WO2006038866A1

WO2006038866A1 - Improved coating comprising a bioadhesive polyphenolic protein derived from a byssus-forming mussel

Info

Publication number: WO2006038866A1
Application number: PCT/SE2005/001458
Authority: WO
Inventors: Magnus Qvist
Original assignee: Bio Polymer Products Of Sweden Ab
Priority date: 2004-10-01
Filing date: 2005-10-03
Publication date: 2006-04-13

Abstract

An improved coating for biomedical surfaces including a bioadhesive polyphenolic protein derived from a byssus- forming mussel, e.g. Mefp-1 (Mytilus edulis foot protein- 1) . The coating reduces the immunogenicity of the coated biomedical surface. The bioadhesive polyphenolic protein may be oxidized or non- oxidized dependent on whether a further layer is to be coated on the surface. The further layer may comprise e.g. heparin, hyaluronic acid or fibrinogen.

Description

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Field of the Invention

The present invention relates to surface treatments, preparations, or coatings which reduce the immunogenicity of medical devices and equipment. More specifically, the invention relates to surface treatments which reduce the immunogenicity of im¬ planted or implantable medical devices.

Background of the Invention

Through advances in biomedical research, it is now possible to perform extraordi¬ nary research and treat or alleviate conditions which were once life-hindering or fa¬ tal. For example, living cells may be cultured in laboratory equipment for medical use or further research; broken, deformed, or missing bones can be repaired with implanted plates, rods or pins; a defective or diseased organ such as a heart may be replaced with a mechanical, chimeric, or animal organ.

Such advances have not come without their own respective challenges. Cell culture equipment can be less than efficient and simple due to problems with undesired ad¬ herence, or insufficient adherence when it is desired. Materials used in bone-setting pins can trigger immune reactions, leading to the use of various alloys of nickel and titanium. Even alloys are imperfect; titanium, for example, is strong and non- immunoreactive but can be prohibitively expensive for many applications. Re¬ placement organs, whether artificial or naturally-occurring, can cause severe adverse reactions in the recipient. Approaches to remedy this problem include pharmaceuti¬ cals, however the long duration of therapy and severe side effects are undesirable.

It is known that the reactions causing the above-described problems can be associ¬ ated with cells and proteins in the blood, cells and proteins that react with foreign bodies. At times this is a naturally-occurring and beneficial reaction, but when oc- curring in relation to the use of a biomedical material the reaction is to the detriment of the patient.

Present research focuses on two main pathways which cause reactions to a foreign body. One is the immune compliment. When activated by material perceived to be foreign, an inflammatory response is triggered and immune system components mi¬ grate to the location of the material that has been identified as foreign. Surface acti¬ vation of the immune compliment system can be associated with a blood bound complement protein called Complement factor 3 (C3), which binds to the foreign surface.

Detecting whether a material will cause such an immune response can therefore be accomplished with assays employing C3. C3, bound to the surface of the proposed material, can be observed for the timing and strength of anti-C3 antibodies. A stronger, faster binding of anti-C3 antibodies from blood indicates a more highly re¬ active material, one that would be less favored in biomedical applications. ELISA and Quartz Crystal Microbalance (QCM) are other commonly-used approaches. For example, QCM dissipation (QCM-D) is an acoustic method where the mass of ad¬ sorbed proteins and the viscosity and elasticity of the protein layer can be deter¬ mined.

The other pathway of reaction to a foreign body is surface-associated blood coagula¬ tion. Blood cells, particularly platelets, can bind to the surface of certain materials. Depending on the size and location, such a coagulated surface can cause a thrombo¬ sis. Assays utilizing platelets can be used to determine the likelihood of reaction of a proposed biomedical material.

