Bifunctional Designed Peptides Induce Mineralization and Binding to

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Bifunctional Designed Peptides Induce Mineralization and Binding to TiO2 Anna Gitelman† and Hanna Rapaport*,†,‡ †

Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, and ‡Ilse Katz Institute for Nano-Science and Technology (IKI), Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel ABSTRACT: A limitation of titanium implants is the rather poor bonding between the metal and the surrounding tissue. In this research, we aimed at developing functional peptides in the form of monomolecular coatings intended to improve adhesion between the native oxide of the metal (TiO2) and the calcium−phosphate mineralization layer with which it is in contact. Accordingly, a bifunctional peptide with a β-strand motif assumed to strongly bind to the oxide through two phosphorylated serine residues, both situated on the same face of the strand, was designed. The β-strand motif was extended by a mineralization “tail” composed of consecutive acidic amino acids capable of adsorbing calcium ions. This peptide was studied together with two additional control peptides, one serving to elucidate the role of the β-strand in stabilizing bonding with the oxide and the other demonstrating the ability of the tail to induce mineralization. The strong adsorption of the three peptides to the oxide surface was revealed by HPLC. That peptide presenting the mineralization tail showed the highest levels of adsorbed calcium and phosphate ions, as well as the largest area of cellular adherence, demonstrating its potential advantages for use with titanium implants in bone tissue. tion.12−14 Along with surface topography modifications and mineral coatings of titanium implants, extensive efforts have concentrated on biological modifications intended to induce specific cell and tissue responses at the bone−implant interface.12,15,16 For instance, it has been shown that immobilization of ECM components on titanium implants can activate osteoblastic differentiation and matrix mineralization.17 ECM proteins may, however, elicit immune reactions and/or nonspecific and sometimes undesired activities.18 As such, recent strategies of implant surface modifications have focused on synthetic peptides that exhibit controllable biological activities. For example, the bioadhesive peptide sequence Arg-Gly-Asp (RGD), present in ECM proteins and recognized by several integrins,19,20 has been deposited on bone implant surfaces.21,22 Another peptide, Gly-Phe-Hyp-Gly-GluArg (GFOGER), found in type I collagen, has been deposited on titanium implants. This peptide motif, recognized by the α2β1 integrin that is highly expressed by osteoblasts, was found to improve osteoblastic differentiation and mineral deposition.15 The adsorption of biomolecules to titanium surfaces has also been studied in various other biological contexts, with an emphasis on elucidating the nature of the chemical bonding between the metal and organic molecules. Indeed, osseointegration entails strong bonding between any coating bridging between the oxide and the bone tissue in contact. The detachment of the coating may lead to bone resorption and,

1. INTRODUCTION Titanium and its alloys are among the most widely used implantable materials in orthopedics and dentistry, thanks to the mechanical strengths and resistance to corrosion of these metals. Successful osseointegration of these implants, i.e., direct contact between the living bone and the implant, is determined by processes that occur at the interface between the implant material and the bone tissue. Specifically, successful integration is largely influenced by the chemical and physical properties of the implant surface1 and their effects on biological processes at the interface. Integration of the implant and bone entails healthy tissue regeneration in that area of the bone in contact with the implant that should promote mineralization events.2 Bone mineralization proceeds through interactions of mainly calcium and phosphate ions with extracellular matrix (ECM) molecules that are facilitated by anionic proteins.3 As such, acidic proteins, rich in aspartic and glutamic acids, are found in mineralized organs and bones.4,5 Titanium implants become spontaneously covered by a 2−6 nm-thick layer of TiO2.1 As compared to other materials, this oxide is considered biocompatible and is highly stable in biological systems. Nevertheless, improved titanium implant osseointegration remains the focus of current research.6 The titanium surface can be modified by electrochemical and physical methods that influence the nanostructure of the metal implant, rendering it more favorable for cell adhesion.7−10 For example, a self-assembled layer of vertically oriented TiO2 nanotubes 15 nm in diameter was found to significantly enhance cell adhesion.11 Similarly, coatings of calcium phosphate in the form of hydroxyapatite widely used in this context show enhanced cell attachment and ECM produc© 2014 American Chemical Society

Received: November 21, 2013 Revised: March 19, 2014 Published: April 2, 2014 4716

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finally, to a loosening of the implant.13,23 In earlier efforts using a microbial cell surface display library, peptides with strong affinity to Ti were selected.16,24−26 Upon conjugation with the RGDS (Arg-Gly-Asp-Ser) integrin recognition motif, these peptides showed high surface binding affinity and enhanced bioactivity toward osteoblast and fibroblast cells.27 Nonetheless, a drawback of such surface display techniques is that they rely on limited adsorption and desorption conditions, necessary to maintain the viability of the peptide-presenting organism.26,28 The binding affinities of peptides adsorbing to titanium, identified on the basis of phage display, were determined using quartz crystal microbalance measurements.25,26 Other studies have employed direct spectroscopic approaches to show that specific chemical groups attached to biomolecules, i.e. carboxyls and phosphates, encourage strong interactions with TiO2 under mild conditions, likely via coordinative bonds.29−31 On the basis of in situ attenuated total reflection (ATR-IR) spectroscopy studies at TiO2 interfaces, it was suggested that phosphates form strong chemical coordinative bonds with the oxide, with a binding constant K ≈ 104 M−1.32 We have previously shown that peptides of random conformation containing two phosphoserine (pSer) amino acids became strongly bound to TiO2 microparticles, again probably by coordinative bonds. By performing HPLC with various loading and eluting buffers, we demonstrated that the phosphorylated peptide became completely chemisorbed to the oxide surface at physiological pH, whereas a similar peptide containing Glu instead of pSer residues did not adsorb under the same conditions. We, moreover, presented evidence showing the effect of peptide secondary structure on the interactions with the oxide layer.29 These results motivated us to test the feasibility of designing bifunctional peptides able to both strongly adsorb to the TiO2 through phosphserine residues within a β-strand secondary structure motif and capable of inducing calcified mineralization via the presence of a “tail” composed of multiple glutamic acids. Such bifunctional peptides are intended to strongly adsorb to titanium oxide, resist dissolution in a physiological milieu, and show enhanced adsorption of calcium−phosphate minerals.

