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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

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 calculations. The formation of such aggregates is believed to change the main oxidative mechanism from Type-II (singlet oxygen) to Type-I (free radical) photosensitization. Remarkably, 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@collagen were able to support fibroblasts proliferation, but only the material containing silver was resistant to S. epidermidis infection.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

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 photochemical 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 strategies 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 protein/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 photocrosslinking properties of rose Bengal (RB), a model dye currently used in sutureless photodynamic tissue welding [17-23].

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Chemicals

Rose Bengal (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein, 99% certified purity), 2-(N-morpholino)ethanesulfonic acid (MES), NaH2PO4, Na2HPO4, 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

Absorption and emission spectra for rose Bengal in the presence of different concentrations of rHC were measured in a M5 Molecular Devices Microplate reader using 200 μL of solution in Corning® 96-well plates. All measurements were carried out in 10 mm MES buffer pH 5.0 at room temperature unless otherwise indicated. Fluorescence emission measurements 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 nanoparticles.

Type-I collagen molecular model

The homology model of Type-I collagen was built using ICM software [25] using the primary sequence obtained from UniProt ID P02452 and the crystallographic data of Collagen-Like peptide (PDB ID: 1CAG) as structural references [26]. The sequence of the chosen model shares a 57% identity with the sequence of the Collagen-Like peptide.

Docking of Rose Bengal with Type-I collagen

The structure of Rose Bengal (RB) was built and optimized using the Molsoft ICM software [25]. For identifying the most probable binding site for RB in Type-I collagen and determining the number of dye molecules that can bind simultaneously, we subsequently performed docking again using Molsoft ICM [25] and using a cubic box of 30 × 90 × 30 Å3 centered on the center of mass of the triplex-helix of Type-I collagen. Default docking parameters were used. Docking progresses by a systematic search of conformations of RB, followed by insertion of RB into the surface of the protein-I model. Subsequently, minimization of the interaction energy between the RB and the collagen as defined by the ECEPP/3 force field [27] was performed. Global optimization was performed by making random conformational changes of the free bonds, torsions and angles according to the Biased Probability Monte Carlo (BPMC) [28]. Finally, the lowest energy configurations were subjected to a Monte Carlo Procedure to refine the conformation of the protein and further reduce the energy, this procedure was performed with each of the 10 RB docked consecutively. All images were generated using the VMD 1.9 software [28].

Zeta potential and circular dichroism measurements

Zeta potential (mV) for mixtures containing different collagen:RB ratios, from 0 to 10, were measured using a dynamic light scattering Malvern Zetasizer Nano ZS. Circular dichroism measurements of rHC in the presence of different RB concentrations were carried out in a similar fashion to the recently described by us [29]. Briefly, CD spectra were measured in a Jasco J-810 spectropolarimeter (1.0 nm data pitch, continuous scanning mode, 20 nm/min scan rate) at 25 ± 0.2°C using 0.1 cm pathlength quartz cells. Changes on the secondary structure of the protein upon RB were analyzed upon calculation of Rpn values (the ratio between 220 and 195.5 nm CD signals).

Time-resolved measurements

Rose Bengal triplet transient absorption measurements were carried out in a LFP 111 laser-flash photolysis system (Luzchem Inc., Ottawa, Canada) equipped with a Surelite OPO Plus (pump with a Nd-YAG 355 nm) operating at 550 nm and 8.0 mJ/pulse in 1.0 cm path length-fused silica cuvettes (Luzchem Inc., Ottawa, Canada). The effect of the protein addition on the generation of RB triplet-excited states was analyzed in terms of ΔOD0, the initial optical density for the sample, without and at different protein concentrations as given in Table 1. Samples were degassed using 99.99% pure N2 where 2.5 mL of each solution was purged for 3 h prior to measurements. Dye degradation was maintained lower than 5% in all cases.

Table 1. Changes of initial absorbance (OD0) and lifetimes for 10 µm RB triplet excited state, OD0 620 nm, and ground state bleaching, OD0 530 nm measured at different protein concentrations upon laser excitation at 550 nm. Singlet oxygen phosphorescence initial intensity at 1270 nm has been also included. OD and Io data have been divided by the sample absorbance at 550 nm. Errors correspond to the standard deviation from the exponential decay fitting
Collagen/µm (occupation number, n)OD0 620 nm/abs 550 nmτ620 nm/µsOD0 530 nm/abs 550 nmτ530 nm/µs1O2 Io/abs
  1. *Values in brackets measured for AgNP@collagen; nd: not detected; †Associate error is considered for this particular case as the response time of the system ≈7.0 ns. All measurements were carried out at room temperature in 10 mm MES buffer pH 5.0 (for 1O2 measurements pD = 5.0, pD = pH + 0.41 (Glasoe, P. K. and F. A. Long (1960) Use of glass electrodes to measure acidities in deuterium oxide. J. Phys. Chem., 64, 188–190). Dye degradation was kept below 5.0% in all cases.

