Noninvasive Probing of the Spatial Organization of Polymer Chains in Hydrogels Using Fluorescence Resonance Energy Transfer (FRET)

摘要

Hydrogels are increasingly used in a variety of biomedical applications owing to their many advantageous features. Physical properties of hydrogels formed from cross-linking between polymer chains are regulated with the cross-linking density, type of cross-linking molecules, and chemistry and molecular weight of polymer chains. It is believed that the spatial conformation and organization of polymer chains in gels are altered by these variables, but this hypothesis has not been examined with gels formed with chemical cross-linking, because of the lack of analytical tools that allow the organization of the polymer chains to be quantified. Several scattering and microscopic techniques have been used to analyze the nano- and microstructure of gels, but these tools do not analyze the spatial intra- and intermolecular arrangements of the single polymer chains in the gel. This study demonstrates a fluorescent resonance energy transfer (FRET)-based technique which allows one to noninvasively monitor the conformation of gel-forming polymer chains and evaluate the intermolecular association of polymer chains in the cross-linked network. In this study, the effects of gelling and the number of cross-links in the hydrogel on the spatial organization of polymer chains were specifically analyzed using alginate hydrogels formed with a varied number of ionic cross-links between polymer chains. Single alginate molecules were labeled with both fluorescein (FITC, donor) and rhodamine (Rho, acceptor) to examine the conformational changes within polymer chains both in solution and in a hydrogel (intrachain FRET) (Figure 1 in Supporting Information). Alternatively, fluorescein and rhodamine were coupled to separate alginate molecules in order to evaluate the intermolecular association of polymer chains in the gels (interchain FRET). The number of fluorophores coupled to a single polymer was varied from one to five, and the size of the single polymer chain and association between polymer chains were minimally changed over this range, as confirmed with the gel permeation chromatography and rheological measurements. The results of these studies suggest that gelling processes and the number of cross-links significantly alter the intermolecular association of polymer chains while leading to minimal change in the intramolecular conformation. This study thus provides better understandings of the hydrogel structure on the molecular scale.