Molecular Dynamics Insights into WaterâParylene C Interface

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Molecular Dynamics Insights into Water-parylene C Interface – Relevance of Oxygen Plasma Treatment for Biocompatibility Monika Golda-Cepa, Waldemar Kulig, Lukasz Cwiklik, and Andrzej Kotarba ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 01 May 2017 Downloaded from http://pubs.acs.org on May 2, 2017

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ACS Applied Materials & Interfaces

Molecular dynamics insights into water-parylene C interface – relevance of oxygen plasma treatment for biocompatibility

Monika Golda-Cepa,1 Waldemar Kulig,2,3 Lukasz Cwiklik,4,5 Andrzej Kotarba1* 1

Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland

2

Department of Physics, University of Helsinki, P.O. Box 64, FI-00014 Helsinki, Finland

3

Department of Physics, Tampere University of Technology, P. O. Box 692, FI- 33101

Tampere, Finland 4

J. Heyrovský Institute of Physical Chemistry, Czech Academy of Sciences, Dolejškova 3,

Prague, 18223, Czech Republic 5

Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo

nám. 2, Prague, 16610, Czech Republic KEYWORDS: molecular dynamics, contact angle, surface free energy, parylene C, biomaterials oxygen plasma

ACS Paragon Plus Environment

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ABSTRACT

Solid–water interfaces play a vital role in biomaterials science since they provide a natural playground for most biochemical reactions and physiological processes. In the study, fullyatomistic molecular dynamics simulations were performed to investigate interactions between water molecules and several surfaces modelling for unmodified and modified parylene C surfaces. The introduction of –OH, –CHO, –COOH to the surface and alterations in their coverage, significantly influence the energetics of interactions between water molecules and the polymer surface. The theoretical studies were complemented with experimental measurements of contact angle, surface free energy, and imaging of osteoblast cells adhesion. Both MD simulations and experiments demonstrate that the optimal, in terms of biocompatibility, interface is obtained when 60% of native –Cl groups of parylene C surface is exchanged for –OH groups. By exploring idealized models of bare and functionalized parylene C, we obtained a unique insight into molecular interactions at the water-polymer interface. The calculated values of interaction energy components (electrostatic and dispersive) correspond well with the experimentally determined values of surface free energy components (polar and dispersive) revealing their optimal ratio for cells adhesion. The results are discussed in the context of controllable tuning and functionalization of implant polymeric coating towards improved biocompatibility.

1. INTRODUCTION Surface engineering being critical for biomedical applications is currently one of the most extensively and dynamically growing areas of research. There are several different strategies to


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functionalize biomaterial surfaces, like coatings made of natural or synthetic polymers for better cells adhesion,1 limited bacterial biofilm formation2 or immobilization of drug-loaded liposomes.3 All the mentioned techniques aim for optimization of surface chemical composition and topography towards enhanced biocompatibility. The biocompatibility concept is based on the so-called ‘race for surface’ phenomenon. Immediately after insertion of any kind of material into the body, tissue cell integration competes with bacterial adhesion to a surface of a medical device.4 In this context, biomaterials surface wettability is considered as one of the most important parameters governing cells adhesion.5 Thus, to design a biomaterial surface of implants, medical devices and sensors, the interaction with water, the universal biological solvent system, consists usually a practical starting point. When a biomaterial surface first comes in contact with a biological fluid, the adsorbed molecules create a conditioned surface which will later govern cell-surface interactions.6 For the cells, it is the conditioned surface covered with the components of the fluid that they first encounter and it governs their attachment, morphology and behavior. Solid–water interfaces play a vital role in biomedicine, since they provide natural playground for most biochemical reactions and physiological processes.7 This is the reason why functionalization of surfaces is brought to the center of interest with the aim of optimizing the implant-tissue interface.1,8,9 One of the general strategies is the coating of various materials (metals, ceramics, composites) with polymer layers or thin films which due to favorable chemical structure are prone to be functionalized.10 In this context, the parylene (poly(pxylylene)) polymer family is widely investigated as a protective coating due to its exceptional properties, such as biocompatibility, excellent mechanical properties, hydrophilicity, and stability in the body fluids.1,10,11 Furthermore, the deposition of such polymers can be performed