Before using materials in biomedical applications, they must be evaluated for reac¬ tivity among other evaluations for, e.g., toxicity, strength, deformability. Particularly in the case of materials that are intended to remain inside the body of a patient, re- ducing or eliminating the immunogenicity of the materials employed is of signifi¬ cant concern. The care that must be taken to avoid triggering of an undesired re¬ sponse or the counteractive measures to suppress that response increases the cost and complexity of research in this area. Some solutions have been presented, for ex¬ ample, the use of titanium in certain applications. But there remains a need to fur¬ ther develop solutions for the various biomedical applications that exist. Particularly felt is the need for new, improved non-immunoreactive coatings for existing and new biomedical materials, devices and equipment.

US 6,497,729 discloses an implant coating comprising a bioactive polymer layer self-assembled with metal cations, wherein the bioactive polymer further contains at least one tissue response modifier. The bioactive polymer is not crosslinked by oxi¬ dation.

WO 01/44401 relates to a bioadhesive composition comprising a polyphenolic pro¬ tein, and how the composition can be applied as an adhesive in ophthalmic therapy. Nothing is disclosed about coating implants in order to reduce immunogenicity.

WO 03/051418 describes a method and kit for providing a bioadhesive binding or coating with polyphenolic mussel proteins in a strongly alkaline solution. Nothing is mentioned about coating implants.

US 2002/0111694 Al relates to a method of joining biopolymers to a metal hydride surface by electrolysis.

Summary of the Invention

It is therefore an object of the present invention to provide an implantable medical device, wherein that at least a part of the surface of the implantable medical device is coated with a bioadhesive composition. The bioadhesive composition comprises a) a bioadhesive polyphenols protein derived from a byssus-forming mussel, which protein comprises 30-300 amino acids and consisting essentially of tandemly linked peptide repeats comprising 3-15 amino acid residues, wherein at least 3 % and pref¬ erably 6-30 % of the amino acid residues of said bioadhesive polyphenols protein are DOPA; and, in case the bioadhesive composition is not going to be coated by a further layer b) a non-enzymatic oxidising agent such as hydrogen peroxide, nitroprusside ions or periodate ions.

Accordingly, the bioadhesive composition may be oxidized or non-oxidized depend¬ ing on whether a further layer is to be coated on the bioadhesive composition.

In a preferred embodiment, the bioadhesive composition may is coated by a further layer comprising at least one compound selected from the group of heparin, hyalu¬ ronic acid, dextransulphate, heparansulphate, sulphated carbohydrates, non- sulphated carbohydrates, polyvinylpyrrolidone, chitosan, modified polyethylene species, fibrinogen and polyimine.

Preferably, the implantable medical device is selected from the group consisting of stents, contact lenses, insulin pumps, pacemakers, implantable defibrillators, re¬ placement organs, dental implants, sutures and prosthetic devices.

The medical device that is covered with the bioadhesive composition may be com¬ prised of a material selected from the group consisting of polyethylene, polyethylene terephthalate, polystyrene, glass, gold, and titanium.

The implantable medical device may, of course be used in medical treatment. For convenience, certain terms employed in the specification, examples, and ap¬ pended claims are collected here.

As used herein the term "animal" refers to mammals, preferably mammals such as live stock or humans. Likewise, a "patient" or "subject" as described herein can mean either a human or non-human animal.

The term "biomedical" refers to a field of work which encompasses the medical arts, biotechnology, and all research and applications which have some relation to medical treatment. The term includes materials research where the materials will be used in an application where they are in contact with living cells or tissues or with secondary materials which will later contact living cells or tissues. Examples of such materials research include contact lenses and syringes.

An "effective amount" refers to an amount of a coating material which, when ap¬ plied according to the present invention, provides a sufficient layer to render the coated surface sufficiently non-reactive as taught by the present invention.