00.0271 ± 50.08584 ± 11.73
0.10 (25)≈0.0049 ± 10.03065 ± 10.80 [0.50]
0.50 (5.0)ndnd0.02229 ± 1nd
2.50 (1.0)nd [0.15]*nd [0.030 ± 0.007]0.02827 ± 1nd

The effect of collagen with and without AgNP on the singlet oxygen generation was quantified by following the singlet oxygen phosphorescence decay at 1270 nm with a Hamamatsu NIR detector (Peltier cooled at −62.8°C operating at 800 V) after laser excitation Surelite OPO Plus (pump with a Nd-YAG 355 nm), 8 mJ/pulse. Data were acquired and processed with customized Luzchem Research software.

Rose Bengal photodegradation and peroxides evaluation

Dye photodegradation was assessed by measuring the decrease in the monomer absorption over time upon exposure to a 525 nm LED (≈4900 mW/m2 nm). A schematic diagram and picture of the actual setup are included in the supplementary information (see Supplementary Material, Figure S1). The irradiations were performed at room temperature in 10 mm MES buffer pH 5.0 in 1.0 cm optical pathlength quartz cuvettes. Samples (2.5 mL) were irradiated individually. Total peroxides and carbonyls were evaluated at different irradiation times. Briefly, peroxide content was determined by using Peroxo-Quant (from Thermo Scientific), that is based on the Fe(II)/(III) oxidation upon Fenton reaction with the peroxides. Thus, the resulting Fe(III) complexes with Xylenol orange/Fe(III) produce a new absorption at 550 nm [30]. Oxygen uptake measurements were also carried out using a MI-730 micro-oxygen electrode bedford (Microelectrodes, Inc.). The presence of anion radical superoxide was determined by using 20 μm hydroethidine fluorescent probe [31].

Lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE) and protein crosslinking

A 10 mm MES solution containing 2.5 μm recombinant human collagen and 2.5 μm rose Bengal was irradiated with a 525 nm LED for incremental time intervals. At each time interval 30 μL aliquots of solution were taken with microeppendorf tubes and stored in the dark at 4°C. To each aliquot 10 μL of LDS solution (Thermo Scientific, Code 84788) was added. Samples were incubated at 95°C for 5 min and 10 μL aliquots were loaded into wells of precast 4–15% polyacrylamide gels (Mini-PROTEAN® TGX™ Precast Gel, Bio-Rad). The remaining volumes of sample were centrifuged in a microcentrifuge (Biofuge pico, Heraeus Instruments, Kendro Laboratory Products, Germany) at 12 000 rpm for 60 s. Then, a 10 μL aliquot from the supernatant was taken and loaded into the gel. The gel was run in a Bio-Rad Mini Protean® 3 cell at variable voltage (50–180 V) for 45 min using a Fisher Biotech Electrophoresis System power source. A broad molecular weight standard (6.5–200 kDa) was employed as ladder (Bio-Rad, 161–0317). Gels were stained using GelCode Blue Safe Protein Stain according to the protocol provided by Bio-Rad. Gel pictures were taken in a gel documentation system (MultiImage® I, model DE450, serial no: 402830, AlphaInnotech) using the AlphaImager® EC by AlphaInnotech software interface. Further imaging processing was carried out using Image J [32]. To examine the light-mediated formation of Tyr–Tyr (dityrosine) –C–C- bonds, which form covalent bridges, the residues were selectively excited at 320 nm [33]. Emission spectra were collected from 330 to 700 nm.

Effect of collagen crosslinking on biocompatibility and anti-infective properties

The cytotoxicity of crosslinked collagen with and without AgNP was evaluated in vitro using primary human adult dermal fibroblasts (ATCC). The fibroblasts were grown in Dulbecco's modified eagle's medium (DMEM, Gibco) supplemented with 10% fetal calf serum and 1.0% penicillin/streptomycin, and cultured at 37°C, 5.0% CO2 and 100% humidity. Solutions (1.0 mL) containing 2.5 μm collagen or AgNP@collagen with and without 2.5 μm RB were irradiated in 24-well plates for 1 h with 525 nm LED and uncrosslinked material removed from the plate by aspiration. Then, 1 × 104 cells were seeded and cultured in 10% FCS-supplemented DMEM. Pictures of the cells in culture were taken by using a digital microscope JuLI and counting carried out using Image J software. Experiments were carried out by duplicate and counting in two different, but representative, areas of the plates carried out.

