Acoustic characterization of ultrasound contrast microbubbles and echogenic liposomes: applications to imaging and drug-delivery
Date
2013
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Micron- 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.