Interaction of a tripeptide with titania surfaces: RGD

adsorption on rutile TiO2(110) and model dental

implant surfaces.

Michael Wagstaffea, Hadeel Hussainb, Mark Taylorb, Matthew Murphyc, Nikolaos

Silikasd, and Andrew G. Thomasc,e*

a. School of Physics and Astronomy, The University of Manchester, Oxford Road, M13 9PL, UK,

b. Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus,

Didcot, Oxfordshire, OX11 0DE, UK,

c. School of Materials, The University of Manchester, Manchester M13 9PL, UK,

d. School of Dentistry, The University of Manchester, Manchester M13 9PL, UK,

and

e. The Photon Science Institute, The University of Manchester, Oxford Road, Manchester, M13

9PL, UK

*Corresponding author. e-mail: andrew.g.thomas@manchester.ac.uk

Abstract

The adsorption of peptides on metal oxides is an area of significant interest, both fundamentally

and in a number of technologically important areas. These range from the integration of

biomaterials in the body, to denaturation of protein therapeutics and the use of biomolecules and

bioinspired materials in synthesis and stabilization of novel nanomaterials. Here we present a

study of the tripeptide arginylglycylaspartic acid (RGD) on the surfaces of vacuum-prepared

single crystalline TiO2(110), pyrocatechol-capped TiO2(110), and model SLA and SLActive

dental implant samples. X-ray Photoelectron Spectroscopy and Scanning Tunneling Microscopy

show that the RGD adsorption mode on the single crystal is consistent with bonding through the

deprotonated carboxylate groups of the peptide to surface Ti atoms of the substrate. Despite the

increased hydrophobicity of the pyrocatechol-capped TiO2(110) surface RGD adsorption from

solution increases following this surface treatment. RGD adsorption on SLA and SLActive

surfaces shows that the SLActive surface has a greater uptake of RGD. The RGD uptake on the

pyrocatechol capped single crystal and the model implant surfaces suggest that the ease with

which surface contaminant hydrocarbons are removed from the surface has a greater influence on

peptide adsorption than hydrophobicity/hydrophilicity of the surface.

1

Introduction.

Titanium is regarded as a highly biocompatible material due to its low modulus of elasticity, high

strength to weight ratio, high capacity for osseointegration and excellent corrosion resistance. Its

ability to withstand the demanding environment within the body stems from the inert and

biocompatible protective titanium dioxide layer which forms at the surface.1-3 These features

combined with the low production cost have led to the widespread use of Ti in the manufacture

of bone anchoring systems, such as dental implants4-6. In both pure Ti and alloy systems the

outermost surface is found to be rich in TiO25,7. The biocompatibility of Ti lies largely with its

surface properties, which heavily influence the biological processes occurring at the interface

between the body and the implant, including protein adsorption and cell/tissue development8,9.

This means the fundamental interaction between biomolecules and the metal oxide surface is of

significant interest10. Over the past twenty years or so, the topography of titanium surfaces has

been investigated to improve implant osseointegration and healing times. For dental implants it

was found that sandblasted and acid-etched SLA, developed by Straumann in 1994 shows

particularly good osseointegration and healing rates compared to titanium plasma sprayed

implants.11-13 More recently, the SLActive surface has been introduced by Straumann. This

implant has a higher surface energy and is more hydrophilic than SLA.14 Whilst the majority of

experimental studies show that the osseointegration of SLA and SLActive shows negligible

differences in the long term, SLActive appears to promote enhanced osseointegration during the

earlier stages of bone regeneration.8,14 The reason for this is not yet fully understood and a

number of factors have been suggested to influence this. Although SLA and SLActive substrates

display very similar morphologies, and Raman measurements suggest the dominant polymorph

of TiO2 is rutile in both cases, the surfaces are found to be hydrophobic and hydrophilic,

respectively5,6,14.

Relatively recently, the SLActive surface has been shown to exhibit elements of

nanotopography6 which appear to have a positive influence on rate of osseointegration15,16.

