Mechanical properties of phospholipid coated microbubbles

Abstract

Phospholipid coated, inert gas filled microbubbles (MBs) are currently in widespread use in medical applications for the enhancement of diagnostic ultrasound images, and they are promising candidates for use in the area of targeted drug/gene delivery and uptake. As phospholipid coated MBs were developed for use with diagnostic ultrasound, their behaviour under acoustic loading is well investigated, however much less is known about their response to direct mechanical loading, which will potentially prove important as the range of uses of MBs expands. This is particularly true of the existing commercially available MB products. In this thesis, atomic force microscopy (AFM) was used to investigate the mechanical behaviour of three types of commercially produced phospholipid coated MBs, Definity®, BR14 and Sonovue®, at small deformations. Force spectroscopy was used to produce force-deformation (F-Δ) curves showing how the MBs deform under mechanical loading. Definity® MBs were deformed with tipless cantilevers at high deformations (though still less than 30% of the initial height of the MB); BR14 and Sonovue® MBs were probed with both tipless and tipped cantilevers to investigate both whole-bubble deformation and also shell indentation. BR14 was limited to low deformations; Sonovue® included both low and high deformations. The F-Δ curves were used to evaluate MB stiffness and also in combination with up to four mechanical models to predict the Young’s modulus of the MBs. The suitability of Reissner, Hertz, Elastic Membrane and De Jong theories for the prediction of the Young’s modulus of the MBs was explored. In the case of Definity® MBs no correlation between MB size and stiffness was observed; however an unexpected size dependence was observed in the Young’s modulus values, possibly due to variations in the thickness of the phospholipid shell. The membrane stretching component of elastic membrane theory was found to be the most applicable model on these MBs in this higher deformation regime. However, in this regime, gas compressibility could play a role and this is not included in the model. We studied the mechanical properties of BR14 MBs at very low deformations using ‘soft’ cantilevers. In this regime, gas compressibility should play a minimal role and there are several mechanical models which may be used. These MBs demonstrated decreasing stiffness with increasing diameter, and little variation in Young’s modulus with diameter. Hertz and De Jong theories showed more realistic Young’s modulus values (compared to other models) with little observable trend. Sonovue® MBs were used for a more comprehensive study of the small and very small deformation regimes using ‘soft’, ‘hard’ and tipped cantilevers. They showed no definitive trend in MB stiffness with MB diameter. Hertz and De Jong theory were again found to be most suitable. Analysis of curves acquired with tipped cantilevers indicated that the stiffness of a localised area of the shell membrane is similar to the overall stiffness of the MB and that the apparent Young’s modulus of the membrane according to the Hertz theory is also similar to that of the MB as a whole. Generally, considering all systems, Reissner theory was found to produce large overestimates of Young’s modulus, exceeding expected values by several orders of magnitude. Hertz and De Jong theories produced underestimates, though by a much smaller margin. Elastic membrane theory worked well and produced realistic Young’s modulus values only at relatively high deformation (the stretching term) in spite of the fact that gas compressibility is not taken into account. The suitability of the models is therefore very dependent on the deformation regime of the experiment. It seems that there is scope for better models at low deformation taking into account the soft shell of the MB and possibly its specific structure. Precise structural information of the MB shells does not exist; it is not trivial to attain and should certainly be a topic of future work with additional instrumentation.