friction coefficient (COF, less than 0.001 as measured using surface force balance).23 However, the grafting-from strategy (such as atom transfer radical polymerization) to modify the substrate with PMPC brush is complicated and often needs harsh reaction conditions. Therefore, a universal and facile surface modification technique that can functionalize the substrate with PMPC brush is required. Mussels can attach to almost all kinds of substrates with high binding strength, and this 4 ACS Paragon Plus Environment

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has been attributed to the 3,4-dihydroxy-L-phenylalanine and lysine-enriched proteins secreted by mussels byssus at the plaque-substrate interface. Dopamine, as an important derivative of dihydroxy-phenylalanine with similar adhesion function to mussels, has received increasing attention since it was first reported by Lee et al. for multifunctional coatings.24-26 Regarding the mechanism of spontaneous attachment, it is well accepted that the catechol groups are susceptible to oxidation to quinone under neutral or alkaline conditions,27,28 and a strong bond is formed between dopamine and the substrate during this process. In the present study, TNTs with a series of length to diameter ratios were prepared by anodization, and simultaneously, motivated by cartilage-inspired superlubricity and mussel-inspired adhesion, a novel copolymer (DMA-MPC) containing PMPC brush and dopamine was synthesized, which could spontaneously attach onto the TNTs. As shown in Figure 1, the copolymer can, on the one hand, enhance lubrication of TNTs and thus reduce wear of the titanium implant, and on the other hand, enable sustained drug release for anti-inflammatory purpose. It is hypothesized that, as a universal and facile technique for enhanced lubrication and sustained drug release, the copolymergrafted TNTs developed here can be used for surface functionalization of titanium to promote the lifetime of orthopedic implants.

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Figure 1. Schematic illustration showing the preparation of the bioinspired copolymer-grafted TNTs for enhanced lubrication and sustained drug release.

MATERIALS AND METHODS Preparation of TNTs. High purity titanium sheet (99 %, Goodfellow Cambridge Ltd, UK) with a thickness of 1 mm was used as the substrate. Initially, the sheet was polished and cleaned by sonication in deionized water for 5 min. The two-electrode system was used in the electrochemical anodization process, with graphite rod as the cathode and titanium sheet as the anode.29 The electrolyte contained 0.5 wt.% ammonium fluoride, 3 vt.% deionized water, and 97 vt.% ethylene glycol. The anodization was performed at room temperature using a direct current power supply, with the anodization voltage of 5 V, 10 V and 15 V sequentially for 12 h. After anodization, the titanium sheets were cleaned by sonication in deionized water and ethanol for 2 min, and dried in vacuum. Synthesis of Bioinspired Copolymers. Dopamine methacrylamide (DMA) was initially synthesized according to the method previously reported.30-32 Briefly, sodium 6 ACS Paragon Plus Environment

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tetraborate (20 g, 52.4 mmol) and sodium bicarbonate (8 g, 95.2 mmol) were dissolved in 200 mL of deionized water under nitrogen atmosphere, followed by rapidly adding dopamine hydrochloride (10 g, 53 mmol, purchased from J&K Scientific Ltd., Beijing, China). Meanwhile, 9.4 mL (63.4 mmol) of methacrylic anhydride was dissolved in 50 mL of tetrahydrofuran and added dropwise to the above solution (pH=8). The mixed solution was stirred overnight under nitrogen atmosphere. Subsequently, the pH value of the solution was adjusted to 2 by hydrochloric acid (0.2 M), followed by extracting with ethyl acetate and filtering with magnesium sulfate. Finally, the filtered solution was recrystallization with petroleum ether to produce a white solid power. The copolymer (DMA-MPC) was synthesized by free radical copolymerization, with the feeding mole ratio of DMA/MPC at 1/4 and azodiisobutyronitrile as the initiator. Briefly, DMA (0.2 g), MPC (0.8 g, purchased from Joy-Nature Institute of Technology, Nanjing, China), and azodiisobutyronitrile (3 mg) were dissolved in 50 mL of N, Ndimethylformamide under nitrogen atmosphere, and then the reaction was proceeded at 65 °C for 24 h. Subsequently, the solution was dialyzed against deionized water and freeze-dried to obtain a flocculent product. Preparation of Copolymer-Grafted TNTs. The DMA-MPC copolymer was grafted onto TNTs using the dripping addition method. Briefly, DMA-MPC was dissolved in phosphate buffered saline (PBS), and 60 μL of the copolymer solution (1 mg/mL, 2 mg/mL, and 5 mg/mL) was dropped onto the TNTs (bare TNTs or drugloaded TNTs) twice. The copolymer-grafted TNTs were washed with deionized water and dried in vacuum. 7 ACS Paragon Plus Environment