The term "immunogenic" refers to items which trigger, cause or exacerbate an im¬ mune response in an animal. It also encompasses a coagulation response to foreign bodies. The term "antigenic" can be used interchangeably. In contrast, the term "non-immunogenic" refers to items which do not independently trigger, cause or ex¬ acerbate in immune response in an animal. A non-immunogenic material, when in contact with living cells or tissues, is chemically and biologically inert to its sur¬ roundings and does not interact or react with the living tissue. There exist degrees of immunogenicity and non-immunogenicity. Products, methods and devices incorpo¬ rating the teachings of the present invention are favourable due to low degrees of immunogenicity and relative inertness. A skilled worker understands that reference to the present inventive materials as non-reactive or inert is a relative comparison and not necessarily an absolute property. As used herein, the terms "materials" and "materials, devices and equipment" re¬ fer to all compounds, preparations, items, and the like, which can be used as de¬ scribed herein. For example, "materials" includes various metals and plastics which may be used to make a medical or biomedical apparatus or tool. The terms also encompass all items which can or are utilized in a biomedical application. Examples include petri dishes, sutures, stents, contact lenses, dental implants, and transplant organs.

As disclosed herein, the terms "polyphenolic protein," "mussel adhesive protein," "MAP," and "Mefp-1" relate to a protein which is synthesized, recombinantly produced, or derived from byssus-forming mussels. Examples of such mussels are mussels of the genera Mytilus, Geukensia, Aulacomya, Phragmatopoma, Dre- issenia and Brachiodontes. Suitable proteins have been disclosed in a plurality of publications, e.g. US 5,015,677, US 5,242,808, US 4,585,585, US 5,202,236, US 5149,657, US 5,410,023, WO 97/34016, US 5,574,134, Vreeland et al., J. Physiol., 34: 1-8, and Yu et al., Macromolecules, 31: 4739-4745. These proteins typically comprise about 30 - 300 amino acid residues and essentially consist of tandemly-linked peptide units comprising 3 — 15 amino acid residues, optionally separated by a junction sequence of 0 - 10 amino acids. A characteristic feature of such proteins is a comparatively high amount of positively charged lysine resi¬ dues, and in particular the unusual amino acid DOPA (L-3,4- dihydroxyphenylalanine). A protein suitable for use in the present invention has an amino acid sequence in which at least 3 % and preferably 6 - 30 % of the amino acid residues are DOPA. Exemplary peptide units are noted below. The amino acid sequences of these proteins are variable; meaning the scope of the present invention is not limited to the exemplified sub sequences below. A skilled worker would recognize that other proteins from different sources, including re¬ combinantly produced proteins, can be regarded as equivalent. a) Val-Gly-Gly-DOPA-Gly-DOPA-Gly-Ala-Lys b) Ala-Lys-Pro-Ser-Tyr-diHyp-Hyp-Thr-DOPA-Lys cO Thr-Gly-DOPA-Gly-Pro-Gly-DOPA-Lys d) Ala-Gly-DOPA-Gly-Gly-Leu-Lys e) Gly-Pro-DOPA-Val-Pro-Asp-Gly-Pro-Tyr-Asp-Lys f) Gly-Lys-Pro-Ser-Pro-DOPA-Asρ-Pro-Gly-DOPA-Lys g) Gly-DOPA-Lys h) Thr-Gly-DOPA-Ser-Ala-Gly-DOPA-Lys i) Gln-Thr-Gly-DOPA-Val-Pro-Gly-DOPA-Lys j) Gln-Thr-Gly-DOPA-Asρ-Pro-Gly-Tyr-Lys k) Gln-Thr-Gly-DOPA-Leu-Pro-Gly-DOPA-Lys

The term "surface" is to be interpreted broadly and may comprise virtually any face of any item. Examples of surfaces for which the invention is particularly well suited include the outer portion of a contact lens, the portion of a metal pin which will con¬ tact or be contacted by bodily substances when in use, and the lining of multi-well plates that will be contacted by test materials.

