Protein-Functionalized Diamond Surfaces in a Water Solvent: A

Article pubs.acs.org/JPCC

Protein-Functionalized Diamond Surfaces in a Water Solvent: A Theoretical Approach Yuan Tian and Karin Larsson* Department of Chemistry − Ångström Laboratory, Uppsala University, Uppsala 75121, Sweden S Supporting Information *

ABSTRACT: In order to improve the performance of a diamond-based implant material, surface functionalization with different proteins is a promising approach. The main goal of the present study has been to theoretically investigate the diamond functionalization by physisorption of different proteins onto different surface planes. The protein candidates selected are growth factors which can promote cell adhesion and growth and subsequent vascularization surrounding the implanted materials. They include Bone Morphogenetic Protein 2 (BMP2), Vascular Endothelial Growth Factor (VEGF), Fibronectin (FN), and Angiopoietin (AGP). Moreover, it is wellknown that diamond surface properties are strongly dependent on diamond surface planes and surface terminations. Therefore, the following two different diamond surface planes [diamond (100)-2 × 1 and diamond (111)] and four different kinds of terminations species (H, OH, COOH, and NH2) were used in the present study. The results from force-field calculations show that the surface wettability is crucial for the protein adhesion onto the diamond surfaces, and the different proteins possess distinct preferences for diamond surface planes and terminations. For the identification of protein functionality, the atomic structures, in addition to corresponding electrostatic maps, were also visualized in the comparison of protein structures before and after adhesion to the diamond surfaces. It could be concluded that the protein structures and binding pocket electrostatic distributions are maintained as a result of the functionalization process, regardless of adhesion energy strength. These results provide a solid base for experimental protein functionalization of the diamond surfaces.

1. INTRODUCTION

mechanisms behind and higher growth factor loading concentrations of bone implant surfaces. Nanodiamonds, with unique chemical and physiological properties, have been widely used in the biomedical research field and possessed satisfactory outcomes.6 Especially in bone implants surface design, the nanocrystalline diamond had exhibited high osseointegration performance and better biocompatibility with BMP2 coating.7 Borisenko et al. investigated adhesions of protein residues on a diamond (111) surface plane theoretically to find a better understanding of the mechanisms behind and showed that substitution of diamond surface atoms would lead to a weaker adhesion of protein residues on diamond surfaces.8 However, adhesions of bigger proteins on diamond surface planes remain blurry and require further research efforts. In this work, four different growth factors approved by the Federal Drug Administration (FDA) and two different diamond surface planes [diamond (100)-2 × 1 and diamond (111)] have been investigated for better performance of bone implant surfaces.9 Thus, the diamond surface planes were terminated with four different kinds of surface terminations [hydrogen

Artificial bone implants and surrogate scaffolds have been widely beneficial for patients with damaged tissues and difficulties of localized cell regeneration. However, failures do occur with a certain amount of patients.1 The current suboptimal bone implant performances are largely attributable to insufficient implant vascularization.2,3 The dominant determinants for bone cell initial adhesion and the following activation of protein cascades are implant surface nature (e.g., wettability, surface roughness)4 and binding integrity between implants and bone cells.2 With the purpose to increase implants’ osseointegration and biocompatibilities, rational design of implant surfaces is of great importance. In the last decades, various approaches were conducted to increase osseointegration by modification of implant surface properties. Functionalization with BMP2 has proven to increase the release of local growth factors significantly, and it has resulted in promotion of bone cell growth (as compared to uncoated implant surfaces). Osseointegration and wound healing performances were also increased with sufficient fibronectin coatings on bone implant surfaces.5 The optimal performances of protein coatings of different growth factors enlightened an increasing need for rational design of bone implant surfaces, with a better understanding of the © 2015 American Chemical Society

Received: November 4, 2014 Revised: March 25, 2015 Published: March 29, 2015 8608

DOI: 10.1021/jp511015m J. Phys. Chem. C 2015, 119, 8608−8618

Article

The Journal of Physical Chemistry C

temperature range. Within each annealing process cycle consisting of 1000 steps (1 fs/step), the temperature increases from 300 to 320 K and is thereafter decreased to 300 K again. In total, 25 cycles were performed, with a total simulation time of 25 ps. The choice of temperature range was based on the fact that the physiological functionalities of the proteins should be maintained. Moreover, the thermodynamic parameter was set to NVT, which means that the total number of atoms, volume, and temperature was held constant. The thermostat algorithm was Nose-Hoover for its constancy with respect to both coordinates and momentum spaces, during the heat cycles.16,17 The charge distributions were assigned by using an atom force field type, which is part of the parameters stored in COMPASS.11 The electrostatic and van der Waals summation methods are Ewald for periodic systems.18 The adhesion energy for the attachment of proteins onto the diamond surface has been calculated by using eq 1, in which ΔEad stands for the adhesion energy; Esystem is the total energy of the system; Eadsorbates is the total energy of the absorbate; and Erest stands for the total energy of the diamond substrate.

