Synthesis of magnetic core
Nanoparticles were synthesized by the decomposition method (24). The flask contains 5.055 g n‑docosane (99 %, Acros Organic), 0.181 g iron oxide hydrate (catalyst grade, 30-50 Mesh, Sigma Aldrich), and 3 g oleic acid (>99 %, Sigma Aldrich). The mixture was put under vacuum for 30 minutes, then heated under reflux to 340°C under argon gas for 1h30m. The mixture was then dissolved in n-pentane (for analysis RPE) and diethyl ether/ethanol (2:1). Nanoparticles were collected by centrifugation and washed with diethyl ether/ethanol 2:1. Finally, all nanoparticles were dissolved in 15 mL CHCl3 (RPE for analysis) and 200 µL oleylamine, then stored at 4°C.
Enwraping of a magnetic core with a mesoporous silica shell
The formation of the silica shell was carried out with tetraethylorthosilicate (TEOS) agent (25). The flask containing 0.125 g cetyltrimethylammonium bromide (CTAB) (≥98 %, Sigma Aldrich), 60 mL pure water (for HPLC, Sigma Aldrich), and 440 µL NaOH 2M was stirred at 70°C for 1h. The iron oxide core in CHCl3 (11 x 100 µL) was then added at 3-4 min intervals. The mixture was stirred at 80°C for 1h30 before the addition of 100 µL of TEOS (≥ 99% (GC), Sigma Aldrich), followed by another addition of 500 µL TEOS 30 min later. The mixture was kept at 80°C for another 1h30 period. Nanoparticles were collected by centrifugation at 20 rpm for 15 minutes (Allegra64R centrifuge, Beckman coulter, USA), then suspended in 0.075M NH4NO3 for one night. Nanoparticles were washed with absolute ethanol (99 %, VWR) and pure water before being dried under vacuum.
Each batch of nanoparticles was examined using Transmission Electronic Microscopy (TEM) and morphometric measurements were carried out with magnification at 15000x and 50000x using a JEOL 1200 EXII microscope (Japan). Magnetic data were collected with a Quantum Design MPMS-XL SQUID magnetometer working in the temperature range 5-300 K.
Silanization of trans-resveratrol
To graft trans-resveratrol on the surface of core/shell nanoparticle, silane-trans-resveratrol derivatives were synthesized by the base-catalyzed reaction of (triethoxysilyl)propyl isocyanate with trans-resveratrol in tetrahydrofuran THF shown in Scheme 3.1.
1.14 g trans-resveratrol (5 mmol, > 99 %, TCI Europe) and 2.50 mL 3-(triethoxysilyl)propyl isocyanate (10 mmol, 95 %, Sigma) were dissolved in 20 mL dry THF, then 70 µL N,N-diisopropylethylamine (> 99 %, TCI Europe) was added. The reaction was stirred under reflux at 70°C for 3 days. The reaction was followed by infrared spectroscopy and thin layer chromatography (TLC) using pre-coated TLC sheets with UV fluorescent silica gel (Merck 60F254). The solvent was then removed under reduced pressure and the mixture of (1), (2), and (3) was purified by flash chromatography (Isolera One, Biotage, Sweden) using a Biotage SNAP ULTRA column and n-hexane/acetone 2:1 as eluent, to give 1 (50%), 2 (30%), 3 (16%).
To elucidate the structures of 1, 2, and 3, NMR spectra were measured in DMSO-d6, using a Bruker Avance 400 MHz spectrometer. Chemical shifts (δ) were expressed in ppm relative to tetramethyl silane (TMS) and coupling constants (J) in Hz. Mass spectra were obtained using a Synapt G2-S high-definition mass spectrometry system (Waters Corp., Milford, MA) equipped with an electrospray ionization (ESI) source. Since compound 1 did not ionize well in the ESI source, its mass spectrum was obtained using a Bruker RapifleX MALDI TOF spectrometer (Bruker Daltonik GmbH, Bremen, Germany) equipped with a Smartbeam 3D laser. The MALDI TOF analysis was performed in positive ion reflectron mode over a mass range of m/z 450 -3000 with or without cationization agent. The matrix (DHB, 10 g.L-1) and the sample were mixed (10:1 v/v) and 1 µL of the mixture was spotted onto the MALDI target plate and air dried. For the experiments with a cationizing agent, 1 μL of 10 g.L-1 sodium trifluoroacetate was added to the mixture before spotting.
