Glycidyl acrylate plasma glow discharged polymers

Glycidylacrylateplasmaglowdischarged polymers FabioTanfani*, AzizA. Durrti, MasayosbiKojima+and DennisChapman Oeparfment of Protein and Molecular Biology, Royal Free Hospital School of Medicine, ZPF, UK [Received 2 1 November 1989; accepted 2 1 November 1989)

Rowland Hill Street, London NW.3

A homogeneous glycidyl acryiate polymer (GAP) has been grafted on to polytetrafluoroethylene (PTFE) and polyethylene (PE) using a modified plasma glow discharge technique with glycidyl acrylate. The polymeric layer appears to be extremely stable to acidic media and to common organic solvents. The modified surface can be derivatized via epoxy groups with hydroxy and amino compounds including sugars and amino sugars. These derivatized surfaces have been characterized by Fourier transform infrared (FTIR) spectroscopy and contact angle measurements. The wide variety of compounds which can be attached provides flexibility in the design of surfaces for the study of a range of biologicaf interactions. Keywords. Polymer grafting. surface chara~terjzation~ cell-polymer

The chemical modification of polymeric surfaces by the plasma glow discharge method is receiving increasing attention Textiles such as synthetic fibres and cotton have been treated by this process to obtain more wettable products’. A polymerized coating has been applied to polypropylene, polyester and Teflon@ surfaces. The modified surfaces (a) became hydrophilic for bonding with adhesives, (b) were coloured by basic dyes, and (c) retained metal ions more easily. There is also a need to produce modified surfaces for the chemical attachment of various bioactive molecules, e.g. giycoproteins and glycolipids which, because of their carbohydrate moities, act as receptors for various ligands*. 3, constitute the blood group of antigens4, and are involved in intercellular adhesion processes. Previous studies have only been possible with a few substrates which contain the appropriate groups for chemical modification, i.e. acrylamide gels5-7. Functionalized surfaces may also be used in the manufacture of medical devices. Many polymers used for their manufacture do not possess appropriate chemical groups on their surfaces. One approach to obtain biocompatible surfaces has been to modify polymeric surfaces byattachmentof certain antithrombogenic moleculess“’ via covalent bonds or ionic interactions. In this paper we describe a method to derivatire polymer surfaces after plasma glow discharge treatment using glycidyl acrylate. Two polymers, PTFE and PE, are used

interacrions

as examples. The aim of the work is to be able to link molecules such as amines and hydroxyl group reactive compounds covalently to the existing polymer surfaces with the potential for studying biological interactions in the presence and absence of blood, e.g. protein adsorptions and coagulation characteristics.

EXPERIMENTAL The polymers used in these experiments were thin sheets (0.1-0.2 mm) obtained from commercial sources. The polymer sheets were cut into 1.2 X 4.5 cm pieces and washed with a liquid detergent and copious volumes of tap water followed by distilled water and finally with absolute ethanol. The polymer pieces were then dried in a vacuum desiccator over silica gel at l-2 mm. Glycidyl acrylate, glucose, galactose, hydrazine and 1.2-diaminoethane were of the purest available grades, purchased from Aldrich, UK. Streptomycin sulphate was supplied by Sigma, UK. Galactosyl hydrazine was prepared according to the reported procedure12.

Plasma acrylate

Q 1990

Butterworth-Heinemann

Ltd. 0142-96

of polymers

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Correspondence to Professor D. Chapman. Present addresses: ‘Institute di Biochimica, Facolta di Medicina e Chirurgla. Unwersrta dt Ancona. Via Monte d’Ago-60100 Ancona, Italy: +2-3-10, Surugadai. Kanda. Chlyoda-Ku, Institute for Medical and Dental Engmeering, Tokyo MedIcal and Dental Unwersity, Tokyo 10 1, Japan.