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by a chemical vapor deposition (CVD) method, which allows the application of the parylene films on biomaterials with complex surface shapes.10 For coating metal implants, parylene C (poly(chloro-p-xylylene), PPX C) proved to have superior properties for anticorrosive protection and efficient limitation of the wear debris formation. Furthermore, this coating demonstrates adequate elastomeric properties, essential to sustain strains during implantation and long–term usage in the body.12 A parylene C film itself is hydrophobic, dense, conformal, and microscopically smooth.13 Therefore, it is necessary to modify the surface towards increased number of peptide adsorption sites stimulating cells adhesion. In order to modify polymeric surfaces, plasma treatment, which is a combination of chemical and physical modifications, was proved to be particularly beneficial.13,14,15 The plasma treatment can be used not only to clean polymeric surfaces, but also to increase adhesion between polymer and implant materials and extend their applications for specific biointerfaces i.e. material-body fluids (blood), material-hard tissue (bone), material-soft tissue (fascia). Surfaces in contact with plasma, independently on the chemical nature of the gaseous agent, are bombarded by large numbers of electrons, photons, free radicals and ions, yielding various effects including surface cleaning, ablation and changes in chemical composition.16 This modification method can be successfully applied to polymers, as the plasma parameters may be precisely adjusted in the broad range: type of plasma gas (oxidizing, reducing, neutral), partial pressure (0.1 – 1000 mbar), power (10 – 1000 W) and exposition time (1 – 103 s).13,15,17,18 For tuning the properties of medical devices surfaces oxygen plasma is the most commonly applied.1,8,19 An apparent benefit is surface cleanness, however, the key issue is the incorporation of oxygen-containing surface functional groups such as –COOH, –OH, –CHO into the originally oxygen free polymers, like parylene C. Such a physicochemical modification transforms the


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hydrophobic polymeric material into a hydrophilic, advanced biomaterial with substantially improved cell adhesion rate, as shown previously.13,17,20,21,22 It has to be emphasized that oxygen plasma treatment significantly modifies the chemical nature of the surface affecting the adsorption and attachment of water, proteins and cells, subsequently. Adsorbed on the surfaces they ultimately impact on the molecular level on all the adhesion centers, bonding forces, surface diffusion, etc. Since the surface hydration is the first step in the interaction of biomaterial with a biological milieu, water molecules play a key role in the conditioning of the exposed surfaces. They mediate protein adsorption (i.e. fibronectins, proteins which adsorb onto implant surface immediately upon contact with physiological fluids) and guide formation of the dynamic interphase between the implant surface and body fluids.7,23 Osteoblast-like cells adhesion is primarily mediated by the integrin family of cell-surface receptors which binds to fibronectin. The latter one adsorbs to the hydrophilic implant surface in the native conformation, thus allowing the cells to recognize the adsorption centers.23 It has to be underlined that hydrophobic surfaces strongly denature adsorbed proteins, revealing the importance of preserving the protein native structure upon adsorption.24 The adsorption, however, is facilitated on hydrophilic surfaces with oxygen-containing functional groups, while their nature and surface coverage determine water wetting characteristics and influence cells adhesion.25 The described observations clearly reveal a primary role of vicinal water molecules on implant surface biocompatibility, rationalizing the wettability measurements (water contact angle) and surface free energy (SFE) calculations as a useful approach for evaluation of polymers biocompatibility.26 There is a well-documented correlation between polymeric biomaterial (such as parylene C, PEEK, PMMA) surface wettability and cells adhesion which has been observed in numerous previous contributions.11,13 ,27,28,29


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The surface hydrophilic properties, which can be quantitatively gauged by surface free energy, play a key role in the biocompatibility concept. This quantity determines the adhesion of both osteoblast and bacteria cells. Since SFE value is not easy to obtain and a vivid discussion on its absolute values determination is taking place,30,31 the most handy method for biocompatibility evaluation is water contact angle measurement. Although this simple practical approach is well established, it provides no direct insight into molecular-level physico–chemical processes taking part at biointerfaces. In recent literature, molecular dynamics (MD) simulations are employed to investigate with atomistic resolution interactions between water molecules and solid surfaces; however, they are typically focused on model systems such as silica32, graphite33 and boron-nitride34, which are far from polymeric surfaces. Certainly, MD simulations of such systems can provide the first rationales for understanding more complex, realistic implant surfaces. However, due to complexity and diversity of processes taking place at the implant surface-water-fibronectinintegrin interphase, a direct insight into specific molecular mechanisms operative in this particular system is required. In this paper, we focus on in silico simulations of atomistic-level interactions between water molecules and polymeric surfaces of parylene C with the idea to compare them with experimental results of surface wettability and osteoblasts adhesion. To our knowledge, such a study is performed for the first time for the realistic implant coating of parylene C which is in practice functionalized by oxygen plasma. By combining energetics of water-surface interaction components calculated from MD simulations with experimentally measured SFE, water contact angle, and osteoblast cells adhesion we aim at explaining how interactions of water with modified parylene C surfaces at the molecular level influence macroscopic wettability and hence, the biocompatibility of the considered surfaces. Such


