Heparin functionalized surfaces and their preparation and use

Abstract

The present invention relates to a method for preparing a multilayer heparin conjugate coating on a substrate, wherein the method promotes better adhesion to the surface of the substrate and increases the number of available heparin.

Description

Technical Field

[0001] This invention relates to a method for preparing a multilayer heparin conjugate coating on a substrate and to an apparatus obtained by the coating method of this invention. The multilayer heparin conjugate coating is biocompatible, and devices comprising such a coating find specific applications in the field of medical implants. Background Technology

[0002] The present invention relates to a method for providing an improved heparin functionalized coating on a surface, such as a synthetic surface or a biological scaffold surface.

[0003] Heparin has long been documented as a clinically acceptable anticoagulant due to its effectiveness as a promoter of antithrombin, a naturally occurring protein in the blood that inhibits many coagulating enzymes, including thrombin. Hirudin is an example of a direct thrombin inhibitor. The use of heparin or hirudin is associated with a negligible risk of bleeding.

[0004] Macromolecular conjugates consisting of multiple heparin chains covalently linked to an inert backbone have been used to modify artificial surfaces (WO93 / 05793) and biological surfaces (WO00 / 45837) to permanently contain surface-bound heparin, mimicking the chemical composition of vascular endothelium carrying surface-localized heparan sulfate.

[0005] However, heparin or heparin conjugates coated on certain surfaces, such as metal surfaces, like nitinol (nickel-titanium alloy), tend to delaminate, thereby losing their antithrombotic properties.

[0006] Therefore, there remains a need in the art to provide improved methods for preparing heparin-containing biocompatible surfaces that retain their functional properties once put into use. Summary of the Invention

[0007] The object of this invention is to overcome the problem of heparin or heparin conjugate coatings delaminating from surfaces, increase the durability and stability of heparin on the surface, and also increase the amount of heparin on the surface. This is accomplished by applying a primer to the surface at elevated temperatures, as further described herein.

[0008] In a first aspect, the present invention relates to the method according to claim 1.

[0009] In a second aspect, the present invention relates to heparin-functionalized surfaces obtained by the method according to the invention.

[0010] In a third aspect, the present invention relates to a device, such as a support or tube, comprising a heparin-functionalized surface according to the invention.

[0011] In a fourth aspect, the present invention relates to heparin-functionalized surfaces as disclosed herein, wherein the coating present on said surface is used to prevent blood clotting and / or to promote anticoagulant properties and / or anticomplement activation properties in subjects in need.

[0012] In a fifth aspect, the present invention relates to the use of a device comprising a heparin-functionalized surface as a medical implant.

[0013] In a sixth aspect, a method is provided for preventing blood clotting and / or promoting anticoagulant and / or anticomplement activation properties in a subject in need, the method comprising the steps of: i) providing a device comprising a heparin-functionalized surface disclosed herein, and ii) implanting the device into a subject in need. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of a coating on the substrate surface according to the present invention.

[0015] Figure 2 This is a diagram illustrating the method according to the present invention. Detailed Implementation

[0016] In this application, the term "sulfated glycosaminoglycan" refers not only to substances commonly included in the term, such as heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate, but also to fragments and derivatives of these substances that are functional for this purpose.

[0017] The terms “heparin macromolecular conjugate” or “heparin conjugate” are used interchangeably herein and refer to a compound comprising a plurality of unsequential glycosaminoglycans (GAGs), preferably heparin molecules, which are preferably covalently linked to an inert backbone via single-point attachment.

[0018] The number of GAG molecules in each conjugate should be at least ten, but preferably 20 to 100, that is, 20 or more, or 50 or more, or 70 or more, or 100 or less, or 80 or less.

[0019] A portion of the GAG ​​molecule will participate in binding to the surface, while the remaining unbound GAG molecules freely exert their biological activity. Due to the multiple binding between the GAG ​​molecule and the surface with affinity for GAG, the binding strength of the macromolecular conjugate of GAG will exceed that of ordinary GAG, thus resulting in superior performance.

[0020] To achieve a favorable combination of strong binding and preservation of biological activity, multiple GAG ​​chains are preferred, allowing them to be freely oriented to interact with surfaces that have an affinity for GAGs, especially heparin. The GAGs should preferably be attached to the main chain via single-point attachment.

