Background: Pain is a complex phenomenon characterized by unpleasant experiences with profound heterogeneity influenced by biological, psychological, and social factors. According to the National Health Interview Survey, 50.2 million U.S. adults (20.5%) experience pain on most days, with the annual cost of prescription medication for pain reaching approximately USD 17.8 billion. Theranostic pain nanomedicine therefore emerges as an attractive analgesic strategy with the potential for increased efficacy, reduced side-effects, and treatment personalization. Theranostic nanomedicine combines drug delivery and diagnostic features, allowing for real-time monitoring of analgesic efficacy in vivo using molecular imaging. However, clinical translation of these nanomedicines are challenging due to complex manufacturing methodologies, lack of standardized quality control, and potentially high costs. Quality by Design (QbD) can navigate these challenges and lead to the development of an optimal pain nanomedicine. Our lab previously reported a macrophage-targeted perfluorocarbon nanoemulsion (PFC NE) that demonstrated analgesic efficacy across multiple rodent pain models in both sexes. Here, we report PFC-free, biphasic nanoemulsions formulated with a biocompatible and non-immunogenic plant-based coconut oil loaded with a COX-2 inhibitor and a clinical-grade, indocyanine green (ICG) near-infrared fluorescent (NIRF) dye for parenteral theranostic analgesic nanomedicine. Methods: Critical process parameters and material attributes were identified through the FMECA (Failure, Modes, Effects, and Criticality Analysis) method and optimized using a 3 × 2 full-factorial design of experiments. We investigated the impact of the oil-to-surfactant ratio (w/w) with three different surfactant systems on the colloidal properties of NE. Small-scale (100 mL) batches were manufactured using sonication and microfluidization, and the final formulation was scaled up to 500 mL with microfluidization. The colloidal stability of NE was assessed using dynamic light scattering (DLS) and drug quantification was conducted through reverse-phase HPLC. An in vitro drug release study was conducted using the dialysis bag method, accompanied by HPLC quantification. The formulation was further evaluated for cell viability, cellular uptake, and COX-2 inhibition in the RAW 264.7 macrophage cell line. Results: Nanoemulsion droplet size increased with a higher oil-to-surfactant ratio (w/w) but was no significant impact by the type of surfactant system used. Thermal cycling and serum stability studies confirmed NE colloidal stability upon exposure to high and low temperatures and biological fluids. We also demonstrated the necessity of a solubilizer for long-term fluorescence stability of ICG. The nanoemulsion showed no cellular toxicity and effectively inhibited PGE2 in activated macrophages. Conclusions: To our knowledge, this is the first instance of a celecoxib-loaded theranostic platform developed using a plant-derived hydrocarbon oil, applying the QbD approach that demonstrated COX-2 inhibition.

Microfluidic devices offer well-defined physical environments that are suitable for effective cell seeding and in vitro three-dimensional (3D) cell culture experiments. These platforms have been employed to model in vivo conditions for studying mechanical forces, cell–extracellular matrix (ECM) interactions, and to elucidate transport mechanisms in 3D tissue-like structures, such as tumor and lymph node organoids. Studies have shown that fluid flow behavior in microfluidic slides (µ-slides) directly influences shear stress, which has emerged as a key factor affecting cell proliferation and differentiation. This study investigates fluid flow in the porous channel of a µ-slide using computational fluid dynamics (CFD) techniques to analyze the impact of perfusion flow rate and porous properties on resulting shear stresses. The model of the µ-slide filled with a permeable biomaterial is considered. Porous media fluid flow in the channel is characterized by adding a momentum loss term to the standard Navier–Stokes equations, with a physiological range of permeability values. Numerical simulations are conducted to obtain data and contour plots of the filtration velocity and flow-induced shear stress distributions within the device channel. The filtration flow is subsequently measured by performing protein perfusions into the slide embedded with native human-derived ECM, while the flow rate is controlled using a syringe pump. The relationships between inlet flow rate and shear stress, as well as filtration flow and ECM permeability, are analyzed. The findings provide insights into the impact of shear stress, informing the optimization of perfusion conditions for studying tissues and cells under fluid flow.

The development of tunable systems for subcutaneous injection is currently the focus of exploratory protein formulation. For the delivery of protein biologics intended for extended release, a high fraction of the drug that escapes from the deposition site ("burst release") may pose safety concerns. Herein, we report an injectable system of bioaffinity zinc-containing peptide coacervates in which recombinant proteins coexpressed with histidine (His)-tags can be captured and released over time. Coacervates are formed by driving self-assembling peptide (SAP), AEAEAKAKAEAEAKAKHHHHHH (EAKH6) β-sheet dimers, into cross-linking fibrils. In the presence of Zn²⁺, the fibrillization of EAKH6 is enhanced through the interaction of metal ions with histidine residues in the peptide. The Zn²⁺:EAKH6 scaffold retains His-tagged proteins both in vitro and in vivo and extends their duration of release. The results present a case study in which the Zn²⁺-[His]₆ interaction can be used to tune the properties of supramolecular structures and the loading of His-tagged proteins.