Brief Description of the Drawing Figures

Figure 1 schematically represents adhesion of Mefp-1 to a hydrophobic QCM sur¬ face;

Figure 2 depicts the dissipation results measured during binding of Mefp-1 to a QCM surface;

Figure 3 depicts further dissipation results measured during binding of Mefp-1 to a QCM surface;

Figure 4 presents in table form the average decrease in dissipation of a QCM surface after crosslinking the Mefp-1 layer with various agents;

Figure 5 depicts the steps in evaluation the formation of an Mefp-1 layer on a gold QCM surface and the reaction of that layer with blood serum and antibodies; Figure 6 schematically represents both oxidized (crosslinked) and non-oxidized

(non-crosslinked) Mefp-1 layers adsorbing fibrinogen, heparin, or hyaluronic acid layers;

Figure 7 depicts QCM mass measured for two control QCM surfaces containing only a gold layer;

Figure 8 depicts QCM mass measured for two test QCM surfaces containing a gold layer and oxidized Mefp-1;

Figure 9 depicts QCM mass measured for two test QCM surfaces containing a gold layer, oxidized Mefp-1 and heparin;

Figure 10 depicts QCM mass measured for two test QCM surfaces containing a gold layer, oxidized Mefp-1 and fibrinogen;

Figure 11 depicts QCM mass measured for two test QCM surfaces containing a gold layer, non-oxidized Mefp-1 and hyaluronic acid;

Figure 12 depicts QCM mass measured for two test QCM surfaces containing a gold layer, oxidized Mefp-1 and hyaluronic acid;

Figure 13 shows QCM mass for a QCM crystal as a Mefp-1 layer is added, a further protein layer is added, then the surface is cross linked;

Figure 14 depicts QCM mass measured with variances in pH during Mefp-1 binding to a QCM surface;

Figure 15 depicts QCM mass for QCM crystals with various coatings;

Figure 16 tabulates the increase in mass measured for various QCM crystals as a percentage increase;

Figure 17 tabulates the increase in mass measured for various QCM crystals as a measured amount increase;

Figure 18 shows tabulated data of mass measured for selected device coatings; and

Figure 19 shows tabulated data of mass measured for Mefp-1 and select common biomedical application materials.

Detailed Description While the present invention is described herein with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. If sources are not specifically described, materials are known and commercially available.

The present invention describes biomedically-acceptable coating compositions and methods which could be used to coat any surface with such compositions. Composi¬ tions according to the present invention are particularly useful when employed for materials which ideally are to be rendered non-immunogenic, non-toxic, non- irritating, and non-allergenic. Such materials are known in the art today and will continue to be developed as technology progresses.

Example 1 QCM with Titanium or Polymer

One of the accepted methods for evaluating potential immunogenicity is Quartz Crystal Microbalance, as noted above. QCM technology employs a piezoelectric quartz sensor that can indicate change in resonance using electric pulses. Adsorbed proteins increase the mass of the sensor surface, which relates to a decrease in reso¬ nance frequency. Properties of the sensor surface can be monitored, providing an indication of the immunogenicity or the likelihood to cause surface-related blood coagulation. These are often measured in terms of higher or lower immunogenicity or coagulability.

Hydrophilic titanium oxide surfaces generally have high activation of coagulation, but low immunogenicity. To create a titanium surface for testing, titanium particles can be deposited on the crystal in a vacuum. Hydrophobic polymer surfaces such as polystyrene, in contrast, have a low activation of coagulation, but high immuno¬ genicity. Hydrophobic polymer coated crystals can be created by dropwise addition of a polystyrene solution on a sensor surface which is rotating at about 5000 rpm. This method deposits a thin layer of polystyrene on the crystal. Example 2 Titanium or Polymer QCM with Optional Mefp-1 and Immunogenicity Thereof

Crystals with titanium or polystyrene coatings were prepared as described above. A thin layer of Mefp-1 was adsorbed onto the titanium or polystyrene layers by incu¬ bating the titanium or polystyrene coated crystal with aqueous Mefp-1 (lOOug Mefp- 1/ml). Sensors, both those modified only with titanium or polystyrene layers and those with a further Mefp-1 layer were tested in QCM equipment. Fresh human blood serum, which contains immune compliment components, was placed on the surfaces for 30 minutes. Afterward, the surfaces were washed. Rabbit anti-human C3 antibodies were added to the crystals. Thirty (30) minutes after the addition of the rabbit antibodies, the resonance frequency of the crystals was measured.

