�ntibacterial Pro�erties o� Titanate Nano�ber Thin Films Formed on a Titanium Plate

A sodium titanate nano�ber thin �lm and a silver nanoparticle/silver titanate nano�ber thin �lm formed on the surface of a titanium plate exhibited strong antibacterial activities against methicillin-resistant Staphylococcus aureus, which is one of the major bacteria causing in-hospital infections. Exposure of the sodium titanate nano�ber thin �lm to ultraviolet rays generated a high antibacterial activity due to photocatalysis and the sodium titanate nano�ber thin �lm immediately aer its synthesis possessed a high antibacterial activity even without exposure to ultraviolet rays. Elution of silver from the silver nanoparticle/silver titanate nano�ber thin �lm caused by the silver ion exchange reaction was considered to contribute substantially to the strong antibacterial activity. e titanate nano�ber thin �lms adhered �rmly to titanium. erefore, these titanate nano�ber thin �lm/titanium composites will be extremely useful as implant materials that have excellent antibacterial activities.

Our group found that a sodium titanate nanotube thin �lm could be formed on the surface of a titanium plate aer hydrothermal treatment of a titanium plate in a 10 mol/L NaOH solution, followed by a �xing treatment [23].Performing a silver ion exchange treatment on the formed thin �lm using a silver acetate solution transformed the sodium titanate nanotube thin �lm into a silver nanoparticle/silver titanate nanotube thin �lm.e obtained thin �lm possessed an excellent antibacterial activity and apatite forming ability [24,25].
In the present paper, we introduce a sodium titanate nano�ber thin �lm.e sodium titanate nano�ber thin �lm was obtained using a 4 mol/L NaOH solution instead of a 10 mol/L NaOH solution.In the synthesis of the sodium titanate nanotube thin �lm, when the sample was washed with water to remove excess NaOH immediately aer the hydrothermal treatment, the nanotube thin �lm was detached from the titanium plate.erefore, �xing treatment was re�uired.On the other hand, the sodium titanate nano�ber thin �lm �rmly adhered to the surface of a titanium plate immediately aer performing the hydrothermal treatment and therefore required no special �xing treatment.e use of titanate thin �lm/titanium composites as biocompatible materials requires �rm adherence between the thin �lm and titanium.erefore, the nano�ber thin �lm, which has excellent adherence to titanium, appears to be a promising biocompatible material.However, only a single study [4] has investigated the antibacterial activity against Escherichia coli of titanate nano�ber thin �lms due to photocatalysis.Further detail is still needed regarding antibacterial activity.In the present study, a sodium titanate nano�ber thin �lm was formed on the surface of a titanium plate by hydrothermal treatment of the plate in a NaOH solution.e obtained thin �lm was then transformed into a silver nanoparticle/silver titanate nano�ber thin �lm by soaking it in a silver acetate solution.Methicillin-resistant Staphylococcus aureus (MRSA), which is one of the major bacteria causing inhospital infections, was then used to evaluate the antibacterial activity of the sodium titanate nano�ber thin �lm due to photocatalysis and the antibacterial activity of the silver nanoparticle/silver titanate nano�ber thin �lm due to elution of silver ions.