[0021] A preferred GAG macromolecular conjugate is the Corline Heparin Conjugate (CHC), which consists of about 70 heparin molecules (available from Corline Biomedical AB in Uppsala, Sweden) linked to an inert backbone. Thus, the number of heparin molecules can be from 40 to 75 per chain. Preferred conjugates are described in US5529986, which is incorporated herein by reference. A macromolecular conjugate is at least substantially water-soluble, biologically active conjugate (macromolecule), preferably in a substantially pure form, comprising a substantially linear organic homopolymer or heteropolymer having multiple functional groups distributed along the polymer backbone chain, with at least about 10 molecules from the inactive moiety of sulfated glycosaminoglycans (GAG) anchored by covalent bonds via these functional groups. Such conjugates can conceptually be described as synthetic proteoglycans whose relative composition can be varied in a controllable manner and adapted to the intended application.

[0022] Of course, after coupling with the glycosaminoglycan or the glycan of interest, the substantially linear polymer chain serving as the backbone of the glycosaminoglycan residues should be substantially bioinert, in that sense it should at least not interfere with biological activity. As readily understood, in order to allow coupling of multiple glycosaminoglycan residues, the backbone should have numerous functional groups, such as amino, amide, sulfate, vinyl, carbonyl, nitro, thiol, hydroxyl, or carboxyl groups, distributed along the chain and capable of coupling the glycosaminoglycan directly or via a coupling sequence after optional modifications. In this case, it should be noted that, depending on the method of preparing the conjugate, the GAG ​​of interest may still have terminal residues of its native conjugate protein with which it is bound, and then, advantageously, binding occurs, for example, through amino acids in those residues.

[0023] Furthermore, the main chain, preferably the polymer chain, should preferably have good water solubility. At least it should be substantially water-soluble after coupling with glycosaminoglycan groups, as previously described regarding conjugates. After understanding the overall inventive concept, specific polymer chains suitable for the purposes of this invention will be apparent to those skilled in the art. This also applies to the degree of branching on the polymer chain that is permissible within the range described as "substantially linear."

[0024] Preferably, the polymer chain is a natural or synthetic polypeptide, polysaccharide, or aliphatic polymer. The backbone may be an inert aliphatic compound. As specific, non-limiting examples, the backbone may be polylysine, polyornithine, chitosan, polyimide and polyallylamine, starch, cellulose, chitin, hyaluronic acid, polyester, polyether, polyamide, polyamine, polyurethane, or combinations or mixtures thereof.

[0025] Regarding the general desire for glycosaminoglycans to retain their bioactivity after binding to the polymer backbone, it is preferable that each glycosaminoglycan molecule is terminally bound to the host polymer and only via single bonds. For example, glycosaminoglycans can be bound to the backbone via amino acids, and then preferably terminal amino acids, and the free amino groups of glucosamine units can also be used. The latter can exist as is or can be released by desulfurization or deacetylation. In particular, when an amino-functionalized polymer is used as the backbone, in some cases, especially when the backbone is sparsely substituted with glycosaminoglycans, it is advantageous to block the remaining free amino groups, which can be, for example, by acetylation. An alternative approach could be to replace the desired number of amino groups with, for example, methyl groups before attaching the glycosaminoglycan.

[0026] The macromolecular heparin conjugates of the present invention preferably have a molecular weight higher than 70 kDa. More preferably, the molecular weight of the macromolecular heparin conjugate is higher than 200 kDa, more preferably higher than 400 kDa, and even more preferably higher than 600 kDa. Other molecular weights are also contemplated herein.

[0027] When added to the surfaces disclosed herein, the conjugate can be dissolved in an aqueous solution at concentrations ranging from 0.001 to 10 mg / ml, for example, 0.001 mg / ml or more, or 0.01 mg / ml or more, or 0.1 mg / ml or more, or 1 mg / ml or more, or 3 mg / ml or more, or 10 mg / ml or less, or 7 mg / ml or less, or 5 mg / ml or less. The aqueous solution can be a buffer solution, and the buffer can be any physiological buffer, such as phosphate buffer, PBS, or acetate buffer.

[0028] refer to Figure 1 This invention relates to a method for preparing a coating on a surface, wherein the method helps to improve the adhesion of the coating to the surface and thereby reduces the risk of delamination. Figure 1 As shown, the coating is a multilayer structure with at least two layers, preferably three layers, wherein each layer comprises a primer and a heparin conjugate. The primer is used to attach the coating to the surface to coat and separate each layer of the heparin conjugate.