Schwarz et al. suggested that the additional step in the preparation process to create SLActive;

i.e. transfer and storage in sterile saline, leads to a hydroxylated/hydrated surface, reducing the

adsorption of atmospheric contamination and thus maintaining a high surface energy17. X-ray

photoelectron spectroscopy (XPS) studies have shown that the thickness of the adventitious

carbon layer on the SLActive surface is ~1 nm less than that on the SLA surface5. It has been

suggested that this may underpin the more rapid osseointegration.8,16,18 Developing a clearer

understanding of osseointegration properties of Ti may lead to an improved understanding of

what exactly contributes to accelerated healing rates and osseointegration from a chemical

2

perspective.4,19 As a result the interaction of peptides with the titania surface is a subject of

considerable interest.3,20-24,

The RGD (Arg-Gly-Asp) sequence, shown in Figure 1, is thought to be important in cell

attachment to biomaterial surfaces and is therefore of interest in the osseointegration process on

titanium-based implant materials25,26. Previous work in this area has studied the adsorption

behaviour and dynamics of the RGD interaction with titania surfaces using molecular dynamics

(MD) simulations on rutile TiO2 surfaces3,10,20. Wu et al3. showed that electrostatic attraction

between the charged groups of RGD and the surface atoms of TiO2 leads to RGD replacing

adsorbed water species and binding directly to the surface titanium atoms through the

deprotonated carboxyl oxygen atoms20. Wu et al.3 suggested that the initial orientation of the

RGD molecule used in the model influences the binding mode and that the strongest interaction

with the TiO2 surface was via hydrogen bonding interactions with the surface oxygen through

NH3+.

In this article, we employ XPS and Scanning Tunneling Microscopy (STM) to investigate the

adsorption of RGD on the surface of titanium dioxide and metallic titanium. The presence of the

Figure 1. Ball and stick models of a). RGD, b) L-arginine c) glycine and d) L-aspartic acid. Black spheres represent carbon, red spheres are oxygen, blue are nitrogen, white are hydrogen.

3

ubiquitous hydrocarbon contamination seen in XPS of materials exposed to ambient atmospheric

conditions makes analysis of “real” implant surfaces difficult, affecting the C 1s and O 1s spectra

to an extent that chemical information concerning the bonding mechanism of the molecule is

hidden. To alleviate this, we study the interaction of RGD with a well-characterised, atomically

clean, single-crystal rutile TiO2(110) surface and compare to commercially pure titanium

substrates subjected to the same surface treatments as SLA and SLActive. This allows us to

determine the adsorption mode of the peptide on a rutile TiO2(110) surface and to make some

qualitative observations regarding peptide adsorption on the SLA and SLActive model materials.

Experimental.

For the model system, a rutile TiO2 (110) single crystal (10 mm x 10 mm x 1 mm, Pikem Ltd.),

was mounted on a tantalum sample plate using tantalum wire. The clean surface was prepared in

vacuum using an established method viz repeated 1 keV Ar ion bombardment and annealing to

~740 °C, until the X-ray photoelectron spectra showed the surface to be free of contamination.

This results in the formation of a well-ordered surface giving rise to a sharp (1 x 1) Low Energy

Electron Diffraction (LEED) pattern and very few surface oxygen vacancies, as determined from

the concentration of surface Ti3+ in the Ti 2p spectra27,28. The surface morphology of TiO2(110)

was studied using AFM-STM (Omicron) in the same chamber. STM data were typically acquired

in the constant current mode at a sample bias of 1.5 - 2.0 V and tunneling currents of 0.1 - 0.5

nA. Image processing was performed using Gwyddion software.29 XPS spectra were recorded at

normal emission and at room temperature using a monochromatic Al k X-ray source (h =

