WO1989011500A1

WO1989011500A1 - Hydrophilic non-swelling multilayer polymeric materials and process for their manufacture

Info

Publication number: WO1989011500A1
Application number: PCT/AU1989/000220
Authority: WO
Inventors: Hans Jorg Griesser; John Gerard Steele; Graham Johnson; Jonathon Howard Hodgkin
Original assignee: Commonwealth Scientific And Industrial Research Or
Priority date: 1988-05-17
Filing date: 1989-05-17
Publication date: 1989-11-30

Abstract

A multilayer polymeric material including a first layer formed from a bulk polymeric material; and at least a second layer deposited by plasma polymerisation on at least a surface of the first polymeric layer to increase the hydrophilic nature of said surface.

Description

HYDROPHILIC NON-SWELLING MULTILAYER POLYMERIC MATERIALS AND PROCESS FOR THEIR MANUFACTURE The present invention relates to hydrophillic non-swelling multilayer polymeric materials and a process for producing such materials.

In the prior art, a number of commercially produced known polymeric materials have been the subject of optimisation of their bulk mechanical and chemical properties. However, for interactions with the environment the same polymeric materials often do not possess good properties. In particular for applications where the polymer surface is in contact with water or other polar liquids and good wetting is desired, the surface of many of the known polymers are too hydrophobic. This is particularly important in biological applications. On the other hand, the polymers that are hydrophilic as a result of their polar constituents, such as poly-hydroxyethylmethacrylate and gelatin, etc., are penetrated by the contacting polar liquid, and the resultant swelling brings about changes in the mechanical properties. When the latter polymers are used in applications where permanent contact with polar liquid exists, such as in many biological and biomedical applications, the mechanical strength is not satisfactory.

For many applications for polymeric materials it is important to be able to optimize both the bulk properties and the surface properties with some degree of independence. In particular the contact with polar liquids must be good while avoiding swelling of the polymer and hydrolysis of groups in the polymer backbone. This invention provides a means for achieving this. Surface properties that govern interactions with the environment are determined by the chemical constituents at the surface and in the sub-surface region of the polymeric material and are due to forces arising from the chemical constituents located within only a few molecular layers of the surface of the polymeric material. To achieve independent control of surface chemical composition a number of surface treatment techniques for polymeric materials are known in the art. Surface treatment techniques for polymeric materials known in the art are Corona Discharge, Flame Treatment, Acid Etching, and a number of other methods intended to perform chemical modification of the surface. Disadvantages of these techniques comprise the use of or production of hazardous chemicals, the often excessive depth of treatment, non-uniformity of treatment at a microscopic level, and often severe etching and pitting that leads to changes in surface topography. The depth of treatment is important because with thin, plastic webs the bulk properties soon become affected to an excessive extent. A glow discharge in air or oxygen has been used to render polymeric surfaces more hydrophilic. The treatment leads to formation of polar, oxygen containing functionalities on the surface. The main disadvantage of this approach is that the treatment is not permanent due to reorientation of the polymer chains with time as a result of unavoidable, thermally driven chain segmental motions. Wettability by water decreases as the hydrophilic surface groups produced by treatment become buried inside the polymer, as described by H. Yasuda et al. in J. Polym. Sci.: Polym. Phys. Ed., 19., 1285 (1981). A glow discharge in an atmosphere comprising water vapour as an essential component, and optionally organic compounds, has also been used (Japan 59-15569). Improved hydrophilicity of the treated surface was obtained: it was, however, not demonstrated that the treated surface was immune to slow reorientation and burial of the created groups.

It is accordingly an object of the present invention to overcome, or at least alleviate, one or more of the difficulties related to the prior art. Accordingly in a first aspect of the present invention, there is provided a multilayer polymeric material including a first layer formed from a bulk polymeric material; and at least a second layer deposited by plasma polymerisation on at least a surface of the first polymeric layer to increase the hydrophilic nature of said surface. The multilayer polymeric material may take the form of a biomedical implant. It will be understood that by provision of the multilayer structure the invention achieves improvement of bulk and surface properties independently.

The bulk polymeric material which forms the first layer may be selected to provide suitable mechanical properties depending on the particular application to which the material is to be put while the second layer may be selected for its surface chemical properties.

Any one of the known polymeric materials may be used for the bulk polymeric material because the process of this invention can proceed without regard for the composition of the bulk polymeric material and in all cases substantially improved wettability of the treated surfaces is obtained. Particularly preferred polymers include perfluorinated polymers including polytetrafluoroethylene, polyethylene, polyesters, polyurethanes, copolymers thereof and mixtures thereof. The invention is particularly applicable to the normally unreactive perfluorinated polymeric materials such as polytetrafluoroethylene or fluorinated ethylene propylene polymers (FEP, Du Pont) .

The second surface layer may be formed from any suitable material amenable to plasma polymerisation techniques that will increase the hydrophilic nature of the multilayer composite. It is preferred that the second surface layer is a relatively thin layer. We have found that such a surface layer may be formed via plasma polymerisation. It has been found advantageously that the second surface layer bonds to the first polymeric layer with substantially no penetration thereinto. As explained above it has been found that significant penetration may affect the desirable bulk properties of the first polymeric layer.

