Enhancement in Sustained Release of Antimicrobial Peptide from

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Enhancement in sustained release of antimicrobial peptide from dual-diameter Structured TiO nanotubes for long-lasting antibacteria and cytocompatibility 2

Yanni Zhang, Lan Zhang, Bo Li, and Yong Han ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00322 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on March 1, 2017

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ACS Applied Materials & Interfaces

Enhancement in sustained release of antimicrobial peptide from dual-diameter structured TiO2 nanotubes for long-lasting antibacteria and cytocompatibility Yanni Zhang, Lan Zhang, Bo Li, Yong Han State Key Laboratory for Mechanical Behavior of Materials, Xi’an Jiaotong University, Xi’an 710049, China KEYWORDS:

dual-diameter nanotube film; antimicrobial peptide; controllable release;

antibacterial activity; cytocompatibility. ABSTRACT:

Novel films on Ti-based orthopedic implants for localized antimicrobial

delivery, which comprised dual-diameter TiO2 nanotubes with the inner layers of compact and Fluorine-free oxide tightly bonding to Ti, were formed by voltage-increased anodization with F− sedimentation procedure. The nanotubes were closely aligned and structured with upper 35 and 70 nm diametric tubes as nanocaps, respectively, and the underlying 140 nm diametric tubes as nanoreservoirs. Followed by loading ponericin G1 (a kind of antimicrobial peptide (AMP)) into the dual-diameter nanotubes with vacuum-assisted physisorption, the resultant films were investigated for loading efficiency and release kinetics of AMP, antibacterial activity against Staphylococcus aureus and osteoblastic compatibility, together with the AMP loaded single-diameter (140 nm) nanotube film. The loaded films were of no statistical difference in the loading efficiency of AMP, and revealed burst release within 6 h followed by steady release of AMP in phosphate buffer solution. At day 42, 

Corresponding author: [email protected] (Y. Han).Tel.:+86 02982665580

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almost all of AMP released out from the single-diameter nanotube film; over which, however, the dual-diameter nanotube films loading AMP still remained sustained release at least up to 60 days, and the sustained efficacy was enhanced with decreasing the diameter of nanocaps. In the case of AMP nominal loading amount of 125 μg, the resultant 35 nm caped dual-diameter nanotube film exhibited significant short-term and long-term (even for 49 days) antibacterial activity not only against planktonic bacteria, which ascribed to the release-killing efficacy of AMP, but also against adhered bacteria, which ascribed to the AMP-derived killing efficacy and the nanocaps derived adhesion resistance. Moreover, this loaded film presented cytocompatibility comparative to Ti but higher than the other AMP-loaded films. Increasing nominal loading amount of AMP to 200 μg improved antibacterial activity, but gave raise to obvious cytotoxicity of the loaded films.

1. Introduction Titanium and its alloys are widely used as orthopedic implants due to their good mechanical property, corrosion resistance and biocompatibility. However, the service life of Ti-based implants is determined by their antibacterial activity, osseointegration and the adhesion of coated films to substrates,1-3 and these concerns are of scientific and clinical significance in the development of medical implants. Implant-related infection caused by bacteria is a healthcare concern, not only impairing osseointegration,2,4 but also increasing mortality and financial expenditure for removal of the infected implants.5,6 To reduce the infection, many antibacterial agents were employed, meanwhile anodized TiO2 nanotube arrays were used as the


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platforms to carry the drugs on Ti-based implants.7,8 For example, the incorporation of inorganic bactericides such as Ag+, Cu2+ and Zn2+ into TiO2 nanotube arrays exhibited high antibacterial activity, but with dose-dependent cytotoxicity.9-11 The antibiotics loaded TiO2 nanotubes could reduce implant-related infections, of which, however, traditional antimicrobial agents easily led to antibiotic resistance.12-14 Antimicrobial peptides (abbr. AMPs) are alternative candidates for the treatment of bacteria invasion, exhibiting potent and broad-spectrum antimicrobial activity, more or less cytotoxicity and low immunogenicity.14-16 It was demonstrated that the TiO2 nanotubes loading HHC-36 peptide showed a much higher bacteria killing ability than the antibiotics loaded ones,12,17 indicating the possibility of TiO2 nanotubes for AMP delivery. Ideally, the release profile of a localized drug delivery implant should exhibit a burst release stage for reducing infection instantly following surgery, and a long-term release stage to prevent potential infection.2,6,17 In the physiological environment, drug release from nanotubes was mainly controlled by a diffusion process.18,19 It was found that the nanotubular diameter affected the loading capacity and release behavior of drugs.20,21 Specifically, larger volume nanotubes were beneficial to trapping drug, but nanotubes with larger diameters could loss more amount of drug during rinse resulting in a lower loading efficiency.20 Moreover, drug release rate increased with tubular diameter but decreased with its length, while mass elution was dependent on the total loading amount.20,21 Currently, the most commonly used nanotube arrays for loading antibacterial agents were vertically aligned and single-diameter patterned. The drug release from



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the nanotubes was spontaneous upon contacting with the physiological solutions, which might cause the cytotoxicity at the early stage of implantation and could not sustain the long-term drug release for antibacteria.7,8,22 To overcome the drawback, various strategies were explored to optimize drug loading amount and release rate of the nanotubes, such as improving the affinity of drug to TiO2 nanotubes and reducing the diameter of nanotubes.17,23-27 Moreover, the drugs-loaded TiO2 nanotubes were further covered with biocompatible polymers, and drug release was thus controlled by the polymeric permeability or degradation.28-31 However, these methods were either sophisticated revealing a limited controllability of drug release or sheltered the nanotubular features. Actually, adjusting the morphology and dimension of nanotubes by anodization is the simplest strategy for the drug release control.18 In this respect, cone-shaped, allihn condenser-shaped, pear-shaped, bamboo-shaped, branched, and double/multiple layered TiO2 nanotube arrays were developed.24,32-36 Among them, cone-shaped and multipodal nanotubes exhibited a promising feature of small mouth and large pocket, but were scarcely used as drug delivery systems owing to the large gaps among the nanotubes.32,37,38 Bamboo-shaped and multiple layered nanotubes were poor in connectivity,34,35 branched nanotubes exhibited an inverted Y-shape with equal diameter,36 unsuitable for drug loading. Notably, allihn condenser-shaped and pear-like nanotubes could improve the loading capacity and slow the drug release rate, however, their adhesion strength was quite low.22,33 As known, the TiO2 nanotube films formed by tranditional anodization are poor in adhesion, easily peeling off from Ti substrates to generate debris, impairing cells