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Lubrication Test. The lubrication test was performed using a universal materials tester (UMT-3, Bruker Nano Inc., Germany). The experiments were completed in a reciprocating mode (oscillation amplitude: 4 mm; sliding frequency: 1 Hz; normal load: 1~3 N) at 25 °C, each for a duration of 10 min. Three kinds of TNTs (bare TNTs or copolymer-grafted TNTs) with different length to diameter ratios (i.e. prepared at the anodization voltage of 5 V, 10 V, and 15 V) were used as the lower specimen, which contacted directly with a polytetrafluoroethylene (PTFE) sphere pin (diameter: 8 mm) as the upper specimen. The lubricant was deionized water for all the experiments to ensure the PMPC brush was well stretched in the medium. Under each test condition, at least three different locations on the TNTs surface were selected to calculate the average COF value to ensure repeatability. Drug Loading and Release. Honokiol is a natural lignin that has been proved for its anti-inflammatory effect in previous study.33 In the present study, honokiol was selected to be loaded to TNTs by physical adsorption and deposition method. Briefly, TNTs were immersed in 2 mg/mL of honokiol solution (dissolved in dimethyl sulphoxide, DMSO), and the dynamic adsorption was allowed by shaking at 37 °C for 24 h. After loading, the TNTs were rinsed thoroughly by PBS to remove the drug remaining on the surface of TNTs. Subsequently, two groups of samples including drug-loaded bare TNTs and drugloaded copolymer-grafted TNTs (six specimens in parallel) were immersed in 5 mL of PBS, and then placed in the shaker for drug release at 37 °C.34,35 At predetermined time intervals, 1 mL of the medium was taken out and mixed with 1 mL of DMSO. The 8 ACS Paragon Plus Environment

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absorbance of the solution was measured by a UV-vis spectrophotometer (UV-8000, Metash Instruments, China) at a wavelength of 296 nm. Meanwhile, 1 mL of fresh PBS was added to the medium for further drug release test. The standard curve of honokiol in DMSO/PBS (volume ratio at 1/1) solution was obtained, and the drug release percentage (wt.%) was calculated based on the following equation (1).

Drug release percentage 

amount of released drug 100 total amount of loaded drug

(1)

Characterizations. The surface and cross-section morphologies of the TNTs were observed by a scanning electron microscope (SEM, SU8220, Hitachi, Japan) associated with an energy dispersive spectrometer (EDS) for surface composition detection. The surface wettability of the TNTs was examined by water contact angle employing a goniometer (OCA25, Dataphysics Instruments, Germany). The 1H NMR spectra of DMA and DMA-MPC were measured using a nuclear magnetic resonance (NMR) spectrometer (JNM-ECS400, JEOL, Japan), with DMSO and D2O as the solvent. The element analysis of the TNTs was performed by an X-ray photoelectron spectroscopy (XPS, PHI Quantera II, Ulvac-Phi Inc., Japan). The Raman spectra of the TNTs were recorded employing an XploRA Plus Raman spectrometer (Horiba, France) under the following conditions: excitation wavelength of 638 nm, laser power of 0.5 mW, and accumulation time of 50 s. RESULTS AND DISCUSSION Characterization of TNTs. Figure 2a-h shows the SEM micrographs of the surface and cross-section morphologies for titanium substrate and ordered TNTs prepared by oxidation at different voltages. The average diameter of the TNTs oxidized at 5 V, 10 9 ACS Paragon Plus Environment