Surfaces coated with polystyrene alone gave a frequency of 800, indicating a high immune response. With polystyrene and the Mefp-1 coating, though, the compara¬ tive result was only 20. This is the same value as was measured for the titanium- only and titanium with Mefp-1 surfaces. These latter coatings provide a low immu¬ nogenicity. One of the useful properties of Mefp-1 in this regard is its ability to fas¬ ten to both hydrophobic and hydrophilic surfaces, making it attractive both as a coating and as an anchor for other molecules.

Example 3 Titanium or Polymer QCM with Optional Mefp-1 and Coagulability Thereof

The same four coatings were evaluated for their ability to initiate coagulation. Fresh human blood was again applied to the surfaces, in addition to a calcium solution. By continually monitoring the resonance frequency, the time point at which 50% of the blood had coagulated could be determined. The polystyrene alone required 90 min¬ utes for 50% coagulation. This indicates a very low coagulation initiation. In con¬ tract, the polystyrene coated with Mefp-1 had 50% coagulation after only 5 minutes. Titanium alone and titanium plus Mefp-1 coatings had 50% coagulation after 10 and 5 minutes, respectively. Thus, both of the titanium coatings and the polystyrene plus Mefp-1 coatings initiate coagulation rapidly.

Example 4 QCM with Gold and Mefp- 1

Based on the foregoing examples, Mefp-1 coatings were considered able to render a surface less immunoreactive but potentially more likely to induce coagulation. The properties of Mefp-1 coatings which could be crosslinked (e.g., oxidized) or non- crosslmked (non-oxidized), and potentially further coated with proteins were evalu¬ ated. Again, QCM-D measurements were conducted. To create the surface, a crystal was coated with a self-assembly monolayer (SAM) of gold. The gold crystal was washed in a UV/ozone chamber for 10 minutes then placed in a 1 :1 :5 solution of 25% H₂O₂, 30% NH₃ and MQ, respectively, for 5 minutes at 7O⁰C. The surface was made hydrophobic by incubating the crystal for at least 12 hours in HS(CH₂)i₇CH₃ dissolved in hexane. Care was taken to ensure the hexane did not evaporate and that the exposure time was sufficient.

The crystal was then placed in a QCM-D chamber and the chamber was pro¬ grammed. Acetate buffer, 0.1 M (75 mM NaCl, pH 5.5) was added. Once a base line with up to 5 Hz frequency had been maintained over 5 minutes, the program was started. Afterward, the surface of the crystal was exposed to 1 ml Mefp-1 (25 μg/ml) dissolved in acetate buffer for 50 minutes. The surface was washed with 1 ml ace¬ tate buffer for 5 minutes.

Optionally, a crosslinking agent (1 ml) dissolved in acetate buffer was added while the crystal was observed for changes in kinetics. The crosslinking agents employed were 1 mM NaIO₄ and 10 mM Cu²⁺Mn²⁺. After approximately 15 minutes with the agent, the surface was washed with ImI acetate buffer for 5 minutes. This provided a crystal surface that had a Mefp-1 layer over the gold, which layer was oxidized or crosslinked. Figure 1 provides a schematic overview of Mefp-1 coating on a QCM surface, both oxidized and non-oxidized, and Figures 2 and 3 depict dissipation measured when various crosslinking agents were employed. Figure 4 shows those dissipation results in tabular form. Figure 5 correlates the stages of Mefp-1 coating, crosslinking and reacting with blood to the measured mass of the QCM crystal.

