WO2010015259A2

WO2010015259A2 - Sterilisation of polymeric products comprising heat and radiation sensitive material

Info

Publication number: WO2010015259A2
Application number: PCT/DK2009/050195
Authority: WO
Inventors: Jimenez Pranov Henrik; Henrik Dörge; Helene Andersen
Original assignee: Inmold Biosystems A/S
Priority date: 2008-08-06
Filing date: 2009-08-05
Publication date: 2010-02-11
Also published as: WO2010015259A3

Abstract

The present invention relates to a polymer article comprising of a thermoplastic compound and at least one material, sensitive to heat and ionising irradiation, embedded at the surface of the article, wherein the polymeric article may be sterilized with ionising irradiation without substantially adversely affecting the embedded material. Another aspect of the present invention regards a process for the production of such a polymer article as well as the sterilised polymer article.

Description

Sterilisation of polymeric products comprising heat and radiation sensitive material

All patent and non-patent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.

Field of invention

Background of invention

In biotechnological and medical applications, it is desirable to apply functional coatings, e.g. proteins, to defined areas of articles for use in laboratories or as medical devices, and subsequently being able to sterilise said articles in order to reduce the likelihood of cross-contamination. A method of producing such articles without substantially affecting the functional coating would be desirable, in particular if such articles can be mass produced at a relative low price as many such articles must be used only once.

WO 2006/097483 discloses a method of providing at least one heat sensitive material on at least part of the surface of a polymer article formed from a heated polymer whose temperature is sufficient to adversely affect the at least one heat sensitive material. The present invention is a further development of the said publication allowing for the subsequent sterilisation of the article without adversely affecting the heat sensitive material.

Summary of invention

The present invention relates to a polymer article comprising of a thermoplastic compound and at least one material sensitive to heat and ionising radiation embedded in or at the surface of the thermoplastic compound, where the polymeric article may be sterilised without causing substantial adverse affect to the embedded material.

In a further aspect the present invention relates to the said polymeric article which has been sterilised to a desired Sterility Assurance Level (SAL), such as a SAL of a log of minus 6 of reduction.

In a still further aspect the present invention relates to a method of producing a polymer article which may be sterilised without causing substantial adverse affect to the embedded material.

In a still further aspect the present invention relates to articles obtained by said method.

Brief description of figures

Figure 1 illustrates the procedure for printing a shim with a desired material and transfer of the material from the shim to the polymer article during injection moulding.

Figure 2 is a schematic diagram of the injection moulding mould.

Figure 3 shows an apparatus for applying the method of an embodiment of the invention too a calendering process.

Figure 4 shows the cell culture experiments on the sterilised surfaces.

Figure 5 shows stem cells grown under serum-free conditions on a coated polystyrene surface.

Detailed description of the invention

The present invention relates to a polymer article comprising of a thermoplastic compound and at least one heat sensitive material embedded in or at the surface of the thermoplastic compound, characterised in that a) the at least one heat sensitive material is sensitive to ionizing irradiation under conditions described in "Sensitivity to ionising radiation", and b) the polymeric article may be sterilised by ionizing irradiation while the embedded heat sensitive material is not substantially adversely affected upon sterilisation to Sterility Assurance Level of a log of minus 6 of reduction.

In a further aspect the present invention relates to the said polymeric article which has been sterilised to a Sterility Assurance Level of a log of minus 6 of reduction.

In a still further aspect the present invention relates to a method of providing at least one heat sensitive material on at least part of the surface of a polymer article formed from a heated thermoplastic polymer whose temperature is sufficient to adversely affect the at least one heat sensitive material, wherein: a) a shaping surface is provided having a smooth surface or an at least partly textured surface, said shaping surface is covered by a layer of organic molecules comprising a reactive group; b) the at least one heat sensitive material is applied to at least part of the shaping surface, the shaping surface being at a temperature at which the heat sensitive material is not adversely affected; c) the heated polymer is brought in contact with the shaping surface; d) the heated polymer is formed by the surface shape of the shaping surface; and e) the at least one heat sensitive material is transferred from the shaping surface to the polymer surface; and the temperature of the shaping surface is maintained sufficiently low that the at least one heat sensitive material is not substantially adversely affected by heat upon or after contact with the heated polymer.

