Phase transformation in Mn-doped titania hollow spheres and their biocompatibility studies
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Mn-doped titania hollow nanospheres were prepared via sacrificial core templating method at room temperature, using carbon spheres as the sacrificial core and template. X-ray diffraction and thermal studies showed the phase transformation of titania from anatase to rutile at temperature as low as 550 °C, when the dopant (i.e., Mn) concentration was increased from 1 to 6 mol % (with respect to Ti). Fourier transform infra red spectroscopic studies have been carried out to determine the surface functional groups, while the spherical and hollow morphology of the titania nanostructures have been confirmed through scanning electron microscopic as well as transmission electron microscopic studies. The chemical composition of the samples has been determined through X-ray photoelectron spectroscopic studies, while their magnetic properties have been studied using superconducting quantum interference device analysis. The biocompatibility and suitability of the nanospheres for intracellular applications has been tested through conventional MTT assay using MDA-MB 231 human breast cancer cell lines.
KeywordsMn-doped Titania Hollow spheres Anatase Rutile Biocompatibility
Recently, there has been a surge of research interest in developing appropriate inorganic nanostructures for different biomedical applications. Various nanostructures of hydroxyapatite (Andronescu et al. 2013; Venkatesan et al. 2011; Wang et al. 2009), magnetite (Daumann et al. 2014; Dorniani et al. 2013), zirconia (Batra et al. 2013; Tang et al. 2010), titania (Hou et al. 2014; Aw et al. 2012), silica (Bernal et al. 2014; Xu et al. 2013) etc., have already gained attention due to their efficacy in the fabrication of biosensors and nanocarriers for targeted delivery of drugs as well as substrates for immobilization of biomolecules. Among the various inorganic materials, titania (TiO2) holds great promise in biomedical applications owing to its wide availability, biological as well as chemical inertness and have so far been reported in various morphological forms such as nanoparticles (Liang et al. 2013), nanorods (Pang et al. 2013), nanowires (Sun and Wu 2013), nanosheets (Liu et al. 2013), nanotubes (Pandikumar and Ramaraj 2013), mesopores (Mascolo 2013), and hollow spheres (Zhao and Middelberg 2013). However, all literature reports on biomedical applications using titania based nanomaterials have been performed using titania (TiO2) films (Kim et al. 2010), mesoporous nanoparticles (Wu et al. 2011), and nanotubes (Aw et al. 2012; Gulati et al. 2012). The hollow nanostructured titania (or, doped titania), which have the potential to create a whole new generation in host–guest chemistry because of their low density, high surface area, tunable pore structure and good surface permeability, have not been much explored for biomedical applications till date, according to the best of our knowledge. In an attempt to contribute in this area, we report a novel methodology for the synthesis of hollow nanospheres of TiO2 which have been tested for their biocompatibility.
From literature reports, it is evident that titania nanostructures can either be functionalized or new properties can be introduced by doping the host material with appropriate dopants. For instance, Ao et al. (2010) investigated the photocatalytic activity of N-doped titania hollow spheres for degradation of reactive Brilliant Red dye X-3B. Xu et al. (2006) investigated the ferromagnetic property evoked in Mn-doped TiO2 thin films and related the structural phase transition of TiO2 to the annealing temperature of the films, while Saponjic et al. (2006) studied the effect of Mn-dopant on the charge separation and surface reconstruction in Mn-doped titania nanoparticles. In the present study, titania hollow spheres have also been doped with Mn(II) to induce magnetic property in the material. The magnetic character developed on doping with manganese is expected to enable the magnetic field induced navigation of the titania nanospheres during biomedical application such as in targeted delivery of drugs. Further, cytotoxicity studies of the synthesized Mn-doped hollow nanospheres of TiO2 have been performed by conventional MTT assay using MDA-MB 231 cells for testing their biocompatibility.
The hollow nanospheres of titania have been prepared under ambient condition by sacrificial core templating method, where carbon spheres were used as the sacrificial core templates, while samples with different doped-Mn concentrations were obtained by varying the mole percent of Mn (i.e., 1, 2, 4, and 6 mol % with respect to Ti). The Mn-doped hollow nanospheres of TiO2, obtained on calcination of the respective precursors at 550 °C, have been characterized in detail.
Chemicals and materials
Titanium tetraisopropoxide was purchased from Spectrochem Pvt Ltd (India), while manganese(II) acetate tetra hydrate and anhydrous dextrose (GR) were purchased from Merck (India). 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma–Aldrich. Human breast cancer cell lines (MDA-MB-231) were obtained from the National Center for Cell Science (NCCS), Pune (India). Absolute ethanol and milli–Q water were used throughout the study.
Preparation of carbon spheres
A 0.7 M aqueous solution of dextrose was hydrothermally treated at 170 °C for 6 h in a Teflon lined autoclave under autogenous pressure. The blackish residue obtained was centrifuged, washed with water and ethanol and finally dried at 60 °C for 12 h to obtain the carbon spheres.