TBS, depositing the solutions on a ZnSe 45° trapezoid prism (REFLEX Analytical Corporation, Ridgewood, NJ), and drying at 37 °C to form films that were macroscopically thick and visible to the naked eye. Spectra in the 4000−600 cm−1 range, at 4 cm−1 resolution, with 224 scans, were recorded using a Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA) fitted with a liquid nitrogen cooled MCT detector. The FTIR data were analyzed with OMNIC software, by applying atmospheric suppression (to minimize infrared absorption by CO2 and water vapor in the ambient air) as well as baseline correction. Reference spectra were measured using the bare ZnSe prism. 2.4. Chromatography. A stainless steel column, 4.6 mm in diameter and 50 mm long, was packed with TiO2 anatase particles (Sachtopore NP 10 μ/100 Å, particle size/pore, and surface area of 62.4 m2 g−1 by Sachtleben, Duisburg, Germany) suspended in methanol. The packed column was washed consecutively with water and 0.1 M NaOH to desorb surface contaminants and pre-equilibrated with loading buffer. Peptides dissolved in the loading buffer (0.4 mg mL−1) were sonicated for 10 min and centrifuged at 14.5 × 103 rpm for 8 min to remove any possible aggregates. Chromatography was performed on an HPLC (1260 Infinity, Agilent Technologies, Berkshire, UK) with solutions filtered using 0.2 μm filters (Millipore, Badford, MA).29 The solutions were then injected onto the TiO2 column at a flow rate of 0.02 mL min−1 to attain a ∼40 min retention time for adsorption. TBS was used as the adsorption media. After 65 min, the flow rate was increased to 0.5 mL/min. Elution was carried out using gradients of 200 mM sodium bicarbonate solution, pH 7.4, followed by 0.1 M NaOH, over 55 and 40 min, respectively. Elution was monitored at 280 nm. 2.5. Adsorption Isotherms. The adsorption isotherms of all three peptides to TiO2 microparticles were measured using peptides dissolved in 10 mM TBS at a range of concentrations from 0.002 to 0.524 mM. The TiO2 particles were also suspended in TBS, to a final concentration of 1.8 mg mL−1. To each test tube containing 0.5 mL of peptide at the different concentrations was added 0.5 mL of the TiO2 particles suspension. The samples were continuously mixed on a tube rotator for 1 h at room temperature to allow for adsorption. The TiO2 particles were subsequently precipitated by centrifugation at 5000 rpm for 3 min. Absorption of the supernatant was measured at 280 nm using a microplate reader (BioTek Instruments, Winoosky, VT). Peptide concentrations were determined on the basis of a calibration curve for each peptide dissolved in TBS. The adsorption isotherms were fitted with the Sips equation, also known as the Langmuir− Freundlich equation:33

2. MATERIALS AND METHODS

S=

2.1. Materials. The peptides Pro-pSer-Phe-pSer-Trp-(Gly)3(Glu)5, denoted as “apSer”, and Pro-pSer-Phe-pSer-Trp-(Gly)3, denoted as “cpSer”, were synthesized and then purified (>95%) by HPLC (Cambridge Research Biochemicals, Billingham, UK). The peptide Pro-pSer-pSer-Phe-Trp-(Gly)3, denoted as “bpSer”, was synthesized and then purified (>95%) by HPLC (Caslo, Lyngby, Denmark). Unless otherwise specified, all other reagents, including sodium hydroxide, sodium bicarbonate, potassium phosphate and calcium chloride dehydrate, were purchased from Sigma−Aldrich (Rehovot, Israel) and were of the highest available purity. Trisbuffered saline (TBS) was prepared with NaCl (154 mM) and Tris base (10 mM) adjusted to pH ∼7.4 with 0.1 M HCl. All solutions were prepared with deionized water (DIW), 18.2 MΩ cm (Direct Q-5 Merck Millipore, Billerica, MA). 2.2. Circular Dichroism (CD) Spectroscopy. CD measurements were performed to characterize the secondary structure of peptides in aqueous solutions. Spectra in the range of 190−260 nm were recorded at room temperature on a JASCO J-715 spectropolarimeter (Tokyo, Japan), using a 1 mm quartz cuvette. Peptide solutions were prepared by dissolving the peptide powder in TBS to concentrations of 0.2 and 0.4 mg mL−1. Data are reported as mean molar ellipticity per residue (deg cm2 dmol−1 residue−1). 2.3. Attenuated Total Reflection (ATR-FTIR) Spectroscopy. Samples (1 mg mL−1) were prepared by dissolving peptide powder in

Smax(kC)α 1 + (kC)α

(1)

where S is the amount of adsorbed molecules (mol m−2), Smax is the maximum amount of molecules adsorbed (mol m−2), C is their concentration is solution at equilibrium (M), k is the association coefficient (M−1), and α is a constant representing the binding energy distribution.33 The Sips equation is a combined form of the Langmuir and Freundlich models and relies on the assumption that a distribution of binding energies to sites on the adsorbing surface exists. For α→1, the Sips equation reduces to the Langmuir equation, representing binding of a monolayer with a homogeneous binding energy. Smaller α values together with low adsorbent concentrations result in Sips reducing to the Freundlich equation, which represents a distribution of binding energies. Peptide adsorption isotherm data could be fitted with the Sips equation up to certain concentrations, as discussed in the Results. 2.6. TiO2 Surfaces. Titanium-coated silicon wafers and borosilicate glass slides (Pgo, Iserlohn, Germany), hereafter termed Ti-silicon and Ti-glass, respectively, were prepared at the Ben-Gurion University Nanofabrication Facility. Titanium (99.995%, Kurt J. Lesker, Hastings, UK) was thermally evaporated at (1.9−7) × 10−6 mbar using a thermal evaporator (Odem, Rehovot, Israel) to a thickness of 40 nm. Prior to coating, the substrates were cleaned in acetone and 2propanol and subjected to oxygen plasma cleaning for 7 min (0.4 mbar 4717

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analyzed using Tukey’s test. Values of p < 0.05 were considered to be statistically significant.

