Freeze-Dried Therapeutic Microbubbles: Stability and Gas Exchange.

Microbubbles (MBs) are widely used as contrast enhancement agents for ultrasound imaging and have the potential to enhance therapeutic delivery to diseases such as cancer. Yet, they are only stable in solution for a few hours to days after production, which limits their potential application. Freeze-drying provides long-term storage, ease of transport, and consistency in structure and composition, thereby facilitating their use in clinical settings. Therapeutic microbubbles (thMBs) consisting of MBs with attached therapeutic payload potentially face even greater issues for production, stability, and well-defined drug delivery. The ability to freeze-dry thMBs represents an important step for their translation to the clinic. Here, we show that it is possible to freeze-dry and reconstitute thMBs that consist of lipid-coated MBs with an attached liposomal payload. The thMBs were produced microfluidically, and the liposomes contained either calcein, as a model drug, or gemcitabine. The results show that drug-loaded thMBs can be freeze-dried and stored for at least 6 months. Upon reconstitution, they maintain their structural integrity and drug loading. Furthermore, we show that their in vivo echogenicity is maintained post-freeze-drying. Depending on the gas used in the original bubbles, we also demonstrate that the approach provides a method to exchange the gas core to allow the formulation of thMBs with different gases for combination therapies or improved drug efficacy. Importantly, this work provides an important route for the facile off-site production of thMBs that can be reformulated at the point of care.

[1]  Damien V. B. Batchelor,et al.  Nested Nanobubbles for Ultrasound-Triggered Drug Release , 2020, ACS applied materials & interfaces.

[2]  M. Versluis,et al.  Foam-free monodisperse lipid-coated ultrasound contrast agent synthesis by flow-focusing through multi-gas-component microbubble stabilization , 2020, Applied Physics Letters.

[3]  K. Maruyama,et al.  Scale-up production, characterization and toxicity of a freeze-dried lipid-stabilized microbubble formulation for ultrasound imaging and therapy , 2020, Journal of liposome research.

[4]  C. Moran,et al.  Probing phospholipid microbubbles by atomic force microscopy to quantify bubble mechanics and nanostructural shell properties. , 2019, Colloids and surfaces. B, Biointerfaces.

[5]  F. Kiessling,et al.  Shelf-Life Evaluation and Lyophilization of PBCA-Based Polymeric Microbubbles , 2019, Pharmaceutics.

[6]  N. Kudo,et al.  Development and evaluation of stability and ultrasound response of DSPC-DPSG-based freeze-dried microbubbles , 2019, Journal of liposome research.

[7]  S. Peyman,et al.  Evaluation of lipid-stabilised tripropionin nanodroplets as a delivery route for combretastatin A4. , 2017, International journal of pharmaceutics.

[8]  Sudeok Kim,et al.  Uniform Drug Loading into Prefabricated Microparticles by Freeze‐Drying , 2017 .

[9]  S. Peyman,et al.  Characterisation of Liposome-Loaded Microbubble Populations for Subharmonic Imaging. , 2017, Ultrasound in medicine & biology.

[10]  S. Kawakami,et al.  Tumor growth suppression by the combination of nanobubbles and ultrasound , 2016, Cancer science.

[11]  A. Lee,et al.  Post-Formation Shrinkage and Stabilization of Microfluidic Bubbles in Lipid Solution. , 2016, Langmuir : the ACS journal of surfaces and colloids.

[12]  Ji-Bin Liu,et al.  Development of an ultrasound sensitive oxygen carrier for oxygen delivery to hypoxic tissue. , 2015, International journal of pharmaceutics.

[13]  Paul S. Sheeran,et al.  Vaporization dynamics of volatile perfluorocarbon droplets: a theoretical model and in vitro validation. , 2014, Medical physics.

[14]  Nico de Jong,et al.  Acoustic behavior of microbubbles and implications for drug delivery. , 2014, Advanced drug delivery reviews.