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pumps were switched on to evacuate the reaction chamber to 3 X ‘i 0-l Ton. This was achieved in 5-10 min during which time the air was replaced by the g~ycidyl acryfate vapours. Plasma formation was commenced at the maximum power. The polymers were treated with the plasma for 2 min, plasma formation discontinued and the surfaces allowed to react with the glycidyl acrylate vapours for up to 10 min. This procedure was repeated twice more and the valve connecting the pump to the reaction chamber was closed followed by the pump. The plastic pieces were reacted with glycidyl acrylate vapours for 6- 18 h, depending upon the thickness of GAP required, after which they were placed under vacuum for 24 h to evaporate any unreacted glycidyl acrylate.

~ariuatizat~o~ of glycidyf acrytate plasma glow dis~har~ad ~oi~mars Reaction ofdeposited GAP WI polymers with dilute suiph~rjc acid (formation of diol~‘~). The GAP-modified polymers were stirred with 3% sulphuric acid for 48 h. Reaction of deposjted GAP on polymers with hydrogen chloride gas (formation of c~iorohydr~ns14). The GAPmodified polymers were treated with anhydrous hydrogen chloride for 30 min. The hydrogen chloride gas was then replaced by dry nitrogen to free the reaction vessel from hydrogen chloride. Reaction of deposited GAP on polymers with amines (formation of amides/amines). The amines used were 1,2diaminoethane, streptomycin, and gafactosyl hydrazine. In general, the GAP-treated surfaces were immersed in the aqueous solutions of amines ( 1 g/ml; 0.5 ml/20 ml for t,2diaminoethane only) for 3 d at room temperature except for streptomycin which was carried out at pli 10.5 and 35°C.

surfaces. The contact angles of the surfaces were measured to show the hydro~hjl~~~~ydrophobjc nature of the surfaces. Scanning electron m~crascopy. The electron micrographs were taken with a Philips scanning electron microscope, Model 501, Specimen surfaces were treated with gold using an Emscope SC.500 sputter coater. Figure la shows untreated PTFE (original magnification x 9750). Figure fb is that of the GAP deposited on PTFE at the same original magnification. The difference in the physical appearance of the two polymers is apparent. The deposited GAP shows a microgranular structure while the commerciat PTFE sheet has a scratchy surface having no resemblance to that of the modified PTFE. FTlR spectroscopy. A Perkin Elmer spectrometer, Model 1750, equipped with a computer, Model 7300 and Perkin Elmer printer, Model PP-1 was used in the present analyses. Multiple internal reflectance (MIR) technique using a KRS-5 crystal with TGS detector and 4cm-’ resolution was employed, The spectrum of the crystal was recorded each time as background. For each sample, 200 scans were taken using two pieces of the polymer, one on each side of the crystal. 1. Glycidyl acrylate plasma glow discharged surfaces. The FTlR spectra in Figures 2e and 4 are those of PE and PTFE, respectively, before modification. In Figures 26 and 5a the absorptions at 850 and 910 cm-” are those from the epoxy groups in the GAP bound on PE and PTFE respectively. A strong peak at 1738 cm -’ in both spectra shows the presence of ester bonds in the acrylate residues.

Reaction of de~osjted GAP on poJymers with sodjum borohydride (formation of akohols). The GAP-treated polymers were placed in a flask containing ethanol (75 ml) and stirred. Sodium borohydride was added in small portions till 2 g was consumed over 15-20 min. The stirring was continued for 24 h. Reaction of the aminated GAP with glucose (formation of saccharide resjdues). The GAP-modified polymer treated with 1,2-djami~oethane as above was stirred with aqueous glucose (7 g/20 ml) at pH 6-7 for 3 d in the presence of sodium cyanoborohydride (ZOO mg). basting and drying procedure for the surfaces treated as above. The polymers were washed under cold running tap water followed by distilled water repeatedly and finally rinsed with absolute alcohol. The drying was performed by placing the polymers in an oven, heated to 6O”C, for 1 O15 min and than in a desiccator over silica gel for 24 h at 0.1 mm.