1486.6 eV) in a vacuum chamber at a base pressure of 1 x 10 -10 mbar. Spectra are aligned on the

binding energy scale relative to the Ti 2p peak at a binding energy of 459.0 eV and quoted to ±

0.1 eV. Fitting of core level spectra was carried out using CasaXPS, with a mixed

Gaussian:Lorentzian (0.7:0.3) line shape and a Shirley background.30

To protect the as-prepared surface against contamination from atmospheric hydrocarbons during

the deposition of RGD, pyrocatechol (1,2, dihydroxy benzene, Sigma Aldrich, 99.5 %) was used

to ‘cap’ the surface. The pyrocatechol powder was thoroughly degassed at 60 °C in a glass tube

attached to the vacuum system and evaporated onto the sample through a high-precision leak

valve. This results in a saturation coverage of one monolayer (1 ML) of pyrocatechol molecules

on the TiO2(110) surface (hereafter referred to as PC-TiO2)31,32. (See figure S1 of the Electronic

Supporting Information (ESI)) This densely packed layer of molecules is thought to reduce

adventitious hydrocarbon contamination from the atmosphere from adsorbing on the surface31,33.

4

Figure S2 of the ESI confirms the ability of PC to reduce the adsorption of atmospheric

adventitious hydrocarbon.

Following the capping procedure, the PC-TiO2 sample was removed from the ultra-high vacuum

(UHV) chamber via a ‘load lock’, which was backfilled with nitrogen gas (Experis R Gases,

99.995%), and a droplet of RGD solution was placed onto the sample. Excess solution was

removed using a positive pressure of nitrogen gas, the load lock was evacuated to 1 x 10 -7 mbar

and the sample returned to the main vacuum system.

Model SLA and SLActive samples were produced by sandblasting 1 cm2 grade 2 titanium ingots

using corundum particles of size 250-500 m. Following this, the samples were etched using a

concentrated mixture of HCl/H2SO4 at elevated temperatures and either cleaned with water in air

and stored in air (SLA) or cleaned with water in an inert (N2) atmosphere and stored in medical

saline (0.89 wt% NaCl).

To study RGD adsorption on these substrates, three 0.1 mg ml-1 RGD solutions were prepared, at

pH 4, 7 and 9.2 using phthalate, phosphate and borate buffers, respectively (Fisher Scientific).

The samples were immersed in these solutions for 24 hours, rinsed and dried under nitrogen and

then mounted on a tantalum sample plate, using carbon tape and transferred to the vacuum

system for analysis by XPS (Kratos Axis Ultra).

Results and Discussion.

1. The interaction of RGD with single crystal TiO2(110) and PC-TiO2(110).

Figure 2(a) shows survey spectra of both surfaces following the deposition and subsequent

rinsing of RGD solution normalized to the intensity of the Ti 2p3/2 peaks. A comparison of the N

1s signal in the survey spectra shows that the PC-capped TiO2 spectrum has a larger uptake of

the peptide. N 1s is a useful marker for the peptide since it is not commonly observed in

adventitious hydrocarbon signals. The difference is more clearly seen in the N 1s spectra shown

in Figure 2(b), which are also normalized to the intensity of the corresponding Ti 2p3/2 spectra.

The PC-TiO2 sample shows a significantly larger N 1s signal compared to the uncapped substrate

confirming that RGD uptake is more significant on this surface.

5

The inset in Figure 2 (a) shows photographs of droplets of the aqueous RGD solution on the two

surfaces. These show that the uncapped surface is more hydrophilic than the PC capped surface.

This is perhaps not surprising since the polar hydroxyl groups of the pyrocatechol molecule are

bonded to the surface of the TiO2 crystal32,34, presenting a densely packed aromatic hydrocarbon

surface to the water droplet. However, the finding is interesting because it is thought that the

Figure 2: (a) Survey photoelectron spectra, (b) N 1s, and (c) C 1s, core-level spectra for (i) the rinsed TiO2-RGD, and (ii) the rinsed PC-TiO2-RGD samples. All spectra are recorded using an Al K source (h = 1486.6 eV) and at normal emission. Inset of (a) shows photographs of the RGD-water droplets on the TiO2 and PC-TiO2 surfaces.

