Thermal and Acoustic Stabilization Of Volatile Phase-Change Contrast Agents Via Layer-By-Layer Assembly

Abstract

Objective: Phase-change contrast agents (PCCAs) are perfluorocarbon nanodroplets (NDs) that have been widely studied for ultrasound imaging in vitro, pre-clinical studies, and most recently incorporated a variant of PCCAs, namely a microbubble-conjugated microdroplet emulsion, into the first clinical studies. Their properties also make them attractive candidates for a variety of diagnostic and therapeutic applications including drug-delivery, diagnosis and treatment of cancerous and inflammatory diseases, as well as tumor-growth tracking. However, control over the thermal and acoustic stability of PCCAs both in vivo and in vitro has remained a challenge for expanding the potential utility of these agents in novel clinical applications. As such, our objective was to determine the stabilizing effects of layer-by-layer assemblies and its effect on both thermal and acoustic stability.

Methods: We utilized layer-by-layer (LBL) assemblies to coat the outer PCCA membrane and characterized layering by measuring zeta potential and particle size. Stability studies were conducted by; 1) incubating the LBL-PCCAs at atmospheric pressure at 37^∘C and 45^∘C followed by; 2) ultrasound-mediated activation at 7.24 MHz and peak-negative pressures ranging from 0.71 - 5.48 MPa to ascertain nanodroplet activation and resultant microbubble persistence. The thermal and acoustic properties of decafluorobutane gas-condensed nanodroplets (DFB-NDs) layered with 6 and 10 layers of charge-alternating biopolymers, (LBL₆NDs and LBL₁₀NDs) respectively, were studied and compared to non-layered DFB-NDs. Half-life determinations were conducted at both 37^∘C and 45^∘C with acoustic droplet vaporization (ADV) measurements occurring at 23^∘C.

Discussion: Successful application of up to 10 layers of alternating positive and negatively charged biopolymers onto the surface membrane of DFB-NDs was demonstrated. Two major claims were substantiated in this study; namely, (1) biopolymeric layering of DFB-NDs imparts a thermal stability up to an extent; and, (2) both LBL₆NDs and LBL₁₀NDs did not appear to alter particle acoustic vaporization thresholds, suggesting that the thermal stability of the particle may not necessarily be coupled with particle acoustic vaporization thresholds.

Conclusion: Results demonstrate that the layered PCCAs had higher thermal stability, where the half-lifes of the LBL_xNDs are significantly increased after incubation at 37^∘C and 45^∘C. Furthermore, the acoustic vaporization profiles the DFB-NDs, LBL₆NDs, and LBL₁₀NDs show that there is no statistically significant difference between the acoustic vaporization energy required to initiate acoustic droplet vaporization.

Keywords: acoustic activation; biopolymers; decafluorobutane; layer-by-layer; lipids; microbubbles; nanodroplets; perfluorocarbon; phase-change contrast agents; thermal activation.