[0029] The surface material can be any suitable material and can be biological (e.g., a biological scaffold material prepared from decellularized tissue) or non-biological (e.g., a metal, polymer, or ceramic). In a preferred embodiment, the surface is a metallic or polymeric material. In one embodiment, the metallic surface is nitinol (nickel-titanium alloy) or a platinum-iridium alloy. The metallic surface may also include steel or gold, or any substance deemed suitable for the intended purpose. In another embodiment, the polymeric material is selected from polyethylene, polypropylene, polystyrene, polyvinyl chloride, polycarbonate, polyurethane, polymethacrylate, polytetrafluoroethylene, cellulose, or carboxymethyl cellulose.

[0030] Furthermore, as described herein, the substrate material to be coated can, in principle, be any material that is expected to be biocompatible, provided that its surface is cationic or can be made cationic. As previously stated, this invention can be applied to body-foreign materials, such as various polymers, metals, and ceramics. This invention can also be applied to tissue-like materials, such as scaffolds prepared from decellularized extracellular matrix, which can offer advantages over fully synthetic materials.

[0031] Various methods for cationizing substrate surfaces are well known. Treatment with polyimides has proven to be a suitable method, but other polyamines, such as polylysine, chitosan, or polyallylamine, can also be used, as will be described in the working examples below.

[0032] The surfaces to be coated using the methods described herein are expected to be collagen. For example, a three-dimensional matrix of hydrated collagen or gelatin (hydrolyzed collagen) is formed by immersion in phosphate-buffered saline (PBS) for at least one hour, followed by immersion in PBS supplemented with CHC at a concentration ranging from 0.01 to 10 mg / ml (such as the heparin conjugate solution presented herein) for 5 to 60 minutes, and then carefully rinsing three times with PBS.

[0033] The types of medical devices that can be coated using this invention include, but are not limited to, stents, tubing, catheters, vascular grafts, membranes, or filters. Therefore, devices or medical devices as disclosed herein can be used as medical implants, sometimes simply referred to as implants. Depending on the purpose, these terms may sometimes be used interchangeably herein. Some implants may be prostheses, i.e., designed to replace missing body parts, while others may provide support to body organs or tissues, deliver drugs, or provide other bodily monitoring functions. Implants may remain permanently in the body or may be removed after a period of time when they are no longer needed.

[0034] Other examples of devices that can be coated with the present invention are ventricular assist devices, extracorporeal membrane oxygenators, mechanical heart valves, or arteriovenous (AV) fistulas.

[0035] The coating devices described herein can be blood-contacting and / or blood-compatible, meaning they possess properties that make them suitable for contact with blood, such as preventing blood clotting by exhibiting anticoagulant properties. In this document, the heparin-functionalized surfaces produced by the methods of this disclosure possess such properties.

[0036] like Figure 1As shown, the coating comprises two or more layers, each layer including a primer layer, on top of which a heparin conjugate layer is disposed. In other words, a primer layer is disposed on the surface of the material, followed by a heparin conjugate layer, thereby forming the first layer.

[0037] According to the present invention, the primer is a polyallylamine having the general structure of Formula 1.

[0038]

[0039] R1 and R2 are independently selected from hydrogen or alkyl groups, preferably C1-C100 alkyl groups. In a preferred embodiment, R1 and R2 are hydrogen. In another preferred embodiment, R1 is hydrogen and R2 is a C1-C100 alkyl group. In a preferred embodiment, the molecular weight of polyallylamine is 30,000-1,000,000 Da.

[0040] As those skilled in the art will understand, even when separate primer layers or heparin conjugate layers are arranged on top of or after each other, the resulting separate layers are at least partially mixed or integrated with the preceding separate layers. The number of primer and heparin conjugate layers is at least two, but preferably three or more.

[0041] Now for reference Figure 2 The method according to the invention is a multi-step approach for coating a substrate surface with a heparin conjugate coating. A substrate surface is provided, and it is preferably cleaned using any method suitable for said surface. When the substrate surface is a polymer material such as polyethylene or polypropylene, it is preferably cleaned using ammonium persulfate and rinsed with an aqueous solution, or when the substrate surface is metallic, it is treated with alcohol in an ultrasonic bath or sonic bath. The cleaned surface is then rinsed with an aqueous solution, preferably two or more times.