oxygen pressure in a chamber). Surfaces were then placed on a heating plate at 180 °C for 8 h to generate the oxide coating.34 Ti-silicon surfaces were 1 × 0.5 cm2, while borosilicate glass surfaces were 1 × 1 cm2. The oxide layer was characterized by X-ray photoelectron spectroscopy (XPS), using an ESCALAB 250 spectrometer (Thermo Fisher Scientific, Loughborough, UK) with an Al X-ray source and a monochromator. General survey and high-resolution spectra of the elements were recorded. Calibration of the peak positions was performed according to the position of the C 1s line (284.8 eV). AVANTAGE software was used for data acquisition and analysis. XPS measurements of TiO2 surfaces identified high-resolution Ti 2p spectra (data not shown).35,36 Two peaks refer to the state of titanium in the oxide and matched those observed for TiO2. A peak corresponding to metal Ti was also identified, but it was less than 2%, relative to all spectra, indicating that the area was largely coated by TiO2. 2.7. Peptide-Coating by Self-Assembly on Surfaces. Oxidized Ti-silicon and Ti-glass surfaces were incubated in flat orientation in TBS for 45 min at room temperature, immersed in 0.4 mg mL−1 peptide in TBS for 1 h at room temperature to allow for self-assembly coating, and subsequently dried under nitrogen. Noteworthy, this weight/volume concentration, corresponding to 0.25 mM for apSer and 0.42 mM for bpSer and cpSer, falls in the saturation region of the adsorption isotherms (see Figure 5). The peptide-coated surfaces were analyzed by XPS. 2.8. Calcium−Phosphate Mineralization on Surfaces. Ti-glass surfaces and peptide-coated oxidized Ti-silicon surfaces were immersed in calcium phosphate (CaP) solution. A modified version of Kokubo’s simulated body fluid (SBF) solution37 was prepared using only the calcium and phosphate salts in the buffer components. The solution was prepared by consecutively dissolving K2HPO4 (1.0 mM), HCI (1 M) and CaCl2·2H2O (2.5 mM) in deionized water at 37 °C, followed by buffering to pH ∼7.4 with 50 mM Tris base. The surfaces were immersed in this solution at 37 °C for 24 h, dried under nitrogen, and analyzed by XPS. 2.9. Cell Culture. The human fetal osteoblast (hFOB) 1.19 cell line (ATCC, Manassas, VA) was used to study cell adhesion to peptide-coated surfaces. These cells have been immortalized using a temperature-sensitive mutant of the SV40 large T antigen that enables cells to proliferate at 33.5 °C and differentiate upon shifting the temperature to 39.5 °C. The cells were cultured in Dulbecco’s modified Eagle’s medium containing Ham’s F-12 (DMEM/F12) nutrient mixture at a 1:1 ratio (GIBCO, Carlsbad, CA), 10% fetal calf serum (Biological Industries, Beit-Haemek, Israel), 0.3 mg mL−1 gentamicin (GIBCO), and 1% penstrep (Biological Industries). Cells were incubated at 33.5 °C in 5% CO2 supplemented air until ∼80% confluence, enzymatically detached from the surface by addition of 1.5 mL of 0.25% trypsin-EDTA solution (Biological Industries), and collected by centrifugation at 1200 rpm for 5 min. Bare Ti-glass surfaces incubated in TBS for 45 min at room temperature, as well as peptide-coated surfaces, were sterilized under UV radiation for 30 min and incubated in the cell medium for 24 h. Subsequently, hFOB cells were seeded on each surface and cultured at 33.5 °C in 750 μL of cell medium. One day after seeding, the surfaces were transferred to a clean 24-well plate. Medium was changed every 2 days. 2.10. Cell Spreading. Cell spreading was assessed using a Live/ Dead Viability Kit (L3224, Invitrogen). A total of 105 cells were seeded on Ti-glass surfaces, each located in a well of a 24-well plate, and cultured for 48 h at 33.5 °C. The cultured surfaces were washed three times with PBS prior to staining, performed according to the manufacturer’s protocol. Surfaces with stained cells were imaged on an inverted fluorescence microscope (Eclipse TE300, Nikon). Surfaces were scanned and analyses were performed on regions in which nonclustered cells appeared adherent to the surface. The images were processed with ImageJ 1.45s software (NIH, Bethesda, MD). Experiments were performed in triplicate, with three images being collected from each surface. 2.11. Statistical Analysis. Quantitative data are represented as mean and standard deviation of the mean and compared by one-way analysis of variance (ANOVA). Results of XPS and cell spreading were

3. RESULTS 3.1. Peptide Design and Characterization. The apSer peptide (Figure 1A) was designed to feature a titanium oxide

Figure 1. Schematic models and sequences of the peptides employed in this study. (A) apSer and (B) cpSer with the binding motif in βpleated structure and (C) bpSer in a possible conformation. Structures were generated with the Cerius2 (Accelrys Inc.) molecular modeling software

binding motif and a stretch of five consecutive negatively charged glutamic acids, intended to induce adsorption of calcium and thus promote calcified mineralization. The titanium oxide anchoring motif comprised Pro followed by pSer residues alternating with hydrophobic amino acids, a motif that was assumed to induce a β-pleated structure at interfaces.38−40 In this structure, the two phosphoserine residues point in the same direction, relative to peptide backbone, and as such were envisaged to create a potentially favorable TiO2 surface-anchoring motif. Trp and Phe were selected as the hydrophobic amino acids introduced along the binding motif so as to enhance peptide detection by light absorption spectroscopy. The cpSer peptide (Figure 1B), lacking the stretch of the Glu residues found in the apSer peptide, was used as a control to assess the effect of the Glu tail on the structure of the peptide and on calcium adsorption. An additional control peptide, bpSer (Figure 1C), was designed with the exact same sequence as cpSer but with pSer and Phe switched in position, a modification expected to hamper appearance of the β-pleated conformation at interfaces. As such, the bpSer peptide was expected to elucidate the contribution of the β-pleated structure to peptide affinity to the TiO2 interface. CD measurements were performed to obtain information on peptide conformation in the solution used for spontaneous adsorption of the peptides to the titanium oxide (Figure 2). All peptides showed a negative absorption at around 196 nm, indicative of a random coil conformation. It can be noted that the two control peptides, cpSer and bpSer, exhibit minima at wavelengths 2−3 nm larger, pointing to a shift in the population secondary structure characteristics, away from the random coil and toward other secondary structures.41 The βsheet conformation is characterized by a negative CD 4718