Definitions

Sterility Assurance Level (SAL) describes the probability of a single unit being non- sterile after it has been subjected to a sterilisation process.

Sterilisation is the process by which living organisms are removed or killed to the extent that they are no longer detectable in standard culture media in which they had previously proliferated. SAL of a six-log reduction (also termed log of minus 6 reduction or ten to the minus six reduction) is considered acceptable for many medical devices, though overkill cycles may be employed to provide greatest assurance of sterility for critical products such as implantable devices.

Embedded material or surface embedded material means in the context of the present application that the heat sensitive material is embedded in or at the surface of the polymer article. The heat sensitive material may be embedded directly in or at the surface of the polymer or it may be embedded in the polymer via a covalently bound linker covalently bound to the heat sensitive material. Preferably only a minor part of the heat sensitive material is embedded in or at the surface so that heat sensitive material maintains its functionality.

Heat sensitive material refers in the context of the present application to material where a structural change in macromolecules is caused by heating the material. Preferred examples of heat sensitive materials are nucleic acids, peptides, proteins, hormones, lipids and carbohydrates.

Nucleic acids comprises ribonucleic acids, deoxyribonucleic acids and nucleic acid analogs.

Ribonucleic acids comprises RNA, imRNA, pre-mRNA, tRNA, rRNA, anti-sense RNA, guide RNA, microRNA, non-coding RNA, piwi-interacting RNA, small-interferring RNA, small-nucleolar RNA and transfer-messenger RNA.

Deoxyribonucleic acids comprises DNA, cDNA, genomic DNA, multi-copy single- stranded DNA and mitochondrial DNA.

Nucleic acid analogs comprises glycerol nucleic acid, locked nucleic acid, peptide nucleic acid, threose nucleic acid and morpholino.

Lipids are any fat-soluble molecule, such as fat, oils, waxes, cholesterol, sterols, fat- soluble vitamins (vitamins A, D, E and K), monoglycerides, diglycerides and phospolipids. In particular the following categories of lipids are considered: fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids and polyketides. Proteins and peptides are any molecules comprising less than approximately 50 amino acids (peptides) or more than approximately 50 amino acids (proteins) linked covalently together by peptide bonds. The proteins and peptides may be associated with non- protein compounds such as prosthetic groups or they may be glycosylated. Proteins and peptides may be synthesised in vivo by translation of mRNA at the ribosomes by naturally occurring or genetically modified organisms (recombinant expression) or they may be chemically synthesised.

Carbohydrates comprises monosaccharides, disaccharides, oligosaccarides and polysaccharides.

Hormones are chemicals released by cells that affect cells in other parts of the body.

Polymer article comprising a heat and ionising irradiation sensitive material.

The polymer article of the present invention is formed from a heated polymer, in particular a thermoplastic compound. Non-limiting examples of thermoplastic compounds that may be used are acrylonitrile butadiene styrene (ABS), acrylic, celluloid, cellulose acetate, Ethylene-Vinyl Acetate (EVA), Ethylene vinyl alcohol (EVAL), Fluoroplastics, Liquid Crystal Polymer (LCP), polyacetal, polyacrylate, polyacrylonitrile, polyamide, polyamide-imide (PAI), polyaryletherketone, polybutadiene, polybutylene, polybutylene terephthalate, polycaprolactone (PCL), polychlorotrifluoroethylene (PCTFE), polyethylene terephthalate (PET), polycyclohexylene dimethylene terephthalate (PCT), polycarbonate (PC), polyhydroxyalkanoates (PHAs), polyketone (PK), polyester, polyethylene (PE), polyetheretherketone (PEEK), polyetherimide (PEI), polyethersulfone (PES), Polyethylenechlorinat.es (PEC), polyimide (Pl), polylactic acid (PLA), Polymethylpentene (PMP), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide (PPA), polypropylene (PP), polystyrene (PS), polysulfone (PSU), polyurethane (PU), polyvinyl acetate (PVA), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC) and styrene-acrylonitrile (SAN), or mixes or copolymers thereof. In a particularly preferred embodiment the polymer article is formed from a biodegradable polymer, such as a biodegradable thermoplast. Biodegradability may be defined by standards well known in the art, such as ASTM D4300, which is a test method designed to be used to determine the susceptibility of polymers to biodegradation.