2.�. Syntheses of a So�ium Titanate Nano�ber in Film an� a Silver
Nanoparticle/Silver Titanate Nano�ber in Film.A titanium plate (20 × 20 × 2 mm) was reacted hydrothermally with 20 mL of a 4 mol/L NaOH solution at 160 ∘ C for 20 h.Aer the hydrothermal treatment, the obtained sample was repeatedly washed with water, resulting in a titanium plate coated with a sodium titanate nano�ber thin �lm.e plate was then soaked in 12 mL of a 0.05 mol/L silver acetate solution for 3 h at 40 ∘ C, repeatedly washed with distilled water, and dried in a dark place to transform the sodium titanate nano�ber thin �lm into a silver nanoparticle/silver titanate nano�ber thin �lm.e antibacterial activity was evaluated by synthesizing a sodium titanate nano�ber thin �lm and a silver nanoparticle/silver titanate nano�ber thin �lm on a titanium plate (50 × 50 × 2 mm) using solutions with the same concentration but different volumes.Transmission electron microscopy (TEM) was performed with a Hitachi H-800MU instrument and a JEOL JEM-1210 instrument.A sample for TEM observation was prepared by sprinkling powders obtained by scaling the thin �lm onto a standard TEM grid.e acceleration voltage for TEM observation was 120 kV.Scanning electron microscopy (SEM) was performed with a Hitachi S-3000N.Energy-dispersive X-ray microanalysis (EDX) was performed with an EDAX Genesis 2000 instrument.X-ray diffraction (XRD) measurements were performed with a Shimadzu XRD-6100 instrument using Cu K radiation.

Antibacterial Activity
Evaluation of the in Film.e antibacterial activity was evaluated using partially modi�ed Japanese Industrial Standard test (JIS Z2801).e samples were disinfected with alcohol for 10 min and dried in air at 25 ∘ C for 18 h.A sterilized polyethylene �lm was used as a blank sample.e environment of infection in an actual living body was mimicked by using a inactivated bovine serum as the solvent for the bacterial suspension, which reproduced eutrophic conditions, and the antibacterial activity was evaluated against MRSA that possessed the bio�lm formation gene.
A 0.2 mL volume of the bacterial suspension was dropped onto the sample plate (50 × 50 × 2 mm), a 40 × 40 mm polyethylene �lm (Elmex Corp.) was placed on the plate, and the sample plate was cultured at 37 ∘ C for 24 h.Subsequently, the number of viable bacteria was counted.e same experiment was partially performed using a different sample plate (20 × 20 × 2 mm), a different quantity of the bacterial suspension, and a different �lm size.Each of the thin �lms (the sodium titanate nano�ber thin �lm, the silver nanoparticle/silver titanate nano�ber thin �lm, and the sterilized polyethylene �lm) was evaluated three times, and the average number of viable bacteria was calculated.e average number of viable bacteria was used to calculate the antibacterial activity value.e antibacterial activity value () for the sample was calculated as follows: Here, , , and  are the average viable MRSA counts just aer inoculation, aer 24 h for a blank and aer 24 h for a sample, respectively.e antibacterial characteristics due to ultraviolet (UV) irradiation were evaluated using a UV irradiation apparatus that was vertically adjusted to set the illumination as 52−92 Lux and the intensity as 133−236 w/cm 2 .e UV irradiation was continued for 24 h.e sample plate was disinfected with alcohol and washed with water between each evaluation.

