The monodispersed dextran coated iron oxide nanoparticles were synthesized by using a method described elsewhere [25
]. Briefly, 15 mL of dextran (MW 10,000 kDa) aqueous solution (15% w/w) was titrated with 4 mL NH4
OH (>25% w/w) to pH 11.7 at room temperature. Five milliliters of freshly prepared FeCl3
O (0.75 g) and FeCl2
O (0.32 g) aqueous solution was gradually injected into the alkali-treated dextran solution after passing through a 0.2 μm pore size filter. After 30 minutes, the black colloidal suspension was centrifuged at 10,000 rpm for 20 minutes to remove the aggregates. The supernatant was dialyzed in a dialysis bag with 25 kDa molecular weight cut off (Spectra/Pro 7, Spectrum Laboratories Inc.) against deionized water for 36 hours to remove ammonia in order to reach a pH value of 7.0. A centrifugal filter device (Ultracel YM-30, Millipore Co.) was used with a relative centrifugal force of 1500 × g to further purify and concentrate the dextran coated iron oxide dispersion. The size of the individual iron oxide nanoparticle cores measured by high resolution transmission electron microscopy (HRTEM) was 5.2 ± 0.8 nm () giving an overall hydrodynamic diameter of about 20 nm () measured by dynamic light scattering (DLS). The induced saturation magnetization of SPIO nanoparticles was measured as 54 emu/gr Fe at 300 K () using a super-conducting quantum interference device (SQUID – Quantum design MPMS).
Figure 1 (a) High-resolution TEM image of the dextran-coated SPIO nanoparticles. (b) Dynamic light scattering (DLS) measurement of hydrodynamic diameter of dextran-coated SPIO nanoparticles. (c) Magnetization of dextran-coated SPIO nanoparticles at different applied (more ...)
Mouse macrophage cells (J774A.1 cell line) were selected for this study due to their high rate of non-specific uptake. To load the cells with dextran-coated magnetic nanoparticles, cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% Fetal bovine serum (FBS) at 37°C in 5% CO2 and then were incubated with the suspension of nanoparticles at the concentration of 0.1 mg/mL Fe (i.e. 4×1014 NPs /mL suspension) for 24 hours. The average number of internalized dextran-coated SPIO nanoparticles was measured using inductively coupled plasma mass spectrometry (ICP-MS). For this purpose the culture was washed with 1× PBS six times to make certain that all non-internalized nanoparticles were removed. Then the labeled cells were removed from the culture, counted using a hemocytometer and dissolved in 35% trace metal-grade Nitric acid (HNO3) and then kept in an oven at 60°C for 12 hours. After baking, the sample was diluted to 1-2% Nitric acid and mass spectroscopy measurements were performed. The results indicated that the cell uptake in culture was (3.2±0.09)×104 particles per cell and were consistent across multiple measurements.
After incubation with NPs, cells were divided for TEM and pMMUS studies. For TEM imaging, cells were fixed in a mixture of 3% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer at pH 7.4 for 30 minutes. Following three buffer rinses, the cells were post-fixed for 30 minutes in reduced osmium, a mixture of 2% osmium tetroxide and 2% potassium ferrocyanide in the cacodylate buffer. The fixed cells were then placed in 2% uranyl acetate for 30 minutes before dehydration in an ethanol series (50-70-95-100%). Dehydration was followed with two changes of absolute acetone after which the cells were infiltrated with a 1:1 mixture of Spurr and EMBed 812 epoxy resins (Electron Microscopy Sciences, Hatfield, PA) which was subsequently polymerized for 2 days at 60°C. Sections with thickness of 60-70 nm were cut from the epoxy blocks and picked up on copper grids for imaging at 80 kV in a Tecnai Spirit BioTwin transmission electron microscope without further staining. As a control, cells not incubated with NPs were prepared with the same protocol.
To demonstrate the ability of pMMUS imaging to detect the intracellular trafficking of SPIO nanoparticles, tissue mimicking phantoms were made out of 6% polyvinyl alcohol (PVA) by weight to mimic the mechanical and magnetic properties of soft tissue. For ultrasound imaging, 0.2% of 15 μm silica particles were added to create acoustic backscattering. Two cylindrical-shape compartments with the diameter of 2.5 mm were created within the phantom. Both compartments were filled with 10% gelatin gel containing either (1) macrophages incubated with dextran-coated SPIO nanoparticles overnight and fixed in 10% formalin solution for 30 minutes, or (2) fixed macrophages mixed with the SPIO nanoparticles. In each inclusion, 5×106 macrophages were used, i.e., the number of cells was the same in each inclusion. Furthermore, the concentration of iron was the same in each inclusion – this was achieved by measuring the amount of iron (or number of SPIO nanoparticles) internalized by cells used in the first inclusion and then adjusting the number of nanoparticles in the second inclusion to match the concentration of iron. Therefore, the inclusions containing an equal number of cells and nanoparticles simulated the two separate states of cell/nanoparticles before and after intracellular trafficking. Similar to the PVA background, 0.5% of 15 μm silica particles were added to the inclusions to act as ultrasound scatters. Once the inclusions solidified, the phantom was placed in a water cuvette for pMMUS imaging.
The diagram of the custom-built US/pMMUS imaging system is presented in . The 10 ms long magnetic excitation pulses were generated by a high-power voltage-controlled current amplifier driving the current to a solenoid magnetic coil. A cone shaped iron core made of ferritic stainless steel was embedded into the center of the coil to increase the magnetic flux density and also to focus it to the desired imaging region. The magnetic flux density of pulses was measured to be about 8000 G at 5 mm above the coil. An active cooling system was used to remove the heat generated within the coil due to the large amount of current passing through it. The ultrasound RF signals before, during and after application of the magnetic pulse were acquired at a high pulse repetition frequency of 1 kHz using a focused single-element ultrasound transducer operating at 25 MHz (focal depth = 25.4 mm, f # 4) interfaced with an ultrasound pulser/receiver. A block-matching motion-tracker algorithm was applied to calculate the magnetically induced displacement.[17
] The cross-section of the phantom with two inclusions was imaged by mechanically moving the water cuvette with the phantom while the ultrasound transducer and the magnetic coil were kept fixed and stationary. The maximum displacement at each position within the imaging plane was then calculated (), normalized to the magnetic pulse strength and displayed in the magneto-motive ultrasound image[17
] – an image that combines both the B-scan ultrasound image and spatially co-registered pulsed magneto-motive displacement image.
Figure 2 (a) Block diagram of a pMMUS imaging system used to image a tissue mimicking phantom with two magnetic inclusions. Both inclusions have approximately identical concentration of iron and macrophages but in one of the inclusions the SPIO nanoparticles are (more ...)