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conformation that the peptides tend to assume in the proximity of a solid surface, in the dry state. All three peptide films showed an absorption band at 1000−1100 cm−1 that is associated with the phosphate groups of the phosphserine amino acids.42,43 In addition, all peptide spectra showed amide I, II, and III peaks at ∼1630, ∼1540, and ∼1300 cm−1, respectively.44 Differences mainly in the amide I but also in the amide II profiles are indicative of differences in secondary structure.45 The amide I peak of the apSer and cpSer peptides containing a β-pleated structure in the anchoring motif appear quite similar and both also show an amide I peak weak split at ∼1692 cm−1, constituting strong evidence for an antiparallel βsheet arrangement.38,44,46−48 The amide I peak of peptide bpSer, however, shows a shoulder at around ∼1645 cm−1 that is more pronounced than for the other two peptides, indicative of the contribution of helical and random structures to the spectra. All three peptides showed an amide II band at ∼1545 cm−1, although in the apSer spectra, this peak appears to partially overlap with the absorption at ∼1520 cm−1 that is characteristic of a β-sheet structure.49 This latter band, which reflects the abundance of β-sheet conformation, is also noted, albeit more weakly, in the cpSer and bpSer spectra. The relatively strong amide I and amide II bands at ∼1520 in apSer suggest that, in the macroscopic film, the Glu-tail motif is also packed in the βsheet structure. The three spectra showed a peak at ∼1400 cm−1 that is attributed to the carboxylate (COO−) symmetric stretching modes.44 In the apSer spectrum, this peak appears slightly stronger, due to the Glu tail motif. The differences in peptide conformations detected in the macroscopic films, generated on the solid support, highlight the stronger tendency of apSer and cpSer to assume the β-pleated structure in thin films, in the proximity of a solid surface. 3.2. Adsorption to Titanium Oxide Micron-Sized Particles. The peptides dissolved in TBS were injected onto a column packed with micrometer-sized particles of the TiO2 anatase phase. At this pH (7.4), the oxide surface is negatively charged.31 Hence, the Tris buffer used was supplemented with saline to screen these repulsion forces and enable the negatively charged peptides to closely approach the oxide surface and generate coordinative bonds that require short-range interaction distances.29,50 Chromatograms indicated that, in general, all three peptides adsorbed to the oxide (Figure 4) and largely resisted the carbonate gradient. Peptides primarily eluted with the applied NaOH gradient, as expected for peptide-binding that had occurred coordinatively through phosphate groups.29 The details of the chromatograms teach that fractions of the apSer and bpSer peptides (19.8% and 36.4%, respectively)

Figure 2. CD spectra of the apSer, bpSer, and cpSer peptides. Each peptide was dissolved in 10 mM TBS (pH 7.4) to a final concentration of 0.4 mg mL−1. The same results were observed with final peptide concentrations of 0.2 mg mL−1 (not shown). Dashed lines are drawn at 196 and 215 nm, where the ellipticity values are used to evaluate the extent of random and β-sheet structures, respectively.

absorption at ∼215 nm and a positive absorption at ∼195 nm. Accordingly, the CD spectra of the two control peptides point to the existence of β-sheet structures in addition to the random conformations, reflected in the decrease (in absolute values) of the ellipticity at ∼195−200 nm and the deeper negative absorption seen at 215 nm. These measurements underscore the contribution of the five Glu residue tail to the random structure of apSer peptide in solution. Peptides were next deposited on ZnSe prisms in the form of macroscopically thick films and ATR-IR spectra were acquired (Figure 3). These measurements provided information on the

Figure 3. ATR-IR spectra of apSer, bpSer, and cpSer peptides on ZnSe prisms. The spectra of the three peptides were normalized according to the phosphate peak at 1035 cm−1.

Figure 4. Chromatograms of apSer, bpSer, and cpSer peptides on a TiO2 anatase column. The chromatograms presented reflect peptide absorption at 280 nm normalized to peptide concentration. Adsorption was performed in 10 mM TBS, pH 7.4, followed by application of sodium bicarbonate (0−200 mM) and NaOH (0−0.1 M) elution gradients. The marked area expanded on the right shows the main elution peaks of the peptides. 4719

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desorbed during the loading and equilibration periods (up to 60 min). These fractions most probably comprise peptides that loosely bound to the oxide groups and thus desorbed during the loading stage. The fact that, of the three peptides, cpSer exclusively desorbed in the presence of NaOH indicates that the β-strand binding motif of this peptide is by itself highly efficient in generating the desired bonding with the oxide. As expected, the tail region of apSer and the replaced pSer residues along the bpSer sequence appear to result in various configurations that yield weaker binding with the oxide. A linear gradient of sodium bicarbonate (0−200 mM, pH 7.4) was applied to the column to assess whether the adsorbed peptides, namely, those resisting the loading stage, could be displaced by bicarbonate ions, which may also become coordinatively bound with the oxide surface, albeit with weaker forces than those of phosphate ions.51 Another small fraction of apSer and bpSer desorbed from the column during elution with bicarbonate (2.2% and 1.7%, respectively). The gradient was then followed by 0−1 M NaOH elution so as to discharge any organic materials from the column. All peptides showed the main elution peak during the NaOH gradient, at ∼84−97 mM. The tailing shapes of apSer and bpSer elution here point to various interactions, existing to small extents, between these peptides and the oxide with apparently higher stability than that of cpSer, considered to contain the optimal oxide-binding motif. Other differences in the elution time profile obtained with the NaOH gradient could not be consistently refined, probably due to the extreme changes in pH that occur along this elution and within the column and that were not adjusted by any buffer. To gain further insight into differences in the binding affinities of the peptides to the oxide, adsorption isotherms were assessed, generated using the same micrometer-sized titanium oxide particles as above, although suspended freely in buffered solution and not packed in a column (Figure 5). The peptides were allowed to adsorb to the suspended particles, with spectroscopic measurements of peptides in solution providing a quantitative measure of the adsorption. The adsorption isotherms could be fitted with the Sips equation up to a certain concentration (∼0.2 mM), after which the experimental data appeared to significantly deviate from the model. It is reasonable to assume that at a ∼0.2 mM concentration, the oxide surface became fully saturated with probably less than monolayer coverage, whereas at higher concentrations a different adsorption mechanism occurred, possibly involving multilayer adsorption. The Smax fitting parameter extracted from the adsorption isotherms corresponds to the amount of molecules adsorbed to the TiO2 surface at saturation. With the assumption of monolayer coverage at the saturation region of the Sips equation-fitted curve, the inverse of Smax provides an estimate of the nominal area each peptide occupies at the oxide surface. Interestingly, despite differences in peptide length, the Smax values of apSer and cpSer were found to be quite similar, (3033 ± 0.03) × 10−7 and (3.93 ± 0.52) × 10−7 mol m−2, respectively, with corresponding nominal areas of ∼498 and ∼422 Å2 molec−1, respectively. These results suggest that, up to monolayer coverage, the Glu tail of apSer does not interfere with the binding of the peptide to the surface. On the basis of previous studies of β-sheet monolayers at interfaces,35 the expected projected area of a peptide in this structure is ∼16.4 Å2 times the number of the peptide residues. Hence, in a β-pleated structure apSer (not including the tail) and cpSer may have occupied 131 Å2, smaller

