Bizarre Effects Of Sound Waves Could Benefit Drug Delivery, Exosome Therapy

By Deborah Borfitz 

January 6, 2021 | Scientists at RMIT University in Melbourne, Australia, have developed a nebulizer for delivering drugs and vaccines to the lungs by generating high-frequency sound waves on a microchip. Serendipitously, the device generates droplets “just the right size”—between 1 and 5 microns—for large molecules such as DNA and antibodies to reach the deep lungs, according to Leslie Yeo, lead researcher and distinguished professor in the school of engineering. 

That makes it a potential game-changer. Conventional nebulizers are “a dime a dozen” but limited to small-molecule drugs due to the energy required to create aerosolized medications, says Yeo. The new technology is suitable for next-generation biologics in addition to being cheap, lightweight, and portable as required for home use and in resource-limited settings.

As Yeo describes it, the high-frequency sound waves being employed are incredibly shallow surface waves traveling “like a 10-nanometer-high earthquake” along the surface of a wafer-sized chip about half a millimeter thick. The high acceleration that it produces, roughly 10 million times that of gravity, turns a fluid or drug into a fine mist of perfectly-sized droplets. 

Size matters if large molecules are to reach the deepest parts of the lungs. Droplets above 5 microns tend to collide against the walls of the upper airways, while those smaller than 1 micron tend to be exhaled, “so we have this really small window we need to optimize for,” says Yeo. 

Ultrasound has been used for decades at low frequencies (around 10 to 100 kHz and up to 3 MHz) to drive sonochemical reactions driven by violent implosion of air bubbles. This process, known as cavitation, results in huge pressures and ultra-high temperatures in the immediate vicinity where the bubbles collapse, explains Yeo. 

Cavitation almost never happens at the power levels (frequencies above 10 MHz) where Yeo and his team operate, thus preserving large molecules as they’re encapsulated into aerosol droplets.

The RMIT University research team recently authored a comprehensive review on the many bizarre effects of high-frequency sound waves on fluids, cells, and materials that published in Advanced Science (DOI: /10.1002/advs.202001983).

Market Dynamics

While the device is not limited to aerosolizing just large molecules, the researchers are focusing their development efforts in this area due to latent demand for a nebulizer to deliver biologics via inhalation, Yeo says. Large-molecule drugs have shown promise in clinical trials as a treatment for many genetic diseases, but the primary delivery route to date has been intravenous infusion that requires administration in a hospital. Study sponsors are unlikely to switch gears unless presented with a suitable, market-ready alternative.

Large molecules are notoriously difficult to administer via inhalation whether for local delivery, to treat lung and respiratory conditions, or for systemic delivery, says Yeo. Until quite recently, the wisdom of using the lungs as a portal for the treatment of systemic conditions has also been a topic of hot debate.

As reported a few years ago in Diabetes Spectrum (DOI: 10.2337/diaspect.29.3.180), waning interest was due in part to Pfizer’s withdrawal of the first inhaled insulin (Exubera) from the market in 2017 after many respiratory adverse effects were reported.

Since then, there have been increasing signs that inhalation as a systemic administration route is becoming acceptable again, says Yeo. MannKind brought this form of therapy back to the market with Afrezza, approved by the U.S. Food and Drug Administration in 2014, but with a boxed warning that the product should not be used by smokers or persons with chronic obstructive pulmonary disorder.

The advantage of a nebulizer over inhalers is that it’s a push-button solution, Yeo continues. Inhalers require hand-breath coordination, which can be problematic for little kids and the seriously ill who are already having a tough time breathing.

Smart Materials 

Separately, the investigative team is attempting to make smart materials using these high-frequency sound waves, says Yeo. Super-porous nanomaterials derived from metal-organic frameworks (MOFs) can be produced by the tiny earthquakes that mysteriously cause molecules to spontaneously self-order along a crystal plane. “Given that the sound wavelengths are thousands or tens of thousands of times bigger than the sound waves themselves, this incredibly puzzling phenomenon is almost like driving a truck into a random scattering of Lego bricks, only for the pieces to neatly stack on top of each other in the process.”

MOFs have a lot of surface area given that they are almost 90% holes. A spoonful of these crystals can be stretched the size of a football field, Yeo explains. They have many applications—for example, for catalysis and loading drugs into smaller materials.

It is thought that MOFs can better protect drugs from harsh environments, including the body’s gastrointestinal tract, so they can reach their intended destination, says Yeo. Another potential use of MOFs, besides stabilizing the concentration of a drug as it is being metabolized by the body, is to slow the release of drugs over prolonged periods, he adds.

The conventional process for making a MOF can take hours or days and requires the use of harsh solvents or intensive energy processes, Yeo says. But the clean, sound wave-driven technique can produce a customized MOF in minutes and can be easily scaled up for efficient mass production.

Good Vibrations

Irradiating cells with the high-frequency sound waves also allows therapeutic molecules to be inserted into those cells without damaging them—a technique that could prove useful with emerging cell-based therapies, says Yeo. Potential applications include CAR-T therapy and CRISPR gene editing. 

The difficulty is in getting past the cell membrane, whose job it is to guard the door, Yeo says. One workaround being explored is to make holes in the cell wall via electroporation or sonoporation, which relies on ultrasonic cavitation. Unfortunately, it is hard for some cells to recover from this, and a substantial number of cells could be killed in the process.

The alternative “trojan horse” approach is to use materials, such as viruses, polymer nanoparticles, or even MOFs, to smuggle the drug into cells. The natural process by which cells uptake these materials, however, takes many hours, he says. In addition, the materials typically end up in the endosomes and lysosomes, where they tend to be degraded, such that only a fraction of the cargo would get to the rest of the cell.

High-frequency sound waves, on the other hand, appear capable of delivering payloads without permanently damaging the cells. Once the sound waves are turned off, they seem to recover almost immediately, Yeo says. The researchers speculate that the high-frequency sound waves, rather than making holes in the cell wall, are gently vibrating lipids in the cell membrane “just enough” that it lets the drug molecules in but relaxes back to its original state when the vibration stops.  

These same vibrations trick the cells into thinking they’re wounded, stimulating them to produce exosomes (extracellular vesicles), Yeo shares. The current bottleneck with exosome therapies and diagnostics is that the cells don’t naturally secrete many exosomes.

Exosomes contain a lot of information with potential diagnostic and therapeutic value, including early-stage cancer detection. For the latter application, RMIT University researchers hope to reengineer the exosomes so they can be used as trojan horses, which would be less likely to provoke an immune response if recognized by the same type of cells they’re produced from, says Yeo. 

The research team is fascinated by the different pathways triggered simply by vibrating cells, Yeo says. Their belief is that some of the ion channels that regulate what goes in and out of cells could be responding to the mechanical vibrations. If those can be controlled, he adds, cell engineering could gain some serious traction.

Interestingly, some of the main achievements of RMIT University scientists were topics of speculation in an opinion piece published last year in Ultrasonics Sonochemistry (DOI: 10.1016/j.ultsonch.2018.07.036). Among the ruminations that the team could confirm from first-hand observations is that sonochemistry in the absence of ultrasonic cavitation can have an “ordering effect” on molecules to induce crystallization, says Yeo, referring back to his Lego brick analogy.