Dotted Core-Shell Nanoparticles for T1 -Weighted MRI of Tumors.

Chemicals: Poly(acrylic acid) (PAA, Mw = 1800), iron (Ⅲ) chloride (FeCl 3 , ≥ 97%), gadolinium (III) nitrate hexahydrate (Gd(NO 3 ) 3 · 6H 2 O, 99.9%), N -(3-Dimethylaminopropyl)- N’ - ethylcarbodiimide (EDC, ≥ 97%), N-hydroxysuccinimide (NHS, 98 %), Rhodamine 6G (R6G), phalloidin-FITC, and Hoechst 33258 were purchased from Sigma-aldrich (USA). Iron (Ⅱ) sulfate heptahydrate (FeSO 4 ·7H 2 O) was purchased from Acros organics. Glu-{Cyclo[Arg-Gly-Asp-(D-Phe)-Lys]} 2 ( i.e. dimeric RGD peptide, or RGD2, 97.92%, Mw = 1318.51) was purchased from C S Bio Co. (CA, USA 94025). Synthesis of ES-MION/GdON hybrid nanoparticles (FeGd-HN): 40 mL of PAA (Mw = 1800) solution (4.0 mg/mL) was first purged with nitrogen (≥ 50 min) to remove oxygen. The polymer solution was then heated to reflux (100 o C). After that, a 0.8 mL mixture of iron precursors (500 mM FeCl 3 plus 250 mM FeSO 4 ) was quickly injected into the heated polymer solution, followed by addition of 12 mL of ammonia solution (28 %). The reaction was kept at 100 o C for 30 min under magnetic stirring to obtain the seeds of extremely small magnetic iron oxide nanoparticles (ES-MIONs). After that, 0.8 mL of Gd(NO 3 ) 3 (62.5 ~ 1000 mM) and 6.0 mL of ammonia solution (28 %) were added into the reaction system. The reaction was continued for 90 min under magnetic stirring at 100 o C to obtain the FeGd-HN. The ES-MIONs were synthesized by a similar method without addition of Gd precursor. [ 18 ] Finally, the solutions were cooled down to room temperature. The obtained FeGd-HN and ES-MIONs were purified by membrane dialysis (MWCO 6–8 kDa) against Milli-Q water for 5 days with a daily change of the water. The purified FeGd-HN and ES-MIONs were concentrated by centrifugal ultrafiltration (Millipore, MWCO 3 kDa). The Fe and Gd concentrations of the solutions were measured by inductively coupled plasma optical emission spectrometry (ICP-OES; Agilent 5100). The Fe or Gd recovery of the FeGd-HN was calculated from the molar ratio of Fe or Gd in the obtained FeGd-HN to the feeding Fe or Gd. In addition, 2.0 mL of the nanoparticle solutions were dried at 70 o C to calculate the mass concentration of the nanoparticles (with Fe 3 O 4 , Gd 2 O 3 and PAA). Characterization of FeGd-HN: Distributions of Fe, Gd, and O in the FeGd-HN nanoparticles were mapped using electron energy loss spectroscopic imaging (EELSI) in a scanning transmission electron microscope (STEM). [ 39 – 41 ] The EELSI data were acquired with a Tecnai TF30 transmission electron microscope (FEI, Inc.) equipped with a Quantum imaging filter (Gatan Inc.), operating at an accelerating voltage of 300 kV. STEM images containing 2048 × 2048 pixels were acquired using a high-angle annular dark-field (HAADF) detector (Fischione Instruments, Inc.). For small selected regions within the HAADF images, EELSI data were acquired with approximately 35 pixels x 35 pixels x 2048 energy channels, spanning energy losses from 445 eV to 1470 eV, with 0.5 eV/channel. The pixel size was approximately 1 nm x 1 nm, and the pixel dwell time 0.1 s. Synthesis of FeGd-HN3-RGD2: The RGD2 were conjugated onto the surface of FeGd-HN3 via the reaction between –COOH and –NH 2 in the presence of EDC/NHS. Typically, 10 μL of EDC (55 μmol) and 50 μL 13 mg/mL of NHS (5.65 μmol) were added into 5.0 mL of FeGd-HN3 solution ( C Fe = 3.10 mM, C Gd = 1.61 mM, ice cold) under magnetic stirring. After that, 100 μL of RGD2 (5.0 mg/mL, 3.8 mM) were added into the mixtures. After 16 h of reaction at room temperature under magnetic stirring, the obtained FeGd-HN3-RGD2 were washed 3 times using Milli-Q water by centrifugal ultrafiltration (Millipore, MWCO 3 kDa) to remove unreacted EDC, NHS, RGD2, and side product EDU, and finally dissolved in 5.0 mL of Milli-Q water. Synthesis of Rhodamine 6G-loaded FeGd-HN3-RGD2: To investigate the internalization of FeGd-HN3 or FeGd-HN3-RGD2 in cells by flow cytometry and laser scanning confocal microscopy (LSCM), Rhodamine 6G (R6G) was loaded onto the surface of FeGd-HN3 or FeGd-HN3-RGD2. Typically, 4.0 mL of FeGd-HN3 ( C Fe = 3.10 mM, C Gd = 1.61 mM), or FeGd-HN3-RGD2 ( C Fe = 3.10 mM, C Gd = 1.61 mM) were mixed with 0.7 mL of Rhodamine 6G (10 μM) under magnetic stirring at room temperature. After 24 h, the obtained R6G-FeGd-HN3 or R6G-FeGd-HN3-RGD2 solution was washed using Milli-Q water by centrifugal ultrafiltration (Millipore, MWCO 3 kDa) to remove free R6G. The resultant R6G-FeGd-HN3 or R6G-FeGd-HN3-RGD2 was dispersed in 4.0 mL of Milli-Q water. Cellular Uptake of the Nanoparticles Measured by ICP: 2.0 mL of U-87 MG cells in complete growth medium were seeded into each well of a 6-well culture plate with a cell density of 1.0×105 cells/mL and allowed to adhere at 37 oC for 24 h. The growth medium was then replaced with fresh media (2.0 mL, without FBS) without or with FeGd-HN3-RGD2, or FeGd-HN3 (CGd = 80 μM). After further 2.0 h incubation, the cells were washed twice with PBS, treated with trypsin for 3.0 min, and then centrifuged at 500 × g for 5 min to remove the extracellular nanoparticles. The obtained cells were used for Gd measurement by ICP.