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and tissues.18,39 To improve their adhesion, some efforts have been undertaken, such as mechanical anchoring, annealing treatment and modified anodization, however, the improvements in adhesion strength were limited.40-43 Besides drug loading capacity and release behavior, nanotubular diameter also affected the behavior of cells. It was demonstrated that a small diameter of nanotubes such as 15-30 nm greatly improved the adhesion, proliferation and differentiation of cells (mesenchymal stem cell and osteoblast etc.), while a large diameter of nanotubes such as 100 nm led to the impairment of cell function or even apoptosis.44,45 Moreover, the 30 nm diametric nanotubes revealed an enhanced new bone formation compared to 100 nm ones.46 Therefore, it is crucial to balance nanotube diameter for loading capacity and sustained release of antimicrobial drugs and osteogenetic ability. In this work, firmly adhered dual-diameter structured TiO2 nanotube films were fabricated, in which the upper nanotubes with diameters of 35 and 70 nm acted as nanocaps for controlling drug release and as the surfaces interacting with cells, the underlying 140 nm diametric nanotubes served as nanoreservoirs for drug storage. As a kind of natural AMPs, ponericin G1 which has an amino sequence of GWKDW AKKAGG WLKKK GPGMA KAALK AAMQ-NH2 and remarkable antibacterial activity,47 was herein employed. The ponericin G1 loaded dual-diameter nanotube films were investigated for their loading efficiency, release kinetics, antibacterial activity and cytocompatibility, together with the AMP-loaded single-diameter one.

2. Materials and Methods 2.1. Fabrication of TiO2 nanotubes



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Commercially pure titanium disks (99.9 wt.% Ti) with size of Φ14×2 mm were mechanically polished and ultrasonically washed in acetone and deionized water. For the formation of TiO2 nanotube films by anodization, a direct current power source (WYK-602, Huatai, China) was employed, with which Ti disks were treated one by one as an anode while a graphite plate was used as a cathode. The electrolyte was an ethylene glycol (EG) solution containing 20 vol% diethylene glycol (DEG), 2 vol% water and 0.08 M NH4F, and was stirred at 20 rps by a magnetic stirrer during anodization. To obtain the dual-diameter and firmly adhered nanotube films, Ti disks were first anodized at a low voltage (stage I), and continued to be anodized at 80 V for proper time by increasing the voltage to 80 V at the rate of 30 V·min-1 (stage II), finally (CH3COO)2Mg was added into the electrolyte at a speed of 0.5 ml·s-1 for F− sedimentation at 80 V for 70 min (stage III), in which the former two stages were for the formation of dual-diameter nanotubes and the later stage was for improving their adhesion. Given that tube diameter is determined by the applied voltage while tube length is dependent on both anodization time and voltage,48 the anodization time of stage I and II was optimized in preliminary experiments. Consequently, two kinds of dual-diameter nanotube films with total nanotubular length of ~12 μm were fabricated according to the parameters listed in Table 1, referred to as D35-3.5 and D70-3.5, exhibiting different diameters and lengths of the nanocaps but the same diameter (~140 nm) of the nanoreservoirs (Table 2). The single-diameter and firmly adhered nanotube films with diameter of ~140 nm and tubular length of ~12 μm (referred to as S140-12) were formed by anodizing the Ti disks at 80 V for 40 min and then


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subjecting to F− sedimentation as above-mentioned stage III. Additionally, the film with the same nanotubular structure as D70-3.5 was formed by anodization without F− sedimentation (termed as T-NT) to identify the effect of F− sedimentation on adhesion. The obtained samples were washed with methanol and deionized water and dried at room temperature. Table 1. The Anodization Parameters for the Fabrication of Single-/Dual-Diameter Structured Nanotube Films

Film Name

Stage I (low voltage)

Stage II (high voltage)

Stage III (F sedimentation) -

Voltage (V)

Time (h)

Voltage (V)

Time (min)

Voltage (V)

Time (min)

Mg2+ concentrations in the electrolyte (M)

S140-12

—

—

80

40

80

70

0.06

D35-3.5

20

8

80

35

80

70

0.06

D70-3.5

30

5

80

20

80

70

0.06

T-NT

30

5

80

35

—

—

—

Table 2. The Sizes of Single-/Dual-Diameter Structured Nanotube Films Nanocap Sizes

Nanoreservoir Sizes

Diameter (nm)

Length (μm)

Diameter (nm)

Length (μm)

Total Nanotubular Length (μm)

S140-12

—

—

140

12

12

D35-3.5

35

3.5

140

8.5

12

D70-3.5

70

3.5

140

8.5

12

T-NT

70

3.5

140

8.5

12

Film Name

2.2. Structure and adhesion characterization of the TiO2 nanotube films The morphologies of this work involved samples were examined using scanning electron microscopy (SEM; Hitachi SU6600, Japan) equipped with an Energy dispersive X-ray spectrometer (EDX; DX-4, Philips, Netherlands). Scratch tests were performed using an auto scratch coating tester (WS-2005, China) to characterize the



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adhesion strengths of the nanotube films. The smallest load at which the recognizable failure occurred was defined as the critical load (abbr. Lc), and determined from the load versus acoustic characteristics and SEM images of the scratches. For each kind of the films, its critical load was averaged by three tests.

2.3. AMP loading into TiO2 nanotubes The AMP, ponericin G1, was loaded into the nanotubes using vacuum-assisted physisorption as described elsewhere.7 Briefly, 10 μl of phosphate buffer solution (PBS) containing 2.5 mg·ml-1 ponericin G1 was pipetted onto the films, which were then set into a vacuum desiccator for 2 h at room temperature. With the aid of vacuum extraction, the solvent inside the nanotubes evaporated quickly, resulting in the fast loading of AMP. Repeating the process, the films were nominally loaded with 125 μg AMP in total. After drying, the AMP residual on the nanotube surfaces was rinsed with 1 ml PBS, and the resultant solutions were collected to calculate the loading efficiency of AMP within the nanotubes according to the method as described elsewhere.7,12 The films loaded with AMP were correspondingly referred to as D35-3.5-AMP, D70-3.5-AMP, and S140-12-AMP, respectively.