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approach, in the broader perspective, provides rationales for designing and tailoring polymeric implant coating surfaces. 2. MATERIALS AND METHODS 2.1 Atomistic molecular dynamics simulations Fully-atomistic molecular dynamics simulations were performed to investigate interactions between water molecules and several surfaces modelling both parylene C and differently modified surfaces. In our model, the slab configuration was used with two model surfaces interacting with a slab of water (Figure 1). The size of the box was 11.622 nm (the x direction), 12.720 nm (the y direction), and height of 8 nm (the z direction). In the middle of the simulation box, two flat surfaces built of carbon atoms separated by 1 nm vacuum slab were placed. 35,000 water molecules (corresponding to the water density of ≈1 g/cm3) were packed in the remaining part of the box. Carbon atoms inside each carbon sheet were arranged in a way that corresponds to their arrangement in the parylene C crystal structure35, namely the distance between carbon atoms in the x direction was equal to 0.149 nm (corresponding to the crystallographic axis a*), while the distance between carbon atoms in the y direction was equal to 0.159 nm (corresponding to the crystallographic axis b). Four different functional groups corresponding to different ways of surface modification were considered, namely chloride (-Cl), hydroxyl (–OH), carbonyl (–CHO), and carboxyl (–COO-) groups. Selection of such chemical groups is based on our previous spectroscopic investigation of oxygen plasma modified parylene C with the use of XPS13 and LDI-MS.36


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Figure 1. Snapshot of the simulation box (A) showing the two surfaces and the water. A fragment of the model surface decorated with aldehyde groups (B). Yellow stars show theoretical positions of the decorated groups assuming 100% surface coverage, while purple parallelogram depicts the crystallographic primitive cell of parylene C.

For each group, seven different concentrations of abovementioned groups corresponding to 0%, 20%, 40% 50%, 60%, 80%, and 100% surface coverage were considered. Initial structures of functionalized parylene systems were obtained by attaching appropriate functional group to randomly chosen surface carbon atom. The systems were first energy minimized using the steepest descent algorithm and then equilibrated for 50 ns. After equilibration, production runs of 200 ns were performed, with the last 100 ns used for analysis. Simulation convergence was controlled by means of the number of water-surface contacts as well as the standard convergence criteria for energy and temperature.


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The atomistic MD simulations were carried out in NVT ensemble using GROMACS 4.6 software.37,38 Temperature was kept at 310 K using the v-rescale thermostat with the temperature coupling constant of 0.5 ps. Water molecules, functional groups, and carbon surface atoms were coupled to separate thermostats. Long-range electrostatic interactions beyond a non-bonded interaction cutoff of 1.0 nm were treated by the Particle Mesh Ewald scheme (PME)39 with a Fourier spacing of 0.12 nm. A long-range dispersion correction to the energy and pressure was added. The LINCS algorithm40 was used to constrain all covalent bonds, allowing a time step of 2 fs. For water, the SETTLE method41 was applied. Periodic boundary conditions were applied in all dimensions. Force field parameters of the carbon surfaces were taken from the Amber03 force field42,43 from the aromatic carbon atom, while functional groups were parametrized using the GAFF force field44 (for details see Supporting Information, Appendix). 2.2 Samples preparation Parylene C (8 μm of thickness) films were prepared by Chemical Vapour Deposition (CVD) technique, provided by ParaTech Coating Scandinavia AB. The dimer of chloro-para-xylylene was used as a precursor and heated up to 690°C for decomposition to monomeric form. The monomers in vapor phase were spontaneously deposited and polymerized at room temperature at 10-3 mbar. The thickness of the coating was controlled by the deposition time. The samples were cleaned with isopropyl alcohol before further investigations. 2.3 Oxygen plasma modification


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In order to modify the parylene C surface, oxygen plasma treatment was carried out using a Diener electronic Femto plasma system (Diener Electronic GmbH, Nagold, Germany) at 50 W and an oxygen partial pressure of 0.2 mbar. The only varied parameter was the time of exposure to the plasma, which was in the range of 0.5–10 min. The detailed parameters are listed in Table 1. According to our previous studies, such range spans over the modification time for increased osteoblast adhesion to parylene C, with the optimal value of 8 min.17 Table 1. The specific oxygen plasma parameters used for functionalization of parylene C. Power Pressure Flow Exposure time [W] [mbar] [cm3/min] [min] 50

