Heparin-functionalized surface, method for preparing the same, and use thereof

Abstract

The present invention relates to a method for preparing a multilayer heparin conjugate coating on a substrate, the method promoting good adhesion to the substrate surface and increasing the number of available heparin molecules.

Description

Technical Field

[0001] The present invention relates to a method for preparing a multilayer heparin conjugate coating on a substrate and a device obtained by this coating method. The multilayer heparin conjugate coating is biocompatible, and devices containing such a coating are particularly useful in the field of medical implants.

Background Art

[0002] The present invention provides an improved heparin-functionalized coating on a surface such as an artificial surface or a biological scaffold surface.

[0003] Heparin has a long track record as a clinically recognized anticoagulant by acting as a powerful promoter of antithrombin, a protein that naturally occurs in the blood and can inhibit many coagulation enzymes such as thrombin. Hirudin is an example of a direct inhibitor of thrombin. The use of heparin or hirudin has a lower risk of associated bleeding than can be ignored.

[0004] Polymeric conjugates composed of multiple heparin chains covalently bonded to an inert backbone have been used to modify both artificial surfaces (Patent Document 2) and biological surfaces to present heparin permanently bound to the surface to mimic the chemical composition of vascular endothelium (carrying heparan sulfate localized on the surface).

[0005] However, heparin or heparin conjugates coated on certain surfaces, such as metal surfaces, for example nitinol (nickel-titanium alloy), tend to peel off, thereby losing their antithrombin properties.

[0006] Therefore, it remains a problem in the art to provide an improved method for preparing a biocompatible surface containing heparin that maintains its functional properties even after the start of use.

Prior Art Documents

[0007] [Patent Document 1] International Publication No. 93 / 05793 [Patent Document 2] International Publication No. 00 / 45837 [Overview of the Initiative]

[0008] The objective of the present invention is to overcome the problem of peeling of heparin or heparin conjugate coatings from surfaces, thereby improving durability and safety, and increasing the amount of heparin on the surface. This is achieved by priming the surface with a primer at an elevated temperature, as described later.

[0009] In a first embodiment, the present invention relates to the method according to claim 1.

[0010] In a second embodiment, the present invention relates to a heparin-functionalized surface obtained by the method according to the present invention.

[0011] In a third aspect, the present invention relates to a device such as a stent or tube that includes a heparin-functionalized surface according to the present invention.

[0012] In a fourth embodiment, the present invention relates to a heparin-functionalized surface disclosed herein, wherein the coating present on the surface is for use in preventing blood coagulation and / or for enhancing anticoagulant properties and / or anticomplement activation properties in an object requiring such use.

[0013] In a fifth embodiment, the present invention relates to the use of a device comprising a heparin-functionalized surface as a medical implant.

[0014] In a sixth embodiment, a method is provided for preventing blood coagulation and / or promoting anticoagulant and / or anticomplement activation properties in an object requiring such property, the method comprising the steps of i) providing a device comprising a heparin-functionalized surface disclosed herein, and ii) implanting the device in an object requiring such property. [Brief explanation of the drawing]

[0015] [Figure 1] This is a schematic diagram illustrating the coating on the surface of a substrate according to the present invention. [Figure 2] This is an explanatory diagram of the method according to the present invention. [Modes for carrying out the invention]

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

[0017] In this specification, "macromolecular heparin conjugate" or "heparin conjugate" as interchangeably referred to herein comprises a plurality of unfractionated glycosaminoglycans (GAGs), preferably heparin, molecules attached to an inactive main chain, preferably by covalent bonds, and preferably via point bonds.

[0018] The number of GAG molecules per conjugate should be at least 10, but preferably 20 to 100, i.e., 20 or more, or 50 or more, or 70 or more, or 100 or less, or 80 or less.

[0019] A certain percentage of GAG molecules are involved in binding to the surface, while the remaining GAG molecules that are not involved in binding are free to exert the biological activity of GAG. Due to the multiple bonds between the GAG ​​molecules and the surface that has affinity for GAG, the binding strength of the GAG ​​polymer conjugate exceeds that of ordinary GAG, resulting in superior performance.

[0020] To obtain a favorable combination of strong binding and residual biological activity, it is desirable that multiple GAG ​​chains be oriented to freely interact with surfaces that have affinity for GAGs, particularly heparin. The GAGs should preferably be bound to the main chain by single-point bonds.