2.4. Release evaluation of AMP from the loaded nanotube films Ponericin G1 was dissolved in PBS to prepare a series of standard solutions with different concentrations. It revealed a maximum absorption at 279 nm (Figure S1a, Supporting Information) as analyzed with a spectrophotometer (Thermo Scientific Multiskan GO). Consequently, a calibration curve could be obtained (Figure S1b,


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Supporting Information) by fitting the measured absorbance values vs corresponding AMP concentrations. In parallel, each of the AMP-loaded films was set in a caped tube with 1 ml of PBS and immersed for 060 days at 37 ºC. Following immersion, the resultant solutions were collected, and measured for the absorbance at wavelength of 279 nm, and the AMP concentrations of the solutions were calculated based on the Figure S1b depicted calibration curve. The AMP concentrations given in μg·ml-1 were converted into amount (μg), and consequently the release profiles of AMP from the loaded films were plotted with cumulative release amount against release time. Each test was repeated three times. 2.5. Antibacterial assay Gram-positive bacteria, Staphylococcus aureus (S. aureus, ATCC 25293) were employed and inoculated twice to obtain the bacteria in the mid logarithmic phase of growth, which were then suspended in Mueller Hinton Broth (MHB) to a final density of ~ 1106 Colony-Forming Units per ml (CFU·ml-1). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ponericin G1 against S. aureus were determined using broth dilution method according to the Clinical and Laboratory Standards Institute.49 Briefly, AMP solution with an initial concentration of 2048 μg·ml-1 was diluted 11 times by means of twofold-dilution, then 1 ml of bacteria solution with density of 1105 CFU·ml-1 was added respectively into 1 ml of one of the above 12 AMP solutions. After incubation at 37 ºC for 24 h, the AMP concentration initially resulting in no visible turbidity was considered as MIC. Afterwards, the above AMP-treated bacterial solutions without visible turbidity



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were inoculated onto Mueller-Hinton agar (MHA) and cultured at 37 ºC for 24 h, and the concentration of AMP initially leading to no bacterial survival on MHA was regarded as MBC. The AMP-loaded nanotube films and those subjected to 30 and 49 days release in PBS were employed to evaluate the short-term and long-term antibacterial activity against S. aureus, respectively, together with bare Ti, D35-3.5, D70-3.5 and S140-12. The samples were placed centrally in 24-well culture plates, then each well was added 1 ml of bacterial solution with density of 1106 CFU·ml-1. After incubation at 37 ºC for the selected period of time, the planktonic survivals in the resultant solutions were counted according to the National Standard of China GB/T 4789.2 protocol, together with the blank well containing bacterial solution as a control. In parallel, the samples subjected to the above-mentioned bacteria incubation were rinsed three times with PBS, the detachment of bacteria adhered on the samples was conducted by ultrasonic vibration in 1 ml PBS for 5 min,10 and the obtained bacterial solutions were sampled to count the viable bacteria on the samples according to GB/T 4789.2 protocol. The antibacterial rates against planktonic bacteria in the solutions (Rp) were calculated as Rp (%) = (B-A)/B ×100%, where A and B are the number of viable bacteria in the solutions incubated with the samples and blank control, respectively; whereas the decrement rates against the bacteria adhered on the samples (Ra) were calculated as Ra (%) = (D-C)/D ×100%, where C and D are the number of viable bacteria on the nanotube films and Ti, respectively. Each test was repeated four times. Furthermore, the samples following incubation were rinsed three times with PBS, fixed in 2.5%


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glutaraldehyde at 4 ºC, dehydrated in ethanol and dried, then coated with platinum for SEM observation of bacterial morphology. In parallel to the long-term antibacterial assay, the release amounts of AMP from the 30 and 49 days immersed samples into bacteria incubation medium were monitored as follows: the samples were immersed in MHB for 024 h at 37 ºC, then the resultant solutions at the selected time points were collected and measured at 279 nm using a spectrophotometer (Thermo Scientific Multiskan GO) to determine AMP amounts. 2.6. Cytocompatibility of the AMP-loaded TiO2 nanotubes. Human fetal osteoblastic cell line, hFOB1.19, was purchased from the Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences (Shanghai, China). The cells were inoculated into complete culture medium, consisting of Dulbecco’s modified Eagle medium (DMEM; HyClone, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, USA), 0.3 mg·ml-1 Geneticine418 (Sigma, USA), 0.5 mM sodium-pyruvate (Sigma, USA) and 1.2 mg·ml-1 Na2CO3, and incubated in a humidified atmosphere incubator supplemented with 5% CO2 at 37 ºC. The complete culture medium was refreshed every 2 days. The samples were placed centrally in 24-well plates. hFOB1.19 cells were seeded on each sample at a density of 1 × 105 cells/well, and incubated for 1, 4 and 7 days. The viability of cells adhered on the samples was investigated using MTT assays. At each time point, the complete culture medium was removed from each well. The cell-adhered samples were washed thrice with PBS then transferred to new 24-well plates. In each well, 30 μl of MTT (Sigma, USA) solution (5 mg·ml-1 MTT in PBS)



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and 500 μl of complete culture medium were added and cultured at 37 ºC for 4 h. Then the medium was gently removed, followed by the addition of 200 μl dimethyl sulfoxide (DMSO; Sigma, USA) into each well, and oscillated for 10 min. Ultimately, 100 μl of the obtained DMSO solution from each well was transferred to new 96-well plates and the absorbance was measured at 490 nm. Each test was repeated four times. Live/dead viability/cytotoxicity kits (Invitrogen, Eugene, OR) were employed to identify viable and dead cells on the samples. After 1 and 4 days of culture, each of the cell-adhered samples was washed thrice with PBS, then incubated with 500 μl of PBS containing ethidium-homodimer-1 (4 μM) and calcein-AM (2 μM) at 37 ºC for 30 min. Epifluorescence images were collected on an Olympus BX52 microscope using a 10× lens. Following culture, the cell-adhered samples were washed with PBS, fixed with 2.5% glutaraldehyde at 4 ºC, dehydrated in ethanol and dried in vacuum, then coated with platinum for SEM observation of cell morphology. 2.7. Statistical analysis The data were analyzed using SPSS 14.0 software (SPSS, USA). A one-way ANOVA followed by a Student–Newman–Keuls posthoc test was used to determine the level of significance. P < 0.05 was considered to be significant and p < 0.01 was considered to be highly significant.

3. Results and discussion 3.1. Formation and features of the single- and dual-diameter TiO2 nanotube films Figure 1 shows schemas and SEM images of the nanotubular layers in S140-12, D35-3.5 and D70-3.5 films anodized with F− sedimentation procedure, the underlying


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were compact and F-free layers (Figure 2c as an example) bonding the nanotubes to Ti. All the nanotubes in the films were smooth and closely aligned with length of about 12 μm (Figure 1), and consisted of amorphous TiO2 (Figure S2, Supporting Information). In S140-12, the nanotubes were single-diameter structured with diameter of about 140 nm. In D35-3.5 and D70-3.5, the nanotubes were dual-diameter structured with 35 or 70 nm ones as nanocaps and the underlying 140 nm ones as nanoreservoirs. The nanocaps revealed lengths of 3.5±0.2 μm for D35-3.5 and 3.5±0.3 μm for D70-3.5, respectively, and the corresponding nanoreserviors had the residual lengths of 12 μm. Notably, the nanocaps and nanoreservoirs in each film were interconnected well as confirmed by the magnified images of their interfaces.

Figure 1. Schema, cross-sectional and surface SEM morphologies of TiO2 nanotube films: (a) S140-12, (b) D35-3.5 and (c) D70-3.5.