While not wanting to be bound by theory, it is believed that of the amino acids of Mefp-1, only alanine and proline are non polar. These are spontaneously attracted to the hydrophobic gold surface, while the rest of the polar and charged groups are re¬ pelled from the surface and hydrolysed. The protein monolayer is built with a large amount of intermingled water molecules, creating a hydrogel-like film. When the crosslinking agent is added, the- water is released creating a rigid protein film. When NaIO₄ is employed as cross linking agent, the DOPA content of Mefp-1 is oxidized by the natural enzyme-like agent to a reactive o-quinone. This group forms di- DOPA bonds in the Mefp-1 protein. When Cu²⁺/Mn²⁺ is used, the copper binds in a complex with DOPA molecules and thereby crosslinks the protein. Even if C3 binds to the crosslinked Mefp-1 coating, there is no conformational change in C3. When C3 binds to an antigen and undergoes a conformational change, the immune re¬ sponse is activated. By presenting a surface to which C3 does not bind, or does bind without changing conformation, immunogenicity is reduced or absent.

Example 5 QCM with Gold, Mefp-1, and Fibrogen, Heparin, or Hyaluronic Acid In addition to creating a gold surface on a QCM crystal and adding a layer of Mefp- 1, further reactions were undertaken to add fibrinogen, heparin, or hyaluronic acid layers on top of the Mefp-1. This resulted in a coating which consisted of two ho¬ mogenous molecule layers. In some circumstances, the Mefp-1 was oxidized prior to the addition of the second layer, in others, not. By oxidizing the Mefp-1 layer, it is rendered relatively non-reactive. It is therefore easier to achieve molecular adher¬ ence to the Mefρ-1 if it is non-oxidized. If the Mefp-1 layer is strongly oxidized, it shrinks and does not stay in contact with the gold surface. The Mefp-1 adheres bet¬ ter to polar surfaces, but will bind well to hydrophobic surfaces such as polystyrene.

Figure 6 provides a schematic overview of the process of adhering fibrinogen, hepa¬ rin, or hyaluronic acid to a Mefp-1 coated gold surface. To the left non-oxidized Mefp-1 is shown, to the right Mefp-1 is oxidized.

Example 6 Comparative Evaluations of QCM with Mefp- 1 Evaluations were made of non-oxidized Mefp-1 on gold, oxidized Mefp-1 on gold, and both oxidized and non-oxidized Mefp-1 on gold with a further layer of fibrino¬ gen, heparin, or hyaluronic acid. Evaluations were generally conducted in duplicate. As control, a plain QCM with gold SAM coating was measured under the same conditions as the actual experimental materials. Results are provided in Figure 7. Measurements were also taken of a QCM with gold and oxidized Mefp-1 surface, see Figure 8.

The QCM which was coated with Mefp-1, oxidized, and reacted to form a heparin layer thereon was evaluated. Data are presented in Figure 9. A slight increase in QCM mass was observed, indicating that heparin does not bind well to oxidized Mefp-1. In contrast, fibrinogen binds well to oxidized Mefp-1, see Figure 10 and the significant increase in mass measured. Hyaluronic acid was also reacted with non- oxidized and oxidized Mefp-1 coated surfaces, see Figures 11 and 12, respectively. The hyaluronic acid utilized with non-oxidized Mefp-1 (Fig. 11) is human hyalu¬ ronic acid, whereas the hyaluronic acid reacted with oxidized Mefp-1 (Fig. 12) is rooster hyaluronic acid. The binding observed with relation to the non-oxidized sur¬ face was greater than that observed on the oxidized surface.

The timing of crosslinking and the pH of the reaction solution play a role in the ad¬ herence of the second monolayer to the Mefp-1 layer. Figure 13 illustrates QCM mass measurements where fibrinogen and hyaluronic acid were bound to non- oxidized Mefρ-1, followed by subsequent oxidation. It illustrates that Mefp-1 will crosslink even if another molecule has bound to the Mefp-1. The effect of pH is de¬ picted in Figure 14, which compares the mass of a QCM during the application of a Mefp-1 layer. One line, that which rises higher, depicts a crystal where the Mefp-1 was deposited under pH 7.4 conditions, the lower line is a measurement with the same factors but for a pH of 3.5 in the reaction solution. With lower pH conditions, lower Mefp-1 protein binding is observed. At the higher pH more binding occurred, although after washing/crosslinking the excess protein dissipated.