The heat sensitive material is in a particular embodiment selected from the group materials where a structural change in macromolecules takes place upon heating of the material to 100°C for 5 seconds. The heat sensitivity is tested in the presence of water, where the presence of water is defined by the level of water present when passively adsorbing the heat sensitive material to a polymer surface from an aqueous solution with subsequent drying for 1 minute in a flow of gaseous, dry nitrogen under conditions that do not adversely affect the heat sensitive material or the polymer substrate. For the material to be considered a heat sensitive material the structural change in the macromolecule preferably leads to denaturation or degradation of the material, i.e. the functionality of the material is impaired upon heating. In a particularly preferred embodiment the heat sensitive material is selected from the group consisting of peptides, hormones, proteins, nucleic acids, lipids and carbohydrates. Other preferred embodiments are components that increase adhesion of cells to surface (including the polypeptide polylysine), antibodies (mono- or polyclonal) and complement factors.

Without being limiting to the present application it is believed that most materials sensitive to ionising irradiation in presence of water are adversely affected by free- radical damage, e.g. lipid peroxidation, denaturation of proteins/peptides and breakdown of polysaccharides to smaller units of carbohydrates. The heat sensitive material, which also is sensitive to ionising radiation under conditions as defined in the section "Sensitivity to ionising radiation", is, when embedded in or at the surface of the thermoplastic compound of the polymer article of the present application, not substantially adversely affected upon sterilisation. In a preferred embodiment the sterilisation is a SAL of a three-log reduction, such as a SAL of a four-log reduction, such as a SAL of a five-log reduction or a SAL of a six-log reduction. In an even more preferred embodiment overkill cycles have been employed without substantially adversely affecting the embedded material.

In a particular embodiment the heat sensitive material has been less than 60% degraded or denatured upon sterilisation to SAL of a six-log reduction, such as less than 50% or 40% degraded or denatured upon sterilisation to SAL of a six-log reduction, in a preferred embodiment the heat sensitive material has been less than 30% degraded or denatured upon sterilisation to SAL of a six-log reduction, such as less than 20% or 10% degraded or denatured upon sterilisation to SAL of a six-log reduction. The ionising irradiation source applied for the sterilisation of the polymer article is preferably alpha radiation, beta radiation, gamma radiation or X-ray radiation. The sterilisation comprises an ionising irradition dose of between 1-100 kGy, such as 2-60 kGy, preferably 3-50 kGy, 3-30 kGy or 3-20 kGy. In a preferred embodiment the irradiation dose is in the range of 5-50 kGy, such as 5-40 kGy, 5-30 kGy or 5-25 kGy. In a particularly preferred embodiment the irradiation dose is about 3 kGy, about 4 kGy, about 5 kGy, about 10 kGy, about 15 kGy, about 20 kGy or about 25 kGy.

The present application also concerns a polymer article as described which has been sterilised to a SAL of a three-log reduction, such as to a SAL of a four-log reduction, such as to a SAL of a five-log reduction or even to a SAL of a six-log reduction.

Process for producing polymer article comprising a heat and ionising irradiation sensitive material.

Typical methods used in the mass production of polymer articles are injection moulding and calendering, the latter process being frequently combined with extrusion. Injection moulding is performed by heating a suitable polymer until molten, injecting the molten polymer into a mould with a special surface coating, allowing the polymer to cool and harden, and removing the moulded article from the mould. This process may be automated and therefore used to produce a rapid succession of identical articles. The mould used may have means for cooling, in order to increase the speed of hardening of the polymer. A removable shim with a special surface coating may be incorporated into the mould, and this shim may bear surface structure and/or texture that is transferred to the polymer article during the moulding process. Alternatively, such structure may be present on the mould.

Calendering is a process used to manufacture polymer sheeting. A suitable polymer in pellet form is heated and forced through a series of heated rollers until the polymer sheet reaches the desired dimensions. The sheeting is then passed through cooling rollers with a special surface coating in order to cool and set the polymer. Frequently, texture is applied to the polymer sheet during the process, or a strip of fabric is pressed into the back of the polymer sheet to fuse the two together. The calendering process may be used in combination with extrusion - the extruded polymer form may be passed through the heated rollers of the calender as above until the required dimensions are obtained, and then passed over cooling rollers with a special surface coating to set the form of the polymer.