[0042] The surface is then incubated with a first borate buffer solution for a first time period. The first borate buffer solution comprises a polyallylamine primer (PAA) as defined above and has a temperature of at least 30°C, preferably at least 37°C, more preferably at least 40°C, but preferably not higher than 70°C, more preferably not higher than 60°C. As shown in the examples, the higher temperature during the first primer step helps improve the adhesion of the coating to the substrate surface and allows for a higher amount of available heparin conjugate. The pH of the first borate buffer solution is greater than 7, preferably 8 to 12, more preferably 8.5 to 11.0, more preferably 8.5 to 10, more preferably 8.5 to 9.5, and more preferably about 9.

[0043] In one embodiment, the concentration of polyallylamine primer (PAA) in the first buffer solution is about 0.15-0.35 mg / ml, preferably about 0.20-0.30 mg / ml, more preferably about 0.25 mg / ml. The first time period is preferably at least 10 minutes, more preferably at least 15 minutes. Optionally, the first primer layer is rinsed to remove any excess primer. Rinsing is preferably performed using an aqueous solution and is preferably repeated at least twice. The first primer layer is incubated in a heparin conjugate solution to form a first heparin conjugate layer. The heparin conjugate solution is preferably a buffer solution, preferably an acetate buffer solution with a pH below 5. The first primer layer is incubated in the heparin conjugate solution for at least 5 minutes, preferably at least 10 minutes. The concentration of the heparin conjugate in the heparin conjugate solution is preferably 0.01-0.1 mg / ml. The first heparin conjugate layer is then preferably rinsed with an aqueous solution, and rinsing is preferably repeated at least twice.

[0044] The second layer is prepared by first incubating the first heparin conjugate layer in a first borate buffer solution at a second primer temperature to produce a second primer layer. The second primer temperature can be the same as or higher than the first primer temperature, but is preferably lower. In one embodiment, the second temperature is below 30°C, preferably 20-25°C. No significant improvement has been observed when using higher temperatures to form the additional primer layer, and using lower temperatures is advantageous from a cost and processing perspective. Preferably, the second primer layer is rinsed with an aqueous solution, and the rinsing is preferably repeated at least twice. The second primer layer is then incubated in a heparin conjugate solution to form the second heparin conjugate layer. The second primer layer may be incubated in the heparin conjugate solution for at least 5 minutes, preferably at least 10 minutes. The second heparin conjugate layer is then preferably rinsed with an aqueous solution, and the rinsing is preferably repeated at least twice.

[0045] To produce a third or additional layer, the steps of incubating the layer in a first borate buffer solution, rinsing, and incubating in a heparin conjugate solution are repeated until the desired number of layers are obtained.

[0046] To further increase the number of heparin groups available on the coating surface, the inventors recognized that acylation coating is preferred. Prior to acylation, the formed coating is incubated in a second borate buffer solution with a pH higher than 7. In one embodiment, the pH of the second borate buffer solution is 8-12, preferably 9-11.5, more preferably 10-11, and even more preferably about 10.5.

[0047] The second borate buffer solution contains no base material. This step is intended to further solidify the layered structure, thereby producing a more stable coating. Incubation in the second borate buffer solution is preferably performed for at least 10 minutes, for example, at least 15 minutes, preferably at least 20 minutes, more preferably at least 30 minutes, for example, about 35, 40, 45, 50, 55 minutes, and at most about 60 minutes, or wherein said incubation is permitted for several hours, such as overnight.

[0048] The coating is then treated with an acylation solution containing an acylation agent, preferably an acid anhydride or acetyl halide, more preferably acetic anhydride or acetyl chloride. The concentration of the acylation agent is at least 0.2% by volume and depends on the number of layers in the coating and the substrate surface. In a preferred embodiment, the concentration is at least 0.3% by volume, but preferably less than 50% by volume, more preferably less than 20% by volume, and more preferably less than 10% by volume. In a preferred embodiment, the concentration is about 0.2% to 0.5% by volume, preferably 0.25% to 0.4% by volume.

[0049] The first and second borate buffer solutions may contain compounds selected from, but not limited to, boric acid, borate esters, sodium tetraborate decahydrate, 1,3-dioxa-5-aza-2,4,6-triborane and / or any combination thereof. The first borate buffer solution is characterized in that it can be used to construct layers comprising a primer layer and a heparin conjugate layer.