Elution of Silver from the Silver Nanoparticle/Silver
Titanate Nano�ber in Film.e silver nanoparticle/silver titanate nano�ber thin �lm formed on a titanium plate (20 × 20 × 2 mm) was then soaked in 15 mL of a fetal bovine serum solution at 37 ∘ C, and then le to stand for 24 h.Fetal bovine serum was used to mimic MRSA environment in order to study the elution behavior of silver [24].e eluate was collected, centrifuged, and �ltrated through a 0.22 m �lter.e Ag concentration in the eluate was measured by inductively coupled plasma (ICP) mass spectroscopy performed with a Seiko SPQ-8000.is experiment was repeated 10 times, and the Ag concentration in the eluate was measured each time.exfoliated from the titanium plate when the thin �lm was washed with a large amount of water immediately aer the hydrothermal treatment [23].In contrast, no exfoliation of the thin �lm was observed in Figure 1(a) (inset); that is, the thin �lm �rmly adhered to the titanium plate.SE� image (Figure 1(a)) con�rmed that the thin �lm was composed of nano�bers.�eng and Chen [3], Dong et al. [4], and Chi et al. [26] also reported formation of similar one dimensional sodium titanate thin �lms, such as sodium titanate nanowire thin �lms [3,4,26] and sodium titanate nanobelt thin �lm [26], although they used different concentrations of NaOH solution at different reaction temperatures.e concentration of the NaOH solution and the reaction temperature were considered to have substantial effects on the structure and the rate of formation of the precursor of sodium titanate and on the morphology of sodium titanate that was �nally formed.TE� image (Figure 1(b)) also revealed that the thin �lm was composed of nano�bers with widths between tens of nm and a little more than 100 nm, and lengths of several m.Cross sections of the thin �lm indicated a �lm thickness of approximately 6 m.e X�D pattern of the thin �lm (Figure 2(a)) showed diffraction peaks attributable to titanate with a layered structure.EDX analysis indicated a molar ratio of Na : Ti : O = 1 : 3.04 : 6.08.Sodium titanates with various compositions have been reported previously.Among the reported sodium titanates, the EDX data from the present study most closely agreed with a molar ratio of Na 2 Ti 6 O 13 .erefore, a Na 2 Ti 6 O 13 nano�ber thin �lm was considered to have formed in the present study.Although their synthesis conditions were different, Peng and Chen [3] reported the synthesis of a Na 2 Ti 6 O 13 nano�ber thin �lm.Chi et al. [26] also suggested that a Na 2 Ti 6 O 13 nano�ber thin �lm had been synthesized.Figure 1(c) (inset) shows the thin �lm �rmly adhered to the titanium plate following the silver ion exchange treatment.e SEM image in Figure 1(c) shows that no large morphological changes at the micrometer level occurred following the silver ion exchange treatment.However, the TEM image (Figure 1(d)) shows the presence of many nanoparticles, between several nm and a little more than 10 nm in size, deposited on the surfaces of nano�bers.Figure 1(e), an enlarged image of Figure 1(d), shows that angular polyhedral nanoparticles appear to have formed.ese nanoparticles were considered to be composed of silver, and were thought to be deposited due to photocatalysis of titanate.As mentioned below, sodium titanate nano�bers possess a photocatalytic capability.Silver ions in silver titanate were reduced to silver by electrons excited by light irradiation.e formed silver, which was electrically neutral, could not remain within the titanate nano�ber, and silver nanoparticles formed by shiing and aggregating on the surface of the nano�ber.�hen the thin �lm was continuously irradiated with light aer performing a silver ion exchange treatment, the color of the sample changed to black.However, when the thin �lm was covered with aluminum foil and stored aer performing a silver ion exchange treatment, the color of the sample changed very little.erefore, the formation of silver nanoparticles was clearly a result of light irradiation, and the mechanism suggested above appeared to explain the formation of these nanoparticles.Since the surfaces of the formed silver nanoparticles were not protected with surfactants or similar compounds, these surfaces might be oxidized or hydroxylated in the solution.e EDX analysis revealed Ag but no Na in the sample aer the silver ion exchange treatment, indicating that an exchange of Na + ions for Ag + ions had taken place in the sodium titanate nano�bers.e molar ratio of Ag/Ti was 0.63�, which was larger than the original Na/Ti = 0.329 in the sample before the silver ion exchange treatment.e reasons for this were probably that Ag + ions were inserted into titanate following the ion-exchange reaction and silver nanoparticles were deposited onto the surfaces of the nano�bers.