0.2

1.0

0.5; 3; 5; 8; 10

2.4 Contact angle measurements and surface free energy (SFE) calculations The oxygen insertion and pore formation have a strong impact on the hydrophilicity of the parylene C film. The changes within the surface were followed by contact angle measurements, using a Surftens universal instrument (OEG GmbH). Static contact angles of water were calculated using Surftens 4.3 — windows image processing software for digital images for determination of contact angles and surface tension. For each sample, five independent 2.5 μL water drops were applied. The mean value was averaged over ten measurements.


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SFE calculations were performed, using Owens-Wendt method (Eq. 1)

g s = g sd + g sp ,

where: (g sd )

1

2

=

g d (cos Qd + 1) - (g dp / g wp )g w (cos Q w + 1)

(g ) p s

2( g dd - g dp (g wd / g wp )

1

2

=

(cos Q w + 1) - 2 g sd g wd 2 g wp

(1)

Where, 𝛾s is the SFE, 𝛾sd is the dispersive component of surface energy, 𝛾sp is the polar component of SFE and 𝛾s𝑑 is the total free energy of diiodomethane. 𝛾𝑑𝑝 and 𝛾𝑑𝑑 are the polar and dispersive components of total free the energy of diiodomethane, respectively. The lower index ‘w’ is for water and 𝜃D and 𝜃W represents the contact angles of diiodomethane and water, respectively. 2.5 Cell culture Cell culture tests were performed with the use of an MG-63 cell line (human osteosarcoma, ATCC 86051601) grown in DMEM (Lonza), supplemented with glutamine, FBS (Biowest) and antibiotic/antimycotic solution (PAN-Biotech). 2.6 F-actin staining The fluorescent staining of MG-63 was performed after 24h of incubation with the evaluated parylene C samples. Cells were washed in PBS, fixed in paraformaldehyde (Sigma-Aldrich) and permeabilized in Triton-X100 (Sigma-Aldrich). In order to stain F-actin and cell nuclei,


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Alexa Fluor 488 dye (Molecular Probes) as well as DAPI (Molecular Probes) were used, respectively. The stained cells were imaged using a fluorescent microscope IX51 (Olympus). 3. RESULTS The MD simulations of water film in contact with variously decorated carbon surfaces were performed to quantify the interactions between water molecules and parylene C surfaces. For the simulations, the surface was built to mimic the ideal structure of the parylene without the chloride atoms, as described in the methodology section. Four decorating groups were considered, resulting in the following decorated surface sites: –Cl (chlorine), –OH (hydroxyl), –CHO (aldehyde), and –COO- (carboxylate ion). The coverage of the decorating groups was varied between 0 and 100%. The 100% coverage by chloride corresponds to an ideal parylene C surface (space group P1, (020) crystallographic plane),45 whereas lower coverages were considered to account for the surface of parylene N (poly(p-xylylene)) non-idealities. The use of hydroxyl, aldehyde, and carboxylate groups was mimicking parylene surface that were observed upon functionalization by oxygen plasma treatment by XPS and LDI-MS.13,36,46 The deprotonated carboxylate group was employed in order to explore consequences of the presence of charged groups that are significantly different from polar, but uncharged, –OH and –CHO.


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Figure 2. Snapshots of water organization next to surface fragments covered with various decorating groups: –Cl (A), –CHO (B), –OH (C), –COO- (D). The snapshots are taken for the representative systems with 50% coverage. For clarity, only carbon atoms and water molecules in the proximity of the visualized group are depicted. Color coding: cyan, carbon; red, oxygen; white, hydrogen; green, chloride. In Fig. 2, typical snapshots taken in the vicinity of the considered surface decorating groups are presented. In the case of –Cl sites (Fig. 2A), water molecules tend to avoid the vicinity of this site which demonstrates the hydrophobic character of the surface. In the case of the other groups (Fig. 2B-D), water molecules are present in the proximity of the decorating group which corresponds to direct, attractive interactions and hence the hydrophilic character of these surfaces. In particular, there is significantly increased water density close to the –COO- moiety, in accord with strongly attractive character of water–COO- interactions.


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Figure 3 presents water binding energy calculated for all the considered functional groups – Cl, –CHO, –OH, –COO- with the surface coverage equals to 50%. Total binding energies together with the specific components of electrostatic and dispersive parts are shown.