[0021] One preferred polymeric conjugate of GAGs is the Corline heparin conjugate (CHC), which consists of about 70 heparin molecules bonded to an inactive main chain (available from Corline Biomedical AB, Uppsala, Kingdom of Sweden). Thus, the number of heparin molecules per chain can be 40 to 75. Preferred conjugates are described in U.S. Patent No. 5529986, which is incorporated herein by reference. The polymeric conjugate is at least substantially water-soluble and a biologically active conjugate (polymer), preferably in a substantially pure form, comprising a substantially linear organic homo or heteropolymer having numerous functional groups distributed along the polymer backbone chain, with at least about 10 molecules of the functional groups of the sulfated glycosaminoglycan (GAG) in its inactive portion fixed via covalent bonds. Such conjugates can conceptually be described as synthetic proteoglycans, and their relative composition can be varied in a controllable manner to adapt to the intended application.

[0022] The substantially linear polymer chain that functions as the main chain of the glycosaminoglycan residue should, of course, be substantially biologically inert in the sense that it should lack at least interfering biological activity after the binding of the glycosaminoglycan or glycans. As can be easily understood, in order to enable the binding of multiple glycosaminoglycan residues, the main chain should be provided with many functional groups, such as amino groups, amide groups, sulfate groups, vinyl groups, carbonyl groups, nitro groups, thiol groups, hydroxyl groups, or carboxyl groups, which are distributed along the chain and can bind glycosaminoglycans directly or through a binding sequence after any modification. It should be noted in this context that the GAG may still retain the terminal residues of its natural conjugate protein to which it is bound depending on the method of manufacturing the conjugate, and in that case, the binding will, of course, be preferably carried out through, for example, amino acids in such residues.

[0023] Furthermore, the main chain (preferably the polymer chain) preferably has good solubility in water. At least, in accordance with what has been described above for the conjugate, it should be at least substantially water-soluble after the binding of the glycosaminoglycan group. Specific polymer chains suitable for the purposes of the present invention will be readily apparent to those skilled in the art who are part of the general inventive concept. This is, of course, also the case for the degree of branching of the polymer chain that can be tolerated within the scope of the expression "substantially linear".

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

[0025] In relation to the fact that it is generally desirable for glycosaminoglycans to maintain their biological activity after being bound to the polymer backbone, it is preferable that each glycosaminoglycan molecule be bound to the main polymer at its terminal and by only a single bond. For example, glycosaminoglycans can be bound to the backbone via amino acids, preferably terminal amino acids, but free amino groups of glucosamine units can also be used. The latter may remain in a free state or may be released by desulfurization or deacetylation. Particularly when an amino-functional polymer is used as the backbone, in situations where glycosaminoglycan substitution to the backbone is sparse, it may be advantageous to block the remaining free amino groups, which can be done, for example, by acetylation. As an alternative approach, a predetermined number of amino groups can be substituted with, for example, methyl groups, before the glycosaminoglycans are bound.

[0026] The polymer heparin conjugate of the present invention preferably has a molecular weight greater than 70 kDa. Preferably, the molecular weight of the polymer heparin conjugate is greater than 200 kDa, more preferably greater than 400 kDa, and even more preferably greater than 600 kDa. Other molecular weights are also conceivable herein.

[0027] When the conjugate is added to the surface disclosed herein, it can be dissolved in an aqueous solution at a concentration range of 0.001 to 10 mg / ml, for example, 0.001 mg / ml or higher, 0.01 mg / ml or higher, 0.1 mg / ml or higher, 1 mg / ml or higher, 3 mg / ml or higher, 10 mg / ml or lower, 7 mg / ml or lower, or 5 mg / ml or lower. The aqueous solution may be a buffer solution, and the buffer may be any physiological buffer such as phosphate buffer, PBS, or acetate buffer.

[0028] Refer to Figure 1. The present invention relates to a method for preparing a coating on a surface, which promotes improved adhesion of the coating to the surface, thereby reducing the risk of peeling. As seen in Figure 1, the coating has a multilayer structure of at least two, preferably three, layers, each layer containing a primer and a heparin conjugate. The primer is used to adhere the coating to the surface to be coated and to separate each layer of the heparin conjugate.

