Supporting informations Glycine-functionalized Fe3O4@TiO2:Er3+,Yb3+ Nanocarrier for Microwave-triggered Controllable Drug Release and Study on Mechanism of Loading/Release Process Using Microcalorimetry Hongxia Peng, Bin Cui*, Weiwei Zhao, Yingsai Wang, Zhuguo Chang, Yaoyu Wang Key Laboratory of Synthetic and Natural Functional Molecule Chemistry (Ministry of Education), Shaanxi Key Laboratory of Physico-Inorganic Chemistry, School of Chemistry & Materials Science, Northwest University, Xi’an 710069, PR China Experimental section Materials and Characterization All the chemical reagents used in this experiment were of analytical grade and were used without further purification. Ferric chloride hexahydrate (FeCl3·6H2O, purity 99%), sodium acetate (CH3COONa, purity >99.0%), ethylenediamine (C2H8N2, purity ≥98.0%), Ethylene glycol (purity 96.0%) were purchased from the Shanghai Chemical Reagent Factory (Shanghai, China). Tetrabutyl titanate (C16H36O4Ti) and isopropyl alcohol ((CH3)2CHOH, purity 99%) were purchased from National Reagent Corporation (Shanghai, China). Glycine was purchased from the Shanghai Chemical Reagent Factory (Shanghai, China). Etoposide (VP-16) was purchased from the Jiangsu Hengrui Medicine Factory. 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from the Sigma Chemical Company. A human breast cancer cell line (MCF-7) was purchased from the Key GEN Biotechnology Company (Nanjing, China). Deionized water was used in all of the experiments. X-ray diffraction (XRD) patterns of the samples were measured using an AXS D8 Advance Diffractometer (Bruker, Bremen, Germany) with Cu Kα radiation (λ = 0.15406 nm) at 40 kV and 40 mA. The morphologies and structures of the as-prepared samples were inspected using a JEM 2010 transmission electron microscope. Room-temperature Fourier-transform infrared (FT-IR) spectra of the samples, mounted on KBr discs, were recorded using a Perkin-Elmer (Waltham, MA, USA) 100 S spectrometer. Ultraviolet–visible light (UV–vis) spectral adsorption values were measured using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The up-conversion emission spectra of the samples were recorded with a Hitachi (Tokyo, Japan) F-4500 fluorescence spectrometer. An adjustable laser diode (980 nm, 2 W) was used as the excitation source. Magnetization measurements were performed at 300 K using an MPMS-XL-7 superconducting quantum interference device (SQUID) magnetometer. Drug release under microwave stimulation was controlled using an HBS-C medical microwave apparatus (Human Biosystems Inc., Palo Alto, CA, USA), at a working frequency of 2450±30 MHz and a microwave power of 0 to 150 W. The thermodynamics of the drug loading and release system were measured using an RD496-2000 Calvet microcalorimeter (Mianyang CAEP Thermal Analysis Instrument Company, Mianyang, China). The microcalorimeter was calibrated using the Joule effect, and its sensitivity was 64.28±0.04 uV/mW at 298.15 K. Absorbance of MTT by treated cells in the MTT assay was measured using an RT-6000 microplate reader (Rayto Life and Analytical Sciences Co. Ltd., Shenzhen, China). The electromagnetic parameters of the Fe3O4@TiO2:Er3+,Yb3+ nanoparticles were measured using a N5230A vector network analyzer (Agilent Technologies, Santa Clara, CA, USA) with the transmission and reflection mode in the 2 to 18 GHz band. All the measurements were performed at room temperature. Synthesis of the Fe3O4nanocomposites Amino-functional magnetic Fe3O4 nanoparticles were prepared by the solvothermal method [17]. Briefly, FeCl3·6H2O (1.0 g) was dissolved in ethylene glycol (30 ml) to form a