Accordingly, the second surface layer may be formed from a plasma in a vapour composition including at least one organic monomeric material capable of polymerisation under plasma polymerisation conditions.

The organic monomeric material may include at least one compound selected from saturated alcohols, amines, and the like, derivatives thereof and precursors therefor. In a preferred aspect according to the present invention the vapour composition utilised to form the second surface layer may further include at least one inorganic gas.

Desirably the vapour composition does not include significant amounts of water vapour.

The at least one organic monomeric material may include saturated alcohols and saturated amines including lower saturated alcohols and amines. The lower saturated alcohols and amines may be selected from substituted or unsubstituted C 1,-Co₀ alcohols and amines. The compounds methanol, ethanol, n-propanol, n-butanol, methylamine, ethylamine, n-propylamine, and n-butylamine are preferred. The lower saturated alcohols and amines may include one or more substituents selected from halogens, amino groups and the like.

The inorganic gas may be of any suitable type. Oxygen, hydrogen, nitrogen (N₂) and the noble gases Helium, Nitrogen, Argon and Neon and the like may be used.

The vapour composition may include one or more additional components. Additional components may include additional monomeric material and/or other components selected to improve the properties of the layer formed.

In a preferred aspect of the present invention, the second surface layer of the multilayer polymeric material may be contacted with a serum component.

The serum component may include an adhesive protein for example an adhesive glycoprotein.

Whilst we do not wish to be restricted by theory, it is postulated that the serum component may provide a serum adhesive protein e.g. a glycoprotein which becomes adsorbed onto the surface, and participate in HUAE cell attachment and spreading onto the surface. Preferred serum components include fibronectin (Fn), vitronectin (Vn) , (see Grinnell (1976, Exp. Cell Res., 97, 265-274; Underwood and Bennett, 1989, J. Cell Sci., in press) and thrombospondin or adhesive gragments thereof.

It is further postulated that the adhesive proteins are adsorbed by the hydrophilic surface layer to form a polymer-protein complex. Such complex assists in the attachment and subsequent growth of animal cells.

The multilayer polymeric material may take the form of a biomedical implant. Accordingly, in a further aspect of the present invention there is provided a biomedical implant including a polymeric article formed from a bulk polymeric material; said polymeric article bearing a surface layer deposited on at least a surface thereof by plasma polymerisation to increase the hydrophilic nature of the surface.

Preferably the bulk polymeric material is selected from perfluorinated polymers, polyethylene, polyesters, polyurethanes, copolymers thereof and mixtures thereof. The polymeric article may be formed into a desired final shape, dependent upon its application prior to plasma polymerisation treatment. As stated above the first polymeric article may take the form of a biomedical implant.

If desired depositing further layers of different composition by plasma polymerisation in vapours of composition different to that used to produce the surface layer.

More preferably the surface layer is formed from a vapour composition including at least one organic monomeric material capable of polymerisation under plasma polymeristaion conditions.

Preferably the surface layer of the biomedical implant is contacted with a serum component. More preferably the serum component is selected from fibronectin, vitronectin, thrombospondin or adhesive fragments thereof.

In a further aspect of the present invention there is provided a process for the preparation of a multilayer polymeric material which process includes providing a first polymeric material formed into a desired final shape from a bulk polymer; depositing from the vapour phase a second layer via a plasma polymerisation reaction in a vapour composition containing at least one monomeric material to form a surface layer on at least one surface of the first polymeric material; if desired depositing further layers of different_^, composition by plasma polymerisation in vapours of composition different to that used to produce the second layer.

The vapour composition desirably includes no more than approximately 20% v/v water vapour more preferably no more than approximately 5% v/v water vapour. The effect of water vapour is to reduce or prevent cross linking of the monomer which appears in the atmosphere, with the result that the vapour created may not be immune to slow reorientation and burial of the created groups and may not be resistant to swelling.

Deposition of the surface layer and optionally subsequent polymeric layers by plasma polymerisation presents the advantage of uniformity on a microscopic scale. A plasma polymerisation (also known as glow discharge) may be produced by electrical discharge in a gas atmosphere at reduced pressure ("vacuum"). It creates a stable, partially ionized gas that may be utilized as a source of reactive species. A wide variety of chemical compounds can be utilized for effecting reactions on the surface of the first polymer layer because the gas plasma environment activates even chemical compounds that are unreactive under normal conditions. Surface topography does not change measurably unless exposure to the plasma is performed for extended periods of time usually much exceeding the time required for achieving the desired formation of the second or subsequent layer. There occurs, therefore, much less alteration of the properties of the bulk polymer than with alternative technologies.

In a preferred aspect of the process of the present invention the process may further include providing a serum component including an adhesive protein; and contacting the second surface layer with the serum component.

Preferably the serum component is selected from fibronectin, vitronectin, thrombospondin or adhesive fragments thereof.