1H-NMR of 1: δ ppm (400 MHz, DMSO-d6, Me4Si): 7.81 (m, 3H, N-H), 7.60 (d broad, J = 8 Hz, 2H, H-3', H-5'), 7.32 (d, J = 16 Hz, 1H, H-α), 7.20 (m, 3H, H-β, H-2', H-6'), 7.11 (d, J = 8 Hz, 2H, H-2, H-6), 6.76 (t, J = 2 Hz, 1H, H-4), 3.77 (q, J = 6 Hz, 18H, -Si-O-CH2-CH3), 3.05 (q, J1 = 4 Hz, J2 = 8 Hz, 6 H, -Si-CH2-CH2-CH2-N), 1.54 (dt, J1 = 4 Hz, J2 = 8 Hz, 6H, -Si-CH2-CH2-CH2-N), 1.17 (m, 27H, -Si-O-CH2-CH3), 0.59 (t, J = 6 Hz, 6H, Si-CH2-CH2-CH2-N). 13C-NMR: δ ppm (101 MHz, DMSO-d6, Me4Si) 154.0 (C=O), 151.7 (C-3, C-5), 150.8 (C-4'), 138.9 (C-1'), 133.5 (C-3', C-5'), 129.1 (Cβ), 127.4 (C-α), 126.7 (C-1), , 122.0 (C-2', C-6'), , 116.2 (C-2, C-6), 114.6 (C-4), 57.7 (Si-O-CH2-CH3), 54.9 (NH-CH2-CH2-CH2-Si), 22.8 (NH-CH2-CH2-CH2-Si), 18.2 (Si-O-CH2-CH3), 7.2 (NH-CH2-CH2-CH2-Si). HRMS (ESI) for C44H75N3O15Si3 [(M +formic acid) – H]-: m/z calc. 1014.4514. MALDI-TOF [M + Na] m/z 992.4.
1H-NMR of 2: δ ppm (400 MHz, DMSO-d6, Me4Si): 9.65 (s, 1H, 3-OH), 7.76 (s broad, 2H, -NH), 7.64-7.56 (m, 2H, H-3', H-5'), 7.18-7.08 (m, H-2', H-6', H-α, H-β), 6.80 (m, 2H, H-2, H-6,), 6.41 (t, J = 2 Hz, 1H, H-4), 3.76 (t, J = 7 Hz, 12H, -Si-O-CH2-CH3), 3.05 (t, J1 = 7.5 Hz, J2 = 4 Hz, 4H, -Si-CH2-CH2-CH2-N), 1.53 (m, 4H, -Si-CH2-CH2-CH2-N), 1.16 (m, 18H, -Si-O-CH2-CH3), 0.59 (m, 4H, Si-CH2-CH2-CH2-N). 13C-NMR:δ ppm (101 MHz, DMSO-d6, Me4Si) 158.2 (C-5), 154.3 (C-3), 154.2 (C=O), 150.7 (C-4'), 138.9 (C-1'), 133.6 (C-1), 128.1 (C-β), 127.7 (C-5'), 127.3 (C-3'), 122.0 (C-α), 115.8 (C-2'), 115.6 (C-6'), 110.4 (C-2), 110.2 (C-6), 108.4 (C-4), 57.7 (Si-O-CH2-CH3), 43.2 (NH-CH2-CH2-CH2-Si), 23.5 (NH-CH2-CH2-CH2-Si), 18.1 (Si-O-CH2-CH3), 7.2 (NH-CH2-CH2-CH2-Si). HRMS (ESI) for C34H54N2O11Si2 [M – H]-: m/z calc. 721.3196. MALDI-TOF [M + Na + H]+ m/z 745.3.
