Microbubbles have been used for several decades as ultrasound
contrast agents in diagnostic ultrasound imaging. However, their application
in gene therapy as delivery vehicles has only recently been realised. The
presence of microbubbles in close proximity to cells during ultrasound
insonation can increase the efficacy of drug or gene delivery by inducing
formation of transient, non-lethal perforations in the cell membrane, a
process termed sonoporation. In order to develop techniques for successful
delivery of therapeutic agents, it is necessary to quantify the composition and
physical characteristics of microbubbles in order to be able to determine how
these affect the sonoporation process as required. Although several
microbubbles are available commercially, the components of the shell of
these proprietary microbubbles have not been disclosed. In order to study
sonoporation and the possibility of delivering drugs and genes it became
necessary to develop a formulation for in-house experimental microbubbles.
These experimental in-house microbubbles have not been previously
investigated with regard to their interaction with cells, their potential for
sonoporation and / or their bioeffects. Characterisation of the in-house
microbubbles was necessary prior to any attempts to use them as delivery
vehicles in vitro, or indeed, in vivo. Confocal laser scanning microscopy
(CLSM) was used in order to determine the size distribution of both in-house
microbubbles and Definity® a commercially available contrast agent.
Confocal imaging and 3-D reconstruction of in-house microbubbles indicated
the structure, morphology and size-distribution of these membrane-bound
microbodies. Microbubbles were later separated according to size using a
density gradient. It was concluded that the distribution of sizes of the microbubbles was in part due to the multi-lamellar nature of the
microbubble shell.
Cells were initially cultured in Petri dishes and insonated in the
presence and absence of in-house microbubbles, in order to assess any
bioeffects emerging from the application of ultrasound alone or in the
presence of the microbubble constructs. Cells were cultured subsequently on
an acoustically-transparent Mylar membrane, which was then “sandwiched”
between two acetal homopolymer (Derlin) rings and placed in a specially
designed ultrasound tank. Ultimately, cells were grown in an OptiCell™, an
acoustically-transparent parallel membrane environment, where delivery of
molecules of various sizes, in the presence of both in-house and Definity®
microbubbles was investigated. Sonoporation was achieved with
insonication of SK Hep-1 cells with a “physiotherapy machine” applying a
power of 2.54 W / cm2 for 2-3 secs in the presence of Definity® microbubbles
and passage of Calcein, an impermeable molecule, into the cells was detected
using flow cytometric analysis. In addition, expression of enhanced green
fluorescent protein (EGFP) was also detected 24 hours after insonication of
SK Hep-1 cells in the presence of Definity® microbubbles and a linearised
plasmid pCS2, encoding EGFP, under the same ultrasonic conditions.
Sonoporation was also investigated with the use of a diagnostic ultrasound
scanner, since it is more clinically relevant. Although several acoustic and
non-acoustic parameters were investigated, sufficient sonoporation was not
attained using this scanner.
The bioeffects of ultrasound on cells both in vivo and in vitro have been
extensively investigated. However, the exact cellular mechanisms that are
affected by the application of ultrasound waves are not understood. In this
study, the effects of ultrasound on a number of pathways were investigated. Expression of Hsp70, a cell stress protein often associated with heat-shock,
during application of continuous wave ultrasound, suggests that cells may
undergo heat stress. During application of continuous wave ultrasound in
the presence of Definity® microbubbles, expression of Hsp70 was shown to
decrease compared to when ultrasound was applied in the absence of
Definity® microbubbles. In addition, expression of HO-1, a protein
associated with hypoxic pathways was also present during application of
ultrasound in the absence of microbubbles. These results suggest that in the
absence of ultrasound contrast agents, insonation can cause the expression of
proteins associated with different forms of cell stress such as heat-shock and
hypoxia, thus initiating the apoptotic process.
In this thesis, it has been shown that the mean size of the in-house
microbubbles is comparable to that of commercially available microbubbles
such as Definity®. In addition, it has been shown that sonoporation and
successful delivery of small molecules in the presence of Definity®
microbubbles is achievable with the equipment and the specific system
which was developed. This reinforces the promising role of in-house
microbubbles as delivery vehicles for therapeutic agents. Finally, an
investigation on the possible bioeffects of ultrasound in the presence and
absence of ultrasound contrast agents, revealed that under acoustic
conditions identical to those used for achieving sonoporation, cells
experience stress, instigating pathways that could potentially lead to cell
death