3.�. Synthesis of a So�ium Titanate Nano�ber in Film an� Its Transformation into a Silver Nanoparticle/Silver Titanate Nano�ber in Film by a Silver Ion E�chan�e Treatment
e XRD pattern of the sample aer the silver ion exchange treatment (Figure 2(b)) showed the disappearance of the diffraction peak at 2   ∘ that was attributed to the sodium titanate layered structure observed in the sample before the silver ion exchange treatment.e diffraction peaks near 2  26 ∘ and 222 ∘ attributed to the crystal structure of sodium titanate also became indistinct, while a broad diffraction peak with high intensity appeared near 2  23 ∘ , and the intensity of a diffraction peak near 2  3 ∘ increased.ese results indicated that the exchange of Na + ions with Ag + ions that took place in the sodium titanate nano�bers changed the crystal structure of titanate from the layered structure to a three-dimensional structure, while maintaining the shape of nano�ber, and conse�uently, silver titanate nano�bers were formed.A change in the crystal structure was also observed following the silver ion exchange treatment due to the transformation of the sodium titanate nanotube into the silver titanate nanotube [24,27].Diffraction peaks attributed to silver were not observed.e reasons for this were considered to be that the diameter of the silver nanoparticles and the amounts of deposited silver nanoparticles were too small to detect. 1 shows the results of evaluation of the sodium titanate nano�ber thin �lm for antibacterial activity due to photocatalysis.e �rst evaluation under UV irradiation showed that 3 × 1 5 CFU/sample viable bacteria were present immediately aer inoculation of MRSA.At 24 h aer the inoculation, the number of viable bacteria increased to 66 × 1  CFU/sample on the blank sample but this number decreased to 2 × 1 2 CFU/sample on the sodium titanate nano�ber thin �lm.ese results indicated that MRSA was not killed by UV irradiation alone but was killed by exposing the sodium titanate nano�ber thin �lm to UV rays.e antibacterial activity value of the sodium titanate nano�ber thin �lm was calculated to be as high as 5.9.In general, an antibacterial activity value of 2 or higher is considered to represent antibacterial activity.erefore, the sodium titanate nano�ber thin �lm possessed a high antibacterial activity under UV irradiation.e antibacterial activity of the sodium titanate nano�ber thin �lm was also evaluated without exposure to UV rays (i.e., in the dark).Table 1 shows a markedly high antibacterial activity value of 5.8.e same sample was repeatedly evaluated for antibacterial activity with UV exposure and showed antibacterial activity values as high as 7.7 and 6.6 for the second and third evaluations, respectively.e antibacterial activity values without UV exposure, for the second and third evaluations, were as low as 1.0 and 0.3, respectively.erefore, the antibacterial activity began to decline at the second evaluation and aerward.In summary: (1) exposure of the sodium titanate nano�ber thin �lm to UV rays generated a high antibacterial activity due to photocatalysis; and (2) the sodium titanate nano�ber thin �lm immediately aer its synthesis possessed a high antibacterial activity even without exposure to UV rays.Dong et al. [4] previously reported the antibacterial activity of a titanate nano�ber thin �lm due to photocatalysis.However, the composition of the thin �lm was not documented and only the antibacterial activity against gram-negative Escherichia coli was investigated.In the present study, the composition of sodium titanate was �rst elucidated and its antibacterial activity was evaluated against gram-positive MRSA, which actually causes inhospital infections.erefore, the present study adds new information to the results reported by Dong et al. [4] and provides a more in-depth understanding of the antibacterial activity of the titanate nano�ber thin �lm due to photocatalysis.

