β-Sheet to Random Coil Transition in Self-Assembling Peptide Scaffolds Promotes Proteolytic Degradation

Abstract

One of the most desirable properties that biomaterials designed for tissue engineering or drug delivery applications should fulfill is biodegradation and resorption without toxicity. Therefore, there is an increasing interest in the development of biomaterials able to be enzymatically degraded once implanted at the injury site or once delivered to the target organ. In this paper, we demonstrate the protease sensitivity of self-assembling amphiphilic peptides, in particular, RAD16-I (AcN-RADARADARADARADA-CONH₂), which contains four potential cleavage sites for trypsin. We detected that when subjected to thermal denaturation, the peptide secondary structure suffers a transition from β-sheet to random coil. We also used Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) to detect the proteolytic breakdown products of samples subjected to incubation with trypsin as well as atomic force microscopy (AFM) to visualize the effect of the degradation on the nanofiber scaffold. Interestingly, thermally treated samples had a higher extent of degradation than non-denatured samples, suggesting that the transition from β-sheet to random coil leaves the cleavage sites accessible and susceptible to protease degradation. These results indicate that the self-assembling peptide can be reduced to short peptide sequences and, subsequently, degraded to single amino acids, constituting a group of naturally biodegradable materials optimal for their application in tissue engineering and regenerative medicine.

Keywords: RAD16-I; degradation; proteolysis; random coil; scaffold; self-assembling peptides; β-sheet.

Figure 1

Schematic model of the nanofiber developed by self-assembling RAD16-I molecules. The peptide alternates hydrophilic (R, arginine and D, aspartic acid) and hydrophobic (A, alanine) amino acids that form a β-sheet structure in aqueous solutions. The nanofiber is formed by a double tape of assembled RAD16-I molecules in antiparallel β-sheet configuration.

Figure 2

Circular dichroism studies of RAD16-I samples diluted in deionized water at a concentration of (a) 25 µM; (b) 50 µM; (c) 75 µM. Spectra were recorded at different temperatures of 20, 40 and 90 °C; (d) Circular dichroism of non-diluted peptide samples (5 mg/mL in water, 2.9 mM). Stock peptide samples (2.9 mM) were incubated at 20 or 90 °C for 10 min before being diluted to 25 µM to record the spectra.

Figure 3

AFM images of the self-assembling peptide RAD16-I. (a) RAD16-I nanofibers at room temperature; (b) higher magnification images of untreated samples; (c) RAD16-I nanofibers after 10 min denaturation at 90 °C; (d) higher magnification images of thermally treated samples. Arrows show small nanofiber fragments.

Figure 4

MALDI spectra of RAD16-I. (a) Potential cleavage sites of trypsin on RAD16-I; (b) MALDI spectrum of untreated RAD16-I; (c) MALDI spectrum of RAD16-I after 1 min trypsin incubation; (d) MALDI spectrum of RAD16-I after 30 min trypsin incubation; (e) MALDI spectrum of thermally denatured RAD16-I after 1 min trypsin incubation; (f) MALDI spectrum of thermally denatured RAD16-I after 30 min trypsin incubation; (g) Evolution of RAD16-I degradation peaks as a function of the incubation time with trypsin; (h) Evolution of RAD16-I peak (m/z 1713) in untreated and thermally treated trypsin digested samples inactivated by the MALDI-TOF sample preparation process.

Figure 5

AFM images of untreated (RT) and thermally treated (90 °C) RAD16-I after trypsin incubation. RAD16-I incubated with trypsin for (a) 10 min and (b) 60 min. Thermally treated RAD16-I incubated with trypsin for (c) 10 min and (d) 60 min. Scale bars represent 500 nm.

Figure 6

Schematic representation of the model for the tryptic degradation of untreated and thermally treated RAD16-I samples. (a) Terminally located peptides are in equilibrium between β-sheet and random coil structure; (b) Thermal treatment accelerates this structural transition, in which the random coil peptide molecules are dissociated from the fiber ending and, thus, becoming susceptible to trypsin cleavage. On the contrary, peptides located in the stable fiber structure are inaccessible to the enzyme.