Overall evaluations of the immunogenicity of various coatings are shown in Figures 15-17. Figure 15 depicts the measured mass of various QCM crystals. Figure 16 presents the percentage increase in mass in tabular form, whereas Figure 17 depicts,, again in tabular form, the increase in mass in terms of actual values measured. All values represent an average of the duplicate experiments conducted for each QCM coating. As can be seen in Figures 15-17, binding of C3 and subsequent binding of anti-C3 antibodies creates an increased mass. Those QCM with higher measured mass values indicate higher relative immunogenicity of the monolayers formed thereon. For example, the gold SAM alone had the highest measured mass value, meaning it was the most immunogenic and therefore the least desirable coating for use in biomedical applications. A gold SAM QCM with Mefp-1 and fibrinogen monolayers also generates a relatively high immune response.

In contrast to the values of the gold SAM QCM and those with Mefp-1 plus fibrino¬ gen, Mefp-1 plus rooster hyaluronic acid monolayers provides less immunoreactiv- ity, Mefp-1 alone even less, heparin and human hyaluronic acid even less. In terms of biomedical applications, then, a Mefp-1 plus fibrinogen coating is preferred to a gold surface alone. But Mefp-1 with rooster hyaluronic acid is more preferred. Mefp-1 alone is even more preferred. Mefp-1 with heparin or human hyaluronic acid is most preferred in terms of providing the least immunogenic coating for a sur¬ face. Example 7 Further Evaluations of gold surfaces coated with some combination of Mefp-1, Heparin, Fibrogen, and Hyaluronic Acid

A gold SAM was used as the base layer. A Mefp-1 layer was adsorbed thereon for 50 minutes followed by NaIO₄ crosslinking. To the Mefp-1 layer one of the mac- romolecules heparin, fibrinogen, human hyaluronic acid and rooster hyaluronic acid were adsorbed for 15 minutes. The surface was exposed to 5% sera for 20 minutes and the immune response evaluated with anti-C3 antibodies. Control surfaces in¬ cluded gold with Mefp-1, gold with heparin, gold with fibrinogen, gold with human hyaluronic acid, and gold with rooster hyaluronic acid. Evaluations were made in duplicate. Figure 18 presents the results of the evaluation.

As shown in Figure 18, a surface coated with Mefp- 1 as an anchor molecule can be used alone or as a base to produce surface coatings with different properties. One contributing factor to the highly immunoreactive results with the control coatings is the relative inability of the large macromolecules to adhere to the hydrophobic cur- face without Mefp-l. This is particularly evident with heparin, where the measured result shows very little ability to bind and this is more indicative of the reactivity of the gold SAM alone than of gold coated with heparin. Where heparin is able to bind to Mefp-1 it can block the gold SAMs immunoreactivity and results in an overall low immunoreactivity, see Figure 18.

Example 8 Immunoreactivity of Mefp-1 as compared to other surfaces The low immunoreactivity of Mefp-1 as a coating was compared to other surfaces commonly-used in biomedical material applications. The binding of anti-C3 anti¬ bodies was used as a measure of the immune response provoked by certain materi¬ als. Results are depicted in Figure 19, where PS represents polystyrene, G represents glass, SAM represents a gold SAM layer, PET represents polyethylene terephtha- late, PE represents polyethylene, Ti is titanium, and Au is gold. This data evidencing the low immunoreactivity of Mefp-1 as compared to other ma¬ terials used in biomedical applications, combined with the additional data provided herein teaching a heparin coating induces low immunoreactivity when its ability to coat a surface is assisted by a Mefp-1 layer will help improve treatment and research in this critical field.

Example 9 Further Coatings on a Gold/Mefp-1 QCM Crystal In addition to the fibrinogen, heparin, and hyaluronic acid discussed above, it is con¬ templated that further molecules could be used to form a second monolayer above the Mefp-1. For example, dextransulphate, heparansulphate, and other relevant sul- phated/non-sulphated carbohydrates, or other molecules or macromolecules such as PVP (polyvinylpyrrolidone), chitosan or modified polyethylene species.