More preferably the serum component is provided in a cell culture medium. The cell culture medium may for example consist of an equal mixture of McCoy 5A (modified) (CSL laboratories, Melbourne) and BM86-Wissler (Boehringer- Mannheim) media supplemented with 30% v/v foetal bovine serum (Cytosystems, Sydney), 40 ng/ml fibroblast growth factor (biomedical Technologies Inc.) 60 ug/ml endothellal cell growth supplement (Collaborative Research) , 20 ug/ml insulin (G1BCO), 60 ug/ml pencillin (Glaxo) and 100 ug/ml streptomycin (Glaxo) .

The biomedical implant according to the present invention may be utilized for the attachment and growth of animal cells in vitro. Accordingly, in a still further aspect of the present invention there is provided a source of animal cells; and a biomedical implant including a first polymeric article formed from a bulk polymer material; said first polymeric article bearing a surface layer deposited on at least a surface thereof by plasma polymerisation to increase the hydrophilic nature of the surface. Preferably the process further includes providing a cell culture medium including a source of animal cells; and a serum component including an adhesive protein; and contacting the biomedical implant with the cell culture medium.

The biomedical implants may further be used for the attachment and growth of animal cells in vitro. Accordingly in a further aspect of the present invention there is provided a biomedical implant including a first polymeric article formed from a bulk polymer material; said first polymeric article bearing a surface layer deposited on at least a surface thereof by plasma polymerisation to increase the hydrophilic nature of the surface;and implanting the biomedical implant into the animal. Preferably the second surface layer of the biomedical implant is contacted with a serum component including an adhesive protein. More preferably the serum component is selected from fibronectin, vitronectin, thrombospondin or adhesive fragments thereof.

The present invention will now be more fully described with reference to the accompanying examples and drawings. It should be understood, however, the following description is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

Figure 1 is a schematic view of a system showing an embodiment of the process of this invention.

Figures la, lb, lc and Id show different possibilities of placement of the bulk polymeric material in the electric discharge zone, and the multilayer structures thus obtained. Figure 2 is a schematic view of a system showing another embodiment of the process of this invention.

Figure 3 is a schematic cross-section of a multilayer polymeric material produced according to this invention. Figure 4 is a schematic cross-section of another multilayer polymeric material produced according to this invention.

Figure 5 is a schematic cross-section of yet another multilayer polymeric material produced according to this invention.

Figure 6 is a schematic cross-section of still another multilayer polymeric material produced according to this invention.

Figure 7 is a schematic cross-section showing an embodiment of the process of this invention using streams of at least two vapours.

Figure 8 is a schematic cross-section showing another embodiment of the process of this invention using streams of at least two vapours. Figure 9 illustrates the difference in morphology of human umbilical artery endothelial cells cultured for e days on tissue culture polystyrene (panel a) or fibronectin-coated tissue culture plastic (panel b), on Example 9 (panel c) or Example 9 precoated with Fibronectin (panel d) , Example 12 (panel 3) or Example 12 precoated with fibronectin (panel f), Example 13 (panel g) or Example 13 precoated with fibronectin (panel h) , Example 14 precoated with fibronectin (panel 1) and Example 15 precoated with fibronectin (panel j).

To overcome the disadvantages associated with the prior art and to produce a stable non-swelling polymeric material with improved wettability by polar solvents, this invention utilizes the technique of plasma polymerization for production of multilayer polymeric material. The multilayer polymeric material of this invention consists of a bulk polymeric material 1 and a thin organic-polymeric layer 2. The bulk pσlymeric material 1 may be of any desired shape and size suitable for the intended application of the finished article. The bulk polymeric material 1 is fashioned into the desired shape of the finished article prior to application of the process of this invention. It is desirable to arrange the geometry of the electric discharge zone such that all desired surfaces of the bulk polymeric material 1 are coated effectively and uniformly with the thin layer 2. By suitable positioning of the bulk polymeric material 1 within or near the electric discharge zone, it is possible to select which surfaces are coated with the thin layer 2 and thereby select an appropriate structure for the multilayer polymeric material of this invention. A few possibilities are shown in Figures la to Id, but the invention is not restricted to those arrangements shown. Neither is the invention restricted to the planar electrode geometry and the planar samples shown for simplicity in Figure 1.

Any one of the known polymeric materials such as polytetrafluoroethylene, polyethylene, polyesters, polyurethanes, etc., may be used for the bulk polymeric material 1 because the process of this invention can proceed without regard for the composition of the bulk polymeric material 1 and in all cases substantially improved wettability of the treated surfaces is obtained. The invention is particularly applicable to the perfluorinated polymeric materials such as polytetrafluoroethylene.

