Preparation and Characterisation of Therapeutic Oxygen Microbubbles

Abstract

Introduction: Tumour hypoxia is a large contributor to radio- and chemo-therapy resistance. Reoxygenation is highly sought after, with oxygen microbubbles (OMBs) seeing increased interest for this purpose. Unfortunately, current research faces problems in OMB manufacture with issues related to low stability, high excipient load and large heterogeneity making these formulations impractical for commercial use. A novel method combining vortexing and gastight extrusion may be able to address these formulation problems. Aim: In this study, a combined approach of vortexing and gastight extrusion was investigated to improve the homogeneity, gas loading and stability of phospholipid-shelled OMB formulations. Methods: First, characterisation techniques were developed to evaluate the size and gas loading content of the OMBs. Light microscopy, dynamic light scattering, oxygen sensors and B-scan ultrasound imaging were investigated. Fabrication protocols were also optimised to achieve high gas loading and homogeneity. This included modulating shell phospholipid concentration, buffer composition, vortexing speed, oxygen loading concentration, extrusion number and experimental temperatures. With techniques optimised, the second phase of the study compared both vortexed only and vortexed plus gas-tight extruded OMB formulations prepared using DSPC and DBPC shells. Formulations prepared using optimised techniques were studied across a 14-day period. Results: Light microscopy and dynamic light scattering were able to identify micro- and nanosized particles, respectively, in-formulation. An oxygen sensor could readily measure the entire formulation oxygen concentration whereas B-scan ultrasound gave an indication of the proportion of the oxygen dose that was present within the OMB. Increasing phospholipid concentration from 2 mg/ml to 5 mg/ml allowed higher oxygen loading. Conversely, adding glycerol to the buffer as a viscosity modifier decreased oxygen loading and was not further investigated. Higher vortexing speeds and times caused the development of larger uninjectable foam layers, as these were irreversible a maximum of 2500 rpm for 5 min was employed to retain formulation stability. Oxygen was also loaded at ambient pressure as higher pressures did not increase final oxygen content. The extrusion number was restricted number was restricted to 11 and performed at room temperature to ensure reasonable homogeneity without compromising oxygen loading. DBPC OMBs performed better than DSPC OMBs in terms of superior size profiles, oxygen loading and long-term stability. Extrusion helped standardise particle size but substantially decreased formulation oxygen concentration and saw peculiar trends of oxygen in-OMBs suggesting the formulation was changing considerably over the course of the 14-day study. Conclusion: This research demonstrated the successful formulation and characterisation of OMBs using the vortexing and gastight extrusion strategy. The longer chained DBPC is superior to DSPC in all tested aspects. The used gastight extrusion strategy requires further optimisation to minimise gas loss and ensure the formulation behaves predictably over its storage period.