Process for applying polymer coating to protein-containing biomaterial

Abstract

The present invention relates to a method of applying a polymeric coating to a protein-containing biomaterial. The method is characterized by the following steps: a) providing a protein-containing biological material (1) isolated or artificially produced from the organism, and b) depositing a plasma polymer coating (8) of at least one polymer (7) on the surface of the protein-containing biological material (1) by plasma-assisted chemical vapor deposition, wherein the polymer coating (8) is covalently bonded to the surface of the protein-containing biomaterial (1). The invention also relates to a polymer-coated protein-containing biomaterial which can be produced by such a method, and to the use of such a material.

Description

Technical Field

[0001] The invention relates to a process for applying a polymer coating to a protein-containing biological material according to claim 1, a material produced by this process according to claim 13 and the use of this material. Background Art

[0002] Xenogeneic materials, especially pericardium, are of great importance in implant technology, for example in pericardial closure surgery, in heart defects, as vascular replacements, in stent-graft systems and in the manufacture of aortic valve implants. Such xenogeneic materials are used both for heart valves implanted by open surgery and for heart valves implanted by catheter (transarterial valve replacement (TAVR)).

[0003] By using stabilizers such as glycerol and / or polyethylene glycol (PEG), in particular pericardium-based heart valve implants are available which can be dried for storage.

[0004] Immobilization of drugs in polymer matrices is challenging for heterogeneous materials.

[0005] The pericardium exhibits outstanding biocompatibility and mechanical properties, in particular due to the combination of collagen and elastin fibers in the pericardium. However, the pericardium and other protein-containing or protein-based materials are susceptible to denaturation processes that destroy the structural properties of these materials. This is particularly relevant when such materials are used as implants. This is because the surface of the implant plays a major role in the organism's response to this foreign body. This includes in particular rejection reactions, thrombosis, calcification tendency, fibrotic reactions, cell colonization (especially endothelialization) and susceptibility to infection. Summary of the invention

[0006] The object of the present invention is to provide a method for improving the properties of biomaterials such as pericardium, in particular with regard to the biocompatibility of these materials. Another object of the present invention is to provide corresponding materials and to indicate possible uses of such materials.

[0007] This object is achieved by a method having the features of claim 1. The method is used for applying a polymer coating to a protein-containing biomaterial. The method is characterized by the steps explained below.

[0008] First, a protein-containing biological material is provided which has been isolated from an organism or artificially produced. The steps of isolation or production are not covered by the scope of the present application. Instead, the process currently claimed is a pure in vitro process carried out outside the human or animal body. The protein-containing biological material has a solid aggregate state, i.e. it is a solid material, and is therefore distinguished from a liquid material such as a body fluid.

[0009] In a further process step, a polymer coating is deposited on the surface of the protein-containing biomaterial. This is done by plasma enhanced chemical vapor deposition (PECVD) or plasma assisted chemical vapor deposition (PACVD). The polymer coating is covalently bonded to the surface of the protein-containing biomaterial during the deposition process. Thus, the process produces a biocomposite material having a covalently bonded polymer coating on its surface.

[0010] By this polymer coating, the improvement of the properties (i.e. biomaterial properties) of protein-containing biomaterials is achieved. When protein-containing biomaterials are used as implants, these improved biomaterial properties are particularly effective. In particular, compared with uncoated protein-containing biomaterials, the improvement of biomaterial properties relates to calcification, inflammatory potential, implant-tissue interaction, homogeneity, biocompatibility, cell colonization and / or fluid mechanical properties of the protein-containing biomaterials of coating. In addition, polymer coatings can be used as the wear protection and / or adhesion promoter layer of another coating. In addition, polymer coatings can be used as the basis of subsequent chemical functionalization, thereby being able to simplify the functionalization of protein-containing biomaterials. In all of these, the characteristic structure of protein-containing biomaterials is retained by the appropriate pretreatment required for vacuum technology. In addition, inherent material properties are not affected, particularly about the mechanical behavior of protein-containing biomaterials.

[0011] The method claimed in the present application thus provides a means of modifying the surface of a protein-containing biomaterial, such as the pericardium, in such a way that undesired reactions with the material during subsequent implantation of the material are avoided, while retaining the mechanical properties and the essentially characteristic surface structure of the material. Thus, by means of the method claimed in the present invention, physical and in particular chemical surface properties are modified in an advantageous manner, rather than structural surface properties.

[0012] In particular, the protein-containing biomaterial is an implantable material, such that it can be used to prepare an implant (before or after its coating) which can subsequently be implanted into a patient.

[0013] In one process variant, the protein-containing biomaterial does not originate from the patient in which it is subsequently implanted as an implant, but rather from another organism. In other words, it is a xenogeneic material. In this respect, protein-containing biomaterials of porcine or bovine origin are particularly suitable for the present method. Materials originating from humans other than the human patient in which the material is subsequently implanted as an implant are also very suitable for coating by the claimed process.

[0014] In another process variant, the protein-containing biomaterial is an autologous material, i.e. a material derived from the patient, which is subsequently implanted in the patient in the form of an implant. Many difficulties that arise when a foreign material is later implanted are avoided by using an autologous material. For example, no immune response to the autologous material is expected. However, surface refinement of the autologous material by the method claimed herein can be well used to allow the protein-containing biomaterial to grow better at its subsequent implantation site. In addition, improvements in the mechanical properties of the protein-containing biomaterial can be achieved by applying a polymer coating, which is also advantageous for the autologous material.