(H), hydroxyl (OH), carboxyl group (COOH), and amino group (NH2)] for their distinct influences on a diamond’s physical and chemical properties.10 A schematic illustration of terminated diamond surfaces, functionalized with proteins in water environment, is presented in Figure 1.

ΔEad = (Esystem − Eadsorbates − Erest)

(1)

19,20

DMol3, a Density Functional Theory based program, is utilized to create the water solvent environment for proteins. The geometries of water solvent and charge distributions are obtained after annealing of 25 cycles from 0 to 500 K in order to obtain a rational water solvent foundation. The local numerical orbital basis set parameter is Gaussian double-ζ plus polarization function basis set (DNP) and has a cut off value of 0.05 Å for the best accuracy. The exchange and correlation functional is the Perdew−Burke−Ernzerhof (PBE) generalized gradient approximation (GGA) due to its good performances with surface calculations compared to the local density approximation (LDA).21 The energy cut off value is 380 eV, and 3 k points are included for the systems.22 The protein structural analyses are conducted by using the programs AutoDock and VMD.23 Moreover, the infrared spectrum analyses were conducted by performing molecular dynamics simulation and using dipole moment autocorrelation24 of the protein structures, before and after the functionalization process. The total simulation time length for the molecular dynamics simulations is 25 ps, consisting of 25 000 steps and 1 fs per step.

Figure 1. Schematic illustration of an X-terminated diamond surface, being functionalized with a protein in a water environment (X = H, OH, COOH, or NH2).

2. METHODS AND METHODOLOGIES 2.1. Theoretical Methods. The calculation program Materials Studio was applied to investigate the physisorption of four different proteins onto differently terminated diamond surface planes. Forcite, a force field method, was utilized for protein solvation in a water environment afterward and physisorption onto diamond surfaces. The utilized force field method, compass, is selected for the systems since it is an ab initio force field.11 The force field parameters within the method used (i.e., COMPASS) have been obtained by using a more accurate ab initio method. These parameters have shown adequate performances for both biological and inorganic systems.12−15 Annealing calculations have been performed in order to simulate the dynamic behavior of systems composed of a diamond substrate, protein, and water molecules in a selected

Figure 2. Models of construction process for diamond−protein interfaces in water solvent: (a) up to down: VEGF structure, water cube, VEGF solvated in water; (b) diamond (100)-2 × 1 surface slab; (c) the water-solvated VEGF attached onto the diamond (100)-2 × 1 surface. 8609


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The Journal of Physical Chemistry C 2.2. Models. Interfacial models composed of a diamond surface plane with an attached biomolecule in water solvent were in the present study modeled as periodic systems (Figure 1). The diamond surface slabs contained seven layers of carbon atoms, with the bottom two layers constrained to simulate the continuation of a bulk structure (Figure 2b).22,25 Four different termination types (H, OH, COOH, and NH2) onto the diamond (111) and diamond (100)-2 × 1 surfaces were investigated for their individual surface reactivity and wettability. The termination coverage rates are selected to be the most energetically favorable ones according to previous theoretical investigations.22 Water cubes are generated in adequate sizes for the protein dimensions and are thereafter annealed from 0 to 500 K. In order to avoid interaction between different layers (in the z-direction), the water cube and diamond surface slab are put together in supercells with the lattice parameters a = 90 Å, b= 90 Å, and c = 250 Å (as shown in Figure 2). For the investigation of surface wettability, as a function of surface termination, pure diamond−water systems are modeled and annealed (from 0 to 500 K). The functionalized proteins investigated within this study are Federal Drug Administration approved growth factors in association with bone cell growth and implant vascularization. They are Bone Morphological Protein 2 (BMP2),26 Vascular Endothelial Growth Factor (VEGF),27 Fibronectin (FN),28,29 and Angiopoietin (AGP).30 The protein molecular structures were downloaded from the protein data bank (www.pdb.org): ID 2QJA (BMP2, 3860 atoms), 1VPF (VEGF, 1531 atoms), 2FN2 (FN, 2944 atoms), and 1Z3U (AGP, 3383 atoms). The proteins are first modeled individually but solvated in water (Figure 2a). They are thereafter annealed in 25 cycles from 300 to 320 K. The hereby geometry-optimized solvated proteins were then (noncovalently) attached to the differently terminated diamond surfaces (Figure 2). The orientation of proteins is positioned to have the functional group up, away from the diamond surface in order to keep the functionality of the proteins. The geometry optimization results showed that the position with the binding pocket up will shorten the distances between the protein atoms from the diamond surface planes, which results in larger van der Waals energies and electrostatic energies, which will hence stabilize the diamondbiomolecule structures. As a result, a combination of larger adhesion energies and maintenance of protein functionalities will be achieved. Test calculations have been performed, where the preferred protein orientation on the diamond surface plane has been looked for. The results show that the protein geometry with the binding pocket up from the surface is energetically more preferred than the orientation with the binding pocket down toward the diamond surface. These results validate the choice of models in the present investigation, where the proteins are attached to the diamond surface with the binding pocket in an upward position (as shown in Figure 2). All adhesion energies that are here discussed have negative values. This indicates that the adhesion processes are exothermic. From here on, the absolute values of these energies will be presented and analyzed.

