Photochemistry and Photobiology, 2013, 89: 1433–1441 Impact of Dye-Protein Interaction and Silver Nanoparticles on Rose Bengal Photophysical Behavior and Protein Photocrosslinking † Madeline J. Simpson 1 , Horacio Poblete 2 , May Griffith 3,4 , Emilio I. Alarcon* 1 and Juan C. Scaiano* 1,4 1 Department of Chemistry and Centre for Catalysis Research and Innovation, University of Ottawa, Ottawa, ON, Canada 2 Center for Bioinformatics and Molecular Simulation, Universidad de Talca, Chile 3 University of Ottawa Eye Institute, Ottawa, ON, Canada 4 Integrative Regenerative Medicine Centre, Department of Clinical and Experimental Medicine, Link€ oping University, Link€ oping, Sweden Received 26 April 2013, accepted 18 June 2013, DOI: 10.1111/php.12119 ABSTRACT The role of recombinant Type-I human collagen in the free form or forming AgNP@collagen on the photophysical and photochemical behavior of rose Bengal was analyzed. The formation of dye aggregates on the protein surface was experimentally observed and corroborated by docking calcu- lations. The formation of such aggregates is believed to change the main oxidative mechanism from Type-II (singlet oxygen) to Type-I (free radical) photosensitization. Remark- ably, the presence of AgNP in the form of AgNP@collagen altered the dynamics of dye triplet deactivation, effectively preventing the dye degradation and reducing the extent of protein crosslinked. Both crosslinked rHC and AgNP@colla- gen were able to support fibroblasts proliferation, but only the material containing silver was resistant to S. epidermidis infection. INTRODUCTION Interactions between low molecular weight molecules and proteins not only play a key role in the biochemistry of living organisms, but are also responsible for changes in the photo- chemical properties of photosensitizers (1–3). Association of a dye within the structure of a protein can promote, for example, direct electron transfer from the dye triplet/singlet-excited states to the nearby amino acid residues (4–8). For the past 3 years, our research team has focused on the development of new strate- gies for the fabrication of hybrid materials for biomedical applications (9–11). Thus, we have explored the photochemical synthesis of new silver nanoparticles protected by proteins, such as Type-I collagen, for tissue regeneration (11). However, little is known on how the presence of nanoparticles affects the pro- tein/dye interaction and more importantly what the impact of the nanoparticles on the photochemical behavior of the dye may be. Light-mediated crosslinking of biomolecules, including collagen, is an example of the use of photogenerated reactive intermediates from a photosensitizer to promote the formation of chemical bonds (12–16). The wide variety of sensitizers and conditions employed to achieve crosslinking plus the intrinsic dye/biomolecule interactions have made it difficult to discern the involvement of pure radical intermediate (Type-I mechanism) or singlet oxygen (Type-II mechanism)-mediated crosslinking. Proteins, including Type-I collagen, have been shown to act as efficient stabilizers for spherical silver nanoparticles (AgNP) and have been used to generate new, stable hybrid nanostructures, AgNP@collagen, with strong biocompatibility and antimicrobial properties (11). Further work has been successfully incorporated these nanoparticles within chemically crosslinked collagen-based hydrogels (unpublished data). However, the effect of silver nanoparticles on the photocrosslinking efficiency in the presence of a dye has not been explored. By exploiting the use of AgNP@collagen as wide-spectrum antimicrobial agents, new photocrosslinked materials will be produced with positive impacts on the prevention of potential infections in tissue regeneration. Thus, in the present work, we have explored the effect of Type-I recombinant human collagen in its free form or as capping agents for AgNP on the photophysical, photochemical and photocross- linking properties of rose Bengal (RB), a model dye currently used in sutureless photodynamic tissue welding (17–23). MATERIALS AND METHODS Chemicals. Rose Bengal (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluoresce- in, 99% certified purity), 2-(N-morpholino)ethanesulfonic acid (MES), NaH 2 PO 4 , Na 2 HPO 4 , sodium chloride and sodium azide were purchased from Sigma Aldrich (Oakville, ON). Type-I recombinant human collagen (rHCollagen Type-1, Fibrogen, FG-5014, 3.02  0.30 mg/mL) was employed as models for the association of RB to collagen-like proteins. Changes in the rose Bengal absorption and emission spectra. Absorp- tion and emission spectra for rose Bengal in the presence of different concentrations of rHC were measured in a M5 Molecular Devices Micro- plate reader using 200 lL of solution in Corning â 96-well plates. All measurements were carried out in 10 mM MES buffer pH 5.0 at room temperature unless otherwise indicated. Fluorescence emission measure- ments were obtained upon 525 nm excitation from the bottom of the plate. In all cases, a 530 nm cut off filter was employed and background signal discounted using previous integration of the emission spectrum. Formation of rose Bengal aggregates was monitored by plotting the changes in the absorbance at 550 nm as a function of Type-I collagen concentration at a constant dye concentration. An increase in absorbance at shorter wavelengths (520 nm) was also observed providing a clear indication of dye aggregation (2, 24). Isotherms were measured for the different Type-I collagens. The same procedure was carried out to explore the association of rose Bengal to rHC-protected silver nano- particles. *Corresponding authors email: scaiano@photo.chem.uottawa.ca (Tito Scaiano), emilio@photo.chem.uottawa.ca (Emilio I. Alarcon) †This article is part of the Special Issue dedicated to the memory of Elsa Abuin. © 2013 The American Society of Photobiology 1433