[0015] In one process variant, the polymer of the polymer coating is produced from at least one monomer during plasma-assisted chemical vapor deposition. The at least one monomer is selected from the group consisting of siloxanes, functionalized allyl compounds, acrylates, pyrroles, thiophenes, furan compounds, anilines and epoxides. All these monomer compounds are particularly suitable for polymerization into polymers by free radical polymerization and thus covalently attached to the surface of the protein-containing biomaterial.

[0016] According to one process variant, the polymer is formed from at least one siloxane during plasma-assisted chemical vapor deposition. Siloxanes have proven to be particularly suitable compounds for forming polymer coatings on protein-containing biomaterials. In this respect, they can change the surface chemistry of protein-containing biomaterials in a particularly advantageous manner and thus significantly improve the quality of implants made of polymer-coated protein-containing biomaterials. For example, according to in vitro tests conducted to date, the risk of undesired calcification of protein-containing biomaterials can be virtually eliminated by using polymer coatings of siloxanes. As a result, the service life of implants made of protein-containing biomaterials coated with such polymers is significantly extended.

[0017] The at least one siloxane may include or be hexamethylene disiloxane (HMDSO), 2,6-diphenylhexamethylcyclotetrasiloxane, octamethyltrisiloxane, decamethylcyclopentasiloxane, tetramethylcyclotetrasiloxane and / or octamethylcyclotetrasiloxane. In one process variant, the at least one siloxane includes hexamethylene disiloxane (HMDSO), in particular, the at least one siloxane is HMDSO.

[0018] According to a process variant, the polymer coating has a layer thickness of 0.1 nm to 10 μm, in particular 1 nm to 1 μm, in particular 200 nm to 900 nm, in particular 300 nm to 800 nm, in particular 400 nm to 700 nm, in particular 500 nm to 600 nm. Such ultrathin polymer coatings are significantly thinner than coatings that can be produced, for example, by dip coating, spraying or electrospraying. In addition, the polymer coating produced according to the method of the invention is more uniformly and more firmly anchored to the substrate (i.e. the protein-containing biomaterial) than in the case of the above-mentioned coating methods. Due to the covalent bonding of the polymer coating with a very small layer thickness at the same time, the polymer coating follows the morphology of the underlying material on the surface of the protein-containing biomaterial.

[0019] According to a process variant, the deposition of the polymer coating on the surface of the protein-containing biomaterial is not carried out in the entire area of ​​the surface, but only in partial areas. For this purpose, for example, a cover or template can be used to achieve a specific structuring of the polymer coating relative to the surface of the protein-containing biomaterial. In this way, certain areas of the protein-containing biomaterial can be changed in terms of its chemical surface properties, while other areas remain in an unchanged form. This can simplify the subsequent processing of the polymer-coated protein-containing biomaterial (for example, when it is fixed to certain holding structures). At the same time, the surface coating on other areas there improves the material properties.

[0020] According to a process variant, the polymer coating is metal-free. In particular, the polymer does not contain titanium, tantalum, nickel, niobium, nitinol, zirconium, hafnium, iridium, gold, lead, platinum, silver and / or copper. According to the solutions described in the prior art, such metals are used for surface coatings of biomaterials. However, this greatly changes the structural surface properties of the corresponding materials. In addition, the metal coatings are very porous and brittle, which means that direct contact between the surface of the protein-containing biomaterial and its environment is still possible despite the corresponding metal coating. Therefore, such metal coatings cannot prevent adverse environmental influences on the protein-containing biomaterial. The situation is completely different from the polymer coating applied to the protein-containing biomaterial according to the method claimed in the present application. This can completely prevent direct contact between the original surface of the protein-containing biomaterial and its environment. Silicon, in particular silicon in the form of siloxane or polysiloxane, is not a metal within the meaning of the present disclosure.

[0021] According to one process variant, the polymer coating has no amorphous carbon.

[0022] According to a process variant, the protein-containing biomaterial comprises collagen. Therefore, in this variant, it is a collagen-containing material. A collagen-containing material is a material comprising collagen. Collagen is the most abundant protein in the human body. A collagen-containing material, in particular a collagen-containing tissue, can be, for example, a connective tissue, such as cartilage, bone, tendon, ligament or skin. Collagen-containing materials of viscera, cornea, blood vessels, heart valves, intestines or intervertebral discs can also be used. Preferably, the collagen-containing tissue can be the pericardium, myocardium, connective tissue, tendon, peritoneal tissue, pleura, dura mater, mucosa (mucosa) or Teller submucosa, in particular the submucosa of the gastrointestinal tract (e.g., the submucosa of the small intestine).

[0023] The protein-containing biological material, in particular the collagen material, may originate from humans, pigs, sheep, goats, horses, crocodiles, kangaroos, ostriches, monkeys, preferably primates, octopuses, rabbits or cows.

[0024] In one process variant, the protein-containing biological material is a biological tissue. The pericardium is a particularly suitable biological tissue for carrying out the method claimed here.

