Dephosphorylation of D-peptide derivatives to form biofunctional, supramolecular nanofibers/hydrogels and their potential applications for intracellular imaging and intratumoral chemotherapy

Abstract

D-Peptides, as the enantiomers of the naturally occurring L-peptides, usually resist endogenous proteases and are presumably insensitive to most enzymes. But, it is unclear whether or how a phosphatase catalyzes the dephosphorylation from D-peptides. In this work, we examine the formation of the nanofibers of D-peptides via enzymatic dephosphorylation. By comparing the enzymatic hydrogelation of L-peptide and D-peptide based hydrogelators, we find that the chirality of the precursors of the hydrogelators affects little on the enzymatic hydrogelation resulted from the removal of the phosphate group from a tyrosine phosphate residue. The attachment of a therapeutic agent (e.g., taxol) or a fluorophore (e.g., 4-nitro-2,1,3-benzoxadiazole) to the D-peptide based hydrogelators affords a new type of biostable or biocompatible hydrogelators, which may find applications in intratumoral chemotherapy or intracellular imaging, respectively. This work, as the first comprehensive and systematic study of the unexpected enzymatic dephosphorylation of D-peptides, illustrates a useful approach to generate supramolecular hydrogels that have both biostability and other desired functions.

Figure 1

The binding of the phosphate precursors (presented as CPK model: yellow, phosphorous; red, oxygen.) to the active site of an ALP (presented as solid ribbons). (A) L-peptide based precursor (1a) and (B) D-peptide based precursor (1b) binding to the phosphatase. (C) Top view and (D) side view of 1a (green) and 1b (dark pink) in the active site. Purple atoms represent the parts of 1a and 1b that would occupy same space in the active site.

Figure 2

The TEM images of the hydrogels formed by using ALP (1.0 U/mL) to treat 1b at pH 7.6 and concentrations of (A) 0.4 wt%, (B) 0.6 wt% (C) 0.8%wt, and (D) 1.0 wt%. Inset: optical images. Scale bar is 100 nm.

Figure 3

The ³¹P NMR shows the conversion of 1.0 wt% of (A) 1a and (B) 1b catalyzed by the phosphatase (0.02 U/mL) at pH 7.6 at 3 minutes and 4, 12, 24, and 48 h; The time dependent rheology study of 1.0 wt% of (C) 1a and (D) 1b catalyzed by the phosphatase (0.02 U/mL) at pH 7.6.

Figure 4

The optical images and TEM images of the hydrogels formed by using ALP (1.0 U/mL) to treat 0.4 wt% of (A) 1a and (B) 1b at pH 7.6. (C) The strain sweep and (D) the frequency sweep of the hydrogels 2a and 2b.

Figure 5

(A) The optical image and TEM image of hydrogel formed by 0.4 wt% of 4b at pH 7.4 upon the catalysis of ALP (20.0 U/ml). (B) The fluorescent confocal microscope image of a HeLa cell incubated with 500 μM of 4b in PBS buffer (scale bar is 10 μm). The fluorescent confocal microscope images of HeLa cells incubated with 500 μM of 4b without (C) or with (D) the PTP1B inhibitor (25 μM) (scale bar is 50 μm).

Figure 6

(A) The optical and TEM images of hydrogel formed by 1.8 wt% of 10b at pH 7.4 with the catalysis of ALP (1 U/mL) with scale of 100 nm; (B) The IC₅₀ values of 6, 9b, and 10b incubated with HeLa cells after 72 h; (C) The relative tumor sizes and (D) relative weights of mice treated with 6, 10a, and 10b for in vivo tests.

Scheme 1

The synthetic route of the precursor of the NBD or taxol-containing hydrogelator based on a D-peptide.