clear solution, followed by the addition of NaAc (2.0 g) and ethylenediamine (6.5 g). The mixture was stirred vigorously for 30 min and then sealed in a teflon-lined stainless-steel autoclave (50mL capacity). The autoclave was heated to and maintained at 200 ◦C for 6 h, and then cooled to room temperature naturally. The black products were washed several times with ethanol and finally dried at 60 ◦C for 6 h. Then, the amino-functional Fe3O4 nanoparticles were obtained. Supplemental figures Figure S1 shows the XRD patterns of the Fe3O4, TiO2:Er3+,Yb3+ and Fe3O4@TiO2: Er3+, Yb3+ nanoparticles. Figure S1 (curves a) shows that the magnetite cores were well indexed to the cubic spinel structure of Fe3O4 JCPDS (No. 65-3107), with good crystallinity. Figure S1 (curves b) shows that the diffraction peaks can be readily indexed to the rutile type TiO2 nanostructure according to the standard JCPDS (No. 21-1272). In the case of Fe3O4@TiO2:Er3+,Yb3+ (Figure S1 curves c), not only show the characteristic diffraction peaks of cubic spinel Fe3O4, but there were also 3+ 3+ obvious diffraction peaks indexed to the anatase phase of TiO502:Er (PDF of phx10Fe3O4TiO point,Yb AA Smoothing ### 21-1272), which suggested the successful crystallization of TiO2:Er3+,Yb3+ on the 20 point AA Smoothing of Smoothed19_Sm surface of the Fe3O4 nanoparticles. In addition, we detected no peaks of other phases, ### indicating that no reaction occurred between the core and the shell during the synthesis process. Fe3O4 TiO2 Intensity (a.u.) (c) (b) (a) 10 Figure S1 20 30 40 50 2-theta (degree) 60 70 80 XRD patterns of the samples: (a) Fe3O4, (b) TiO2:Er3+,Yb3+ and (c) Fe3O4@TiO2:Er3+,Yb3+ To investigate the hydrodynamic sizes and stability of magnetic nanocarriers, the dried magnetic nanocarriers were redispersed in physiological saline to make aqueous dispersions, and hydrodynamic sizes of nanocarriers were determined by dynamic light scattering (DLS). The zeta potential of Fe3O4@TiO2:Er3+,Yb3+ and Fe3O4@TiO2:Er3+,Yb3+-Glycine is shown in Figure S2. The Fe3O4@TiO2:Er3+,Yb3+-Glycine nanocarriers have been proposed to have excellent stability at pH 7 and to provide negative charge at the surface that can be used to attach other biomolecules. The dispersion of nanocarriers in aqueous solution mainly depends on the particle surface zeta potential, the particles have certain zeta potential which produces electrostatic repulsion between particles. It will have a better dispersion and dispersion stability when its absolute value is higher. For Fe3O4@TiO2:Er3+,Yb3+-Glycine, owing to its several intrinsic properties, the Glycine not only provides –NH2 but also gives them excellent water solubility, long time stability against aggregation and biocompatibility. The hydrodynamic sizes of nanocarriers at pH 5, pH 6 and pH 7 and mean particle sizes measured by TEM are reported in Table 1. It was observed from DLS analysis that the hydrodynamic size was only slightly altered in different pH values leading to suspensions with a wider pH range of stability. The hydrodynamic size of the nanocarriers is gradually decreased with decreasing pH value, and the hydrodynamic sizes obtained from the DLS method were larger than the actual size of the nanocarriers obtained with TEM results, which can be attributed to the presence of few aggregates due to a lack of charge at this pH and the hydrophobic capping effect of nanocarrier nuclei when dispersed in aqueous media 40 30 Zeta potential (mv) 20 10 0 -10 -20 3+ 3+ Fe3O4@TiO2:Er ,Yb -Glycine 3+ 3+ Fe3O4@TiO2:Er ,Yb -30 -40 2 3 4 5 6 7 8 9 10 11 pH Figure S 2. Zeta potential