energy over the diamond−water interface is presented in Figure 3. For the diamond (111) surface plane, the water adhesion

Figure 3. Water layer absolute adhesion energies for the attachment to diamond (111) and diamond (100)-2 × 1, respectively. Four different diamond surface termination species are represented: H, OH, NH2, and COOH.

energy was found to be most pronounced for the COOH diamond surface termination (8713 kcal/mol). The order of adhesion energies for all of the terminating species was: COOH (8713) > OH (3311) > H (3105) > NH2 (2768) kcal/mol. Since the present calculations are based on force field calculations, the largest number represents the strongest adhesion energy. For the diamond (100) surface plane, the corresponding order of adhesion energies was: COOH (5721) > OH (4548) > NH2 (2903) > H (961) kcal/mol. Hence, the two different surfaces only differ with respect to H and NH2 termination. The adhesion energy for an attached water adlayer onto a solid surface is generally regarded to be an indication of the surface wettability. On the basis of the present calculations, it can thereby be concluded that the surface termination type that renders the most hydrophilic diamond surface is COOH, while the surface termination type that renders the most hydrophobic diamond surface is NH2 for diamond (111) and H for diamond (100). These theoretical results correlate well with previous experimental results. The measured contact angle, by using atomic force microscopy (AFM), for a water droplet positioned onto a diamond (100) surface was 74.9° for the H-terminated surface and 64.7° for the O-terminated diamond surface.31 Moreover, the water droplet contact angle onto diamond (111), measured by performing a sessile drop technique, showed that the wettability will increase when more hydrogen gas was present during the synthesis process.32 3.2. Effect of Surface-Terminating Species on Protein Adhesion Energies for a Diamond (100)-2 × 1 Surface. The calculated adhesion energies for four different proteins attached to a diamond (100)-2 × 1 surface are visualized in Figure 4a, and the adhesion energy values are shown in Table S1 (Supporting Information). The present study has focused on proteins in water solvents, but the obtained results will also be compared with earlier results (by present authors) for the adhesion of nonsolvated proteins. The proteins were BMP2, VEGF, FN, and AGP. The diamond surface terminating species included H, OH, COOH, and NH2. Since the models not only consist of diamond surfaces and attached proteins but also H2O molecules in the solvation medium, the calculated adhesion energy will be larger than for the nonsolvated situations.

3. RESULTS AND DISCUSSIONS 3.1. Effect of Surface-Terminating Species on Diamond Surface Wettability. The adhesion energies for the water adlayer onto the different terminated diamond (111) and (100)-2 × 1 surface planes were calculated, and compared, in the present study (see model in Figure 2). The interaction 8610


