542. Development and Characterization of Ultrasound Responsive Microbubbles for Gene Delivery

Ultrasound in combination with microbubbles (sonoporation) has recently acquired much attention in the field of gene delivery. The mechanism by which ultrasound mediates cellular delivery has been ascribed as cavitation. Cavitation is the alternate growing and shrinking of microbubbles as a result of the high and low pressure waves of the ultrasound. Eventually, these cavitating microbubbles can also implode due to these high pressure waves.The cavitation and especially the implosion of microbubbles generate local shock waves and microjets that can temporally perforate the cell membrane. However, a major limitation of the currently available microbubbles is that they have a short lifetime, and neither bind or protect the therapeutic DNA against nuclease. Consequently, the aim of this work was to develop ultrasound responsive microbubbles which can bind and protect DNA against nucleases. We developed new microbubbles by coating classic albumin microbubbles with a cationic charged polymer via the layer-by-layer technique. Albumin microbubbles were prepared by sonicating a dextrose- albumin solution with perfluorobutane gas. Poly(allylamine hydrochloride) (PAH) coated microbubbles were prepared starting from the microbubbles above using the Layer-by-Layer technique. About 90% of the uncoated and coated microbubbles had a size between 1 and 5 μm. Coating of the microbubbles with PAH, turned the surface charge positive. The albumin shell (green) of the uncoated microbubbles and the PAH coat (red) of the coated microbubbles was subsequently visualized using CSLM (figure 1A and 1B ). The appearance of a red colored ring around the microbubbles further proved that the microbubbles are indeed coated with PAH. Incubation of unlabeled, uncoated microbubbles with green labeled pDNA resulted in a rather homogeneous distribution of the fluorescence (Figure 1C). However, an accumulation of the pDNA around the microbubbles occurred when the unlabeled PAH coated microbubbles were incubated wih the green labeled pDNA (Figure 1D). Similar conclusions could be drawn from zeta potential measurements. The maximum loading capacity of the PAH coated microbubbles was estimated around 0.1 pg/ microbubble. A 5-fold increase of the half-life of the microbubbles was obtained after coating them with PAH. The ability of the microbubbles to protect pDNA against nuclease cleavage was tested using gel electrophoresis. The uncoated microbubbles were not able to protect pDNA from degradation by DNase I. Oppositely the PAH coated microbubbles were able to prevent degradation.