1H-NMR of 3: δ ppm (400 MHz, DMSO-d6, Me4Si): 9.24 (s, 2H, 3-OH, 5-OH), 7.76 (t, J = 6 Hz, 1H, -NH-CO), 7.57 (t, J = 8 Hz, 2H, H-3', H-5'), 7.09-7.02 (m, 4H, H-α H-β, H-2', H-6'), 6.44 (m, 2H, H-2, H-6), 6.17 (t, J = 2 Hz, 1H, H-4), 3.77 (t, J = 7 Hz, 6H, -Si-O-CH2-CH3), 3.05 (t, J = 7 Hz, 2H, N-CH2-CH2-CH2-Si), 1.53 (q, J = 6 Hz, 2H, -N-CH2-CH2-CH2-Si), 1.18 (t, J = 7 Hz, 9H, -Si-O-CH2-CH3), 0.59 (t, J = 6 Hz, 4H, N-CH2-CH2-CH2-Si). 13C-NMR:δ ppm (101 MHz, DMSO-d6, Me4Si) 159.2 (C-3, C-5), 154.9 (C=O), 151.0 (C-4'), 139.4 (C-1'), 134.5 (C-1), 129.3 (C-β), 127.8 (C-3', C-5'), 127.6 (C-α), 122.5 (C-2', C-6'), 105.3 (C-2, C-6), 102.9 (C-4), 58.4 (Si-O-CH2-CH3), 43.8 (NH-CH2-CH2-CH2-Si), 23.5 (NH-CH2-CH2-CH2-Si), 18.8 (Si-O-CH2-CH3), 7.8 (NH-CH2-CH2-CH2-Si). HRMS (ESI) for C24H33NO7Si [M – H]-: m/z calc. 474.1947. MALDI-TOF [M +Na + H]+ m/z 498.2
Functionalized nanoparticles preparation
Three flasks, each containing 100 mg of core/shell (CS) nanoparticles, were added with 1 mL toluene (ACS reagent, Sigma Aldrich). Three other flasks contained 150 mg of 1, 2, and 3, respectively, in 1 mL toluene. The 1 mL suspensions of CS nanoparticles were added to the latter flasks along with 60 µL of ultrapure water(18.5 MΩ) then stirred and heated up to 70°C for 15 hours. The functionalized CS1, CS2 and CS3 nanoparticles were then collected by washing with ethanol and drying under vacuum for 3 – 4 hours.
The successful grafting of 1, 2 and 3 on CS was verified by recording the UV and IR absorption spectra of CS1, CS2, and CS3 using a UV-1800 spectrometer (Shimadzu, Japan) with 10 mm quartz cuvettes (101-QS, Hellma™, Germany), and a Spectrum Two FT-IR spectrometer (Perkin Elmer, England) in transmittance mode with KBr discs.
The distribution of hydrodynamic diameter (dH) of the nanoparticles was studied at 25°C by Dynamic Light Scattering (DLS) using a Zetasizer Nano-ZS (Malvern Instruments Limited, UK), adapted to assess particle sizes between 5 and 1000 nm. The instrument was equipped with a 633 nm diode laser and a DTS1070 cell. DLS was analyzed at the back scattering angle of 173°. In this configuration, the contributions of rotational diffusion effects in the observed autocorrelation profiles can be neglected and the translational diffusion coefficient, D, can be assessed (26). Five replicates of each colloidal suspension were homogenized by an ultrasound bath for 5 min and were immediately analyzed by Zetasizer Nano-ZS over 3 min.
Zeta potential was also measured with the Zetasizer Nano-ZS. An electric field of 120V was applied across the DTS1060C zeta cell and the electrophoretic mobility of the colloidal suspension was then measured by Laser Doppler Velocimetry (LDV). The zeta potential is referred to as the electrostatic charge at the splining plane boundary separating ions within the diffuse layer which moves with the nanoparticles, and ions that remain with the bulk water, corresponding to the Stern shell. Zeta potential is related to the surface electrostatic charge of nanoparticles 241.
For extinction cross-section determination, all nanoparticles were initially suspended in absolute ethanol and were then diluted at the final suspension concentration of 50 ppm (50 µg/mL) in aqueous tris (hydroxymethyl)aminomethane (Tris) buffer (50 mM), previously adjusted to pH 3.0, 5.0, 7.0, and pH 10.0 with HCl 37% and HCl 0.1 N. Extinction was recorded by Enspire microplate reader (PerkinElmer, Singapore) at 1 nm step in front face configuration using 96 wells black microplates (UV-Star µClear, Greiner-Bio-One, Germany) set up at 27 °C.