The antimicrobial activity of the crosslinked materials was assessed against the gram (+) Staphylococcus epidermidis (strain SE19). Samples were prepared as described for biocompatibility. Growth inhibition was carried out following standard protocols as described in the standard Clinical and Laboratory Standards Institute (CLSI) protocol [34]. Exponentially growing cultures were incubated at 37°C in 24-well plates (Falcon™) at a bacteria density of ≈1.0 × 105 cfu/mL in 25% Lysogeny broth (LB). Bacteria growth was monitored in a SpectraMax 5 from Molecular Devices as the optical density at 630 nm (OD630), which corresponds to the light scattering from the bacteria, in each well.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Association of RB with Type-I collagen

Rose Bengal (RB, scheme 1) has been used in the light-assisted crosslinking of collagen [18, 35], because of its high singlet oxygen quantum yield and photostability [36]. The photophysical properties of rose Bengal have been studied in homogeneous [37-39] and microheterogeneous systems including micelles [40-42], liposomes [3] and proteins [1, 2, 43]. These studies revealed that the photophysical properties of the dye are strongly dependent on the aggregation state and microenviroment where the dye is located. The formation of rose Bengal aggregates in microheterogenous systems including proteins and lipids are widely documented in the literature [1-3]. The formation of aggregates (dimers/trimers) produces an increase in the absorption intensity at shorter wavelengths, most likely due to the formation of H-aggregates [24]. This increase is also accompanied by a broadening of the absorption spectrum of the dye [2, 24].

image

Scheme 1. Chemical structure for rose Bengal.

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Figure 1A shows that the absorption spectrum of a 10 μm rose Bengal solution is modified upon addition of submicromolar concentrations of Type-I recombinant human collagen, rHC. In particular, the 10 nm redshift plus the broadening of the absorption spectrum upon rHC addition are clear indications of the dye association in the aggregate state, see inset of Fig. 1A. In fact, Fig. 1B displays the normalized absorption spectra for 10 μm RB at different concentrations of collagen, where a 200% increase in the aggregate band absorption can be seen, denoted as (1) in the figure, plus the above mentioned 10 nm redshift (2) at 2.5 μm collagen. In this matter, we have recently reported the use of the monomer/dimer ratio, defined as gamma (γ), as a tool to determine the aggregation state of ZnPc bound to bovine serum albumin [6]. As shown in the inset of Fig. 1B, it can be seen that γ for RB decreases ≈40% upon addition of 2.5 μm rHC, most likely due to the formation of dye aggregates within the protein.

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Figure 1. (A) Effect of human recombinant Type-I collagen addition on the absorption spectrum of 10 μm rose Bengal. Collagen concentrations employed were 0 μm (i), 0.1 μm, 0.3 μm, 0.75 μm, and 2.5 μm (ii) as indicated by the arrow in the plot. The black dashed area in the plot corresponds to the integrated area for rose Bengal in the absence of protein, whereas the blue dashed area is the corresponding extra area obtained upon addition of 2.5 μm of collagen. Inset: Changes on the absorption spectrum width measured at different protein concentrations. (B) Normalized absorption spectra for 10 μm rose Bengal in the presence of different concentrations of collagen as described in 1A; however, there is an overlap between the data measured for 0 and 0.1 μm. The arrows in the figure denote the relative increase in the aggregate form (i) and the redshift upon protein addition into the solution (ii). Inset: Variation in the gamma (γ) parameter for rose Bengal calculated at different concentration of protein. Gamma was calculated from the 520/554 nm ratio in each case (see main text). (C) Changes on rose Bengal absorption at 550 nm expressed as A/A0 at different protein/dye ratio obtained using different initial concentrations of dye: 2.5 μm (●), 5.0 μm (○) and 10 μm (□). The data point obtained for 2.5 μm collagen and rose Bengal has been included in the text with a black oval. (D) Fluorescence emission spectra of 10 μm rose Bengal in the presence of different concentrations of human recombinant Type-I collagen. Collagen concentrations 0 μm (i), 0.1 μm (ii), 0.3 μm (iii), 0.75 μm (iv), and 2.5 μm (v) Inset: Variation on the corrected rose Bengal Fluorescence intensity at 570 nm, plotted as F/F0, measured as function of protein concentrations. All measurements were carried out at room temperature in 10 mm MES buffer pH 5.0. Error bars correspond to a 10% associated experimental standard error.

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When the A/A0 ratio at 550 nm, depicting monomer absorption, is plotted as function of the protein/dye ratio, similar behavior is observed regardless of the initial rose Bengal concentration (Fig. 1C). This value reaches a plateau around 0.6 at occupation numbers ≈10 similar to those previously reported for binding of multiple rose Bengal molecules to human serum albumin [2]. No changes on the A/A0 value of 0.6 were observed even at occupation numbers above one. Addition of RB to 2.5 μm rHC solutions leads to an increase in the zeta potential values of the protein from +16 ± 1.3 to +21 ± 1.6 mV (P < 0.001) at 0 and 12.5 μm RB, respectively (see Figure S2 top). These data indicate a modification in the protein surface charge upon binding of up to 5.0 RB molecules per triple helix of rHC molecule, which is fully congruent binding of the dye on the protein surface. Note that no changes in the protein structure were observed throughout the whole range of RB concentrations employed in our work as revealed by CD spectroscopy (Figure S2 bottom), thus ruling out changes in the protein conformation as a cause for the differences in protein charge. Rose Bengal has a relatively low, but still measurable, fluorescence quantum yield (~0.018) in aqueous solutions. Upon addition of rHC, it is seen that the fluorescence intensity decreases by a factor of 10, see Fig. 1D inset, at occupation numbers of 5.0. This fluorescence quantum yield is considerably lower when compared to the 0.013 reported for occupation numbers ≈5.7 in the RB/human serum albumin system [6]. These differences could be explained in terms of the formation of aggregates on the protein surface as described below.