[0050] The second borate buffer is characterized in that it can be used in the final stage of the method disclosed herein, i.e., when the final layer comprising the primer layer and the heparin conjugate layer has been added to the surface of one or more layers comprising the primer layer and the heparin conjugate layer.

[0051] As previously mentioned herein, one of the final steps in a coating apparatus using one or more layers of a primer and heparin conjugate as disclosed herein is the acylation of the remaining amine in the primer, regardless of the number of layers. Through acylation, the positively charged amine is exchanged for acyl groups, which are not attracted to the negatively charged heparin chains on the heparin conjugate, resulting in more freely bound, non-covalently bonded heparin chains. The acylation step is preferably performed rapidly to avoid hydrolysis of the acylating agent.

[0052] One object of the present invention is to provide an improvement to a method for coating a medical device with a heparin conjugate layer, which, if implemented, would result in an increase in the concentration of heparin and antithrombin available on the surface, thereby improving the coating process of the medical device.

[0053] Another objective is to provide a heparin-functionalized surface as disclosed herein, wherein the coating is intended to prevent blood clotting and / or to promote anticoagulant and / or anticomplement activation properties in subjects in need. Such a coating may also be described as antithrombotic. In summary, these properties promote healing in the area where the device of this disclosure is implanted. Subjects implanted with the device of this disclosure may have cardiovascular disease or other diseases or conditions requiring implant assistance.

[0054] This disclosure also provides heparin-functionalized surfaces as disclosed herein, wherein the coating is used to promote tissue growth, healing, and / or regeneration in subjects.

[0055] The use of devices comprising heparin-functionalized surfaces as disclosed herein as medical implants is also provided.

[0056] A method for preventing blood clotting and / or promoting anticoagulant and / or anticomplement activation properties is also provided in subjects in need, the method comprising the steps of: i) providing a device comprising a heparin-functionalized surface disclosed herein, and ii) implanting the device into a subject in need.

[0057] This disclosure will now be illustrated through the following experimental section, but it is not intended to be limited thereto.

[0058] Experimental Section

[0059] Example 1

[0060] The study investigated the effect of solution temperature during the coating process.

[0061] Three different sample materials were tested: nickelinol (nickel-titanium alloy), platinum-iridium alloy, and polyethylene.

[0062] All tests were performed in a flow chamber, where the solution was recirculated through or past the test sample for 15 minutes, and all samples were coated with three layers of primer and heparin conjugate. The heparin conjugate used in the examples was Corline heparin conjugate (CHC).

[0063] For each test, a first borate buffer solution (pH 9.0–9.1) containing an alkylated polyallylamine primer (0.25 mg primer / ml) was prepared, a second borate buffer solution (pH 10.5–10.6) was prepared, and a heparin conjugate solution (pH 4.0) containing a heparin conjugate dissolved in an acetate buffer was prepared (0.051 mg heparin conjugate / ml).

[0064] All materials were first washed with ethanol in an acoustic bath and then rinsed with Milli-q water before coating.

[0065] 1) The first borate buffer solution at temperature T1 is recirculated through the sample to apply a primer to the sample surface.

[0066] 2) Rinse four times with Milli-q water at temperature T2 (without recirculation).

[0067] 3) The first heparin conjugate solution at temperature T3 is recycled through the sample.

[0068] 4) Rinse twice with Milli-q water at temperature T4 (without recirculation).

[0069] 5) Recirculate the first borate buffer solution at temperature T5 through the sample.

[0070] 6) Rinse four times with Milli-q water at temperature T6 (without recirculation).

[0071] 7) Recycle the first heparin conjugate solution at temperature T7 through the sample.

[0072] 8) Rinse twice with Milli-q water at temperature T8 (without recirculation).

[0073] 9) Recirculate the first borate buffer solution at temperature T9 through the sample.

[0074] 10) Rinse four times with Milli-q water at a temperature of T10 (without recirculation).

[0075] 11) The first heparin conjugate solution at temperature T11 is recycled through the sample.

[0076] 12) Rinse twice with Milli-q water at temperature T12 (without recirculation).

[0077] 13) Recirculate the second borate buffer solution at temperature T13 for 5 minutes, and continue for 30 minutes.

[0078] 14) Prepare an acylated borate buffer solution at a temperature of T14 (pH approximately 10.5, acetic anhydride concentration of 0.3 vol%), and recirculate the acylated buffer solution through the sample for 5 minutes.