Characterization

nf treated surfaces

We set out to demonstrate that GAP is indeed present on the modified polymers by using scanning electron microscopy. GAP-treated PTFE was used as an example. The treated and untreated polymers were also examined by FTIR spectroscopy to determine the chemical structures present on the

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that under the present reaction conditions ester bonds remained stable. 3. Hydrogen chloride gas-treated GAP on PE. The FTIR spectrum (Figure 2~) show that the majority of the epoxide bands have disappeared while the band at 3400 cm-’ (-OH) has intensified. A new band at 61 O-cm-’ (C-Cl) occurs. 4. Amine-treated GAP on PTFE. (a) Reaction with 1,2-diaminoethane. Comparing the FTIR spectrum in Figure 56 of the amine-treated surface, with the spectrum of GAP-deposited PTFE (Figure 5a), it is evident that as a result of the reaction a split band at 34003200 cm-’ has appeared. Primary amino, hydroxyls and NH bands due to the secondary amides occur in this region. However, it is also clear that two new peaks at 1635 cm-’ (amide I) and 1560 cm-’ (amide II) have also been formed. These structural features of the 1,2-diaminoethane-treated GAP suggest that amide formation has predominated. This reaction is similar to that which occurs with ammonia with the electron-deficient carbon atoms of acrylate esters to form amides. (A cleavage of the epoxide bonds by 1,2-diaminoethane is also a possibility.) The primary and secondary amino groups bands (1650-l 550 cm-‘; NH deformation and 3398-3310 cm-‘; NH stretching of primary and secondary amines) may have been overlapped by the more intense amide vibrations in these regions. The presence of the free-NH2 groups has been demonstrated by attaching glucose molecules to the surface. (b) Reaction with galactosyl hydrazine and streptomycin. The spectra in Figures 6a and 66 were obtained after GAPmodified PTFE surfaces were reacted with galactosyl hydrazine and streptomycin respectively. Both of these molecules have sugar and amino moities. There are similarities in the spectra. In both the cases the epoxy bonds disappear after the reaction and new peaks at 3400-3200,1635-and 1560 cm-’ appear. Weak peaks at 1045 cm-’ (C-O of COH) also emerge, indicative of the presence of sugar residues. The band at 1635 cm-’ in Figure 6a is stronger than the band at 1560 cm-’ (amide II) showing that there is more contribution in this instance from free amino components than is seen in the spectrum shown in Figure 5. The 1600- 1700 cm-’ region of the spectrum in Figure 6b is

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2. Dilute sulphuric acid-treated GAP on PE. In Figure 3, the FTIR spectrum of dilute sulphuric acid-treated GAP on PE shows the appearance of a strong absorption at 3400 cm-’ (H-bonded-OH groups) at the expense of the epoxy absorptions (850 and 910 cm-‘). This indicates that successful hydroxylation of the PE surface has taken place. No decrease in the intensity of the ester band (1738 cm-‘) is indicative

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Plasma glow discharged polymers: E Tanfani et al.

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Contact angle measurements. The contact angle measurements before and after the treatments show marked changes in the surface wettabifity characteristics. The contact angle of untreated PTFE (124”) is reduced to 70” after glycidyi acrylate plasma glow discharge treatment.This value is further reduced to only 28” when reacted with 1,2diaminoethane. Similar changes were observed when gaiactosyi hydrazine (50“) and streptomycin (20”) treated surfaces were examined. Owing to the granular nature of GAPdeposits, the real values may, however, differ from these values. Polyethylene was also found to be more hydrophobic (82”) than the GAP-treated surface (68”). Dilute acid treatment of this surface reduced this value to 50”.

DISCUSSIDN

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FTtR spectra of GAP deposited on PTFE after reaction with f8j hydrazine and fb] streptomycin.

more complex; an indication of the presence of free amino groups. It therefore follows that stronger bases, i.e. 1,2diaminoethane, form the majority of the amide groups when reacted with GAP, while weaker basic substances form largely amino alcohol components. 5. Sodium borohydride-treated GAP on PE. A sharp decrease in the ester band (1738 cm-‘) and the appearance of a new peak at about 3400 cm-’ (H-bonded-OH) (Figure 7) showed that a successful reduction of ester bonds in the GAP has taken place. 6. Saccharide-treated GAP on PTFE. The spectrum shown in Figure 5c was obtained when the 1,2_diaminoethanetreated surface was reacted with glucose. Similarities in the spectrum with those in Figures 6a and 66 (galactosyl hydrazine and streptomycin-treated surfaces) are expected. The bands at 3400-3200, 1635 and 1045 cm-’ are indeed common. Only a small increase in absorption in the 3400-3200 cm-’ region indicates that a very small amount of glucose has been attached to the surface, supporting the earlier conclusion that the reaction of 1,2-diaminoethane with GAP forms the majority of the amide groups.