In biotechnological and medical applications, it is desirable to apply heat sensitive coatings to defined areas of articles and subsequently being able to sterilise the product by ionizing radiation, preferably alpha, beta or gamma radiation or X-ray radiation after the application of the heat sensitive coating. In a certain embodiment the heat sensitive material is also sensitive to ionizing irradiation when water is present. The presence of water is defined by the level of water present when passively adsorbing the heat sensitive material to a polymer surface from an aqueous solution with a subsequent drying for 1 minute in as flow of gaseous, dry nitrogen under conditions that does not adversely affect the heat sensitive material or the polymer substrate.

It has been discovered by the applicants that modelling the temperature of the mould in an injection moulding apparatus during injection of the molten polymer indicates that the molten polymer cools from around 300 °C to 5-10 °C above the mould temperature within 1 micro second of contact between the mould and the molten polymer. The possibility of applying a heat-sensitive material to the surface of a mould covered by a layer of organic molecules comprising a reactive group, or other cooled shaping surface with such a surface coating of a polymer-forming device, and transferring that material intact to the surface of the polymer article, is therefore raised. Accordingly, in one aspect, the invention provides a method of providing at least one heat sensitive material on at least part of the surface of a polymer article formed from a heated polymer whose temperature is sufficient to adversely affect the at least one heat sensitive material, wherein: a shaping surface is provided having a smooth surface or an at least partly textured surface; the at least one heat sensitive material is applied to at least part of the shaping surface with a layer of organic molecules comprising a reactive group, the shaping surface with said surface coating being at a temperature at which the heat sensitive material is not adversely affected; the heated polymer is brought in contact with the shaping surface with said surface coating; the heated polymer is formed by the surface shape of the shaping surface with the said surface coating; and the at least one heat sensitive material is transferred from the shaping surface with said surface coating to the polymer surface; and the temperature of the shaping surface with said surface coating is maintained sufficiently low that the at least one heat sensitive material is not substantially adversely affected by heat upon or after contact with the heated polymer.

The surface coating of organic molecules comprising a reactive group ensures that the heat sensitive material may be deposited on the surface coating, and furthermore ensures that the heat sensitive material may be transferred to the polymer surface. The surface coating shall furthermore ensure that the temperature of the heat sensitive material is lowered to a temperature that does not adversely affect the heat sensitive material within a time short enough that an otherwise (long term) adversely affecting (high) temperature does not affect the heat sensitive material. This may be realised by fulfilling one of two conditions:

1. The thickness of the organic layer comprising a reactive group is sufficiently thin (preferably below 10 nm), as the heat transport properties in the injection moulding apparatus will therefore only be marginally affected, or

2. The heat transfer properties of the organic layer comprising a reactive group are more similar to the heat transfer properties of the mould (high heat capacity, heat conductance and density) than the heat transfer properties of the polymer mould (low heat capacity, heat conductance and low density).

In a preferred embodiment the layer of organic molecules is formed by alkanes, such as halogenated alkanes. In an even more preferred embodiment the layer of organic molecules is a self assembling monolayer.

The layer of organic molecules comprising a reactive group may be bound directly to the shaping surface or bound to an adhesion layer on the shaping surface via the comprised reactive group. The adhesion layer is selected from the group consisting of metal or semimetal oxides, metal or semimetal nitrides, metal or semimetal carbides or diamond-like carbon. The reactive group may be selected from the group consisting of halogenated silanes, hydroxylated silanes, or other groups capable of forming covalent bonds to the mould material or adhesion layer.

The at least one heat sensitive material is maintained at a temperature such that it is not more than 60%, such as not more than 50%, 40% or 30% degraded or denatured by contact with the heated polymer, for example not more than 20% degraded or denatured, or not more than 10% degraded or denatured. Preferably, the at least one heat sensitive material is not detectably degraded or denatured by the heated polymer. Preferably, the at least one heat sensitive material has selective binding properties. Preferably, the selective binding affinity of the at least one heat sensitive material is maintained after contact with the heated polymer.