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

[0030] Furthermore, the substrate material to be coated according to this specification can, in principle, be any material to which biocompatibility is desired, as long as its surface is cationic or can be cationized. As described above, the present invention is applicable to various xenobiotic materials such as polymers, metals, and ceramics. It is also applicable to tissue-like materials such as scaffolds prepared from decellularized extracellular matrix, which may offer advantages over fully synthetic materials.

[0031] Various methods for cationizing the surface of a substrate are well known. Treatment with polyimines has proven to be an appropriate method, but as will be explained in the examples below, other polyamines such as polylysine, chitosan, or polyallylamine can also be used.

[0032] The surface to be coated by the method described herein is collagen. For example, a three-dimensional matrix of collagen or gelatin (hydrolyzed collagen) is hydrated by immersion in phosphate buffer solution (PBS) for at least 1 hour, then immersed for 5 to 60 minutes in PBS supplemented with CHC (heparin conjugate solution as shown in this specification) in a concentration range of 0.01 to 10 mg / ml, and then carefully rinsed three times with PBS.

[0033] Examples of medical devices that can be coated using the present invention include, but are not limited to, stents, tubes, catheters, vascular grafts, membranes, or filters. The devices or medical devices disclosed herein can be used as medical implants (also simply called implants). These terms may be used without distinction in meaning within this specification depending on the purpose. Some implants may be artificial organs, i.e., intended to replace missing body parts, while others may provide support to body organs or tissues, deliver drugs, or provide other body monitoring functions. Implants may be permanently placed in the body or removed at some point when they are no longer needed.

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

[0035] The coated devices described herein may be blood-contactable and / or blood-compatible, meaning they possess properties that make them suitable for contact with blood, for example, they may prevent blood coagulation by exhibiting anticoagulant properties. In this specification, heparin-functionalized surfaces manufactured by the methods of this disclosure possess such properties.

[0036] As shown in Figure 1, the coating comprises two or more layers, each layer including a primer layer on which a heparin conjugate layer is placed. That is, a primer layer is placed on the material surface, and a heparin conjugate layer is placed on top of it to form the first layer.

[0037] According to the present invention, the primer is a polyallylamine having the general structure shown in Formula 1. [ka] In the formula, R1 and R2 are individually selected from hydrogen or alkyl groups, preferably C1-C100 alkyl groups. In one 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 the polyallylamine is 30,000 to 1,000,000 Da.

[0038] As those skilled in the art will understand, even if individual primer layers or heparin conjugate layers are arranged as upper or lower layers, the formed individual layers are at least partially mixed or integrated with the preceding individual layers. The number of primer and heparin conjugate layers is at least two, but preferably three or more.

[0039] Refer to Figure 2. The method according to the present invention is a multi-step method for coating a substrate surface with a heparin conjugate coating. A substrate surface is provided and preferably cleaned using any method suitable for the surface. If the substrate surface is a polymer material such as polyethylene or polypropylene, it is preferably cleaned with ammonium persulfate and rinsed with an aqueous solution, or if the substrate surface is metal, the surface is treated with alcohol in an ultrasonic bath or ultrasonic cleaning tank. The cleaned surface is preferably rinsed with an aqueous solution two or more times.

[0040] Next, the first borate buffer solution is incubated on the surface for a first period of time. The first borate buffer solution contains the polyallylamine primer (PAA) as defined above, and the temperature is at least 30°C, preferably at least 37°C, more preferably at least 40°C, but preferably not exceeding 70°C, more preferably not exceeding 60°C. As shown in the examples, the high-temperature treatment in the first priming step improves the adhesion of the coating layer to the substrate surface and increases the amount of available heparin conjugate. The first borate buffer solution has a pH higher than 7, preferably 8-12, more preferably 8.5-11.0, even more preferably 8.5-10, more preferably 8.5-9.5, and most preferably about 9.