6

hydrophilicity of the titania surface is one of the key properties of the SLActive surface that

accelerates healing time. Here we see that the surface, which is initially more hydrophobic has a

higher uptake of the RGD peptide suggesting that hydrophilicity is not necessarily the main

factor governing adsorption of RGD peptide at the surface of titanium-based implants with a

native oxide layer. We note that unlike carboxylic acids, which are thought to be the main

constituents of the adventitious carbon layer that forms on rutile TiO2(110)35, that pyrocatechol

flips between a bidentate and monodentate adsorption mode suggesting a weaker interaction.36

The N 1s spectra in Figure 2(b) for both samples are fitted with two components at binding

energies of 400.6 eV and 402.4 eV. The former is assigned to amide N–C=O linkages and the

protonated guanidine group, and the latter to the protonated arginine -NH3+.37 We note that the

FWHM of the peak at 400.6 eV is relatively wide, this is not unexpected since although the

nitrogen species in the peptide bonds are in similar chemical environments they are not identical

and are also likely to be involved in H-bonding between peptide molecules to varying extents

leading to a broadening of the peak. In order to quantify the uptake of RGD on each surface, we

consider the relative atomic ratio of (NN/(NN+NTi)). NN and NTi represent the number density of

nitrogen and titanium atoms, calculated from the measured peak intensity divided by the relative

sensitivity factor. Analysis of the N 1s core-level spectra gives values for the TiO2 and the PC-

TiO2 surface of 8.8 % and 17.2 %, respectively, i.e. an increase in RGD uptake of 95 % on the

capped surface.

The C 1s spectra shown in Figure 2(c) are complex and can be fitted with several components.

We know however that the spectrum will be composed from a combination of pyrocatechol

(giving rise to two peaks) and RGD (five peaks) with the remaining spectra weight of the C 1s

spectrum arising from adventitious hydrocarbon contamination. This residual spectral weight is

fitted with three components at binding energies of 285.0 eV, 286.8 eV and 288.5 eV, assigned

to aliphatic C, C-OH and adsorbed carboxylate, respectively. Prior knowledge of the molecular

structure and photoelectron spectra34,37 of these species allow us to constrain peak intensities and

binding energies of the components for each of the molecules contributing to the C 1s spectra.

The assignment, contributions and shape of these peaks is described in detail in Figure S3 of the

ESI. In order to simplify the fits to the spectra we only show the envelope for each molecular

species in Figure 2 (and subsequent figures). It is clear when fitting these envelopes that when

the RGD solution droplet is added to the surface the uncapped surface shows more

contamination from hydrocarbons. These results further suggest that the pyrocatechol capping

7

layer is readily displaced by RGD compared to the adventitious hydrocarbon layer which forms

spontaneously in air, or water exposed to air, from trace organic acids35.

In order to attempt to understand the reasons for the increased uptake of RGD and to elucidate

the bonding mechanism of RGD on the TiO2(110) surface, the adsorption of RGD from aqueous

solution onto the PC capped TiO2 surface was investigated further. Core-level photoelectron

spectra for Ti 2p, O 1s, C 1s and N 1s recorded from the PC-TiO2-RGD system are shown in

Figure 3 (a) – (d). Adsorption of RGD from 1 mgml-1 of aqueous RGD solution, onto the PC-

TiO2(110) crystal, results in attenuation of the Ti 2p signal by nearly 50%, suggesting that

several multilayers of RGD are adsorbed, or precipitated onto the surface in the vacuum system.

