ABSTRACT

In this work we have developed an analytical method to measure potential titanium debris released from TiO₂ nanotube layers devices immersed in biological fluids. This quantitative study is highly required to ensure both the security and non toxicity of the nanostructured surfaces used as future implantable medical devices in the living. A one-pot synthesis process is developed to produce high quality standard solutions of titanium dioxide nanoparticles in aqueous medium. The elaborated dispersion is then used to fabricate standard solutions in both aqueous and human blood plasma media. The synthesized nanoparticles dispersion was characterized by granulometry. The nanoparticles structure and morphology were then observed using Transmission Electron Microscopy (TEM). Thermogravimetric Analysis (TGA) was used to evaluate the concentration of TiO₂ in the suspension. A quantitative routine by the use of Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) is developed. The quantification threshold of titanium species is found to be in the 30 - 40 ppb range. None interference is detected between the particles and the human blood plasma. Using the established quantitative routine, the titanium species release from titania nanotube layers in human blood plasma is evaluated.

1. Introduction

Nanotextured materials with the required surface properties are promising candidates to develop the implantation of new kind of biocompatible platforms in the living. Titanium dioxide nanotube layer is an active field of research concerning antibacterial efficacy [1], orthopedic [2], and dental surgery [3], drug delivery platforms [4], tissue regeneration [5-9] and implants [10]. The behavior of the nanostructured implants in the living is currently not well known. Numerous studies relate the toxicity of titanium dioxide nanoparticles in the living [11-13]. However, as far as we know, few studies are dedicated to the quantification of potential metallic release from the implanted device surface and their underlying potential toxicity. Works need to be done in order to ensure the biological security of such future nanostructured medical devices implanted in the living [14]. Debris from a likely wear in the living of these devices could be hazardous to human health. In this work, we develop a quantitative method to measure possible metallic debris released from the use of TiO₂ nanotube layers as future nanostructured medical implants. This quantitative method using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) requires well defined TiO₂ standard solutions commercially unavailable. So we have developed a onepot synthesis of nanoparticles dispersion in an aqueous medium. The physico-chemical properties of the nanoparticles in dispersion are investigated by granulometry, transmission electron microscopy and thermogravimetry. Different standard solutions are made by dilution from the native dispersion and assayed by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Calibration lines are obtained and the method is checked in term of linearity, accuracy and repeatability. The possible influence of the human blood plasma matrix on the ICP analytical results is also investigated. To simulate the release, nanotube layers samples were immersed in human blood plasma under magnetic stirring at 37˚C, and the titanium species concentration in the liquid is followed by the established quantification method.

2. Experimental Section

2.1. Synthesis of Stable Suspension of TiO₂ Nanoparticles

The starting precursor solution was 5 mL titanium IV isopropoxide 97% supplied by Sigma Aldrich mixed with 15 mL isopropanol (from Rhônes Poulenc) under a vigorous stirring. A 200 mL HNO₃ 0.01 M solution was used as an acid hydrolysis catalyst. The nanoparticles preparation started up with the quick addition of the hydrolysis solution heated to 80˚C in the precursor solution under vigorous stirring. The spontaneous hydrolysis of the titanium isopropoxide leads to a turbid white solution which was heated under reflux for almost 48 hours (peptidization process). After this peptidization step, some tartric acid powder supplied by Rhônes Poulenc was added to the white mixture in order to graft a carboxylic acid as surfactant on the oxide nanoparticles surface. The white, opaque resulting mixture was cooled down to room temperature. A few drops of Rhônes Poulenc triethylamine were added under stirring: the opaque, settling solution turned to a clear, soft blued aqueous suspension of TiO₂ nanoparticles.

2.2. Granulometry Study of the Suspension

Granulometry studies are achieved by using the Nano Sizer S90 model (Malvern Instrument ltd.). In order to define the influence of the pH of the carrying liquid on the nanoparticles aggregate states, different samples were analysed by varying the pH from 3 to 10. A first sample with the highly measurable concentration is prepared and then different amounts of acid or base are added in order to vary the pH.

2.3. Transmission Electron Microscopy Analysis of the Suspension

The morphology and the particles sizes were characterized using a Philips CM 20 transmission electron microscope (TEM). The accelerating voltage was 200 kV. The samples were dispersed in methanol by ultrasonication. A drop of the suspension was then laid on a carbon-coated grid and dried. Selected area electron diffraction (SAED) was performed to determine the crystallinity of the structure. The interplanar distances were evaluated from the SAED patterns using the following formula:

(1)

where λL is a constant of the microscope, R is the ring radius, and d is the interplanar distance. The constant of the microscope was calculated by measuring the radius of a gold standard pattern which interplanar distances are well documented in scientific publications.