[0041] In one embodiment, the concentration of polyallylamine primer (PAA) in the first buffer solution is about 0.15 to 0.35 mg / ml, preferably about 0.20 to 0.30 mg / ml, and more preferably about 0.25 mg / ml. The first period is preferably at least 10 minutes, more preferably at least 15 minutes. Optionally, the first primer layer is rinsed to remove excess primer. Rinsing is preferably done using an aqueous solution and is preferably repeated at least twice. When the first primer layer is incubated in the heparin conjugate solution, a first heparin conjugate layer is formed. The heparin conjugate solution is preferably a buffer solution, preferably an acetate buffer solution having a pH lower than 5. The first primer layer can be incubated in the heparin conjugate solution for at least 5 minutes, preferably at least 10 minutes. The concentration of heparin conjugate in the heparin conjugate solution is preferably 0.01 to 0.1 mg / ml. Subsequently, the first heparin conjugate layer is rinsed, preferably with an aqueous solution, and this rinsing is preferably repeated at least twice.

[0042] The second layer is prepared by first incubating the first heparin conjugate layer in a first borate buffer solution at a second priming temperature to form the second primer layer. The second priming temperature may be the same as or higher than the first priming temperature, but is preferably lower. In one embodiment, the second temperature is less than 30°C, preferably 20-25°C. No significant improvement is observed when using higher temperatures during the formation of the additional primer layer, and it is advantageous to use a lower temperature from a cost and handling perspective. Preferably, the second primer layer is rinsed with an aqueous solution, and this rinsing is preferably repeated at least twice. Next, the second primer layer is incubated in a heparin conjugate solution to form the second heparin conjugate layer. The second primer layer can be incubated in the heparin conjugate solution for at least 5 minutes, preferably at least 10 minutes. After that, it is preferable to rinse the second heparin conjugate layer with an aqueous solution, and this rinsing is preferably repeated at least twice.

[0043] To form a third layer or further layers, the incubation step in the first boric acid buffer solution, the rinsing step, and the incubation step in the heparin conjugate solution are repeated until the desired number of layers is obtained.

[0044] To further increase the number of available heparin groups on the coating surface, the inventors found it preferable to acylate the coating. Prior to acylation, the formed coating is incubated in a second borate buffer solution having a pH higher than 7. In one embodiment, the pH of the second borate buffer solution is 8 to 12, preferably 9 to 11.5, more preferably 10 to 11, and more preferably about 10.5.

[0045] The second boric acid buffer solution does not contain a primer. This step is thought to further harden the layered structure and result in a more stable coating. Incubation in the second boric acid buffer solution is preferably carried out for at least 10 minutes, for example, at least 15 minutes, preferably at least 20 minutes, more preferably at least 30 minutes, for example, up to about 35, 40, 45, 50, 55 minutes and about 60 minutes, or incubation can be continued for several hours (for example, overnight).

[0046] Subsequently, the coating is treated with an acylation solution containing an acylation agent, preferably an anhydride or acetyl halide, preferably an anhydride acetic acid or acetyl chloride. The concentration of the acylation agent is at least 0.2 vol%, and depends on the number of coating layers and the substrate surface. In one preferred embodiment, the concentration is at least 0.3 vol%, preferably less than 50 vol%, preferably less than 20 vol%, and preferably less than 10 vol%. In one preferred embodiment, the concentration is about 0.2 vol% to 0.5 vol%, preferably 0.25 to 0.4 vol%.

[0047] The first and second borate buffer solutions may contain, but are not limited to, compounds selected from the group consisting of boric acid, borate esters, sodium tetraborate decahydrate, 1,3-dioxa-5-aza-2,4,6-triborinan, and / or any combination thereof. The first borate buffer solution is characterized in that it can be used to construct each layer, including the primer layer and the heparin conjugate layer.

[0048] The second boric acid buffer solution is characterized in that it can be used in the final step of the method of the present disclosure, i.e., when the final layer, which includes a primer layer and a heparin conjugate layer, has been added to a surface containing one or more layers of the primer layer and the heparin conjugate layer.

[0049] As previously described herein, one of the final steps in coating a device with one or more layers of the primers and heparin conjugates disclosed herein is acylation of residual amines in the primers, regardless of the number of layers. By acylation, positively charged amine groups are replaced with acyl groups that are not attracted to the negatively charged heparin chains on the heparin conjugate, thereby creating more freely non-covalent heparin chains. To avoid hydrolysis of the acylating agent, the acylation step is preferably performed rapidly.

[0050] One objective of the present invention is to demonstrate an improvement in the method of coating medical devices with a heparin conjugate layer, thereby increasing the available concentrations of heparin and antithrombin on the surface and improving the coating process for medical devices.