Based on the curves of acoustic output versus load and scratch images of the nanotube films (Figures 2b and S3a-c, Supporting Information), their adhesion strengths as characterized by Lc are shown in Figure 2a. All the single-/dual-diameter nanotube films anodized with F− sedimentation procedure exhibited a similar Lc value



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to that of D70-3.5 as high as 13.2±0.4 N (Figure 2b). In contrast, T-NT anodized with the same nanotubular formation procedure as D70-3.5 but without F− sedimentation procedure revealed Lc as low as 3.0±0.8 N, although it showed the same tubular structure as D70-3.5; moreover, T-NT easily peeled off from substrate during the preparation of cross-sectional sample for SEM observation, leaving dimples on substrate (Figure 2d).

Figure 2. (a) Critical loads of the single-/dual-diameter nanotube films, (b) representative curve of acoustic output versus load and scratch morphology (the insert) of D70-3.5, the cross-sectional morphologies of (c) D70-3.5 (inset showing elemental profile) and (d) T-NT.

Admittedly, the formation of nanotubes was mainly governed by the field-assisted oxidation and F− etching to TiO2, as described elsewhere.48 Field-assisted oxidation: Ti + 2H2O → TiO2 + 4H+ +4e−

(1)

F− etching: TiO2 + 6F− + 4H+ → [TiF6]2- + 2H2O

(2)

In the EG-based electrolyte supplemented with DEG, F− ions were found to have lower conductance and mobility than O2− ions due to the proton jump mechanism.32,50 At the low applied voltage (stage I in Table 1), the etching of F− to TiO2 could be slow down and gradually reached a balance with the field-assisted oxidation, leading to the generation of fine diameter nanotubues (nanocaps).36,48 However, with increasing the


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applied voltage to 80 V, the electric field was intensified and O2− ions from the electrolyte migrated to the TiO2/Ti interface at a higher speed than F− ions, causing the increase of active area for oxidation with oxidation frontier at the tip of nanocap bottoms.32,51 As nanotubes competed for growth, some oxidation range was deeper (abbr. strong tube) and the other was relatively shallower (abbr. weak tube).51 Strong tubes with higher oxidation rate produced more H+ ions (reaction (1)), resulting in the acceleration of F− etching to deepen the strong tube (reaction (2)). On the contrary, weak tubes generated less H+ ions, and their growth were preferentially terminated.51 Consequently, strong nanotubes kept on growing under the guidance of nanocap bottoms to occupy the neighboring space, leading to the formation of larger-diameter nanotubes,32,36,52 and thereby an inter-connected dual-diameter tubular structure. However, the reaction (2) formed [TiF6]2- ions were difficult to diffuse quickly into the viscous electrolyte under Coulombic attraction to the anode, owing to their large hydrodynamic radius and low mobility.32 Instead, they accumulated at the TiO2/Ti interface to form a water-soluble fluoride-rich layer and weakened adhesion,50 resulting in the separation of TiO2 nanotubes from Ti substrate at a low critical load, even during the preparation of cross-sectional sample for SEM observation (Figure 2d). Noticeably, the addition of F− sedimentation procedure significantly enhanced the adhesion strengths of the anodized nanotube films, as revealed by D70-3.5 vs T-NT, ascribing to the formation of a F-free and compact oxide layer to tightly bond the nanotubular layer to Ti substrate (Figure 2b). 3.2. Loading feature of AMP within the nanotube films and its in vitro release



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For the single-/dual-diameter nanotube films nominally loaded with 125 μg of ponericin G1, the loading efficiencies of AMP within the films were of no statistical difference (p=0.469) in the range of 64.9±12.372.6±1.1% (Figure 3a), independent on the nanotubular structure of the films in the present case. As detected by EDX on the cross-sections of the AMP-loaded nanotube films (Figure 3b-d), the characteristic element N from ponericin G1 was quite similar in content at the same distance from the nanotubular surfaces. Moreover, N contents were relatively lower at the regions adjacent to the surfaces than the underlying regions, where N content was essentially unchangeable with the distance away from the surface for each of the AMP-loaded nanotube films. This ascribed to the AMP loss during rinse for removing the residual AMP on surfaces. As shown in Figure 3c as an example, the AMP appeared as spherical particles to exist in the nanotubes of the films. The adhesion strengths of the AMP-loaded nanotube films were also examined, as shown in Figure S3e-f, revealing no obvious change in Lc values compared to the AMP-free ones.

Figure 3. (a) Loading efficiencies of AMP within the nanotube films; cross-sectional SEM images and EDX-detected N content profiles of the AMP-loaded nanotube films: (b) D35-3.5, (c) D70-3.5 and (d) S140-12, the amplified views of I- and II-marked square regions in (c) showing the morphologies of the trapped AMP (as marked by arrows) within the nanotubes.


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The release profiles of AMP from the AMP-loaded films were evaluated in PBS up to 60 days, as shown in Figure 4a, revealing a burst release stage within the initial 6 h followed by a slow and steady release from 6 h to 60 days, e.g., sustained release stage. D35-3.5-AMP, D70-3.5-AMP and S140-12-AMP, during the burst release stage, presented AMP cumulative release amounts of about 18.3±1.3, 20±1.2 and 26.7±3.3 μg, respectively; At days 30, about 56.4±0.3% (45.8±0.2 μg), 64.4±0.2% (54.8±0.7 μg) and 93.1±0.2% (77.5±2.5 μg) of AMP released out respectively from the aforementioned corresponding films. At days 42, almost all (97.8±2.1%) of AMP released out from S140-12-AMP; over which, however, the AMP loaded within the dual-diameter structured nanotube films still remained sustained release at least up to 60 days, and the release amount of AMP from D70-3.5-AMP was more than D35-3.5-AMP all the time. Given that the initial 6 h following surgery is a peak period of bacteria invasion, while localized rapid release of antimicrobial agents from implant at this stage and long lasting release for the prevention of potential infection are strongly desired.13,53 The release profiles of AMP presented by the dual-diameter structured nanotube films (Figure 4a) are more appreciated compared to the single-diameter structured one, owing to the slower and longer-term release efficacy of the former as schematically illustrated in Figure 4b and c. As known, drug release from nanotubes was mainly controlled by molecular diffusion.20 In the initial case of immersion of AMP-loaded nanotube films in PBS, there existed a high concentration gradient of AMP between the tube interior and the surrounding PBS, beneficial to fast release of AMP. On the



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other hand, the nanotube derived capillary force also affects drug diffusion and the inhibited efficacy is related to nanotube diameter, tending to increase with diameter refinement.18,20 Consequently, the diameter-refined nanocaps (such as D35-3.5-AMP vs D70-3.5-AMP) of dual-diameter structured nanotube films could act as strong barriers for delaying AMP diffusion to reduce burst release and slowing the sustained release from the nanoresevoirs, also as compared to the single-diameter structured one such as S140-12-AMP (Figure 4b and c).