The approximate thickness of the RGD overlayer was calculated to be 18 Å, assuming a uniform

RGD coverage (see ESI). The O 1s spectrum from this sample is comprised of 4 components at

binding energies of 530.3 eV (lattice oxygen (O2-)), 531.6 eV (deprotonated oxygen from

pyrocatechol, Ti-OH and COO- species in RGD), 532.4 eV (O=C-N species in RGD) and 533.3

eV (C-OH species). The latter arises from the intact carboxylic acid group in the RGD molecule

suggesting that some portion of the RGD remains protonated.38,39 The measured O 1s

stoichiometry for COO-:O=C–N:CO(OH) is 2.0:1.0:0.2, compared to the theoretical

stoichiometry of 1.0:1.0:1.0 for RGD in the neutral state, or 2.0:1.0:0.0 if peptide oxygen is

completely deprotonated. The measured O 1s stoichiometry therefore suggests that the RGD is

predominantly adsorbed with the carboxylic acid groups of aspartic acid deprotonated. We note

for the uncapped surface that the O 1s spectra (see ESI Figure S4) for the unwashed and washed

surface look very similar to those for the capped surface. For the unwashed, uncapped surface the

substrate oxide peak is more prominent, suggesting a lower uptake of RGD. In addition there is

some evidence of a peak associated with C-OH suggesting the molecule is not completely

deprotonated. After soft rinsing with deionized water the ratio of the RGD derived O 1s peaks

for COO:O=C-N is again 2:1 with no evidence of C-OH, suggesting that adsorption on the

uncapped surface is also via the carboxylic acid group. However, as shown in the ESI there is

also a significant amount of C-O signal arising from contamination, which makes it difficult to

separate the contribution from RGD and contamination in the O 1s spectrum.

8

In an attempt to remove RGD that was not bound directly to the crystal surface, the PC-TiO 2-

multilayer RGD sample was transferred to the load lock, vented to N2 and rinsed. Work on

glutathione on Au(111) surfaces have suggested that soft-rinsing with pure water allows the

peptide to retain the ionic form it has in solution. Longer rinsing on the other hand results in a

change to the state that would exist in the rinsing solution40. For this reason, we used two drops

of ultrapure water to minimize the chance of a change in ionic state. The Ti 2p, O 1s, C 1s and N

1s spectra following rinsing are displayed in Figure 3(a)(ii) - (d)(ii). Following rinsing the

thickness of the RGD is calculated to be ~6 Å, indicating that at least some precipitated RGD has

been removed from the surface. The oxide O 1s photoelectron peak is seen to increase in

intensity with respect to the molecular O 1s peaks, relative to the non-rinsed film. In addition, the

peak at a binding energy of 533.3 eV, due to C-OH groups, is no longer observed. This suggests

that the peptide carboxylate groups are fully deprotonated. This then implies that bonding in the

first layer involves these deprotonated carboxylate groups. After rinsing no changes, other than a

reduction in intensity, can be seen in the N 1s spectrum and the C 1s spectrum can be fitted with

the same components as before rinsing, but there is a reduction in RGD relative to the

adventitious carbon.

Figure 3: Core-level photoelectron spectra, normalized to the most intense peak in each case, for (a) Ti 2p, (b) O 1s, (c) C 1s and (d) N 1s following (i) exposure to PC in vacuum followed by a droplet of 1 mgml-1 RGD solution and (ii) the same sample following rinsing with deionized water. All XPS spectra are recorded using an Al K source (h = 1486.6 eV) and at normal emission.

9

In order to determine the contributions to the C 1s peak, and confirm the removal of the PC layer

for the rinsed RGD-TiO2(110) surface a difference spectrum was obtained by subtracting the PC-

TiO2 spectrum from that following exposure to the rinsed PC-TiO2-RGD sample. The difference

spectrum in Figure 4 clearly shows a "negative" contribution from the pyrocatechol envelope,

suggesting pyrocatechol is lost from the surface. It is possible that the decrease in signal from the

Figure 4: C 1s spectrum (1) after deposition of a 1 mgml-1 solution of RGD and rinsing in ultra-pure water, (2) the PC-TiO2 surface. The blue line is a difference spectrum (1)-(2). The red curves at the bottom of the plot show the contributions of the envelopes of RGD, adventitious carbon and pyrocatechol required to fit to the difference spectrum. Spectra (1) and (2) are normalised to the intensity of the corresponding Ti 2p spectra.

