Real‐Time, In Situ Imaging of Macrophages via Phase‐Change Peptide Nanoemulsions

Abstract

Macrophages are specialized phagocytes that play central roles in immunity and tissue repair. Their diverse functionalities have led to an evolution of new allogenic and autologous macrophage products. However, realizing the full therapeutic potential of these cell‐based therapies requires development of imaging technologies that can track immune cell migration within tissues in real‐time. Such innovations will not only inform treatment regimens and empower interpretation of therapeutic outcomes but also enable prediction and early intervention during adverse events. Here, phase‐changing nanoemulsion contrast agents are reported that permit real‐time, continuous, and high‐fidelity ultrasound imaging of macrophages in situ. Using a de novo designed peptide emulsifier, liquid perfluorocarbon nanoemulsions are prepared and show that rational control over interfacial peptide assembly affords formulations with tunable acoustic sensitivity, macrophage internalization, and in cellulo stability. Imaging experiments demonstrate that emulsion‐loaded macrophages can be readily visualized using standard diagnostic B‐mode and Doppler ultrasound modalities. This allows on‐demand and long‐term tracking of macrophages within porcine coronary arteries, as an exemplary model. The results demonstrate that this platform is poised to open new opportunities for non‐invasive, contrast‐enhanced imaging of cell‐based immunotherapies in tissues, while leveraging the low‐cost, portable, and safe nature of diagnostic ultrasound.

Recent Advancements in Ultrasound Contrast Agents Based on Nanomaterials for Imaging

“…31 In addition, the team of Medina et al reported a liquid PFC nanoemulsion via spontaneous assembly, undergoing a US directed liquid-to-gas phase transition to convert into echogenic microbubbles in situ and enabling on-demand, real-time, and continuous acoustic imaging of macrophages within porcine coronary arteries. 32 In conclusion, PFC gasfilled microbubbles show great potential in in vivo vascular imaging.…”

Life at the interface: Engineering bio‐nanomaterials through interfacial molecular self‐assembly

“…Work from our lab has shown that non-canonical amino acids can be directed to assemble at fluorous-water interfaces to develop mechanically responsive thin films and viscoelastic synthetic mucus (Miller & Medina, 2024;Sloand, Culp, et al, 2021;Sloand, Rokni, et al, 2021). Mechanistic insights gained from this work enabled the development of fluorine-rich small molecules and peptides that act as emulsifiers to stabilize perfluorocarbon nanodroplets (Kim et al, 2023;Lawanprasert et al, 2021;Medina et al, 2017;Sloand et al, 2020;Sloand, Culp, et al, 2021;Sloand, Rokni, et al, 2021). By controlling the physicochemical properties of the emulsifier, we have shown that the thermodynamic stability of phase separated emulsions can be rationally tailored to enable thermally and mechanically responsive nanomaterials.…”

“…In our group, we have exploited these unique properties to create ultrasound-sensitive liquid nanoemulsions for diagnostic and drug delivery applications (Medina et al, 2017;Sloand et al, 2020;Sloand, Culp, et al, 2021;Sloand, Rokni, et al, 2021). Through this work, we have demonstrated that controlling the assembly pathway of the peptide emulsifier at the liquid-liquid emulsion interface can yield nanoparticles with tunable acoustic sensitivity, cellular internalization, and in cellulo stability (Figure 8) (Kim et al, 2023). In particular, Pickering emulsions, which are particles stabilized by the accumulation of solid matter at the liquid-liquid interface (Aveyard et al, 2003;Zhao et al, 2020), can be generated by creating peptide-based fibers and sheets at the interphase.…”

“…Our lab has also developed targeted PCCAs stabilized by a de novo peptide surfactant, permitting incorporation of the targeting ligand directly into the emulsifier sequence itself (Medina et al, 2017;Sloand et al, 2020). We demonstrated these particles could be internalized into immune cells to enable real-time B-mode and Doppler ultrasound imaging of cell migration in situ within tissues (Figure 10) (Kim et al, 2023). Additional development of platelettargeting formulations allowed for contrast enhanced imaging of blood clots in deep vein thrombosis models (Sloand, Culp, et al, 2021;Sloand, Rokni, et al, 2021).…”