According to a preferred practice of the invention, the polymer article is formed by injection moulding, said shaping surface covered by a layer of organic molecules comprising a reactive group forming an internal surface of the mould. Alternatively, a different shaping process is used such as compression moulding or calendering. The melting temperature for the polymers typically used in injection moulding or calendering processes is generally between 100°C and 350°C. A suitable temperature for a melt for injection moulding may therefore be up to 350°C, for example 250°C. A suitable temperature for a melt to be formed by calendering may be up to 350°C, for example 250°C.

The temperature of the mould surface must be maintained during polymer injection at a temperature below the denaturation temperature of the heat sensitive material. This may be achieved by cooling the mould. Preferably, cooling means cooling the mould so that the temperature of the shaping surface is kept at or below 40°C, e.g. by keeping the temperature of the mould below approximately 30°C. This may for instance be a cooling fluid passed through cooling channels in a mould or chilling a calendering roller.

The at least one heat sensitive material is preferably applied to the shaping surface covered by a layer of organic molecules comprising a reactive group by contact with a solution of the heat sensitive material, for which one may use a liquid dispensing machine or a submersion bath. The areas in which the shaping surface and the solution of the heat sensitive material is in contact may be an array of smaller homogenous areas or one homogeneous area. The solvent in which the heat sensitive material is dissolved in is preferably water, or water with additives (e.g. a buffer solution). The contact lasts for a time being preferably lower than one hour and preferably more than one second. More preferably the contact lasts for more than 30 s and less than 10 minutes. After the contact the excess solvent is preferably removed by mechanical means, preferably pressurised gas or a combination of a liquid, preferably water, and pressurised gas. The time after removal of the excess solvent to the time of contact with the polymer is preferably less than 10 minutes, more preferably less than one minute.

The solution contact method and subsequent injection molding process is shown in figure 1. The heat sensitive material (e.g. Collagen) is dissolved in an appropriate solvent (e.g. de-ionised water) in a concentration of e.g. 10 μg/ml. The solution is pipetted onto the shim in an array of drops (Fig1A). After being in contact for 5 minutes, the drops are removed from the shim by blowing dry, compressed air parallel to the shim surface (Fig1 B). The shim now contains a corresponding array of surface adsorbed Collagen (Fig1C). The shim may be textured or flat, and may form a removable or a permanent part of the mould. The shim and/or the mould may additionally have macroscopic surface structure, in addition to the surface texture. For example, the shim and/or mould may be shaped to form a multi-well plate from the polymer, in which the bottoms of the wells are smooth or textured. The shaping forming the wells is the macroscopic structure, and the texture of the shim forms the texture of the bottom of the wells, and may be termed the microscopic structure. Once the desired combination of materials has been applied to the shim, the shim is inserted into the mould. A molten polymer is then introduced into the mould according to the usual injection moulding technique. The time from the removal of the excess solvent to the introduction of the molten polymer is preferably as short as possible, and in our embodiment less than 30 s. This is shown in Figures 1 D and 1 E; a schematic diagram of the mould is shown in Figure 2.

The molten polymer is allowed to remain in the mould until it has cooled and set. This time may be of the order of a minute or less. After setting, the materials have transferred from the shim to the surface of the polymer according to the pattern and topography with which the materials were placed on the shim. The polymer article is removed from the mould (Fig1 F), and the materials deposited on its surface may be detected by appropriate methods.

After the removal of the part, the part is packaged in an inert atmosphere, e.g. nitrogen. When packaged it is subject to ionizing radiation (e.g gamma or beta or X-ray radiation with at dose capable of ensuring a sterility assurance level of at least a four-log reduction, such as a five-log or six-log reduction. The dose depends on the sterility level of the production, but normally doses between 3 and 60 kGy are used. The packaged parts may be subjected to the irradiation at room temperature, or at a lowered temperature, e.g temperature about 0°C or lower, such as about -40°C, - 50°C, -60°C, -70°C or -80°C or even lower temperatures such as about -90°C, -100°C, -1 10⁰C, -120⁰C, -140⁰C, -160⁰C, -180°C or -200°C

It may also be encompassed in the present invention to use a method of forming the polymer other than injection moulding. For example, Figure 3 shows an apparatus for incorporation into a calender, preferably between the heated rollers and the cooling rollers. The desired material may be transferred to the surface of a shaping roller (C) by submersing a part of this in a bath containing the solution of the heat sensitive material. Immersion of the surface of the shaping roller B in a solution of the desired material D, transfers the material to the surface of the polymer. The surfaces of the shaping roller may be textured, and so different topographies of the material (A) may be obtained. As many biologically-active molecules' biological activity is destroyed by exposure to high temperatures - for example, some proteins are denatured at temperatures above 40°C - the shaping part is preferably maintained at a temperature of about 30°C or lower during contact with the heated polymer. Alternatively, compounds that modify the surface characteristics of the polymer may be used, such as polymer coatings that reduce or enhance the residence time of a substance on the polymer surface without reacting with that substance.