Figure 3. Energetics of water binding at surfaces with 50% coverage of all the considered decorating groups. The total binding energies and the contribution from electrostatic and dispersive interactions are presented. Energy values are reported per mole of total surface sites.

The strength of water binding increases from approximately -1.8 to -22 kJ/mol (i.e., the binding energy is more negative) while going from –Cl toward more oxidized –OH, –CHO, and –COO- groups. In the case of –Cl groups which correspond to the unmodified parylene C, the binding is mostly due to dispersive interactions (denoted as Edispersive). For both –OH and – CHO groups, the Edispersive component is similar to that of –Cl but the electrostatic term (Eelectrostatic) is significant and hence responsible for stronger water binding. The energetic


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picture in the case of the carboxylate group is totally different. Namely, the electrostatic interaction component is dominant being ~one order of magnitude larger than that of other surface groups. At the same time, the dispersion energy term appears slightly repulsive (~1 kJ/mol). Of note, the dispersion energy reported here based on MD simulations contain all terms from Lennard-Jones interaction potential which include both dispersive energy (always attractive) and Pauli repulsion (acting at much shorter interatomic distances). The repulsive dispersion energy in the case of –COO- originates from Pauli repulsion, as electrostatic interactions bring water molecules to such a close distance from –COO- group that the Pauli energy dominates over purely dispersive energy terms. It should be noted, that the binding energy calculated from MD simulations does not directly correspond to the experimentally measured SFE. Simulations, because of their atomistic resolution and the length scale limited to nanometers, probe local interactions between water and surface sites. The energetics of these interactions can be studied in detail as a function of surface sites chemical identity, coverage, arrangement, etc. This energetics is one of the factors governing the macroscopically observed behavior at the water-solid interface; in particular, it can be directly related to surface hydrophilicity.


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Figure 4. Water binding energy vs. coverage by the decorating groups for –Cl and –OH (for – CHO and –COO- see Figure S1 in Supporting Info). Energy values are reported per mole of total surface sites.

In Fig. 4A, water binding energy and its components are presented as a function of varying coverage of the chloride sites. The zero coverage corresponds to a pure polymer surface of


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parylene N which weakly binds water (~-2 kJ/mol) by purely dispersive interactions. As the concentration of –Cl atoms at the surface increases, the water binding strength is somewhat reduced (to ~-1.5 kJ/mol). The presence of –Cl also leads to slightly more pronounced electrostatic interactions with water; however, these are negligible in comparison with the dispersive energy term. It should be noted, that the water-surface interaction energy of the macroscopically hydrophobic surfaces, such as that of ~-2 kJ/mol for the non-functionalized polymer surface, is negative; this is due to the always-present attractive dispersive interactions. Nevertheless, the water binding energy at hydrophobic surfaces is weaker than in the case of macroscopically hydrophilic ones as the latter interact with water also by Coulombic interactions that occur due to the presence of surface polar groups. Figure 4B presents water binding energy and its components for varying coverage of –OH surface sites. As the coverage increases, water binding gets stronger, up to over -5 kJ/mol. This is because of electrostatic interactions which are more pronounced with the increasing presence of the decorating polar groups. The same trends are observed for other considered surface groups (Fig. S1 in Supporting Info). In each case, water binding energy is well below 2 kJ/mol, i.e., the water binding is stronger than at the hydrophobic polymer surface. In other words, the change of the water binding energy observed in Fig. 4B corresponds to the increase of surface microscopic hydrophilicity which can be related to the macroscopic hydrophilicity of the polymeric material. The latter can be quantified by SFE calculated from contact angle measurements of liquids composed of molecules with different polar and dispersive components. For polymeric surfaces, the Owens-Wendt method using water and diiodomethane is often applied1,30.


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Figure 5. Density profiles of water oxygen (Owater) and hydrogen (Hwater) for parylene C surface coverage of 50% with various functional groups in panels A and B, respectively. The density of both atoms (oxygen – full lines, hydrogen – dashed lines) are compared for short distances in panel C. Density profiles only for one half of the simulation box are presented (the other halves provided the same results). In (C), the density of hydrogen is reduced by the factor of 0.5 to allow for an easier comparison with the oxygen profiles.