The thin layer 2 of this invention is formed by the plasma polymerization method, a method for deposition of thin layers onto surfaces exposed to the constituents of the vapour phase activated by the electric discharge. In plasma polymerization the polymerization is performed by introducing vapour comprising one or more organic compounds, henceforth called the monomer vapour, into an electric discharge region. The monomer vapour is directly electrically dissociated and the partially ionized vapour brings about the formation of a polymeric layer 2 on the exposed surfaces of the bulk polymeric material 1. Plasma polymer layers are known in the art to present a uniform, defect free surface to the environment as a result of the molecular nature of the layer formation. The plasma polymerization method is thus a uniquely suitable coating method for formation of a thin "skin" layer that enables the multilayer polymeric material to retain the mechanical properties of the bulk polymeric material while uniformly presenting the chemical groups of the plasma polymer film to the environment. The inadequate, adhesion between the bulk 1 and the thin layer 2 that often is obtained with conventional coating methods based on solvent coating, is overcome in the plasma process because the bulk polymeric material 1 and the thin plasma polymer layer 2 are connected by covalent bonds across the interface. The high cohesion and extensively crosslinked structure of plasma polymer films prevent rotational reorientation of the structure of the plasma polymer and thereby prevent re-emergence of the original surface of the bulk polymeric material, which remains buried underneath the mechanically strong plasma polymer thin layer. The slow loss of wettability with time that is observed after plasma treatment in non-depositing oxidative atmospheres is thus avoided. Furthermore, it is also known in the art that plasma polymers are highly crosslinked in a three-dimensional random structure; such a polymer network is resistant to swelling by contacting liquids. Therefore the process presents a method of achieving properties in the thin layer 2 not matched by conventional solvent-based coating methods.

The vapour composition and the plasma conditions together determine the composition and properties of the thin layer 2.

The present invention also prescribes a novel composition for the monomer vapour suitable for the plasma process of this invention. One component of the vapour phase of this invention may include vapour of a saturated alcohol or amine compound. The vapour phase of this invention also may include a mixture of vapours of several saturated alcohol or amine compounds and optionally other organic compounds._ In another embodiment of the invention the monomer vapour is mixed with an inorganic gas. The vapour from a single alcohol or amine compound may be used. Alternatively, several vapour streams from different compounds may be mixed. Examples of useful saturated compounds include methanol, ethanol, propanol, methylamine, ethylamine, propylamine, etc., and generally any compound of the structure R-OH or R-NH₂ where R is a saturated alkyl group, R may optionally contain one or several halogen atoms, amine groups, etc.

Other organic compounds may optionally be used in the vapour phase of the invention, to give a mixed monomer vapour, provided that at least one of the compounds in the mixed vapour phase of the invention is a saturated alcohol or amine compound.

In another embodiment of the process of this invention, an inorganic gas is added to the organic monomer vapour phase. Examples of the inorganic gas useful for assisting the plasma process and controlling the resultant thin layer composition include oxygen, argon, hydrogen, etc. Inorganic vapours, in particular water vapour, are, however, not essential in this invention.

When vapour of more than one chemical compound is used in the process of the invention, the mixing of vapours can be done in several ways. The streams of two or more vapour and gas components may be united prior to reaching the electric discharge zone in an embodiment as shown in Figure 1. In other embodiments of the invention at least one of the streams passes through an electric discharge zone and becomes excited by partial ionization, while at least one other stream does not pass through a discharge zone and is admixed in an unexcited state to the other excited vapour stream, and the mixture is then directed to the bulk polymeric material 1 for thin layer formation, as shown in Figures 7 and 8. When three or more chemical components are used, any combination is possible provided at least one vapour becomes electrically excited.

The thin layer produced by plasma polymerization from a monomer vapour containing vapour of at least one saturated alcohol compound and optionally other organic compounds and/or inorganic gases has an excellent wettability by polar solvents (hydrophilicity) and effects a permanent modification of the surface of the bulk polymeric material 1 by the application of the second layer which is stable with time and does not swell. The multilayer polymeric materials of the invention thus retain good mechanical and other bulk properties while exhibiting improved and permanent wettability in hydrophilic environments.

The invention will be further explained by referring to the embodiments shown in the accompanying drawings. Figure 1 is a schematic view showing the essential parts of a plasma polymerization apparatus 3 for performing the process of this invention. A vacuum chamber 4 is evacuated by a pumping system 5 connected by an adjustable throttle valve 6 and an exhaust pipe 7. The pressure in the chamber is monitored by a pressure gauge 8. Vapour and gas streams are supplied to a mixing device 9 and reach the vacuum chamber 4 via an inlet pipe 10.

Organic monomer liquids are stored in thermostatted containers 11. Evaporation by boiloff supplies organic vapour. Inorganic gases, such as argon and oxygen, are supplied from cylinders 12. All streams are individually controlled by flow control equipment 13 prior to reaching the mixing device 9. All streams may be used singly or in any combination. High frequency electric power is provided by a source 14 via a matching network 15 to the electrodes 16. On application of electric power a discharge is established in the discharge zone 18 between the electrodes and the vapour activated. From the gas plasma the activated products of organic monomer or monomers are polymerized as a thin layer 2 on to one or multiple surfaces of the bulk polymeric material 1.

Figure 2 is a schematic view of a modification of the process of this invention. The apparatus is the same as in Figure 1 except that the high frequency electric power is supplied to a coil 17 and no electrodes are provided.

