Promoting Cell Adhesion and Antifouling Properties via a Wet

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Tailoring novel PTFE surface properties: Promoting cell adhesion and anti-fouling properties via a wet chemical approach Matthias Gabriel, Kerstin Niederer, Marc Becker, Christophe Michel Raynaud, Christian Vahl, and Holger Frey Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00047 • Publication Date (Web): 03 Apr 2016 Downloaded from http://pubs.acs.org on April 9, 2016

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Bioconjugate Chemistry

Tailoring novel PTFE surface properties: Promoting cell adhesion and anti-fouling properties via a wet chemical approach. Matthias Gabriel1,4*, Kerstin Niederer2, Marc Becker3, Christophe Michel Raynaud1, Christian-Friedrich Vahl4, Holger Frey2 1

Sidra Medical and Research Center, Cardiovascular Division, QCRC, Doha, Qatar

2

Department of Organic Chemistry, Johannes Gutenberg-University Mainz, Mainz, Germany

3

Center for Musculoskeletal Surgery, Department of Orthopedic Surgery, Johannes-

Gutenberg-University School of Medicine 4

Department of Cardiothoracic and Vascular Surgery, Johannes Gutenberg-University School

of Medicine, Mainz, Germany.

*

Corresponding author:

Matthias Gabriel Sidra Medical and Research Center, Cardiovascular Division Qatar Cardiovascular Research Center, Doha, Qatar Qatar Science & Technology Park, Tech 2, Room 104 PO box 5825 Tel. +974 3371 9757 Email: [email protected]

Keywords polytetrafluoroethylene, polyethylene glycol, surface modification, endothelialization, antifouling

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Abstract Many biomaterials used for tissue engineering applications lack cell-adhesiveness and in addition, are prone to nonspecific adsorption of proteins. This is especially important for blood-contacting devices such as vascular grafts and valves where appropriate surface properties should inhibit the initial attachment of platelets and promote endothelial cell colonization. As a consequence the long-term outcome of the implants would be improved and the need for anticoagulation therapy could be reduced or even abolished. Polytetrafluoroethylene (PTFE), a frequently used polymer for various medical applications, was wet-chemically activated and subsequently modified by grafting the endothelial cell (EC) specific peptide arginine-glutamic acid-asparic acid-valine (REDV) using a bi-functional polyethylene glycol (PEG)-spacer (known to reduce platelet and nonspecific protein adhesion). Modified and control surfaces were both evaluated in terms of EC adhesion, colonization and the attachment of platelets. In addition, samples underwent bacterial challenges. The results strongly suggested that PEG-mediated peptide immobilization renders PTFE an excellent substrate for cellular growth while simultaneously endowing the material with antifouling properties.


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Introduction Foreign materials in contact with blood generally suffer from thrombogenicity which limits their use in certain applications, such as small calibre vascular grafts1. Polytetrafluoroethylene (PTFE) in the form of expanded PTFE (ePTFE, GoreTex) along with knitted polyethylene terephthalate (PET, Dacron) are the most common synthetic materials for the fabrication of vascular prostheses. Because of the highly hydrophobic nature of PTFE, unspecific adsorption of blood components readily occurs resulting in adverse effects such as complement activation, platelet adhesion and activation of the coagulation cascade2. These events may result in the occlusion of the vascular graft3. Attempts were made to alter PTFE properties, mainly employing two strategies: anti- (or non)-fouling surface modification and endothelialization. The antifouling modification aims at inhibiting the unspecific adsorption of proteins. This is mostly achieved by covalently immobilizing polyethylene glycol (PEG) to the substrate4. As a consequence the highly swollen layer prevents macro molecules from interactions with the surface of the base material5. In addition, PEGylated surfaces may reduce colonization with bacteria and the formation of biofilms6. In the case of vascular prostheses the complete endothelialization of the device’s inner surface is highly desirable, thus mimicking the luminal endothelial layer which has the best known natural non-thrombogenic characteristics7. Unfortunately most synthetic polymers, including PTFE and PET, do not support cellular adhesion, making modification necessary. Celladhesive surfaces can be created by both, physisorption and chemical binding of an entire extracellular matrix (ECM) protein or by conjugation of adhesive peptides. The three-aminoacid motif Arg-Gly-Asp (RGD) is frequently used in this context8 and its covalent coupling to PTFE was recently demonstrated9. For blood-contacting devices, RGD-modification is hampered by the fact that thrombocytes also possess an integrin that recognizes this peptide sequence10 requiring pre-colonization with endothelial cells (ECs) to avoid the occlusion of the device by platelet adhesion and subsequent thrombosis. As an example of adhesion peptides that interact solely with a specific cell type, the Arg-Glu-Asp-Val (REDV) motif selectively binds to an integrin expressed by ECs11. REDV supports EC adhesion, spreading and growth over other cell types12. In this work we propose to combine the beneficial anti-fouling properties of a PEG-spacer molecule and the EC-specific REDV peptide to achieve endothelialization while preventing platelet and microbial adhesion. 3 ACS Paragon Plus Environment


