In Vitro Cytotoxicity Study of Pt Nanoparticles Decorated TiO2 Nanotube Array

Titanium dioxide nanotubes were synthesized by anodizing Ti sheets in the ethylene glycol solution and were covered in Pt nanoparticles onto the surface of TiO2NTs using electrodeposition method from using five derivatives of Mannich base Pt complexes which have been used as precursor of platinum. The mean size, shape, elemental composition of the titanium dioxide nanotubes and platinum deposited on the template were evaluated by different techniques such as field emission scanning electron microscope (FE-SEM), transmission electron microscopy (TEM), X-ray diffraction pattern (XRD), and energy dispersive X-ray (EDX) technique. From all these analyses, the TiO2NTs prepared and Ptnanoparticles deposited on it were identified. The diagnoses proved that all the Pt nanoparticles have a size less than 50 nm. The MCF-7 cancer cell lines and WRL68 normal cell lines were treated with concentration 800, 400,200,100, 50, 25, 12.5μg\ml of TiO2NTs and Pt\TiO2NTs(1) and (2) for 48hours using MTT assay.IC50 and inhibition rate were calculated. The result shows that the Pt\TiO2NTs have more inhibition effect on cancer cell lines than TiO2NTs array.


Introduction:
Most recent research on titanium dioxide nanotubes interested in the doping or deposition of metal ions like chromium, iron, cobalt, nickel ,copper, palladium, platinum, sliver, and zirconium (1) and like boron ,carbon, nitrogen and fluoride as non-metal in addition to using the metal oxides such as manganese dioxide had increased its applications in several fields (2). Among the varied nanostructured oxide materials, special attention has been directed toward TiO 2 nanotubes as result of developing some feature like low-cost and it has a large surface area compared to the volume(3). The titanium nanotubes are used as catalysts in accumulation boiling, photocatalysis (4), electrochromic device (5), resistant to corrosion (6), H 2 gas generation(7), solar cells (8), sensors, memory device (9), catalyst support (10), wastewater. Conjointly consistent with several researchers, the titanium oxide nanotubes could have been employed in drug-eluting stents and for the native unleash of antibiotics, drug delivery in cancer and tumor therapy. It is also widely used in dental implants and bones (11). Using titanium nanotubes in nanomedicine has a promising future in treating many diseases because improving cell adhesion, growth and differentiation (12,13), as well as its use in drug delivery. Later findings proved the strong relation between the cell responses and nanotube dimensions(14).Some of methods used to improve the performance of TiO 2 NTs are to load nanotubes with some antibiotics such as vancomycin(15) or decorate the surface of TiO 2 NTs with different nanoparticles such as gold(16) and sliver(17). Platinum medicine is still one of the most important treatments for

Preparation of Complexes
Platinum (IV). Dichloride.Dihydrate (PtL 5 ). The Pt complexes were prepared according to the literature (21, 22) The Mannich bases reaction occurs in ethanol with platinum salt , 1:1 and 1:2 molar ratio for L 1 ,L 2 ,L 3 and L 4, L 5 respectively. The mixture was then refluxed for (3 hours.); the color solid complexes were formed, and then filtered, washed with ethanol and dried in dissector .

Preparation of TiO 2 Nanotubes
Titanium dioxide nanotubes was prepared according to the literature (8) .Titanium foils were cut into the suitable size (1×2 cm 2 ). A direct current power supply (matrix E3612A) was utilized as the voltage source for the anodization. The anodization process was executed in a homemade plexiglass cell with two electrode arrays; titanium foil as the working electrode and Pt mesh utilized as the counter electrode in constant potential at 25 0 C. The distance between the substrates and the counter-electrode was approximately 1.5 cm. Degreased by sonication in detergent, deionized (DI) water, ethanol and acetone respectively for 10 minutes dried in an oven at 100 °C for 15 minutes. For the anodization process, the electrolyte used was 0.5 wt% ammonium fluoride (NH 4 F), (99.5%) in anhydrous ethylene glycol (99.8% of purity at room temperature. The anodized substrate was then soaked in a water bath at 40 °C for 20 mintes to remove the organic electrolyte. The anodization was performed for one hour at 40 V. After the occurrence of the anode, annealing in the oven with a temperature at 550 0 C was done (8).