Use of sterilised polymeric products

The polymeric products of the invention may be sterilised without adversely affecting the surface embedded material. They may be used, e.g., in the following applications:

• Implants

• Diagnostics

• Ligand binding processes • Enzyme immobilisation

• Cell culture

• Personalised medicine

• Cell based bioreactors • Enzyme based bioreactors

• Lab on chip systems

• Drug screening

• Biosensors

The present invention is further described in the following examples which should not be construed as limiting the scope of the invention.

Experimental

Sensitivity to ionising irradiation

A test for sensitivity to ionizing radiation may in the context of the present application be conducted the following way: The material is passively adsorbed to a polymer surface from an aqueous solution with a subsequent drying for 1 minute in a flow of gaseous, dry nitrogen under conditions that does not adversely affect the heat sensitive material or the polymer substrate. Subsequently the material is irradiated with a dose of 20 kGy of an ionising radiation.

The material is considered sensitive to ionising radiation if it is has been denatured or degraded. Tests for denaturation or degradation are available in the art; see e.g. reference 1 and 2.

Examples

Example 1 General procedure for injection moulding

Injection moulding was performed on an Engel 25 tonnes machine fitted with a water-cooled mould with a replaceable shim. The dimensions of the shim were radius 85 mm, with a 300 μm thickness. The shim was supported by a highly heat-conducting backplate. The nickel shims used were flat and coated by a self assembled monolayer of a fluoro-alkane bond covalently through a reactive silane group to an adhesion layer of silicon oxide with a thickness of 10 nm. After application of the media to be transferred, the shim was mounted in the mould and the polymer injected into the mould. The water cooling was set to the minimum temperature, yielding a mould temperature of 26 °C before injection of the molten polymer. The mould temperature was monitored via a thermistor in the backplate and increased to approximately 30 °C during injection of the molten polymer. The polymer article was removed from the mould after a cooling time of 60 s. The melt temperature used for polystyrene was 250 °C.

Example 2 Procedure for coating the entire shaping surface with Collagen 1 and subsequent irradiation sterilisation

The shape generating surface of a petri dish mold was defined on a metallic inlay of the mould cavity. The metallic inlay, made from nickel coated with first an adhesion layer of silicon oxide and second a self assembled monolayer of a covalently bond fluoro-alkane comprising a halogenated silane group, was removed from the cavity and immersed in a solution of the material. Coating by Collagen 1 , by immersion in a 10 μg/ml solution in water for 5-7 minutes. The surface was dried in a nitrogen flow. The injection molding procedure in example 1 was performed. After removal of the final part the parts were packaged in a nitrogen atmosphere. The parts were then sterilised using 20 kGy gamma radiation. Human fibroblast cells were cultured serum- free on the surface, with a comparable study of commercially available collagen 1 surfaces (not radiation sterilized) and a commercially available tissue culture polystyrene (gamma-sterilized, but not containing any biological molecules). After 24 hours the images displayed in figure 4 were taken. Figure 4 shows that the cell density on InMolds petri dish is similar to or exceeds that of the both non-biological surfaces (Corning Cell Bind) and the non-sterilized biological surfaces (Greiner Cell Coat and BD BioCoat Collagen 1 surfaces).

Example 3 Transfer of arrays of Superfibronectin to PS, basic fibroblast growth factor and a mixture thereof with subsequent irradiation sterilisation

The transfer of superfibronectin (SF) /basic fibroblast growth factor (bFGF) /mixture of SF and bFGF was carried out according to the general procedure using a flat shim coated by using a solution of 10 micro-g/ml SF in water, 10 micro-g/ml bFGF in water or 10 micro-g/ml SF + 10 Dg/ml bFGF in water. An array of drops was brought in contact with the shim. Injection moulding using the shim with adsorbed SF/bFGF/mixture was carried out according to the general procedure, with the replica being removed from the mould after a cooling time of 60 s. After the part was removed from the mold the part was packed in a nitrogen atmosphere, and irradiated by 25-50 kGy of gamma or beta irradiation. Afterwards human stem cells were cultured on the surface showing good adhesion and no toxic effects (Figure 5).