Molecular dynamics simulations of the interaction between RB and a collagen-like peptide [26] were also carried out to gather further information on the possible binding modes and multiple-occupation sites on Type-I collagen as seen in Fig. 2, left (top). The computational simulation revealed in fact that multiple-occupation binding for RB up to only 10 dyes per protein is energetically feasible (see Figure S3). Notably, we have found that RB binding energies favor multioccupation at numbers 3, 6, 8, and 10 as shown in Fig. 2 right (bottom), most likely due the formation of thermodynamically stable aggregates on the protein surface. These observations are compatible with those experimentally observed for the formation of aggregates upon RB binding to Type-I collagen discussed above. Note that this binding model differs from what has been reported in the literature for the interaction between rose Bengal and serum albumins due to the involvement of binding pockets within the protein [1, 2, 43]. In the presence of Type-I collagen, the binding follows a cooperative multioccupation process on the actual surface of the triple helix, as seen in Fig. 2 left (top), which leads to the formation of local dye aggregates around the collagen.

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Figure 2. Top: Docking simulation of the interaction of different numbers of RB molecules, as indicated by the n in the left with a Type-I collagen protein. Lateral and front views have been incorporated to facilitate the visualization of the arrangement of the RB molecules on the protein surface. Bottom: ICM scores calculated for different numbers of RB molecules per collagen from 1 to 10. The asterisks in the figure denote the most favorable occupation numbers for the system under our computational setup (see experimental for further details).

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Effect of silver nanoparticles on RB association with Type-I collagen

The effect of AgNP on the rose Bengal–collagen interaction was explored by preparing Type-I collagen protected AgNP. Thus, 4.4 ± 1.0 nm AgNP were prepared according to the methodology previously reported using Type-I recombinant human collagen. Fig. 3 top shows the changes in the absorption spectra, upon UVA irradiation of a solution containing 0.2 mm AgNO3, 0.2 mm I-2959 plus 2.5 μm rHC. The formation of a new absorption band around 400 nm after only 20 s of irradiation is a clear indication of the presence of AgNP in the system as described for porcine Type-I collagen [11], see inset, and human serum albumin [9]. Figure 3 bottom displays the changes in the RB absorption intensity at 550 nm as function of protein addition for AgNP@collagen, (Note: AgNP@collagen concentration corresponds to total collagen concentration). No significant differences were noticed between pure collagen and AgNP@collagen after plotting the 550 nm dye absorption at different protein concentrations, inset Fig. 3 bottom. This confirms that the AgNP do not modify the protein structure upon formation of AgNP@collagen, as previously reported [29]. Electrophoretic mobility of collagen was not affected by the presence of nanoparticles; see Figure S4, a fact fully compatible with the nonmodification on the protein conformation.

image

Figure 3. Top: Changes on the absorption spectrum of an aqueous solution containing 0.2 mm AgNO3, 0.2 mm I-2959 and 2.5 μm rHC upon UVA irradiation, as shown in the purple area in the figure, at 25 ± 0.5°C up to 15 min. Inset (top): Integrated area under the curve between 350 and 600 nm obtained using rHC or porcine Type-I collagen, Theracol, as protecting agents for the nanoparticles. The data obtained using 1.0 mm citrate are also included for comparison purposes; (bottom) 1 × 1 cm cuvettes before, left, and after 15 min UVA irradiation, right, of the solutions containing rHC. Bottom: Changes on 5.0 μm rose Bengal absorption spectrum as a function of AgNP@collagen concentration, as indicated in the figure. The arrow indicates the growth of the dimer absorption. Inset: Variation on rose Bengal absorption at 550 nm measured at different collagen concentrations for AgNP@collagen or collagen, rHC was used in all cases. 10% error bars are included for data comparison. All measurements were carried out at room temperature.

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Photophysical and photochemical properties of RB bound to Type-I collagen

The effect of the dye association on the photophysical properties of rose Bengal was explored by measuring the dye's triplet transient absorption at 620 nm, see Fig. 4, and the ground state recovery at 530 nm upon laser excitation. Table 1 summarizes the main photophysical properties for RB as either a free dye or when in a complex with Type-I collagen with and without AgNP. This table shows that the addition of rHC at concentrations higher than 0.1 μm causes the triplet signal to vanish and reduces the ground state bleaching to almost a quarter of the value measured in 10 mm MES without protein. Interestingly, for 2.5 μm rHC, it was not possible to detect any RB triplet absorption, probably due to fast intramolecular reaction, while for 2.5 μm AgNP@collagen an eight-fold enhancement of the triplet state was observed, see inset Fig. 4. This transient had an exceptionally short lifetime, which resembles the state recently reported by the group for methylene blue@AuNP [44], see Fig. 4. Singlet oxygen emission was also affected by the presence of rHC in a similar fashion to the observed effect for the RB triplet as seen in Table 1. This could either indicate a lack of singlet oxygen generation or a fast intraprotein reaction of this intermediate within the collagen structure, as seen with human serum albumin [1, 2].

