Cell penetrating peptide functionalized perfluorocarbon nanoemulsions for targeted cell labeling and enhanced fluorine-19 MRI detection
Dina V. Hingorani
Department of Radiology, University of California San Diego, California
Search for more papers by this authorFanny Chapelin
Department of Bioengineering, University of California San Diego, California
Search for more papers by this authorEmma Stares
Department of Radiology, University of California San Diego, California
Search for more papers by this authorStephen R. Adams
Department of Pharmacology, University of California San Diego, California
Search for more papers by this authorHideho Okada
Department of Neurological Surgery, University of California San Francisco, California
Search for more papers by this authorCorresponding Author
Eric T. Ahrens
Department of Radiology, University of California San Diego, California
Correspondence
Eric T. Ahrens, Department of Radiology, University of California San Diego, 9500 Gilman Dr. #0695, La Jolla, CA 92093-0695.
Email: [email protected]
Search for more papers by this authorDina V. Hingorani
Department of Radiology, University of California San Diego, California
Search for more papers by this authorFanny Chapelin
Department of Bioengineering, University of California San Diego, California
Search for more papers by this authorEmma Stares
Department of Radiology, University of California San Diego, California
Search for more papers by this authorStephen R. Adams
Department of Pharmacology, University of California San Diego, California
Search for more papers by this authorHideho Okada
Department of Neurological Surgery, University of California San Francisco, California
Search for more papers by this authorCorresponding Author
Eric T. Ahrens
Department of Radiology, University of California San Diego, California
Correspondence
Eric T. Ahrens, Department of Radiology, University of California San Diego, 9500 Gilman Dr. #0695, La Jolla, CA 92093-0695.
Email: [email protected]
Search for more papers by this authorAbstract
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 19F MRI detection, is a non-invasive 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 ~1012 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 19F 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.
CONFLICT OF INTEREST
ETA is founder, consultant, member of the advisory board and shareholder of Celsense, Inc.
Supporting Information
| Filename | Description |
|---|---|
| mrm27988-sup-0001-FigS1-S8.pdfPDF document, 625 KB |
FIGURE S1 Synthesis scheme of F68-TAT co-surfactant. F68 is functionalized with a maleimide group to enable addition of the TAT peptide with a terminal cysteine (Cys-TAT) FIGURE S2 Size stability of TAT-F68-PFC nanoemulsions. The effect of % TAT incorporation on size (A) and polydispersity index (PDI) (B) of nanoemulsions is shown. The nanoemulsion size (C) and PDI (D) of nanoemulsions over time while stored at 4°C is displayed FIGURE S3 Optimization of lipid-TAT-PFC incubation time in Jurkat cells. Incubation times of 2, 4, and 18 h are tested as shown in (A), and the highest uptake is observed at 18 h. Jurkat cell viability is not altered by labeling for different durations (B) FIGURE S4 Cy5-TATA,P-F68-PFC synthesis scheme. Scheme shows synthesis of fluorescently labeled co-surfactants 8 and 9 consisting of Cy5 dye attached to the respective fluorous anchors 6 and 7 for incorporation into TATP-F68-PFC and TATA-F68-PFC nanoemulsions FIGURE S5 Localization impact of incorporation of fluorescent dye into surfactant layer during nanoemulsion preparation. (A) 19F uptake for cells treated with nanoemulsions prepared with and without anchored Cy5 at 10 mg/mL and 20 mg/mL doses; no significant differences are observed. Additionally, CAR T cell viability is not affected as shown in (B). (C) Intracellular localization of the nanoemulsion (Cy5 in red) in CAR T cells via confocal microscopy. Hoechst dye (nuclei, blue) and Alexa488 dye (cell membrane, green) is used to delineate cell structures FIGURE S6 Fluorescent dye conjugate nanoemulsions without TAT do not get internalized into CAR T cells. Panels show that dye compounds 8 and 9 do not induce non-specific internalization into live cells. Hoechst dye (nuclei, blue) and Alexa488 dye (cell membrane, green) are used to delineate the cells FIGURE S7 CAR T cell killing assay in vitro. Co-incubation of human U87-EGFRvIII-Luc glioma cells with TATP-F68-PFC-labeled or unlabeled CAR T cells, or untransduced T cells results in significant cell death at 12 and 24 h. CAR T cells exhibit significant tumor killing ability (~98%) compared to untransduced T cells (~60%). Killing efficacy is unaltered by nanoemulsion labeling of the cells FIGURE S8 Ex vivo 3D microimaging of excised glioma tumors harboring PFC-labeled CAR T cells. Contiguous images show overlays of 19F (pseudo-color) and 1H (grayscale) slices of right tumor receiving an intratumoral injection of 107 TATP-F68-PFC-labeled CAR T cells (A) and the left tumor with the same number of F68-PFC-labeled CAR T cells (B) |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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