Ligand Distribution and Lipid Phase Behavior in Phospholipid-Coated Microbubbles and Monolayers

Abstract

Phospholipid-coated targeted microbubbles are ultrasound contrast agents that can be used for molecular imaging and enhanced drug delivery. However, a better understanding is needed of their targeting capabilities and how they relate to microstructures in the microbubble coating. Here, we investigated the ligand distribution, lipid phase behavior, and their correlation in targeted microbubbles of clinically relevant sizes, coated with a ternary mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), with PEG40-stearate and DSPE-PEG2000. To investigate the effect of lipid handling prior to microbubble production in DSPC-based microbubbles, the components were either dispersed in aqueous medium (direct method) or first dissolved and mixed in an organic solvent (indirect method). To determine the lipid-phase behavior of all components, experiments were conducted on monolayers at the air/water interface. In comparison to pure DSPC and DPPC, the ternary mixtures had an additional transition plateau around 10–12 mN/m. As confirmed by infrared reflection absorption spectroscopy (IRRAS), this plateau was due to a transition in the conformation of the PEGylated components (mushroom to brush). While the condensed phase domains had a different morphology in the ternary DPPC and DSPC monolayers on the Langmuir trough, the domain morphology was similar in the coating of both ternary DPPC and DSPC microbubbles (1.5–8 μm diameter). The ternary DPPC microbubbles had a homogenous ligand distribution and significantly less liquid condensed (LC) phase area in their coating than the DSPC-based microbubbles. For ternary DSPC microbubbles, the ligand distribution and LC phase area in the coating depended on the lipid handling. The direct method resulted in a heterogeneous ligand distribution, less LC phase area than the indirect method, and the ligand colocalizing with the liquid expanded (LE) phase area. The indirect method resulted in a homogenous ligand distribution with the largest LC phase area. In conclusion, lipid handling prior to microbubble production is of importance for a ternary mixture of DSPC, PEG40-stearate, and DSPE-PEG2000.

The Impact of Lipid Handling and Phase Distribution on the Acoustic Behavior of Microbubbles

“…The addition of cholesterol to the indirect DSPC-based microbubble coating affected both the ligand and the lipid phase distribution. Indirect DSPC microbubbles without cholesterol had a mostly homogeneous ligand distribution as shown by fluorescence microscopy imaging, which is in agreement with results from Langeveld et al [ 16 ]. However, all types of microbubbles with cholesterol had significantly more heterogeneous and variable ligand distribution than those without cholesterol.…”

“…Biotinylated lipid-coated microbubbles with a C 4 F 10 gas core were made as described previously [ 27 ], by probe sonication at 20 kHz with a Sonicator ultrasonic processor XL2020 at a power setting 10 (HeatSystems, Farmingdale, NY, USA) for 10 s. Three types of microbubbles were made by altering the production method or adding cholesterol to the microbubble coating. For microbubbles without cholesterol, the coating components (84.8 mol% DSPC; 8.2 mol% PEG40-stearate; 5.9 mol% DSPE-PEG2000; 1.1 mol% DSPE-PEG2000-biotin) were prepared with either an indirect or a direct method as described previously [ 16 ]. In short, for the indirect method, the components were dissolved in chloroform/methanol (9:1 vol/vol), the solvent was evaporated using argon gas, and the obtained lipid film was dried overnight under vacuum.…”

“…The streptavidin-conjugated microbubbles were imaged by microscopy as described by Langeveld et al [ 16 ]. In short, the microbubbles were placed between quartz glass in 87% glycerol ( v / v in phosphate-buffered saline) to reduce Brownian motion and imaged with a Leica TCS 4Pi confocal laser-scanning microscope [ 30 ].…”

“…Quantitative analysis was performed on the 4Pi microscopy data using custom-developed image analysis software in MATLAB (Mathworks, Natick, MA, USA), based on the method described by Langeveld et al [ 16 ]. The microbubble coating was subdivided into 32 parts, of which the mean fluorescence pixel intensity ( I part for the green channel and I part-rhod for the red channel) was calculated.…”

“…The microbubble coating can also be altered by the distribution of the phospholipid and PEGylated-emulsifier molecules over the microbubble coating, depending on the lipid handling prior to microbubble production by probe sonication. The use of organic solvent resulted in a more homogeneous ligand distribution than the use of aqueous solutions only [ 16 ]. The effect of lipid handling on the acoustic response of microbubbles, however, has not been investigated.…”