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Figure 4. Rose Bengal triplet transient traces measured at 620 nm for 2.5 μm RB in nitrogen purged 10 mm MES buffer pH 5.0. The inset depicts the transient trace obtained when 2.5 μm AgNP@collagen is added into the solution. All measurements were carried out at room temperature using a 550 nm OPO laser source at 8.0 mJ/pulse.

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The data for free rose Bengal photobleaching seen in the insets of Fig. 5A and B have been incorporated to show the differences in degradation rates between the free and bound dye. The degradation mechanism observed in a protein-free solution cannot be extrapolated to the trend observed in the presence of rHC. Thus, the faster photodegradation of RB in the presence of 2.5 μm rHC, see Fig. 5A, in “oxygen free” solutions as seen in inset Fig. 5A and the almost null effect of D2O suggest a Type I [45, 46], electron transfer mechanism, as primarily responsible for RB degradation in the presence of rHC. In agreement with this, oxygen uptake experiments did not reveal a measurable oxygen consumption over 30 min of irradiation. Furthermore, the superoxide anion radical detection assay did not reveal the presence of this intermediate under our experimental conditions (data not shown). Peroxides were only detectable for rHC in the 2–8 μm range at longer irradiation times exclusively under air and in the D2O samples. No peroxides were detected for any of the samples prepared using AgNP@collagen (data not shown). Interestingly, almost no dye photodegradation was observed when the irradiation was performed in the presence of AgNP@collagen as seen in inset Fig. 5B. This behavior can be understood if the nanoparticle acts as a controller for the dynamics of the dye within the protein. Similar behavior has been described for the lowest triplet excited state of methylene blue in the near vicinity of spherical gold nanoparticles [44]. Thus, our data may be explained if the quenching of the nascent triplets by the nanoparticle competes favorably with the initial electron transfer that is responsible for dye degradation.

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Figure 5. Rose Bengal, 2.5 μm, photodegradation upon 525 nm LED irradiation for up to 180 min, as the arrows indicate, in air-equilibrated solutions, 10 mm MES pH 5.0, in the presence of 2.5 μm rHC (A) or AgNP@collagen (B). Insets show the profile degradation expressed as the changes on the 550 nm absorbance measured under air or argon and in deuterated MES buffer; the profile obtained for RB in the absence of protein is also included. (C) Representative LDS electrophoresis images for selected time points, indicated at the top of the figure, of the samples shown in A and B measured under air. Bottom shows LDS pictures for two time points 5 and 180 min of samples measured under Argon for rHC and AgNP@collagen. Ladder is included at the left for the two cases.

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LDS electrophoresis data in Fig. 5C show that the rHC is effectively crosslinked under air to form dimers and likely trimers within the first 5 min of irradiation. Irradiation performed in deuterated MES buffer did not show any significant differences to the degradation in aqueous solutions under air. It was observed that the intensity and number of bands for larger molecular weights obtained for AgNP@collagen was considerably lower than the results obtained when rHC is employed. It can be clearly seen when the data measured under Ar are compared at 180 min irradiation as shown in Fig. 5C. These results are fully compatible with the above results regarding the role of AgNP in the photophysical behavior of rose Bengal; additionally, we can also see that the protein crosslinking is also affected. This observation implies that RB photodegradation promotes the crosslinking of rHC. Although further characterization of the crosslinked collagen material is needed prior to achieve any conclusion on the mechanism for collagen photocrosslinking. Our work reveals that the photocrosslinking efficacy of rose Bengal is in fact modified upon incorporation of silver nanoparticles within the protein structure. Preliminary work on this matter has shown the lack of dityrosine bonds under our experimental conditions (data not shown), which indicates the involvement of other amino acids during the formation of high molecular weight collagen aggregates.

Biocompatibility and antimicrobial activity of crosslinked rHC and AgNP@collagen

The biocompatibility of our crosslinked rHC was assessed by measuring the proliferation of human skin fibroblasts on plates containing the crosslinked materials and their respective controls without RB, but with rHC. Figure 6 (left) displays the cell proliferation profiles where it can be clearly seen that the crosslinking of the material does not affect the biocompatibility, when compared to the uncrosslinked rHC control, but retains the antibacterial properties as depicted in Fig. 6 right up to 18 h of culture. These results are fully compatible with those reported by our group for the biocompatibility and antimicrobial performance of photochemically prepared AgNP protected with Type-I porcine collagen [11] and they reflect that AgNP are still active as antibacterial agents within the photocrosslinked material.

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Figure 6. Left: Number of human skin fibroblasts per cm2 counted on plates irradiated in the presence of the different sources of rHC with and without rose Bengal, see experimental for further details; asterisk indicates cells which were confluents. Right: Growth inhibition profile for 1 × 105 cfu/mL S. epidermidis seeded on 24-well plate containing irradiated solutions of the different sources of collagen employed in this work. Absorption at 630 nm was measured every 15 min over an 18 h time interval.