[15]  S. Evans,et al.  High-frequency subharmonic imaging of liposome-loaded microbubbles , 2013, 2013 IEEE International Ultrasonics Symposium (IUS).

[16]  S. Peyman,et al.  Research spotlight: microbubbles for therapeutic delivery. , 2013, Therapeutic delivery.

[17]  M. Borden,et al.  Lipid monolayer collapse and microbubble stability. , 2012, Advances in colloid and interface science.

[18]  Steven Freear,et al.  Expanding 3D geometry for enhanced on-chip microbubble production and single step formation of liposome modified microbubbles. , 2012, Lab on a chip.

[19]  Stephen Meairs,et al.  Self-assembled liposome-loaded microbubbles: The missing link for safe and efficient ultrasound triggered drug-delivery. , 2011, Journal of controlled release : official journal of the Controlled Release Society.

[20]  S. D. De Smedt,et al.  Tumor cell killing efficiency of doxorubicin loaded microbubbles after ultrasound exposure. , 2010, Journal of controlled release : official journal of the Controlled Release Society.

[21]  M. Wheatley,et al.  Preserving enhancement in freeze-dried contrast agent ST68: Examination of excipients. , 2010, International journal of pharmaceutics.

[22]  George M Whitesides,et al.  Formation of bubbles in a multisection flow-focusing junction. , 2010, Small.

[23]  M. Borden,et al.  Microbubble dissolution in a multigas environment. , 2010, Langmuir : the ACS journal of surfaces and colloids.

[24]  A. Katiyar,et al.  Growth and dissolution of an encapsulated contrast microbubble: effects of encapsulation permeability. , 2009, Ultrasound in medicine & biology.

[25]  Niek N. Sanders,et al.  Drug loaded microbubble design for ultrasound triggered delivery , 2009 .

[26]  Sosaku Ichikawa,et al.  A comparative study of microbubble generation by mechanical agitation and sonication , 2008 .

[27]  Y. Baba,et al.  Effect of surfactant type on microbubble formation behavior using Shirasu porous glass (SPG) membranes , 2008 .

[28]  P. Sontum,et al.  Physicochemical characteristics of Sonazoid, a new contrast agent for ultrasound imaging. , 2008, Ultrasound in medicine & biology.

[29]  H. M. Nielsen,et al.  alpha,alpha'-trehalose 6,6'-dibehenate in non-phospholipid-based liposomes enables direct interaction with trehalose, offering stability during freeze-drying. , 2008, Biochimica et biophysica acta.

[30]  Paul A Dayton,et al.  Maintaining monodispersity in a microbubble population formed by flow-focusing. , 2008, Langmuir : the ACS journal of surfaces and colloids.

[31]  Yanjing Chen,et al.  Encapsulation and Releasing of Calcein by Spontaneously Formed Zwitterionic/Anionic Vesicle without Separation , 2007 .

[32]  S. Stainmesse,et al.  Freeze-drying of nanoparticles: formulation, process and storage considerations. , 2006, Advanced drug delivery reviews.

[33]  Alexander L. Klibanov,et al.  Microbubble Contrast Agents: Targeted Ultrasound Imaging and Ultrasound-Assisted Drug-Delivery Applications , 2006, Investigative radiology.

[34]  S. Feinstein,et al.  The powerful microbubble: from bench to bedside, from intravascular indicator to therapeutic delivery system, and beyond. , 2004, American journal of physiology. Heart and circulatory physiology.

[35]  Jonathan R. Lindner,et al.  Microbubbles in medical imaging: current applications and future directions , 2004, Nature Reviews Drug Discovery.

[36]  E. Unger,et al.  Therapeutic applications of lipid-coated microbubbles. , 2004, Advanced drug delivery reviews.

[37]  E Stride,et al.  Microbubble ultrasound contrast agents: A review , 2003, Proceedings of the Institution of Mechanical Engineers. Part H, Journal of engineering in medicine.

[38]  L. Hoff,et al.  Acoustic properties of NC100100 and their relation with the microbubble size distribution. , 1999, Investigative radiology.