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Electronic Supplimentry Information for Manuscript #L13-04048
Engineering of Gadofluoroprobes: Broad-spectrum applications from
cancer diagnosis to therapy
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Ranu Dutta1*, Prashant K. Sharma1,2, Vandana Tiwari3, Vivek Tiwari4, Anant B Patel4,
Ravindra Pandey5 and Avinash C. Pandey1,6
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Nanotechnology Application Centre, University of Allahabad, Allahabad 211002, India.
Indian School of Mines, Dhanbad, India.
Department of Pathology, KGMU, Lucknow, India.
Centre for Cellular and Molecular Biology, Hyderabad, India.
Department of Physics, Michigen Technological University, Michigen, United States.
Bundelkhand University, Jhansi, India.
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*E-mail: ranu.dutta16@gmail.com, Tele/Fax: +91-532-2460675 (O)
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Keywords: Quantum dots; Gadofluoro probes; Photoluminescence; Magnetism.
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PACS: 75.50.Tt, 75.75.Cd, 78.67.-n, 87.15.mq, 87.80.Qk, 87.85.J-
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Supplementary Information
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In the first method stock solutions of metal nitrates were prepared by dissolving
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appropriate amount of metal nitrates in de-ionized water. 40 mM aqueous solution of Na2S
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was prepared separately. 40 mM Gd(NO3)3.6H2O and 160 mM Eu(NO3)3.6H2O solutions
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were allowed to mix together homogeneously for 45 minutes in a condenser flask. Now 40
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mM of Na2S solution was added to the above metal nitrate solution. After 30 minutes of
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stirring at room temperature the solution was heated at 30º C with constant stirring till visible
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precipitate appeared. The reaction mixture was left overnight under stirring conditions.
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Precipitate was centrifuged, washed several times with absolute ethanol and de-ionized water.
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Obtained slurry was dried in vacuum oven at room temperature for 24 hours to get powder
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sample. In the second method 50 mL of 40 mM Gd(NO3)3.6H2O solution was prepared by
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dissolving Gd(NO3)3.6H2O in de-ionized water. The weight equivalent of 4 mM
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Eu(NO3)3.6H2O was added to it. The solution was allowed to mix together homogeneously
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for 45 minutes. 40 mmol solid Na2S was added to the above metal nitrate solution mixture.
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Precipitate was seen after some time. After 30 minutes of stirring at room temperature the
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solution was heated at 30º C with constant stirring. Precipitate was centrifuged, washed
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several times with absolute ethanol and de-ionized water. Obtained slurry was kept in
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vacuum oven for 24 hours to get powder sample.
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The prepared magnetic nanoparticles were thoroughly characterized by X-ray
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diffraction (XRD) and transmission electron microscopy (TEM) in order to elaborate
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structural properties in precise manner. XRD was performed on Rigaku D/max-2200 PC
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diffractometer operated at 40 kV/40 mA, using CuKα1 radiation with wavelength of 1.54 Å
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in the 2θ angle ranging from 10 to 80. The size and morphology of prepared nanoparticles
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were characterized using a transmission electron microscope (Model Tecnai 30 G2S-Twin
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electron microscope) operated at 300 kV accelerating voltage. For TEM study a drop of the
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colloidal solution obtained by dissolving of nanoparticles in ethanol was placed on the
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surface of a carbon coated copper grid. Room temperature magnetization measurement was
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carried out in pellets of nanoparticles using a vibrating sample magnetometer (EV9, ADE
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Magnetics, USA) in the applied field up to 1.75 T. All the bioimaging related experiments
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were carried out using a Bruker Avance DRX 400 MHz FT-NMR Spectrometer with Micro-
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Imaging facility.
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Fig. ESI 1 shows XRD spectra of the prepared Gd2S3:Eu3+ nanomagnet obtained from
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both the methods adopted, which seem to be quite similar with respect to most of the peak
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positions. The XRD spectra showed excellent similarity with standard JCPDS file for Gd2S3
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(JCPDS no 76-0265; cell parameters a = 10.74 Å, b = 3.898 Å, c = 10.54 Å) and can be
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indexed as orthorhombic system with primitive lattice having space group P nma. The few
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peaks of standard JCPDS pattern were found missing in the experimental XRD data. This is
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due to the broadening in experimental XRD spectra due to the formation of nanostructure.
