Multiple Linear Regression Predictive Modeling of Colloidal and Fluorescence Stability of Theranostic Perfluorocarbon Nanoemulsions

Abstract

Perfluorocarbon nanoemulsions (PFC-NEs) are widely used as theranostic nanoformulations with fluorescent dyes commonly incorporated for tracking PFC-NEs in tissues and in cells. Here, we demonstrate that PFC-NE fluorescence can be fully stabilized by controlling their composition and colloidal properties. A quality-by-design (QbD) approach was implemented to evaluate the impact of nanoemulsion composition on colloidal and fluorescence stability. A full factorial, 12-run design of experiments was used to study the impact of hydrocarbon concentration and perfluorocarbon type on nanoemulsion colloidal and fluorescence stability. PFC-NEs were produced with four unique PFCs: perfluorooctyl bromide (PFOB), perfluorodecalin (PFD), perfluoro(polyethylene glycol dimethyl ether) oxide (PFPE), and perfluoro-15-crown-5-ether (PCE). Multiple linear regression modeling (MLR) was used to predict nanoemulsion percent diameter change, polydispersity index (PDI), and percent fluorescence signal loss as a function of PFC type and hydrocarbon content. The optimized PFC-NE was loaded with curcumin, a known natural product with wide therapeutic potential. Through MLR-supported optimization, we identified a fluorescent PFC-NE with stable fluorescence that is unaffected by curcumin, which is known to interfere with fluorescent dyes. The presented work demonstrates the utility of MLR in the development and optimization of fluorescent and theranostic PFC nanoemulsions.

In vitro Quality Assessments of Perfluorocarbon Nanoemulsions for Near-infrared Fluorescence Imaging of Inflammation in Preclinical Models

“…Defined quality control attributes (CQAs) and quality control (QC) measures listed in Table 1 encompass routine assessments of the manufactured nanoemulsions to ensure colloidal stability over time after refrigerated (4 °C) storage. We present procedures adapted from Herneisey and Janjic (2023) . Nanoemulsion droplet size, polydispersity index (PDI), and fluorescence signal are measured throughout the lifetime of the nanoemulsion.…”

“…Our lab has utilized this battery of tests in numerous publications for nanoemulsion quality assurance. CQAs and QC for our formulations are listed in Table 1 , which was adapted from Herneisey and Janjic (2023) . Figure 2 summarizes the representative results of CQAs and QC obtained after manufacturing two-color fluorescently labeled PFC-NEs.…”

“…Our lab has reported multiple examples of triphasic theranostic NEs used to encapsulate and deliver non-steroidal anti-inflammatory drugs (e.g., celecoxib) ( Herneisey et al, 2019 ; Liu et al, 2020 ) and natural products (resveratrol, curcumin) ( Herneisey et al, 2016 ; Herneisey and Janjic, 2023 ), rendering them multimodal ( 19 F MR/NIRF) imaging and drug delivery agents or theranostics. When theranostic NEs are used in vivo, NIRF imaging tracks macrophages as they respond to the delivered drug by monitoring changes in their infiltration patterns at the site of inflammation caused by injury, surgery, or other insults.…”

“…Figure 1 shows the conceptual framework for the presented protocols and how they are utilized in validating PFC-NEs ex vivo for the purpose of in vivo macrophage tracking in preclinical models, followed by ex vivo immunofluorescence in tissues of interest. Based on our prior work and by utilizing quality-by-design (QbD) methodologies, we established critical quality attributes (CQAs) that, when met, render PFC-NEs effective in vitro and in vivo as NIRF imaging agents for macrophage tracking ( Herneisey et al, 2019 ; Saleem et al, 2019 ; Liu et al, 2020 ; Nichols et al, 2021 ; Herneisey and Janjic, 2023 ). In the current protocol, we describe stress tests and associated instrumental measurements that can be easily performed to obtain reliable results on colloidal and fluorescence stability of PFC-NEs under varied conditions: filtration, storage at physiological temperatures, and centrifugation.…”

Cardiovascular Molecular Imaging With Fluorine-19 MRI: The Road to the Clinic

“…Robust mathematical modelling approaches were developed to better control nanoemulsions colloidal and fluorescence properties by customizing their composition and manufacturing processing parameters. 30 Furthermore, several approaches have been described to improve the relaxation times of the designed nanoemulsions by incorporating paramagnetic agents such as gadolinium or iron ions (Figure 2). [31][32][33] In practice, tracers are either produced in-house with wellestablished quality control procedures, acquired through collaborations, or bought from various small companies.…”