Figure 4. (a) Cumulative release of AMP from nanotube films up to 60 days, inset showing the details within the period of 024 h. All the data are expressed as means ± SD (n = 3). Schematic diagrams showing AMP release from (b) dual- and (c) single- diameter structured nanotube films.

3.3. Antibacterial activity S.aureus is known to be a major strain for implant-associated infection, which caused infection rate is as high as 2025%.15,17,47 Against the strain, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ponericin G1 were herein examined as shown in Figure 5, to be approximately 8 and 32 μg·ml-1, respectively. While the AMP cumulative release amounts during initial 6 h from D35-3.5-AMP, D70-3.5-AMP and S140-12-AMP into 1 ml PBS reached about 18.3±1.326.7±3.3 μg (Figure 4), higher than MIC but lower than MBC, indicating that the AMP cumulative release amounts from the nanotube films were sufficient to promptly inhibit S.aureus growth, but insufficient to completely kill all the bacteria.


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Figure 5. Determination of (a) minimum inhibitory concentration and (b) minimum bactericidal concentration of ponericin G1 against S.aureus. Here A = 0, B = 0.5, C = 1, D = 2, E = 4, F = 8, G = 16, H = 32, I = 64, J = 128, K = 256, L = 512, and M = 1024 μg·ml-1.

Figure 6a shows the short-term antimicrobial activities of the AMP-loaded nanotube films against planktonic S. aureus in the samples-immersed solutions. At hours 6 and 24 of incubation, their antibacterial rates (Rp) against planktonic bacteria were all higher than 85% and no statistical difference in Rp was observed between D35-3.5-AMP and D70-3.5-AMP, while S140-12-AMP revealed a statistically higher Rp than D35-3.5-AMP and D70-3.5-AMP due to a larger amount of AMP releasing from the former. This result indicated a release-killing manner of the AMP-loaded films against bacteria, as given that AMP could penetrate into bacterial membrane, leading to the leakage of intracellular substance and eventual death of bacteria.15 Following immersion in PBS for 30 days, S140-12-AMP almost lose the antibacterial ability against planktonic bacteria (Figure 6b), owing to that AMP has released out essentially from the single-diameter carrier (Figure 4); however, D35-3.5-AMP and D70-3.5-AMP still kept Rp of 56.7±0.9% and 58.6±1.9%, although they revealed a further decrease in Rp to 47.8±1.1% and 50.9±2.1% at day 49, respectively (Figure 6b). Notably, D70-3.5-AMP seemed to show a slightly higher Rp than D35-3.5-AMP following immersion for 30 and 49 days, but no statistical difference could be drawn.



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This is because the cumulative release amounts of AMP within 24 h from the 30 days immersed D35-3.5-AMP and D70-3.5-AMP into bacteria incubation media were quite close at each of the selected time points and so did the 49 days immersed samples (Figure 6c), all lower than MIC. It is highlighted that the nanocaps-nanoreservoirs structured nanotube films could promise long-term prevention of potential infection by the controllable sustained release of AMP against planktonic bacteria.

Figure 6. Number of S. aureus colonies in the sample-immersed solutions after incubation with (a) the AMP-loaded nanotube films (nominal loading amount of 125 μg) for 6 and 24 h, respectively, and (b) the AMP-loaded nanotube films subjected to 30- and 49-day release of AMP for 24 h. The blank well containing S. aureus solution was served as the control in (a) and (b). (c) The cumulative release of AMP within 24 hours from nanotube films subjected to 30- and 49-day immersion in PBS. All the data are expressed as means ±SD (n = 4). **p < 0.01 compared with Ti, $ p < 0.05 and $$p < 0.01 compared with S140-12-AMP.

As a common source of infection, biofilm is known to form via bacteria adhesion, proliferation and subsequent peptidoglycan encapsulation on implant surfaces, while the critical step for preventing biofilm is to inhibit bacteria adhesion.53-55 To this end,


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the current antimicrobial material surface design fall into three strategies: (1) adhesion resistance (e.g. altering surface properties such as topography and hydrophobility to reduce adhesion of viable bacteria), (2) release-killing (e.g. biocide leaching to induce death either of planktonic bacteria or of adhered bacteria), and (3) contact-killing (e.g. to biochemically induce death of bacteria that have adhered to material surface).53-55 Herein, we firstly investigated the nanotopography effect on antibacterial ability of the anodized nanotube films. As shown in Figure 7a, D35-3.5, D70-3.5 and S140-12 showed a statistically lower number of adhered bacteria compared to Ti control at 6 and 24 h of incubation. Although the anodized films contained fluorine (F), which has been proven to possess antibacterial ability,56-58 the F contents of the films were quite similar either in the anodized state or in the state of subjecting to 30 and 49 days immersion in PBS (Table 3), suggesting that the difference in antibacterial ability of the films was contributed to their topographies, e.g., nanotubular diameters rather than F ions. It is therefore indicated an enhanced resistance of the anodized films against bacteria adhesion (at 6 h) and thus subsequent proliferation (at 24 h) with the decrease of nanotube diameter, appearing nanotopography-dependent, although the underlying mechanism remained unclear. Moreover, Figure 7a also reveals that the loading of AMP into the anodized nanotube films further improved their antibacterial ability. At 6 h, D35-3.5-AMP, D70-3.5-AMP and S140-12-AMP presented Ra (e.g. decrement rate against adhered bacteria compared to Ti) values of 88.7±0.4%, 79.8±1.7% and 83.2±0.1%, of which 47.9±2.1%, 51.6±1.8% and 67.5±1.9% ascribed to the killing efficacy of AMP, in accordance with the amounts of AMP released from the films, and



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the others were correspondingly contributed to the adhesion resistance derived by their topographies (e.g. nanotube diameters) , respectively. At 24 h of incubation, all the tested samples revealed increased amounts of adhered bacteria compared to the corresponding ones at 6 h. This may be partially owing to bacteria proliferation and on the other hand due to the perspective that the dead bacteria derived proteins and DNA are favorable to the subsequent bacteria adhesion.59,60 However, D35-3.5-AMP, D70-3.5-AMP and S140-12-AMP depicted Ra values of 78.1±2.0%, 71.8±0.8% and 74.5±1.7%, respectively, greater than those derived by the corresponding AMP-free films (59.2±1.6%, 53.5±2.0% and 47.7±0.7%, respectively), further indicating the killing efficiency of AMP against bacteria and suggesting that the difference in Ra values of the AMP-loaded films is given raise to the synthetic effects of topography derived adhesion resistance and AMP derived killing efficacy. Figure 7b shows the long-term antibacterial activities of the AMP-loaded films against adhered bacteria, depicting a similar trend to that against planktonic bacteria with prolonging the time of immersion in PBS to 49 days. Notably, even immersed for 49 days, D35-3.5-AMP, D70-3.5-AMP and S140-12-AMP still kept Ra values of 65.6±0.9%, 61.8±1.1% and 47.9±1.7%, respectively. It is indicated that the AMP-loaded nanocaps/nanoreservoirs structured nanotube films could provide higher long-term antibacterial ability against adhered bacteria compare to the single-diameter structured one.


