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Peptides are widely used within biomaterials to improve cell adhesion, incorporate bioactive ligands, and enable cell-mediated degradation of the matrix. While many of the peptides incorporated into biomaterials are intended to be present throughout the life of the material, their stability is not typically quantified during culture. In this work, we designed a series of peptide libraries containing four different N-terminal peptide functionalizations and three C-terminal functionalizations to better understand how simple modifications can be used to reduce the nonspecific degradation of peptides. We tested these libraries with three cell types commonly used in biomaterials research, including mesenchymal stem/stromal cells (hMSCs), endothelial cells, and macrophages, and quantified how these cell types nonspecifically degraded peptides as a function of terminal amino acid and chemistry. We found that peptides in solution which contained N-terminal amines were almost entirely degraded by 48 h, irrespective of the terminal amino acid, and that degradation occurred even at high peptide concentrations. Peptides with C-terminal carboxylic acids also had significant degradation when cultured with the cells. We found that simple modifications to the termini could significantly reduce or completely abolish nonspecific degradation when soluble peptides were added to cells cultured on tissue culture plastic or within hydrogel matrices, and that functionalizations which mimicked peptide conjugations to hydrogel matrices significantly slowed nonspecific degradation. We also found that there were minimal differences in peptide degradation across cell donors and that sequences mimicking different peptides commonly used to functionalize biomaterials all had significant nonspecific degradation. Finally, we saw that there was a positive trend between RGD stability and hMSC spreading within hydrogels, indicating that improving the stability of peptides within biomaterial matrices may improve the performance of engineered matrices.
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Designing Coiled Coils for Heterochiral Complexation to Enhance Binding and Enzymatic Stability
Coiled coils, commonly found in native proteins, are helical motifs important for mediating intermolecular interactions. While coiled coils are attractive for use in new therapies and biomaterials, the lack of enzymatic stability of naturally occurring l-peptides may limit their implementation in biological environments. d-peptides are of interest for biomedical applications as they are resistant to enzymatic degradation and recent reports indicate that stereochemistry–driven interactions, achieved by blending d- and l-peptides, yield access to a greater range of binding affinities and a resistance to enzymatic degradation compared to l-peptides alone. To our knowledge, this effect has not been studied in coiled coils. Here, we investigate the effects of blending heterochiral E/K coiled coils, which are a set of coiled coils widely used in biomaterials. We found that we needed to redesign the coiled coils from a repeating pattern of seven amino acids (heptad) to a repeating pattern of 11 amino acids (hendecad) to make them more amenable to heterochiral complex formation. The redesigned hendecad coiled coils form both homochiral and heterochiral complexes, where the heterochiral complexes have stronger heats of binding between the constituent peptides and are more enzymatically stable than the analogous homochiral complexes. Our results highlight the ability to design peptides to make them amenable to heterochiral complexation, so as to achieve desirable properties like increased enzymatic stability and stronger binding. Looking forward, understanding how to engineer peptides to utilize stereochemistry as a materials design tool will be important to the development of next-generation therapeutics and biomaterials.
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- Award ID(s):
- 2143647
- PAR ID:
- 10596078
- Publisher / Repository:
- American Chemical Society
- Date Published:
- Journal Name:
- Biomacromolecules
- Volume:
- 25
- Issue:
- 8
- ISSN:
- 1525-7797
- Page Range / eLocation ID:
- 5273 to 5280
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1021/acs.biomac.4c00661
