Method for providing an improved heparin-functionalized surface

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 on the surface.

Description

Technical Field

[0001] The present invention relates to the field of methods for coating a device, more specifically a medical device, with a heparin-functionalized surface. The present invention also relates to a device having such a surface produced by the method according to the present invention. Further, the present invention relates to the use and method of such a coated device 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 potent accelerator 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 is less than that with an acceptable risk of accompanying bleeding.

[0004] Polymeric conjugates composed of multiple heparin chains covalently bound 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 the vascular endothelium (carrying heparan sulfate localized on the surface).

[0005] Patent Document 3 relates to a heparin conjugate solution for treating blood vessels. This document further relates to the use of the heparin conjugate as a medicine and a method for coating a tissue using the conjugate.

[0006] There is still a need in this field to provide improved methods for coating the surfaces of medical devices with biocompatible heparin-containing coatings. Such coatings would either assist the healing process of such surfaces or, if the surface introduced into the human body is a non-biological surface, simply help avoid bodily reactions. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 93 / 05793 [Patent Document 2] International Publication No. 00 / 45837 [Patent Document 3] International Publication No. 2013 / 095270 [Overview of the project]

[0008] The above-mentioned problems are overcome, or at least mitigated, by providing a method for providing a heparin-functionalized coating on a surface in this specification.

[0009] Therefore, in the first aspect, the present invention is a method for providing a heparin-functionalized coating on a surface, i) A step of providing a surface having a coating comprising one or more layers, wherein each layer comprises a polyallylamine (PAA) primer layer and a heparin conjugate layer, ii) A step of incubating the above coating in a second boric acid buffer solution, iii) Incubating the coating from step ii) with an acylation solution containing an acylation agent. Includes, The second boric acid buffer solution described above has a pH higher than 7; The above acylation solution contains an acylation agent at a concentration of at least 0.2 vol%; and In step ii), the incubation of the coating in the second boric acid buffer solution is carried out for at least 10 minutes. Regarding the method.

[0010] In a second embodiment, heparin-functionalized surfaces including coatings obtained by the methods disclosed herein are also provided herein.

[0011] In a third embodiment, devices such as stents, catheters, vascular grafts, or tubes, which include a heparin-functionalized surface obtained as disclosed herein, are also provided herein.

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

[0013] In a fifth embodiment, the use of the device disclosed herein as a medical implant is provided.

[0014] In a sixth embodiment, a method is provided for preventing blood coagulation and / or promoting anticoagulant and / or anticomplement activation properties in a subject 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 a subject requiring such property.

[0015] In a seventh embodiment, a method for providing an improved heparin-functionalized coating on a surface, the coating comprising one or more layers, each layer comprising a primer layer and a heparin conjugate layer, the method comprising: i) providing a surface; ii) optionally cleaning the surface 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) priming the surface by incubating the surface in a first boric acid buffer solution containing a polyallylamine (PAA) primer, wherein the first boric acid buffer solution forms a primer layer (leaves); iv) optionally rinsing the primer layer; v) incubating the primer layer obtained in the preceding step in a heparin conjugate solution forming a heparin conjugate layer; vi) optionally rinsing the surface in step v); vii) optionally, The process includes: incubating the heparin conjugate layer obtained in step (1) in a first boric acid buffer solution containing a PAA primer to form an additional primer layer; vii) optionally rinsing the surface of step vii); ix) optionally incubating the additional primer layer in a heparin conjugate solution to form an additional heparin conjugate layer; x) optionally rinsing the surface of step ix); xi) optionally repeating steps vii) to x) to produce any third or more layers to form a coating; xii) incubating the coating in a second boric acid buffer solution, wherein the second boric acid buffer solution has a pH higher than 7, preferably 8 to 12, preferably 9 to 11.5, more preferably 10 to 11, more preferably about 10.5; and xiii) incubating the coating from step xii) by treating the surface with an acylation solution containing an acylation agent. The above-mentioned first boric acid buffer solution has a pH higher than 7, preferably 8 to 12, more preferably 8.5 to 11.0, still more preferably 8.5 to 10, still more preferably 8.5 to 9.5, and still more preferably about 9. The above-mentioned acylation solution contains an acylating agent at a concentration of at least 0.2 vol%, and the incubation of the above-mentioned coating in the second boric acid buffer solution in step viii) is carried out for at least 10 minutes, and a method is provided.