[0051] A further object is to provide a heparin-functionalized surface disclosed herein, wherein, in subjects requiring such a coating, the coating is for use in preventing blood coagulation and / or for promoting anticoagulant and / or anti-complement activation properties. Such a coating is also described as thrombotic-resistant. Overall, such properties may promote healing within the area where the device of the disclosure is implanted. Subjects receiving the device of the disclosure may have cardiovascular disease or other disease or disorder requiring implantation.

[0052] This disclosure also provides heparin-functionalized surfaces disclosed herein, wherein the coating is intended for use in promoting tissue growth, healing, and / or regeneration in a subject.

[0053] The use of a device including a heparin-functionalized surface as disclosed herein as a medical implant is also provided.

[0054] A method for preventing blood coagulation and / or promoting anticoagulant properties and / or anticomplement activation properties in a subject requiring such properties is also provided, the method comprising the steps of i) providing a device comprising a heparin-functionalized surface disclosed herein, and ii) implanting the device in a subject requiring such properties.

[0055] This disclosure is described herein by the following experimental section, but is not intended to be limited thereto.

[0056] [Experiment Section] Example 1 Study on the effect of solution temperature during the coating process Three different sample materials were tested: Nitinol (nickel-titanium alloy), platinum-iridium alloy, and polyethylene.

[0057] All tests were performed in a flow chamber in which the solution was recirculated or passed through the test samples 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).

[0058] For each test, a first borate buffer solution (pH 9.0-9.1) containing an alkylated polyallylamine primer was prepared (0.25 mg primer / ml), and a second borate buffer solution (pH 10.5-10.6) and a heparin conjugate solution (pH 4.0) (0.051 mg heparin conjugate / ml) containing heparin conjugate dissolved in acetate buffer were prepared.

[0059] All materials were first cleaned with ethanol in an ultrasonic bath, and then rinsed with milli-q water before coating.

[0060] 1) To prime the sample surface, the first boric acid buffer solution at temperature T1 is recirculated to the sample. 2) Rinse four times with milli-q water at temperature T2 (no recirculation). 3) Recirculate the first heparin conjugate solution at temperature T3 back into the sample. 4) Rinse twice with milli-q water at temperature T4 (no recirculation). 5) Recirculate the first boric acid buffer solution at temperature T5 back into the sample. 6) Rinse four times with milli-q water at temperature T6 (no recirculation). 7) Recirculate the first heparin conjugate solution at temperature T7 back into the sample. 8) Rinse twice with milli-q water at temperature T8 (no recirculation). 9) Recirculate the first borate buffer solution at temperature T9 back into the sample. 10) Rinse four times with milli-q water at temperature T10 (no recirculation). 11) Recirculate the first heparin conjugate solution at temperature T11 back into the sample. 12) Rinse twice with milli-q water at temperature T12 (no recirculation). 13) Recirculate the sample with the second borate buffer solution at temperature T13 for 5 minutes or 30 minutes. 14) Prepare acylated borate buffer solutions at a temperature of T14 (pH ~10.5, with acetic anhydride concentrations of 0.3 vol% or 0.1 vol%, respectively), and recirculate the acylated buffer solutions through the sample for 5 minutes or 30 minutes, respectively. 15) Rinse four times with milli-q water at temperature T15 (no recirculation). 16) Allow the PE sample to dry overnight.

[0061] [Table 1]

[0062] Analysis and Results To determine whether the heparin concentration on coated surfaces is affected by temperature, a test is performed using toluidine blue (TB) according to a well-known protocol. The amount of heparin available in each sample was determined using an appropriate optical spectrometer (polyethylene sample).

[0063] [Table 2]

[0064] While higher temperatures in the process yielded a larger amount of heparin (see Sample 1b, Table 2), the effect of using higher temperatures throughout the entire process was not significant (see Sample 1b vs. Sample 3b, Table 2). The higher temperature (T1) in the first priming step was sufficient to increase the amount of heparin compared to priming at room temperature (see Sample 3b, Table 2).

[0065] To investigate the coating's resistance to peeling, samples prepared in 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 measured. 2 This was investigated to assess the amount of heparin available (Table 3).

