Enhancement of Gas-filled Microbubble Magnetic Susceptibility by Iron Oxide Nanoparticles

INTRODUCTION Gas-filled microbubbles were originally developed as an intravascular contrast agent to enhance backscattering in ultrasound imaging. Microbubbles possess the ability to be an MR susceptibility contrast agent due to the induction of large local magnetic susceptibility differences by the gas-liquid interface. Feasibility of microbubbles as an MR pressure sensor, based on the susceptibility change caused by pressure-induced microbubble size change, has been explored through theoretical and phantom studies. Gas-filled microbubbles have also been shown as an MR susceptibility contrast agent in vivo. However, microbubble susceptibility effect is relatively weak when compared with other intravascular MR susceptibility contrast agents. By optimizing the microbubble size distribution and choice of shell coating material and core gas, it is possible to substantially enhance the microbubble susceptibility effects and reduce the dosage requirement for MR applications. In this study, we aim to demonstrate that microbubble susceptibility effects can be improved by embedding and entrapping iron oxide nanoparticles. METHODS Synthesis of iron oxide nanoparticles embedded albumin-coated microbubbles: Iron oxide nanoparticles embedded albumin-coated microbubbles (AMB) were produced by an adapted sonication method. Briefly, 18 mg of monocrystalline iron oxide nanoparticles (MION; MGH) was added into a 5% solution of bovine serum albumin (10857, USB Corporation). The mixture was preheated to about 70C and sonicated under aseptic conditions using an ultrasound frequency of 20 kHz. Synthesis of iron oxide nanoparticles entrapped polymeric microbubbles: Iron oxide nanoparticles entrapped polymeric microbubbles (polymeric MB) were produced by an adapted double emulsion method. Briefly, 0.5g poly(D,L-lactide-co-glycolic acid 50:50, PLGA; Sigma) was dissolved in 10 mL of ethyl acetate (Sigma). 1 mL of MION solution (1.164mg/mL) was added to the polymer solution and probe sonicated for 30 s. The W/O emulsion was then poured into a 5% poly(vinyl alcohol) (PVA; Sigma) solution and homogenized for 5 min. The double (W/O)/W emulsion was then poured into a 2% isopropyl alcohol (Sigma) and stirred at room temperature for 1 hour. The capsules were collected by centrifugation, washed once with deionized water, centrifuged at 15°C for 5 min. at 3000g and the supernatant discarded. The capsules were then washed three times with hexane (Sigma). The capsules were frozen in a -80°C freezer and lyophilized using a freeze dryer to fully dry the capsules and sublime the encapsulated water. MRI and Data Analysis: All MRI experiments were performed on a 7 T Bruker MRI scanner. Microbubble phantom study was performed with 38-mm quadrature resonator for RF transmission and receiving. AMB were diluted from a well-mixed microbubble suspension to 4% volume fraction with the addition of saline, while polymeric MB were prepared by adding saline of 2 mL to 50 mg of the lyophilized powder. The microbubbles were then placed in separate 2-mL cylindrical phantom tubes. Each phantom tube was slowly warmed to room temperature and gently mixed for 2 min outside the magnet prior to MR measurements. To ensure uniform suspension of microbubbles, the phantom was then continuously stirred by rotation inside the magnet. It was then arrested in horizontal position immediately before the start of MR acquisition sequence. Apparent transverse relaxation rate enhancement (ΔR2 ) was measured by acquiring multi-echo gradient-echo (GE) signals continuously without phase encoding for 2 min from an axial 1-mm slice at middle of the phantom. The measurement was repeated six times for each microbubble phantom. The parameters were TR = 1000 ms, TE = 3.5, 7, 10.5, 14, 17.5, 21, 24.5, 28 ms, flip angle = 30o and NEX = 1. Phantom R2 * values were computed by monoexponential fitting of the peak magnitudes of the multi-echo GE signals using a software toolkit developed in MATLAB (MathWorks). Initially, there was a uniform suspension of microbubbles. As GE signals were acquired, microbubbles started to migrate upward; therefore, in the final state the microbubbles aggregated in the upper part of the tube. Microbubble induced ∆R2 * was then calculated as the difference between R2 * in the initial state and that in the final state. To demonstrate that MION were embedded and entrapped, R2 * was measured before and after cavitation, which was performed by applying ultrasound of frequency 40 kHz. R2 * maps of the suspending solutions were acquired before and after cavitation with multiple gradient echo sequences. In vivo Demonstration: Normal SD rats (~200-250 g) were injected intravenously with 0.2 mL of microbubble suspension (~4% volume fraction; N = 1 for AMB with MION and N = 1 for polymeric MB with MION) at a rate of 1.2 mL/min to avoid possible microbubble destruction due to high pressure and shear stress under femoral vein catheterization. Dynamic susceptibility weighted liver MRI was performed with respiratory-gated single-shot GE-EPI sequence under inhaled isoflurane anaesthesia using TR ≈ 1000 ms, TE = 10 ms, FA = 90o, FOV = 50 mm × 50 mm, slice thickness = 2 mm, acquisition matrix = 64 × 64, and NEX = 1. RESULTS AND DISCUSSIONS Values of R2 * were plotted against time in Figure 1 for different microbubbles. As GE signals were acquired, microbubbles started to migrate upward; therefore, in the final state values of R2 * were due to the suspending solution. The different amounts of free MION in the suspended solution accounted for the difference in the R2 * of the suspending solution. Microbubble induced ∆R2 * of different microbubbles were depicted in Figure 2. The MION embedded and entrapped would enhance the susceptibility effect and increased the values of ∆R2 *