[0079] 15) Rinse four times with Milli-q water at a temperature of T15 (without recirculation).

[0080] 16) Dry the sample overnight.

[0081] Temperature gauges for each step

[0082] sample T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 1a 40 35 35 36 36 34 35 36 36 36 36 38 36 38 37 1b 40 35 35 36 36 34 35 36 36 36 36 38 36 38 37 2a 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 2b 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 2c 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 3a 41 20 20 20 20 20 20 20 20 20 20 20 20 20 20 3b 41 20 20 20 20 20 20 20 20 20 20 20 20 20 20

[0083] Table 1: Sample 1a = Nitinol, Sample 1b = Polyethylene; Sample 2a = Nitinol, Sample 2b = Polyethylene, Sample 2c = Platinum-iridium; Sample 3a = Nitinol, Sample 3b = Polyethylene.

[0084] Analysis and Results

[0085] The test to observe whether the amount of heparin on the coated surface is affected by temperature was performed using toluidine blue (TB) according to a known protocol. The amount of available heparin in each sample (polyethylene sample) was determined using a suitable spectrometer.

[0086] Table of temperature effects in each step of the method

[0087] sample Heparin (index) can be used. StdEv 1b (All High Temperatures) 165 0.14 2b (All ambient temperatures) 100 0.03 3b (Highest First Temperature) 147 0.03

[0088] Table 2: Sample 2b is the reference sample (all steps were performed at RT, 20°C), and the indexed amount of available heparin in the sample was set to 100.

[0089] Higher temperatures were obtained when higher temperatures were used in this method (see Sample 1b, Table 2); however, the effect of using higher temperatures throughout the method appeared to be insignificant (see Sample 1b vs. Sample 3b, Table 2). Higher temperatures (T1) in the first primer step appeared sufficient to increase the amount of heparin compared to primer applied at room temperature (Sample 3b, Table 2).

[0090] To test the coating's resistance to delamination, samples prepared at RT were stirred (250 rpm) at different times and temperatures. The stirring solution used contained 0.15 M NaCl + 4% HSA. Subsequently, the AT binding capacity (AT = antithrombin, pmol / cm³) was tested. 2 To observe how much heparin is available (Table 3).

[0091] AT binding capacity table at different times and temperatures

[0092] sample T0 (Control) T24 / 37℃ T62 / 37℃ T24 / 6-7℃ 2a 100 34.5 0.014 69.0 2b 100 96.5 1.06 1.11 2c 100 99.8 100 N / A

[0093] Table 3: Each sample has been individually indexed, with the AT value at T0 set to 100.

[0094] For the Nitinol samples, a significant loss of AT bonding capacity was observed over time during stirring, while other samples did not show this reduction, at least at T24. When using the improved method of this disclosure, which includes the increased primer temperature in step 1, the Nitinol samples retained most of their AT bonding capacity (Table 4).

[0095] sample T0 (Control) T24 / 37℃ T18 / 6-7℃ 3a 100(n＝2) 134(n＝4) 108(n＝3)

[0096] AT binding capacity table at different times and temperatures

[0097] Table 4: Each sample has been individually indexed, with the AT value at T0 set to 100.

Claims

1. A method for providing a heparin-functionalized coating on a surface, wherein the coating comprises one or more layers, wherein each layer comprises a primer layer and a heparin conjugate layer, wherein the method comprises the following steps: i) Provide a surface; ii) Optionally, the surface is cleaned, and the cleaning is optionally performed by treating the surface with ammonium persulfate and rinsing the surface with an aqueous solution or by treating the surface with alcohol in an ultrasonic bath; iii) The surface is undercoated by incubating the surface in a first borate buffer solution containing a polyallylamine (PAA) primer at a first primer temperature of at least 30°C, wherein the first borate buffer solution leaves a primer layer; iv) The primer layer obtained in the previous step is incubated in a heparin conjugate solution to form a heparin conjugate layer; v) Optionally, the heparin conjugate layer obtained in the previous step is incubated in the first borate buffer solution at the second undercoating temperature, thereby leaving an additional undercoating layer; vi) Optionally, the additional primer layer is incubated in the heparin conjugate solution to form an additional heparin conjugate layer; vii) Optionally, repeat steps v) to vi) to form an optional third or more layers of heparin conjugate; viii) Optionally, the coating obtained from steps i) to vii) is processed, the processing comprising the following steps: a. Incubate the coating in a second borate buffer solution, wherein the pH of the second borate buffer solution is higher than 7, and b. Incubate the coating from step a) with an acylation solution containing an acylation agent. Optionally, a rinsing step is performed prior to steps iv), v), vi), and / or vii), the rinsing step being optionally performed with an aqueous solution, and wherein the pH of the first borate buffer solution is higher than 7.