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adhesion energy for the various diamond surface termination species is approximately up to 2300 kcal/mol. The largest difference in adhesion energy for the other proteins is ∼300 kcal/mol. For the situation with adsorbed molecular proteins (i.e., nonsolvated), much weaker binding energies were observed. In addition to the results presented for H-, OH-, and NH2terminated diamond (100)-2 × 1 surfaces, the adhesion of the proteins onto a COOH-terminated surface has also been included in the present study. The adhesion energies were in the range 168−1816 kcal/mol, as compared with the situation of solvated proteins (2088−8876 kcal/mol). However, even though the water solvation will create much strong biomolecular binding energies, there is a resemblance in adhesion energy order for the various systems (i.e., solvated and nonsolvated). For both systems, the BMP2 molecule showed the strongest binding to the diamond (100)-2 × 1 surface, for all different termination types. Molecular VEGF, FN, and AGP did though bind to the terminated diamond (100)-2 × 1 surface with very similar adhesion energy, while a certain order in bond energy could be detected for the solvated proteins. This is understandable since the solvated ones were calculated to have a much larger adhesion energy compared to the nonsolvated proteins. Thus, the COOH termination was observed to result in large adhesion energies for all protein candidates in the present study, which is most probably caused by the high degree of hydrophilicity of the COOH-terminated surface (as discussed in Section 3.1). 3.3. Effect of Surface-Terminating Species on Protein Adhesion Energies for a Diamond (111) Surface. Similar to the situation with variously terminated diamond (100)-2 × 1 surfaces, the adhesion energies for different proteins in either a nonsolvated or water-solvated environment are shown in Figure 4b and Table S2 (Supporting Information). A similar strong binding of water-solvated BMP2 (7100−8900 kcal/mol) was observed for the diamond (111) surface, which was also found to be independent of type of surface termination. The order of adhesion energies for the rest of the proteins was VEGF (2000−2500) < FN (3800−4300) < AGP (3750−5300) kcal/ mol. This order of energies and independence of surface termination type are identical to the calculated results for the diamond (100)-2 × 1 surface. This independence of type of terminating species indicates that the adhesion (being noncovalent) of proteins onto the diamond (111) surface is, in addition to the diamond (100)-2 × 1 surface, mainly influenced by the individual proteins, instead of being influenced by surface termination type. As was the situation with the diamond (100)-2 × 1 surface, even though water-solvated VEGF molecules show the weakest adhesion energies among the chosen proteins, it experiences a strong binding to the diamond (111) surface, with a numerical value of about 2000 kcal/mol for the different termination types. Moreover, the adhesion energies for the various proteins were very similar for the two surface planes and for the various proteins. There is, hence, a large similarity between the results for the diamond (111) and (100)-2 × 1 surfaces, but there are also dissimilarities in that the effect of surface termination is different for the two surfaces. For the BMP2 adhesion to the diamond (111) surface plane, the OH termination was preferred, while the H termination was the least preferred one. Moreover, the situation with an adsorbed AGP showed its strongest binding to an NH2-terminated diamond (111) and weakest for the corresponding H-terminated surface. For

Figure 4. Absolute values of the adhesion energies for attached proteins (BMP2, VEGF, FN, and AGP) onto a (a) 2 × 1reconstructured diamond (100) surface and a (b) diamond (111) surface. The surface was terminated with either of four different species (H, OH, COOH, or NH2), and the proteins were either nonsolvated or solvated in water.

For the proteins positioned in water solvents, the strongest binding was, independent of type of termination species, observed for the BMP2 species (6500−8900 kcal/mol, as visualized in Figure 4a). This result correlates perfectly with the nonsolvated results and previous experimental observations. Strong binding of BMP2 was observed for all surface planes and terminations for nonsolvated calculations as well. Previous experimental studies by Steinmuller-Nethl et al. have shown strong adhesion energies for BMP2 attached onto H- and Oterminated nanocrystalline diamond surfaces. These observations were made by using X-ray photoelectron spectroscopy. In addition, biochemistry assays show that the bioactivity of BMP2 was preserved during the functionalization processes.21 The order of binding energies (i.e., absolute values of the negative adhesion energy) for the rest of the proteins was AGP (5200− 5400 kcal/mol) > FN (3500−4500 kcal/mol) > VEGF (2000− 2500 kcal/mol). All of these binding energy differences were independent of termination species types, which indicate that the adhesion (with noncovalent bonds) of proteins onto the diamond (100)-2 × 1 surface is mostly influenced by the individual proteins, instead of influenced by the termination species. It should be noted that the weakest adhesion is observed for VEGF attached onto a NH2-terminated diamond (100)-2 × 1 surface: 2088.1 kcal/mol. Thus, the preferences for different termination species for BMP2 are more notable than for the other three proteins (AGP, FN, and VEGF). For the situation with BMP2 adhesion, the largest difference in 8611


