A chemically labeled cytotoxic agent: two-photon fluorophore for optical tracking of cellular pathway in chemotherapy

Abstract

Chemotherapy is commonly used in the treatment of cancers. However, the mechanism of action of many of these agents is not well understood. We present the synthesis of a two-photon fluorophore (C625) and its biological application when chemically linked to a chemotherapeutic agent (AN-152). By using two-photon laser-scanning microscopy, the drug:fluorophore conjugate can be observed directly as it interacts with receptor-positive cell lines. The results of this project visually show the receptor-mediated entry of AN-152 into the cell cytoplasm and subsequently into the nucleus. These observations will allow for better understanding of the drug's therapeutic mechanism, which is a subject of ongoing research aimed at improving present methods for cancer therapy.

Figure 1

Synthetic pathway of C625. DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide.

Figure 2

Excitation spectrum (measured at emission wavelength of 500 nm) (a) and emission spectrum (measured at excitation wavelength of 400 nm) (b) of C625 in chloroform. AU, atomic units.

Figure 3

Coupling of C625 to AN-152. DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; BOP, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate.

Figure 4

Fluorescence images of a single MCF-7 cell (LH-RH receptor-positive), taken 4 min (a), 20 min (b), 40 min (c), and 50 min (d) after treatment with AN-152:C625 (0.6 μM). The staining of the cell occurred within 5 min, and the entry into the nucleus occurred after 20 min, with an increase of the fluorescence intensity over time.

Figure 5

Reflection image (a) and fluorescence image (b) of a UCI-107 cell (LH-RH receptor-negative) 40 min after treatment with AN-152:C625 (0.6 μM). Compared with the MCF-7 cell, the UCI-107 cell showed very little drug uptake.