Investigation of microbubble-cell interaction and development of an ultrasound delivery system
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.