Figure 5. Adsorption isotherm plots of (A) apSer, (B) bpSer, and (C) cpSer peptides onto TiO2 particles in 10 mM TBS, pH 7.4. The continuous line represents the Sips equation adsorption fitting, while the dots represent the amount of peptide adsorbed on TiO2 particles normalized to the particles surface area versus the final peptide concentration in the bulk solution, following the adsorption. Values represent means ± standard deviation.

than the nominal areas extracted from the fit to the adsorption isotherms, pointing to less than monolayer coverage for both these peptides. The Smax of bpSer on the oxide [(7.48 ± 0.918) × 10−7 mol m−2] was found to be about 2-fold larger than those of the two other peptides. Accordingly, the inverse of Smax (∼221 Å2 molec−1), representing the nominal area per molecule, is about twice as small as the same value of the two other peptides, in agreement with an expected smaller area projected by bpSer with its neighboring pSer residues in the binding region (one possible conformation of bpSer is illustrated in Figure 1C). The apSer peptide seems to adsorb relatively little beyond the ∼0.2 mM point, whereas cpSer continues binding, revealing an apparently second saturation curve beyond this concentration. At a concentration of ∼0.25 mM, bpSer seems to switch from adsorption following the Sips equation to a different mode, represented by an additional notable increase in adsorption. The distribution of binding energies to sites on the oxide is represented by the Sips equation α value that was found to be 0.8 for apSer and 0.4 for the two other peptides. These values point to a more homogeneous binding energy distribution for 4720

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Figure 6. XPS measurements describe the atomic ratio of phosphorus to titanium, indicative of peptide adsorption to the oxide surface (A), as well as the atomic ratio of phosphorus and calcium to titanium (B), for surfaces coated with the peptides and uncoated surfaces incubated in CaP solution for 24 h at 37 °C. Values represent means ± SEM (n = 5). Asterisks denote significant difference (according to the Tukey’s test): *p < 0.01.

Figure 7. Quantitative analysis and qualitative representative images of hFOB cell spreading on coated and uncoated Ti surfaces, determined using a fluorescent live/dead viability assay. Cell spreading was analyzed by estimating the area occupied by live cells (A) based on fluorescence images taken 48 h postseeding. Representative images show cells spreading on an uncoated surface (B), on a Ti surface coated with apSer peptide (C), on a Ti surface coated with bpSer peptide (D), and on a Ti surface coated with cpSer peptide (E). Green fluorescence (calcein-AM) indicates live cells, while red fluorescence (ethidium homodimer-1) indicates dead cells. The scale bar corresponds to 40 μm. Values in part A represent means ± SEM. Asterisks denote significant difference (according to the Tukey’s test): *p < 0.05.

temperature, and the coated surfaces were analyzed by XPS. Figure 6A shows the atomic concentration of phosphorus atoms to that of titanium, with the only source of phosphorus atoms being the phosphoserine amino acids of the peptides. The results show significantly higher peptide concentrations for the control peptides, cpSer and bpSer, compared to apSer, suggesting that the poly-Glu tail interferes with binding to the surface. To examine the influence of the Glu tail on the calcified mineralization process, the peptide-coated and uncoated TiO2 surfaces were immersed for 24 h in CaP solution. Figure 6B shows that surfaces that were coated with apSer peptide adsorbed significantly higher calcium and phosphate concentrations than did surfaces that were uncoated or coated with cpSer or bpSer peptides. Both of the control peptides adsorbed approximately the same amount of calcium and phosphate, probably via by the charged peptide termini. A similar trend in calcium adsorption was observed for surfaces immersed in 0.01 M CaCl2 solution. 3.4. Cell Spreading. Cell spreading was assessed using a live/dead assay carried out 48 h after seeding hFOB cells on coated and uncoated oxidized Ti-glass surfaces. Live cells

apSer, which generates a profile with resemblance to Langmuir isotherm behavior that describes uniform monolayer adsorption. The relatively uniform binding energies of apSer may be associated with the tail motif that is interfering with the possibility of multilayer formation. Both bpSer and cpSer lack the tail motif and are thus expected to have higher propensities for bilayer formation (see Discussion). Peptides apSer and cpSer, which showed similar Smax values, were found to also exhibit similar binding affinities, k values, to the oxide [(21.06 ± 0.6) × 103 and (21.29 ± 14.49) × 103 M−1, respectively]. Peptide bpSer showed different behavior, with a k value significantly lower than those of the other two peptides [(3.76 ± 2.15) × 103 M−1], albeit with a rather large standard deviation. These differences in k values underscore the advantage of the β-pleated structure binding motif found in apSer and cpSer (but not in bpSer) in binding to the oxide surface. The affinity values found here are quite similar to the 40 × 103 M−1 previously reported by Connor and McQuillan for adsorbed phosphate ions on TiO2 at low pH values.32 3.3. Adsorption to Titanium Oxide Surfaces. Each peptide was adsorbed to oxidized Ti-silicon surfaces at room 4721