[0025] According to one process variant, the protein-containing biomaterial is dried / subjected to a drying step (i.e. the water content of the provided protein-containing biomaterial is reduced by at least 50 wt%, preferably at least 90 wt%) prior to depositing the polymer coating. Thus, the protein-containing biomaterial, in particular collagen-containing tissue, such as the pericardium, has a water content of less than 50 wt%, in particular less than 10 wt%, more in particular less than 7 wt%. Drying prior to deposition effectively prevents damage or destruction of the protein-containing biomaterial during plasma-assisted chemical vapor deposition (PACVD). Since such vapor deposition takes place under reduced pressure (compared to atmospheric pressure), when using materials with a high water content, spontaneous evaporation of water occurs when the pressure is reduced to the operating pressure of the process. This may be accompanied by (irreversible) material damage. By pre-drying the material, such damage can be effectively avoided.

[0026] According to one process variant, the protein-containing biomaterial is stabilized with a stabilizer before the polymer coating is deposited. This stabilization allows the material to be dried in a particularly gentle manner. This is also referred to as stabilized dry material or stabilized dry tissue. This stabilized drying of a protein-containing biomaterial is explained in more detail below using collagen-containing tissue as a non-limiting example of a protein-containing biomaterial.

[0027] In collagen-containing tissues, a distinction is made between structural water, bound water and free water. Structural water, also called tightly bound water, is associated with the stabilization of the triple helical structure of collagen. The intermolecular water molecules between the triple helix and the microfibrils are called bound water or contact water. Between the microfibrils and the fibrils is free, unbound water. When the water is removed, the collagen fibers lose their flexibility. If the removal of water is limited to free, unbound water, the process is reversible. However, if the drying process leads to the removal of bound water molecules, the fiber network is irreversibly damaged. The lack of water causes changes in the tissue conformation, which are accompanied by an increase in intramolecular and possibly also intermolecular hydrogen bonds. Complete rehydration of the fiber composite is therefore excluded. The elastic properties of the collagen fibers are lost and the tissue becomes brittle and fragile.

[0028] In order to achieve that the structure of the tissue remains dry, ie stabilizes the dryness, at least one stabilizer is used, in particular a hygroscopic exchange substance, which stabilizes the collagen structure when removing water. In this way, rehydration makes it possible to restore the original state of the tissue.

[0029] Suitable stabilizers are all biocompatible and nonvolatile hygroscopic exchange substances, in particular hydrophilic hydrocarbons having a large number of hydroxyl groups, for example water-soluble sugar alcohols, such as glycerol, ethylene glycol or polyethylene glycol.

[0030] The hygroscopic exchange substance can be, for example, glycerol, or a mixture of two or more hygroscopic exchange substances, for example glycerol and polyethylene glycol (PEG) (possibly PEG with different molecular weights). The hygroscopic exchange substances can be added individually, for example one after the other, or used as a mixture of two substances, for example glycerol dissolved in polyethylene glycol. Particularly suitable is the sequence of glycerol in the first stabilization step, followed by a first PEG stabilization (for example with PEG200, i.e. a PEG with a molecular weight of about 200 g / mol), followed by a second PEG stabilization (for example with PEG400, i.e. a PEG with a molecular weight of about 400 g / mol).

[0031] The second mentioned variant of two or more hygroscopic exchange materials also includes embodiments in which the same exchange material is used, for example polyethylene glycol, but in different forms, for example with different molecular weights.

[0032] All of the above substances are characterized by their hygroscopicity, low toxicity values ​​and excellent water solubility. The ability to absorb and bind water from the environment opens up a wide range of applications for glycerol and polyethylene glycol in various suitable mixing ratios for the stabilization of collagen-containing tissues.

[0033] In a particularly suitable stabilization step, at least one solution, e.g., an aqueous solution, comprising glycerol and / or polyethylene glycol is added to the fabric. In this step, either or both of these solutions are added to the tissue sequentially in any order and composition, as a first and second solution, or both solutions are added simultaneously as a solution mixture. When drying the tissue, e.g., for storage or transportation of the tissue, the stabilization process is preferably performed prior to drying.

[0034] Optionally, decellularization of the collagen-containing tissue can be performed, for example, using surfactin and deoxycholic acid, DNase, α-galactosidase treatment, etc., to produce a collagen-containing tissue with a reduced immune response.

[0035] As a non-limiting example, a stabilization process can be performed, for example, after decellularization by immersing the collagen-containing tissue in a series of one or more stabilizing solutions of glycerol and / or polyethylene glycol to fully saturate the tissue with the stabilizer and ultimately introduce the stabilized tissue into a subsequent drying process.

[0036] In the method claimed herein, covalent bonds are formed between the polymer coating and the surface of the protein-containing biomaterial. On the other hand, if the material surface is completely formed by stabilizers such as glycerol or polyethylene glycol, the polymer coating may not be covalently bonded to the surface of the material, but to these stabilizers. When the material is rehydrated, the polymer coating will subsequently be lost together with the stabilizer flushed out by the rehydration. On the other hand, if the entire material surface is not completely formed by stabilizers, only the kinetics of rehydration are affected by the polymer coating. Therefore, the quantity and quality of the subsequent polymer coating can be influenced by adaptive process control during the stabilization drying period.

[0037] In order to make the surface of the protein-containing biomaterial as close as possible to the polymer coating, it is useful that the surface of the material to be coated has as little aggregation stabilizer as possible (as described above, such stabilizers are used in the process of drying the material). In a process variant, only one or two low molecular weight stabilizers are used to stabilize the material. Glycerol and / or PEG with a molecular weight of less than 200 g / mol are particularly suitable. In this process variant, the stabilizer is only used in a concentration significantly lower than the concentration described in US10,390,946B2 or WO2022 / 090419A1. In the following, as an example of a protein-containing biomaterial, an exemplary process control for stabilizing and drying a protein-containing biomaterial starting from a fixed or native tissue is explained.

