A new acoustically-active delivery vehicle was developed by conjugating liposomes and microbubbles, using the high affinity interaction between avidin and biotin. Binding between microbubbles and liposomes each containing 5% DSPE-PEG2kBiotin was highly dependent on avidin concentration and observed above an avidin concentration of 10 nM. With an optimized avidin and liposome concentration, we measured and calculated as high as 1000 to 10,000 liposomes with average diameters of 200 and 100 nm, respectively, attached to each microbubble. Replacing avidin with neutravidin resulted in 3-fold higher binding, approaching the calculated saturation level. Highspeed photography of this new drug delivery vehicle demonstrated that the liposome-bearing microbubbles oscillate in response to an acoustic pulse similar to microbubble contrast agents. Additionally, microbubbles carrying liposomes could be spatially concentrated on a monolayer of PC-3 cells at the focal point of ultrasound beam. As a result of cell-vehicle contact, the liposomes fused with the cells and internalization of NBD-cholesterol occurred shortly after incubation at 37°C, with internalization of NBD-cholesterol substantially enhanced in the acoustic focus.

We report on a novel application of the layer-by-layer (LbL) assembly technique to attach multiple layers of DNA and poly-l-lysine (PLL) onto preformed lipid-coated microbubbles to increase the DNA loading capacity. We first measured the effects of the cationic lipid fraction and salt concentration on the microbubble stability. Microbubble production and stability were robust up to a cationic lipid fraction of 40 mol % in 10 mM NaCl. DNA adsorption was heterogeneous over the microbubble shell and occurred primarily on the condensed phase domains. The amount of adsorbed DNA, and subsequently adsorbed PLL, increased linearly with the fraction of cationic lipid in the shell. DNA loading was further enhanced by the LbL assembly method to construct polyelectrolyte multilayers (PEMs) of DNA and PLL. PEM buildup was demonstrated by experimental results from zeta potential analysis, fluorescence microscopy, UV spectroscopy, and flow cytometry. The PEMs exhibited two growth stages and were heterogeneously distributed over the microbubble surface. The DNA loading capacity onto the microbubbles was enhanced by over 10-fold by using five paired layers. However, the PEM shell did not prevent oscillation or destruction during ultrasound insonification. These results suggest that the surface can be compartmentalized to make multifunctional, high-payload ultrasound contrast agents for targeted gene therapy.

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