10

pyrocatechol actually arises as it is simply buried beneath the adsorbed RGD. The attenuation of

the pyrocatechol C 1s and Ti 2p signals, however, indicates a reduction in signal of 25% for Ti

2p3/2 and 85% for the pyrocatechol C1s respectively, when compared to the capped surface. This

confirms that some pyrocatechol is being removed from the surface. This loss of pyrocatechol

occurs due to the dynamic nature of the equilibrium between the adsorbed pyrocatechol

molecules on the TiO2 surface and RGD in solution. Similar approaches to the adsorption of N3

dye, and fluorescein using other small capping molecules have been employed previously33,41

STM images were obtained from a freshly prepared TiO2(110) crystal, capped with PC, brought

to atmospheric pressure under N2 gas, and then exposed to a droplet of a 0.01 mgml-1 RGD

solution. This reduced concentration appears to result in adsorption of less than a monolayer of

RGD on the surface on the time scale of the experiment and removed the need for rinsing.

Figure 5 shows a 200 x 200 nm2 STM image and line profile recorded from this surface. The

dotted line fitted to the profile simulates a ‘clean’ stepped surface showing a

monoatomic step of height 0.33 nm between neighbouring terraces42. A feature with a height of

around 0.6 nm is also observed and a number of these 0.5-0.6 nm high features can be seen in the

STM image in Figure 5 as white specks on the TiO2(110) surface with an apparent alignment

along the [110] direction. The measured height is in good agreement with the thickness of the

RGD layer calculated from XPS spectra recorded from the rinsed surface following deposition of

a 1 mgml-1 solution of RGD, suggesting these features are due to the presence of adsorbed RGD

molecules. This appears to suggest that the "thick" film, obtained by adding a drop of 1 mgml -1

RGD solution to the surface and drying, is composed of an adsorbed monolayer with a weakly

bound multilayer above it.

Figure 5: STM image of the capped TiO2 surface following deposition of 0.01 mgml-1 of RGD, 200 x 200 nm2. Left: Height profile along the line shown by the arrow.

(b)

11

To summarise the findings for the adsorption of RGD on the pyrocatechol capped single crystal

rutile TiO2(110) surface, deposition of RGD from a 1 mgml-1 solution onto the surface leads to

formation of a thin layer of RGD, comprised of a chemisorbed layer covered by weakly bound

RGD which is precipitated by drying in vacuum. This precipitate layer can be removed by

rinsing with deionized water. The first monolayer of RGD is chemisorbed through the

carboxylate groups. This is supported by the O 1s spectra where after rinsing C-OH species are

no longer observed. Hydrogen lost from the carboxylic acid groups are likely to form surface

hydroxyl species. This adsorption mode is widely reported for simple carboxylic acids and amino

acids on TiO2 surfaces in vacuum43-45. Such an adsorption geometry also agrees with molecular

dynamics simulations of the RGD/TiO2 surface reaction which suggests that RGD bonds to the

titania surface through three carboxyl oxygen atoms20. Deposition of RGD from a 0.1 mgml-1

solution results in a chemisorbed submonolayer. Chemisorption is inferred from the fact that this

surface can be imaged by STM. For a weakly bound molecule, one would anticipate that the tip

would move the peptides across the surface resulting in streaking in the images.

