Designing Visible Light‐Cured Thiol‐Acrylate Hydrogels for Studying the HIPPO Pathway Activation in Hepatocellular Carcinoma Cells

Abstract

Various polymerization mechanisms have been developed to prepare peptide-immobilized poly(ethylene glycol) (PEG) hydrogels, a class of biomaterials suitable for studying cell biology in vitro. Here, we report a visible light mediated thiol-acrylate photopolymerization scheme to synthesize dually degradable PEG-peptide hydrogels with controllable crosslinking and degradability. We systematically evaluated the influence of immobilized mono-thiol pendant peptide on the crosslinking of these hydrogels. We further proposed methods to modulate hydrogel crosslinking, including adjusting concentration of co-monomer or altering the design of multifunctional peptide crosslinker. Due to the formation of thioether ester bonds, these hydrogels were hydrolytically degradable. If the di-thiol peptide linkers used were susceptible to protease cleavage, these thiol-acrylate hydrogels could be designed to undergo partial proteolysis. The differences between linear and multi-arm PEG-acrylate (i.e., PEGDA vs. PEG4A) were also evaluated. Finally, we explored the use of the mixed-mode thiol-acrylate PEG4A-peptide hydrogels for in situ encapsulation of hepatocellular carcinoma cells (Huh7). The effects of matrix stiffness and integrin binding motif (e.g., RGDS) on Huh7 cell growth and HIPPO pathway activation were studied using PEG4A-peptide hydrogels. This visible light polymerized thiol-acrylate hydrogel system represents an alternative to existing light-cured hydrogel platforms and should be useful in many biomedical applications.

Comparative study of visible light polymerized gelatin hydrogels for 3D culture of hepatic progenitor cells

“…Reverse transcription of isolated RNA (8–130 ng) into cDNA was achieved using PrimeScript RT reagent kit (Clontech). Gene expression level was assessed by quantitative real‐time polymerase chain reaction (PCR) on an Applied Biosystems 7500 fast real‐time PCR machine and analyzed following a published protocol . In short, samples were run at 95 °C for 30 s, followed by 45 cycles of 95 °C for 5 s, 55 °C for 30 s, and at 72 °C for 30 s. Within each experimental group, the detected levels of the genes of interest were normalized to the level of house‐keeping gene GAPDH.…”

A fast-degrading thiol–acrylate based hydrogel for cranial regeneration

“…The thiyl radicals attack vinyl groups on PEGDA macromers to form carbonyl radicals and allow their propagation through other acrylates by chain-growth homopolymerization or by reacting with other thiols to allow step-growth addition, resulting in a mixed-mode step-chain growth polymerization mechanism ( Figure 1A) . The incorporation of thiol groups into PEGDA hydrogel imparts a thiol-etherester bond that makes bulk hydrolytic degradation possible and increases gel swelling ratio over time (Lin et al, 2015). The mechanism of PEGDA hydrogel gelation, stiffness, and degradation depend on several factors, including the concentrations of the NVP monomers and the CRGDS peptide.…”

“…However, a gradual decrease in the shear moduli is observed over time due to hydrolytic degradation. Previous studies have shown that the shear moduli of hydrogels are affected by the percentage of NVP macromers , Lin et al, 2015, Lee et al, 2014. Based on these studies, we chose 0.2% NVP peptide, which was shown to increase the viability and proliferation of hepatic cells over time .…”

“…as the backbone macromer, non-cleavage type photosensitizer eosin Y as the initiator, and bifunctional dithiothreitol (DTT) as the co-initiator and crosslinker ). In this system, the incorporation of thiol groups into the PEGDA hydrogel resulted in a thiolether-ester bond that allows bulk hydrolytic degradation (Lin et al, 2015). Because the PEGDA backbone lacks biological motifs, the cell adhesion peptide, Cys-Arg-Gly-Asp-Ser (CRGDS), was added to the prepolymer solution for incorporation into the network structure (Hern and Hubbell, 1998, Sokic and Papavasiliou, 2012, Li and Kao, 2003, Yang et al, 2005.…”

“…The advantage of using a synthetic hydrogel is the ability to design and control the degradation rate based on a target application. In previous studies, several parameters such as the concentrations of the co-monomers NVP, DTT, and the types of pendent peptide were shown to affect the degradation behaviors of hydrolytically degradable PEGDA hydrogels , Lin et al, 2015. It is known that the cellmaterial interaction is dependent on the type of cells encapsulated within the hydrogels.…”