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systems were selected. More precisely, the combinations of each protein type with surface plane and termination type, rendering a maximum and minimum in adhesion energy, respectively, were chosen. Specifically, systems with extremes are (i) BMP2 [OH-terminated diamond (100)-2 × 1 (maximum) and H-terminated diamond (100)-2 × 1 (minimum)]; (ii) AGP [NH2-terminated diamond (100)-2 × 1 (maximum) and H-terminated diamond (111) (minimum)]; (iii) fibronectin [COOH-terminated diamond (111) (maximum) and NH2-terminated diamond (111) (minimum)]; and (iv) VEGF [OH-terminated diamond (111) (maximum) and H-terminated diamond (111) (minimum)]. The respective absolute adhesion energy values are presented in Table 1. These specific systems were thereafter considered for protein structural analysis, as presented below.

VEGF, the strongest adhesion was observed for OH termination, and the weakest adhesion energy was observed for H termination. The weak performances for H and NH2 termination can be explained by their larger degree of hydrophilicity, as presented and discussed in section 3.1. When comparing the results for the two diamond surface planes, the deviations in BMP2 adhesion energy for the different termination species are more pronounced for the 2 × 1-reconstructured (100) surface (∼1800 kcal/mol) than for the (111) surface (∼1050 kcal/mol). For the situation with AGP, the adhesion energies for the diamond (100)-2 × 1 surface, 5200−5400 kcal/mol, were larger than for the diamond (111) surface, 3750−5300 kcal/mol, and also showed smaller variations in numerical values for the different termination species. This indicates that AGP will energetically prefer a diamond (100)-2 × 1 surface. For fibronectin, the adhesion energies were positioned in approximately the same energy range, but there were minor differences. The deviation in adhesion energy was more pronounced for the diamond (100)2 × 1 surface (2500−3500 kcal/mol) than for diamond (111) (3800−4300 kcal/mol). This observation indicates that FN is more sensitive to type of surface termination when attached to a 2 × 1-reconstructured diamond (100) surface, as compared to diamond (111). For VEGF, the range of adhesion energies was 2000−2500 kcal/mol for both diamond (100)-2 × 1 and diamond (111) surfaces, which indicates that no pronounced preference for diamond surface planes was observed for the adhesion of VEGF. When comparing the adhesion energies for the nonsolvated proteins attached to variously terminated diamond (111) surfaces, the range of adhesion energies for the individual proteins [BMP2 (550 to 1850 kcal/mol), AGP (550 to 680 kcal/mol), FN (280 to 680 kcal/mol), and VEGF (410 to 600 kcal/mol)] was not as pronounced as for the solvated ones [BMP2 (7100 to 8900 kcal/mol), AGP (3700 to 5300 kcal/ mol), FN (3700 to 4300 kcal/mol), and VEGF (2000 to 2600 kcal/mol)]. As was the situation with diamond (100)-2 × 1, even though the water solvation will create much stronger biomolecular adhesion energies, there is a resemblance in order of adhesion energy for the various systems (i.e., solvated vs nonsolvated). For both systems, the BMP2 molecule showed the strongest binding to the diamond (111) surface for all different termination types. Nonsolvated VEGF, FN, and AGP did though bind to the terminated diamond (111) surface with very similar adhesion energies, while a certain order in bond energy could be detected for the solvated proteins. Compared to the nonsolvated situation, in which larger adhesion energies were observed for diamond (111) than for the 2 × 1-reconstructured diamond (100), the differences in adhesion energies for solvated proteins were not observed to differ notably for the two surface planes. 3.4. Protein Structural Analysis before and after Adhesion to a Diamond Surface. 3.4.1. General. Protein structural changes, as a result of adhesion to a diamond surface, are determinant for successful protein-mediated surface design. In order to investigate the surface-mediated effect on these protein structural changes, geometrical structures, functional groups, infrared spectrum, and surface electrostatics have been calculated and displayed, before and after physisorption to the diamond surfaces. In order to ensure the maintenance of protein functionality after adhesion to a diamond surface, regardless of adhesion energy strength, some representative

Table 1. Absolute Adhesion Energies for the Proteins BMP2, AGP, FN, and VEGF, Respectivelya adhesion energy [kcal/mol] proteins BMP2 AGP FN VEGF

surface_term. ΔEads surface_term. ΔEads surface_term. ΔEads surface_term. ΔEads

maximum

minimum

100_OH 8875.58 100_NH2 5358.39 111_COOH 4280.20 111_OH 2515.69

100_H 6600.10 111_H 3798.12 111_NH2 3766.86 111_H 2095.77

a

The systems diamond plane and surface termination are chosen depending on their capacity in rendering a maximum, or minimum, in adhesion energy. The diamond planes include diamond (111) and (100)-2 × 1. The surface-terminating species are H, OH, COOH, or NH2. All proteins were solvated in water.