Brief Description of the Drawings

[0016] [Figure 1] It is a diagram showing the layer structure of the coating according to the present disclosure. [Figure 2] It is a diagram explaining the method according to the present disclosure. [Figure 3] It is a diagram showing the sampling scheme for the antithrombin (AT) and thrombin (TB) analyses of Example 1. [Figure 4] It is a diagram showing the available heparin vs. block positions. The average of all positions is drawn as a black straight line. (Part 1) [Figure 5] It is a diagram showing the AT concentration vs. block positions. The average of all positions is drawn as a black straight line. (Part 1) [Figure 6A] Available heparin vs. block positions at 5 minutes and 30 minutes of incubation of boric acid buffer 10.5 and 0.3 vol% acetic anhydride. Two different runs are shown. The average of all positions in the first run is shown as a solid line for 5 minutes and a dotted line for 30 minutes. (Parts 1 and 2) [Figure 6B] AT concentration vs. block positions at 5 minutes and 30 minutes of incubation of boric acid buffer 10.5 and 0.3 vol% acetic anhydride. Two different runs are shown. The average of all positions in the first run is shown as a solid line for 5 minutes and a dotted line for 30 minutes. (Parts 1 and 2) [Figure 7]Figure showing the available heparin of PE-pegs coated with blocks. The average value is represented by a straight line and is for the PE samples in incubation with acetic anhydride at a concentration of 0.3 vol% and the second boric acid buffer solution for 5 minutes and 30 minutes. Various lots of PE samples were coated with one block using incubation with acetic anhydride at 0.1 vol% and boric acid buffer for 5 minutes using a previous protocol (data not shown).

Mode for Carrying Out the Invention

[0017] Definition As used herein, the term "sulfated glucosaminoglycan" or simply "glucosaminoglycan" refers not only to substances normally 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.

[0018] The "macromolecular conjugate of heparin" or "heparin conjugate" referred to interchangeably herein includes molecules in which a plurality of unfractionated glycosaminoglycans (GAGs), preferably heparin, are bound to an inert backbone, preferably by a covalent bond, preferably via a single point bond.

[0019] The number of GAG molecules per conjugate should be at least 10, 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.

[0020] A certain proportion of the GAG molecules is involved in binding to the surface, and the remaining GAG molecules not involved in binding are free to exert the biological activity of the GAG. Due to the multiple bonds between the GAG molecules and the surface having an affinity for the GAG, the binding strength of the macromolecular conjugate of the GAG exceeds the binding strength of the normal GAG, resulting in excellent performance.

[0021] 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.

[0022] 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.

[0023] The substantially linear polymer chain that functions as the backbone of the glycosaminoglycan residues should, naturally, be substantially biologically inactive in the sense that, after the binding of the glycosaminoglycan or glycans, it should lack at least interfering biological activity. As is easily understood, in order to enable the binding of multiple glycosaminoglycan residues, the backbone 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, after any modification, can bind glycosaminoglycans directly or via a binding sequence. It should be noted in this context that, depending on the method of producing the conjugate, the GAG ​​may still retain the terminal residues of its native conjugate protein to which it is bound, in which case the binding is, naturally, favorably carried out, for example, via amino acids in such residues.

[0024] Furthermore, the main chain (preferably the polymer chain) preferably has good solubility in water. At the very least, following the above-mentioned characteristics of the conjugate, it should be at least substantially water-soluble after the bonding 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 familiar with some of the general concepts of the invention. This, of course, also applies to the degree of branching of the polymer chain that is acceptable within the scope of the expression "substantially linear".

[0025] Preferably, the polymer chain is a natural or synthetic polypeptide, polysaccharide, or aliphatic polymer. The main chain may be an inert aliphatic compound. Specific non-limiting examples include the main chain being polylysine, polyornithine, chitosan, polyimines and polyallylamines, starch, cellulose, chitin, hyaluronan, polyester, polyether, polyamide, polyamine, polyurethane, or a combination or mixture thereof.

[0026] 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.

[0027] 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.

[0028] 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 or PBS.

[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). The surface may be a metallic or polymer material. The metallic surface may be, but is not limited to, 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. The polymer material may be 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] Examples of medical devices that can be coated using the present invention include, but are not limited to, tubes, stents, catheters, vascular grafts, membranes, and 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 of 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.

[0033] Other examples of devices that can be coated using the present invention include ventricular assist devices, extracorporeal membrane oxygenators, stents, artificial heart valves, or arteriovenous (AV) fistulas. Such coated devices may be blood-contactable and / or blood-compatible, meaning they have 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 produced by the method of this disclosure have such properties. The surface envisioned to be coated by the method of this specification is collagen. For example, a three-dimensional matrix of collagen or gelatin (hydrolyzed collagen) is hydrated by immersion in phosphate-buffered 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.