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1990, Vol 7 I October

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During plasma treatment of organic polymers, free radicals are produced on the polymer surface15. When these plasmatreated polymers are immediately immersed in an acryiate monomer, the free radicals which are still present act as sites for ~l~e~zation and produce an acrylate polymer, ap~ren~y covaiently attached to the underlying surface” 15. Plasma polymerization of acrylate monomers, including giycidyl methacrylate and ally1 giycidyl ether, has been previously performed by using the conventional technique where the substrates are treated with the plasma alone and a thin film of the deposit is obtained~6-‘E. The chemical composition of these films is mixed and several organic functional groups are present’g-23. The basic principle used in the present method to obtain a moderatelythick film of GAP is similar to this but we have introduced a new modification to the process. When plasma generation is stopped, the radicals present on the polymer surface induce polymerization of the vaporized glycidyi acryiate monomer to produce GAP. Unlike the procedure where the activated surfaces are immersed in the monomer, in our process the activated surface is brought instantly into contact with the glycidyl acryiate vapours. This minimizes the loss of preformed free radicals. Other advantages in the present procedure are the formation of thick layers of GAP free from other functional groups, e.g. oxidation products”. This procedure allows the activated surface sufficient time to react with the glycidyi acryiate vapours, thereby eliminating such side reactions. The presence of GAP on PTFE is illustrated in the electron micrographs (Figure 7).This shows a complete and homogeneous deposit of the acrylate polymer. The evidence for the presence of GAP is also provided by FTI R spectroscopy. Absorption bands due to the epoxy group and acrylate ester bonds are clearly present (Figures 26 and 5a). Furthermore the epoxy groups have been cleaved with a variety of reagents. The resultant surfaces may be reacted with molecules of biological importance to obtain derivatized surfaces for different kinds of biological studies. A probable mechanism for the deposition of GAP on polymers is given below: Provided that enough glycidyi acryiate is present, a fairly thick layer of GAP may be obtained. The deposited layer is stable towards common organic solvents. Further derivatization procedures of GAPdo not decrease the intensity of the ester bonds, showing the stability of the deposit under different reaction conditions. We have also shown that sodium borohydride reduction of GAP diminished the intensity of the ester bond in the FTIR spectrum but the detectability of GAP posed no problems. These observations

Plasma glow discharged polymers: E Tanfani et al.

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REFERENCES

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coupled with the given probable mechanism of GAP formation, which involves the polarization of carbon atoms under glow discharge conditionsz4, strongly suggest that the GAP deposit on polymers is covalent in nature. Its detection by FTIR also indicates that it is several layers deep. Because of the high energetic nature of the reactive species formed under plasma conditionsz4, most polymers, regardless of their chemical reactivity, can be treated using this su~ace-modification method.

CONCLUSION Polymeric surfaces have been modified by means of a layer of GAP using plasma glow discharge conditions with a modified technique. This layer is stable towards common organic solvents and is probably covalently attached to the underlying surface. The structure of the GAP layer has been demonstrated by FTIR spectroscopy. The epoxide groups in the deposited layer have been used for reactions with several molecules to produce novel surfaces having useful biological properties. Uneven activation of the polymer surfaces causes more GAP deposition on some activated sites, resulting in the granular structure of the deposit. A smoother surface may be obtained by moderating the GAP thickness.

12

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ACKNOWLEDGEMENTS F. T. gratefully acknowledges financial support from the Wellcome Trust. D. C. and A. A. D. are grateful to SERC, Medical Engineering Committee, for their financial support.

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