3.4.2. Protein Structures before and after Adhesion to a Diamond Surface. 3.4.2.1. BMP2 Structures. Structural changes, before and after physisorption of BMP2 onto a terminated diamond surface, are visualized in Figure 5a,c,e. The corresponding surface electrostatic maps, illustrating the charge distributions, are shown in Figure 5b,d,f. A solvated molecular BMP2 structure, with its binding pocket marked in yellow (i.e., visualizing the area of bioactivity), is shown in Figure 5a. The solvated BMP2 structures after adhesion to an OH- and Hterminated diamond surface are presented in Figure 5c and e. The corresponding electrostatic maps are shown in Figure 5b,d,f. The negatively charged areas are displayed in red, and the positively charged areas are displayed in blue. Generally, there is a large structural similarity between a free, solvated BMP2 molecule and solvated BMP2 molecules adsorbed onto OH- or H-terminated diamond (100)-2 × 1 surfaces. As visualized in Figure 5c and e, the structural geometries are almost identical for the two termination types and also show a resemblance to the geometrical structure of molecular BMP2 in a solvent environment. This result has been found to be independent of adhesion energy strength since BMP2 adhesion to the OH-terminated surface showed the strongest binding for this type of biomolecule, while the adhesion to the H-terminated surface showed the weakest binding. It shall here be stressed that also the protein functionalities and functional groups on BMP2 seem to be maintained after the adhesion to the diamond (100)-2 × 1 surface. 8612


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Figure 5. Solvated BMP2 structures and surface electrostatic maps, before and after adhesion onto the diamond surfaces. The position of the functional groups is marked in yellow and with orange circles: (a) atomic BMP2 structure in water solvent; (b) surface electrostatic map of BMP2 in water solvent; (c) BMP2 structure after adhesion onto an OH-terminated (100)-2 × 1 surface; (d) BMP2 surface electrostatic map after adhesion onto an OH-terminated (100)-2 × 1 surface; (e) BMP2 structure after adhesion onto an H-terminated diamond (100)-2 × 1 surface; (f) BMP2 surface electrostatic map after adhesion onto an H-terminated diamond (100)-2 × 1 surface.

to the NH2-terminated diamond (100)-2 × 1 surface, is shown in Figure 6c and d. The situation with the weak adhesion to the H-terminated diamond (100)-2 × 1 surface is shown in Figure 6e and f. It can be concluded that the overall atomic structures, including the binding pocket structure, are kept after the interaction with the diamond surface. As can be seen from the displayed electrostatic maps, the slightly negatively charged areas of the binding pockets (as visualized with orange circles) are kept as well. 3.4.2.3. Fibronectin Structures. The atomic structure of molecular FN in a water solvent is visualized in Figure 7a. Moreover, the structures of an adhered and solvated FN molecule to either a COOH- or NH2-terminated diamond (111) surface are shown in Figure 7c and e. The corresponding electrostatic maps are presented in Figure 7b,d,f. By visualizing the atomic structures before and after surface functionalization, it can be concluded that the binding pocket structures are

As can be seen in Figure 5b,d,f, the electrostatic potentials became, for both type of terminations, overall slightly reduced (in absolute values) as an effect of adhesion to the diamond (100)-2 × 1 surface. In addition, the region close to the functional group showed a slightly more negative electrostatic potential for both OH- and H-terminated diamond surfaces, as compared to a nonadsorbed BMP2 molecule. Moreover, the positive electrostatic potential (to the left of the functional groups in Figure 5 in blue boxes) almost disappeared as a result of adhesion to any of the terminated diamond (100)-2 × 1 surfaces. 3.4.2.2. Angiopoietin (AGP) Structures. Accordingly, angiopoietin atomic structures and electrostatic maps are visualized in Figure 6. The molecular AGP structure (i.e., prior to adhesion) is shown in Figure 6a, with the corresponding electrostatic map in Figure 6b. The AGP structure, with electrostatic map, after the strongest adhesion 8613