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Apart from water binding energy, the atomistic-scale MD simulations allow for detailed analysis of water behavior in the vicinity of the variously decorated surfaces. In Fig. 5A-C, density profiles of water oxygen and hydrogen atoms are depicted as a function of the distance from the surface with the coverage of 50% by different groups. In each case, water density is perturbed by the presence of the surface up to the distance of ~1.2 nm (see Fig. 5AB). This perturbation is the least pronounced in the case of a –Cl-covered surface, as the amplitude between maxima and minima at the corresponding curves is the lowest. The similarly positioned oxygen and hydrogen maxima (see Fig. 5C) show that both atoms reside on average at the same distance from the –Cl-covered surface. This indicates that rotational orientation of water is approximately uniform next to this surface, i.e., there is no orientation preference or any geometrical specificity in water binding in this system. Both the weak water density perturbation and the lack of a specific water orientation reveal the hydrophobic character of the parylene surface. Among the polar decorating groups, the perturbations of water density increase in the order –CHO < –OH < –COO- (based on the height of the first peak at density profiles) which corresponds to the hydrophilic character of these surfaces and is in accord with the water-surface interaction energy shown in Fig. 3. In the case of –CHO, there is only a little difference between the position of oxygen and hydrogen maxima, pointing to the lack of water orientation preference (Fig. 5C, red). In the case of –OH, the maxima of oxygen and hydrogen are located relatively close to each other, but hydrogen density has a significant tail toward the surface (Fig. 5C, orange). This indicates some orientational preference as well as hydrogen bonds formation between water and the –OH groups. In the case of –COO-, the hydrogen maximum is shifted toward the surface (Fig. 5C blue); this is due to the formation of relatively strong hydrogen bonds between water and the carboxylate groups.


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The influence of decorating group’s concentration on the structure of interfacial water layer for –Cl- and –OH-covered surfaces is demonstrated in Fig. 6. In the case of –Cl, with increasing concentration of the decorating group, the perturbation in the density is diminished; this is in accord with the interaction energy shown in Fig. 4A. On the contrary, in the case of – OH, the concentration effect is much less pronounced and results in a shift of the density toward the surface. This is caused by the increasing presence of water-surface hydrogen bonds at the surfaces with –OH sites. The trends similar to those observed in –Cl-decorated case, can also be observed in the presence of –CHO and –COO- (Fig. S2 in Supporting Info). The main difference observed in the two latter cases is restructuring of the second density band (between 0.5 and 0.7 nm) which is due to the ability of these groups to form complex hydrogen bond networks with water.


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Figure 6. Density profiles of water oxygen for parylene C surfaces functionalized by –Cl and – OH at varying decorating group coverage (for –CHO and –COO- see Figure S2 in Supporting Info). Concluding the MD simulations, the presented results demonstrate that the initially hydrophobic surface of parylene C turns into hydrophilic when functionalized with oxygencontaining groups. As shown above, the changes in the water-parylene C interactions depend on the chemical nature of surface groups as well as their surface coverage. The explored functionalization results in substantial increase in total surface-water binding energies and alters both dispersive and electrostatic contributions. The –OH functional groups are of special interest since they are formed as the most probable product of oxygen plasma functionalization of polymers. Furthermore, it was demonstrated before that osteoblasts preferentially bind to – OH surface groups.17,47 The obtained calculated data were confronted with the experimental results of oxygen plasma treated parylene C. The corresponding results of contact angle measurements, SFE calculations and biocompatibility evaluation are presented below.

Table 2. Summary of contact angle measurements with H2O (ΘW) and CH2I2 (ΘD) together with calculated values of SFE determined by the Owens-Wendt method. 𝛾s𝑝 and 𝛾s𝑑 are the polar and dispersive components of SFE for the unmodified and oxygen plasma modified (8 min) parylene C, respectively.

sample

ΘW [deg]

ΘD [deg]

𝛾sp [mJ/m2]

𝛾sd [mJ/m2]

SFE [mJ/m2]