Figures 3 to 6 schematically show several embodiments of the multilayer polymeric products of this invention. ₅ Figures 3 to 5 show a surface layer 2 applied to one or several faces of a bulk polymeric material 1 of rectangular cross-section. Figure 6 shows a schematic cross-section through a tube-shaped bulk polymeric material 1 that has a thin plasma polymer layer 2 both on the inside and the o outside. _

Figure 7 is a schematic view of an embodiment of the process of this invention using two streams of vapour or gas. Each of the streams may direct one or several vapour components to the vacuum chamber via its inlet pipe. As in 5 Figure 1 the organic vapours and inorganic gases are controlled and mixed prior to the inlet pipe. One of the two streams is fed through the electric discharge zone 18 and the other stream is added to the vacuum chamber downstream of the discharge zone. Figure 8 shows another embodiment of the process where one vapour stream is activated by a coil 17. The process of the invention is not restricted to the particular embodiments shown; three or more inlet pipes may be provided. Examples of the multilayer polymeric materials of this invention will be explained below together with comparative examples. Comparative Examples 1 to 4

Comparative example 1 was untreated sheeting of polytetrafluoroethylene. Comparative examples 2 to 4 were prepared using an apparatus as shown in Figure 1 by plasma treatment according to the prior art, that is, with no saturated alcohol vapour provided. Only air was supplied to the vacuum chamber via the flow control 13. Exposure to a plasma of an air atmosphere produces the oxidative plasma treatment known in the art. For example 2 the pressure in the chamber was 0.70 Torr and the density of the applied

2 high frequency power was 0.57 W/cm . Treatment duration was 1 minute. For example 3 the pressure was 0.81 Torr, the

2 power density 0.99 W/cm and the duration 2 minutes. For example 4 the pressure was 0.59 Torr, the power density 1.28

2

W/cm and the duration 3 minutes. Several drops of distilled water were applied onto the surface of each sample and the receding contact angles (RCA) measured according to standard procedures on a standard contact angle measurement device Kernco Model G-II) . To eliminate inaccuracies due to the slow time dependence reported elsewhere, measurements were performed within half an hour of treatment. The results are shown in Table 1. Examples 5 to 7

Multilayer polymeric materials were prepared using the apparatus as shown in Figure 1. Sheet material of polytetrafluoroethylene was used as the bulk polymeric material 1 and attached to the electrodes as in Figure la. Vapour of ethanol (absolute, BDH Chemicals) was supplied by evaporation from the liquid held in a container. Before ignition of the plasma, the vapour was pumped through the reactor and system for several minutes. Plasma polymer layers 2 were then deposited from ethanol vapour onto the surface of the bulk material 1 to a thickness of the layer 2 in the range of 50 to 500 n . Sample 5 was prepared under

2 the conditions 0.76 Torr, power density 1.06 W/cm and duration 40 seconds. Sample 6 was prepared under the

2 conditions 0.56 Torr, power density 0.67 W/cm and duration

5 minutes. Sample 7 was prepared under the conditions 0.50

Torr, power density 1.15 W/cm 2 and duration 2 minutes.

The receding contact angles of several drops of distilled water on each sample were measured in the same way as for the comparative examples. The results are listed in Table 1. From the results shown it was confirmed that the multilayer polymeric materials of this invention had superior^ wettability compared with the comparative examples prepared by the known art when using comparable input of plasma power. Table 1. Receding contact angles of freshly prepared samples. Example 1 RCA (degrees) : 100+10

2 75+5

3 55±5 4 37.5+2.5

5 25±5

6 7.5±5

7 7+5

The receding contact angles of samples 5 to 7 were measured again after 13 months of storage under ambient conditions of temperature and humidity. Results are shown in

Table 2 and prove that the hydrophilic wettability of the surface is permanent.

Table 2. Receding contact angles of samples after 13 months storage.

Example 5 RCA(degrees) : 23±3

6 5±5

7 5±5

Example 8

Plasma polymerisation was performed as in examples 5 to 7, but with a vapour source of 96% ethanol and 4% water_^ (spectroscopic grade 96% ethanol, Aldrich) . Sample 8 was prepared under the conditions 0.60 Torr, power density 0.84

2 W/cm and duration 1 minute. The receding contact angles of several drops of distilled water on the samples were measured in the same way as for the other examples. The average value was 12.5+7 degrees. It showed that the addition of 4% water to the source liquid ethanol^" does not have a marked influence on the hydrophilic properties of the plasma polymer layer 2 made from ethanol, and that water is an optional, but not necessary, component of the plasma gas. In other words, the presence of water vapour is not required

- for production of hydrophilic surfaces by the plasma process. In contrast to a prior patent (JP 59-15569) which used atmospheres comprising water as an essential component. Comparative examples 9 to 11

Comparative example 9 was untreated sheet material of

10 fluorinated ethylene propylene (FEP, Du Pont) . Comparative examples 10 and 11 were prepared from FEP sheet material by plasma treatment according to the prior art, as in examples 2 to 4, that is, with no saturated alcohol vapour provided. For example 10, air was supplied to the vacuum chamber via

15 the flow control 13 and for example 11 oxygen gas was supplied. For example 10 the pressure in the chamber was

0.60 Torr and the density of the applied high frequency power

₂ was 0.76 W/cm . Treatment duration was 30 seconds. For example 11 the pressure was 0.625 Torr, the power density

2 20 0.76 W/cm and the duration 30 seconds. The receding contact angles, determined in the same way, are listed in

Table 3.

