Delivery of chlorambucil using an acoustically-triggered perfluoropentane emulsion

Abstract

Ultrasound-mediated delivery systems have mainly focused on microbubble contrast agents as carriers of drugs or genetic material. This study uses micron-sized, perfluoropentane (PFP) emulsions as carriers of chlorambucil (CHL), a lipophilic chemotherapeutic. The release of CHL is achieved via acoustic droplet vaporization (ADV), whereby the superheated emulsion is converted into gas bubbles using ultrasound. Emulsions were made using an albumin shell and soybean oil as the CHL carrier. The ratio of the PFP to soybean oil phases in the droplets and the fraction of droplets that vaporize per ultrasound exposure were shown to correlate with droplet diameter. A 60-min incubation with the CHL-loaded emulsion caused a 46.7% cellular growth inhibition, whereas incubation with the CHL-loaded emulsion that was exposed to ultrasound at 6.3 MHz caused an 84.3% growth inhibition. This difference was statistically significant (p < 0.01), signifying that ADV can be used as a method to substantially enhance drug delivery.

Figure 1

Left: Experimental setup used to perform ADV experiments with OptiCells™. Right: Exposure conditions utilized during experiments. The emulsion was added to the chamber containing adherent CHO cells and subsequently exposed to US to cause ADV of the introduced droplets.

Figure 2

A visible image (left) and its corresponding fluorescent image (right) of the dual-phase emulsion containing PFP and soybean oil stained with a fluorescent dye. The spacing between the two horizontal lines on the hemacytometer is 200 μm and a 20 μm scale is included in each image. Although large droplets appear in this figure, droplets larger than 10 μm in diameter account for only 4.9% (by number) of total droplets. Refer to Figure 4 for a size distribution, obtained via Coulter counter, for the emulsion.

Figure 3

Left: For n = 338 droplets, the ratio of the inner to outer droplet diameter is plotted as function of the outer droplet diameter. The raw data is plotted using open, gray circles and the averaged, binned (1 μm) data is presented as closed, black circles. Right: A histogram of the raw data.

Figure 4

The emulsion size distribution, obtained using a Coulter counter, plotted as the number percent of total droplets. The 50 μm aperture enables the sizing of particles whose diameters are between 1 and 30 microns. The mean droplet diameter is 3.06 ± 0.21 μm with 4.9% (by number) of the droplets larger than 10 μm in diameter. The CHL loading in the droplets is 3.12±0.01 mg/mL emulsion.

Figure 5

A comparison of the mean (n = 3) ADV thresholds, plotted with standard deviations, for single-phase and dual-phase droplets as a function of average droplet diameter. The single-phase ADV thresholds are taken from a previous study (Fabiilli et al. 2009). The dual-phase droplets with a mean diameter of 6.96 μm were obtained by centrifuging the emulsion. The data labeled ‘dual-phase (shifted)’ has been corrected for the estimated mean PFP core diameter, using Figure 3. The horizontal error bars were obtained based on the standard deviation in Figure 3 (right). The following experimental conditions were used: degassed water at 37°C, 3.5 MHz single e lement transducer, 83 Hz pulse repetition frequency, and 13 cycles.

Figure 6

Left: Cytotoxicity of CHL initially dissolved in DMSO on CHO cells for 15 and 60-minute exposures. Right: The mean CHO cell diameter as a function of the %GI. For both the left and right plots, each data point is the average of six wells, from three independent experiments. Additionally, error bars are standard deviations of the means.

Figure 7

Mean (n = 5) %GI and standard deviation for each of the eight experimental groups. The presence (+) or absence (-) of each parameter – droplets, CHL, and US – is indicated above each group number. The same exposure procedure, acoustic parameters, droplet concentration, and CHL concentration (100 μM whether in DMSO or emulsified) was used for all groups.

Figure 8

Mean (n = 5) cell diameter and standard deviation for each of the eight experimental groups. The presence (+) or absence (-) of each parameter – droplets, CHL, and US – is indicated above each group number. The same exposure procedure, acoustic parameters, droplet concentration, and CHL concentration (100 μM whether in DMSO or emulsified) was used for all groups.

Figure 9

The number and volume weighted distributions for the dual-phase droplets are plotted along with the mean (n=3) fraction of droplets vaporized, as a function of droplet size, for different number of exposure passes by the ultrasound array. The standard deviation is plotted for the single pass case, with similarly sized standard deviations obtained for the 2 and 5 pass cases. The small decrease in efficiency between 1 and 2 μm is due to the subtraction errors between the treated (with US) and untreated (without US) cases.