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Over 80% of biologic drugs, and 90% of vaccines, require temperature-controlled conditions throughout the supply chain to minimize thermal inactivation and contamination. This cold chain is costly, requires stringent oversight, and is impractical in remote environments. Here, we report chemical dispersants that non-covalently solvate proteins within fluorous liquids to alter their thermodynamic equilibrium and reduce conformational flexibility. This generates non-aqueous, fluorine-based liquid protein formulations that biochemically rigidify protein structure to yield thermally stable biologics at extreme temperatures (up to 90 °C). These non-aqueous formulations are impervious to contamination by microorganismal pathogens, degradative enzymes, and environmental impurities, and display comparable pre-clinical pharmacokinetics and safety profiles to standard saline protein samples. As a result, we deliver a fluorochemical formulation paradigm that may limit the need for cold chain logistics of protein reagents and biopharmaceuticals.

 

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.

 

Non‐invasive imaging modalities that identify rupture‐prone atherosclerotic plaques hold promise to improve patient risk stratification and advance early intervention strategies. Here, phase‐changing peptide nanoemulsions are developed as theranostic contrast agents for synchronous ultrasound detection and therapy of at‐risk atherosclerotic lesions. By targeting lipids within atherogenic foam cells, and exploiting characteristic features of vulnerable plaques, these nanoemulsions preferentially accumulate within lesions and are retained by intraplaque macrophages. It is demonstrated that acoustic vaporization of intracellular nanoemulsions promotes lipid efflux from foam cells and generates echogenic microbubbles that provide contrast‐enhanced ultrasound identification of lipid‐rich anatomical sites. In Doppler mode, stably oscillating peptide nanoemulsions induce random amplitude and phase changes of the echo wave to generate transient color imaging features, referred to as ‘twinkling’. Importantly, acoustic twinkling is unique to these peptide emulsions, and not observed from endogenous tissue bubble nuclei, generating diagnostic features that offer unprecedented spatial precision of lesion identification in 3D.

 

Although rarely used in nature, fluorine has emerged as an important elemental ingredient in the design of proteins with altered folding, stability, oligomerization propensities, and bioactivity. Adding to the molecular modification toolbox, here we report the ability of privileged perfluorinated amphiphiles to noncovalently decorate proteins to alter their conformational plasticity and potentiate their dispersion into fluorous phases. Employing a complementary suite of biophysical, in‐silico and in‐vitro approaches, we establish structure‐activity relationships defining these phenomena and investigate their impact on protein structural dynamics and intracellular trafficking. Notably, we show that the lead compound, perfluorononanoic acid, is 10⁶times more potent in inducing non‐native protein secondary structure in select proteins than is the well‐known helix inducer trifluoroethanol, and also significantly enhances the cellular uptake of complexed proteins. These findings could advance the rational design of fluorinated proteins, inform on potential modes of toxicity for perfluoroalkyl substances, and guide the development of fluorine‐modified biologics with desirable functional properties for drug discovery and delivery applications.