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Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

In summary, we have proven that the presence of rHC modifies the photophysical and photochemical behavior of rose Bengal by promoting the formation of aggregates on the protein surface. This leads to a change on the main oxidative mechanism of the dye from being a pure Type-II (singlet oxygen) to a Type-I (free radical) photosensitizer. It was, however, observed that the presence of AgNP in the form of AgNP@collagen are able to modify such behavior not by changing the association of the dye to the protein in the ground state, but rather by altering the dynamics of the triplet state deactivation. The behavior is consistent with the rapid excited dye deactivation observed in laser-flash photolysis experiments. Although, further work is needed to have a better understanding on the RB and AgNP dynamics to the best of our knowledge, the foregoing is the first report of nanoparticle control of dye photophysical properties in hybrid materials.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors dedicate this work to the memory of Elsa Abuin, a mentor, teacher and friend. This work was supported by NSERC (Canada). H.P. thank the Doctoral Program of Applied Sciences of Talca University, as well as CONICYT-Chile for a doctoral fellowship. EIA thanks Professor Irene Kochevar whose talk at ASP meeting in Montreal 2012 inspired this project.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information
  • 1
    Alarcon, E., A. M. Edwards, A. Aspee, D. Gonzalez-Nilo, F. E. Moran, C. D. Borsarelli, E. A. Lissi, H. Poblete and J. C. Scaiano (2010) Photophysics and photochemistry of dyes bound to human serum albumin are determined by the dye localization. Photochem. Photobiol. Sci. 9, 93102.
  • 2
    Alarcon, E., A. M. Edwards, A. Aspee, C. D. Borsarelli and E. A. Lissi (2009) Photophysics and photochemistry of rose bengal bound to human serum albumin. Photochem. Photobiol. Sci. 8, 933943.
  • 3
    Hugo, E., E. Abuin, E. Lissi, E. Alarcon and A. M. Edwards (2011) Effect of temperature on the photobehavior of rose bengal associated with dipalmitoylphosphatidyl choline liposomes. J. Lumin. 131, 24682472.
  • 4
    Foley, M. S. C., A. Beeby, A. W. Parker, S. M. Bishop and D. Phillips (1997) Excited triplet state photophysics of the sulphonated aluminium phthalocyanines bound to human serum albumin. J. Photoch. Photobiol. B. 38, 1017.
  • 5
    Alarcon, E., A. Aspee, M. Gonzalez-Bejar, A. M. Edwards, E. A. Lissi and J. C. Scaiano (2010) Photobehavior of merocyanine 540 bound to human serum albumin. Photochem. Photobiol. Sci. 9, 861869.
  • 6
    Alarcon, E., A. M. Edwards, A. M. Garcia, M. Munoz, A. Aspee, C. D. Borsarelli and E. A. Lissi (2009) Photophysics and photochemistry of zinc phthalocyanine/bovine serum albumin adducts. Photochem. Photobiol. Sci. 8, 255263.
  • 7
    Davila, J. and A. Harriman (1990) Photoreactions of macrocyclic dyes bound to human serum albumin. Photochem. Photobiol. 51, 919.
  • 8
    Bracchitta, G., A. Catalfo and G. De Guidi (2012) Photoinduced protein modifications by methylene blue and naproxen. Photoch. Photobio. Sci. 11, 18861896.
  • 9
    Alarcon, E., C. Bueno-Alejo, C. Noel, K. Stamplecoskie, N. Pacioni, H. Poblete and J. C. Scaiano (2013) Human serum albumin as protecting agent of silver nanoparticles: Role of the protein conformation and amine groups in the nanoparticle stabilization. J. Nanopart. Res. 15, 114.
  • 10
    Bueno-Alejo, C. J., C. D'Alfonso, N. L. Pacioni, M. Gonzalez-Bejar, M. Grenier, O. Lanzalunga, E. I. Alarcon and J. C. Scaiano (2012) Ultraclean derivatized monodisperse gold nanoparticles through laser drop ablation customization of polymorph gold nanostructures. Langmuir 28, 81838189.
  • 11
    Alarcon, E. I., K. Udekwu, M. Skog, N. L. Pacioni, K. G. Stamplecoskie, M. A. Gonzalez-Bejar, N. Polisetti, A. Wickham, A. Richter-Dahlfors, M. Griffith and J. C. Scaiano (2012) The biocompatibility and antibacterial properties of collagen-stabilized, photochemically prepared silver nanoparticles. Biomaterials 33, 49474956.
  • 12
    Shen, H.-R., J. D. Spikes, P. Kopecekova and J. Kopecek (1996) Photodynamic crosslinking of proteins. I. Model studies using histidine- and lysine-containing n-(2-hydroxypropyl) methacrylamide copolymers. J. Photochem. Photobiol., B 34, 203210.
  • 13
    Shen, H.-R., J. D. Spikes, P. Kopecekova and J. Kopecek (1996) Photodynamic crosslinking of proteins ii. Photocrosslinking of a model protein-ribonuclease a. J. Photochem. Photobiol., B 35, 213219.
  • 14
    Spikes, J. D., H.-R. Shen, P. Kopecekova and J. Kopecek (1999) Photodynamic crosslinking of proteins. Iii. Kinetics of the fmn- and rose bengal-sensitized photooxidation and intermolecular crosslinking of model tyrosine-containing n-(2-hydroxypropyl)methacrylamide copolymers. Photochem. Photobiol. 70, 130137.
  • 15
    Snibson, G. R. (2010) Collagen cross-linking: A new treatment paradigm in corneal disease – a review. Clin. Exp. Ophthal. 38, 141153.
  • 16
    Verweu, H. and J. v. Steveninck (1982) Model studies on photodynamic cross-linking. Photochem. Photobiol. 35, 265267.
  • 17
    Kamegaya, Y., W. A. Farinelli, A. V. Vila Echague, H. Akita, J. Gallagher, T. J. Flotte, R. R. Anderson, R. W. Redmond and I. E. Kochevar (2005) Evaluation of photochemical tissue bonding for closure of skin incisions and excisions. Lasers Surg. Med. 37, 264270.
  • 18
    Ibusuki, S., G. J. Halbesma, M. A. Randolph, R. W. Redmond, I. E. Kochevar and T. J. Gill (2007) Photochemically cross-linked collagen gels as three-dimensional scaffolds for tissue engineering. Tissue Eng. 13, 19952001.