[0038] First, the tissue is rinsed in a saline solution (eg, 0.9% saline solution) with gentle agitation. All percentage concentrations mentioned in this example refer to mass percentage (w / v).

[0039] The tissue is then rinsed in a 1% to 5%, in particular 3% glycerol solution (preferably in ultrapure water) for 5 to 60 minutes, in particular 10 to 50 minutes, in particular 20 to 40 minutes, in particular 15 to 30 minutes, in particular 15 minutes, with gentle agitation. If the tissue has a sample size of, for example, 6 cm×8 cm, a volume of 100 ml of glycerol solution is very suitable for this process step.

[0040] Subsequently, the tissue is rinsed in a 2% to 6%, especially 4% PEG200 solution (again in ultrapure water) for 5 to 60 minutes, especially 10 to 50 minutes, especially 20 to 40 minutes, especially 15 to 30 minutes, especially 15 minutes, while gently agitating. If the tissue has a sample size of, for example, 6 cm×8 cm, a 100 mL volume of PEG200 solution is very suitable for this process step.

[0041] The stabilized fabric is then dried in a climate chamber by reducing the relative humidity from 80 to 100%, in particular 90 to 95%, to 5 to 20%, in particular 10%, at 30 to 50°C, in particular 35 to 40°C.

[0042] Tissue stabilized and dried in this way can be removed and stored.

[0043] As described above, stabilization with only one stabilizer is possible. Thus, if desired, rinsing the tissue in glycerol or rinsing the tissue in PEG200 can be omitted. The concentration of the stabilizer used according to the above examples is one order of magnitude lower than that described in the works US 10,390,946 B2 or WO 2022 / 090419 A1. As a result, the matrix of the protein-containing biomaterial is not filled with stabilizer, but can still be used for subsequent polymer coating.

[0044] In order to further increase the accessibility of the stabilized dry fabric for subsequent polymer coating, the stabilizer present on the surface of the stabilized dry fabric can optionally be removed by a brief rinse with anhydrous ethanol or anhydrous methanol. Instead of this rinse, the material sample can also be wiped with a cloth soaked in anhydrous ethanol or anhydrous methanol. Any residual ethanol or methanol will evaporate very quickly. What is left is then the surface of the protein-containing biomaterial free of stabilizer. The deposition of the polymer coating on the surface of the protein-containing biomaterial by PECVD can then be carried out in a particularly advantageous manner.

[0045] In one process variant, the stabilizer exposed on the surface is selectively removed only locally. This makes it possible to provide the protein-containing biomaterial with a polymer coating only in partial areas. Thus, in this way, a structuring of the polymer coating can be achieved.

[0046] According to a process variant, the complete encapsulation of the surface of the protein-containing biomaterial coated with a polymer is carried out, on the one hand, to protect the functional groups applied by the polymer coating and to maintain the chemical and morphological properties thereof, and on the other hand, to protect the protein-containing biomaterial for subsequent storage and stabilization. Such encapsulation is achieved by wetting the polymer-coated material with a stabilizer such as polyethylene glycol (PEG400) having a molecular weight of about 400 g / mol. The material so wetted is then dried in air or in a climate chamber. This results in the complete encapsulation of the material functionalized by means of the polymer coating.

[0047] According to one process variant, the polymer-coated protein-containing biomaterial is intended to be fully rehydrated and then dried by a suitable drying process, such as described in US 10,390,946 B2 or WO 2022 / 090419 A1. These process steps are explained in more detail below.

[0048] First, the polymer-coated protein-containing biomaterial is rinsed several times in a saline solution (eg, 0.9% saline solution) while gently stirring.

[0049] The material is then rinsed in a 20 to 40%, in particular 30%, glycerol solution (in particular ultrapure water) for 5 to 60 minutes, in particular 10 to 50 minutes, in particular 20 to 40 minutes, in particular 15 to 30 minutes, in particular 15 minutes, with gentle stirring. For material samples with a size of about 6 cm×8 cm, 100 ml of glycerol solution are very suitable for this purpose.

[0050] Subsequently, the material is rinsed in a 30 to 50%, in particular 40% PEG200 solution (in particular ultrapure water) for 5 to 60 minutes, in particular 10 to 50 minutes, in particular 20 to 40 minutes, in particular 15 to 30 minutes, in particular 15 minutes, with gentle stirring. For the given exemplary sample size, 100 ml of PEG200 solution is also very suitable for this purpose.

[0051] Subsequently, the material is rinsed in a 30 to 50%, especially 40% PEG400 solution (especially ultrapure water) for 5 to 60 minutes, especially 10 to 50 minutes, especially 20 to 40 minutes, especially 15 to 30 minutes, especially 15 minutes, while gently stirring. For this purpose, if the exemplary sample size remains constant, a 100 mL PEG400 solution is also very suitable.

[0052] The material stabilized in this way is then dried in a climate chamber by reducing the relative humidity from 80% to 100%, in particular 90% to 95%, to 5% to 20%, in particular 10%, at 30° C. to 50° C., in particular 35° C. to 40° C., for a period of 6 to 24 hours, in particular 10 to 18 hours, in particular 12 hours.

[0053] Finally, the polymer-coated and stabilized dry material is removed and stored.