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Figure 6. Solvated AGP structures, and surface electrostatic maps, before and after adhesion onto the diamond surfaces. The position of the functional groups is marked in yellow and with orange circles: (a) atomic AGP structure in water solvent; (b) surface electrostatic map of AGP in water solvent; (c) AGP structure after adhesion onto an NH2-terminated (100)-2 × 1 surface; (d) AGP surface electrostatic map after adhesion onto an NH2-terminated (100)-2 × 1 surface; (e) AGP structure after adhesion onto an H-terminated diamond (111) surface; (f) AGP surface electrostatic map after adhesion onto an H-terminated diamond (111) surface.

maintained and positioned in desired sterical places, with some surrounding bonds rotated or stretched. More importantly, the electrostatic maps showed that the binding pocket charges are maintained as rather neutral after the functionalization process. This is regardless of high (with COOH termination) or low (with NH2 termination) adhesion energies. In addition, the electrostatic environment surrounding the binding pocket was kept red, which indicate that the negatively charged area was maintained during the adhesion process. All in all, these findings suggest that the FN functionality will be maintained after physisorption onto the diamond surfaces. 3.4.2.4. VEGF Structures. For VEGF, the molecular atomic structure, and its electrostatic map, are presented in Figure 8a and b. After physisorption onto the OH-terminated diamond (111) surface, with its maximum in adhesion energy, the atomic structure looks like the one in Figure 8c. The corresponding

electrostatic map is shown in Figure 8d. For the adhesion energy minimum (i.e., for VEGF adsorbed onto the Hterminated diamond (111) surface), the atomic structure and electrostatic map are presented in Figure 8e and f. A good structural resemblance can be concluded by comparing the various VEGF atomic structures (including the binding pocket marked in yellow). The corresponding electrostatic maps do also strongly support this conclusion, with slightly negatively charged binding pockets and with a good resemblance of the overall electrostatic distribution. 3.4.3. Infrared Spectrum Analysis. With the purpose to more in detail analyze the binding of proteins to different terminated diamond surfaces, various IR spectra have been calculated (see Figures S1−S4, Supporting Information). As was the situation previously, the molecular protein structures, as well as the corresponding adsorbed ones, have here been 8614


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Figure 7. Solvated FN structures and surface electrostatic map before and after adhesion onto the diamond surfaces. The positions of the functional groups are marked in yellow and with orange circles: (a) atomic FN structure in water solvent; (b) surface electrostatic map of FN in water solvent; (c) FN structure after adhesion onto a COOH-terminated diamond (111) surface; (d) FN surface electrostatic map after adhesion onto a COOHterminated diamond (111) surface; (e) FN structure after adhesion onto an NH2-terminated diamond (111) surface; (f) FN surface electrostatic map after adhesion onto an NH2-terminated diamond (111) surface.

in surface N−H related groups is not affected by the adhesion to the diamond surfaces. For the situation with an AGP spectrum, the peak around 2600 cm−1 for the S−H stretch vibrations was also observed to disappear after the AGP molecule had physisorbed onto the Hterminated diamond (111) surface. This was, however, not the situation for the NH2-terminated diamond (100)-2 × 1 surface. Again, these results can most probably be explained by the hydrogen bond formation between sulfur (within AGP) and the H-terminated diamond (111) surface. Moreover, the C−N stretching around 2000−2200 cm−1 for VEGF and the broad hydroxyl O−H stretching around 2500 cm−1 were observed to disappear for the situation with VEGF adhered onto the OH-terminated diamond (111) surface. This was, however, not the situation for the H-terminated diamond (111) surface. This observation is most probably due to the formation of hydrogen bonds with the OH-terminated

studied and compared more in detail. As can be seen in Figures S1−S4 (Supporting Information), the peaks around 3600 cm−1, for the O−H stretching within the COOH group, were either weakened or disappeared upon adhesion. This can be explained by the possibility to form hydrogen bonds between the proteins and the diamond surface termination groups. This type of hydrogen bond formation is expected to weaken the COOH group peak and move it to a lower region in the IR spectra. Similarly, the CO group vibration near 1600 cm−1 was also observed to be weakened as an effect of adhesion, and the underlying cause to this observation is also most probably the formation of hydrogen bonds between the proteins and the surfaces. Also here, the hydrogen bond formation is expected to weaken the CO group peak and move it to a lower region in the IR spectra. On the contrary, the broad N−H stretch region (3000−3800 cm−1) appeared in most spectra (i.e., for both molecular and adsorbed proteins). The observation indicates that the nitrogen 8615


