Freeze-dried therapeutic microbubbles: stability and gas exchange

Microbubbles (MBs) are widely used as contrast agents for ultrasound imaging and have been shown to enhance therapeutic delivery. However, they are only stable in solution for a few hours after production, which limits their potential application. Freeze-drying provides long term storage, ease of transport and consistency for therapeutic preparations thereby facilitating their use in clinical settings. The ability to freeze-dry therapeutic MBs is an important development in the translation of therapeutic MBs for clinical application. Here we show that it is possible to freeze-dry, and reconstitute, therapeutic MBs (thMBs) that consist of a lipid-coated microbubble with an attached liposomal payload. These thMBs have been produced using a microfluidic approach, with either calcein as a model drug or gemcitabine (gem) encapsulated inside the liposomes. The results show that freeze-dried powder of thMBs can be stored for at least 6 months, and upon reconstitution maintain their structure, composition and drug loading as well as their in vivo echogenicity and stability. We have also demonstrated that this approach offers the ability to exchange the gas core in the bubble to allow thMBs with different gases to be constructed. This approach is advantageous for the translation process of thMBs as it allows precise control of drug and MB concentration and ease of production at the point of care.