Examples 12 to 15

Multilayer polymeric materials were prepared with an

25 apparatus as shown in Figure 1 and using sheet material of

FEP as the bulk polymeric material 1 attached to the electrodes. For examples 12 and 13, vapour of ethanol

(absolute, BDH Chemicals) was supplied. For examples 14 and

15, vapour of isobutyl alcohol (BDH Chemicals, AnalaR grade) was supplied. Before ignition of the plasma, the vapour was pumped through the reactor and system for several minutes . Plasma polymer layers 2 were then deposited onto the surface of the bulk material 1 to a thickness of the layer 2 in the range of 50 to 500 nm, under conditions in the same range as those used in examples 5 to 8. The receding contact angles of several drops of distilled water on each sample were measured in the same way as for the comparative examples. The results are listed in Table 3. From the results shown it was confirmed that the hydrophilic surface layer 2 can be produced by the present process with saturated alcohol compounds other than ethanol, and that the presence of water vapour is not required for production of the hydrophilic layer 2 by the plasma process, in contrast to a prior patent (JP 59-15569) which used atmospheres comprising water as an essential component.

Table 3. Receding contact angles of samples 9 to 15. Example 9 RCA(degrees, ±3): 95

10 21 11 * 25

12 30

13 20

14 28

15 45 Examples 9 and 12 to 15 were used in experiments involving attachement and growth of human endothelial cells. Results showed that the hydrophilic layer 2 strongly promoted the attachment and growth of the cells. Examples 16 to 18 Multilayer polymeric materials were prepared with an apparatus as shown in Figure 1 and using sheet material of polytetrafluoroethylene as the bulk polymeric material 1 attached to the electrodes. For examples 16 to 18, vapour of methanol (BDH Chemicals, AnalaR grade) was supplied. Plasma polymer layers 2 were then deposited onto the surface of the bulk material 1 to a thickness of the layer 2 in the range of 50 to 500 nm, under conditions in the same range as those used in the previous examples. The receding contact angles of several drops of distilled water on each sample were measured in the same way as for the comparative examples. The results are listed in Table 4.

Table 4. Receding contact angles of samples 16 to 18. Example 16 RCA(degrees,+3) : 20 17 16

18 3

Comparative Example 19

Comparative example 19 was a sheet of poly-dimethylsiloxane (Silastic ) washed in a mixture of n-hexane and methylene chloride, but not plasma treated. Its receding contact angle is listed in Table 5. Examples 20 and 21

Multilayer polymeric materials were prepared with an apparatus as shown in Figure 1 and using sheet material of Silastic , washed as in example 19, as the bulk polymeric material 1 attached to the electrodes. Vapor of ethanol (absolute, BDH Chemicals) was supplied. Plasma polymer layers 2 were then deposited onto the surface of the bulk material 1 to a thickness of the layer 2 in the range of 50 to 500 nm, under conditions in the same range as those used in the previous examples. The receding contact angles of several drops of distilled water on each sample were measured in the same way as for the comparative example. The results are listed in Table 5. Example 22

Multilayer polymeric materials were prepared with an apparatus as shown in Figure 1 and using as the bulk material 1 polytetrafluoroethylene sheet attached to the electrodes as in Figure la. Vapour of heptylamine (Fluka purum grade) was supplied by evaporation from the liquid held in a container. Before ignition of the plasma, the vapour was pumped through the reactor and system for several minutes. Plasma polymer layer 2 were then deposited onto the surface of the bulk material 1 to a thickness of 50 to 500 nm under a pressure of 0.50 Torr, a power density of 0.9 w/cm2 and durations 2 minutes. Receding contact angles (RCA) of several drops of distilled water were measured in the same way as for the previous examples. The results are listed in Table 5. The RCA value of 33 ± 3 degrees for this material contrasted with the value of 100 ± 10 degrees determined in Comparative Example 1 for untreated polytetrafluorethylene again demonstrates the hydrophilic nature of materials of this invention. Table 5. Receding contact angles of samples 19 to 21. Example 19 RCA(degrees,+3) : 70

20 32

21 5 22 33

Example 23

Multilayer polymeric materials were prepared with an apparatus as shown in Figure 1 and using as the bulk material 1 a multilayer structure consisting of a polyimide film and a Co„Cr layer applied by evaporation on one side of the polyimide film. The Co_Cr layer was on the side opposite to the electrode and the plasma polymer layer 2 was applied on top of the Co_Cr layer by supplying vapour of ethanol (absolute, BDH Chemicals). The plasma polymer layer 2 was deposited to a thickness of the layer 2 of 40 nm, under conditions in the same range of those used in the previous examples. By applying the plasma polymer layer onto a metal film, a brilliant colour due to interference was obtained. Drops of water were then applied onto the finished multilayer material as in the standard contact angle measurement method. When the drops of water were carefully removed by soaking them up with a tissue, the interference coloured, fringes were restored immediately and with no discontinuity at the locations where the edges of the droplets had been. These observations proved that the layer 2 had not swelled to any significant and measurable extent by absorption of substantial quantities of water. Thus the thin layers of this invention are wetted well but do not suffer from penetration of water. Further, using a custom built contact angle device with a video recording camera and an enclosed cabinet, the time dependence of the contact angle was determined. Observation of the sessile drop of distilled water on the surface of sample 22 immediately following application and continuously over five minutes showed no change at all in the drop shape and contact angle, indicating negligible absorption of water into the plasma polymer film. This observation proved the non-swelling properties of the plasma polymer layer 2 made from ethanol vapour.

