Photofunctional Co-Cr Alloy Generating Reactive Oxygen Species for Photodynamic Applications

1 Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-Gu, Seoul 120-749, Republic of Korea 2 Cellbiocontrol Laboratory, Department of Medical Engineering, College of Medicine, Yonsei University, 250 Seongsanno, Seodaemun-Gu, Seoul 120-752, Republic of Korea 3 Brain Korea 21 Project for Medical Science, College of Medicine, Yonsei University, Seoul 120-752, Republic of Korea 4Cardiology Division, Cardiovascular Center, College of Medicine, Yonsei University, Seoul 120-752, Republic of Korea


Introduction
Co-Cr alloy is a type of metallic alloy that has been used as a biomaterial for the human application.Co-Cr alloy is rapidly gaining attention for a wide variety of its applications in cardiology, orthopedics, and dentistry [1][2][3][4][5].Compared with other metallic implant materials, Co-Cr alloy has several outstanding properties that make it attractive.It has good wear resistance, crevice corrosion resistance, and pitting resistance.It also has high abrasion resistance, fatigue strength along with good workability and ductility [6,7].Due to such superior properties, Co-Cr alloy is currently used for coronary artery stents [8].However, on the other hand, Co-Cr alloy stents are limited by the occurrence of restenosis induced by smooth muscle cell proliferation, neointimal hyperplasia, and thrombus formation [9][10][11].To overcome the problems, polymer based drug eluting stents (DESs) were developed and reduced the occurrence of major cardiac event [12].However, DES has also several drawbacks such as polymer fracture during stent expansion and inflammatory reaction to the polymer, which leads back to restenosis.Moreover, it is not easy to have precise control on the releasing rate of the drug, inducing problematic side effects [13][14][15].DES also loses its functionality because of a limited amount of the drug included.Therefore, in a field of DES, a new semipermanent biomaterial that does not have side effect and cytotoxicity is in a great demand for the development.Furthermore, it must have the control function for the rate and time of drug elution.
In recent years, it has been reported that suppression of smooth muscle cells proliferation and destruction of Scheme 1: Fabrication procedure of the photofunctional Co-Cr alloy plate.
thrombus, the major factors for the control of restenosis, are achieved by reactive oxygen generated from photodynamic inactivation [16,17].In details, the photosensitizer hematoporphyrin (Hp) in its ground state absorbs light and undergoes intersystem crossing (ISC) with high efficiency to its triplet state.Then the reactive oxygen species (ROS) can be generated from the triplet state of photosensitizer by energy transfer or charge transfer processes [18].Photodynamic inactivation for therapy has been applied for many years and it is now becoming widely accepted and utilized.Photodynamic therapy (PDT) can be defined as a therapy that involves the administration of a nontoxic drug or dye known as a photosensitizer (PS) either systemically, locally, or topically to a patient bearing a lesion, which is frequently, but not always used for cancer treatment [18].After an incubation period, the specific lesion is then usually illuminated with a visible light in the presence of oxygen, which leads to the generation of cytotoxic species of reactive oxygen and consequently causes cell death and tissue devastation [19,20].The use of reactive oxygen species (ROS) in PDT is particularly attractive because of its high efficiency for deactivation of harmful organism and with less side effect.Therefore, ROS can be applied in water disinfection, air cleaning, and thrombolysis [21][22][23].
In this study, we have successfully fabricated the photofunctional Co-Cr alloy plate by a simple process.Photoinduced functionality of the fabricated photofunctional Co-Cr alloy plate is provided by the photosensitizer of hematoporphyrin (Hp) that can generate reactive oxygen species (ROS) such as superoxide anion radical and/or singlet oxygen.The generation efficiency of ROS from the fabricated photofunctional Co-Cr alloy plate is confirmed with photocatalytic experiment.Furthermore, photodynamic inactivation of cells by the photofunctional Co-Cr alloy plate is investigated with rat aorta smooth muscle cells (RAOSMCs) for the potential bioapplication.