�.�. Anti�acte�ial Acti�ity o� the Sodium �itanate �ano��e� in Film against MRSA due to Photocatalysis. Table
Our group has previously reported that the antibacterial activity value of a sodium titanate nanotube thin �lm without exposure to UV rays was 1.7, and that of a sodium titanate thin �lm with a porous network structure was 1.8 [24].A sodium titanate nanosheet thin �lm was hydrothermally synthesized using a 1 mol/L NaOH solution at 160 ∘ C for 20 h.e antibacterial activity value of this synthesized thin �lm was 2.1.ese results indicated that the sodium titanate nano�ber thin �lm alone had a markedly high antibacterial activity.As shown in Table 2, the antibacterial activity values of the sodium titanate nano�ber thin �lm against gramnegative E. coli and gram-positive Staphylococcus aureus, evaluated without UV exposure, were 5.5 and 4.7, respectively.is indicated that the sodium titanate nano�ber thin �lm had high antibacterial activity against different bacteria.e reproducibility of the antibacterial activity evaluation against MRSA, without UV exposure, was con�rmed by investigating the antibacterial activities of sodium titanate nano�ber thin �lms that were synthesized following different reaction times.e SEM images in Figure 3 show that a nano�ber thin �lm was clearly formed as the reaction time proceeded.e XRD pattern in Figure 4 shows that the intensity of each diffraction peak or the crystallinity increased as the reaction time proceeded.Table 3 shows that the antibacterial activity values of thin �lms synthesi�ed with the reaction times of 1 h and 5 h were below 2. erefore, these �lms had no clear antibacterial activity.However, extending the reaction time to 12 h resulted in an antibacterial activity value of 3.1.When the reaction time was 20 h, the value was �.5.erefore, increases in the crystallinity of the nano�bers were accompanied by increases in the antibacterial activity of the thin �lm.At this moment, we cannot clearly explain the reason for these high antibacterial activity values without UV exposure.However, the following hypothesis can be proposed.Sodium titanate nanotubes, nano�bers, and nanosheets were composed of sheets consisting of titanate.A larger area of the edge of the titanate sheet was exposed in nano�bers than in nanotubes and in nanosheets.Since the surface and the edge of the titanate sheet possessed different properties, as reported by Matsumoto et al. [28], the edge of the titanate sheet might become a chemically active site.e chemically active site had a speci�c effect on bacteria, and consequently a high antibacterial activity was expressed.We are currently attempting to elucidate the detailed mechanism underlying the antibacterial activity.immediately aer inoculation of MRSA was 2.7 × 10 5 CFU/sample.At 24 h aer the inoculation, the number increased to 8.6 × 10 8 CFU/sample on the blank sample, but decreased to below 1 × 10 1 CFU/sample on the silver nanoparticle/silver titanate nano�ber thin �lm.Based on these values, the antibacterial activity value of the silver nanoparticle/silver titanate nano�ber thin �lm was calculated as 7.9, which indicated an extremely high antibacterial activity.

Antibacterial Activity of the
As shown in Figure 5, a large amount of silver (110 ppm) was eluted on the �rst day.erefore, the elution of silver was suggested as a cause of the high antibacterial activity.However, the amount of eluted silver had decreased to 77 ppm on the second day and this amount continued to decrease gradually over time, to 20 ppm by the 10th day.is elution curve was similar to that reported previously by us for a silver nanoparticle/silver titanate nanotube thin �lm [24].However, the amount of silver eluted on the �rst day was larger for the silver nanoparticle/silver titanate nano�ber thin �lm (110 ppm) than for the previously reported silver nanoparticle/silver titanate nanotube thin �lm (98 ppm) [24].e amount of eluted silver was considered to be greatly related to the amount of silver in the thin �lm.e amount of silver in the silver nanoparticle/silver titanate nano�ber thin �lm might differ from that in the silver nanoparticle/silver titanate nanotube thin �lm.However, since the amount of silver in nano�ber and nanotube thin �lms is extremely difficult to measure correctly and no effective measuring technique exists at present, the amount of silver in the thin �lms was not measured.Instead, we provide the minimum information on the amount of silver in these thin �lms by showing the results of EDX analysis of the thin �lm sample before the silver elution test.e molar ratio of Ag/Ti for the silver nanoparticle/silver titanate nano�ber thin �lm was 0.638, which was similar to 0.67 of that for the silver nanoparticle/silver titanate nanotube thin �lm [24].For the actual amount of silver in the thin �lm, the nanostructure and the microstructure of the thin �lm including its density and thickness must be taken into consideration, in addition to the results of EDX analysis.e antibacterial activity value was also larger for the silver nanoparticle/silver titanate nano�ber thin �lm (7.9) than for the previous silver nanoparticle/silver titanate nanotube thin �lm (6.3).e reason for this difference was probably that a larger amount of silver was eluted from the silver nanoparticle/silver titanate nano�ber thin �lm.
e elution of silver from the silver nanoparticle/silver titanate nano�ber thin �lm is considered to be due to the elution from silver nanoparticle and the elution from silver titanate nano�ber caused by an ion exchange reaction due to high affinity between Ag + ions and proteins.e fetal bovine serum used as the solvent in the present study contains large amounts of proteins, which have very high affinity with Ag + ions through the -SH group or -NH group in the proteins, and appreciable concentrations of cations.Ion exchange reactions are generally known to proceed more rapidly than dissolution reactions of metals.Furthermore, we have already revealed that at the silver elution test of the silver nanoparticle/silver titanate nanotube thin �lm although silver at a concentration of tens of thousands of ppb was eluted in fetal bovine serum, silver at a concentration of only several hundreds of ppb could be eluted in physiological saline and PBS solutions [24].Since the silver nanoparticle/silver titanate nano�ber reported in this study possessed a crystal structure that was basically similar to that of the silver nanoparticle/silver titanate nanotube, although the nanomorphology differed, the elution behavior of silver was presumed to be similar in both structures.Actually, as shown by the repeated silver elution test in Figure 5, the elution behavior of silver from the silver nanoparticle/silver titanate nano�ber thin �lm was similar to that from the silver nanoparticle/silver titanate nanotube thin �lm [24].erefore, the elution of silver from silver titanate nano�ber caused by the silver ion exchange reaction due to high affinity between Ag + ions and proteins was considered to contribute substantially to the large amount of eluted silver on the �rst day and the high antibacterial activity.When the elution time was extended, this contribution then would become smaller and the contribution of silver eluted from silver nanoparticles would become larger as time progressed.