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V, and 15 V is 25 nm, 30 nm, and 40 nm respectively, and the length is 0.6 μm, 1.0 μm, and 1.0 μm respectively, resulting in the length to diameter ratio to be 24, 33, and 25 for the three kind of TNTs. This indicates that increasing the anodization voltage within a certain range can increase the diameter of the TNTs as a result of a faster anodization process. The EDS analysis of the TNTs is shown in Figure 2e, and clearly the O element is detected on the surface. The formation of TNTs by anodization is attributed to the equilibrium between the electrochemical process (oxidation reaction at the metal/oxide interface, equation (2)) and the chemical process (etching reaction at the oxide/solution interface, equation (3)).36,37 Briefly, the titanium substrate reacts with water molecules under the action of electric field to generate an oxidation TiO2 layer. Subsequently, the TiO2 layer was chemically attacked by fluoride ions to form the [TiF6]2- complexes, which are water-soluble, and therefore a series of nanopores are produced and the oxidation film becomes thinner. As a consequence, in these areas the ion migration rate will be greater and the etching of the nanopores will be further accelerated to finally generate TNTs. Ti + 2H2O → TiO2 + 4H+ + 4e-

(2)

TiO2 + 6HF → [TiF6]2- + 2H2O + 2H+

(3)

Figure 2i-l shows the water contact angle of the titanium substrate and ordered TNTs prepared by oxidation at different voltages, which is measured after the water droplets stay on the sample surface for 5 sec. The TNTs surface (contact angle: 2.6~3.5°) is more hydrophilic than the titanium substrate (contact angle: 84.5°). The higher hydrophilicity of TNTs can be attributed to two aspects. One is that the special tubular 10 ACS Paragon Plus Environment

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structure itself allows for seeping of the water droplet into the TNTs because of capillary action.38 The other is that the Ti-OH group remains on the surface of TNTs (formed owing to the cathode process consumes H+ to produce H2 during anodization), thus increasing the hydrophilicity of TNTs.

Figure 2. Characterization of TNTs: (a-d) SEM micrographs of the surface morphology for (a) titanium substrate and TNTs anodized at (b) 5 V, (c) 10 V, and (d) 15 V; (e) typical EDS analysis of TNTs with the detection of O element; (f-h) SEM micrographs of the cross-section morphology for TNTs anodized at (f) 5 V, (g) 10 V, and (h) 15 V; (i-l) water contact angle of the (i) titanium substrate and TNTs anodized at (j) 5 V, (k) 10 V, and (l) 15 V.

Characterization of DMA-MPC Copolymer. The 1H NMR spectra of DMA and DMA-MPC are displayed in Figure 3. As shown in Figure 3a, the signal at 7.93 ppm is assigned to the -NHCO-group of DMA, which indicates the success of the amidation reaction between dopamine hydrochloride and methacrylic anhydride. Additionally, the signals at 8.74 ppm and 8.62 ppm are attributed to the catechol group of DMA, which maintains similar adhesion property to dopamine. As shown in Figure 3b, the signals at 11 ACS Paragon Plus Environment

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6.83 ppm and 6.71 ppm are assigned to the 3, 4, 6-trihydrophenyl groups of DMA in the copolymer. The signals at 4.23 ppm, 4.03 ppm, 3.60 ppm, and 3.16 ppm belong to the groups of MPC in the copolymer. It is calculated via integration that the content of DMA in the DMA-MPC copolymer is ~20.3 wt.%, which conforms to the feeding mole ratio of DMA/MPC (1/4) in the copolymerization reaction.

Figure 3. 1H NMR spectra of (a) DMA monomer and (b) DMA-MPC copolymer.