Preparation of the Photofunctional Co-Cr Alloy Plate.
Overall synthetic procedure of the photofunctional Co-Cr alloy plate is presented in Scheme 1. Hematoporphyrin (Hp, Aldrich) was used as a photosensitizer.All solvents from Merk were used without further purification.Co-Cr alloy plate (Tae-woong medical, 5 × 3 × 0.2 mm) was washed and sonicated with acetone for 30 min.It was then dipped into piranha solution (H 2 O 2 : H 2 SO 4 = 1 : 3 (vol%)) for 2 h at 100 ∘ C. To enhance the hydroxyl groups on the surface of Co-Cr alloy plate, the oxidized Co-Cr alloy plate was immersed in boiling H 2 O 2 solution for 50 min.The fully oxidized Co-Cr alloy plate was reacted with Hp/ethanol solution in the presence of EDC/NHS (wt%, 1 : 1) for 3 h at room temperature.Finally, it was washed with ethanol and then dried.The washing solution and reactant solution were collected and measured with UV-Vis spectrophotometer for the quantization of Hp bonded to the surface of the oxidized Co-Cr alloy plate.Amount of Hp bonded to the surface of the oxidized Co-Cr alloy plate was estimated to be 1.1 × 10 −8 mol/cm 2 .with an energy dispersive X-ray spectroscopy (EDS).The functional groups on the surface of the Co-Cr alloy plate were confirmed with X-ray photoelectron spectroscopy (XPS, Thermo VG, Escalab 220i-XL).XPS was used to determine the atomic composition of the sample surface.Compositional survey and detailed scans were acquired using pass energy of 50 eV.The binding energies were corrected by referencing the C 1s binding energy to 285 eV.Steady-state absorption and emission spectra were obtained with a diffuse reflectance UV-Vis spectrophotometer (Jasco V-550) equipped with an integrating sphere (Jasco ISV-469) and a spectrofluorimeter (Hitachi, F-4500), respectively.

Detection of Reactive Oxygen Generation from the
Photofunctional Co-Cr Alloy Plate.Degradation of 1,3diphenylisobenzofuran (DPBF), a reactive oxygen quencher [24], was studied with the photofunctional Co-Cr alloy plate.In a typical experiment, 3.5 mL of EtOH solution containing the photofunctional Co-Cr alloy plate and DPBF (1.0 × 10 −5 M) was introduced into a 1 cm quartz cell in the dark.The light source for irradiation photofunctional Co-Cr alloy plate was a nanosecond Nd-YAG pumped optical parametric oscillator (OPO) laser (OPOLETT, 20 Hz, 5 ns FWHM pulse).The total output power for the irradiation was measured with a laser power meter (Ophir-optronics Ltd., Nova, 10A-P-V2-SH), and the irradiated laser power was 15 mW at 510 nm.At every 10 min of irradiation, the absorption spectra of the DPBF were monitored with a UV-Vis spectrophotometer (Hitachi, U-2800).cytoskeleton and nuclei were counterstained with Hoechst 33528.The morphological observation of RAOSMC was evaluated using an Olympus fluorescence microscope (Melville, NY) equipped with a DP-71 digital camera (Olympus, Japan).

