Lysosome-Targeted Bioprobes for Sequential Cell Tracking from Macroscopic to Microscopic Scales

Abstract

Longitudinal tracking of living cells is crucial to understanding the mechanism of action and toxicity of cell-based therapeutics. To quantify the presence of administered cells in the host tissue without sacrifice of animals, labeling of the target cells with a nontoxic and stable contrast agent is a prerequisite. However, such long-term live cell tracking is currently limited by the lack of fluorophores with steady optical and physicochemical properties in the near-infrared (NIR) window. Herein, for the first time, the design of fixable cell-tracking NIR fluorophores (CTNFs) with high optical properties, excellent cell permeation and retention, and high stability against chemical treatments is reported. Efficient cellular labeling and tracking of CTNFs using intraoperative optical fluorescence imaging by following the fate of NIR-labeled cells from the time of injection into animals to ex vivo cellular analysis after resection of the target tissue is demonstrated. Due to the lipophilic cationicity and primary amine docking group, CTNF126 outperforms the other tested fluorophores with rapid diffusion into the cytoplasmic membrane and sequestration inside the lysosomes, which prevents cellular efflux and improves cellular retention. Thus, CTNF126 will be useful to track cells in living organisms for the mechanism of action at the single cell level.

Keywords: fixable bioprobe; histological analysis; in vivo cell tracking; near-infrared fluorescence; optical imaging.

Figure 1.. Design of lysosome-targeted NIR fluorophores:

(a) Synthetic scheme of CTNF126 composed of two functional groups and (b) optical properties of three representative NIR fluorophores. Absorbance (Abs) and fluorescence (Fl) spectra were measured in FBS at 5 μM. (c) Longitudinal cell tracking process of NIR fluorophores and lysosomal sequestration of CTNF126. The thickness of red arrow represents ionization tendency.

Figure 2.. Physicochemical and optical stability of lysosome-targeted NIR fluorophores:

(a) Long-term cellular stability of CTNF126, IR786, and CTNF103 in live PC3 cells and (b) their stability after formaldehyde fixation and detergent washing was calculated (n = 5, mean ± s.d., *P <0.05; ***P <0.001). Scale bars = 25 μm.

Figure 3.. In vivo tracking of lysosomal fluorophore-labeled cells using the intraoperative optical imaging system.

(a) CTNF126 and IR786-stained cells were trapped in the lung with strong fluorescent signal at 5 min post-intravenous injection. The cells labeled with CTNF126 were found in the liver as an evidence of cell migration from the lung capillaries at 24 h post-injection. (b) The cells injected into the liver orthotopically were found in the lung after 6 h, implicated liver-to-lung tracking of the cells. All NIR fluorescence images are identically normalized. Abbreviations used are: He, heart; In, intestine; Ki, kidney; Li, liver; Lu, lung. Scale bars = 3 mm.

Figure 4.. Stability of histological processes, including formaldehyde fixation and H&E staining:

(a) Cell pellets mimicking the tissue structure and (b) the lung tissue sections harvested from C57BL/6 mice after intravenous administration of the cells labeled with lysosomal-targeted fluorophores. (c) Lysosomal sequestration of CTNF126 (left), LysoTracker (middle), and merged image of the two (right) in PC3 cells. All NIR fluorescence images have identical exposure and normalization. Scale bars = 25 μm.