[0054] In principle, it is not important at which stage the protein-containing biomaterial to be coated is applied for the method claimed herein. For example, the polymer coating can be applied immediately after the removal of the biological protein-containing material (and, if necessary, after appropriate purification). However, according to a process variant, it is envisaged that the protein-containing biomaterial is first made into a form for later use. For example, the material can be made into the shape of a heart valve by a pressing process. According to this process variant, the polymer coating is not performed until the material to be coated has assumed the desired shape. For example, a heart valve can also be formed from the protein-containing biomaterial and attached to a stent structure. The polymer coating is then applied to the shaped protein-containing biomaterial that has been attached to the stent structure.

[0055] According to a process variant, the polymer coating is functionalized with an active ingredient in a process step after the polymer coating is deposited. The functionalization of the polymer coating can be carried out, for example, by wet chemical means. In other words, in this process variant, the polymer coating is used as a substrate coating for subsequent functionalization with an active ingredient. Suitable active ingredients are, for example, active ingredients for improving subsequent implant-tissue interactions, for example, for controlling or improving cell proliferation. Particularly suitable active ingredients are, for example, active ingredients from the class of mTOR inhibitors, in particular rapamycin derivatives, such as sirolimus, everolimus, zotarolimus or biolimus.

[0056] In a process variant, the active ingredient is applied to a polymer coating in a polymer matrix with the active ingredient. The polymer matrix can be composed of one or more polymers selected from the group consisting of polyester, polyamide, polyurethane, polycarbonate, polysaccharide, polyether, polyethylene and halogenated polyethylene. However, in another variant, it is also possible to use an active ingredient without such a polymer matrix, i.e. as an active ingredient without a polymer matrix. In particular, the active ingredient is used for local release of the active ingredient on the surface of a protein-containing biomaterial. When the material is subsequently used as an implant, the implant adjusts the local release of the active ingredient.

[0057] According to one process variant, drying of the coated material takes place after deposition of the polymer coating, in particular by means of stabilized drying with a stabilizer, in order to protect the polymer-coated protein-containing biomaterial and the functional groups to which the coating is applied (i.e. contained in the polymer coating) in a particularly gentle and sustainable manner.

[0058] According to one process variant, the protein-containing biomaterial is fixed by a suitable fixative before the polymer coating is deposited. Glutaraldehyde is a particularly suitable fixative. This fixation achieves a particularly high tissue compatibility, but generally also the biodegradability of the biomaterial. Since the polymer coating applied to the protein-containing biomaterial also already imparts a high tissue compatibility, this fixation is generally not required. Therefore, according to another process variant, the corresponding fixation, in particular the use of glutaraldehyde, is explicitly omitted. In other words, in this process variant, a protein-containing biomaterial is used, which has not been previously treated with a fixative, in particular with glutaraldehyde, and has not been treated with such a fixative, in particular with glutaraldehyde, during the method claimed herein.

[0059] Whether or not a fixative is used, alternative or additional pretreatment of the protein-containing biomaterial may be performed. For example, decellularization may be performed. Alternatively or additionally, removal of free aldehyde groups (so-called capping) may be performed to reduce the calcification potential. Such pretreated materials are particularly suitable or even necessary for long-term stable implants, in which enzymatic degradation of the protein-containing biomaterial is not possible.

[0060] By selecting the process parameters of the vapor phase 12 deposition, the stoichiometry of the applied polymer coating as well as its structural composition, for example its layer thickness, can be varied.

[0061] According to one process variant, the vapor deposition is carried out at a temperature in the range of 15°C to 40°C, in particular 20°C to 37°C, especially 25°C to 30°C.

[0062] According to one process variant, vapor deposition is carried out at a pressure in the range of 0.1 mbar to 1.5 mbar, in particular in the range of 0.2 mbar to 1.4 mbar, in particular in the range of 0.3 mbar to 1.3 mbar, in particular in the range of 0.4 mbar to 1.2 mbar, in particular in the range of 0.5 mbar to 1.1 mbar, in particular in the range of 0.6 mbar to 1.0 mbar, in particular in the range of 0.7 mbar to 0.9 mbar, in particular in the range of 0.75 mbar to 0.8 mbar.

[0063] According to a process variant, the vapor deposition is carried out within a reaction time in the range of 1 minute to 60 minutes, in particular in the range of 2 minutes to 50 minutes, in particular in the range of 3 minutes to 40 minutes, in particular in the range of 4 minutes to 30 minutes, in particular in the range of 5 minutes to 20 minutes, in particular in the range of 6 minutes to 15 minutes, in particular in the range of 7 minutes to 14 minutes, in particular in the range of 8 minutes to 13 minutes, in particular in the range of 9 minutes to 12 minutes, in particular in the range of 10 minutes to 11 minutes.

[0064] According to one process variant, the vapor deposition is carried out in an atmosphere saturated with monomers which react during the vapor deposition to form polymers which form the polymer coating.

[0065] According to one process variant, the vapor deposition is carried out in an atmosphere comprising oxygen as an oxidant. According to a process variant, oxygen is introduced into the reaction chamber at a gas flow of 1 standard cubic centimeter per minute (sccm) to 20 sccm, in particular 2 sccm to 15 sccm, in particular 3 sccm to 10 sccm, in particular 4 sccm to 9 sccm, in particular 5 sccm to 8 sccm, in particular 6 sccm to 7 sccm.

