Peptide Sequence and Backbone Conjugation Chemistry Direct Alginate Hydrogel Bioactivity for Human Mesenchymal Stem...

Peptide-functionalized alginate hydrogels are a promising biomaterial candidate for supporting the expansion of adherent human mesenchymal stem cells (hMSCs) toward therapeutic applications. Despite widespread RGD-modified alginates, critical aspects of peptide conjugation chemistry, including verification of the coupling orientation and role of amino acids flanking the core RGD motif, remain underexplored. Through systematic material characterization and peptide sequence design, this work addresses these important research gaps. We first thoroughly characterized the alginate backbone composition via 1H NMR spectroscopy using peak deconvolution, providing more distinct quantitation of alginate backbone composition and conjugation sites. We then coupled 4 peptide sequences systematically designed to probe the effect of the spacer N-terminal to RGD, either glycine-glycine-phenylalanine-threonine (GGFT) or cysteine-glycine-phenylalanine-threonine (CGFT), and the presence or absence of a C-terminal serine-proline (SP) motif. As a spacer, we capitalized on using phenylalanine (F), as opposed to the previously published tyrosine (Y), to mitigate side reactions with the phenolic hydroxyl group while retaining aromatic protons (δ ∼7.2-7.4 ppm) for a quantitative degree of substitution and coupling efficiency characterization via 1H NMR. Further included in our design was polar threonine (T), to enhance the aqueous solubility of the overall peptide spacer. Critically, we provided evidence (previously only assumed) that amide bond formation occurred through the peptide N-terminus as intended (versus the unwanted side reaction of coupling the guanidinium group of arginine (R) to alginate's carboxyl) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) coupling. The attachment, spreading, and proliferation of hMSCs to hydrogel films of this well-characterized alginate series (cross-linked with calcium chloride) were then examined to understand the role of peptide sequence on performance. Alginates functionalized with GGFTRGD or CGFTRGD failed to support hMSC expansion, whereas inclusion of the C-terminal SP sequence (yielding GGFTRGDSP or CGFTRGDSP) promoted robust cell attachment and a >10-fold increase in the final cell number, demonstrating the essential role of SP immediately C-terminal to RGD, which more closely resembles the sequence of native fibronectin. Together, the characterization methods and sequence/activity relationships established herein provide a foundation for the rational design of well-characterized and reproducible peptide-alginate hydrogels for scalable hMSC expansion and therapeutic applications.