Bifunctional Poly(acrylamide) Hydrogels through Orthogonal Coupling Chemistries

Abstract

Biomaterials for cell culture allowing simple and quantitative presentation of instructive cues enable rationalization of the interplay between cells and their surrounding microenvironment. Poly(acrylamide) (PAAm) hydrogels are popular 2D-model substrates for this purpose. However, quantitative and reproducible biofunctionalization of PAAm hydrogels with multiple ligands in a trustable, controlled, and independent fashion is not trivial. Here, we describe a method for bifunctional modification of PAAm hydrogels with thiol- and amine- containing biomolecules with controlled densities in an independent, orthogonal manner. We developed copolymer networks of AAm with 9% acrylic acid and 2% N-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)acrylamide. The covalent binding of thiol- and amine-containing chromophores at tunable concentrations was demonstrated and quantified by UV spectroscopy. The morphology, mechanical properties, and homogeneity of the copolymerized hydrogels were characterized by scanning electron microscopy, dynamic mechanical analysis, and confocal microscopy studies. Our copolymer hydrogels were bifunctionalized with polylysine and a laminin-mimetic peptide using the specific chemistries. We analyzed the effect of binding protocol of the two components in the maturation of cultured postmitotic cortical neurons. Our substrates supported neuronal attachment, proliferation, and neuronal differentiation. We found that neurons cultured on our hydrogels bifunctionalized with ligand-specific chemistries in a sequential fashion exhibited higher maturation at comparable culture times than using a simultaneous bifunctionalization strategy, displaying a higher number of neurites, branches, and dendritic filopodia. These results demonstrate the relevance of quantitative and optimized coupling chemistries for the performance of simple biomaterials and with sensitive cell types.

Photoactivatable Adhesive Ligands for Light‐Guided Neuronal Growth

“…The photoactivatable IK‐12 peptides were covalently bound to poly(acrylamide‐co‐acrylic acid) P(AAm‐AA) hydrogel films. These gels have recently been demonstrated to be valuable platforms for study of neuron differentiation and maturation . Gels of 2 kPa stiffness were selected for the neuronal cultures, on the basis of the current understanding that neurons prefer soft substrates for development in vitro .…”

“…Centrifugation at 123 g was performed for 5 min, cell pellets were re‐suspended in differentiating medium DMEM/F12 (GIBCO, 1 mL) and B27 (Invitrogen, 2 %), and cell density was counted. Substrates functionalized with different ligands were sterilized with ethanol, washed with PBS, and placed in a 24‐well cell culture plate; 5×10 4 cells were seeded in each well, cell growth was followed by regular intervals, and cells were fixed after 24–48 h …”

“…Cell viability and proliferation was calculated from time‐lapse videos. Lengths of axons were measured by using FIJI software, whereas numbers of neurites, dendrites, and dendritic filopodia were counted manually …”

Arylierungschemie für die Biokonjugation

“…Die Reaktionsgeschwindigkeit korrelierte mit dem pH-Wert der Lçsung,w as mit der Vorstellung übereinstimmt, dass Thiolate gegen MSBT eine grçßere Reaktivitäta ufweisen als Thiole. [83] Die ausgeprägte Thiolspezifitätd ieser Sulfonreagentien wurde außerdem durch selektive Modifikation komplexerer Moleküle wie Polyacrylamidhydrogele [84] und Proteinenzyme [85] demonstriert. [80] Mindt und Mitarbeiter stellten 18 F-markierte Phenyloxadiazolsulfon-Reagentien her und zeigten, dass diese füre ine rasche Markierung von Peptiden und Proteinen fürd ie Anwendung in der Positronenemissionstomographie (PET) geeignet sind.…”

“…[82] Xian und Furdui entwickelten ein neues heteroaromatisches Alkylsulfon, 4-(5-Methylsulfonyl-[1,2,3,4]tetrazol-1-yl)phenol (MSTP), das fürd ie selektive Blockade von Protein-Thiolen ohne eine Kreuzreaktivitätm it Sulfensäuren geeignet ist. [83] Die ausgeprägte Thiolspezifitätd ieser Sulfonreagentien wurde außerdem durch selektive Modifikation komplexerer Moleküle wie Polyacrylamidhydrogele [84] und Proteinenzyme [85] demonstriert.…”