Photoinduced Hydrogel-Forming Caged Peptides with Improved Solubility

Abstract

Self-assembling peptides are attractive alternatives in the field of biomaterial science due to their variability and biocompatibility. Unfortunately, such peptides have poor solubility, and their purification, synthesis, and overall handling are challenging. Our main objective was to develop a cage peptide design with full control over self-assembly. Theoretically, aggregation can be suppressed by temporally masking the amino acid side chains at critical positions. Taking into account several biological and synthetic requirements, a photosensitive protecting group, p-hydroxy-phenacyl (pHP), was chosen as the "masking" moiety. To test our theory, EAK16-II was chosen as a model self-assembling peptide, and a caged derivative containing photosensitive pHP groups was synthesized. Both spectroscopic and in vitro experiments on A2058 melanoma cells confirmed our hypothesis that the caged-EAK16-II peptide has good solubility and that the hydrogel formed after photolysis results in similar viability and cell aggregate formation of melanoma cells as the native EAK16-II-based hydrogel.

Scheme 1. Synthesis of Fmoc-l-Lys(pHP(tBu))–OH

(i) I₂, TBHP, DMSO in H₂O/MeCN, 1.5 h; (ii) Na₂Cr₂O₇/H₂SO₄ in MeTHF, 30 min; (iii) Na₂NO₂ in DMF, 30 min, 0 °C; (iv) DSC, DIPEA in DMF, 3 h, (v) Fmoc-l-Lys-OH, DIPEA in DMF.

Scheme 2. Scheme of Caged Peptide Synthesis with a “Two-Step” pHP Protection

Figure 1

Optical transmittance of native (nonprotected) EAK16-II peptide and caged-EAK16-II (300 μM) in PBS, 25 °C, 532 nm, 8 h.

Figure 2

(A) Analytical RP-HPLC of caged peptide (caged-EAK16-II) before and after photolysis (illumination). Peaks were detected at 220 nm, and the peptide solutions (300 μM) in PBS were illuminated at 254 nm for 15 min. (B) Size of aggregates of native (nonprotected) EAK16-II peptide and caged-EAK16-II (60 μM) by dynamic light scattering (DLS) measurement (at 25 °C with 532 nm for 5 h) before and after illumination in PBS.

Figure 3

In vitro cytotoxicity of p-hydroxyphenylacetic acid (HPAA, 1 mM) on A2058 human melanoma cell line growing in EAK16-II (300 μM) hydrogel (3D) or in a monolayer (2D) for 96 h. Cell viability measurements were performed using CellTiterGlo assay (Promega, Madison, WI, USA). Viability data were compared and normalized to 2D growing control cells (normalized live cells = 100%). Data are presented as mean values ± standard deviation (SD) from three independent experiments.

Figure 4

(A) Viability of the A2058 human melanoma cell line in native EAK-16-II and caged peptide hydrogels as well as in monolayer (2D) after 96 h. Cell viability was measured using CellTiterGlo assay (Promega, Madison, WI, USA) and normalized to 2D growing cells (control cells) (normalized live cells = 100%). Data are presented as mean values ± standard deviation (SD) from three independent experiments. (B) 3D representation of cellular arrangements in both types of hydrogels. Cells were stained with orange acridine (0.1 mM). The cellular aggregates were visualized by Zeiss Cell Discoverer 7 using 10× magnification and acquisition of Z-stacks with 10 μm intervals between individual planes.