Dual Mode Nanoemulsions for 19F MRI and Fluorescence Detection

DESCRIPTION (provided by applicant): Recently, scientists at Carnegie Mellon University (CMU) have invented a compelling technology that makes transplanted cells visible with magnetic resonance imaging (MRI). Cells of interest are labeled ex vivo with a novel perfluoropolyether (PFPE) nanoemulsion composition and then introduced into an animal or human subject; cell migration is subsequently monitored in vivo using fluorine-19 (19F) MRI. The 19F images are extremely selective for the labeled cells, and the co-registered conventional proton (1H) MR images acquired in the same scanning session places the labeled cells into their precise anatomical context. A commercial version of the enabling PFPE reagent, called 'Cell Sense' (CS), has recently been developed and manufactured by Celsense, Incorporated. In further developments, recently published research from CMU has demonstrated the formulation of robust, 'dual mode' nanoemulsions for 19F MRI and fluorescence detection. Key innovation lies in the fact that fluorescent dye is present in the fluorocarbon oil in low amount, yet covalently conjugated to PFPE oil remains in fluorocarbon phase throughout processing. These new reagents are stable, non-toxic and self-deliverable to a wide range of cell types. An apparent linear correlation between the 19F NMR signal and the fluorescence intensity of labeled cells is observed. Building on these results, the goal of this application is to develop a commercially-scalable fluorescent version of Cell Sense. The current results, including biological testing, are on small pilot scale. Therefore, necessary steps towards the development and production on a larger scale are proposed. The technical challenges underlying these scale up procedures are addressed in great detail in this revised application. Unusual physicochemical properties of PFPE oils, high hydrophobicity and significant lipophobicity, make the synthetic modifications and formulations of these oils more difficult than traditional emulsification of hydrocarbons. In the synthesis issues such as PFPE conjugate blending, unconjugated dye removal and purification of final product, nanoemulsion preparations, are also addressed in this proposal. This true dual mode agent is critical for the adoption of the Cell Sense platform in both preclinical and clinical studies. In preclinical studies, it will allow investigators to positively identify the fate and phenotype of labeled cells using histology or flow cytometry following 19F MRI, days and weeks after transfer. In the clinical domain, the dual mode agent will be used to ensure consistent CS labeling ex vivo in an aliquot of patient- or donor- derived cells using quantitative fluorescent measurements. Fluorescent CS will be able to validate the amount of CS reagent delivered to cells using a low cost, rapid multi-well plate reader. This Phase I proposal has three Specific Aims: (1) Synthesis. Develop protocols for the synthesis of fluorescent blended PFPE amide (FBPA) molecules on large-scale, sufficient to produce gt1 L of nanoemulsion; (2) Formulation. Develop protocols for the production of dual mode nanoemulsions using the FBPA produced in Aim 1;.(3) Validation. Evaluate cell labeling efficacy, safety and linear validation of fluorescence versus 19F signal of cells labeled with dual mode nanoemulsions. The synthetic and formulation work (Aims 1,2) will be performed at CMU, while the biological testing (Aim 3) will be performed at the laboratories at Celsense, Inc. Successful completion of this project in a one-year time frame will facilitate our transition to testing safety and efficacy of dual mode nanoemulsions in human therapeutic cells, such as stem cells, in vitro in a Phase II project. PUBLIC HEALTH RELEVANCE: Currently, more than 700 clinical trials involving the transplantation of therapeutic cells (e.g., lymphocytes and stem cells) are underway to treat major diseases such as cancer and heart failure. The proposed project will help accelerate these trials, as well as the routine clinical use of cellular therapeutics, by providing unique reagents and tools for magnetic resonance imaging (MRI) and optical imaging that will enable one to follow cells after transplant into patients.