unmodified parylene C modified parylene C

90.1 0.1

32.1 35.0

0.6 46.6

43.1 26.5

43.7 74.2


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In Table 2, the detailed results of contact angle measurements and SFE calculations are summarized. Originally, the hydrophobic surface of parylene C (which corresponds to the surface with –Cl groups, as presented in Fig. 4A) with water contact angle ΘW = 90º turns hydrophilic upon oxygen plasma treatment. As can be inferred from Table 2 as well as from Fig.7, the treatment results in the dramatic decrease of water contact angle value to ΘW = 0.1º (super hydrophilic surface). The presented results of modified parylene C are representative for 8 min exposure time, determined as a compromise between biocompatibility and polymer bulk degradation in our previous studies.1,17 Significant differences can also be observed in the case of the calculated values of SFE as well as their corresponding polar (𝛾sp) and dispersive (𝛾sd) components. Initially, SFE of unmodified parylene C is 43.7 mJ/m2 and consists mostly of dispersive component (43.1 mJ/m2) with minimal polar influence (0.6 mJ/m2). Modification of parylene C with oxygen plasma and incorporation of oxygen-containing surface functional groups cause significant increase of the SFE value to 74.2 mJ/m2 as well as the 𝛾sd/𝛾sp ratio, as presented graphically in upper panel of Fig. 7. The role of the dispersive component diminishes to 26.5 mJ/m2 while the polar component becomes dominant with 46.6 mJ/m2. This founding has an important significance, for the modified parylene C surface, the experimentally obtained ratio of dispersive and polar components 𝛾sd/𝛾sp = 0.5 is in-line with the theoretically determined 60% surface coverage for –OH, where the corresponding ratio of dispersive and electrostatic energies Edispersive/Eelectrostatic = 0.56 (Fig. 4). Additionally, the surface coverage of –OH groups of 60% compares quite well with the XPS results, where 40 – 60% of native chlorine is substituted with oxygen upon the plasma treatment. This agreement reveals that the direct insight obtained from MD in silico modelling is predictive not only regarding the optimal surface coverage of functional groups but also regarding the key role of


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balance between electrostatic and dispersive components. This finding may be of more general importance for biomaterial surfaces, since the electrostatic component plays primary role for adhesion of bacteria as explicitly shown elsewhere.48

Figure 7. The summary of the experimental data obtained for unmodified (A) and oxygen plasma modified for 8 min (B) parylene C. Upper panels present results of water contact angle measurements and SFE calculation (blue: dispersive, grey: polar component); in lower panels the images of fluorescently stained MG-63 cells on the corresponding surfaces are shown. The biological response to biomaterials is largely controlled by their surface chemical identity and water-surface interactions. The performance of the biomaterial surface in the body can be predicted with the use of wettability measurements, SFE calculations, and biological tests with selected cell lines. In Fig. 7A-B the results of water contact angle measurements and the resulting calculated SFE values together with osteoblasts cells-like morphologies on the unmodified and oxygen plasma modified parylene C are presented. The water contact angle


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measurement is a method to probe surface hydrophilic/hydrophobic properties and predict how the surface will interact with the liquid environment. The determined contact angle probe directly surface wettability and indirectly surface energy. The observed changes in wettability and SFE values have a significant impact on the MG-63 morphology on the investigated surfaces. In the lower panels of Fig.7, the images of fluorescently stained MG-63 cells are presented. The osteoblast-like cells were not well spread and displayed high motility on the unmodified parylene C (Fig. 7A), whereas on the oxygen plasma-modified samples the cells were well spread with either bipolar or tripolar morphology, as indicated by the parallel orientation of actin filaments with the long cell axis (Fig. 7B). These results show the dramatic influence of the presence of surface oxygen-containing functional groups on the biocompatibility of the polymeric material. Taking into consideration that less motile cells form adhesive structures, such as focal adhesion sites, which is crucial for successful osseointegration, oxygen plasma modified parylene C is a suitable surface for bone tissue formation. Summarizing the obtained results, the parylene C surface can be successfully modified using mild oxygen plasma modification methods. Upon exposure to plasma, oxygen-containing functional groups are formed on parylene C surface which is a key for its biocompatibility.1,13,17 The effect is observed at the macroscopic level, biocompatibility is related to the surface hydrophilicity and can be determined by the means of contact angle measurements accompanied by SFE calculations.7 These values give an experimental measure of waterpolymer surface interactions and can predict some surface free energy-related processes, i.e. protein adsorption and cells attachment. Nevertheless, hydrophobicity/hydrophilicity is a complex property, depending both on molecular-level interactions and on macroscopic


