Noninvasive probing of the spatial organization of polymer chains in hydrogels using fluorescence resonance energy transfer (FRET).

Hydrogels are increasingly used in a variety of biomedical applications1-3 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 crosslinking 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,4 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 techniques5 have been used to analyze the nanoand microstructure of gels, but these tools do not analyze the spatial intraand intermolecular arrangements of the single polymer chains in the gel. This study demonstrates a fluorescent resonance energy transfer (FRET)-based technique5,6 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. In the first study, the ability of the FRET technique to monitor the conformational changes of polymer chains was examined using polymers labeled with both fluorescein and rhodamine (denoted FITC-Rho-alginate). The conformation of the polymer chains in solution was first altered with the pH of the solution, because alginate, which consists of uronic acids, is known to change its hydrodynamic radius in response to pH changes. Polymer solutions were prepared in the dilute regime, at which physical contacts between polymer chains were minimized. Interestingly, at pH 5, the emission intensity of fluorescein (IFITC) in the FITC-Rhoalginate, maximized at a wavelength (λ) of 520 nm, was greatly reduced, as compared with the emission intensity of alginate molecules labeled solely with fluorescein and analyzed under the same condition (Figure 1a). The emission intensity of the coupled rhodamine (IRho), maximized at λ of 580 nm, was greatly increased, indicating significant energy transfer at this condition. Increasing the pH to 9 led to a smaller decrease of IFITC and a smaller increase of IRho as compared to pH 5 (Figure 1a). The distance between fluorophores (r) was calculated from the emission intensity of the donor in the absence of the acceptor (IFITC,0), the presence of the acceptor (IFITC), and the critical Förster radius (R0) using the equation r ) R0[IFITC,0,/IFITC 1]-1/6. IFITC,0 and R0 were measured in parallel with IFITC at varied pH (SI, Table 2).6 Interestingly, the distance between fluorophores was linearly related to the pH of the solution (Figure 1b). The ratio between IRho and IFITC was also linearly related to changes of pH (SI, Figure 2a). The variation of DFRET with pH could also be visually demonstrated by a change of fluorescence when solutions were illuminated with an ultraviolet light (SI, Figure 2b). Control experiments were also performed in parallel to confirm that the FRET signal in these experiments resulted solely from the intramolecular association of FITC and Rho, and not from intermolecular interactions. Equal volumes of polymers labeled with fluorescein (FITC-alginate) and rhodamine (Rho-alginate) were individually mixed while maintaining solutions in the dilute regime and analyzed. No significant change of IFITC and IRho was detected with changes of pH (SI, Figure 3). These results clearly indicated that the FRET signal was solely related to the conformational change of single polymer chains in this dilute concentration. However, increasing the polymer concentration would eventually lead to interchain energy transfer because of the increase of physical contacts between polymer molecules. † Harvard University. ‡ University of Illinois. § Harvard-MIT Division of Health Sciences and Technology. Figure 1. FRET experiment to analyze the conformation of polymers in dilute polymer solutions: (a) emission spectrum from alginate solution with pH varied from 9 to 5. (b) relation of distance between FITC and Rho coupled to a single polymer chain to pH. Published on Web 03/24/2007