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The crystallite size‘d’ was estimated using Debye-Scherrer’s equation by fitting all the
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available reflections of the experimental XRD phases with a Gaussian distribution and then
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calculating Scherer’s broadening (FWHM). The calculated crystallite size was ~ 5 nm. The
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XRD data from both the synthetic methods adopted in the present study seem to be quite
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similar with respect to most of the peak positions.
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In fig. ESI 2 (a), TEM image of the nanoparticles formed by the first synthesis
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method is shown. It is evident that the nanorods are around 60 nm in length and 7 nm in
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diameter. In this case the reaction parameter promotes the growth of rod shaped
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nanoparticles. However the exact mechanism for the formation of these rod shaped
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nanoparticles is not known. Fig. ESI 2 (b) shows TEM image of uniformly monodispersed
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nanospheres synthesized by the second method followed, where the mean particle size is
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around 10 nm. Fig. ESI 3 shows the dependence of magnetization with applied magnetic field
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(M - H loop) for as synthesized Gd2S3:Eu3+ nanomagnets at room temperature. A clear
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hysteresis loop with, susceptibility ×was observed. The values of coercitivity and
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remanence are 7.74 × 10-3 T and 2 × 10-3 emu/g, respectively. The noticeable coercivity of
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M-H loop could be due to strong ferromagnetism at room temperature. The strong magnetic
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behaviour can be attributed to the presence of small magnetic dipoles located at the surface of
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nanocrystals, which interact with their nearest neighbours inside the nanocrystal.
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Consequently, the interchange energy in these magnetic dipoles makes other neighbouring
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dipoles oriented in the same direction. In nanocrystals, surface to volume ratio increases, so
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the population of magnetic dipoles oriented in the same direction increase at the surface.
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Thus, the sum of the total amount of dipoles oriented along the same direction also increases
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subsequently. In short the crystal surface becomes usually more magnetically oriented. The
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narrow hysteresis implies a small amount of dissipated energy in repeatedly reversing the
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magnetization which is important for quick magnetization and demagnetization of the
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nanomagnet synthesized; this property could be employed for generation of heat for
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hyperthermia applications. From the magnetic characterization results of the nanoparticles
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obtained by the second method, a clear and strong paramagnetic nature with paramagnetic
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term, susceptibility ×was reported [14]. So a transition from the ferromagnetic
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state to the paramagnetic state of this system was observed.
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Furthermore, the origin and root cause of such strong magnetic behaviour of the host
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Gd2S3 nanoparticles were studied with the help of electronic structure calculations of Gd2S3
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cluster and fragments/nanostructures of -Gd2S3 of various sizes. Nanoparticles of Gd2S3
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were simulated by considering the bulk fragments of -Gd2S3 which are spherical for two
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different radii, resulting in Gd8S16 and Gd12S20 (sub nanometer) clusters. Spin polarized
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geometry optimization calculations were carried out on the Gd2S3, Gd8S16, and Gd12S20
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clusters without any symmetry constraints in the framework of generalized gradient
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approximation to density functional theory (GGA-DFT) using the DMol3 software package
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[15]. Perdew-Wang (PW91) [16] functional form, the generalized gradient approximations
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for exchange and correlation potential, is used in these calculations. The core electrons of Gd
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atoms are represented by norm-conserving Density functional Semi-core Pseudo Potentials
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(DSPP) [17] while the valence electrons are described by double numeric basis sets with
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polarization functions (DND).
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The S atoms are represented by the all electron DND basis set. During the self
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consistent field (SCF) calculations, tolerance for density was set to 106 e/bohr3, while the
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convergence criterion for energy was set to 106 Hartree. In the geometry optimization
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procedure, the structural parameters of Gd2S3 clusters were completely optimized for various
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spin states without any symmetry constraints. The geometries were considered to be fully
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optimized when the energy converged to 105 Hartree and the gradient to 104 Hartree/Å. For
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Gd2S3 and Gd8S16 clusters the AFM and FM states are energetically degenerate with AFM
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being lower in energy ( = 0.01 – 0.02 eV), while in case of Gd12S20 cluster, the FM state is
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more favorable ( = 0.11 eV). The preference of AFM coupling between the Gd atoms in
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Gd2S3 cluster is similar to the recently reported theoretical study [18] of Gd2O3, where the Gd
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atoms prefer to couple anti-ferromagnetically. The molecular orbital (MO) analysis of Gd2S3
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cluster show that the highest occupied molecular orbital (HOMO) is dominated by sulfur – p
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orbitals with a minor contribution from the Gd – f orbitals.