[0066] According to one process variant, the vapor deposition is carried out in an atmosphere containing ammonia as a reducing agent. According to a process variant, ammonia is introduced into the reaction chamber with a gas flow of 1 to 20 sccm, in particular 2 to 15 sccm, in particular 3 to 10 sccm, in particular 4 to 9 sccm, in particular 5 to 8 sccm, in particular 6 to 7 sccm.

[0067] According to one process variant, the vapor deposition is carried out using a pulsed plasma.

[0068] According to one process variant, the vapor deposition is carried out with an excitation frequency for the plasma to be generated, which is in the range of 13.56 MHz, 2.45 GHz or 1 kHz to 100 kHz, in particular 10 kHz to 400 kHz, in particular 50 kHz to 300 kHz, in particular 100 kHz to 200 kHz, in particular 200 kHz to 300 MHz, in particular 300 kHz to 400 kHz, in particular 400 kHz to 500 MHz, in particular 500 kHz to 600 kHz, in particular 600 kHz to 700 kHz, in particular 700 kHz to 800 kHz, in particular 800 kHz to 900 kHz, in particular 900 kHz to 1 MHz.

[0069] Such an excitation frequency can be generated, for example, using a kilohertz generator, a radio frequency generator or a microwave generator.

[0070] One aspect of the present invention relates to a polymer-coated protein-containing biomaterial, which can be prepared by the method according to the above explanation, and more specifically by the method according to the above explanation. Such a polymer-coated protein-containing biomaterial can also be called a composite biomaterial. It comprises a protein-containing biomaterial and a polymer coating deposited on the surface of the protein-containing biomaterial, the polymer coating being covalently bonded to the material surface.

[0071] According to one variant, the polymer coating prevents volume changes of the polymer-coated protein-containing biomaterial during rehydration. This results in particularly good stability of the material and objects made therefrom, such as implants.

[0072] According to one embodiment, the polymer coating reduces the risk of calcification of the protein-containing biomaterial or completely prevents such calcification because the polymer coating acts like a firmly anchored hydrophobic top layer through its covalent bonds to the surface of the protein-containing biomaterial, preventing calcium ions from penetrating the biomaterial.

[0073] According to one embodiment, the polymer coating provides functional groups on the surface of the composite material, which improves the cell colonization ability of the composite material. This is because such functional groups on the surface of the composite material improve the adhesion of cells to the surface.

[0074] According to one embodiment, the polymer-coated protein-containing biomaterial exhibits anti-thrombotic properties. This can be achieved by the polymer coating reducing or preventing surface binding of thrombotic proteins and / or platelet adhesion. Thus, by such embodiments of the polymer coating, interruption of the thrombotic cascade is achieved, resulting in the anti-thrombotic properties of the composite material.

[0075] According to one variant, the composite material has anti-infective properties. This can be achieved, for example, by reducing and preventing the surface binding of biofilm proteins through the polymer coating. Alternatively or additionally, bacterial adhesion can also be reduced or prevented. As a result, biofilm formation is prevented or the risk of such biofilm formation is significantly reduced.

[0076] According to one variant, the surface morphology of the polymer-coated protein-containing biomaterial corresponds to the surface morphology of the still uncoated protein-containing biomaterial, i.e. the material before the step of depositing the polymer coating. Alternatively or additionally, the polymer-coated protein-containing biomaterial exhibits structural-mechanical properties, in particular elastic modulus, elongation at break and / or breaking force, which correspond to the structural-mechanical properties of the still uncoated protein-containing biomaterial. This correspondence of structural-mechanical properties can be assumed if the quantitative values ​​used to describe the structural-mechanical properties correspond to the corresponding values ​​of the uncoated protein-containing biomaterial by at least 80%, in particular by at least 85%, in particular by at least 90%, in particular by at least 95%, in particular by about 100%.

[0077] Another aspect of the invention relates to the use of such polymer-coated protein-containing materials in the form of implants in the human or animal body. The implant can be designed, for example, as a so-called implant graft. Particularly suitable examples of implants are heart valve replacement prostheses (such as pericardial-based heart valve replacement prostheses) and (coated) stent systems, which are used to treat acute vascular perforations or (abdominal) aortic aneurysms (endovascular aortic repair (EVAR) and thoracic endovascular aortic repair (TEVAR)), respectively.

[0078] According to another aspect, the present invention relates to a medical method for implanting an implant into a human or animal body, wherein the implant comprises a protein-containing biomaterial coated with a polymer according to the above explanation or consists of a protein-containing biomaterial coated with a polymer according to the above explanation. This medical method is directed to patients who need corresponding implants. In this implantation method, for example, before an implant formed with or by a protein-containing biomaterial coated with a polymer as described herein is implanted into a patient's body, an existing endogenous structure or element can be first removed. The implantation of this implant is no different from the implantation of a conventional implant. However, due to the improved surface properties of the implant, the biocompatibility is significantly improved compared to an implant made of or by a material known in the prior art. Therefore, this implantation surgery provides medical advantages over prior art surgery.

[0079] Another aspect of the invention relates to an implant comprising or consisting of a protein-containing biomaterial coated with a polymer as previously explained. Particularly suitable examples of such implants are heart valve replacement prostheses (e.g. pericardium-based heart valve replacement prostheses) and (covered) stent systems for the treatment of acute vascular perforations or (abdominal) aortic aneurysms.