Results and Discussion
The color of Co-Cr alloy plate is changed when hematoporphyrin is introduced to surface of the oxidized Co-Cr alloy plate as shown in Figure 1(a).alloy plate are observed at 576.8, 580.0, and 586.2 eV and these peaks are not changed when the pure Co-Cr alloy plate is oxidized and Hp is bonded to the surface of Co-Cr alloy plate as shown in Figure 2. It means that Hp is dominantly bonded to Co elements.Such tendency is in a good agreement with the reported values of the oxidized Co-Cr alloy and other organic compounds bonded Co-Cr alloy [25][26][27].
Diffuse reflectance UV-Vis absorption spectrum of the photofunctional Co-Cr alloy plate is presented in Figure 3(a) along with the absorption spectrum of the pure Hp molecules in an ethanol solution.Diffuse reflectance spectrum is a type of the absorption spectrum measured by the scattering from the surface of the sample.This diffuse reflectance spectrum is translated into the absorption spectra by the Kubelka-Munk method as follows: where  indicates the absorption coefficient, and  and  represent the scattering coefficient and the absolute reflectance, respectively [28].
The spectrum of Hp solution shows a B band (413 nm) and four Q bands (500, 531, 572, 622 nm).Diffuse reflectance absorption spectrum of the photofunctional Co-Cr alloy plate also presents B and Q bands at the similar wavelengths but with broad shape and slightly red shifted peak position.The difference in the peak width and position is possibly due to the self-coupling of Hp molecules bonded to the surface and the inhomogeneous bonding nature of Hp molecules on the surface of Co-Cr alloy plate.The fluorescence spectrum of Hp ( ex = 510 nm) solution in Figure 3 ground states [29][30][31].Therefore, the results suggest that Hp molecules are bonded to the surface of Co-Cr alloy through the esterification reaction without a significant change of its photophysical properties.
Decomposition study of DPBF was performed with the photofunctional Co-Cr alloy plate that generates ROS upon International Journal of Photoenergy irradiation.As a ROS quencher, DPBF readily undergoes 1,4-cycloaddition reaction with singlet oxygen to form an endoperoxide that decomposes into the irreversible product (1,2-dibenzoylbenzene) [24].It is also reported that DPBF can be decomposed by superoxide anion radical of ROS [32,33].
As shown in the inset of Figure 4, the optical densities of DPBF absorption peak at 415 nm are not changed by the irradiation of light and, also, by the photofunctional Co-Cr alloy without the light irradiation condition.Therefore, the decomposition rate induces the decrease of the optical density of the DPBF absorption peak at 415 nm as shown in Figure 4, which indicates the generation of ROS from the photoirradiated surface of the photofunctional Co-Cr alloy plate.
Figure 5 shows the fluorescence microscope image for the comparative morphologies of the cells after laser irradiation on the photofunctional Co-Cr alloy plate by fluorescence microscope.RAOSMC had well organized plasma membrane (Red) and the actin cytoskeleton (Green) on the pure Co-Cr alloy plate.The morphology of the cells on the pure Co-Cr alloy plate was not changed even with the laser irradiation compared to the nontreated cells (Figures 5(a)-5(c)).Also, the morphology of the cells on the photofunctional Co-Cr alloy plate was not changed in the dark condition.However, the cells appeared loosely to be adherent and lost their normal morphology after laser irradiation of 15 mW/cm 2 for 15 min on the photofunctional Co-Cr alloy plate (Figure 5(e)).Also, the cells get rounded and detached from the surface with the irradiation of 30 min (Figure 5(f)).These results reveal that the cells are damaged by ROS from the photo-irradiated surface of the photofunctional Co-Cr alloy plate upon irradiation of light [34,35].

Conclusion
The photofunctional Co-Cr alloy plate is successfully fabricated by a simple surface modification process of oxidation and esterification reaction with Hp.The surface of the Co-Cr alloy plate homogeneously consists of Co and Cr elements.The X-ray photoelectron, UV-Vis, and emission spectra indicate that the Hp molecules are covalently bonded to the surface of the Co-Cr alloy plate through the carboxyl group.The DPBF decomposition study indicates that ROS is efficiently generated from the photoexcited Hp molecules that are bonded to the surface of the Co-Cr alloy plate.Also, RAOSMC is effectively damaged and detached from surface of the Co-Cr alloy plate due to PDI.The results suggest that such ROS generated from photofunctional Co-Cr alloy plate give the possibility of such biomaterials to be used for coronary artery stent and antithrombogenic applications.

Figure 2 :
Figure 2: XPS analysis of (a) the pure Co-Cr alloy plate, (b) the oxidized Co-Cr alloy plate, and (c) the Hp bonded Co-Cr alloy plate.

Figure 1 (Figure 3 :
Figure 3: (a) Absorption and (b) emission spectra of Hp in EtOH (dashed line) and of the Hp bonded to Co-Cr alloy plate (solid line).

Figure 4 :
Figure 4: Reaction time dependent UV-Vis spectra of DPBF in the presence of the photofunctional Co-Cr alloy plate in EtOH solution with a 510 nm laser irradiation.The inset represents the ratio between the after decomposition reaction O.D. () and the initial O.D. ( 0 ) of DPBF as function of irradiation time: (a) DPBF only under the light, (b) DPBF with the photofunctional Co-Cr alloy plate in the dark condition, (c) DPBF with the photofunctional Co-Cr alloy plate under the light.

Figure 5 :
Figure 5: Morphology of RAOSMC on the pure Co-Cr alloy plate (a-c) and the photofunctional Co-Cr alloy plate (d-f) without laser irradiation (a, d) and with laser irradiation for 15 min (b, e) and 30 min (c, f) of 15 mW/cm 2 ( ex = 510 nm).Green, red, and blue represent cytoskeleton, plasma membrane, and nuclei, respectively.Magnification was 200x.