Protein-loaded microbubbles to investigate ultrasound-mediated microbubble-cell membrane interactions

The immune response is regulated by the dynamic spatiotemporal regulation of signalling proteins at the interface between immune cells and their targets. The broadened knowledge of immunology and antitumour immune responses has led to the development of a novel cancer therapy avenue: immunotherapy. Unfortunately, the immune system's response to growing cancers is often weakened, and cancer cells have the distinct ability to evade the immune response. From this, two avenues to tackle this problem could readily be identified: (i) increasing the potency of immune cells or (ii) making diseased target cells, such as cancer cells, more visible to the immune system.
This work applies a novel strategy employing protein-loaded microbubbles to ‘tag’ cell membranes of interest with a relevant protein and investigates cavitation-induced microbubble-cell membrane interactions. To this end, microbubbles functionalised with a model protein were successfully produced and characterised, using a metallochelating complex through an NTA(Ni) and histidine residues interaction as an efficient loading strategy.
Proof-of-principle confocal microscopy experiments showed protein/dye-lipid material transfer, or ‘membrane tagging’ to a model cellular membrane using ultrasound in adherent A549 cells. This material transfer was subsequently investigated quantitatively in suspension using flow cytometry. It was observed for two cell types - A549 cells and Jurkat T cells - that cells undergo a morphological change upon ultrasound exposure, increasingly so with increasing pressures and duty cycles. Interestingly, a similar dependence of the ‘membrane tagging’ bioeffect on ultrasound was not observed in the suspension cell models. Potential evidence of a recovery mechanism was observed for both cell types after ultrasound exposure, indicating sonoporation and subsequent resealing of the cell membrane.
Lastly, these protein-functionalised microbubbles were modified to selectively bind microbubbles to cells of interest to improve the specificity of the proposed ‘membrane tagging’ and limit potential off-target interaction. Microbubbles were functionalised with His-transferrin, as A549 cells overexpress transferrin receptors on their outer membranes. The results show a significant increase in microbubble-cell attachment of His-transferrin functionalised microbubbles compared to control His-human serum albumin microbubbles. There was also limited binding of His-transferrin functionalised microbubbles to A549 cells with transferrin receptors blocked by excess free transferrin, confirming targeting specificity. Again, ultrasound did not significantly affect the fraction of cells showing ‘membrane tagging’.
In conclusion, this Thesis shows the successful fabrication and characterisation of NTA(Ni) microbubbles to which His-tagged proteins can be conjugated. Subsequently, the use of the microbubbles to ‘tag’ cellular membranes with these proteins was assessed both qualitatively with adherent A549 cells and quantitatively with A549 and Jurkat T cells in suspension. Lastly, this effect was greatly enhanced by functionalising microbubbles with the targeting protein His-transferrin to specifically target A549 cells expressing the equivalent receptor. This Thesis lays the groundwork for the use of custom-made protein- functionalised microbubbles in immunomodulation and, if successfully developed, may provide an inexpensive, low-risk alternative to CAR T cell therapy.

Keywords: microbubbles
recombinant proteins
cell membrane tagging
microbubble-cell interactions
metallochelation
ultrasound
flow cytometry
confocal microscopy
Advisor: Stride, E
Sezgin, E