2. The method according to claim 1, wherein the temperature of the first primer is at least 37°C, preferably at least 40°C, but preferably not higher than 70°C.

3. The method according to claim 1 or 2, wherein the surface is a material selected from the group consisting of: metals, such as nickel-titanium alloys, preferably nickel-titanium; plastic or polymeric materials, such as PVC (polyvinyl chloride), PE (polyethylene), PP (polypropylene), PC (polycarbonate), PU (polyurethane), PTFE (polytetrafluoroethylene), PMMA (poly(methyl methacrylate)); cellulose materials; and ceramic materials; and biological scaffold materials, such as scaffolds containing decellularized tissue.

4. The method according to any one of the preceding claims, wherein the heparin conjugate is a substantially water-soluble bioactive conjugate comprising a backbone chain of an inert aliphatic compound having a plurality of functional groups distributed along the backbone chain, wherein at least about 20 and at most about 100, for example about 40 or 75 heparin molecules in the inactive portion of the molecule are covalently anchored via the functional groups.

5. The method according to any one of the preceding claims, wherein the acylating agent is an acid anhydride or acetyl halide, preferably acetic anhydride.

6. The method according to any one of the preceding claims, wherein the acylation solution comprises an acylation agent at a concentration of at least 0.2 vol%, preferably at least 0.3 vol%, optionally wherein the concentration is from about 0.2 vol% to 0.5 vol%.

7. The method according to any one of the preceding claims, wherein the first borate buffer solution and the second borate buffer solution comprise compounds selected from the group consisting of: boric acid, borate esters, sodium tetraborate decahydrate, 1,3-dioxa-5-aza-2,4,6-triborane and / or any combination thereof.

8. The method according to any one of the preceding claims, wherein the incubation of the coating in the second borate buffer solution in step vii) is performed for at least 10 minutes, for example at least 15 minutes, preferably at least 20 minutes, more preferably at least 30 minutes, for example about 35, 40, 45, 50, 55 minutes, and at most about 60 minutes; or wherein the incubation is allowed to continue for several hours, for example overnight.

9. The method according to any one of the preceding claims, wherein the incubation of the surface in the heparin conjugate solution is performed for at least 15 minutes in each step.

10. The method according to any one of the preceding claims, wherein the concentration of polyallylamine primer (PAA) in the first buffer solution is about 0.15-0.35 mg / ml, preferably about 0.20-0.30 mg / ml, more preferably about 0.25 mg / ml.

11. The method according to any one of the preceding claims, wherein the pH value of the second borate buffer solution is preferably 8 to 12, preferably 9 to 11.5, more preferably 10-11, and even more preferably about 10.

5.

12. The method according to any one of the preceding claims, wherein the pH value of the first borate buffer solution is 8 to 12, more preferably 8.5 to 11.0, more preferably 8.5 to 10, more preferably 8.5-9.5, and more preferably about 9.

13. A heparin-functionalized surface obtained by the method according to any one of claims 1 to 10.

14. An apparatus comprising a heparin-functionalized surface according to claim 13.

15. The apparatus of claim 14, wherein the apparatus is a blood contact and / or blood compatibility device.

16. The device according to claim 14 or 15, wherein the device is a vascular graft, catheter, membrane, filter, ventricular assist device, extracorporeal membrane oxygenator, mechanical heart valve, or arteriovenous (AV) fistula.

17. The heparin-functionalized surface of claim 13, wherein the coating is used to prevent blood clotting and / or promote anticoagulant and / or anticomplement activation properties in subjects in need.

18. Use of the device according to any one of claims 14 to 16 as a medical implant.

19. A method for preventing blood clotting and / or promoting anticoagulant and / or anticomplement activation properties in subjects in need, the method comprising the steps of: i) Providing an apparatus comprising a heparin-functionalized surface according to any one of claims 14 to 16, and ii) Implant the device into the subject in need.

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