Preparation of hollow Mn-doped titania nanospheres
Initially, undoped titania hollow spheres were prepared as follows: 20 mmol of titanium tetraisopropoxide was added to 40 mL ethanol. This was then mixed with 0.08 g of freshly prepared carbon spheres and the mixture was sonicated for 30 min. After 6 h of undisturbed standing, the solution mixture was centrifuged and the collected solids were washed with ethanol, dried at 60 °C for 12 h, and finally calcined at 550 °C for 4 h in air. These samples have been named as HT in the text. For preparing the Mn-doped nanospheres, the procedure was repeated with the addition of the required amount of manganese(II) acetate tetra hydrate in the starting solution mixture of titanium tetraisopropoxide and carbon spheres. Four different samples with different dopant (i.e., Mn) concentrations were prepared by changing the mole percentage of Mn (i.e., 1, 2, 4, and 6 mol % with respect to Ti) in the samples. The dried Mn-doped titania samples, containing 1, 2, 4 and 6 % mole percent of Mn were calcined at 550 °C for 4 h in air and were indexed as HMT-1, HMT-2, HMT-4, and HMT-6, respectively, in the text.
Procedure for the cytotoxicity studies
X-ray diffraction (XRD) was employed for the crystalline phase identification of the prepared hollow spheres using Cu-Kα radiation over 2θ range of 10°–70° at a scan rate of 3° min−1 and with a sampling interval of 0.05 at 40 mA and 40 kV by using Bruker AXS Diffractometer D8 Powder XRD. The functional group analysis was done by Fourier transform infrared (FT-IR) spectroscopy using Perkin-Elmer Spectrum RXI instrument, within the scan range 4,000–400 cm−1. The thermogravimetric analysis (TGA) was performed using Netzsch STA 409 PC Luxx (Germany). The morphology of prepared hollow spheres were analysed by scanning electron microscopy (SEM) using SUPRA 40 Field Emission Scanning Electron Microscope (Carl Zeiss SMT AG, Germany) and transmission electron microscopy (TEM) using JEM-2100 HRTEM (JEOL, Japan) operating at 200 kV. The chemical composition of the samples were determined by X-ray photoelectron spectroscopy (XPS), carried out on PHI 5000 VersaProbe II Scanning XPS Microprobe (Φ ULVAC- PHI, INC.) using monochromatic AlKα radiations (1,486 eV). The carbon 1 s peak at a standard value of 284.5 eV has been used to calibrate the binding-energy scale for the XPS measurements. Magnetic measurements were carried out using EverCool SQUID VSM DC magnetometer (Quantam Design, USA).
Results and discussion
Crystalline phase analysis
Functional group analysis
The XPS spectra of O 1s core level for HMT-1 and HMT-6 are shown in Fig. 6b. The binding energy peak for O 1s was observed at 529.29 and 529.4 eV for HMT-1 and HMT-6, respectively. These binding energy values correspond to the normal lattice sites in the titania (TiO2) structure with oxygen in O2− state in the hollow spheres.
The XPS spectra of Mn 2p core levels for the samples were shown in Fig. 6c. The binding energy peaks corresponding to Mn 2p3/2 and Mn 2p1/2 peaks were observed at 641.3 and 652.9 eV, respectively, for HMT-1 and 641.7 and 653.4 eV, respectively, for HMT-6 with a doublet separation of 11.6 and 11.7 eV for HMT-1 and HMT-6, respectively. The peak position and doublet separation values for the samples correspond to the presence of Mn2+ state in the prepared spheres. In addition, it was observed that the binding energy peaks shows a positive shift for all the three elements in HMT-6 as compared to HMT-1, which indicates the more positive charged surface of HMT-6 which is due to the high concentration of dopant (i.e., Mn) ions in HMT-6 as compared to HMT-1.
In conclusion, Mn-doped titania hollow spheres have been successfully prepared through sacrificial core templating method using carbon spheres as the sacrificial core templates. The hollow spherical structures of the calcined samples, with diameters of about 400 nm and wall thickness of 40 nm, were confirmed through SEM and TEM studies. XRD studies revealed the anatase phase for undoped titania sample which got transformed into the rutile phase with increase in the dopant (i.e., Mn) concentration at temperature as low as 550 °C. XPS studies shows the presence of Ti4+ state for Ti, O2− state for O, and Mn2+ state for Mn. Magnetic studies confirmed the presence of room temperature ferromagnetism in the Mn-doped samples. Further, the samples were found to be biocompatible with IC50 values higher than 200 μg/mL for all the samples, thereby validating their applicability for different biomedical applications.
The authors are thankful to Prof. Mahitosh Mandal, School of Medical Science and Technology, IIT Kharagpur India, for helping with the cytotoxicity studies. The authors would also like to acknowledge the DST-FIST funded XPS facility at the Department of Physics and Meteorology, IIT Kharagpur India and University Grants Commission (UGC) New Delhi, India for financial support.
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