Surface Grafting of TNTs with DMA-MPC Copolymer. The SEM micrographs of DMA-MPC copolymer-grafted TNTs (TNTs@DMA-MPC) are presented in Figure 4a-c. The tubular structure of TNTs disappears, and it is covered by a dense copolymer layer after surface grafting. The water contact angle of TNTs@DMA-MPC is shown in 12 ACS Paragon Plus Environment

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Figure 4d-f (in the range of 8.9~10.4°), indicating that the DMA-MPC copolymer is superhydrophilic. Moreover, the presence of the copolymer layer grafted onto the TNTs is evaluated by XPS. As shown in Figure 4g, the constituents of O 1s, Ti 2p, C 1s, and Ti 3p with corresponding binding energies at 531 eV, 459 eV, 285 eV, and 40 eV are observed in the titanium substrate and the TNTs@DMA-MPC. Compared with that of the titanium substrate, the spectra of TNTs@DMA-MPC demonstrate new peaks of N 1s (402 eV, corresponding to -N+(CH3)3 and –NHCO-), P 2s, and P 2p (190 eV and 132 eV, corresponding to -OPOCH2). Furthermore, the Raman spectra of the bare TNTs and TNTs@DMA-MPC are shown in Figure 5. It is clear that a few new peaks (such as P-O, P=O, and C=O) are observed for the TNTs@DMA-MPC compared with the bare TNTs, indicating the presence of the DMA-MPC copolymer on the TNTs surface. More importantly, the TiO bond splits sharply for the TNTs@DMA-MPC, forming a chemical bond at 650 cm-1 between the Ti atoms (in TNTs) and the hydroxyl of catechol groups (in DMA-MPC copolymer). This confirms that the DMA-MPC copolymer has been successfully grafted onto the TNTs surface via an ion-dipole interaction.39

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Figure 4. Characterization of copolymer-grafted TNTs: (a-c) SEM micrographs and (d-f) water contact angles of the TNTs@DMA-MPC anodized at (a, d) 5 V, (b, e) 10 V, and (c, f) 15 V; (g) XPS of the titanium substrate and TNTs@DMA-MPC.

Figure 5. Raman spectra of (a) the bare and (b-d) DMA-MPC copolymer-grafted TNTs anodized at (b) 5 V, (c) 10 V, and (d) 15 V, respectively.

Lubrication Performance. The lubrication test is performed to characterize the lubrication performance of the DMA-MPC copolymer-grafted TNTs. Figure 6a shows 14 ACS Paragon Plus Environment

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the schematic experimental setup of the lubrication test, and Figure 6b-d displays the COF-loading plots for the TNTs prepared by oxidation at different voltages. Obviously, all the COF values of the TNTs@DMA-MPC are lower than those of bare TNTs. For instance, the COF value decreases evidently from 0.16 for the bare TNTs to 0.10 for the TNTs@DMA-MPC (~37 %) prepared at 10 V under 3 N (Figure 6c). Additionally, the COF value of the TNTs@DMA-MPC slightly increases under 3 N than that under 1 N and 2 N. It is considered that the PMPC brush in the DMA-MPC copolymer (grafted on the TNTs) is well stretched in aqueous medium under low load, but the brush can be crushed under relatively higher load, resulting in the increase of COF value. However, on the other hand, the crush of the brush may be beneficial for accelerated release of the anti-inflammatory drug, when the increased friction causes generation of more wear debris at the sliding interface and correspondingly inflammation response of the joint.

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Figure 6. Lubrication performance of copolymer-grafted TNTs: (a) schematic diagram showing the experimental setup of the lubrication test; (b-d) COF-loading plots for the bare TNTs and TNTs@DMAMPC anodized at (b) 5 V, (c) 10 V, and (d) 15 V.