2.4. Thermogravimetric Analysis of the Nanoparticles Suspension

The thermogravimetric analysis was carried out using the TGA 4000 thermogravimetric analyser (Perkin Elmer instrument). The analysis was performed at a heating rate of 5˚C/min under a 40 mL/min N₂ gas flow. Prior to the TGA tests, a designated volume of 50 mL of the white, opaque acid tartric grafted mixture was centrifuged at 7000 rpm during 10 minutes. The clear supernatant was removed and the wet TiO₂ grafted tartric acid nanopowder was dried at 110˚C overnight. As a reference 20 mg TiO₂ nanopowder supplied by Sigma Aldrich has also been analyzed.

2.5. Synthesis of TiO₂ Nanotube Layers

The main goal of this part is to obtain TiO₂ nanostructured surface in order to study the potential titanium species release in biological fluids. To fabricate anodic TiO₂ nanotube layers, Ti foils, 99.6% purity, 0.25 mm thick, supplied by Good-fellow are used. The foils are successively degreased in trichloroethylene, methyl alcohol, acetone and deionized water baths under sonication for 5 minutes. The drying is made under nitrogen flush. The anodization process is carried out at 20˚C in 0.4 wt% HF aqueous solution, under an anodizing voltage maintained at 20 volts during 20 minutes. Just after the anodization, samples are rinsed in deionized water, flushed under nitrogen and dried in an oven at 100˚C for 10 minutes. Finally, the anodized foils are cooled down to room temperature in a desiccator.

2.6. TiO₂ Nanotube Layer Surface Characterization

The morphological study of the nano-structured surfaces was carried out using a Zeiss Supra 55 VP scanning electron microscope (SEM) with secondary emission and in lens detector. The accelerating voltage and working distance are respectively 5 kV and 3 mm.

2.7. Inductively Coupled Plasma Atomic Emission Spectroscopy

Inductively Coupled Plasma Atomic Emission Spectroscopy analysis was carried out using a Varian Liberty II apparatus under the operating conditions given in Table 1. To increase the results accuracy, three emitting wavelengths of the titanium are used for quantification at 334.941, 336.121 and 337.280 nm respectively.

2.8. Titanium Species release Quantification from TiO₂ Nanotube Layers

In order to quantify the potential titanium release in human blood plasma, 3 anodized samples (0.5 cm × 2 cm)

are immersed in 25 mL of human blood under magnetic stirring at 70 rpm. Each closed vial is kept at 37˚C in a thermostatic bath. A non-anodized titanium foil is used as a reference and dipped in the same conditions. A volume of 3 mL of solution is collected after the 2-day stirring and assayed by ICP in order to obtain the kinetic of release.

3. Results and discussion

3.1. Synthesis of Homogeneous Suspension of TiO₂ Nanoparticles

Functionalized crystalline TiO₂ nanoparticles in suspension have been synthesized by a derivated sol-gel process at low temperature. Figure 1 shows the proposed method for a one-pot synthesis of homogeneous suspension of TiO₂ nanoparticles in an aqueous medium. The process begins by hydrolysis and condensation of titanium alkoxide specie, which the inorganic TiO₂ core is made of. This step was followed by grafting a carboxylic acid as surfactant on the oxide nanoparticle surface. Finally the organic treatment was carried out by adding triethylamine into the mixture. This chemical functionalization of the surface provides the required stable dispersion of the particles in the aqueous medium, leading to a soft blued aqueous suspension of TiO₂ nanoparticles.

Different synthesis methods lead to nanopowders with sharp sized distribution. The main problem occurs at the dispersion step of these nanopowders into water or water based solutions. The aggregative state is so high that it leads to turbid, opalescent and fairly stable solutions. Such settling solutions are not suitable for developing rigorous quantitative measurements. On the other hand, the use of commercial dispersion is not appropriate: the chemical compatibility between the turbid commercial product and the aqueous medium is low, so the dilutions cannot be used for quantitative analysis. In order to cope with this key problem of the required stability of the standard solution, we used a one-pot synthesis of TiO₂ nanoparticles dispersion in an aqueous medium. This method solves the problem of the turbidity and settlement generated by the nanopowder dispersion in liquid. This gives rise to stable clear nanoparticles suspension, ready to induce rigorous standard solution for the development of the quantitative analysis contrary to those made from nanopowder or commercial formulation. The difference between the formulations is evidenced in Figure 2.

Figure 2(a) shows a try of TiO₂ nanopowder dispersion in H₂O: without chemical functions grafted on the nanoparticles surfaces, the TiO₂ nanopowder settled quickly in the bottom of the tube: a white TiO₂ paste is formed, the clear supernatant does not carry nanoparticles in suspension. Figure 2(b) demonstrates the high quality of our synthesized suspension; the suspension is not turbid, soft blued, with high transparency thanks to the chemical surface functions which maintained the TiO₂ nanoparticles in a non aggregated state and highly dispersed in the carrying liquid. Figure 2(c) illustrates that the commercial suspension is totally turbid, the white color of the liquid shows that the TiO₂ nanoparticles are aggregated: such dispersions can not be used as standards.