[0034] Referring to Figure 1, the present invention relates to a method for preparing a coating on a surface, which facilitates an improved heparin conjugate coating with higher abrasion resistance. As seen in Figure 1, the coating is 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 heparin conjugate.

[0035] 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.

[0036] 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 polyallylamine has a molecular weight of 30,000 to 1,000,000 Da.

[0037] 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.

[0038] Referring 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.

[0039] 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. Here, the concentration of the polyallylamine primer (PAA) in the first buffer solution can be 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.

[0040] Subsequently, the first primer layer is incubated in a heparin conjugate solution to form the first heparin conjugate layer. 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. The first heparin conjugate layer is then rinsed, preferably with an aqueous solution, and this rinsing is preferably repeated at least twice.

[0041] 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 a second primer layer. Preferably, the second primer layer is rinsed with an aqueous solution, and this rinsing is 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 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 repeated at least twice.

[0042] 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.

[0043] To further increase the number of available heparin groups on the coating surface, the inventors found it preferable to acylate the coating. Before acylation, the formed coating is incubated in a second borate buffer solution having a pH higher than 7, preferably 8–12, preferably 9–11.5, more preferably 10–11, and more preferably about 10.5. The second borate 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 borate 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 about 35, 40, 45, 50, 55 minutes and about 60 minutes, or incubation can be continued for several hours (e.g., overnight).

[0044] 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%, depending on the number of coating layers and the substrate surface. The concentration may be at least 0.3 vol%, but is preferably less than 50 vol%, preferably less than 20 vol%, and preferably less than 10 vol%. Preferably, the concentration is about 0.2 vol% to 0.5 vol%, preferably 0.25 to 0.4 vol%.

[0045] 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.

[0046] 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.

[0047] Therefore, this specification provides a method for providing a heparin-functionalized coating on a surface: i) A step of providing a surface having a coating comprising one or more layers, wherein each layer comprises a polyallylamine (PAA) primer layer and a heparin conjugate layer, ii) A step of incubating the above coating in a second boric acid buffer solution, iii) Incubating the coating from step ii) with an acylation solution containing an acylation agent. Includes, A method is disclosed wherein the second borate buffer solution has a pH higher than 7; the acylation solution contains an acylation agent at a concentration of at least 0.2 vol%; and the incubation of the coating in the second borate buffer solution in step ii) is carried out for at least 10 minutes. 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.

[0048] The incubation of the coating in the second boric acid buffer solution in step ii) 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, up to about 35, 40, 45, 50, 55 minutes and about 60 minutes, or the incubation is carried out over several hours, for example, overnight.

[0049] As otherwise described herein, the acylation solution contains an acylation agent. The concentration of the acylation agent is at least 0.2 vol%, and depends on the number of layers in the coating and the substrate surface. In one preferred embodiment, the concentration is at least 0.3 vol%, but 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%.

[0050] The acylating agent can be selected from acetic anhydride and acetyl chloride, and preferably from acetic anhydride.

[0051] The first and second borate buffer solutions disclosed herein may, but are not limited to, compounds selected from the group consisting of borate esters, borate and / or its variants, sodium tetraborate decahydrate, 1,3-dioxa-5-aza-2,4,6-triborinane and / or any combination thereof. The first borate buffer solution is characterized in that it can be used in step i) of the method disclosed herein, which includes constructing layers comprising a primer layer and a heparin conjugate layer, i.e., providing a surface having a coating comprising one or more layers, each layer comprising a polyallylamine (PAA) primer layer and a heparin conjugate layer.

[0052] 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 the primer layer and the heparin conjugate layer, is added to a surface containing one or more layers, which include the primer layer and the heparin conjugate layer.

[0053] As illustrated elsewhere herein, the heparin conjugate preferably has an inert aliphatic compound as its main skeleton having numerous functional groups distributed along the skeleton, and at least about 20 and up to about 100, for example about 50 or 70, heparin molecules are immobilized via covalent bonds through these functional groups.

[0054] The step of providing a surface having a coating comprising one or more layers, wherein each layer comprises a polyallylamine (PAA) primer layer and a heparin conjugate layer, further 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 treating the surface with alcohol in an ultrasonic bath. iii) A step of priming the surface by incubating it in a first boric acid buffer solution containing a polyallylamine (PAA) primer, wherein the first boric acid buffer solution forms a primer layer. iv) Incubating the primer 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, incubate 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 second or more layers. Includes, Here, the first boric acid buffer solution has a pH higher 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.

[0055] In the above method, optionally, a rinsing step may be performed with an aqueous solution after steps iii), iv), v) and / or vi).