Interaction of RGD with SLA and SLActive

In order to investigate the interaction of RGD with the native oxide formed on Ti implant

materials, model SLA and SLActive substrates were left to soak in 0.1 mgml-1 RGD solution for

24 hours at pH 4, 7 and 9.2. Prior to transfer to the XPS system they were rinsed with deionized

water under N2 and dried with N2 gas to prevent exposure to atmospheric O2 or water. Figure 6

shows the Ti 2p and the N 1s (normalized to the Ti 2p3/2 peak) XPS spectra for the samples. The

C 1s and O 1s spectra are too complex to deconvolute reliably due to the presence of

adventitious carbon, which is present on the model implant surfaces before immersion in RGD

solution, as well as contributions from the buffers and hydroxides. We can however gain a semi-

quantitative measure of the adsorption of RGD on these materials from the intensities and

structure of the N 1s and Ti 2p spectra, since N species are rarely found in adventitious carbon

and are not present in any of the buffers used. The Ti 2p spectra are fitted with four components

(two spin-orbit doublets) arising from Ti(IV) and trace amounts of Ti(0), the 2p3/2 components of

which are located at 459.0 eV and 454.1 eV respectively. The doublet for the metallic (Ti(0))

component is fitted with an asymmetric Lorentzian peak shape due to the asymmetric broadening

associated with the conduction electrons in metals. The N 1s spectra are comprised of two

components separated by 1.8 eV, in agreement with adsorption on the single crystal surface.

12

The uptake of RGD is quantified from the relative intensities of the N 1s and Ti 2p signals before

normalisation and indicates a noticeable increase in the amount of RGD adsorbed on the

SLActive surface compared to the SLA surface. This increase is consistent for all solution pH

investigated as shown in Figure 6 (c). We also see a variation of the uptake of RGD at different

pH. At pH 7 the adsorption is lowest with a slight increase at pH 4 and a larger increase at pH 9.

It is also interesting that the ratio of protonated arginine -NH3+ varies for the two materials. For

SLA it remains relatively constant compared to the rest of the nitrogen signal (1:9) at all pH but

for the SLActive sample the degree of protonation of the arg -NH3+ is drastically reduced at pH

4 and 9, whereas at pH 7 it is similar to that for the SLA. The 1:9 ratio would correspond to

roughly 50 % of the arg -NH3+ moieties being protonated, since there are 5 nitrogen atoms

(three in guanidine and two in peptide bonds) per RGD molecule. It is difficult to pinpoint the

reasons for this behavior for a combination of reasons. Firstly, the isoelectric point (IEP) of TiO2

Figure 6. Core-level photoelectron spectra for (a) Ti 2p, (b) N 1s recorded for both SLA and SLActive following soaking in solutions of RGD at pH, 4 , 7 and 9. Spectra are normalized to the intensity of the largest peak of the fitted components in the N 1s. Panel c) shows the relative uptake of RGD on SLA (red) and SLActive (green) at each pH and d) shows photographs of water droplets on the surfaces indicating the wetting of the two surfaces.

pH

13

has a very wide range of values depending on the crystal structure and the specific surface plane

that is exposed46. This makes it difficult to judge the charge of the surface at the various pH

investigated here. Secondly, the degree of protonation of the guanidine group cannot easily be

deconvoluted in the N 1s spectra, since it overlaps with that from the peptide backbone so the

degree of protonation of the molecule as a whole is difficult to determine. Thirdly, the O 1s

spectra are difficult to deconvolute. This latter issue arises because as mentioned above the

buffers, peptide, solvent (water/OH) and surface oxide all contain oxygen, thus obtaining the

degree of deprotonation of the carboxylic acid from these data is not possible. Finally, we note

that it has been shown via molecular dynamics (MD) calculations that the isoelectric points of

the surface and biomolecules are important in the adsorption mode, since Coulombic interactions

will come into play. In addition, it has been shown that cations, such as Ca2+, Na+ and K+, in

solution can also play a part in mediating the interaction between the peptides and the metal

oxide surface. Both the charge and size of the ion having an effect on the adsorption of Asp 47 and

the mode of adsorption of peptides21,48 on rutile TiO2(110). This is important here since the

buffers used here contain either K (pH 4 and 9) or Na (pH 7). Therefore the observed variations

in uptake at the different pH may in fact be modified by cations in solution, rather than pH alone.

