Rozans Samuel J, Moghaddam Abolfazl Salehi, Wu Yingjie, Atanasoff Kayleigh, Nino Liliana, Dunne Katelyn, Pashuck E Thomas
Lehigh University.
bioRxiv. 2024 Apr 24:2024.04.19.590329. doi: 10.1101/2024.04.19.590329.
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 functionalization to better understand how simple modifications can be used to reduce non-specific 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 non-specifically degraded peptide 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 hours, 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 cells. We found that simple modifications to the termini could significantly reduce or completely abolish non-specific 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 non-specific degradation. We also found that there were minimal differences across cell donors, and that sequences mimicking different peptides commonly-used to functionalized biomaterials all had significant non-specific 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.
肽广泛应用于生物材料中，以改善细胞黏附、引入生物活性配体并实现细胞介导的基质降解。虽然许多掺入生物材料的肽旨在在材料的整个生命周期中存在，但它们在培养过程中的稳定性通常未被量化。在这项工作中，我们设计了一系列肽库，包含四种不同的N端肽功能化和三种C端功能化，以更好地了解如何通过简单修饰来减少肽的非特异性降解。我们用生物材料研究中常用的三种细胞类型测试了这些肽库，包括间充质干/基质细胞（hMSC）、内皮细胞和巨噬细胞，并量化了这些细胞类型如何将肽作为末端氨基酸和化学性质的函数进行非特异性降解。我们发现，溶液中含有N端胺的肽在48小时内几乎完全降解，无论末端氨基酸如何，即使在高肽浓度下也会发生降解。与细胞一起培养时，具有C端羧酸的肽也有显著降解。我们发现，当将可溶性肽添加到在组织培养塑料上或水凝胶基质中培养的细胞中时，对末端进行简单修饰可以显著减少或完全消除非特异性降解，并且模仿肽与水凝胶基质缀合的功能化显著减缓了非特异性降解。我们还发现不同细胞供体之间差异最小，并且模仿常用于功能化生物材料的不同肽的序列都有显著的非特异性降解。最后，我们发现RGD稳定性与hMSC在水凝胶中的铺展之间存在正相关趋势，这表明提高生物材料基质中肽的稳定性可能会改善工程化基质的性能。
