Biofunctionalization of Poly(acrylamide) Gels

Abstract

Engineering novel biomaterials that mimic closer in vivo scenarios requires the simple and quantitative incorporation of multiple instructive signals to gain a higher level of control and complexity at the cell-matrix interface. Poly(acrylamide) (PAAm) gels are very popular among biology labs as 2D model substrates with defined biochemical and mechanical properties. These gels are cost effective, easy to prepare, reproducible, and available in a wide range of stiffness. However, their functionalization with bioactive ligands (cell adhesive proteins or peptides, growth factors, etc.) in a controlled and functional fashion is not trivial; therefore reproducible and trustable protocols are needed. Amine or thiol groups are ubiquitous in natural or synthetic peptides, proteins, and dyes, and hence routinely used as handles for their immobilization on biomaterials.We describe here the preparation of mechanically defined (0.5-100 kPa, range that approximates the stiffness of most tissues in nature), thin PAAm-based hydrogels supported on a glass substrate and covalently functionalized with amine- or thiol-containing bioligands via simple, robust, and effective protocols.

Thiol-Methylsulfone-Based Hydrogels for 3D Cell Encapsulation

“…Thiols effectively react with aromatic rings bearing MS groups under physiological conditions via a nucleophilic aromatic substitution mechanism. , This reaction is characterized by high yield, high chemoselectivity, and the formation of a stable thio-ether product . Moreover, its reaction rate can be regulated by adjusting the pH in the range 6–9 and by adjusting the electron-deficient character of the aromatic substrate bearing the MS group. , The thiol-MS reaction, originally developed for the selective blocking of thiol-containing proteins and for protein bioconjugation in solution, has been recently applied by our group to biofunctionalize poly­(acrylamide) hydrogels for 2D cell culture. − The observed high reaction conversion and moderate reaction rate of the thiol-MS chemistry as well as the good stability , and proven cytocompatibility of formed adducts are ideal properties for cross-linking cell-laden hydrogels. In this manuscript, hydrogels based on the thiol-MS reaction are reported for the first time.…”

Tailorable biofunctionalization of poly(acrylamide) hydrogels via firefly luciferin-bioinspired click ligation accelerates cell attachment, spreading and proliferation

“…An adapted protocol was followed. [25] Round glass coverslips (15 mm diameter) were acrylsilanized by overnight incubation with 1% v/v solution of 3-acryloxypropyl-trimethoxysilane (APM, dissolved in ethanol (95% v/v) and water (4% v/v)). To stabilize the interface, the coverslips were washed with ethanol and water and then exposed to at 80 ˚C in vacuum for 1 h.…”

Current strategies for ligand bioconjugation to poly(acrylamide) gels for 2D cell culture: Balancing chemo-selectivity, biofunctionality, and user-friendliness

“…Selective dual functionalization of PAM gels has been reported combining methylsulfonyl/thiol coupling with NHS ester/amine coupling ( Farrukh et al, 2017a ; Paez et al, 2018 ). PAM gels were copolymerized with AA and MS comonomers.…”

“…Mechanically tunable PAM gels (for example, with regulable elasticity characterized by the Young’s modulus E in the range of 0.1–100 kPa) can be obtained by adjusting the bis-AM crosslinker concentration and/or the total content of acrylamide monomers in the precursor solution, ( Denisin and Pruitt, 2016 ), and numerous standard protocols can be found in the literature. ( Tse and Engler, 2010 ; Paez et al, 2018 ) In contrast, precise modulation of the biochemical properties (e.g., cell-adhesiveness to promote cell-material interactions) remains challenging because biofunctionalization of these materials is not straightforward. The reason is that PAM hydrogels are intrinsically protein repellent and chemically inert, with the lateral amide groups being poorly reactive under mild aqueous conditions.…”