Cell penetrating peptide functionalized perfluorocarbon nanoemulsions for targeted cell labeling and enhanced fluorine‐19 MRI detection

Abstract

Purpose: A bottleneck in developing cell therapies for cancer is assaying cell biodistribution, persistence, and survival in vivo. Ex vivo cell labeling using perfluorocarbon (PFC) nanoemulsions, paired with 19 F MRI detection, is a noninvasive approach for cell product detection in vivo. Lymphocytes are small and weakly phagocytic limiting PFC labeling levels and MRI sensitivity. To boost labeling, we designed PFC nanoemulsion imaging probes displaying a cell-penetrating peptide, namely the transactivating transcription sequence (TAT) of the human immunodeficiency virus. We report optimized synthesis schemes for preparing TAT co-surfactant to complement the common surfactants used in PFC nanoemulsion preparations. Methods: We performed ex vivo labeling of primary human chimeric antigen receptor (CAR) T cells with nanoemulsion. Intracellular labeling was validated using electron microscopy and confocal imaging. To detect signal enhancement in vivo, labeled CAR T cells were intra-tumorally injected into mice bearing flank glioma tumors. Results: By incorporating TAT into the nanoemulsion, a labeling efficiency of ~10 12 fluorine atoms per CAR T cell was achieved that is a >8-fold increase compared to nanoemulsion without TAT while retaining high cell viability (~84%). Flow cytometry phenotypic assays show that CAR T cells are unaltered after labeling with TAT nanoemulsion, and in vitro tumor cell killing assays display intact cytotoxic function.The 19 F MRI signal detected from TAT-labeled CAR T cells was 8 times higher than cells labeled with PFC without TAT. Conclusion: The peptide-PFC nanoemulsion synthesis scheme presented can significantly enhance cell labeling and imaging sensitivity and is generalizable for other targeted imaging probes.

Assessing Oximetry Response to Chimeric Antigen Receptor T-cell Therapy against Glioma with 19F MRI in a Murine Model

“…Oximetry Response to CAR T-cell Therapy against Glioma by 19 F MRI: A Murine Model An alternative PFC nanoemulsion formulation displayed a cell-penetrating peptide as a component of the surfactant to boost cell labeling in T cells and other weakly phagocytic cell types (21), namely, the transactivating transcription sequence (TAT) of the human immunodeficiency virus. Details on the synthesis of the TAT conjugate are found in Appendix E2 (supplement).…”

The sensitivity of magnetic particle imaging and fluorine-19 magnetic resonance imaging for cell tracking

“…19 F may not be suitable for tracking labeled cancer cells to metastatic sites. Further improvements to PFPE nanoemulsions will enhance 19 F cellular sensitivity, such as incorporation of paramagnetic agents 16,17 , or the addition of surface modifications to enhance the uptake of PFPE nanoemulsions 18 .…”

Prospect of cell penetrating peptides in stem cell tracking

“…To explore the biodistribution of cells, CPPs had also been used to help label and track CAR-T cells. In experiments on mice, TAT increased the MRI signal intensity of perfluorocarbon nano-emulsions by 8 times, and the tumor cell killing analysis showed that the markers did not affect cell function and viability [ 92 ]. Unfortunately, the application of CPPs to enhance MRI labeling effect of MSCs is still in the early experiments in vitro, although CPPs have been proven to be widely effective in increasing the signal strength of magnetic particles.…”