Cytosolic Delivery of Nanolabels Prevents Their Asymmetric Inheritance and Enables Extended Quantitative in Vivo Cell Imaging

Abstract

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 labelling 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 vapour 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 labelling by photoporation resulted in a marked improvement in long-term cell visibility in vivo where an insulin producing cell line (INS-1E cell line) labelled with fluorescent dextrans could be tracked for up to two months in Swiss Nude mice compared to two weeks for cells labelled by endocytosis.

Gold Nanoparticle-Mediated Photoporation Enables Delivery of Macromolecules over a Wide Range of Molecular Weights in Human CD4+ T Cells

“…A wide range of therapeutically relevant cargoes are within this size range, including transcription factors, antibodies, and genome-editing nucleases [15]. Our results are in agreement with a previous study from our group on HeLa cells [55], and suggested that the VNB-induced pores can have a size of up to 30 nm, as inferred by the observed influx of 500 kDa dextrans. For the efficient delivery of cargoes of this size, the generated membrane pores should be big enough and remain open sufficiently long for these larger and slowly diffusing molecules to be able to reach the cell cytoplasm.…”

Rotatable Aggregation‐Induced‐Emission/Aggregation‐Caused‐Quenching Ratio Strategy for Real‐Time Tracking Nanoparticle Dynamics

“…The micelles in early endosomes then quickly transformed into a dissociated state from 5 to 25 min, which was consistent with the classical endocytosis process. [ 34 ] At 4 °C, the strong blue fluorescence signal from TPE in early endosomes was maintained over 25 min, suggesting that the dispersing process of PTI micelles in the early endosome was delayed due to the decreased molecule velocity in the low temperature. [ 35 ] The PTI micelles were gradually delivered to lysosomes from 25 to 60 min (Pearson's coefficient of ICGD/lysosome: 0.584, Figure 3b).…”

Accomplishments and challenges in stem cell imaging in vivo

“…To maximize reporter labeling, fluorescent polymeric NPs and quantum dots (QDs) have been used to label and monitor SCs [15], as well as monitor their accumulation in wound healing using a chemoattractants [100]. An interesting method of cell labeling is the direct introduction of reporter into the cytoplasm using sonoporation with US and MB, or photoporation with light and gold nanoparticles [101]. Using photoporation, Xiong et al showed a several-fold increase in labeling efficiency, symmetric signal distributed to daughter cells and the reporter remained visible for 2 months rather than 2 weeks [101].…”

“…An interesting method of cell labeling is the direct introduction of reporter into the cytoplasm using sonoporation with US and MB, or photoporation with light and gold nanoparticles [101]. Using photoporation, Xiong et al showed a several-fold increase in labeling efficiency, symmetric signal distributed to daughter cells and the reporter remained visible for 2 months rather than 2 weeks [101].…”