Defence of doctoral thesis: Ksenia Loskutova – Perfluorocarbon microdroplets stabilized by cellulose nanofibers

Ultrasound contrast agents consist of gas-filled micrometer-sized bubbles that are injectedinto the blood stream. Ultrasound contrast agents are an invaluable tool for ultrasound imaging of the cardiac muscle and highly vascularized structures such as kidneys and the liver. The ability of gas-filled microbubbles to enhance contrast in ultrasound imaging comes from their increased scattering ability due to significantly lower compressibility compared to surrounding soft tissues.

The discovery of acoustic droplet vaporization, the phase-transition of liquid-filleddroplets into gas-filled microbubbles upon ultrasound exposure, has expanded the potential utility of ultrasound-mediated diagnostics and therapy to include applications such as gas embolization, histotripsy, and localized drug delivery. Multiple requirements are put onto both gas-filled microbubbles and phase-change contrast agents: they have to be non-toxic, acoustically active at clinically relevant pressure amplitudes, and their dynamic behavior has to be predictable to maximize the therapeutic or diagnostic effect while minimizing mechanical damage to surrounding healthy tissue. Novel designs of phase-change contrast agents that are able to undergo acoustic droplet vaporization could enable improved in vivo stability compared to conventional gas-filled ultrasound contrast agents.

Pickering emulsions, with solid particles used as stabilizing agents instead of surfactants, have an increased stability compared to conventional emulsions. Cellulose-based Pickering emulsions in particular have previously been investigated for biomedical applications. Cellulose is a suitable material in biomedical applications as it originates from renewable sources, is biocompatible, and the surface can be easily modified. To the author’s current knowledge, cellulose-based Pickering emulsions have not previously been investigated for ultrasound-mediated applications. It is necessary to know the mechanical and acoustic properties of novel formulations and their impact on biological cells for their translation into in vivo research and future clinical use.

In this thesis, the acoustic, mechanical, and biological properties of cellulose nanofiber(CNF)-shelled perfluoropentane (PFP) droplets, a type of Pickering emulsion, were investigated for ultrasound-mediated medical applications. Firstly, the current state-of-the-art and development of phase-change contrast agents, the mechanism behind acoustic droplet vaporization, and potential ultrasound-mediated medical applications were investigated. Secondly, a theoretical model that would describe and predict the acoustic response of CNF-shelled PFP droplets undergoing acoustic droplet vaporization was developed. Thirdly, the compressibility of CNF-shelled PFP droplets using an acoustophoretic setup was measured. Later, the effect of the geometry of the surrounding medium and acoustic parameters on the acoustic response of CNF-shelled PFP droplets was explored. Finally, the biocompatibility of CNF-shelled PFP droplets cells was investigated through a hemolysis assay and measurement of change in cell viability of breast cancer cells.

The CNF shell has a significant impact on the predicted resonance behavior and compressibility of CNF-shelled PFP droplets, as it has significantly larger bulk and Young’s modulus than previously reported shell materials. The predicted linear resonance behavior was in the upper range of medical ultrasound (5-8 MHz), making harmonic imaging at optimal conditions difficult. However, it was demonstrated that CNF-shelled PFP droplets could be imaged using a nonlinear ultrasound imaging sequence at a frequency regularly used in clinics. Thus, CNF-shelled PFP droplets were able to undergo acoustic droplet vaporization at clinically relevant conditions. The peak negative pressure of the incident acoustic wave had a significant impact on the acoustic response of CNF-shelled PFP droplets, as higher acoustic pressure amplitudes resulted in a more disruptive behavior. Finally, CNF-shelled PFP droplets did not influence the cell viability of breast cancer cells. This was true regardless of whether or not a non-encapsulated cytotoxic drug with a known impact on cell viability was present. In summary, the results of this work showed that CNF-shelled PFP droplets are biocompatible and acoustically active at clinically relevant conditions, which shows that cellulose-based Pickering emulsions have potential in ultrasound-mediated diagnostics and therapy.

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