[0080] All variants and embodiments of the described processes can be combined with one another in any way and can be transferred to polymer-coated protein-containing biomaterials, their uses, implants or corresponding other processes, individually or in any combination. Similarly, all variants and embodiments of polymer-coated protein-containing biomaterials can be combined with one another in any way and can be transferred to methods, uses of materials and implants, individually or in any combination. In addition, all variants and embodiments of the uses of polymer-coated protein-containing biomaterials can be combined with one another in any way and can be applied to methods, polymer-coated protein-containing biomaterials and implants, individually or in any combination. In addition, all variants and embodiments of implants can be combined with one another in any way and can be transferred to the described methods, polymer-coated protein-containing biomaterials and their uses, individually or in any combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Further details of various aspects of the invention are described in more detail below with reference to the examples and figures.

[0082] Figure 1 Schematic diagram of an embodiment of a process for depositing a polymer coating on a protein-containing biomaterial by plasma-assisted chemical vapor deposition;

[0083] Figures 2A-2D Scanning electron microscope images of pig pericardium samples with different treatments

[0084] Figure 3 Spectra of the pericardium obtained by energy dispersive X-ray spectroscopy (EDX) after deposition of a plasma polymer coating on the pericardium;

[0085] Figure 4 Two spectra of plasma polymer coated porcine pericardium obtained by attenuated total reflection infrared spectroscopy, with coated aluminum as reference;

[0086] Figure 5A-5B Scanning electron microscopy images of porcine pericardium coated with plasma polymer before and after continuous stress test

[0087] Figure 6 EDX-derived spectra of plasma polymer-coated pericardium after continuous stress testing

[0088] Figure 7 Stress-strain curves of pig pericardium samples treated with different methods

[0089] Figure 8 Experimental results of in vitro calcification of porcine pericardial samples treated differently. DETAILED DESCRIPTION

[0090] Figure 1A schematic diagram of the surface finishing of a heterogeneous material is shown as an example of a process for applying a plasma polymer coating to a protein-containing biomaterial. The protein-containing biomaterial used in this embodiment example is a porcine pericardium 1, which is formed into a valve leaflet and sutured to a stent 2 as a supporting structure. The pericardium 1 formed into a valve leaflet and the stent 2 can be used as an artificial aortic valve prosthesis and implanted with the aid of a catheter as part of a transcatheter aortic valve implantation (TAVI) procedure. Via a gas supply device 3, a monomer 4 is introduced into a reaction chamber in which a plasma-assisted chemical vapor deposition is performed. For this purpose, a low-pressure plasma 6 is generated with the aid of a plasma source 5. The low-pressure plasma 6 leads to the formation of free radicals and ions of the monomer 4, which subsequently recombine and form a polymer 7. The polymer 7 then forms a plasma polymer coating 8 on the pericardium 1.

[0091] like Figure 1 As shown, plasma enhanced chemical vapor deposition can be performed in situ, that is, after the pericardium 1 to be coated is actually formed. Therefore, the plasma enhanced chemical vapor deposition process is suitable for coating complex materials. In this case, certain areas or sections of the pericardium 1 and / or the stent 2 can be masked to avoid the formation of the plasma polymer coating 8 on these sections or areas.

[0092] This is explained in more detail with reference to the example embodiments shown below. Figures 2A to 8 .

[0093] First, porcine pericardium was stabilized as a protein-containing biomaterial using glycerol and polyethylene glycol (PEG200) with an average molecular weight of 200 g / mol. Subsequently, pre-drying was performed at 37°C overnight under ambient pressure and ambient humidity. Further drying was then performed within 48 hours by successively reducing the pressure to 60 mbar.

[0094] The material obtained in this way is coated on both sides with a polymer coating. In principle, it is also possible to coat only one side of the pericardium. The polymer coating is applied by plasma enhanced chemical vapor deposition (PECVD). Figure 1 It is explained that monomers excited by the low-pressure plasma polymerize on the pericardial surface and are deposited there as a polymer coating.

[0095] In this example, hexamethylene disiloxane (HMDSO) was used as a monomer, which was then deposited on the surface of the pericardium in the form of a polymer layer in the form of polymerized HDMSO (pHMDSO). PECVD was performed at a process pressure of 0.2 to 0.6 mbar and a chamber temperature of 25° C. to 30° C. with a reaction time of 10 minutes. A 10-watt radio frequency generator was used to generate a low-pressure plasma; the excitation frequency was 13.56 MHz. The coated pericardium thus obtained will be referred to as pHMDSO-coated pericardium in the further explanation of the figures.

[0096] Figure 2A Shown is a SEM image of porcine pericardium dried using hexamethylenedisilazane (HMDS). Figure 2B Shown is an SEM image of porcine pericardium stabilized by drying using glycerol / PEG200. Figure 2C SEM images of pHMDSO-coated pericardium are shown (after polymer coating with HMDSO using PECVD as described above, according to Figure 2B Finally, Figure 2D SEM images of stabilized dried pericardium after plasma coating are shown (according to Figure 2C ), where the stabilizer is subsequently washed out and the pericardium is freeze-dried. It can be clearly seen that different treatments of the pericardium result in different surface structures. In particular, it is easy to see that drying the pericardium using HMDS results in a significantly different surface structure than drying the pericardium using glycerol / PEG200 and then coating it with HMDSO using PECVD.