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The lowest occupied molecular orbital (LUMO), on the other hand, is due to the
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empty f orbitals of Gd atom. In case of larger clusters, namely Gd8S16 and G12S20, the HOMO
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has contributions only from the sulfur-p orbitals, thereby indicating the localized
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characteristics of Gd-f electronic states. The most important feature of our calculations
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however, is the large spin magnetic moments of these clusters exhibited in their FM state.
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The total spin magnetic moment of the Gd2S3 cluster in FM spin alignment is calculated to be
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14 B, with the magnetic moment on each Gd atom being 7.09 B, thus giving a clear
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indication of the formation of Gd3+ ions. In case of Gd8S16 and G12S20 clusters, the total spin
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magnetic moment was found to be 56 B and 84 B, respectively for their FM state. In both
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these clusters, every Gd is carrying a spin magnetic moment of ~7.03 B, again indicating the
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presence of Gd3+ ions in these clusters. Thus, the 7.0 B spin magnetic moment on each Gd3+
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is due to the f electrons. It is noteworthy here that, the spin magnetic moment of free Gd atom
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is 9 B, while the total magnetic moment is 6.53 B. In order to further confirm the origin of
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such a high spin magnetic moment in the FM states of these clusters, we have carried out spin
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density analysis of Gd8S16 and Gd12S20 clusters, which are given in fig. ESI 4. The spin
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density plots of Gd8S16 and Gd12S20 clusters clearly show that the spin magnetic moment
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originate from the highly localized Gd – f electrons, with no contribution from sulfur atoms.
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The spin density plots for the AFM states of these clusters (not shown here) also illustrate
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that the AFM coupling between the Gd is resulting from the formation of Gd3+ and their
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corresponding localized f electron spins.
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MRI experiments were carried out to calculate the relaxivities of the nanoparticles.
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The following contrast agent preparations were placed in a series of 1.5 ml tubes and diluted
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with saline to concentrations ranging from 2.56 µM to 1.6 mM. Final preparations had pH
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values ranging from 7 to 7.2. Saline was taken as a reference. All data and images were
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acquired using a Bruker Avance DRX 400 MHz FT-NMR Spectrometer with Micro-Imaging
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facility. The signal decay time constants T1 (spin-lattice relaxation time) and T2 (spin-spin
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relaxation time) of each sample were measured using a spin-echo saturation-recovery (time to
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echo (TE) = 15ms; repetition time (TR) = 1900, 1700,1500, 1300, 1100, 900, 700 and 500
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ms) and a Carr-Purcell-Meiboom-Gill spin-echo train (TE = 15-40 ms in 5 ms increments;
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TR = 1900 ms) respectively. The recording parameters were, TR (Repetition Time) = 500,
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700, 900, 1100, 1300, 1500 ms TE (Echo Time) = 13 ms, FOV (Field of view) = 40 mm,
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Matrix Size = 256 × 256, Slice thickness as well as inter-slice thickness = 2mm, NEX
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(Number of Excitation) = 4.
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SAR is defined as the amount of heat released by a unit weight of the material per unit
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time during exposure to an oscillating magnetic field of defined frequency and field strength.
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It is determined by the “rate of temperature rise” and expressed as mean absorbed power per
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unit mass (W/g): The SAR (W g-1) value was calculated by using the formula.
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SAR = c (T/t)
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Where, Specific heat capacity is the amount of heat energy required to raise the
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temperature of a body per unit of mass. T/t is the temperature increase per unit time, initial
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slope of the temperature versus time dependence. SAR should be as large as possible for
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hyperthermia application.