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parameters of the surface such as the degree of crystallinity, surface defects and roughness, etc. Therefore, in this study we employ molecular-level MD simulations for understanding how surface chemical modification of parylene C influences interaction with water (observed macroscopically as changes in contact angle). Such MD simulations, employing a simple, idealized model of functionalized and non-functionalized parylene C, give a unique insight not only into surface energy components (electrostatic/polar and dispersive) but also their contribution in total energy. The complementary (theoretical and experimental) approach allows relating molecular-level interactions with macroscopically-measured SFE. Moreover, the key role of a balance between electrostatic and dispersive interaction components was demonstrated at both molecular and macroscopic levels. This confirms that application of in silico molecular modelling in tandem with experimental measurements allows understanding the water-surface interactions. Importantly, such an insight can be further employed to design polymeric surfaces with intended biological function by introducing controlled amounts of polar groups of various chemical identities. This opens new possibilities for more rational surface modifications and tailoring of its properties for optimal cells–biomaterial interactions. 4. CONCLUSIONS We present a combined theoretical and experimental approach for evaluating the role of functional groups at the water-parylene C interface for improved biocompatibility. The energetics of interactions between water molecules and polymer surface was evaluated upon changing the chemical nature of the surface (introduction of –OH, –CHO, –COOH) and varying surface coverage. The molecular-level in silico approach was supported by macroscopic experimental measurements of contact angle, SFE, and osteoblast adhesion employing plasmamodified parylene C surfaces. Influence of the presence of polar surface groups on microscopic


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water-surface interactions as well as macroscopic wetting and cell adhesion was described in detail. Most importantly, it was found in simulations and further supported by experimental measurements that the optimal surface, in terms of biocompatibility, is obtained when 60% of native –Cl groups is exchanged for –OH groups. Such modification results in balanced dispersive and polar components of the overall interaction between the implant surface and the biological milieu. Our results are promising in the context of controllable tuning and functionalization of implant polymeric coating. Supporting information Additional plots illustrating the water binding energy vs. coverage and density profiles of water oxygen for surfaces covered by –CHO and –COO-; detailed description of parametrization of the functional groups with force field parameters. Acknowledgments Lukasz Cwiklik thanks the Czech Science Foundation for support via grant 17-06792S. CSC – Finnish IT Center for Scientific Computing (Espoo, Finland) is acknowledged for computer resources. European Research Council (Advanced Grant project CROWDED-PROLIPIDS) and the Academy of Finland (Centre of Excellence and FiDiPro programs) are thanked for financial support. Monika Golda-Cepa thanks the Polish National Science Centre for financial support, decision number DEC-2012/05/N/ST8/03321


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Figure 1. Snapshot of the simulation box (A) showing the two surfaces and the water. A fragment of the model surface decorated with aldehyde groups (B). Yellow stars show theoretical positions of the decorated groups assuming 100% surface coverage, while purple parallelogram depicts the crystallographic primitive cell of parylene C. 91x89mm (300 x 300 DPI)


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Figure 2. Snapshots of water organization next to surface fragments covered with various decorating groups: –Cl (A), –CHO (B), –OH (C), –COO- (D). The snapshots are taken for the representative systems with 50% coverage. For clarity, only carbon atoms and water molecules in the proximity of the visualized group are depicted. Color coding: cyan, carbon; red, oxygen; white, hydrogen; green, chloride. 111x117mm (300 x 300 DPI)



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Figure 3. Energetics of water binding at surfaces with 50% coverage of all the considered decorating groups. The total binding energies and the contribution from electrostatic and dispersive interactions are presented. Energy values are reported per mole of total surface sites. 105x105mm (300 x 300 DPI)


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Figure 4. Water binding energy vs. coverage by the decorating groups for –Cl and –OH (for –CHO and – COO- see Figure S1 in Supporting Info). Energy values are reported per mole of total surface sites. 202x392mm (300 x 300 DPI)



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Figure 5. Density profiles of water oxygen (Owater) and hydrogen (Hwater) for parylene C surface coverage of 50% with various functional groups in panels A and B, respectively. The density of both atoms (oxygen – full lines, hydrogen – dashed lines) are compared for short distances in panel C. Density profiles only for one half of the simulation box are presented (the other halves provided the same results). In (C), the density of hydrogen is reduced by the factor of 0.5 to allow for an easier comparison with the oxygen profiles. 158x262mm (300 x 300 DPI)


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Figure 6. Density profiles of water oxygen for parylene C surfaces functionalized by –Cl and –OH at varying decorating group coverage (for –CHO and –COO- see Figure S2 in Supporting Info). 103x113mm (300 x 300 DPI)



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Figure 7. The summary of the experimental data obtained for unmodified (A) and oxygen plasma modified (B) parylene C. Upper panels present results of water contact angle measurements and SFE calculation (blue: dispersive, grey: polar component); in lower panels the images of fluorescently stained MG-63 cells on the corresponding surfaces are shown. 95x50mm (300 x 300 DPI)


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Graphical abstract 246x80mm (96 x 96 DPI)


Molecular Dynamics Insights into WaterâParylene C Interface

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