The surfaces including the extra cellular matrix protein, the growth factor or the mixture thereof, have been sterilized at room temperature with either beta or gamma radiation at doses 25 kGy (beta or gamma) or 50 kGy (gamma only). The uncoated polystyrene sterilized at 25 kGy beta is shown as reference. It is clearly seen (figure 5) that the cells adhere well to the extracellular matrix containing areas, the growth factor containing areas and the mixture containing areas forming a dense cell layer, whereas the cells on the untreated polystyrene do not adhere well.

References

(1 ) Gutteridge JM: Int. J Biochem. 1982; 14(7): 649-53: Free-radical damage to lipids, amino acids, carbohydrates and nucleic acids determined by thiobarbituric acid reactivity.

(2) Rael, L. T. et al: Journal of Biochemistry and Molecular Biology, 2004; 37(6): 749- 752: Lipid Peroxidation and the Thiobarbaturic Acid Assay: Standardization of the assay when using saturated and unsaturated fatty acids.

Claims

1. A polymer article comprising of a thermoplastic compound and at least one heat sensitive material embedded at the surface of the thermoplastic compound, characterised in that a. the at least one heat sensitive material is sensitive to ionizing irradiation under conditions described in "Sensitivity to ionising radiation", and b. the polymeric article may be sterilised by ionizing irradiation while the embedded heat sensitive material is not substantially adversely affected upon sterilisation to Sterility Assurance Level of a six-log reduction.

2. The polymer article of claim 1 wherein the thermoplastic compound is selected from the group consisting of acrylonitrile butadiene styrene (ABS), acrylic, celluloid, cellulose acetate, Ethylene-Vinyl Acetate (EVA), Ethylene vinyl alcohol (EVAL), Fluoroplastics, Liquid Crystal Polymer (LCP), polyacetal, polyacrylate, polyacrylonitrile, polyamide, polyamide- imide (PAI), polyaryletherketone, polybutadiene, polybutylene, polybutylene terephthalate, polycaprolactone (PCL), polychlorotrifluoroethylene (PCTFE), polyethylene terephthalate (PET), polycyclohexylene dimethylene terephthalate (PCT), polycarbonate (PC), polyhydroxyalkanoates (PHAs), polyketone (PK), polyester, polyethylene (PE), polyetheretherketone (PEEK), polyetherimide (PEI), polyethersulfone (PES), Polyethylenechlorinat.es (PEC), polyimide (Pl), polylactic acid (PLA), Polymethylpentene (PMP), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide (PPA), polypropylene (PP), polystyrene (PS), polysulfone (PSU), polyurethane (PU), polyvinyl acetate (PVA), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), styrene- acrylonitrile (SAN), or mixes or copolymers thereof.

3. The polymer article of claim 1 or 2, wherein the thermoplastic compound is biodegradable.

4. The polymer article of claim 1 , wherein the at least one heat sensitive material is selected from the group consisting of peptides, proteins, nucleic acids, lipids and carbohydrates.

5. The polymer article of claim 4, wherein the peptide or the protein is expressed recombinantly or synthesized chemically.

6. The polymer article of claim 4, wherein the nucleic acid is selected from the group consisting of ribonucleic acids, deoxyribonucleic acids and nucleic acid analogues.

7. The polymeric article of claim 4, wherein the lipid is selected form the group consisting of fatty acyls, glycerolipids, glycerophospolipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids and polyketides.

8. The polymeric article of claim 4, wherein the carbohydrate is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides and polysaccharides.

9. The polymer article of claim 1 , wherein the ionizing irradiation is selected from the group consisting of alpha radiation, beta radiation, gamma radiation and X-ray radiation.

10. The polymer article of claim 1 , wherein the heat sensitive material is less than 60% degraded or denatured upon sterilisation to Sterility Assurance Level of a six-log reduction.