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Results

PTFE modification and characterization

Highly hydrophobic PTFE can readily be converted into a hydrophilic material by means of sodium naphthalenide treatment9, as schematically represented in figure 1.

Figure 1: Reaction scheme of the PTFE modification.

Oxidation and the introduction of amino-functionalities rendered the samples slightly less hydrophilic (but still significantly more hydrophilic than untreated material). The immobilization of the REDV-peptide either directly (REDV) or via a PEG-spacer (PEGREDV) resulted in surfaces with similar excellent wetting behavior (Figure 2).


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Figure 2: Contact angle measurement of untreated PTFE and modified PTFE Compared to untreated PTFE (pristine PTFE), sodium naphthalenide treatment (PTFE NaNaph) induced a minimum hydrophobicity of PTFE, but oxidation (PTFE oxidized) or amination (PTFE-NH2) also significantly diminished the hydrophobicity of PTFE. The coupling of REDV or PEG-REDV (PTFE-REDV and PTFE-PEG-REDV respectively) resulted in similar hydrophobicity as oxidation and amination. The IR-spectra of pristine PTFE (blank) exhibits only two prominent signals at 1200 cm−1 and 1140 cm−1 that correspond to C-F bonds (sym. stretching) specific for PTFE but no additional signals at higher wave numbers. Upon treatment with sodium naphthalenide on PTFE additional signals appear indicating the presence of C=C double bonds (1300-1500 cm−1) and the presence of OH-moieties (1600 cm−1 and broad band 3400 cm−1 respectively). The ether signal (~ 1000 cm-1) expected in the presence of PEG is buried in the peak corresponding to the fluoromethylene groups. Bands indicating N-H bonds (amine, amide) fuse with the extremely broad O-H peak between 3400 and 3500 cm-1 due to hydrogen bonding (Figure 3).



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Figure 3: ATR-FTIR-Spectra. A. Survey of the full ATR-FTIR spectrum of blank PTFE (light blue), oxidized PTFE (orange), sodium naphthalenide treated PTFE (grey), REDV coupled PTFE (yellow) and PEG-REDV coupled PTFE (dark blue). In all spectra two prominent signals at 1140 cm−1 and 1200 cm−1 are present and correspond to C-F bonds of PTFE material. B. Detail of the different spectra in higher resolution reveal additional signals on modified PTFE in contrast to the blank material. Sodium naphthalenide treated and oxidized PTFE display new signals (1300-1500 cm−1) indicating the presence of C=C double bonds and a large signal (broad band between 3400 and 3500 cm-1) indicative of OH-moieties. When PEGREDV and REDV are present on the surface of the material ether signal is expected at ~ 1000 cm-1 but is buried under the strong CF2 signals of the PTFE. Additionally, N-H signal is expected as well but is overlapping with the broad O-H signal.

The sulfo-SDTB colorimetric assay yielded values that correspond to 2.7 ± 0.073 amino groups/nm2 after HDMI treatment and hydrolysis (analysis were performed in triplicate using the pristine material as baseline).

EC, platelet and bacterial adhesion The influence of REDV-modification on cellular colonization is presented in Figure 4.


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Figure 4: Endothelial-cell adhesion and proliferation on modified PTFE. Column one (A1-3): untreated PTFE (PTFE). Column two (B1-3): PTFE modified with PEGspacer and REDV (PTFE-PEG-REDV). Column three (C1-3): REDV directly immobilized on PTFE (PTFE-REDV). First row (A1, B1, C1): proliferation after 24 h. Second row (A2, B2, C2): proliferation after one week. Third row (A3, B3, C3): proliferation after two weeks. Quantification of the surface coverage is shown as mean ± standard deviation (D). Pictures were taken at 100X magnification.