Drug Loading and Release Property. Figure 7a demonstrates the standard curve of honokiol in the DMSO/PBS mixed solution, and Figure 7b-d shows the drug release behavior of the bare and copolymer-grafted TNTs prepared by oxidation at different voltages. Clearly, all the curves have two stages including the initial burst release stage followed by a relative plateau stage. The drug release of bare TNTs is remarkably higher than that of TNTs@DMA-MPC (especially at the higher concentration), indicating that the DMA-MPC copolymer layer can effectively block the drug from burst release and prolong the drug release duration. For instance, the drug release reduced from 60 % (at 11.5 h) for the bare TNTs to less than 40 % (at 50 h) for the TNTs@DMA-MPC (5 mg/mL) prepared at 15 V (Figure 7d). Moreover, it is noted that 16 ACS Paragon Plus Environment

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the burst drug release and the total drug release are the highest for the bare TNTs anodized at 15 V, which is caused due to the largest diameter of the tubular structure. Additionally, the total drug release cannot reach 100 % during the test period owing to the slow diffusion rate of the drug molecules entrapped inside the TNTs. These results indicate that the TNTs@DMA-MPC can achieve a sustained release of the loaded drug, which is desirable for the implanted orthopedic biomaterials.

Figure 7. Drug loading and release property of copolymer-grafted TNTs: (a) standard curve of honokiol in the DMSO/PBS mixed solution; (b-d) drug release curves of the bare TNTs and TNTs@DMA-MPC anodized at (b) 5 V, (c) 10 V, and (d) 15 V.

Mechanism for Enhanced Lubrication and Sustained Drug Release. Figure 8 schematically demonstrates the mechanism for enhanced lubrication and sustained drug release of the copolymer-grafted TNTs, which is attributed to the zwitterionic charges 17 ACS Paragon Plus Environment

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(N+(CH3)3 and PO4–) contained in the PMPC brush and the catechol groups contained in the adhesive DMA. Water molecule is overall neutral, but it has a large electric dipole due to the residual charges on the H and O atoms. Therefore, the zwitterionic charges in the PMPC brush can attract water molecules to form a tenacious hydration shell due to the charge-dipole interaction. The water molecules in the hydration shell can support high pressures without being squeezed out, and exchange rapidly with the nearby “free” water molecules, resulting in a fluidlike response when being sheared and consequently an enhanced lubrication at the interface. This mechanism, proposed by Klein et al., is called hydration lubrication.23 Additionally, the adhesive copolymer layer can reduce the diffusion rate of the drug previously encapsulated into the TNTs, thus inhibiting burst drug release into the solution and achieving sustained drug release property.

Figure 8. Schematic illustration showing the mechanism for enhanced lubrication and sustained drug release of copolymer-grafted TNTs, which can be attributed to hydration lubrication of the phosphorylcholine groups in PMPC brush and spontaneous adhesion of the catechol groups in DMA.

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CONCLUSIONS In the present study, bioinspired by superlubricity of articular cartilage and spontaneous adhesion of mussel, a novel DMA-MPC copolymer was synthesized and used to modify the TNTs for enhanced lubrication and sustained drug release. The characterizations of surface morphology, surface wettability, and surface composition all confirmed that the copolymer was successfully grafted onto the TNTs. Additionally, the lubrication test showed that the COF value of the copolymer-grafted TNTs was lower than that of the bare TNTs, which was attributed to the hydration lubrication mechanism of the PMPC brush with zwitterionic charges. The drug release test demonstrated that the copolymer layer grafted on the surface of TNTs effectively inhibited burst drug release and thus achieved sustained drug release property. The copolymer-grafted TNTs, as a promising lubricated drug nanocarrier, can be used to functionalize orthopedic biomaterials (such as titanium) for enhanced lubrication and sustained drug release.

ASSOCIATED CONTENT The Supporting Information of the deconvolution study of XPS spectra (DMA-MPC and TNTs@DMA-MPC) for C 1s, O 1s, N 1s and P 2p, and the drug release test of honokiol from the DMA-MPC copolymer.

ACKNOWLEDGEMENTS This study was financially supported by National Natural Science Foundation of China (grant no. 51675296), Ng Teng Fong Charitable Foundation (grant no. 202-276-13219 ACS Paragon Plus Environment

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13), and Research Fund of State Key Laboratory of Tribology, Tsinghua University, China (grant no. SKLT2018B08). The authors thank Dr. Yilin Lu for his help in Raman spectrum characterization.

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