FITC characterization of a cathepsin B-responsive nanoprobe for report of differentiation of HL60 cells into macrophages.

A cathepsin B (Cat B)-responsive optical nanoprobe is designed and prepared for report of HL60 differentiation into macrophage. A peptide sequence FRFK is linked to fluorescein (FITC) via the distant amino group of its lysine and N-terminated with acrylic acid (AA) to yield a molecular fluorescent probe AA-FRFK (FITC). The molecular probe is further embedded in poly(lactic-co-glycolic acid) (PLGA) to form a fluorescent nanoprobe AA-FRFK (FITC)@PLGA. The resultant optical nanoprobe is degradable by lysosomal Cat B, which is expressed in macrophages with a level of 5-10 times of that in HL60 cells. As a result, a significant decrease in fluorescence intensity is associated with the differentiation process of HL60 to macrophage and can be used as an indication of the differentiation process. The findings may pave a way toward the development of a universal in vitro labeling strategy of exogenous stem cells for report of in vivo cell differentiation by a dual-mode imaging modality involving optical imaging and magnetic resonance imaging.

Naphthalene-facilitated self-assembly of a Gd-chelate as a novel T2 MRI contrast agent for visualization of stem cell transplants.

Naphthalene is coupled with DOTA via a peptide sequence to yield an amphipathic MRI probe Nap-CFGKTG-DOTA-Gd (Nap-Gd) that can self-assemble into nanofibers. Incubation of NSCs, hMSCs and L929 cells in the presence of Nap-Gd in the µM level can introduce a significant amount of Nap-Gd into the cells as nanoclusters or nanofibers. The resultant intracellular Gd content is 10-60 times that achieved by incubation with Dotarem at the same concentration. The labelled cells exhibit a significant hyperintensive effect under T1-weighted MRI and a significant hypointensive effect under T2-weighted MRI. The hypointensive effect is more persistent than the hyperintensive effect, which allows in vivo tracking of labelled hMSCs for over 12 days under T2-weighted MRI. A comprehensive interpretation of the MRI signal intensity and the associated relaxation times reveals the structure-function relationship between the binding status of Nap-Gd in cells (structure) and the magnetic relaxation processes (function) toward a full understanding of the observed hyperintensive and hypointensive effects.

IQF characterization of a cathepsin B-responsive nanoprobe for report of differentiation of HL60 cells into macrophages.

Tracking of in vivo fates of exogenous cell transplants in terms of viability, migration, directional differentiation and function delivery by a suitable method of medical imaging is of great significance in the development and application of various cell therapies. In this contribution directional differentiation of HL60 cells into macrophages and granulocytes, and a difference in the associated expression level of cathepsin B (Cat B) among the parent and daughter cells is used as a model to guide and evaluate the development of a Cat B-responsive Abz-FRFK-Dnp@PLGA nanoprobe for an optical report of the differentiation process. A well-documented internally quenched fluorescence (IQF) pair coupled with a peptide substrate FRFK of Cat B was synthesized and imbedded in PLGA to form the nanoprobe. The nanoprobe is resistant to leakage when dispersed in water for 10 days. Degradation of the nanoprobe is dominated by Cat B. HL60 cells were then labelled with the Abz-FRFK-Dnp@PLGA nanoprobe to track the differentiation process. Differentiation of labelled HL60 cells into macrophages exhibited a significantly higher fluorescence relative to the granulocytes or the labelled parent cells. The fluorescence difference allows the differentiation process to be followed. The established characterization and assessment procedure is to be used for the development and evaluation of nanoprobes for other imaging modalities.