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Figure 7. Number of S. aureus colonies on the sample surfaces after incubation with (a) the AMP-loaded nanotube films (nominal loading amount of 125 μg) for 6 and 24 h, respectively, and (b) the AMP-loaded nanotube films subjected to 30- and 49-day release of AMP for 24 h. SEM images of S.aureus colonies on the as-received samples after (c) 6 and (d) 24 h of incubation. Bare Ti, D35-3.5, D70-3.5 and S140-12 were served as the control surfaces in (a)(d). All the data are expressed as means ± SD (n = 4). **p < 0.01 compared with Ti, ++p < 0.01 compared with ▼▼ S140-12, %%p < 0.01 compared with D70-3.5, p < 0.01 compared with D35-3.5, $p < 0.05 and ★★ $$ p < 0.01 compared with S140-12-AMP, p < 0.01 compared with D70-3.5-AMP. Table 3. Fluorine Content in Single-/Dual-Diameter Structured Nanotube Films after Immersion in PBS for Different Time. All the data are expressed as means ±SD (n = 3). Immersion Time (days)

F Content (at.%) D35-3.5

D70-3.5

S140-12

0 30 49

7.19±0.52 6.83±0.31 6.36±0.30

7.96±0.42 6.97±0.30 6.49±0.26

8.08±0.19 7.37±0.35 7.18±0.57

Figures 7c and 7d show the SEM images of S. aureus on Ti, AMP-unloaded and loaded nanotube films at 6 and 24 hours of incubation, respectively. Besides the samples derived variation in the number of adhered bacteria in line with that depicted by viable bacteria counting method (Figure 7a), noticeably, the samples also gave raise to the change in morphology of adhered bacteria. S. aureus appeared ball-shaped with diameter of about 300400 nm on Ti, slightly irregular ball shaped on AMP-unloaded nanotube films and seriously sunk on AMP-loaded ones at hour 6, and the sunk extent was further aggravated at 24 h. It is indicated that AMP disrupted the



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integrity of bacterial membrane, leading to the leakage of intracellular substance and eventually losing ball-shaped morphology and viability. 3.4. Cytocompatibility of the AMP loaded nanotube films Figure 8a shows the MTT assessed mitochondrial activity of osteoblasts on the AMP unloaded and loaded nanotube films together with Ti, while Figure 8b displays the number of live/dead cells on the samples assessed by counting based on Figure 8c depicted fluorescent observations of stained cells. At each time point of days 1, 4 and 7, the anodized films gave raise to a nanotubular diameter dependence of cell viability and number, following the order: D35-3.5 > D70-3.5 > S140-12 > Ti, and the number of cells on the samples increased with prolonging incubation time. It is indicated that the cell adhesion and proliferation on the anodized films enhanced with decreasing nanotubue diameter. As known, cell attachment to a material surface was mediated by focal adhesion, whose formation involved the clustering of transmembrane integrins at nanoscale.61 Given that nanotubes with diameter of 15-30 nm could provide more suitable length scale for integrin clustering and focal contact formation compared to those with diameter larger than 50 nm.44,45 Thus, the decrease in diameter of our caped nanotubes from 140 to 35 nm resulted in enhanced adhesion and proliferation of cells. Moving to the AMP loaded nanotube films, they gave raise to more or less decrease in the viability and number of osteoblasts compared to the corresponding AMP-free films at each time point (Figure 8), dependent on their nanocap diameters and consequent AMP release behaviors as given that ponericin G1 could be of potential to cause cytotoxicity.62 In details, D35-3.5-AMP exhibited the number of


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viable cells similar to Ti, more than D70-3.5-AMP, and much pronounced than S140-12-AMP, indicating that D35-3.5-AMP was cytocompatible and more beneficial to adhesion and proliferation of cells compared to the other AMP loaded films. On the other hand, the numbers of dead cells on the films and Ti at each time point of 1 and 4 days were in the following order: S140-12-AMP > D70-3.5-AMP > D35-3.5-AMP > S140-12 > D70-3.5 ≈ Ti > D35-3.5, and tended to increase with culture time from 1 to 4 days (the inserted colorimetric schema in Figure 8b and visual images in Figure 8c). Figure 8d reveals the morphologies of cells on the AMP unloaded and loaded films. At each incubation time point of 1 and 4 days , D35-3.5 promoted cell spread dramatically compared to D70-3.5, and more pronounced compared to S140-12. However, cells spread worse on the AMP loaded films relative to the corresponding AMP-free ones. Also as depicted by the magnified images, cells on D35-3.5-AMP and the AMP-free films presented lamellipodia and filopodia to stretch out and tightly anchor to the films. However, the curled lamellipodia and filopodia were observed on S140-12-AMP, suggesting an unstable bond of cells to the surface. Summarily, in the case of AMP nominal loading amount of 125 μg, D35-3.5-AMP not only exhibited significant short-term and long-term antibacterial activity against both planktonic and adhered bacteria, but also presented cytocompatibility comparative to Ti but higher than D70-3.5-AMP and S140-12-AMP.



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Figure 8. (a) MTT assays of cells incubated on the nanotube films unloaded and loaded with nominal amount of 125 μg AMP for 1, 4 and 7 days. (b) The number of live and dead cells on the samples. The top inset in (b) shows the number of dead cells on the samples after culture for 4 d. (c) Fluorescent images (live cells were stained green while dead cells were stained red, scale bar is 200 μm) and (d) SEM images of cells incubated on as-received samples for 1 and 4 days. Data are presented as the mean ±SD, n = 4; *p < 0.05 and **p < 0.01 compared with Ti, +p < 0.05 and ++p < ▼ ▼▼ 0.01 compared with S140-12, %%p < 0.01 compared with D70-3.5, p < 0.05 and p < 0.01 ★ ★ compared with D35-3.5, $p < 0.05 and $$p < 0.01 compared with S140-12-AMP, p < 0.05 and ★ p < 0.01 compared with D70-3.5-AMP.

