Regulating the Rate of Molecular Self-Assembly for Targeting Cancer Cells

Abstract

Besides tight and specific ligand-receptor interactions, the rate regulation of the formation of molecular assemblies is one of fundamental features of cells. But the latter receives little exploration for developing anticancer therapeutics. Here we show that a simple molecular design of the substrates of phosphatases-tailoring the number of phosphates on peptidic substrates-is able to regulate the rate of molecular self-assembly of the enzyme reaction product. Such a rate regulation allows selective inhibition of osteosarcoma cells over hepatocytes, which promises to target cancer cells in a specific organ. Moreover, our result reveals that the direct measurement of the rate of the self-assembly in a cell-based assay provides precise assessment of the cell targeting capability of self-assembly. This work, as the first report establishing rate regulation of a multiple-step process to inhibit cells selectively, illustrates a fundamentally new approach for controlling the fate of cells.

Keywords: cancer; enzymes; rate regulation; selective inhibition; self-assembly.

Figure 1

Molecular structures of the tetrapeptide derivatives/precursors with two or one phosphotyrosines (TPD-2p and TPD-1p) and their fluorescent analogs (fTPD-2p and fTPD-1p). These precursors turn into the self-assembling D-peptidic hydrogelators (i.e., TPD and fTPD) after enzymatic dephosphorylation by alkaline phosphatase (ALP).

Figure 2

TEM images of aggregates/nanofibrils in the solutions of different precursors (TPD-2p, TPD-1p and fTPD-2p, fTPD-1p) or nanofibrils in the hydrogels formed by treating the solutions of the precursors with alkaline phosphatase (ALP). C = 0.5 wt%, pH = 7.4, [ALP] = 1 U/ml. The scale bar is 100 nm.

Figure 3

Confocal microscopy images of HepG2 and Saos-2 cells after ALPL antibody staining. Nuclei are stained by Hoechst 33342.

Figure 4

(A) 48-hour cytotoxicity of TPD-2p and TPD-1p on HepG2 and Saos-2 cells at different concentration. The viability differences between the two cell lines are labelled as well. (B) 48-hour cell viability of HepG2 and Saos-2 cells incubated with TPD-2p and TPD-1p (300 μM) with or without ALPL inhibitors (DQB, 2 μM) for 48 h. The initial cell number is 1 × 10⁴ cells/well.

Figure 5

Confocal microscopy images of (A) HepG2 and (B) Saos-2 cells treated with fTPD-2p and fTPD-1p at the concentration of 500 μM for 12 hours. Nuclei are stained by Hoechst 33342.

Figure 6

The time-dependent curves for dephosphorylation process of TPD-2p and TPD-1p (500 μM) after incubation with the cell lysate of HepG2 and Saos-2 at 37 °C in PBS buffer.

Scheme 1

Schematic illustration of the use of the rate of molecular self-assembly (controlled by numbers of enzymatic site) to amplify the difference of the expression level of ALPs in different cell lines.