  • 19
    O'Neill, A. C., J. M. Winograd, J. L. Zeballos, T. S. Johnson, M. A. Randolph, K. E. Bujold, I. E. Kochevar and R. W. Redmond (2007) Microvascular anastomosis using a photochemical tissue bonding technique. Lasers Surg. Med. 39, 716722.
  • 20
    Yao, M., A. Yaroslavsky, F. P. Henry, R. W. Redmond and I. E. Kochevar (2010) Phototoxicity is not associated with photochemical tissue bonding of skin. Lasers Surg. Med. 42, 123131.
  • 21
    Gu, C., T. Ni, E. E. Verter, R. W. Redmond, I. E. Kochevar and M. Yao (2011) Photochemical tissue bonding: A potential strategy for treating limbal stem cell deficiency. Lasers Surg. Med. 43, 433442.
  • 22
    Wang, Y., I. E. Kochevar, R. W. Redmond and M. Yao (2011) A light-activated method for repair of corneal surface defects. Lasers Surg. Med. 43, 481489.
  • 23
    Yang, P., M. Yao, S. L. DeMartelaere, R. W. Redmond and I. E. Kochevar (2012) Light-activated sutureless closure of wounds in thin skin. Lasers Surg. Med. 44, 163167.
  • 24
    Xu, D. and D. C. Neckers (1987) Aggregation of rose bengal molecules in solution. J. Photochem. Photobiol., A 40, 361370.
  • 25
    Abagyan, R., M. Totrov and D. Kuznetsov (1994) Icm—a new method for protein modeling and design: Applications to docking and structure prediction from the distorted native conformation. J. Comp. Chem. 15, 488506.
  • 26
    Bella, J., M. Eaton, B. Brodsky and H. M. Berman (1994) Crystal and molecular structure of a collagen-like peptide at 1.9 a resolution. Science 266, 7581.
  • 27
    Nemethy, G., K. D. Gibson, K. A. Palmer, C. N. Yoon, G. Paterlini, A. Zagari, S. Rumsey and H. A. Scheraga (1992) Energy parameters in polypeptides. 10. Improved geometrical parameters and nonbonded interactions for use in the ecepp/3 algorithm, with application to proline-containing peptides. J. Phys. Chem. 96, 64726484.
  • 28
    Abagyan, R. and M. Totrov (1994) Biased probability monte carlo conformational searches and electrostatic calculations for peptides and proteins. J. Mol. Biol. 235, 9831002.
  • 29
    Alarcon, E., A. Aspee, E. A. Abuin and E. A. Lissi (2012) Evaluation of solute binding to proteins and intra-protein distances from steady state fluoresence measurements. J. Photoch. Photobiol. B. 106, 17.
  • 30
    Jiang, Y., J. V. Hunt and S. P. Wolff (1992) Ferrous oxidation in the presence of xylenol orange for detection of lipid hydroperoxides in low density lipoproteins. Anal. Biochem. 202, 384389.
  • 31
    Gomes, A., E. Fernandes and J. L. F. C. Lima (2005) Fluorescence probes used for detection of reactive oxygen species. J. Biochem. Biophys. Methods 65, 4580.
  • 32
    Rasband, W. S. (1997–2009) Imagej, U.S. National Institutes of Health, Bethesda, MA.
  • 33
    Borsarelli, C. D., L. J. Falomir-Lockhart, V. Ostatná, J. A. Fauerbach, H. H. Hsiao, H. Urlaub, E. Paleček, E. A. Jares-Erijman and T. M. Jovin (2012) Biophysical properties and cellular toxicity of covalent crosslinked oligomers of a-synuclein formed by photoinduced side-chain tyrosyl radicals. Free Radic. Biol. Med. 53, 10041015.
  • 34
    Wikler, M. A. (2005) Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth Informational Supplement. Clinical and Laboratory Standards Institute, Wayne, PA.
  • 35
    Milne, P. J. and R. G. Zika (1992) Crosslinking of collagen gels: Photochemical measurements. Proc. SPIE 1644, 115124.
  • 36
    Neckers, D. C. (1989) Rose bengal (review). J. Photochem. Photobiol. A. Chemistry 47, 129.
  • 37
    Fleming, G. R., A. W. Knight, J. M. Morris, R. J. S. Morrison and G. W. Robinson (1977) Picosecond fluorescence studies of xanthenes dyes. J. Am. Chem. Soc. 99, 43064311.
  • 38
    Cramer, L. E. and K. G. Spears (1978) Hydrogen bond strengths from solvent dependence lifetimes of rose bengal dye. J. Am. Chem. Soc. 100, 221222.
  • 39
    Islam, S. D.-M. and O. Ito (1999) Solvent effects on rates of photochemical reactions of rose bengal triplet state studied by nanosecond laser photolysis. J. Photochem. Photobiol., A 123, 5359.
  • 40
    Rodgers, M. A. J. (1981) Picosecond fluorescence studies of rose bengal in aqueous micellar dispersions. Chem. Phys. Lett. 78, 509514.
  • 41
    Bilski, P., R. Dabestani and C. F. Chignell (1991) Influence of cationic surfactant on the photoprocesses of eosine and rose bengal in aqueous solution. J. Phys. Chem. 95, 57845791.
  • 42
    Rodgers, M. A. J. (1984) Picosecond studies of rose bengal fluorescence in reverse micellar systems. In In Reverse Micelles. (Edited by E. P. Press), pp. 164173. Plenum Press, New York.
  • 43
    Abuin, E., A. Aspee, E. Lissi and L. Leon (2007) Binding of rose bengal to bovine serum albumin. J. Chil. Chem. Soc. 52, 11961197.
  • 44
    Pacioni, N. L., M. Gonzalez-Bejar, E. Alarcon, K. L. McGilvray and J. C. Scaiano (2010) Surface plasmons control the dynamics of excited triplet states in the presence of gold nanoparticles. J. Am. Chem. Soc. 132, 62986299.
  • 45
    Fuenteabla, D., M. Galvez, E. Alarcon, E. A. Lissi and E. Silva (2007) Photosensitizing activity of advanced glycation endproducts on tryptophan, glucose 6-phosphate dehydrogenase, human serum albumin and ascorbic acid evaluated at low oxygen pressure. Photochem. Photobiol. 83, 563569.
  • 46
    de La Rochette, A., I. S. Birlouez-Aragon, E. Silva and P. Morliere (2003) Advanced glycation endproducts as uva photosensitizers of tryptophan and ascorbic acid: Consequences for the lens. Biochim. Biophys. Acta, 1621, 235241.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
php12119-sup-0001-PageS1-S5.pdfapplication/PDF777K

