Acoustic characterization of ultrasound contrast microbubbles and echogenic liposomes: applications to imaging and drug-delivery

Sarkar, KausikMicron- to nanometer - sized ultrasound agents, like encapsulated microbubbles and echogenic liposomes, are being actively developed for possible clinical implementations in diagnostic imaging and ultrasound mediated drug/gene delivery. Contrast microbubbles (1-10 micron in diameter) contain a low solubility gaseous core stabilized by an encapsulation made of lipids/proteins/polymers/surfactants. Echogenic liposomes (ELIPs), which combine the advantages of liposomes such as biocompatibility and ability to encapsulate both hydrophobic and hydrophilic drugs with a strong reflection of ultrasound, are also excellent candidates for concurrent ultrasound imaging and drug delivery applications. The primary objective of this thesis is to characterize the acoustic behavior and the ultrasound-mediated content release of these contrast agents for developing multi-functional ultrasound contrast agents. The first part of this thesis reports the investigation of encapsulated microbubbles utilized as ultrasound contrast agents, whereas the second part reports the experimental characterizations of echogenic liposomes (ELIPs) and echogenic polymersomes. Contrast microbubbles are nonlinear systems capable of generating a subharmonic response i.e., response at half the excitation frequency, which can improve image quality by providing a higher signal to noise ratio. However, design and development of contrast microbubbles with favorable subharmonic behavior requires accurate mathematical models capable of predicting their nonlinear dynamics. To this goal, ‘strainsoftening’ viscoelastic interfacial models of the encapsulation were developed and subsequently utilized to formulate a modified form of the Rayleigh-Plesset equation to model the nonlinear dynamics of these encapsulated microbubbles. A hierarchical twopronged approach of modeling — a model is applied to one set of experimental data to obtain the model parameters (material characterization), and then the model isvalidated against a second independent experiment — is demonstrated in this thesis for two lipid coated (Sonazoid™ and Definity®) and a few polymer (polylactide) encapsulated microbubbles. We performed in vitro acoustic characterization with these contrast microbubbles, i.e., determined the material properties of their encapsulations and compared model predictions with experimental observations. The nonlinear elastic models developed were successful in predicting several experimentally observed behaviors e.g., low subharmonic thresholds and “compression-only” radial oscillations. Results indicate that neglecting the polydisperse size distribution of contrast agent suspensions, a common practice in the literature, can lead to inaccurate predictions and unsatisfactory results. Recent numerical investigations of the nonlinear dynamics of encapsulated microbubbles from our group contradicted previously published experimental results on the dependence of subharmonic behaviors on ambient pressure. We wanted to investigate this issue through new in vitro acoustic experiments by designing a modified experimental setup. Preliminary results indicate that the previously published conclusion that subharmonic response from contrast microbubbles linearly decreases with increasing ambient pressure might not be correct under all excitation conditions; it may both increase or decrease under appropriate excitations in conformity with the results of numerical investigations. Experimental characterization of the ELIPs and polymersomes was performed with the goal of demonstrating their potential as ultrasound agents with simultaneous imaging and drug/gene delivery applications — ‘dual-purpose’ contrast agents. Carefully performed experiments conclusively demonstrate the ultrasound reflectivity (echogenicity) of the liposomes prepared using an established protocol. Although, no subharmonic response from these ELIPs was observed, altering the constituents of the lipid bilayer and polymerizing it generated a subharmonic response indicating that the echogenic properties of ELIPs can be controlled by altering the preparation protocol. Our results indicate that the freeze-thaw cycle and lyophilization in presence of mannitol followed by reconstitution in a buffer was critical for generating echogenic response from these liposomes. A finite amount of mannitol (above 100 mM) proved critical for echogenicity, but increasing the mannitol concentration above that amount did not change the echogenicity. Lyophilized powders create a polydisperse suspension of liposomes upon reconstitution, which in turn results in a response without a distinct resonance peak. We believe that the echogenicity of the liposomes results from the larger diameter liposomes present in this polydisperse suspension. In spite of the conclusive experimental evidence of echogenicity, the underlying mechanisms are not completely understood primarily due to the uncertainty regarding the exact location of the gas pockets. An accurate knowledge of the locations and dimensions of the gas pockets is critical for developing improved mathematical models of their acoustic behaviors. For the experimental validation of the concept of ‘dual-purpose’ contrast agents, four novel formulations were investigated—a lipopeptide conjugated ELIP formulation that can be triggered by the extracellular enzyme matrix metalloproteinase-9 (MMP- 9), a polymer coated redox triggered ELIP formulation for cytosolic drug delivery, pH sensitive liposomes with tunable echogenicity capable of drug-release in mildly acidic micro-environment and redox sensitive echogenic polymersomes. Both in vitro acoustic studies and ultrasound imaging (the latter performed in NDSU by our collaborators) demonstrated the echogenicity of each of these formulations. Although, ultrasound excitation (< 5 MHz) alone was incapable of causing optimal release of contents, a dualtriggering strategy proved successful. Application of ultrasound in conjunction of other triggers (e.g., enzyme, pH, redox) showed significant enhancements (10-20%), which resulted in a total release of up to 80-90%. Considering these experimental results, it can be concluded that these novel formulations have the potentials for simultaneous imaging and therapeutic applications. These contrast agents hold the potential of providing powerful treatment strategies for many diseases, including cardiovascular ones and various cancers.University of Delaware, Department of Mechanical EngineeringPh.D