Conclusion
e present study revealed that a sodium titanate nano�ber thin �lm and a silver nanoparticle/silver titanate nano�ber thin �lm formed on the surface of a titanium plate exhibited strong antibacterial activities against MRSA, which is one of the major bacteria causing in-hospital infections.e high antibacterial activity seen for the sodium titanate nano�ber thin �lm under UV irradiation indicated that bacteria could be prevented from growing and adhering to a sample when the sample was stored under UV irradiation.In contrast, the silver nanoparticle/silver titanate nano�ber thin �lm would be able to act efficiently as an antibacterial material in a living body where no light could penetrate.e titanate nano�ber thin �lm/titanium composites adhered �rmly to titanium.erefore, these composites will be extremely useful as implant materials that have excellent antibacterial activities.
Figure 1(a) (inset) shows a digital photograph of the synthesized thin �lm, which appeared as a uniform thin �lm formed on the titanium plate.A sodium titanate nanotube thin �lm

F 2 :
XRD patterns of the thin �lm before and aer the silver ion exchange treatment; the XRD pattern immediately aer the hydrothermal reaction and prior to the silver ion exchange treatment (a) and the XRD pattern following the silver ion exchange treatment (b).Peak assignment: •, sodium titanate; ▴, titanium; ○, silver titanate.

T 1 :
Evaluation of the antibacterial activity of the sodium titanate Nano�ber thin �lm against MRSA with and without UV exposure.

F 3 :F 4 :
S�M images of sodium titanate nano�ber thin �lms synthesi�ed following different reaction times; the titanium plate before the synthesis (a), and aer 1 h (b), 5 h (c), and 12 h (d) reaction times.�R� patterns of sodium titanate nano�ber thin �lms following different reaction times; the titanium plate before the synthesis (a), and aer 1 h (b), 5 h (c), 12 h (d), and 20 h (e) reaction times.Peak assignment: •, sodium titanate; ▴, titanium.

F 5 :
Repeated silver elution test performed on the silver nanoparticle/silver titanate nano�ber thin �lm.
Evaluation of the antibacterial activity of the sodium titanate nano�ber thin �lm against E. coli and S. aureus.
Silver Nanoparticle/Silver �itanate Nano�ber �in �il�.e antibacterial activity of the silver nanoparticle�silver titanate nano�ber thin �lm against MRSA was investigated.e number of viable bacteria