Amyloid-Based Injectable Hydrogel Derived from Hydrolyzed Hen Egg White Lysozyme

Abstract

Injectable hydrogels based on synthetic peptides have shown great promise in many biomedical applications. Yet, the high cost generally associated with synthetic peptides hinders the practical use of such peptide-based injectable hydrogel. To overcome this drawback, here, we propose to use the peptides from hydrolyzed low-cost natural protein as an economical and convenient peptide source to prepare an injectable hydrogel. We demonstrate the effectiveness of this alternative strategy using hen egg white lysozyme (HEWL) as an example. We used the peptide fragments from hydrolyzed HEWL as the gelator, and the magnesium ion as the performance enhancer to prepare the injectable hydrogel. We showed that the hydrogel is an amyloid gel as it was formed by a dense network of amyloid fibrils. We also showed that the hydrogel possesses a thixotropic property and displays a low cytotoxicity. The hydrolysis extent of HEWL was found to be a critical factor that influences the performance of the hydrogel. A fluorescence assay based on 8-anilinonaphthalene-1-sulfonic acid was proposed as a mean to precisely and conveniently control the hydrolysis extent of HEWL to enable the best injectability performance. At last, using doxorubicin as a model compound, we explored the potential of this amyloid-based hydrogel as an injectable drug carrier.

Figure 1

Strategy of making an injectable hydrogel from hydrolyzed HEWL.

Figure 2

Hydrolysis mechanism of HEWL. (A) C-terminally partial hydrolysis of the peptide bond at the aspartic acid site (Asp-X type hydrolysis). (B) N-terminally partial hydrolysis of the peptide bond at the aspartic acid site (X-Asp type hydrolysis). Red star denotes the peptide bond to be cleaved.

Figure 3

Amino acid sequence of HEWL.

Figure 4

(A) AFM characterization of HEWL fibril; (B) ThT fluorescent assay; (C) CR assay; and (D) FTIR characterization of HEWL fibril. a.u.: arbitrary unit.

Figure 5

(A) Gelation of the amyloid fibril powder derived from hydrolyzed HEWL. (B) Injectability of the hydrogel with drug entrapment.

Figure 6

Frequency sweep rheological analysis of the hydrogel with 3% (w/v) (i.e., 30 mg/mL) HEWL concentration at a constant strain of 0.1%.

Figure 7

Step-strain time-dependent rheological analysis of the hydrogel with 3% (w/v) HEWL concentration with a fixed angular frequency of 3 rad/s.

Figure 8

Cell viability of L929 cells after 24 and 48 h incubations with gel extracts.

Figure 9

Cumulative percentage release plot of different concentrations of Dox entrapped in the hydrogel in PB buffer with pH = 7.4 at 37 °C.

Figure 10

ANS assay during the hydrolysis of HEWL.