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Table 1. Sips Equation-Fitting Parameters and Standard Deviations for the Three Peptides Sips equation constant

apSer

bpSer

cpSer

R2 Smax (mol m−2) K (M−1) α

1 (3.3 ± 0.032) × 10−7 (21.06 ± 0.6) × 103 0.79 ± 0.01

0.999 (7.48 ± 0.918) × 10−7 (3.76 ± 2.15) × 103 0.41 ± 0.02

0.998 (3.93 ± 0.524) × 10−7 (21.29 ± 14.49) × 103 0.41 ± 0.04

(Figure 7) were quantified by measuring fluorescently green areas. The apSer-coated surfaces were found to exhibit significantly higher areas of live cells, as compared to the two other peptide-coated surfaces.

both the affinity measurements, based on adsorption isotherms, and the β-sheet structure, demonstrated by ATR-IR, it can be concluded that the β-pleated structure in both apSer and cpSer exhibits higher affinity to TiO2 surface than does the binding region of bpSer. Furthermore, the nominal area per molecule values (based on the Smax Sips equation parameters) of apSer and cpSer were found to be similar and higher than the value obtained for bpSer. This suggests that the Glu tail of apSer does not adsorb to the surface, as otherwise the nominal area of apSer would have been bigger than that of cpSer. This also suits the presumed smaller binding region of bpSer generated through its two adjacent pSer residues. The tail region appeared to be also effective in preventing multilayer peptide adsorption. Monolayers of adsorbed bpSer and cpSer peptides could result in energetically less favorable and rather hydrophobic interfaces pointing to the surrounding solution. For this reason, these two peptides may have higher propensities toward the formation of multilayer adsorbed structures such that the hydrophilic residues of a second layer would point to solution. This behavior is corroborated by the CD spectra detecting β-sheet assemblies generated by bpSer and cpSer even in TBS solution. Finally, the peptides were adsorbed to titanium oxide solid substrates. XPS measurements detected lower adsorption of apSer, as compared to the other two peptide coatings. This difference was not detected when measuring adsorption to the microparticles, possibly due to differences in the oxide phase which was not determined for the oxidized titanium solid substrates. Nevertheless, apSer-coated oxide exhibited higher CaP concentrations, relative to the other peptide coatings and to uncoated TiO2 surfaces, pointing to the functionality of the tail region in apSer. Although the negatively charged tail would be expected to be neutralized only by adsorbed calcium ions, XPS measurements indicated that approximately an equal atomic ratio of calcium to phosphate ions adsorbed. A similar result was also observed with the two other peptide-coated surfaces. This suggests that prenucleating CaP particles, forming first in solution (Scheme 1), became adsorbed to the peptide-coated TiO2 surfaces. Indeed, nanometer scale calcium phosphate clusters have been described in various studies as playing a role in mineralization evolvement.53,54 Such CaP clusters may also become adsorbed directly to the TiO255 and with time transform to hydroxyapatite, which is the most stable

4. DISCUSSION Here we studied a bifunctional peptide designed to strongly bind through phosphate groups to titanium oxide in addition to inducing CaP mineralization at the oxide surface. Two other control peptides were used to demonstrate each of the two functionalities. CD measurements indicated that all three peptides exist essentially in random conformation in solution. Yet, both control peptides, bpSer and cpSer, exhibited a small fraction of β-sheet structure. This indeed may be expected for the two control peptides, both being shorter and relatively more hydrophobic than is apSer and, hence, having higher propensity toward intermolecular interactions, such as those leading to β-sheet bilayer and fibril formation.52 Differences between the three peptides became more evident in the assembled dry state, on solid support. ATR measurements highlighted the tendency of both apSer and cpSer, sharing the same binding motif, to assume a β-sheet structure in the proximity of the solid support, in the solid state. HPLC and adsorption isotherms were carried out to assess the influence of both the tail and the β-sheet binding motif, on the affinity of apSer to titanium oxide. The two phosphoserine residues, common to all three peptides, enabled strong peptide−oxide interactions evident by a main elution peak at pH ∼12.6. Small differences in the HPLC chromatograms were, however, evident during the adsorption stage. A portion of the apSer pool desorbed as early as the adsorption stage, highlighting the interfering effect the tail may exert on peptide adsorption. Indeed, Glu residues, composing the apSer tail, may also adsorb coordinatively to TiO2, although in 10 mM sodium acetate buffer, pH 5.29 In designing apSer, we took advantage of these differences in the tendencies and binding strengths of Glu and pSer to TiO2. On the basis of the apSer chromatogram, 78% of the peptide became bound to the oxide through the phosphoserine residues, while the remainder was probably bound less effectively, as it became desorbed during the loading and the sodium bicarbonate gradient elution phases. Although the 0−0.1 M NaOH gradient effectively desorbed all the three peptides off the column, this rather harsh elution did not provide information on differences between the peptides’ binding strengths. To elucidate possible differences in peptide affinities to titanium oxide, adsorption isotherms were obtained in 10 mM TBS, using the same particles as employed in the HPLC studies. Differences in adsorption of the three peptides were indeed detected in the isotherms. Fitting the data to the Sips equation revealed that the apSer and cpSer peptides displayed similar binding affinities to TiO2 (expressed as similar k values, Table 1) that were higher than that of bpSer. This result confirms the differences observed by ATR-IR between these two peptides with the β-sheet binding motif and bpSer. Taking

Scheme 1. A Schematic Model for CaP Adsorption on (A) Uncoated and (B) apSer-Coated TiO2 Surfaces

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phase of calcium and phosphate minerals.51 Hydroxyapatite coatings in direct contact with titanium have previously been shown to be susceptible to detachment from the metal surface.13 The apSer peptide described in this study thus serves as a mineral-nucleating matrix that is bound by strong chemical interactions with the oxide. The protruding peptide tails of apSer could then further act as anchoring sites to the mineralizing tissue expected to form around an implant. In addition, hFOB cell spreading was examined on the different peptide-coated TiO2 surfaces. Uncoated and apSercoated surfaces were found to exhibit increased area of viable, adhered cells, relative to those obtained on surfaces coated with the control peptides, bpSer and cpSer. This result is in accordance with previous studies showing correlation between CaP levels and migration and proliferation of preosteoblasts.56

