The Role of Ultrasound-Driven Microbubble Dynamics in Drug Delivery: From Microbubble Fundamentals to Clinical Translation.

In the last couple of decades, ultrasound-driven microbubbles have proven excellent candidates for local drug delivery applications. Besides being useful drug carriers, microbubbles have demonstrated the ability to enhance cell and tissue permeability and, as a consequence, drug uptake herein. Notwithstanding the large amount of evidence for their therapeutic efficacy, open issues remain. Because of the vast number of ultrasound- and microbubble-related parameters that can be altered and the variability in different models, the translation from basic research to (pre)clinical studies has been hindered. This review aims at connecting the knowledge gained from fundamental microbubble studies to the therapeutic efficacy seen in in vitro and in vivo studies, with an emphasis on a better understanding of the response of a microbubble upon exposure to ultrasound and its interaction with cells and tissues. More specifically, we address the acoustic settings and microbubble-related parameters (i.e., bubble size and physicochemistry of the bubble shell) that play a key role in microbubble-cell interactions and in the associated therapeutic outcome. Additionally, new techniques that may provide additional control over the treatment, such as monodisperse microbubble formulations, tunable ultrasound scanners, and cavitation detection techniques, are discussed. An in-depth understanding of the aspects presented in this work could eventually lead the way to more efficient and tailored microbubble-assisted ultrasound therapy in the future.

Temperature-Induced Self-Assembly of the Group B Streptococcus (GBS) Fusion Antigen GBS-NN.

Group B Streptococcus (GBS) is a leading cause of serious bacterial neonatal infections worldwide, which provides an unmet medical need for a globally effective vaccine. The recombinant GBS fusion antigen GBS-NN contains the N-terminal regions of the GBS Rib and Alpha C proteins. It shows promising immunogenicity eliciting protective immunity in mice and encouraging results in early human clinical trials. Understanding the physical stability of GBS-NN containing conformational B-cell epitopes is crucial to ensure optimal vaccine stability, efficacy, and safety. We initially discovered that GBS-NN is prone to form higher-order structures at elevated temperatures. We therefore investigated the self-assembly behavior of GBS-NN and characterized the higher-order conformational structures as a function of temperature. In the native state, GBS-NN exists as a monomer and has a secondary structure containing α-helix and ß-sheet. Langmuir studies demonstrated that the native protein is highly surface-active and forms a monolayer film at the air-water interface because of its amphipathic properties. The conformational stability of GBS-NN was measured as a function of temperature. GBS-NN has an unusual thermal behavior with a phase transition of approximately 61 °C, which is not accompanied by any major changes in the secondary structure. However, the antigen showed irreversible self-assembly as a function of temperature into higher-order structures with a hydrodynamic diameter of approximately 100 nm. Cryo-transmission electron microscopy analyses demonstrated that these self-assemblies consist of vesicular, ring-like structures with a hollow aqueous interior. Therefore, GBS-NN is a physically stable monomeric protein but is prone to temperature-induced self-assembly above 61 °C.

Ultrasound-Responsive Cavitation Nuclei for Therapy and Drug Delivery.

Therapeutic ultrasound strategies that harness the mechanical activity of cavitation nuclei for beneficial tissue bio-effects are actively under development. The mechanical oscillations of circulating microbubbles, the most widely investigated cavitation nuclei, which may also encapsulate or shield a therapeutic agent in the bloodstream, trigger and promote localized uptake. Oscillating microbubbles can create stresses either on nearby tissue or in surrounding fluid to enhance drug penetration and efficacy in the brain, spinal cord, vasculature, immune system, biofilm or tumors. This review summarizes recent investigations that have elucidated interactions of ultrasound and cavitation nuclei with cells, the treatment of tumors, immunotherapy, the blood-brain and blood-spinal cord barriers, sonothrombolysis, cardiovascular drug delivery and sonobactericide. In particular, an overview of salient ultrasound features, drug delivery vehicles, therapeutic transport routes and pre-clinical and clinical studies is provided. Successful implementation of ultrasound and cavitation nuclei-mediated drug delivery has the potential to change the way drugs are administered systemically, resulting in more effective therapeutics and less-invasive treatments.

Enhancing Nucleic Acid Delivery with Ultrasound and Microbubbles.

For gene therapy to work in vivo, nucleic acids need to reach the target cells without causing major side effects to the patient. In many cases the gene only has to reach a subset of cells in the body. Therefore, targeted delivery of genes to the desired tissue is a major issue in gene delivery. Many different possibilities of targeted gene delivery have been studied. A physical approach to target nucleic acids and other drugs to specific regions in the body is the use of ultrasound and microbubbles. Microbubbles are gas filled spheres with a stabilizing lipid, protein, or polymer shell. When these microbubbles enter an ultrasonic field, they start to oscillate. The bubbles' expansion and compression are inversely related to the pressure phases in the ultrasonic field. When microbubbles are exposed to high-intensity ultrasound the microbubbles will eventually implode and fragment. This generates shockwaves and microjets which can temporarily permeate cell membranes and blood vessels. Nucleic acids or (non)viral vectors can as a result gain direct access to either the cytoplasm of neighboring cells, or extravasate to the surrounding tissue. The nucleic acids can either be mixed with the microbubbles or loaded on the microbubbles. Nucleic acid loaded microbubbles can be obtained by coupling nucleic acid-containing particles (i.e., lipoplexes) to the microbubbles. Upon ultrasound-mediated implosion of the microbubbles, the nucleic acid-containing particles will be released and will deliver their nucleic acids in the ultrasound-targeted region.

Sonoprinting of nanoparticle-loaded microbubbles: Unraveling the multi-timescale mechanism.

Ultrasound-triggered microbubble-assisted drug delivery is a promising tool for localized therapy. Several studies have shown the potential of nanoparticle-loaded microbubbles to effectively enhance the delivery of therapeutic agents to target tissue. We recently discovered that nanoparticle-carrying microbubbles can deposit the nanoparticles in patches onto cell membranes, a process which we termed 'sonoprinting'. However, the biophysical mechanisms behind sonoprinting are not entirely clear. In addition, the question remains how the ultrasound parameters, such as acoustic pressure and pulse duration, influence sonoprinting. Aiming for a better understanding of sonoprinting, this report investigates the behavior of nanoparticle-loaded microbubbles under ultrasound exposure, making use of three advanced optical imaging techniques with frame rates ranging from 5 frames per second to 10 million frames per second, to capture the biophysical cell-bubble interactions that occur on a multitude of timescales. We observed that non-spherically oscillating microbubbles release their nanoparticle payload in the first few cycles of ultrasound insonation. At low acoustic pressures, the released nanoparticles are transported away from the cells by microstreaming, which does not favor uptake of the nanoparticles by the cells. However, higher acoustic pressures (>300 kPa) and longer ultrasound pulses (>100 cycles) lead to rapid translation of the microbubbles, due to acoustic radiation forces. As a result, the released nanoparticles are transported along in the wake of the microbubbles, which eventually leads to the deposition of nanoparticles in elongated patches on the cell membrane, i.e. sonoprinting. We conclude that a sufficiently high acoustic pressure and long pulses are needed for sonoprinting of nanoparticles on cells.