3.5. Effect of AMP nominal loading amount on antibacterial activity and cytocompatibility We also investigated the effect of ponericin G1 nominal loading amount of 200 μg on the loading efficiencies, release behaviors, antibacterial activity and osteoblast viability of the AMP-loaded nanotube films. In this case, the loading efficiencies of AMP within the single- and dual-diameter nanotubes were of no statistical difference


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(p=0.096) in the range of 59.6±4.5%67.8±0.8% (Figure S4a, Supporting Information), independent on the tubular structure, but revealed a decrease compared to the case of AMP nominal loading amount of 125 μg. In details of both the cases, S140-12-AMP revealed the reduction of 10.5% in loading efficiency, while D35-3.5 and D70-3.5 depicted the reduction of 4.2% and 4.8%, respectively. Generally, with increasing the nominal loading amount, the pre-entrapped AMP could occupy the loading space of the nanotubes and obstruct the subsequent AMP entrance, leading to more AMP on the nanotube surfaces and thereby lower loading efficiency. However, the measured loading amounts of AMP within the single- and dual-diameter nanotubes were higher in the case of nominal loading amount of 200 μg than in the case of 125 μg. Each kind of the nanotube films nominally loaded with 200 μg of AMP showed a similar AMP release trend to that depicted by the 125 μg AMP nominally loaded one, but an increased AMP cumulative release amount compared to the later at each release time point (Figure S4b, Supporting Information). In details, the cumulative release amounts from D35-3.5-AMP and D70-3.5-AMP at day 30 were 89.9±0.3 and 106.6±0.2 μg, respectively, in the case of nominal loading amount of 200 μg, higher than those in the case of 125 μg. Consequently, they revealed higher short-term and long-term antibacterial activities against both planktonic and adhered bacteria (Figure 9) compared to the 125 μg AMP nominally loaded ones. For example, the nanotube films nominally loaded with 200 μg of AMP gave raise to the Rp and Ra in the ranges of 99.7±0.1%99.8±0.1% and 88.9±0.6%94.1±0.2% at 6 h of incubation, and



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91.3±0.6%95.5±0.9% and 77.2±0.6%83.5±0.4% at 24 h, respectively, obviously higher than those revealed by the corresponding films nominally loaded with 125 μg of AMP. Notably, the improvement of long-term antibacterial activities is owing to the fact that the cumulative release amounts of AMP from the 200 μg AMP nominally loaded films subjected to immersion for 30 and 49 days were higher than those from the corresponding 125 μg nominally loaded ones at each of the selected time points within 24 h (Figure S5, Supporting Information). However, the 200 μg AMP nominally loaded films exhibited quite lower cytocompatibility as characterized by mitochondrial activity of osteoblasts (Figure 10) compared to the corresponding 125 μg nominally loaded. Given that ideal strategies should endow orthopaedic implants with long-lasting antibacterial activity and no or minimal toxicity for host cells and tissues (ideally enhance bone growth),63,64 the AMP nominal loading amount of 200 μg inside the single-/dual-diameter nanotube films was of overdose.

Figure 9. Number of S. aureus colonies in the sample-immersed solutions (a and b), and on the sample surfaces (c and d) after incubation with (a and c) the AMP-loaded nanotube films (nominal loading amount of 200 μg) for 6 and 24 h, respectively, and (b and d) the AMP-loaded nanotubes subjected to 30- and 49-day release of AMP for 24 h. The blank well containing S. aureus solution


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was served as the control in (a) and (b); bare Ti was served as the control surface in (c) and (d). All the data are expressed as means ± SD (n = 4). *p < 0.05 and **p < 0.01 compared with Ti, $$p < ★ ★★ 0.01 compared with S140-12-AMP, p < 0.05 and p < 0.01 compared with D70-3.5-AMP.

Figure 10. MTT assays of cells incubated on the nanotube films loaded with nominal amount of 200 μg AMP for 1 day. Data are presented as the mean ± SD, n = 4. **p < 0.01 compared with Ti, ★ $$ p < 0.01 compared with S140-12-AMP, p < 0.05 compared with D70-3.5-AMP.

As mentioned above, TiO2 nanotubes were good platforms for AMP delivery with the purpose of reducing bacteria infection. There are also other strategies of implant surface modification for antibacteria. For instance, Jiang et al. developed zwitterionic surfaces which effectively reduced bacteria adhesion and resisted biofilm formation by electrostatic or hydrogen bonding interactions;65,66 Zhang et al. reported slippery coatings and quaternary ammonium salt functionalized liquid-repellent coatings, which exhibited significantly antibacterial activity due to slippery surfaces or contact killing.67,68 Nevertheless, the cytocompatibility and long-term antibacterial activities of those coatings were not addressed. In the present study, the dual-diameter structure of D35-3.5-AMP not only presented cytocompatibility comparative to Ti, but also exhibited significant short- and long-term (up to 7 weeks) antibacterial activity against planktonic bacteria via release killing and against adhered bacteria via nanocaps derived adhesion resistance. This is actually ascribed to the dual-diameter structures which not only reduced the burst release amount by decreasing the nanocap diameters but also exhibited long-term (on the order of months) controllable and



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sustained AMP release. Moreover, the sustained release efficacies of AMP presented herein were better than those in previous studies, where the release of drug from the as-anodized TiO2 nanotubes last for only couple of hours to about 2 weeks.7,20-22 However, due to the complex interactions of transport mechanisms in vivo,13,69 the release kinetics of AMP from dual-diameter nanotubes would be quite different from that in vitro. Thus, further investigation need to be carried out in the future. Given that the AMP loaded on Ti alloy can still survive after sterilization by ethylene oxide for in vivo implantation,70 the dual-diameter structure of D35-3.5-AMP presents great potential for real-life clinical applications. 4. Conclusion Novel films, comprised dual-diameter TiO2 nanotubes with the inner compact and F-free oxide to tightly bond to Ti substrates, were fabricated by voltage-increased anodization with F− sedimentation procedure. The nanotubes were closely aligned and structured with upper 35 and 70 nm diametric tubes as nanocaps, respectively, and the underlying 140 nm diametric tubes as nanoreservoirs which were interconnected well with the nanocaps. Employing vacuum-assisted physisorption, a kind of natural AMP (ponericin G1) was successfully loaded into the dual-diameter nanotube films and the anodized single-diameter nanotube film with tubular diameter of 140 nm, appearing as spherical particles to exist in the nanotubes of the films. The loaded films were of no statistical difference in the loading efficiency of AMP, and revealed burst release within 6 h followed by steady release of AMP. At day 42 of immersion in PBS, almost all of AMP released out from the single-diameter nanotube film; over which, however,


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the dual-diameter nanotube films loading AMP still remained sustained release at least up to 60 days, and the sustained efficacy was enhanced with decreasing the diameter of nanocaps. In the case of AMP nominal loading amount of 125 μg, the resultant 35 nm caped dual-diameter nanotube film exhibited significant short- and long-term (up to 49 days) antibacterial activity not only against planktonic bacteria, which ascribed to the release-killing efficacy of AMP, but also against adhered bacteria, which were contributed to the AMP-derived killing efficacy and the nanocaps derived adhesion resistance. Moreover, this loaded film presented cytocompatibility comparative to Ti but higher than the other AMP-loaded films. Increasing nominal loading amount of AMP to 200 μg improved antibacterial activity, but caused obvious cytotoxicity of the loaded films. The finely caped dual-diameter nanotubes show a great potential as a sustained release platform on Ti orthopedic implant for local antimicrobial delivery.