Pristine PTFE does not induce cellular adhesion or growth even after prolonged incubation. However grafting of the REDV-peptide resulted in pronounced colonization (7 ± 2 %, one day; 14 ± 3 % one week; 18 ± 4 %, two weeks coverage for PTFE-PEG-REDV and 8 ± 2 %, one day; 11 ± 2 %, one week; 21 ± 4 %, two weeks coverage for PTFE-REDV respectively). In contrast, untreated polymer was prone to pronounced unspecific thrombocyte attachment after only 45 minutes as shown in Figure 5 (7 ± 1.5 % coverage). The number of adhered platelets is drastically reduced by REDV-immobilization (0.15 ± 0.02 % coverage) and PEGmediated REDV modification (0.2 ± 0.05 % coverage).



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Figure 5: Thrombocyte adhesion on modified and pristine PTFE. Significant thrombocyte adhesion can be observed on unmodified material (left), while PTFE modification with PEG-spacer and REDV (middle) or REDV alone (right) almost totally abolished thrombocyte adhesion. Quantification of the surface coverage is shown as mean ± standard deviation. Pictures were taken at 100X magnification.

The biological effect of the surface modification was additionally investigated by challenging the samples with bacterial suspensions (Figure 6). Whereas bare polymer was readily colonized (4.2 ± 0.7 % coverage), the presence of REDV-peptide considerably reduced bacterial adhesion (1.8 ± 0.5 % coverage). In addition the usage of PEG as a spacer for REDV conjugation almost completely obviated microbial attachment (0.1 ± 0.1 % coverage).

Figure 6: Adhesion of Staphylococcus aureus on pristine and modified PTFE surfaces. Staphylococus aureus demonstrate a strong affinity for untreated PTFE (left). The coupling of REDV reduced significantly this adhesion but did not abrogate it entirely (right). On the other hand, use of PEG spacer in combination with REDV almost entirely abolished the attachment of the bacteria (middle). Quantification of the surface coverage is shown as mean ± standard deviation. Pictures were taken at 200X magnification.


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Discussion Combined non-fouling and cell-adhesive properties were achieved by grafting the REDVpeptide onto PTFE substrate using PEG as an intermediate spacer molecule. In this wetchemical approach the activation of the substrate and subsequent conjugation was accomplished using a step-wise procedure. A similar concept was used previously by adding the RGD-sequence to a substrate involving dextran as the fouling reducing component13. In another example14 the utilization of PEG-mediated RGD-modification was demonstrated in order to achieve a surface that was readily recognized by cells but not adhesive towards bacteria and proteins. In order to add cell-specificity to peptide-mediated adhesiveness we chose one of the known sequences that interact exclusively with one specific cell-type. The present coupling strategy was based on the utilization of a bifunctional PEG-derivative and the utilization of the EC-specific adhesive peptide REDV. By using homobifunctional PEGs, possibly both ends may react with the surface leaving less reactive functionalities for the peptide coupling. Because of the extremely high excess of PEG (3.3 x 10-5 mol PEG or 6.6 x 10-5 mol NHS groups respectively) compared to the available primary amino groups (1.11 x 10-9 mol), ratio of approximately 1 : 3 x 104, it is most likely not the preferred reaction due to probability considerations. Anyway, if such would occur, “short circuit” immobilized PEG would simply contribute to the antifouling effect. Homobifunctional PEGs were succesfully employed in a similar fashion in a previous report15. Amino-functionalized PTFE prepared as recently reported9 served as a platform for subsequent conjugation chemistry. Activation as well as grafting was monitored by static contact angle measurement. Hydrophobic PTFE was thus converted into hydrophilic surfaces in accordance with the highly polar molecules grafted. Preferential EC-adhesion over other cell types has previously been described by Ceylan et al16. In this study, grafting of REDV resulted in selective promotion of EC adhesion and growth over smooth muscle cells and fibroblasts16. It is generally agreed that one of the most desirable features for vascular devices is low thrombogenicity, ideally achieved via endothelialization17. Our results suggest that the very presence of covalently coupled peptide renders the polymer an excellent substrate for endothelial cell adhesion and proliferation. The results obtained point to the fact that the usage of PEG as a spacer did not influence EC interaction with the material. Abberant from the concept of PEG-mediated antifouling we found that REDV coupling alone reduced platelet adhesion drastically but that this response was less pronounced with bacteria. A plausible explanation of the former finding might 9 ACS Paragon Plus Environment


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simply be the increased hydrophilicity of peptide decorated substrate that impedes unspecific adhesion. The distinguished property of PEGylated surfaces to prevent fouling by proteins and cells is well-established18. This was confirmed in our experiments to such a degree that unfavorable attachment of platelets and bacteria can be inhibited to a large extent while promoting cell-adhesion. In summary the present work demonstrated a feasible strategy for combining two desirable surface properties, i.e. (specific) cell-adhesiveness and antifouling features.