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SAR depends on many factors:
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Magnetic field amplitude (H)
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Frequency (f)
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Particles permeability ()
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Particle size and shape’
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All the AC magnetic field experiments (to determine SAR values and for MHT) were
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conducted using a radio frequency (RF) generator in a magnetic field with a frequency of 380
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kHz and at an amplitude of 200 Gauss. For SAR experiments, the suspensions with five
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different concentrations of nanoparticles were prepared in water, 5 µg/ml, 10 µg/ml, 15
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µg/ml, 20 µg/ml, and saline was used as control. To measure the SAR values of these
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suspensions, 2 mL of each suspension was taken in identical double walled test tubes where
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the space between the outer and inner walls were evacuated to minimize the heat loss. These
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tubes were then placed in a thermo-cool cylinder for thermal insulation, which were then kept
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inside the coil generating the magnetic field. An alcohol thermometer was used to measure
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the increase in temperature in the suspension, which avoids electrical and magnetic effects of
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the generator on the thermometer. As a control experiment, only water was kept in the test
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tube and exposed to the above field and the temperature increase was measured with respect
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to time.
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The SAR values for the suspensions having 20, 15, 10 and 5µgml-1 of the LMQDs
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were estimated to be 25.1, 24.2, 23.3 and 15.9 W g-1 respectively at 37 °C. The temperature
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versus time graphs, for various suspensions containing different concentrations of the
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synthesized LMQDs are shown in fig. ESI 5 (a). The temperature rise was faster in the case
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of higher amount of MNPs. In the controlled experiment, where only water (saline) was kept
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in the RF-generator coil, the temperature did not rise significantly, even after 50 min of AC
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magnetic field application.
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To determine the nanoparticles’ cytotoxicity to cells, 3-[4,5-dimethylthiazol- 2yl]-2,5-
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diphenyltetrazolium bromide (MTT) assay was carried out. MTT assay is a standard
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colorimetric assay that measures the activity of enzymes in mitochondria. 3-[4,5-
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dimethylthiazol- 2yl]-2,5-diphenyltetrazolium bromide (MTT), is reduced to formazan in the
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mitochondria of living cells, changing from yellow to purple. The absorbance was recorded at
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an excitation of 570 nm. The viability of MCF 7 cells was determined using a standard
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methyl thiazol tetrazolium bromide (MTT) assay (Sigma, St Louis, USA).
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Briefly, 24 h after incubation with nanoparticles, MTT was added to each well (the
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final concentration of MTT in medium was 50 μg ml-1) for 4 h at 37 ◦C. The formazan that
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formed in the cells was dissolved adding 0.5 ml of DMSO in each dish, and the optical
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density was evaluated at 570 nm in an Elisa reader. Cell survival was expressed as the
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percentage of absorption, of treated cells in comparison to a control, as shown in fig. ESI 5
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(b). It is evident that the drug molecule conjugated nanomagnets show higher efficieny of
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cancer cell killing compared to cells treated with the drug only.
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To observe the effect of nanoparticles on the cellular morphologies, electron
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microscopic studies were carried out. The cells were incubated with nanoparticles (0.025 mg
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to 0.2 mg/ml) for 24 h. After incubation, the cover slips were washed with PBS, fixed with
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paraformaldehyde (1% wt/vol), and washed with Phosphate buffered saline (PBS). The cells
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were observed under the scanning electron microscope to visualize the nanoparticles treated
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cells.
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Scanning electron microscopy was performed to visualise the effect on the
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morphology of the breast cancer cells upon treatment with these nanoparticles. Fig. ESI 6
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shows the Environmental Scanning Electron Microscopic (ESEM) images of (a) 200 µg/mL
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of LMQDs treated MCF 7 cell lines (b) 100 µg/mL LMQDs treated MCF 7 cell lines (c)100
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µg/mL Methotrexate conjugated to LMQDs treated MCF 7 cell lines and (d) 100 µg/mL
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Methotrexate treated MCF 7 cell lines. Fig. ESI 6 c showing complete destruction of the cells
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compared to fig. ESI 6 (d) with only partial destruction of cells. Fig. ESI 7 shows the higher
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magnification ESEM images: of (a) untreated MCF 7 cell lines (b) 100 µg/mL Methotrexate
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conjugated
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LMQDs treated MCF 7 cell lines.
