Utilizing D-Amino Acids to Engineer Peptides and Hydrogels with Stability and Controlled Degradability for Biological Applications

Inspired by Nature, peptides with different amino acid sequences have been designed for controlled enzymatic hydrolysis in range of unique applications in medicine and biology, including in the fabrication of biomaterial scaffolds¹. For example, hydrogels are often formed or modified with peptides to impart desired biophysical or biochemical properties. While degradation of engineered peptide linkers (e.g., sequence VPMSMRGG (VPMS)) often is desired (e.g., scaffold degradation in 3D cell delivery and culture), this degradation can be rapid depending on the level of matrix metalloproteinase (MMP) secreted by cell types, including macrophages, and even deleterious (e.g., undesired degradation in 2D culture). Accessible approaches are needed for tuning peptide degradation profiles for extended cell culture and other biological applications. Previous studies have shown that substituting L-amino acids with D-amino acids increases the resistance of peptides to degradation in the presence of proteases². In this study, we systematically examined how substituting different numbers and positions of L-amino acids with D-amino acids within the VPMS peptide impacted its degradation and the degradation of hydrogels made with it. For the different peptide designs, we quantified their degradation profiles and cytotoxicity in the presence of relevant enzymes and cells. Analysis by tandem liquid chromatography-mass spectrometry allowed us to confirm the identity of the degraded peptide fragments and the time scale of degradation of peptides in the presence of Collagenase II and IV at various concentrations. We report that, by switching more L-amino acids with D-amino acids, we observed slower degradation profiles and ultimately a non-degradable sequence within the experiment time course. Further, when hydrogels are formed using these different amino acid sequences, mass loss profiles correlated with associated peptide degradation rates, suggesting that the degradation profile of hydrogels can be tuned with systematic D-amino acid substitutions. Finally, we studied the cytotoxicity of the different peptide sequences and observed that D-amino acid substitutions at specific sites resulted in increased cytotoxicity in select cases. Overall, our results support the importance of peptide design and the opportunity that D-amino acid substitutions provide for achieving tunable degradation profiles while maintaining low cytotoxicity.

Reference:

1. Bomb et al., Biomaterials Science, 2022
2. Lueckgen et al., Biomaterials 2019