The difference in the amount of RGD present on the SLA and SLActive samples are simpler to

explain. For SLA the surface is already populated with adventitious hydrocarbon from the air,

resulting in a hydrophobic surface5,16. It has recently been demonstrated that atmospheric

adventitious hydrocarbons are mainly comprised of small organic acids, such as formic or acetic

acid, which are strongly adsorbed with a degree of ordering. These bind via the deprotonated

acid group in a bridging mode to under-coordinated cations at the surface35. Since these same

cations are most likely to be involved in the bonding to amino acids the uptake of RGD will be

reduced on this surface. This may seem at odds with the observation on the single crystal where

the pyrocatechol covered surface, despite being hydrophobic, resulted in a higher uptake of RGD

than for the clean rutile (110) surface. This difference most likely occurs because pyrocatechol

is only present as a monolayer and that pyrocatechol is more weakly adsorbed than carboxylic

acids35,36. Although these organic acids are also found to be dissolved in water that has been

exposed to atmospheric conditions, the amount of aliphatic component adsorbed on TiO2 is much

smaller than for air-exposed surfaces. This is perhaps not surprising since hydrophobic

molecules will not easily dissolve in water and explains why the SLActive surface is hydrophilic

and why these molecules are more easily displaced from the surface in RGD solution. Previous

studies of SLA and SLActive materials have shown that the adventitious hydrocarbon overlayer

on the SLA material is only around 1 nm thicker than that on the SLActive surface5 so it was

14

unclear why this would make such a large difference to the healing rate and hydrophilicity of the

surfaces. The data here comparing adsorption on the model TiO2 surface capped with

pyrocatechol, and the RGD – implant interactions suggest that it is the ease with which this

surface layer of hydrocarbons can be displaced by the peptides which governs the adsorption of

RGD. We plan to investigate this further by looking at capping layers, which have different

adsorption strengths and different peptides and small proteins in future work, in order to

determine how the chemistry of the adventitious C may affect uptake of peptides. In addition, we

plan to investigate the effect of the point of zero charge for the oxide and isoelectric points of the

oxide and biomolecules, and the role of ions in the solutions to enhance our understanding of

peptide adsorption on metal oxide surfaces since these appear to play a major role in adsorption

in aqueous solutions.

Conclusions

The interaction of arginylglycylaspartic acid with the surface of vacuum-prepared single crystal

rutile TiO2(110), SLA and SLActive has been investigated using a combination of X-ray

photoelectron spectroscopy and scanning tunneling microscopy. At low concentration, RGD is

found to bond to the single crystal surface in vacuum via deprotonated carboxylate groups

attached to surface Ti atoms. Results suggest that prior capping of the surface with pyrocatechol

leads to the adsorption of more RGD on the surface, when compared to the pristine TiO2 surface.

This is thought to be due to the adsorption of airborne or dissolved small carboxylic acids on the

uncapped TiO2 (110) surface. Results from this study also show that SLActive has an increased

uptake of the tripeptide RGD, relative to SLA at different pH. Comparing the results of

adsorption of RGD on the surface of rutile TiO2(110) with and without capping, and the two

model implant surfaces suggests that hydrophilicity alone may not be a prime factor in the

uptake of RGD. The data here suggest that RGD adsorption on these two materials is related to

the ease with which the adventitious carbon layer can be removed by the peptide, which itself

will be related to the solubility of the adventitious carbon overlayer.

Acknowledgements

MW acknowledges a Doctoral Training Award studentship from EPSRC, funding Beamline I07

Diamond Light Source, and a University of Manchester President’s Scholarship.

Supporting Information Available

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Supplementary information regarding this work is available online at

https://doi.org/10.1016/j.msec.2019.110030.

The raw data in .vms format and log book are also available for download from

http://dx.doi.org/10.17632/65kv687spt.1

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