LMQDs treated MCF 7 cell lines (c) 200 µg/mL Methotrexate conjugated
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Gadolinium nanoparticles were synthesized by a simple reduction method. For
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synthesizing Gd nanoparticles, 0.4M of aqueous Gd chloride solution was added to 0.25mM
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of citric acid solution in 100ml of distilled water. To this a 100ml solution of 0.01M sodium
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borohydride (NaBH4) in distilled water was added as a reducing agent. After mixing for
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about 4 hours, 1N NaOH at pH 7 was added and the solution was kept for stirring for about
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another 4 hours. The Colour development was observed. This solution was centrifuged and
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the pellet collected was washed 3 times in distilled water and once in ethanol to remove all
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impurities and unreacted salts. They were synthesized and similarly characterized by several
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techniques, some of which have been discussed here.
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The longitudinal relaxation time (T1) was measured using saturation recovery method
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where axial images of the microfuge tubes filled with Gd-nanocrystals solution were
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obtained. The MRI contrast property of Gd-nanocrystals was measured by determining the
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longitudinal (T1) and transverse relaxation time (T2) of water at 25°C. Rapid acquisition of
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Multiple slice multiple echo protocol was used for determination of T2. Typical parameters
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used for T1 measurements are given in experimental section. The T1 value was determined
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by fitting the function STR= STR()(1-exp(-TR/T1)) to the signal intensity versus repetition
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time graph (Figure not shown here).
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The longitudinal (T1) relaxation times along with bio-imaging studies were carried
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out at various solutions of different Gd-nanocrystals concentrations. The solutions of the
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nanoparticles were imaged using a clinical MRI scanner at 3T. The vials containing
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nanoparticles were placed in a human wrist coil during the scanning process. All data and
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images were acquired using a 3T Siemens MRI instrument. The recording parameters were,
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TR (Repetition Time) = 500,700,900,1100,1300,1500 ms TE (Echo Time) = 13 ms, FOV
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(Field of view) = 40 mm, Matrix Size = 256 X 256, Slice thickness as well as inter-slice
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thickness = 2mm, NEX (Number of Excitation) = 4. Further to demonstrate their applicability
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for MRI, imaging was performed in mice models.
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For in vivo studies, mice induced with skin cancers were taken and imaged before and
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after injection of Gd nanocrystals suspension. Mice were anesthetized for imaging with the
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use of a general anaesthesia administrated (intra peritoneal I.P. injection of a mixture of 12
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mg/kg xylazine and 80 mg/kg ketamine.). MR imaging was performed with a 3 T imager (GE
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Sigma Exite Twin-speed, GE Health Care, Milwaukee, WI) using a human wrist coil. Groups
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of three mice each were used to evaluate contrast enhancement efficacy for the contrast
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agent. Following acquisition of baseline images, nanoparticles were administered via tail vein
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and mice were repositioned in the microimager. Contrast agent was administered via tail vein
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at a dose of 0.01 mmol Gd/kg for both standard Gd-DTPA and synthesized Gd nanocrystals
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suspension. The mice were placed in a human wrist coil and scanned in a Siemens 3T MRI
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scanner at preinjection and at 2 min and after 30 min post injection using a fat suppression
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3D FLASH sequence (TR ) 7.8 ms, TE ) 2.74 ms, 25° flip angle, 0.5 mm slice thickness).
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Axial tumor MR images were also acquired using a 2D spin-echo sequence (TR ) 400 ms, TE
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) 8.9 ms, 90° flip angle, 2.0 mm slice thickness). The images in Fig. ESI 8(a-c) show axial
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MRI sections of the mice. These images reveal that the tumours on the back of the mice have
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been resolved after application of the contrast agent. It is quite clear from the studies the Gd-
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nanocrystals based contrast agent very clearly resolves the cancer lesions and shows the
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growth of cancer much better than existing commercial product Gd-DTPA.
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For curcumin conjugation, the pellet formed after the Gadolinium salt reduction by
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the method discussed above was taken. The schematic diagram is shown in fig. ESI 9. The
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pellet was resuspended in 20 ml water 10 ml curcumin in ethanol was added to the
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Gadolinium nanoparticles suspended in water at pH 6 and the solution was kept in continuous
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stirring condition for 24 hours. After that the curcumin coated Gd nanoparticles were
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obtained. The solution was centrifuged and the pellet was collected after washing as
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described earlier. The sample obtained was resuspended in water and used for further studies.