of (a) Fe3O4 and (b) Fe3O4@TiO2:Er3+,Yb3+-Glycine Table 1. Experimental Results on Mean Particle Sizes Measured by TEM, Hydrodynamic Sizes at pH 5, pH 6 and pH 7 by DLS. Sample mean particle size by TEM (nm) hydrodynamic size at pH 5 (nm) hydrodynamic size at pH 6 (nm) hydrodynamic size at pH 7 (nm) 49.6 50.6 51.8 52.2 50.8 50.1 51.4 51.8 Fe3O4@TiO2:Er3+, Yb3+ Fe3O4@TiO2:Er3+, Yb3+- Glycine To investigate the up-conversion luminescent properties of Fe3O4@TiO2: Er3+, Yb3+, Fe3O4@TiO2:Er3+,Yb3+-Glycine and Fe3O4@TiO2:Er3+,Yb3+-Glycine-VP16 nanocomposites, the up-conversion luminescent spectra was recorded at room temperature. Figure S3 shows that the green emission band centered at 500~560 nm is attributed to the 4S3/2→4I15/2 electronic transition of the Er3+ ions. The 660 nm red emission is ascribed to the 4F9/2 → 4I15/2 transition of Er3+ ions [25]. Here, the role of Yb3+ is sensitizer. The sensitization of Yb3+ ions can improve the conversion efficiency of Er3+ ions significantly. This is because Yb3+ ions have large absorption cross section near a wavelength of 980 nm and they can transfer the excitation energy to the Er3+ ions nearby through resonance. The decreased up-conversion emission of Fe3O4@TiO2:Er3+,Yb3+-Glycine (curves b) and Fe3O4@TiO2:Er3+,Yb3+-Glycine-VP16 (curves c) nanocomposites could be caused by introduced reactive amino groups and drug at their surfaces, and leading to energy loss on the nanoparticles surface and cause fluorescence quenching by the introduced organic groups [27]. Although the up-conversion luminescent intensity of the Fe3O4@TiO2:Er3+,Yb3+-Glycine and Fe3O4@TiO2:Er3+,Yb3+-Glycine-VP16 nanocomposites decreased, the up-conversion luminescent emission intensity was still strong enough to permit bio-imaging [28]. ex=980 nm 4 4 F9/2 - I15/2 Intensity/a.u. a b c 4 4 S3/2 - I15/2 450 500 550 600 650 700 Wavelength/nm Figure S3 Upconversion emission spectra of (a) Fe3O4@TiO2:Er3+,Yb3+ , (b) Fe3O4@ TiO2:Er3+,Yb3+-Gly and (c) Fe3O4@TiO2:Er3+,Yb3+-Gly-(VP-16) Magnetic measurements showed that pure Fe3O4, Fe3O4@TiO2:Er3+,Yb3+, Fe3O4@TiO2:Er3+,Yb3+-Glycine and Fe3O4@TiO2:Er3+,Yb3+-Glycine-VP16 had magnetization saturation values of 71.9, 41.2, 31.3 and 26.7 emu/g, respectively (Figure S3). It is noteworthy that the Fe3O4@TiO2:Er3+,Yb3+-Glycine and Fe3O4@TiO2:Er3+,Yb3+-Glycine-VP16 retains strong magnetization, indicating its suitability for targeting and separation as a drug carrier. The saturation magnetization of Fe3O4@TiO2:Er3+,Yb3+-Glycine and Fe3O4@TiO2:Er3+,Yb3+-Glycine-VP16 nanocomposites occurred at 31.3 and 26.7 emu/g, which is smaller than that of the Fe3O4@TiO2:Er3+,Yb3+ nanoparticles. This is likely due to the decreased proportion of Fe3O4 in the nanocomposites. The magnetization was still strong enough to permit targeting and bio-separation. Moreover, the Fe3O4@TiO2: Er3+, Yb3+-Glycine-VP16 nanocomposites show fast response to the external magnet and can be readily re-dispersion occurred quickly with a slight shaking once the magnetic field was removed (Figure S4, inset). These results show that the nanocomposites possess excellent magnetic responsivity and redispersibility, which is important in terms of their practical manipulation [28]. 100 60 60 -1 M (emu g ) 40 M (emu g-1) 80 40 a 20 b c d 0 a b c d -20 -40 20 -60 -200 -150 -100 -50 0 50 100 150 200 H (Oe) 0 -20 -40 -60 -80 -100 -10000 -5000 0 5000 10000 H (Oe) Figure S4 Hysteresis Loops of (a) Fe3O4, (b) Fe3O4@TiO2:Er3+,Yb3+, (c)Fe3O4@TiO2: Er3+,Yb3+-Gly and (d) Fe3O4@TiO2:Er3+,Yb3+-Gly-(VP-16) O O O H OH O H O O O H O HO OH O O Figure S5 Anticancer Drug Etoposide (VP-16)