(9) Lincks, J.; Boyan, B.; Blanchard, C.; et al. Response of MG63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition. Biomaterials 1998, 19, 2219. (10) Martin, J.; Schwartz, Z.; Hummert, T.; et al. Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63). J. Biomed. Mater. Res. 1995, 29, 389−401. (11) Park, J.; Bauer, S.; von der Mark, K.; Schmuki, P. Nanosize and vitality: TiO2 nanotube diameter directs cell fate. Nano Lett. 2007, 7, 1686−1691. (12) Dekker, R. J.; de Bruijn, J. D.; Stigter, M.; Barrere, F.; Layrolle, P.; van Blitterswijk, C. A. Bone tissue engineering on amorphous carbonated apatite and crystalline octacalcium phosphate-coated titanium discs. Biomaterials 2005, 26, 5231−5239. (13) Ogiso, M.; Yamamura, M.; Kuo, P. T.; Borgese, D.; Matsumoto, T. Comparative push−out test of dense HA implants and HA-coated implants: Findings in a canine study. J. Biomed. Mater. Res. 1998, 39, 364−372. (14) Heimann, R. B.; Kurzweg, H.; Ivey, D. G.; Wayman, M. L. Microstructural and in vitro chemical investigations into plasmasprayed bioceramic coatings. J. Biomed. Mater. Res. 1998, 43, 441−450. (15) Reyes, C. D.; Petrie, T. A.; Burns, K. L.; Schwartz, Z.; García, A. J. Biomolecular surface coating to enhance orthopaedic tissue healing and integration. Biomaterials 2007, 28, 3228−3235. (16) Vidal, G.; Blanchi, T.; Mieszawska, A. J.; et al. Enhanced cellular adhesion on titanium by silk functionalized with titanium binding and RGD peptides. Acta Biomater. 2013, 9, 4935−4943. (17) García, A.; Reyes, C. Bio-adhesive surfaces to promote osteoblast differentiation and bone formation. J. Dent. Res. 2005, 84, 407−413. (18) Lutolf, M.; Hubbell, J. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat. Biotechnol. 2005, 23, 47−55. (19) Ruoslahti, E. RGD and other recognition sequences for integrins. Annu. Rev. Cell Dev. Biol. 1996, 12, 697−715. (20) Siebers, M.; Ter Brugge, P.; Walboomers, X.; Jansen, J. Integrins as linker proteins between osteoblasts and bone replacing materials. A critical review. Biomaterials 2005, 26, 137−146. (21) Elmengaard, B.; Bechtold, J. E.; Søballe, K. In vivo study of the effect of RGD treatment on bone ongrowth on press-fit titanium alloy implants. Biomaterials 2005, 26, 3521−3526. (22) Schliephake, H.; Scharnweber, D.; Dard, M.; Sewing, A.; Aref, A.; Roessler, S. Functionalization of dental implant surfaces using adhesion molecules. J. Biomed. Mater. Res. Part B: Appl. Biomater. 2005, 73, 88−96. (23) Rakngarm, A.; Mutoh, Y. Characterization and fatigue damage of plasma sprayed HAp top coat with Ti and HAp/Ti bond coat layers on commercially pure titanium substrate. J. Mech. Behav. Biomed. Mater. 2009, 2, 444−453. (24) Tamerler, C.; Sarikaya, M. Molecular biomimetics: Utilizing nature’s molecular ways in practical engineering. Acta Biomater. 2007, 3, 289−300. (25) Khoo, X.; Hamilton, P.; O’Toole, G. A.; Snyder, B. D.; Kenan, D. J.; Grinstaff, M. W. Directed assembly of PEGylated-peptide coatings for infection-resistant titanium metal. J. Am. Chem. Soc. 2009, 131, 10992−10997. (26) Sano, K.; Shiba, K. A hexapeptide motif that electrostatically binds to the surface of titanium. J. Am. Chem. Soc. 2003, 125, 14234− 14235. (27) Yazici, H.; Fong, H.; Wilson, B.; et al. Biological response on titanium implant grade surface functionalized with modular peptides. Acta Biomater. 2012, 9, 5341−5352. (28) Chen, H.; Su, X.; Neoh, K.; Choe, W. Probing the interaction between peptides and metal oxides using point mutants of a TiO2binding peptide. Langmuir 2008, 24, 6852−6857. (29) Gertler, G.; Fleminger, G.; Rapaport, H. Characterizing the adsorption of peptides to TiO2 in aqueous solutions by liquid chromatography. Langmuir 2010, 26, 6457−6463.

5. CONCLUSION In this study, we demonstrated that a bifunctional peptide, apSer, is capable of adsorbing strongly to TiO2 surfaces, while leaving an anionic functional “tail” free for inducing calcium phosphate mineralization. Understanding the principles of peptide structures and their mechanisms of binding to TiO2 enabled us to create this bifunctional peptide. Conformational characterizations in solution and on solid supports were performed and substantiated the functionality of the binding region and the functional tail of the designed peptide. This bifunctional peptide is expected to improve interactions between the interface of Ti implants and bone tissue regenerated around such implants by inducing implant osseointegration and durability. This type of design, comprising an oxide-binding region extended by a functional tail, can, in principle, be extended to additional desired activities, such as, for example, antibacterial and cell adhesion uses.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +972 8 647 9043. Fax: +972 8 647 7188. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by Kamin Foundation. REFERENCES