Adhesion testing was done by the standard sticky tape test. In no case was detachment of the thin layer observed, proving that a permanent and strong interfacial bond had been produced between the bulk material and the thin layer. General understanding of the plasma polymerisation process suggests covalent bonding across the interface. Such bonding is superior in strength and durability to the bonding of polar nature (van der Waals forces) obtained by alternative processes for deposition of the thin layer. Example 24

To test the value of the multilayer polymeric material according to the present invention for potential use_^ in biomedical and biotechnological applications, human endothelial cells derived from umbilical arteries (HUAE cells) were seeded onto the surface in an in vitro assay of biological cell attachment and growth. HUAE cells were seeded onto surfaces of polymeric articles according to the present invention formed as described in examples 12, and 13, 14 and 15 and compared to HUAE cell attachment and growth on unmodified fluorinated ethylene propylene (FEP), example 9, and attachment and growth on hydrophilic "tissue culture" polystyrene (TCP, commercially obtained) .

The samples were prepared for cell attachment assays by sonication in a solution of 70% Ethanol at 24 urn for 20 seconds per sample, then rinsed in sterile phosphate-buffered saline solution. Samples were then transferred to serum-free McCoys culture medium for equilibration (at least 1 hr incubation) prior to commencing the cell assay. The modified surface of the film was exposed to the cells in the assay. The cells were routinely maintained in a growth medium consisting of an equal mixture of McCoy 5A (modified) (csL laboratories, Melbourne) and BM86-Wissler (Boehringer-Mannheim) media supplemented with 30% v/v foetal bovine serum (Cytosystems, Sydney), 40 ng/ml fibroblast growth factor (Biomedical Technologies Inc.), 60 ug/ml endothelial cell growth supplement (Collaborative Research), 20 ug/ml insulin (GIBCO), 60 ug/ml penicilin (Glaxo) and 100 ug/ml streptomycin (Glaxo) . A cell culture medium that contained a serum component was used to assist the JUAE cells to become attached onto either TCP or any of the multilayer polymeric articles according to the present invention. In the cell attachment assays, the suitability of the multilayer polymeric articles according to the present invention for cell attachment was determined both in culture medium containing serum and also where the surfaces had also been precoated with serum Fn purified from bovine serum. Coating with Fn was achieved by incubating the flasks with 5 ml solution of 40 ug/ml Fn in PBS at 37°C for 1 hr prior to cell seeding. Excess solution was removed before cells were added. Fn-coated TCP was also included as a control surface, as this surface is known to support good HUAE cell attachment and growth, and the HUAE cells were rountinely maintained on Fn-coated TCP. The morphology of the attached cells when attached to multilayer polymeric articles according to the present invention was taken as an important indicator of the suitability of the surfaces for cell attachment and growth, as only hydrophilic surfaces that do permit HUAE cells to spread well also permit good cell growth.

The HUAE cells failed to attach to Example 9, the unmodified FEP film. Furthermore, HUAE cells failed to attach to Example 9 film which had been precoated with Fn. Thus any cell attachment that occurred to Examples 12, 13, 14 or 15, whether with or without precoating with purified Fn, would indicate that the multilayer polymeric articles according to the present invention was more suitable for cell attachment than the unmodified surface. The morphology of the HUAE cells was determined after

3 and 5 days of culture, and a semi-quantitative assessment of the suitability of the surfaces for HUAE cell attachment and growth, based upon the number and morphology of the attached cells, was made: The results are listed in Table 6. Table 6. Morphology of Attached Cells Surface Fn precoating Assessment

TCP no 3 + yes 5 + Example 9 no 0 yes 0 Example 12 no 2 to 3 + yes 4 to 5 + Example 13 no 3 + yes 5 + Example 14 no 0 yes 3 to 4 + Example 15 no 0 yes 3 + The morphology of HUAE cells attached to the surfaces of Examples 12 and 13, as viewed after 3 days after cell seeding, was equivalent that on TCP for the Fn coated Examples 12 and 13 the number of cells attached and the morphology of the attached cells was equivalent to that on the Fn-coated TCP.* This morphology indicated that although the HUAE cells had attached to the surface of Examples 12 and 13 when seeded in the presence of serum, the cells had not formed the well spread morphology that is typical of HUAE cells that have been seeded onto Fn-coated TCP. However the morphology of the HUAE cells that attached to the Fn-coated Example 12 and 13 films was well spread and the cell morphology was similar to HUAE cells growing on Fn-coated TCP. ^• Examples 14 and 15 failed to support HUAE cell attachment when the cells were seeded in culture medium containing serum. However, when the modified surfaces were precoated with purified Fn and the cells then seeded in culture medium containing serum, HUAE cells attached and spread to a well-spread morphology equivalent to that of Fn-coated TCP. It should be noted that the unmodified FEP (Example 9) did not show this suitability for cell attachment following precoating with purified Fn.