1 1. A method of providing at least one heat sensitive material on at least part of the surface of a polymer article formed from a thermoplastic polymer whose temperature is sufficient to adversely affect the at least one heat sensitive material, wherein: a. a shaping surface is provided having a smooth surface or an at least partly textured surface, said shaping surface is covered by a layer of organic molecules comprising a reactive group; b. the at least one heat sensitive material is applied to at least part of the shaping surface, the shaping surface being at a temperature at which the heat sensitive material is not adversely affected; c. the heated polymer is brought in contact with the shaping surface; d. the heated polymer is formed by the surface shape of the shaping surface; and e. the at least one heat sensitive material is transferred from the shaping surface to the polymer surface; and the temperature of the shaping surface is maintained sufficiently low that the at least one heat sensitive material is not substantially adversely affected by heat upon or after contact with the heated polymer.

12. The method of providing a polymer article according to claim 11 , wherein the polymer article is formed by injection moulding, said shaping surface forming an internal surface of the mould.

13. The method of providing a polymer article according to claim 11 , wherein the polymer article is formed by a process comprising calendering.

14. The method of providing a polymer article according to any previous claim, in which cooling means cooling the shaping surface maintains the temperature of the shaping surface at or below 30 °C.

15. The method of providing a polymer article according to claim 11 , in which the material applied to the shaping surface is applied to the shaping surface by contact printing.

16. The method of providing a polymer article according to claim 15, in which the heat sensitive material applied to the shaping surface is applied to the shaping surface using a smooth or at least partly textured stamp.

17. The method of providing a polymer article according to any previous claim, in which the material applied to the shaping surface is applied to the shaping surface having an at least partly textured surface such that only elevated parts of at least part of the textured surface are coated with the material.

18. The method of providing a polymer article according to any preceding claim, wherein, after application of a first material, at least part of the first material is removed from the shaping surface and at least a second material is applied to at least some of the parts of the shaping surface from which the first material has been removed.

19. The method of providing a polymer article according to claim 11 , wherein the topographical replication efficacy is modulated by choice of replication process parameters, selected from varying the speed of injection of the molten polymer into the mould, and spatially selectively varying the heat transport properties of the shaping part surface.

20. The method of providing a polymer article according to claim 19, wherein the microscopic topography of the shaping part is replicated with different detail from the macroscopic topography.

21. The method of providing a polymer article according to claim 11 , wherein the layer of organic molecules is formed by alkanes.

22. The method of providing a polymer article according to claim 21 , wherein the alkanes forming the layer are halogenated.

23. The method of providing a polymer article according to claim 21 , wherein the layer of organic molecules is a self assembling monolayer.

24. The method of providing a polymer article according to claim 11 , wherein the layer of organic molecules is bound directly to the shaping surface.

25. The method of providing a polymer article according to claim 11 , wherein the layer of organic molecules is bound to an adhesion layer on the shaping surface.

26. The method of providing a polymer article according to claim 25, wherein the adhesion layer is selected from the group consisting of metal or semimetal oxides, metal or semimetal nitrides, metal or semimetal carbides or diamond-like carbon

27. The method of providing a polymer article according to claim 11 , wherein the reactive group is selected from the group consisting of halogenated silanes, hydroxylated silanes, or other groups capable of forming covalent bonds to the mould material or adhesion layer.

28. The method of providing a polymer article according to claim 11 further comprising sterilisation to Sterility Assurance Level of a six-log reduction following ionizing irradiation.

29. The method of providing a polymer article according to claim 28, wherein the ionizing irradiation is selected from the group consisting of alpha radiation, beta radiation, gamma radiation and X-ray radiation.

30. The method of providing a polymer article according to claim 28, wherein the sterilisation comprises a dose of at least 3 kGy.

31. The method of providing a polymer article according to claim 28 further comprising the step of freezing the polymer article prior to irradiation.

32. The method of providing a polymer article according to claim 31 , wherein the freezing temperature is about -O⁰C or lower.

33. A product obtained by the process of claim 1 1 through 32.

34. Use of any of the products of claim 1 , 10 or 33 for implants, diagnostics, ligand binding processes, enzyme immobilisation, cell culturing, personalised medicine, cell based bioreactors, enzyme based bioreactors, lab on chip systems, drug screening or biosensors.