(1) Puleo, D.; Nanci, A. Understanding and controlling the bone− implant interface. Biomaterials 1999, 20, 2311−2321. (2) Boskey, A. L. Matrix proteins and mineralization: An overview. Connect Tissue Res. 1996, 35, 357−363. (3) Boskey, A. L. Biomineralization: Conflicts, challenges, and opportunities. J. Cell Biochem. 1998, 72, 83−91. (4) Addadi, L.; Weiner, S. Control and design principles in biological mineralization. Angew. Chem. Int. Ed. Engl. 1992, 31, 153−169. (5) Addadi, L.; Weiner, S. Interactions between acidic proteins and crystals: Stereochemical requirements in biomineralization. Proc. Natl. Acad. Sci. U. S. A. 1985, 82, 4110−4114. (6) Elias, C. N.; Meirelles, L. Improving osseointegration of dental implants. Expert Rev. Med. Devices 2010, 7, 241−256. (7) Variola, F.; Brunski, J. B.; Orsini, G.; de Oliveira, P. T.; Wazen, R.; Nanci, A. Nanoscale surface modifications of medically relevant metals: State-of-the art and perspectives. Nanoscale 2011, 3, 335−353. (8) Bagno, A.; Di Bello, C. Surface treatments and roughness properties of Ti-based biomaterials. J. Mater. Sci. Mater. Med. 2004, 15, 935−949. 4723

dx.doi.org/10.1021/la500310n | Langmuir 2014, 30, 4716−4724

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Article

(52) Zhang, S. Fabrication of novel biomaterials through molecular self-assembly. Nat. Biotechnol. 2003, 21, 1171−1178. (53) Dey, A.; Bomans, P. H.; Müller, F. A.; Will, J.; Frederik, P. M.; Sommerdijk, N. A. The role of prenucleation clusters in surfaceinduced calcium phosphate crystallization. Nat. Mater. 2010, 9, 1010− 1014. (54) Posner, A. S.; Betts, F. Synthetic amorphous calcium phosphate and its relation to bone mineral structure. Acc. Chem. Res. 1975, 8, 273−281. (55) Hanawa, T.; Ota, M. Calcium phosphate naturally formed on titanium in electrolyte solution. Biomaterials 1991, 12, 767−774. (56) Chai, Y. C.; Carlier, A.; Bolander, J.; et al. Current views on calcium phosphate osteogenicity and the translation into effective bone regeneration strategies. Acta Biomater. 2012, 8, 3876−3887.

(30) Roddick-Lanzilotta, A. D.; McQuillan, A. J. An in situ infrared spectroscopic study of glutamic acid and of aspartic acid adsorbed on TiO2: Implications for the biocompatibility of titanium. J. Colloid Interface Sci. 2000, 227, 48−54. (31) Dobson, K. D.; Connor, P. A.; McQuillan, A. J. Monitoring hydrous metal oxide surface charge and adsorption by STIRS. Langmuir 1997, 13, 2614−2616. (32) Connor, P.; McQuillan, A. J. Phosphate adsorption onto TiO2 from aqueous solutions: An in situ internal reflection infrared spectroscopic study. Langmuir 1999, 15, 2916−2921. (33) Sips, R. On the structure of a catalyst surface. J. Chem. Phys. 1948, 16, 490. (34) Vaquila, I.; Vergara, L.; Passeggi, M.; Vidal, R.; Ferron, J. Chemical reactions at surfaces: Titanium oxidation. Surf. Coat. Technol. 1999, 122, 67−71. (35) Luo, Y.; Wang, X.; Xu, D.; Wang, Y. Preparation and characterization of poly(lactic acid)-grafted TiO2 nanoparticles with improved dispersions. Appl. Surf. Sci. 2009, 255, 6795−6801. (36) Armstrong, N. R.; Quinn, R. K. Auger and X-ray photoelectron spectroscopic and electrochemical characterization of titanium thin film electrodes. Surf. Sci. 1977, 67, 451−468. (37) Kokubo, T.; Ito, S.; Huang, Z.; et al. Ca, P-rich layer formed on high-strength bioactive glass−ceramic A−W. J. Biomed. Mater. Res. 1990, 24, 331−343. (38) Rapaport, H.; Kjaer, K.; Jensen, T. R.; Leiserowitz, L.; Tirrell, D. A. Two-dimensional order in β-sheet peptide monolayers. J. Am. Chem. Soc. 2000, 122, 12523−12529. (39) DeGrado, W.; Lear, J. Induction of peptide conformation at apolar water interfaces. 1. A study with model peptides of defined hydrophobic periodicity. J. Am. Chem. Soc. 1985, 107, 7684−7689. (40) Osterman, D.; Mora, R.; Kezdy, F.; Kaiser, E.; Meredith, S. C. A synthetic amphiphilic β-strand tridecapeptide: A model for apolipoprotein B. J. Am. Chem. Soc. 1984, 106, 6845−6847. (41) Greenfield, N. J.; Fasman, G. D. Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry (N. Y.) 1969, 8, 4108−4116. (42) Fowler, B. Infrared studies of apatites. I. Vibrational assignments for calcium, strontium, and barium hydroxyapatites utilizing isotopic substitution. Inorg. Chem. 1974, 13, 194−207. (43) Rey, C.; Shimizu, M.; Collins, B.; Glimcher, M. Resolutionenhanced fourier transform infrared spectroscopy study of the environment of phosphate ion in the early deposits of a solid phase of calcium phosphate in bone and enamel and their evolution with age: 2. Investigations in the ν3 PO4 domain. Calcif. Tissue Int. 1991, 49, 383−388. (44) Barth, A.; Zscherp, C. What vibrations tell us about proteins. Q. Rev. Biophys. 2002, 35, 369−430. (45) Sreerama, N.; Venyaminov, S. Y.; Woody, R. W. Estimation of protein secondary structure from circular dichroism spectra: Inclusion of denatured proteins with native proteins in the analysis. Anal. Biochem. 2000, 287, 243−251. (46) Miyazawa, T.; Blout, E. The infrared spectra of polypeptides in various conformations: Amide I and II bands. 1. J. Am. Chem. Soc. 1961, 83, 712−719. (47) Atkinson, R. J.; Parfitt, R. L. Smart RSC. Infra-red study of phosphate adsorption on goethite. J. Chem. Soc., Faraday Trans. 1 1974, 70, 1472−1479. (48) Krimm, S. Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. Adv. Protein Chem. 1986, 38, 181. (49) Pelton, J. T.; McLean, L. R. Spectroscopic methods for analysis of protein secondary structure. Anal. Biochem. 2000, 277, 167−176. (50) Topoglidis, E.; Campbell, C. J.; Cass, A. E.; Durrant, J. R. Factors that affect protein adsorption on nanostructured titania films. A novel spectroelectrochemical application to sensing. Langmuir. 2001, 17, 7899−7906. (51) Chen, H.; Zahraa, O.; Bouchy, M. Inhibition of the adsorption and photocatalytic degradation of an organic contaminant in an aqueous suspension of TiO2 by inorganic ions. J. Photochem. Photobiol., A 1997, 108, 37−44. 4724

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