These results clearly demonstrate that the multilayer polymeric articles according to the present invention support human endothelial cell attachment, spreading and growth preferably when the culture medium includes serum components such as fibronectin or vitronectin. The materials are therefore suitable for inclusion as components in implants where where good cell interactions with the surface is required, and for use in in vitro cell culture applications.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims

C L A I MS :

1. A multilayer polymeric material including a first layer formed from a bulk polymeric material; and at least a second layer deposited by plasma polymerisation on at least a surface of the first polymeric layer to increase the hydrophilic nature of said surface.

2. A multilayer polymeric material according to claim 1 wherein the second surface layer is bonded to the first polymeric layer with substantially no penetration thereinto. *

3. A multilayer polymeric material according to claim 2 wherein the first layer is formed from a bulk polymeric material selected from perfluorinated polymers, polyethylene, polyesters, polyurethanes, copolymers thereof and mixtures thereof.

4. A multilayer polymeric material according to claim 3 wherein the first layer is formed from polytetrafluoro¬ ethylene or fluorinated ethylene propylene polymers.

5. A multilayer polymeric material according to claim 2 wherein the second surface layer is formed from a vapour composition including at least one organic monomeric material capable of polymerisation under plasma polymerisation conditions. _

6. A multilayer polymeric material according to claim 5 wherein the at least one organic monomeric material is selected from saturated alcohols, saturated amines, derivatives thereof and precursors therefor.

7. A multilayer polymeric material according to claim 5 wherein the vapour composition further includes at least one inorganic gas selected from oxygen, hydrogen, nitrogen, helium, argon and neon.

8. A multilayer polymeric material according to claim 7 wherein the vapour composition includes no more than approximately 20% v/v water vapour.

9. A multilayer polymeric material according to claim 2 wherein the second surface layer is contacted with a serum component including an adhesive protein.

10. A multilayer polymeric material according to claim 9 wherein the serum component is selected from fibronectin, vitronectin, thrombospondin or adhesive fragments thereof.

11. A biomedical implant including a first polymeric article formed from a bulk polymer material; said first polymeric article bearing a surface layer deposited on at least a surface thereof by plasma polymerisation to increase the hydrophilic nature of the surface.

12. A biomedical implant according to claim 11 wherein the bulk polymeric material is selected from perfluorinated polymers, polyethylene, polyesters, polyurethanes, copolymers thereof and mixtures thereof.

.

13. A biomedical implant according to claim 12 wherein the_ surface layer is formed from a vapour composition including at least one organic monomeric material capable of polymerisation under plasma polymerisation conditions.

14. A biomedical implant according to claim 11 wherein the surface layer is contacted with a serum component including an adhesive protein.

15. A biomedical implant according to claim 14 wherein the serum component is selected from fibronectin, vitronectin, thrombospondin or adhesive fragments thereof.

16. A process for the preparation of a multilayer polymeric material which process includes providing a first polymeric article formed into a desired final shape from a bulk polymer; depositing from the vapour phase a second layer via a plasma polymerisation reaction in a vapour composition containing at least one monomeric material to form a surface layer on at least one surface of the first polymeric material; if desired depositing further layers of different composition by plasma polymerisation in vapours of composition different to that used to produce the second surface layer.

17. A process according to claim 16 wherein the first layer is formed from a bulk polymeric material selected from perfluorinated polymers, polyethylene, polyesters, polyurethanes, copolymers thereof and mixtures thereof.

18. A process according to claim 17 wherein the first layer is formed from polytetrafluoroethylene or fluorinated ethylene propylene polymers.

19. A process according to claim 18 wherein the vapour composition includes at least one organic monomer material selected from saturated alcohols, saturated amines, derivatives thereof and precursors therefor; and at least one inorganic gas selected from oxygen, hydrogen, nitrogen, helium, argon and neon.

20. A process according to claim 16 which process further includes providing a serum component including an adhesive protein; and contacting the second surface layer with the serum component.

21. A process according to claim 20 wherein the serum component is selected from fibronectin, vitronectin, thrombospondin or adhesive fragments thereof.

22. A process according to claim 21 wherein the serum component is provided in a cell culture medium.

23. A process for culturing animal cells in vitro which process includes providing a source of animal cells; and a biomedical implant including a first polymeric article formed from a bulk polymer material; said first polymeric article bearing a surface layer deposited on at least a surface thereof by plasma polymerisation to increase the hydrophilic nature of the surface; and contacting the biomedical implant with the animal cells.

24. A process according to claim 23 which process further includes providing a cell culture medium including a source of animal cells; and a serum component including an adhesive protein; and contacting the biomedical implant with the cell culture medium.

25. A process for the treatment of animals, including humans, which process includes providing a biomedical implant including a first polymeric article formed from a bulk polymer material; said first polymeric article bearing a surface layer deposited on at least a surface thereof by plasma polymerisation to increase the hydrophilic nature of the surface; and implanting the biomedical implant into the animal.

26. A process according to claim 25 wherein the second surface layer of the biomedical implant is contacted with a serum component including an adhesive protein.

27. A process according to claim 26 wherein the serum component is selected from fibronectin, vitronectin, thrombospondin or .adhesive fragments thereof.