Cytosolic Delivery of Nanolabels Prevents Their Asymmetric Inheritance and Enables Extended Quantitative in Vivo Cell Imaging.
Long-term in vivo imaging of cells is crucial for the understanding of cellular fate in biological processes in cancer research, immunology, or in cell-based therapies such as beta cell transplantation in type I diabetes or stem cell therapy. Traditionally, cell labeling with the desired contrast agent occurs ex vivo via spontaneous endocytosis, which is a variable and slow process that requires optimization for each particular label-cell type combination. Following endocytic uptake, the contrast agents mostly remain entrapped in the endolysosomal compartment, which leads to signal instability, cytotoxicity, and asymmetric inheritance of the labels upon cell division. Here, we demonstrate that these disadvantages can be circumvented by delivering contrast agents directly into the cytoplasm via vapor nanobubble photoporation. Compared to classic endocytic uptake, photoporation resulted in 50 and 3 times higher loading of fluorescent dextrans and quantum dots, respectively, with improved signal stability and reduced cytotoxicity. Most interestingly, cytosolic delivery by photoporation prevented asymmetric inheritance of labels by daughter cells over subsequent cell generations. Instead, unequal inheritance of endocytosed labels resulted in a dramatic increase in polydispersity of the amount of labels per cell with each cell division, hindering accurate quantification of cell numbers in vivo over time. The combined benefits of cell labeling by photoporation resulted in a marked improvement in long-term cell visibility in vivo where an insulin producing cell line (INS-1E cell line) labeled with fluorescent dextrans could be tracked for up to two months in Swiss nude mice compared to 2 weeks for cells labeled by endocytosis.
Laboratory of General Biochemistry and Physical Pharmacy, Ghent University , 9000 Ghent, Belgium.|Centre for Nano- and Biophotonics, Ghent University , 9000 Ghent, Belgium.
Ranhua
R
Xiong
Laboratory of General Biochemistry and Physical Pharmacy, Ghent University , 9000 Ghent, Belgium.
Freya
F
Joris
Biomedical NMR Unit, Faculty of Medicine, Katholieke Universiteit Leuven , 3000 Leuven, Belgium.
Sayuan
S
Liang
Inflammation Research Center, Image Core Facility, VIB , 9052 Ghent, Belgium.|Department of Biomedical Molecular Biology, Ghent University , 9052 Ghent, Belgium.
Riet
R
De Rycke
Inflammation Research Center, Image Core Facility, VIB , 9052 Ghent, Belgium.|Department of Biomedical Molecular Biology, Ghent University , 9052 Ghent, Belgium.
Saskia
S
Lippens
Laboratory of General Biochemistry and Physical Pharmacy, Ghent University , 9000 Ghent, Belgium.
Jo
J
Demeester
Department of Molecular Biotechnology, Ghent University , 9000 Ghent, Belgium.|Max-Planck Institute of Colloids and Interfaces , 14424 Potsdam, Germany.
Andre
A
Skirtach
Laboratory of General Biochemistry and Physical Pharmacy, Ghent University , 9000 Ghent, Belgium.
Koen
K
Raemdonck
Biomedical NMR Unit, Faculty of Medicine, Katholieke Universiteit Leuven , 3000 Leuven, Belgium.
Uwe
U
Himmelreich
Laboratory of General Biochemistry and Physical Pharmacy, Ghent University , 9000 Ghent, Belgium.
Stefaan C
SC
De Smedt
Laboratory of General Biochemistry and Physical Pharmacy, Ghent University , 9000 Ghent, Belgium.|Centre for Nano- and Biophotonics, Ghent University , 9000 Ghent, Belgium.|Univ Lille 1, Univ Lille Nord France, IEMN, UMR 8520, 59652 Villeneuve Dascq, France.|Univ Lille 1, Univ Lille Nord France, Lab Phys Lasers Atomes & Mol, UMR 8523, 59655 Villeneuve Dascq, France.
Kevin
K
Braeckmans
Cell labeling; cytosolic delivery; in vivo cell tracking; nanoparticle inheritance; quantum dots; vapor nanobubble photoporation