Taurine Boosts Cellular Uptake of Small D-Peptides for Enzyme-Instructed Intracellular Molecular Self-Assembly

Abstract

Due to their biostability, D-peptides are emerging as an important molecular platform for biomedical applications. Being proteolytically resistant, D-peptides lack interactions with endogenous transporters and hardly enter cells. Here we show that taurine, a natural amino acid, drastically boosts the cellular uptake of small D-peptides in mammalian cells by >10-fold, from 118 μM (without conjugating taurine) to >1.6 mM (after conjugating taurine). The uptake of a large amount of the ester conjugate of taurine and D-peptide allows intracellular esterase to trigger intracellular self-assembly of the D-peptide derivative, further enhancing their cellular accumulation. The study on the mechanism of the uptake reveals that the conjugates enter cells via both dynamin-dependent endocytosis and macropinocytosis, but likely not relying on taurine transporters. Differing fundamentally from the positively charged cell-penetrating peptides, the biocompatibility, stability, and simplicity of the enzyme-cleavable taurine motif promise new ways to promote the uptake of bioactive molecules for countering the action of efflux pump and contributing to intracellular molecular self-assembly.

Figure 1

(A) Molecular structures of precursors 1a and 1b, and the corresponding hydrogelators 2a and 2b after enzymatic transformation. (B) Taurine conjugation boosts cellular uptake of d-peptide precursors and subsequent enzyme-instructed self-assembly to form nanofibers, accumulating inside cells.

Figure 2

TEM images of the solutions of 1a in PBS buffer (A) before and (B) after addition of esterase. Scale bar = 100 nm. (C) SLS signals of the solution of 1a in PBS buffer without and with addition of esterase. Inset: corresponding analytical HPLC traces. (D) The confocal microscope image shows the appearance of fluorescence in the solution of 1a after esterase treatment (<10 min). Inset: corresponding image before esterase treatment. Scale bar = 100 μm. [1a] = 100 μM in PBS.

Figure 3

Fluorescent confocal microscope images showing the fluorescence emission in HeLa cells with the treatment of (upper) 1a and (bottom) 3a at 200 μM concentration in culture medium for 24 h and co-stained with Hoechst 33342 (nuclei). Scale bar = 50 μm.

Figure 4

(A) Uptake concentration (C_u) of 1a, 3a, and 4 inside HeLa cells after 24-h incubation. The incubating concentration is 200 μM. (B) C_u of 1a (200 μM) at different time points and temperatures. (C) C_u of 1a at different incubating concentration after 24 h. (D) C_u of 1a and 4 after washing the cells with PBS buffer at each wash.