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These could be employed simultaneously for the imaging as well as therapy for several
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cancers. Owing to the role of curcumin in nervous system disorders, this conjugate could be
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used for imaging and therapeutic applications for various brain lesions.
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In the optical absorption spectrum of the curcumin conjugated Gd nanoparticles fig.
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ESI 9 (b), a peak is seen at around 420 nm (this corresponds to the absorption of the
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curcumin molecule) which is not seen in the Gd nanoparticles UV absorption spectrum (fig.
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ESI 10 (a). This is indicative of the fact that curcumin has bound to the Gd nanoparticles. The
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optical absorption spectrum of the Gd-nanocrystal-water suspension, exhibits a broad
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absorption band edge in the UV spectral range. The energy gap (Eg) can be estimated by
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assuming direct transition between the conduction band and the valence bands. Theory of
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optical absorption gives the relationship between the absorption coefficients α and the photon
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energy hυ for direct allowed transition as,
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(αhυ)2 = A(hυ – Eg) where , α is the absorption coefficient of the material and
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A is a function of index of refraction and hole/electron effective masses. From
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this equation, the direct band gap is determined using Tauc Plot, when straight portion of the
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(αhυ)2 against hυ plot is extrapolated to intersect the energy axis at α =0,. The calculated
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band gap is around 5 eV which is quite close to the reported band gap of Gd2O3. This
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suggests that the suspension of Gd-nanocrystals in water also contain some Gadolinium oxide
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nanoparticles, due to oxidation of some Gd-nanocrystals. These results also support the
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SAED and TEM results shown in fig. ESI 4 in main manuscript, pertaining to the Gd
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nanoparticles formation.
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Figure 1
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Fig. ESI 1: XRD spectra of the synthesized luminescent nanomagnets of Gd2S3:Eu3+.
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Figure 2
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Fig. ESI 2: TEM images of few nanorods: scale bar 100 nm, (a) and (b) showing
uniformly monodispersed nanoparticles of spherical nature.
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Figure 3
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Fig. ESI 3: Room temperature M-H loop for LMQDs (b) shows the magnified M-H loop
at the origin. Observed clear hysteresis indicates ferromagnetic nature of the LMQDs at
room temperature.
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Figure 4
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Gd12S20
Gd8S16
FM state: Spin 56
Gd: 7.03 B
FM state: Spin 84
Gd: Avg. 7.04 B
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Fig. ESI 4: The spin density plots of Gd8S16 and G12S20 clusters in their FM state. The
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yellow spheres represent the sulfur atoms. The spin density surfaces around Gd atoms
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are denoted by semi-transparent blue spheres.
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Figure 5
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Figure 5: (a) The temperature versus time graphs. (b) MTT assay of MCF 7 cell lines.
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Figure 6
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Fig. ESI 6: ESEM images: 200 µg/mL of (a) LMQDs treated MCF 7 cell lines (b) 100
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µg/mL LMQDs treated MCF 7 cell lines (c) Methotrexate conjugated LMQDs treated
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MCF 7 cell lines and (d) 100 µg/mL Methotrexate treated MCF 7 cell lines.
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Figure 7
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Fig. ESI 7: Higher magnification ESEM images: of (a) untreated MCF 7 cell lines (b)
100 µg/mL Drug conjugated LMQDs treated MCF 7 cell lines (c) 200 µg/mL Drug
conjugated LMQDs treated MCF 7 cell lines.
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Figure 8
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Fig. ESI 8: Axial view MRI images of mice having advanced stage cancer (a) Pre
contrast (b) 2 min post injection of Gd-DTPA (c) 2 post injection of Gd-nanocrystals
and (d) coronal image of the tumour bearing mice post injection of Gd-nanocrystals.
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Figure 9
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Fig. ESI 9: (Scheme A): Schematic diagram for Synthesis process and growth of
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colloidal citrate stabilized nanocongregates. (Scheme B): Strategy of curcumin
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modifications.
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Figure 10
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Fig. ESI 10 Absorption spectra of (a) Gd nanoparticles and (b) curcumin conjugated to
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Gd nanoparticles.
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