Conclusion Endowing materials with specific cell-adhesive and concomitantly antifouling characteristics can be regarded as a useful tool for various tissue engineering applications, especially where the devices are in blood contact. Here we report a wet-chemistry based, stepwise, immobilization of the EC-specific peptide REDV to the medically well-established polymer PTFE. Inherent thrombo-adhesive properties were overcome by the usage of PEG-mediated conjugation. This concept bears great potential for related fields in biomedical research where specific interaction of cells is required.

Experimental procedure

Sample preparation PTFE was obtained from Cadillac Plastic (Sulzbach, Germany). Cys-Arg-Glu-Asp-Val (REDV) was synthesized by standard solid phase Fmoc-chemistry (Genecust, Luxemburg). PEG-(NHS)2 was obtained from Rapp-Polymere (Tübingen, Germany). Samples 12 mm in diameter punched from 0.5 mm PTFE material were used in the experiments. Surface activation was accomplished as detailed recently 9. In brief, PTFE specimen were treated with sodium naphthalenide in dry tetrahydrofuran and oxidized with acidic hydrogen peroxide. Conversion of hydroxy functionalities to amino-groups was carried out with hexamethylene diisocyanate and subsequent hydrolysis. Amine-bearing material was treated with PEG(NHS)2 (0.5 mg/ml in 50 mM phosphate buffer, pH 7.4) or ethyleneglycol diglycidyl ether (250 µL, 1.6 mmol in 5 mL carbonate buffer) respectively, for 1.5 h at room temperature. Homobifunctional PEG used was from Fluka (MW 3000 g/mol, Fluka 15961).


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After washing, the samples were incubated in REDV solution (0.5 mg/ml in 50 mM carbonate buffer, pH 9) for 3 h or overnight (Figure 1). Cleaned discs were sterilized in 50 % isopropanol for 30 min prior to use in cell culture experiments.

Characterization Contact angles were measured on a Krüss DSA 10-MK2 (Krüss Optronic, Hamburg, Germany). Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectra were recorded on a Nicolet Magna-IR 850 (Dreieich, Germany). Newly created surface amino groups were quantified with a colorimetric assay using the reagent sulfo-SDTB according to the manufacturer’s instructions. Sulfosuccinimidyl-4-O-(4,4-dimethoxytrityl) butyrate (SulfoSDTB) was purchased from VWR (Darmstadt, Germany). In brief, samples were incubated in 50 mM carbonate buffer, pH 8.5 containing 3 mg sulfo-SDTB in 50 ml for 45 min. After thorough washing with water, each well was incubated with 1 ml 35 % perchloric acid until colour development was complete (appr. 15 min). Surface-bound amino group density was calculated from the OD498 values using the Lambert-Beer Law with ε = 70000 l mol-1 cm-1 and a surface area of 360 mm2. Mesurements presented here were performed in triplicate and is presented as mean ± standard deviation.

Endothelial cell culture Human Umbilical Vein Endothelial Cells (HUVEC) were provided by PAN-Biotech (Aidenbach, Germany). Cell culture plastic was purchased from Greiner-Bio-One (Frickenhausen, Germany). ECs were grown in EC-growth medium containing appropriate supplements as recommended by the manufacturer. Cells harvested with accutase enzyme were seeded at 5 x 104 cells in 1 ml medium on the modified or blank control PTFE-samples placed in 24 well plates and incubated for 3 h at 37°C, 5% CO2 and 100 % humidity. Proliferation was monitored after 24 h, one week and two weeks by staining the cells with Calcein-AM. For staining, cell-seeded samples were rinsed twice in PBS and subsequently incubated with a solution of Calcein-AM (2 µg/mL in PBS) in the dark for 30 minutes at 37 °C. After additional washing with PBS, pictures were taken with a Keyance microscope using standard FITC filters. Surface coverage was quantified with the Image-J software (NIH) and expressed as percentage of total area.



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Every surface area coverage mesurement presented here was performed in triplicate and is presented as mean ± standard deviation.