Preparation of PtNPs/TiO 2 NTs
Platinum nanoparticles were deposited onto the annealed TiO 2 by using an electrochemical (reduction) method at a constant potential in a typical two-electrode system with the prepared TiO 2 nanotube as the working electrode, Pt sheet as the counter electrode. The electrolyte solution was prepared by dissolving the 2mM from five complexes PtL 1 , PtL 2 , PtL 3 , PtL 4 and PtL 5 in 100 (1:1:1)). Electrodeposition time was set at 3 minutes, while the PtL 4 at 6minutes while the electrodeposition voltage was fixed at 7 V and pH=5.5. The prepared Pt modified TiO 2 NTs was washed several times with deionized water for 3 minutes to remove the residue of the solutions that are not deposited above the template, and then dried in air.

Cytotoxic Assays
Cytotoxicity effect of TiO2NTs and PtNPs when deposition on TiO2NTs on MCF-7 and WRL68 cancer cell line, and normal cell lines were done in a sterile area using the biosafety conditions of the airflow cabinet, MCF-7, WRL68 cell lines used in this study were equipped from Biotechnology Center/Al-Nahrain University. The cells were cultured in (MEM) modified eagles medium with serum ((100 U\ml) of antibiotic, ((100 μg)) of streptomycin/ml in incubator with (5% CO 2 at 37 °C). The survival or death of cells were determined using (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl Tetrazolium bromide ((MTT)) which is diagnosed by using spectrophotometer. Plated in 96-well sterilized microliter-plates at a density of (1×105 cells/well). After twenty-four hours, Cells were treated with different concentrations of prepared compounds starting from the lowest concentration and incubated in (5% CO 2 ) atmosphere with high humidity. After forty eight hours of compounds exposure, the cells were incubated with (0.5 mg/ml, MTT) distilled water for another four hours at thirty-seven degrees.10% of salt (sodium dodecyl sulphate) then incubated for two hours. Absorption was measured at the wave length 620 nm on a multi-well ELISA plate reader (23).

Results and Discussion
Hitachi S-4160 Field emission scanning electron microscope (FE-SEM) was utilized to diagnose the surface morphology of TiO 2 nanotubes template Fig. 1(A,B,C,D,E,F). Template was scratched with a steel blade so as to observe the nanotubes of the side, as shown in Fig. 1 (  A1,B1,C1,D1,E1,F1). The process of anodizing led to the arrangement of nanotubes vertically. Generally, the nanotubes had lengths in the range 3 -5 µm, and average diameters 83 nm, range from (51.8-95.7) .There were no differences when compared the observed morphology of the annealed crystalline nanotubes and transmission electron microscopy Fig. 2. TiO 2 NTs may serve as the active sites or platform to deposit nanocrystals and able to promote unidirectional charge transport due to the one dimensional feature of the nanotubes. The aggregated Pt nanoparticles formed for (PtL 4 ) were larger than the other particles upon electrodeposition at 6minutes as depicted as in Fig.  1 (B, B1). While other which observed in Fig. 1 (C,C1,D,D1,F,F1,E,E1), the Pt nanoparticles were dispersed uniformly on the tube mouth of the TiO 2 NTs at 7V, 2 mM and 3minutes, some Pt nanoparticles were found to have embedded into the TiO 2 NTs. However, Pt nanoparticles prepared at 7 V, 2 mM for 6 minutes, became larger than Pt synthesized at 3minutes (24). The EDX unmistakably demonstrates that Pt, Ti and O are the major elements of composition which assures the existence of Pt decorated on TiO 2 NTs substrate as appear in Fig. 3 Fig. 2. both scans, show similar results in size and shape of nanotubes in average diameter (83nm) and nanoparticles (less than 50 nm) and deposition of platinum nanoparticles on the internal and external walls of TiO 2 NTs, Fig. 2 a and b.

