In Vitro and In Vivo Cell Uptake of a Cell-Penetrating Peptide Conjugated with Fluorescent Dyes Having Different Chemical Properties

Abstract

In molecular imaging, a targeting strategy with ligands is widely used because specificity can be significantly improved. In fluorescence imaging based on a targeting strategy, the fluorescent dyes conjugated with ligands may affect the targeting efficiency depending on the chemical properties. Herein, we used a cell-penetrating peptide (CPP) as a ligand with a variety of fluorescent cyanine dye. We investigated in vitro and in vivo cell uptake of the dye-CPP conjugates when cyanine dyes with differing charge and hydrophilicity/lipophilicity were used. The results showed that the conjugates with positively charged and lipophilic cyanine dyes accumulated in cancer cells in vitro, but there was almost no accumulation in tumors in vivo. On the other hand, the conjugates with negatively charged and hydrophilic cyanine dyes did not accumulate in cancer cells in vitro, but fluorescence was observed in tumors in vivo. These results show that there are some cases in which the cell uptake of the dye-peptide conjugates may differ significantly between in vitro and in vivo experiments due to the chemical properties of the fluorescent dyes. This suggests that attention should be paid to the chemical properties of fluorescent dyes in fluorescence imaging based on a targeting strategy.

Structure‐Activity Studies of Nitroreductase‐Responsive Near‐Infrared Heptamethine Cyanine Fluorescent Probes

“…The inherent "stickiness" of a cationic heptamethine cyanine dye, such as probe 5, suggests that it is unlikely to exhibit the favorable in vivo biodistribution properties needed for high contrast imaging of living subjects. [38] Even the anionic benzoindolinene probe 7, with two appended sulfonate groups, was highly self-aggregated in the NTR assay solution and the rate of NTR-catalyzed cleavage and concomitant fluorescence "turn on" was unacceptably slow. Many literature studies obviate this type of dye self-aggregation problem by including DMSO in the testing solution, but this strategy had no effect on the self-aggregation of probes 5 and 7 (data not shown).…”

“…[41] Looking to the future, we expect that the two alkyne groups at each end of probe 9 can be used as conjugation sites for attachment of prosthetic groups that further improve probe NIR fluorescence imaging performance by enabling helpful molecular functions such as cell targeting, cell permeation, or ratiometric signalling. [38] Experimental Section…”

“…Despite the probe self‐aggregation, cell microscopy studies using A549 cells (human lung adenocarcinoma) showed that both probes produced statistically higher NIR fluorescence in hypoxic cells compared to the normoxic cells (Figure 3). The inherent “stickiness” of a cationic heptamethine cyanine dye, such as probe 5 , suggests that it is unlikely to exhibit the favorable in vivo biodistribution properties needed for high contrast imaging of living subjects [38] . Even the anionic benzoindolinene probe 7 , with two appended sulfonate groups, was highly self‐aggregated in the NTR assay solution and the rate of NTR‐catalyzed cleavage and concomitant fluorescence “turn on” was unacceptably slow.…”

Tuning the affinity of amphiphilic guest molecules in a supramolecular polymer transient network

“… 28 However, solely cy5 also shows cellular uptake, with large intracellular clusters being visible intracellular, as shown in previous studies. 29 This indicates that the cy5 can have an effect on the cellular uptake of the cy5–bound guests. Still, clear differences are observed within the cy5-labelled guest molecules, with UPy–cy5 showing no uptake, and c12–cy5 and chol–cy5 displaying cellular uptake, with the latter one also displaying membrane binding.…”

Oligonucleotide Therapies in the Treatment of Arthritis: A Narrative Review

“…Cell-penetrating peptides (CPPs) such as TAT, penetratin and antennapedia are short cationic peptide sequences that can translocate across cell membranes [ 113 , 114 ]. Therefore, a number of studies have demonstrated the utility of CPPs to transport biological cargo into cells in vitro and into tissues in vivo, including oligonucleotide cargo.…”