On-demand engineering of bimodal nanoimaging agents for monitoring inflammation using quality-by-design

Inflammation is a complex immune response to injuries, infections, or tissue damage. Incomplete or dysregulated inflammation resolution accelerates disease progression and impairs functional recovery following trauma-induced injuries. Current clinical methods to monitor inflammation rely on quantifying soluble biomarkers, performing biopsies, or using non-specific nuclear imaging to detect downstream inflammation consequences. These techniques lack sensitivity for early detection and provide limited spatiotemporal information. This necessitates the development of a scalable, non-invasive nanoimaging agent capable of monitoring inflammation using clinically available imaging modalities to improve patient prognosis. Given macrophages are the key immune cells recruited at early stages and activated across different inflammatory pathologies, they function as a cellular-level readout that informs the severity of inflammation. In this work, we demonstrate the application of the quality by design (QbD) framework to develop a bimodal imaging nanoemulsion engineered for preferential size-dependent uptake by macrophages and that can be imaged using a near-infrared fluorescence optical imager and a ¹⁹F magnetic resonance imaging system. We integrated face-centered central composite design-based response surface methodology (FCCD-RSM) with desirability function-based optimization to simultaneously achieve multiple critical quality attributes (CQAs) with competing goals. Thus, providing a quantitative framework to anticipate trade-offs in CQAs if factor settings are fine-tuned according to the imaging needs. Furthermore, the presented formulation strategy extended the functional utility of indocyanine green (ICG) beyond short-term imaging procedures (< 24 h) toward multi-day inflammation monitoring using a single low dose. Overall, the study focuses on QbD application for developing a scalable nanoimaging agent and its application for imaging inflammation.