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Figure 8. Solvated VEGF structures and surface electrostatic maps, before and after adhesion onto the diamond surfaces. The position of the functional groups are marked in yellow and with orange circles: (a) atomic VEGF structure in water solvent; (b) surface electrostatic map of VEGF in water solvent; (c) VEGF structure after adhesion onto an OH-terminated diamond (111) surface; (d) VEGF surface electrostatic map after adhesion onto an OH-terminated diamond (111) surface; (e) VEGF structure after adhesion onto an H-terminated diamond (111) surface; (f) VEGF surface electrostatic map after adhesion onto an H-terminated diamond (111) surface.

diamond (111) surface but not with the H-terminated diamond (111) surface.

the protein binding. As a general conclusion, hydrophilic surface planes tend to possess a higher affinity for proteins. Within the present study, it was found that the physisorption of BMP2 to the various diamond surface planes will exceed the physisorption of the other three proteins (AGP, FN, and VEGF). For BMP2, the adhesion to the OH-terminated diamond (100)-2 × 1 surface was observed to possess the highest adhesion energy among all combinations of surface planes and terminations, while adhesion to an H-terminated diamond (100)-2 × 1 surface was found to be the lowest one. For AGP, the largest adhesion energy was found to occur for the NH2-terminated diamond (100)-2 × 1 surface and the lowest one for the H-terminated diamond (111) surface. Moreover, FN tends to adsorb onto a COOH-terminated diamond (111) surface (with the largest adhesion energy) but not preferentially to an NH2-terminated diamond (111) surface. For VEGF, the largest adhesion energy appears for

4. SUMMARY AND CONCLUSIONS In order to promote implant vascularisation, diamond surface functionalizations with four different growth factors (BMP2, AGP, FN, and VEGF) have been theoretically investigated using force field simulations. The chosen diamond surface planes include diamond (100)-2 × 1 and (111) surfaces. Furthermore, the modeled systems were solvated in a water medium. As a result, strong bindings of the different proteins were observed for the different diamond surface planes. These binding affinities were mainly attributed to the type of protein. As the second most important influence on the protein binding, the type of surface termination was also decisive, and as the third order of influence, the type of surface plane did influence 8616


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an OH-terminated diamond (111) surface and the weakest one for the H-terminated diamond (111) surface. The structural analyses also prove good protein structural similarities, before and after adhesion to the diamond surfaces. These results indicate a high probability for the bioactivity to be kept as a result of the adhesion processes, independent of adhesion strength. By visualizing the protein structures, it can be concluded that the atomic structures (including the binding pocket structures) were maintained after the adhesion onto differently terminated diamond surfaces. The corresponding electrostatic maps do support this conclusion with similar charging situations (i.e., before and after adhesion) for the overall protein structures, including especially the binding pocket position. Moreover, the infrared spectrum analysis was conducted. From the results, there are some common routes concluded for identifying protein structural changes by infrared spectra. First of all, the O−H stretch vibrations from the carboxylic group (around 3600 cm−1) tend to disappear as a result of adhesion to the diamond surface. The most plausible explanation for these observations is hydrogen bond formations between the proteins and the diamond surfaces. Second, the N−H stretch peaks in the broad range of 3000−3800 cm−1 come from amine-related groups that are usually not affected by functionalization. The reason why they are here visualized is most probably due to their abundance in the atomic structures and the embedding rather deep within the protein structures. Third, the vibration of the CO group around 1600 cm−1 is often weakened due to the formation of hydrogen bonds as well. In addition, it can be concluded that the hydrogen bond formations between the protein adsorbates and the diamond surfaces are more frequently observed for OH termination but not for H termination. However, a good overall structural resemblance was observed when comparing the protein structures before and after diamond functionalization. These theoretical results elucidate the protein adhesion dependences on surface terminations and provide a solid base for implant surface design by controllable surface chemistry.

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ASSOCIATED CONTENT

S Supporting Information *

Tables of absolute adhesion energy values of four proteins on diamond (100)-2 × 1 and diamond (111) surfaces and simulated infrared spectra of proteins before and after functionalization processes. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The presented work was financially supported by the European Union Seventh Frame programme (FP7) with the project name Vascubone. The grant number was 242175. The computational program was developed by Accelrys, Inc.

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REFERENCES

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