[0066] [Table 3]

[0067] In the nitinol sample, a clear decrease in AT binding capacity was observed over time during stirring, whereas other samples did not show such a decrease, at least at T24. When the improved process of this disclosure, including the increased priming temperature in Step 1, was used, the nitinol sample retained most of its AT binding capacity (Table 4).

[0068] Table 4

Claims

1. A method for providing a heparin-functionalized coating on a surface, wherein the coating comprises one or more layers, each layer comprising a primer layer and a heparin conjugate layer. i) A step of providing a surface, ii) A step of optionally cleaning the surface by optionally treating the surface with ammonium persulfate and rinsing the surface with an aqueous solution, or by optionally treating the surface with alcohol in an ultrasonic bath. iii) A step of priming the surface by incubating the surface in a first boric acid buffer solution containing a polyallylamine (PAA) primer at a first priming temperature of at least 30°C, wherein the first boric acid buffer solution forms a primer layer. iv) Incubating the primer layer obtained in the previous step in a heparin conjugate solution that forms a heparin conjugate layer. v) Optionally, incubate the heparin conjugate layer obtained in the previous step in a first boric acid buffer solution at a second priming temperature to form an additional primer layer. vi) Optionally, incubating the additional primer layer in a heparin conjugate solution to form an additional heparin conjugate layer. vii) Optionally, repeat steps v) to vi) to produce any third or more heparin conjugate layers. viii) Optionally, a step of processing the coating obtained from steps i) to vii), wherein the processing is a. A step of incubating the coating in a second boric acid buffer solution, wherein the second boric acid buffer solution has a pH higher than 7, and b. A step comprising incubating the coating from step a) with an acylation solution containing an acylation agent. Includes, Optionally, the rinsing step optionally precedes steps iv), v), vi) and / or vii) with an aqueous solution having a pH higher than 7. method.

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

4. The method according to any one of claims 1 to 3, wherein the heparin conjugate is a substantially water-soluble, biologically active conjugate comprising a main skeleton of an inert aliphatic compound having a number of functional groups distributed along the skeleton, wherein at least about 20 and up to about 100, for example about 40 or 75, heparin molecules are immobilized via covalent bonds in the inactive portion of the molecule by the functional groups.

5. The method according to any one of claims 1 to 4, wherein the acylating agent is selected from acetic anhydride and acetyl chloride, and preferably selected from acetic anhydride.

6. The method according to any one of claims 1 to 5, wherein the acylation solution contains an acylation agent at a concentration of at least 0.2 vol%, preferably at least 0.3 vol%, and optionally the concentration is about 0.2 vol% to about 0.5 vol%.

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

8. The method according to any one of claims 1 to 7, wherein the incubation of the coating in the second boric acid buffer solution in step vii) is carried out 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 about 60 minutes, or the incubation is carried out over several hours, for example overnight.

9. The method according to any one of claims 1 to 8, wherein the incubation of the surface in the heparin conjugate solution is carried out for at least 15 minutes in each step.

10. The method according to any one of claims 1 to 9, wherein the concentration of the polyallylamine primer (PAA) in the first borate buffer solution is about 0.15 to 0.35 mg / ml, preferably about 0.20 to 0.30 mg / ml, and more preferably about 0.25 mg / ml.

11. The method according to any one of claims 1 to 10, wherein the pH of the second boric acid buffer solution is preferably 8 to 12, preferably 9 to 11.5, more preferably 10 to 11, and more preferably about 10.

5.

12. The method according to any one of claims 1 to 11, wherein the pH of the first boric acid buffer solution is 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.

13. A heparin-functionalized surface comprising a coating obtained by the method described in any one of claims 1 to 10.

14. A device such as a stent or tube comprising the heparin-functionalized surface described in claim 13.

15. The device according to claim 14, wherein the device is a blood-contact and / or blood-compatible 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, artificial heart valve, or arteriovenous (AV) fistula.

17. The heparin-functionalized surface according to claim 13, wherein the coating is for use in preventing blood coagulation in an object requiring it, and / or for promoting anticoagulant properties and / or anti-complement activation properties.

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

19. A method for preventing blood coagulation and / or promoting anticoagulant properties and / or anticomplement activation properties in subjects requiring such properties, i) A step of providing a device comprising a heparin-functionalized surface according to any one of claims 14 to 16, and ii) The process of embedding the device into an object that requires it. Methods that include...

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