Dipalmitoyl phosphatidylcholine (DPPC), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol) 2000] (DSPE-PEG2000) were bought from Avanti Polar Lipids (Alabaster, AL). Mag (gadopentate dimeglumine) was bought from Bayer HealthCare Pharmaceuticals Inc. (Wayne, NJ). All solvents used were of analytical grade.
Preparation and characterization of Mag-Lnps
The fabrication of Mag-Lnps was adopted from previous methods and other publications (Affram et al. 2015; Ghaghada et al. 2008, 2009). In brief, DPPC, MPPC and DSPE-PEG2000 in the ratio 90:10:4 respectively were weighed to yield a total weight of 100 mg. Subsequently, lipids were dissolved in chloroform followed by the removal of chloroform by drying the lipid mixture solution in a stream of dry nitrogen gas under a fume hood.
Residual chloroform in the thin lipid mixture was further removed by incubating the mixture in a vacuum chamber for 2 h. The thin film was then hydrated by vortexing it with 2 mL (an aliquot fashion) of PBS solution containing 37.5 mM Mag to yield a suspension of multilaminar vesicles (MLV). The lipid suspension was then extruded (11 times) through a 200-nm polycarbonate membrane placed between two filter supports.
This process was repeated using 100-nm polycarbonate membrane. The temperature of the heating block was kept below 80 °C while the liquid suspension was kept between 55 and 60 °C (above the phase transition of the liquid) during hydration and extrusion.
The formed liposomes solution was diluted with distilled water according to the manufacturer’s instructions and the particle size and zeta potential determined on the NICOMP™ Particle Sizing Systems (Santa Barbara, California, USA).
Transmission electron microscopy (TEM) measurement of Mag-Lnps
As part the physicochemical characterization of Mag-Lnps, the structural morphology of Mag-Lnps was determined by TEM (JEOL) at 120 kV. A drop of the Mag-Lnps prepared as described above, serially diluted and placed on a carbon-coated copper grid and negatively stained with 1% ammonium molybdate. The grid was dried and viewed by JEM-ARM200cF TEM.
MRI scans were carried out on the ultra-wide bore 21.1 T (900 MHz) vertical magnet built at the National High Magnetic Field Laboratory (NHMFL) (Fu et al. 2005). The magnet is equipped with a Bruker Avance III console and data acquisition was performed with ParaVision 6.0.1 acquisition and processing (BioSpinCorp, Billerca, MA) together with a 64-mm inner diameter high-performance gradient (Resonance Research Inc, MA) capable of producing 0.6 T/m peak gradient strength.
Mag-Lnp phantom imaging via MRI
Mag-Lnps phantoms were prepared as described by others (Affram et al. 2017; Agyare et al. 2014). In brief, Mag-Lnps solution was diluted by thoroughly mixing it with distilled water in a ratio of 1:1, 1:5, 1:10 and 1:100. The various Mag-Lnps diluted solutions were further mixed with 1% agarose solution in the ratio 1:1 to yield concentrations 14.3 mM (1:1), 4.8 mM (1:5), 2.6 mM (1:10) and 0.3 mM (1:100), warmed slightly and carefully loaded into microcapillary tube. After solidification, the ends of each tube were sealed with wax.
For control, equal volume of distilled water and 1% agarose was prepared, loaded into microcapillary tube and allowed to solidify. All test samples and control were stored at 4 °C overnight prior to MR imaging (Agyare et al. 2014). Using a 10-mm birdcage radio frequency (RF) coil, Mag-Lnps phantoms together with control were loaded together and measurements were made to determine 1/T1 (R1) and as 1/T2 (R2) relaxation rates. Data acquisition was achieved with a 100 × 100 matrix in a plane resolution of 100 × 100 µm with a slice thickness of 1 mm. A spin echo (SE) sequencing using nine (9) incrementing repetition times (TR) between 26 and 15,000 ms and 16 incrementing echo time (TE) between 10 and 160 ms was performed to obtain R1 and R2, respectively. Signal intensity was used to determine R2 relaxation rates by mapping regions of interest (ROIs) from the sample scan against TE using a single exponential decay function.
Female PDX mice (NOD/Scid-IL2rg) models with tumor planted in the left flank were used and all procedures with mice were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Florida A & M University Animal Care and Use Committee. Mice were put up in a virus-free, indoor, light and temperature controlled barrier environment with unlimited access to water and food.
In vivo imaging of pancreatic PDX via MR
The mice bearing pancreatic PDX were injected with single dose (4 mg/kg) of Mag or Mag-Lnps equivalent dose of Mag through intraperitoneal (IP) route after the average tumor size has reached 12 mm. Prior to imaging, mice were sedated with isoflurane in an enclosed chamber. Following sedation, tumor-bearing mouse was secured with a home-built 33-mm inner diameter RF birdcage coil with tumor positioned at center of the coil. Data acquisition (T1 maps) was acquired with 250 × 210 mm in-plane resolution and 0.75 mm slice thickness (Fig. 6). TR was incremented 6 times between 170 and 4000 ms and TE was 6 ms. SNR was measured over time with a turbo spin echo using TE/TR = 5/1500 ms and 90 × 90 mm in plane resolution and a 1-mm slice. Both acquisitions took 7 min.
Data characterizing Mag and Mag-Lnps was presented as mean ± standard deviation and statistical difference between Mag and Mag-Lnps was determined using Student’s t test and considered significant at p < 0.05. With the exception of in vivo MRI and MRI phantoms, all other experiments were performed at least in triplicate and analyzed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA).