Deep-freezing preparation of erythrocyte-derived carriers associated with naked perfluorocarbon nanodroplets for ultrasound-triggered phase change

Ultrasound-activatable nanodroplets represent a class of agents for diagnostic and therapeutic applications, but most formulations use synthetic stabilizing shells. Here, we describe a proof-of-concept platform in which erythrocyte-derived whole-cell/ghost structures serve as biomimetic carriers for perfluorocarbon (PFC) nanodroplets. Naked (surfactant-free) nanodroplets of perfluorobutane (PFB), perfluoropentane (PFP), and perfluorohexane (PFH) were fabricated using a simplified Ouzo emulsification method and combined with erythrocytes by deep-freeze processing. Bright-field and confocal fluorescence microscopy provided supportive qualitative evidence of droplet-like PFC-associated structures within projected cell contours and at erythrocyte membranes. Upon ultrasound exposure in a gel phantom, the treated erythrocyte preparations generated bubbles, with lower apparent activation pressures for the more volatile PFCs. We propose, as a working hypothesis, that freeze-thaw injury may facilitate droplet entry or association through transient membrane defects; ice-crystal-induced membrane disruption, ethanol-induced membrane fluidization, osmotic stress, and other freeze-thaw effects may all contribute. These preliminary findings establish feasibility while leaving loading efficiency, membrane integrity, biological function, stability, and in vivo performance to be quantified in future studies.