Biofunctionalization of Magnetite Nanoparticles with Stevioside: Effect on the Size and Thermal Behaviour for use in Hyperthermia Applications.

The crystalline structure and the approximate size of the particles were determined by Bruker X-ray diffraction (XRD) system using Cu Kα radiation with a wavelength of 1.5404 Å and operated at 40 kV and 25 mA. A coarse scan from 20 to 80° (2θ) was performed at a scan rate of 0.020 to acquire an overall spectrum to identify the crystal structure. The mean diameter of the particles were also calculated from the XRD spectra according to the linewidth of the (311) plane refraction peak using Scherrer equation (1), given as:

spectrum to identify the crystal structure. The mean diameter of the particles were also calculated from the XRD spectra according to the linewidth of the (311) plane refraction peak using Scherrer equation (1), given as: Where θ is the angle at which the reference peak occurs, is the X-ray wavelength (1.5418 Å), b is the FWHM of the XRD peak and K is the shape factor whose value is 0.9 for magnetite.
Transmission electron microscopy (TEM) was done to study the morphology and size of the nanoparticles using JEOL JEM 2100 transmission electron microscope operating at the acceleration voltage of 200 KV. A drop of diluted nanoparticle suspension in methanol (100 μg/ml) was casted onto a 300 mesh formvar-carbon coated copper grids and air-dried before measurements. The hydrodynamic diameter of the magnetite nanoparticles were measured using Malvern Zetasizer Nano-ZS (λ = 632.8 nm, T = 25°C). The concentration of each sample analysed was 10 μg/ml DMSO.
Fourier transform infrared spectroscopy (FTIR) analysis was done using Agilent Technologies Cary 600 series spectrometer. The spectrum for all samples was recorded in the wavelength range of 400-4000 cm -1 . The powder samples of the coated nanoparticles were ground with KBr and compressed into a pellet using manual hydraulic press. These generated pellets were used to obtain the spectrum. The spectrum of neat OA and P-80, and pure STE powder was recorded as standard reference for the coated nanoparticles. To further confirm the STE coating onto the surface of Fe3O4 nanoparticles, FTIR spectra of bare Fe3O4 nanoparticles mixed with pure STE powder was also compared with that obtained from STE-coated Fe3O4 nanoparticles.
The magnetic measurements of the coated and uncoated Fe3O4 nanoparticles were performed with a Quantum Design Dynacool PPMS magnetometer in the temperature range 5-300 K and magnetic fields up to 5 T. The zero-field-cooled (ZFC) curves were obtained by cooling the magnetite samples from 300 to 5 K in the absence of an external magnetic field, followed by the magnetization measurement under a magnetic field of 100 Oe as the temperature was raised back to room temperature. The fieldcooled (FC) measurements were carried out in a similar way, except for the cooling process, which was performed under an external magnetic field of 100 Oe. The hysteresis loop measurements were performed at 300 K and 5 K under magnetic fields of up to 5 T.  To understand the adsorption mechanism of the surfactant moieties onto the surface layer of magnetite nanoparticles, FTIR analysis were performed on bare Fe3O4 nanoparticles; OA-, P-80-and STE-coated Fe3O4 nanoparticles; neat drop of OA, P-80 and STE powder ( Figure S1). In Figure S1(A),  Figure S1(

Effect of MNP concentration on SAR value
The SAR value with particle concentration has been evaluated by using the method suggested by Natividad et al 2013 [1], using the equation (Table 3A): Where T is maximum rise in temperature achieved;  is the relaxation time of the nanosystems calculated from the complete T-vs-t curve fit to the exponential trend characteristic of isoperibol conditions.
Where dT/dt is the initial slope of the temperature versus time graph taking into consideration first few seconds only. As seen, the SAR value increases with increasing particle concentration only until it reaches the optimal particle concentration. Beyond this concentration, SAR value starts decreasing with further increase in particle concentration. This effect could be attributed to increased relaxation time of the nanosystems due to enhanced dipolar interaction and agglomeration with increasing particle concentration. Similar trend in SAR variation with particle concentration has also been reported in various other studies, both experimental [2] and theoretical [3][4].