Smart Thixotropic Hydrogels by Disulfide-Linked Short Peptides for Effective Three-Dimensional Cell Proliferation

Abstract

Supramolecular assembly of short peptides is a crucial process and has shown numerous potential applications as biomaterials. In the present work, the hydrogelation process of short peptides containing C-terminal “Lys–Cys” (KC) residues have been studied in detail. The N-terminal capping is found to be essential for effective gelation. Out of 12 peptides we studied, two of them could form hydrogels efficiently: Ac-VVKC-NH2 and Ac-FFKC-NH2. In both cases, the monomer-to-dimer formation through disulfide linkages by Cys residues controls the aggregation process. Interestingly, the presence of H2O2 facilitated the dimerization and thereby reduced the gelation time but could not impart much effect on the mechanical properties of the gels. Detailed rheological study revealed that both hydrogels are thixotropic in nature. Moreover, they are responsive to glutathione (GSH) due to the presence of disulfide linkages. However, the hydrogel of Ac-FFKC-NH2 is found to be stronger and more effective for biological applications. The thixotropic nature as well as a model drug release study in response to varying GSH concentration indicates the possible use of the hydrogel as an injectable local drug delivery vehicle. The hydrogel of Ac-FFKC-NH2 is noncytotoxic in nature. Three-dimensional cell proliferation has been found to be more effective than 2D, as it mimics the in vivo situation more closely if not exactly. In the present study, we have shown that both differentiated RAW macrophages and undifferentiated THP-1 monocytes could proliferate significantly within the 3D matrix of the hydrogel, without depicting any apparent cytotoxicity. Thus, the hydrogel of Ac-FFKC-NH2 has potential for application in localized drug administration and as a supporting biomaterial to study basic phenomena involving cell behavior.

Rational Design of Peptide-based Smart Hydrogels for Therapeutic Applications

“…Additionally, the ease of incorporation of stimuli sensitivity and tunability of mechanical properties of these materials make peptide based hydrogels inevitable choices for therapeutic and biomedical applications. Indeed, the last few decades have witnessed exhilarating advancement in the development of peptide hydrogels for a variety of therapeutic applications that range from, drug delivery ( Kuang et al, 2014 ; Thota et al, 2016 ), tissue engineering ( Kumar et al, 2017 ; Chowdhuri et al, 2020 ; Yang et al, 2020 ), wound healing ( Xiao et al, 2016 ; Cui et al, 2020 ), biofabrication ( Singh et al, 2008 ; Hedegaard et al, 2018 ) to name a few.…”

Short Peptide-Based Smart Thixotropic Hydrogels

“…Cysteine (Cys) is one of the vital amino acids and is prone to form disulfide bonds (–S–S–) in basic pH or under aerobic conditions, or by oxidation of dissolved oxygen in solvents ( Figure 5 A) [ 40 , 82 , 102 , 103 ]. Disulfide chemistry has been found to be of significant importance in biological systems in order to stabilize the protein’s secondary and tertiary structures [ 3 ].…”

Mechanics of 3D Cell–Hydrogel Interactions: Experiments, Models, and Mechanisms

“…Nonetheless, this finding suggests that stress relaxing hydrogels can support proliferation in stiffer hydrogels. Chowdhuri et al 355 reported rapid cell proliferation by ∼4.5-fold and ∼5.5-fold in 72 h of macrophages and monocytes, respectively, that were encapsulated in a self- assembling peptide hydrogel with a stiffness of ∼2 kPa (G). Interestingly, proliferation was higher in the 3D fibrous hydrogels compared to cells cultured on the same hydrogels in 2D.…”