Uptake and Metabolism of a Dual Fluorochrome Tat-nanoparticle in HeLa Cells

Abstract

The ability to use magnetic nanoparticles for cell tracking, or for the delivery of nanoparticle-based therapeutic agents, requires a detailed understanding of probe metabolism and transport. Here we report on the development and metabolism of a dual fluorochrome version of our tat-CLIO nanoparticle termed Tat(FITC)-Cy3.5-CLIO. The nanoparticle features an FITC label on the tat peptide and a Cy3.5 dye directly attached to the cross-linked coating of dextran. This nanoparticle was rapidly internalized by HeLa cells, labeling 100% of cells in 45 min, with the amount of label per cell increasing linearly with time up to 3 h. Cells loaded with nanoparticles for 1 h retained 40-60% of their FITC and Cy3.5 labels over a period of 72 h in label-free media. Over a period of 144 h, or approximately 3.5 cell divisions, the T2 spin-spin relaxation time of cells was not significantly changed, indicating retention of the iron oxide among the dividing cell population. Using confocal microscopy and unfixed cells, both dyes were nuclear and perinuclear (broadly cytoplasmic) after Tat(FITC)-Cy3.5-CLIO labeling. Implications of the rapid labeling and slow excretion of the Tat(FITC)-Cy3.5-CLIO nanoparticle are discussed for cell tracking and drug delivery applications.

Magnetosome-like ferrimagnetic iron oxide nanocubes for highly sensitive MRI of single cells and transplanted pancreatic islets

“…To precisely investigate cell-level events, it is highly desirable to have the ability to image a very small number or single cells. To improve the cellular MRI capability, there have been various attempts to increase the cellular uptake of nanoparticles, such as conjugation with cell-penetrating peptides (27), encapsulation with dendrimers (28), and coincubation with transfection agents such as PLL (29). However, these approaches involve complicated conjugation steps, which sometimes lead to cell death by generating transient holes in the cell membrane (30).…”

A newly identified bacterial cell-penetrating peptide that reduces the transcription of pro-inflammatory cytokines

“…Interestingly, endocytosed CPPs are able to escape from endosomes and enter the cell cytosol (Magzoub et al, 2005). Currently, the nature of this endosomal escape mechanism remains elusive, but translocation across the endosomal lipid bilayer appears to be driven by endosome acidification (Potocky et al, 2003;Koch et al, 2003;Fischer et al, 2005).…”

Enhanced heparan sulfate proteoglycan-mediated uptake of cell-penetrating peptide-modified liposomes

“…Most studies involving PTDmediated cellular uptake have been carried out with Tat peptide. Tat-modified nanoparticles, such as cross-linked iron oxide for cell tracking by magnetic resonance [18] or polymeric drug carriers [19] showed improved uptake properties when compared to unmodified particles. The Tat peptide was also used to investigate PTD-mediated liposome uptake.…”