Interpretation of Cytotoxic Assay Results
Cells toxicity was evaluated by (3-(4,5dimethylthiazole-2-yl)-2,5-diphenyl Tetrazolium bromide ((MTT)) method. Cultured MCF-7 were treated with TiO 2 NTs and Pt\TiO 2 NTs at concentration (800, 400, 200, 100, 50, 25 and 12.5μg/ml) for 48 hours. Table 1 shows the statistical results, and the value of IC 50 for MCF-7 cancer cell lines and WRL68 normal cell lines. According to IC 50 test, the concentration of Pt \TiO 2 NT that was required for 50% inhibition of MCF-7 and WRL68 cell inhibition was calculated. All data were expressed as means±standard deviations (SD). The statistical analysis was performed using Independent Samples Test (2tailed (t-test )) at confidence levels of 95%.
The results in Table 1 when deposited the Pt nanoparticles have different grain size on the surface of titanium nanotubes to modify it, and when we compare the values of IC 50 for the three compounds Pt\TiO 2 NTs (1), Pt\TiO 2 NTs (2), and TiO 2 NTs, the following are concluded: 1-The nanomaterial Pt\TiO 2 NTs (1) has platinum of particle size between 22-32 nm which has an inhibitory effect more than platinum of a particle size between 30-45 nm on MCF-7 cell line. 2-When comparing values IC 50 of the three nanomaterials Pt\TiO 2 NTs (1), Pt\TiO 2 NTs (2) and TiO 2 NTs, it has been observed that the modification of the titanium-nanotubes surface by different nanoparticles size of platinum, which has a particle size of less than 50 nm, has toxicity against MCF-7 higher than titanium nanotubes alone Pt\TiO 2 NTs(1)>Pt\TiO 2 NTs (2)> TiO 2 NTs. 3-When comparing values IC 50 for the two cell lines MCF-7 and WRL68 of the three nanomaterials Pt\TiO 2 NTs (1), Pt\TiO 2 NTs (2) and TiO 2 NT, it was observed that the toxicity of these nanomaterials towards cancer cells were much higher than that of normal cell lines The viability of the cell depends on the environment or the dominant medium in order to achieve the best response, including cell adhesion or migration and proliferation. Biological effectiveness depends largely on several factors, the most important of which are chemical and physical properties, including surface area, particle size shape and purity of the phase in addition to the concentration of nanoparticles (28,29). Therefore a number of reasons have been suggested to inhibit the growth of cancer cell lines, including the Pt-high surface density of nanoparticles which was found to be incompatible with MCF-7 cell adhesion and proliferation (28,29). Therefore, it is important and desirable to find an optimal surface density of Pt nanoparticles to be decorated on TiO 2 NTs including the nanoparticle and nanotube diameters that effectively kill bacteria, cancer cells and remains favorable to the normal cells. The reason may be releasing platinum nanoparticles from Pt/TiO 2 NTs and the breakdown of DNA (30), or maybe attributed to inhibition of cancer cells incorporated the nanostructure into the cells; form aggregates in the cells and inhibit migration and proliferation of cancer cells (31).

Conclusions:
Electrodeposition was applied to synthesize Pt\TiO2NTs. The regular crystalline with single-phase formation (anatase). The experiential methods Powder XRD, FE-SEM, TEM, EDX analytical techniques confirmed the presence of TiO 2 NTs in anatase phase and Pt nanoparticles decorated on it. In vitro cytotoxicity test has been carried out using the MTT assay method in wave length 620 nm. The study proved that the toxicity of the titanium nanotubes toward cancer cell lines (MCF-7) increased by deposition platinum nanoparticles on it. Also from IC 50 Value proved that these prepared nanomaterials have very low toxicity toward normal cell lines.