[0097] Figure 3 The spectrum of pHMDSO-coated pericardium obtained by energy dispersive X-ray spectroscopy (EDX) is shown. The NaCl signal comes from the residue of the washing solution used to remove the stabilizer. The unlabeled signal comes from the gold in the sputtered layer used to perform SEM. Based on the clearly discernible silicon signal, EDX was able to demonstrate the successful coating of the pericardium with pHMDSO.

[0098] Figure 4 Two spectra obtained by attenuated total reflection infrared spectroscopy (ATR-IR spectroscopy) are shown. The upper spectrum was recorded on pHMDSO-coated pericardium, while the lower spectrum was recorded on an aluminum support coated with pHMDSO as a reference. The characteristic pHMDSO bands are marked with the corresponding wavenumbers.

[0099] Figure 5A Another SEM image of pHMDSO-coated pericardium is shown. Figure 5BThe same pericardial sample after cyclic loading in a continuous load test is shown. Prior to the load test, the pericardial sample was rehydrated with isotonic saline solution. The continuous load test was performed on an Instron E1000 cyclic continuous loading machine. A rectangular sample geometry was used (b=5 mm, l=40 mm). Isotonic saline solution at a temperature of 37°C was used as the medium for the continuous load test. The loading frequency was 10 Hz. The maximum deflection was 10% and the maximum force was 10 N. 50,000 cycles were performed. This continuous load test resulted in surface changes, such as Figure 5B However, after this continuous loading test, the pericardium remained intact and operable.

[0100] Figure 6 Shown is a spectrum obtained by EDX from a pHMDSO-coated pericardium after completion of a continuous stress test of 50,000 cycles. Again, the gold signal is from the gold of the sputtered layer used to perform the SEM. Figure 3 Compared to the EDX spectrum of pHMDSO-coated pericardium before the continuous stress test, the silicon signal becomes smaller and broader. This reflects the changed surface structure due to the continuous stress test. However, at the same time, the EDX spectrum also shows that the pHMDSO coating is still bound to the surface of the pericardium after the continuous stress test.

[0101] Figure 7 Three stress-strain curves are shown for each of i) porcine pericardium 11 fixed with glutaraldehyde, ii) pericardium 12 stabilized with glycerol / PEG200, and iii) pHMDSO-coated pericardium 13. These stress-strain curves were obtained by tensile testing, wherein the different pericardial samples were each rehydrated in isotonic saline solution before the tensile testing. The tensile test was performed on a Zwick Z2.5 universal testing machine. The failure strain was determined. The sample geometry was based on ISO 527-2 (rectangular specimen, b=5 mm, l=40 mm). An isotonic saline solution at a temperature of 37° C. was used as the medium. The preload was set to 0.05N.

[0102] Figure 7 It is shown that the pHMDSO-coated pericardium 13 exhibits the most favorable stress-strain curve. Due to the polymer coating, even at relatively high strains, unfavorably high stresses are not accumulated in the pericardium.

[0103] Figure 8 The results of the in vitro calcification test are shown. The left bar shows the calcium ion content of the calcium phosphate deposited on the sample in the case of stabilized dry pericardium (control). In the case of pHMDSO-coated pericardium, no deposited calcium ions can be detected. This shows that the polymer coating is an effective means to combat pericardial calcification.

Claims

1. A process for applying a polymer coating to a protein-containing biomaterial comprising or consisting of the following steps: a) Providing protein-containing biological materials (1) isolated from organisms or artificially produced; and b) depositing a polymer coating (8) of at least one polymer (7) on the surface of the protein-containing biomaterial (1) by plasma-assisted chemical vapor deposition, wherein the polymer coating (8) is covalently bonded to the surface of the protein-containing biomaterial (1), Therein, the protein-containing biological material (1) is subjected to a drying step before depositing the polymer coating (8).

2. The process according to claim 1, wherein The at least one polymer (7) is formed from at least one monomer (4) during plasma-assisted chemical vapor deposition, wherein the at least one monomer (4) is selected from siloxanes, functionalized allyl compounds, pyrroles, thiophenes, furanic compounds, anilines and epoxides.

3. The process according to claim 1 or 2, wherein: The at least one polymer (7) is formed during the plasma-assisted chemical vapor deposition from at least one siloxane, in particular hexamethylenedisiloxane.

4. A process according to any one of the preceding claims, wherein The polymer coating (8) has a layer thickness in the range from 0.1 nm to 10 μm.

5. A process according to any one of the preceding claims, wherein: The polymer coating (8) is free of metal.

6. A process according to any one of the preceding claims, wherein The protein-containing biomaterial (1) comprises collagen.

7. A process according to any one of the preceding claims, wherein The protein-containing biological material (1) is biological tissue, in particular pericardium.

8. A process according to any one of the preceding claims, wherein The protein-containing biological material (1) is an autologous or xenogeneic protein-containing biological material.

9. A process according to any one of the preceding claims, wherein: Prior to drying and / or prior to depositing the polymer coating (8), the protein-containing biomaterial (1) is stabilized with a stabilizer.

10. A process according to any one of the preceding claims, wherein: Prior to depositing the polymer coating (8), the protein-containing biomaterial (1) is brought into a predetermined shape by a molding process.

11. A process according to any one of the preceding claims, wherein In a further process step, the polymer coating (8) is functionalized with active substances.

12. A polymer-coated protein-containing biomaterial obtainable by a process according to any one of the preceding claims.

13. The polymer-coated protein-containing biomaterial according to claim 12 for use as an implant in the human or animal body.

14. An implant comprising the polymer-coated protein-containing biomaterial according to claim 13.

Images