Actual picture and power spectrum for the 525 nm LED employed in the samples irradiations in this study. Changes in the Zeta potential values, mV, for 2.5 μm Type-I collagen in the presence of different concentrations of rose Bengal. Variation on the Rpn value of 2.5 μm rHC upon addition of different concentrations of rose Bengal up to 20 μm. Docking simulation for the interaction of different numbers of RB molecules, as indicated by the n number with a Type-I collagen-like protein. LDS electrophoresis for pure human recombinant Type-I collagen, rHC, and AgNP@collagen, AgNP, before irradiation of the samples under different experimental conditions are available free charged on DOI:10.1562/2006-10.1111/php.12119.s1.

Figure S1. Left: Actual picture for the 525 nm LED employed for the irradiations in this study. The system was composed by an LZ4-00G110 LED unit (LedEngin, Inc.) mounted on a PAR25 LED Cooler 32W Synjet that helps to dissipate the heating produced by the LED. Right: Power spectrum of the LED measured at the exact same distance of the center of the cuvette shown in the left picture.

Figure S2. Top: Zeta potential values, mV, for 2.5 μm Type-I collagen in the presence of different concentrations of rose Bengal. P value was calculated from Student's t-test statistic analysis. Bottom: Variation on the Rpn value of 2.5 μm rHC upon addition of different concentrations of rose Bengal up to 20 μm.

Figure S3. Docking simulation for the interaction of different numbers of RB molecules, as indicated by the n number with a Type-I collagen-like protein (further information is included in the experimental main text).

Figure S4. LDS electrophoresis for pure human recombinant Type-I collagen, rHC, and AgNP@collagen, AgNP, before irradiation of the samples under different experimental conditions (see main text for further details).

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