Platelet adhesion assay Platelet rich plasma (PRP) was obtained from blood, donated by a healthy volunteer. Citrated blood was centrifuged for 15 min at 1500 rpm, the clear supernatant platelet rich plasma (PRP) removed and supplemented with 1 µg/ml Calcein-AM. PTFE samples were incubated in the dark for 45 min at 37 °C19. After rinsing with PBS, pictures were taken and surface coverage was quantified with the Image-J software (NIH) as described above. Every surface area coverage mesurement presented here was performed in triplicate and is presented as mean ± standard deviation.

Bacterial adhesion assay The fluorescent dye Syto9 was obtained from Invitrogen (Karlsruhe, Germany). LB-broth was provided by Sigma-Aldrich. Staphylococcus aureus (ATCC 2564) was kindly donated by Matthias Husmann (Institute of Medical Microbiology and Hygiene, Medical School Mainz). Bacteria were grown in LB-broth to an optical density OD600 of ca. 0.5, harvested by centrifugation and re-suspended in medium. Samples were incubated in the bacterial suspension for 3 h under rotation and after replenishing with fresh broth statically for an additional 3 h at 37 °C. Samples were stained with Syto9 (2 µg/ml in PBS, 30 minutes) and after rinsing with PBS, pictures were taken and surface coverage was quantified with the Image-J software (NIH) as described above. Every surface area coverage mesurement presented here was performed in triplicate and is presented as mean ± standard deviation.

Ackowledgements The authors would like to thank Walter Scholdei (Max Planck Institute for Polymer Research, Mainz, Germany) and Birgit Hohmann (Center for Musculosceletal Surgery, University School of Medicine, Mainz, Germany) for technical assistance.


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Figure 1: Reaction scheme of the PTFE modification. 51x27mm (300 x 300 DPI)

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Figure 3: ATR-FTIR-Spectra. A. Survey of the full ATR-FTIR spectrum of blank PTFE (light blue), oxidized PTFE (orange), sodium naphthalenide treated PTFE (grey), REDV coupled PTFE (yellow) and PEG-REDV coupled PTFE (dark blue). In all spectra two prominent signals at 1140 cm−1 and 1200 cm−1 are present and correspond to C-F bonds of PTFE material. B. Detail of the different spectra in higher resolution reveal additional signals on modified PTFE in contrast to the blank material. Sodium naphthalenide treated and oxidized PTFE display new signals (1300-1500 cm−1) indicating the presence of C=C double bonds and a large signal (broad band between 3400 and 3500 cm-1) indicative of OH-moieties. When PEG-REDV and REDV are present on the surface of the material ether signal is expected at ~ 1000 cm-1 but is buried under the strong CF2 signals of the PTFE. Additionally, N-H signal is expected as well but is overlapping with the broad O-H signal. 340x186mm (150 x 150 DPI)


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Figure 4: Endothelial-cell adhesion and proliferation on modified PTFE. Column one (A1-3): untreated PTFE (PTFE). Column two (B1-3): PTFE modified with PEG-spacer and REDV (PTFE-PEG-REDV). Column three (C1-3): REDV directly immobilized on PTFE (PTFE-REDV). First row (A1, B1, C1): proliferation after 24 h. Second row (A2, B2, C2): proliferation after one week. Third row (A3, B3, C3): proliferation after two weeks. Quantification of the surface coverage is shown as mean ± standard deviation (D). Pictures were taken at 100X magnification. 349x156mm (150 x 150 DPI)



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Figure 5: Thrombocyte adhesion on modified and pristine PTFE. Significant thrombocyte adhesion can be observed on unmodified material (left), while PTFE modification with PEG-spacer and REDV (middle) or REDV alone (right) almost totally abolished thrombocyte adhesion. Quantification of the surface coverage is shown as mean ± standard deviation. Pictures were taken at 100X magnification. 364x82mm (150 x 150 DPI)


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Figure 6: Adhesion of Staphylococcus aureus on pristine and modified PTFE surfaces. Staphylococus aureus demonstrate a strong affinity for untreated PTFE (left). The coupling of REDV reduced significantly this adhesion but did not abrogate it entirely (right). On the other hand, use of PEG spacer in combination with REDV almost entirely abolished the attachment of the bacteria (middle). Quantification of the surface coverage is shown as mean ± standard deviation. Pictures were taken at 200X magnification.

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Promoting Cell Adhesion and Antifouling Properties via a Wet

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