[0056] As otherwise exemplified herein, incubation of the surface in the heparin conjugate solution can be carried out for at least 15 minutes in each step. Preferably, the concentration of the polyallylamine (PAA) primer 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.

[0057] As illustrated elsewhere in this specification, the surface is a material, which can be selected from, but is not limited to, 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.

[0058] Heparin-functionalized surfaces, including coatings obtained by the methods disclosed herein, are also provided herein.

[0059] Devices such as stents or tubes, which include a heparin-functionalized surface as disclosed herein, are also provided herein. Such devices may be vascular grafts, catheters, membranes, filters, ventricular assist devices, extracorporeal membrane oxygenators, artificial heart valves, or arteriovenous (AV) fistulas.

[0060] The above-mentioned devices may be blood-contact and / or blood-compatible devices, as described elsewhere in this specification.

[0061] Disclosed herein are heparin-functionalized surfaces, wherein, in subjects requiring such functionalization, the coating is for use in preventing blood coagulation and / or for promoting anticoagulant and / or anti-complement activation properties. Such coatings are also referred to as thrombosis-resistant. Overall, such properties may promote healing within the area where the device of this disclosure is implanted. Subjects receiving the device of this disclosure may have cardiovascular disease or other disease or disorder requiring implantation.

[0062] Heparin-functionalized surfaces disclosed herein, wherein the coating is intended for use in promoting tissue growth, healing, and / or regeneration in a subject.

[0063] The use of a device including a heparin-functionalized surface as disclosed herein as a medical implant is also provided.

[0064] 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.

[0065] More specifically, a method for providing an improved 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 it in a first boric acid buffer solution containing a polyallylamine (PAA) primer, wherein the first boric acid buffer solution forms a primer layer. iv) Optionally, a step of rinsing the primer layer. v) Incubating the primer layer obtained in the previous step in a heparin conjugate solution that forms the heparin conjugate layer. vi) Optionally, a step of rinsing the surface of step v), vii) Optionally, incubate the heparin conjugate layer obtained in the previous step in a first boric acid buffer solution to form an additional primer layer. viii) Optionally, a step of rinsing the surface of step viii), ix) Optionally, incubate the additional primer layer in a heparin conjugate solution to form an additional heparin conjugate layer. x) Optionally, the step of rinsing the surface in step ix. xi) optionally repeat steps vii) to x) to produce any third or more layers that form a coating. xii) A step of incubating the above coating in a second boric acid buffer solution, wherein the second boric acid buffer solution has a pH higher than 7, preferably 8 to 12, preferably 9 to 11.5, more preferably 10 to 11, and more preferably about 10.5, and xiii) A step of incubating the coating from step xii) by treating the above surface with an acylation solution containing an acylation agent, Includes, A method is also provided wherein the first boric acid buffer solution has a pH higher than 7, preferably 8-12, more preferably 8.5-11.0, more preferably 8.5-10, more preferably 8.5-9.5, and more preferably about 9, the acylation solution contains an acylation agent at a concentration of at least 0.2 vol%, and the incubation of the coating in the second boric acid buffer solution in step viiii) is carried out for at least 10 minutes.

[0066] This disclosure is described herein by the following experimental section, but is not intended to be limited thereto.

[0067] [Experiment Section] material and method The following relates to the optimization of a coating process in which a medical device having a heparin conjugate-containing layer as disclosed herein involves increasing the concentration of the acylation solution (acetic anhydride) and the incubation time of a second boric acid buffer solution having a pH of 10.5.

[0068] Example 1 Polyethylene (PE) samples were coated into blocks according to the following procedure.

[0069] The heparin conjugate used in the examples is Corline heparin conjugate (CHC).

[0070] The following tests were conducted. 1. Incubation time for the second borate buffer solution at pH 10.5: 5 minutes + concentration of acetic anhydride (acylated solution) 0.3 vol%. 2. Incubation time for the second borate buffer solution at pH 10.5: 30 minutes + concentration of acetic anhydride (acylated solution) 0.3 vol%. 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 (each layer containing one primer and one heparin conjugate).

[0071] 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.

[0072] All materials were first cleaned with ethanol in an ultrasonic bath, and then rinsed with milli-q water before coating.