Example 10 Mefp-1 and Secondary Monolayers on a Medical Device Surface As noted above, surfaces coated with Mefp-1, followed by optional crosslinking and then optional depositing of a second monolayer of heparin or hyaluronic acid pro¬ vide surprisingly good results in terms of low immunogenicity. This makes the ap¬ plication of such coatings to medical device surfaces desirable. For example, artifi¬ cial stents, which are typically open-ended tubular structures, are commonly used to support tubular body conduits. Because their intended use includes long-term pres¬ ence inside a patient's body, and because their use is often in conjunction with treatment of a medical condition, it is critical that they are as biologically and chemically inert as possible as regards their bodily surroundings.

A commercially-available stent could be obtained, such as one made of titanium or a titanium alloy. The stent could be coated with Mefp-1 as taught in the examples above. Such a stent could then be further reacted to form, for example, a heparin monolayer engulfing the outer surface of the stent. After coating, the stent could be used in a patient. Although commercially-available stents could be so coated, the invention is particularly applicable to stents made from materials heretofore less suitable or unsuitable for medical applications because of their reactivity with living tissue. Such materials may be more readily available, cheaper, and/or easier to use than titanium, and would offer the same degree of inertness as titanium when pro¬ vided with a coating according to the present invention.

Other examples of coatable devices include, but are not limited to, contact lenses, insulin pumps and other implantable pumps, pacemakers, implantable defibrillators, replacement organs, including synthetic, xenographic and allographic organs, dental implants, and prosthetic devices such as implants used in restorative or cosmetic surgery.

It is most preferred that all surfaces of a medical or biomedical device which could contact living cells or tissues are coated according to the present invention. How¬ ever, coating only portions of the exposed surfaces may reduce immunogenicity to an acceptable level. Further, there may be applications where it is desired to have a device which is inert with regard to certain of the surrounding tissues and non-inert with regard to others. In such circumstances, coatings on part of a surface or on cer¬ tain surfaces excluding others would be employed to achieve the desired result. A select semi- or non-inert underlying material would also be used in this regard. Fur¬ ther, certain medical devices, such as contact lenses, continuously contact living tis¬ sue only with a portion of their outer surface. In such applications, coating of the surfaces that do not regularly maintain contact with living tissue would be optional.

The foregoing disclosure has been set forth merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments in¬ corporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.

Claims

Claims:

1. An implantable medical device, characterised in that at least a part of the surface of the implantable medical device is coated with a bioadhesive composition com¬ prising a) a bioadhesive polyphenolic protein derived from a byssus-forming mussel, which protein comprises 30-300 amino acids and consisting essentially of tandemly linked peptide repeats comprising 3-15 amino acid residues, wherein at least 3 % and pref¬ erably 6-30 % of the amino acid residues of said bioadhesive polyphenolic protein are DOPA; and, in case the bioadhesive composition is not going to be coated by a further layer b) a non-enzymatic oxidising agent such as hydrogen peroxide, nitroprusside ions or periodate ions.

2. An implantable medical device according to claim 1, characterised in that a further layer comprising at least one compound selected from the group of heparin, hyaluronic acid, dextransulphate, heparansulphate, sulphated carbohydrates, non- sulphated carbohydrates, polyvinylpyrrolidone, chitosan, modified polyethylene species, fibrinogen and polyimine has been coated on the surface of the bioadhesive composition.

3. An implantable medical device according to claim 1, characterised in that the medical device is selected from the group consisting of stents, contact lenses, insulin pumps, pacemakers, implantable defibrillators, replacement organs, dental implants, sutures and prosthetic devices.

4. An implantable medical device according to claim 1, characterised in that the surface of the medical device that is covered with the bioadhesive composition is comprised of a material selected from the group consisting of polyethylene, polyeth¬ ylene terephthalate, polystyrene, glass, gold, and titanium.

5. An implantable medical device according to anyone of claims 1 - 4, for medical use.