ASSOCIATED CONTENT

Supporting Information Broad spectra of ponericin G1 solutions with different concentrations and the obtained calibration curve; XRD patterns of D35-3.5, D70-3.5 and S140-12 before and after annealing at 400 °C for 2 h in vacuum; representative curves of acoustic output versus load and corresponding scratch morphologies of D35-3.5, S140-12 and T-NT; loading efficiencies of AMP within the nanotube films (nominal loading amount of 200 μg) and cumulative release of AMP from nanotube films up to 60 days; cumulative release of AMP within 24 hours from nanotube films subjected to 30- and



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49-day immersion in PBS. AUTHOR INFORMATION Corresponding Author *Tel.: +86 02982665580. Fax: +86 02982663453. Email: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS

We appreciate the National Natural Science Foundation of China (NSFC; Grant No. 51631007 and 51371137), National Program on Key Basic Research Project of China (Grant No. 2012CB619103) and NSFC (Grant No. 51201129) for financially supporting this work. REFERENCES (1) Geetha, M.; Singh, A. K.; Asokamani, R.; Gogia, A. K. Ti Based Biomaterials, the Ultimate Choice for Orthopaedic Implants - A Review. Prog. Mater. Sci. 2009, 54, 397−425. (2) Peng, W.; Grainger, W. D. Drug/Device Combinations for Local Drug Therapies and Infection Prophylaxis. Biomaterials 2006, 27, 2450−2467. (3) Narayanan, R.; Kwon, T. Y.; Kim, K. H. TiO2 Nanotubes from Stirred Glycerol/NH4F Electrolyte: Roughness, Wetting Behavior and Adhesion for Implant Applications. Mater. Chem. Phys. 2009, 117, 460−464. (4) Tan, A. W.; Pingguan-Murphy, B.; Ahmad, R.; Akbar, S. A. Review of Titania Nanotubes: Fabrication and Cellular Response. Ceram. Int. 2012, 38, 4421−4435.


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Table of Contents



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Figure 2. (a) Critical loads of the single-/dual-diameter nanotube films, (b) representative curve of acoustic output versus load and scratch morphology (the insert) of D70-3.5, the cross-sectional morphologies of (c) D70-3.5 (inset showing elemental profile) and (d) T-NT. 97x67mm (300 x 300 DPI)



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Figure 3. (a) Loading efficiencies of AMP within the nanotube films; cross-sectional SEM images and EDXdetected N content profiles of the AMP-loaded nanotube films: (b) D35-3.5, (c) D70-3.5 and (d) S140-12, the amplified views of I- and II-marked square regions in (c) showing the morphologies of the trapped AMP (as marked by arrows) within the nanotubes. 73x67mm (300 x 300 DPI)


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Figure 4. (a) Cumulative release of AMP from nanotube films up to 60 days, inset showing the details within the period of 0∼24 h. All the data are expressed as means ± SD (n = 3). Schematic diagrams showing AMP release from (b) dual- and (c) single- diameter structured nanotube films. 50x18mm (300 x 300 DPI)



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Figure 6. Number of S. aureus colonies in the sample-immersed solutions after incubation with (a) the AMPloaded nanotube films (nominal loading amount of 125 µg) for 6 and 24 h, respectively, and (b) the AMPloaded nanotube films subjected to 30- and 49-day release of AMP for 24 h. The blank well containing S. aureus solution was served as the control in (a) and (b). (c) The cumulative release of AMP within 24 hours from nanotube films subjected to 30- and 49-day immersion in PBS. All the data are expressed as means ± SD (n = 4). **p < 0.01 compared with Ti, $p < 0.05 and $$p < 0.01 compared with S140-12-AMP. 129x279mm (600 x 600 DPI)



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Figure 7. Number of S. aureus colonies on the sample surfaces after incubation with (a) the AMP-loaded nanotube films (nominal loading amount of 125 µg) for 6 and 24 h, respectively, and (b) the AMP-loaded nanotube films subjected to 30- and 49-day release of AMP for 24 h. SEM images of S.aureus colonies on the as-received samples after (c) 6 and (d) 24 h of incubation. Bare Ti, D35-3.5, D70-3.5 and S140-12 were served as the control surfaces in (a)∼(d). All the data are expressed as means ± SD (n = 4). **p < 0.01 compared with Ti, ++p < 0.01 compared with S140-12, %%p < 0.01 compared with D70-3.5, ▼▼p < 0.01 compared with D35-3.5, $p < 0.05 and $$p < 0.01 compared with S140-12-AMP, ★★p < 0.01 compared with D70-3.5-AMP. 89x57mm (300 x 300 DPI)


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Figure 8. (a) MTT assays of cells incubated on the nanotube films unloaded and loaded with nominal amount of 125 µg AMP for 1, 4 and 7 days. (b) The number of live and dead cells on the samples. The top inset in (b) shows the number of dead cells on the samples after culture for 4 d. (c) Fluorescent images (live cells were stained green while dead cells were stained red, scale bar is 200 µm) and (d) SEM images of cells incubated on as-received samples for 1 and 4 days. Data are presented as the mean ± SD, n = 4; *p < 0.05 and **p < 0.01 compared with Ti, +p < 0.05 and ++p < 0.01 compared with S140-12, %%p < 0.01 compared with D70-3.5, ▼p < 0.05 and ▼▼p < 0.01 compared with D35-3.5, $p < 0.05 and $$p < 0.01 compared with S140-12-AMP, ★p < 0.05 and ★★p < 0.01 compared with D70-3.5-AMP. 142x145mm (300 x 300 DPI)



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Figure 9. Number of S. aureus colonies in the sample-immersed solutions (a and b), and on the sample surfaces (c and d) after incubation with (a and c) the AMP-loaded nanotube films (nominal loading amount of 200 µg) for 6 and 24 h, respectively, and (b and d) the AMP-loaded nanotubes subjected to 30- and 49day release of AMP for 24 h. The blank well containing S. aureus solution was served as the control in (a) and (b); bare Ti was served as the control surface in (c) and (d). All the data are expressed as means ± SD (n = 4). *p < 0.05 and **p < 0.01 compared with Ti, $$p < 0.01 compared with S140-12-AMP, ★p < 0.05 and ★★p < 0.01 compared with D70-3.5-AMP. 106x80mm (600 x 600 DPI)


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Figure 10. MTT assays of cells incubated on the nanotube films loaded with nominal amount of 200 µg AMP for 1 day. Data are presented as the mean ± SD, n = 4. **p < 0.01 compared with Ti, $$p < 0.01 compared with S140-12-AMP, ★p < 0.05 compared with D70-3.5-AMP. 42x30mm (600 x 600 DPI)



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Enhancement in Sustained Release of Antimicrobial Peptide from

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