[0073] The following steps were taken: 1) Recirculate the first borate buffer solution to the sample surface to prime it. 2) Rinse four times with Milli-Q water (no recirculation). 3) Recirculate the first heparin conjugate solution to the sample. 4) Rinse twice with Milli-Q water (no recirculation). 5) Recirculate the first boric acid buffer solution back into the sample. 6) Rinse four times with Milli-Q water (no recirculation). 7) Recirculate the first heparin conjugate solution to the sample. 8) Rinse twice with Milli-Q water (no recirculation). 9) Recirculate the first boric acid buffer solution to the sample. 10) Rinse four times with Milli-Q water (no recirculation). 11) Recirculate the first heparin conjugate solution through the sample. 12) Rinse twice with Milli-Q water (no recirculation). 13) Recirculate the second borate buffer solution through the sample for 5 or 30 minutes at ambient temperature. 14) Prepare acylated borate buffer solutions (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 (no recirculation). 16) Allow the PE sample to dry overnight.

[0074] Figure 3 shows the sampling scheme used for antithrombin (AT) and toluidine blue (TB) analysis.

[0075] result Coating of PE samples Part 1) The available heparin and AT concentrations of the PE-samples analyzed using 0.3 vol% acetic anhydride are shown in Figures 4 and 5, respectively; see Figure 3 for block locations. The available heparin concentrations range from 0.84 to 1.09 μg / cm³. 2 It was during that period.

[0076] In Figure 4, comparing the results by location, the available heparin concentrations at inlet locations 5 and 6, and outlet locations 39 and 40, are above or below the average value, but do not deviate significantly from the average value.

[0077] In Figure 5, the available AT concentration is 25 pmol / cm³. 2 Exceeding 65 pmol / cm³ 2 It falls within the range below [value]. The AT values ​​at positions 1 and 2 (near the entrance) and positions 43 and 44 (near the exit) deviate significantly from the average value compared to other positions, indicating a clear difference.

[0078] Part 2) Refer to Figures 6A-6B for results using 0.3 vol% acetic anhydride and a pH 10.5 borate buffer (second borate buffer) with an extended incubation time (30 minutes). The results for the PE samples above were combined with the results for increased acetic anhydride (0.3 vol%) and an extended incubation time from 5 to 30 minutes. This resulted in a 12% increase in available heparin (Figure 6a) and a 13-15% increase in AT values ​​(Figure 6b), and also showed a tendency for reduced variability in results due to location.

[0079] Consideration As shown in the PE sample herein, both increasing the acetic anhydride concentration (from 0.1 vol% to 0.3 vol%) and extending the incubation time in the second borate buffer at pH 10.5 may increase the concentrations of heparin and AT, while maintaining low variability in the block's position.

[0080] See Figure 5 for an illustration of the optimized process with respect to available heparin. Figure 5 compares the previously available data for PE-pegs coating in blocks with 0.1 vol% and a second borate buffer for 5 minutes incubation with the optimized CHS for PE-pegs coating in 0.3 vol% acetic anhydride for either 5 minutes incubation or extended incubation for 30 minutes. TMThis demonstrates the effectiveness of the process. Here, the combination of increasing the acetic anhydride concentration and extending the incubation time with a second borate buffer at pH 10.5 resulted in increases in available heparin of 42% and 59%, respectively.

Claims

1. A method for providing a heparin-functionalized coating on a surface, i) A step of providing a surface having a coating comprising one or more layers, wherein each layer comprises a polyallylamine (PAA) primer layer and a heparin conjugate layer, ii) A step of incubating the coating in a second boric acid buffer solution, iii) Incubating the coating from step ii) with an acylation solution containing an acylation agent. Includes, The second boric acid buffer solution has a pH higher than 7; The acylation solution contains an acylation agent at a concentration of at least 0.2 vol%; and In step ii), the incubation of the coating in the second boric acid buffer solution is carried out for at least 10 minutes. method.

2. The method according to claim 1, wherein the incubation of the coating in the second boric acid buffer solution in step ii) is carried out for 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 the incubation is carried out over several hours, for example overnight.

3. The method according to claim 1 or 2, wherein the acylation solution contains an acylation agent at a concentration of about 0.2 vol% to about 0.5 vol%, for example, about 0.3 vol%.

4. The method according to claim 1, 2, or 3, wherein the acylating agent is selected from acetic anhydride and acetyl chloride, and preferably selected from acetic anhydride.

5. The method according to any one of claims 1 to 4, 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 50 or 70 heparin molecules are immobilized via covalent bonds in the inactive portion of the molecule by the functional groups.

6. A step to provide a surface having a coating comprising one or more layers, wherein each layer comprises a polyallylamine (PAA) primer layer and a heparin conjugate layer, i) A process 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, wherein the first boric acid buffer solution forms a primer layer. iv) Incubating the primer layer from the previous step in a heparin conjugate solution that forms the 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 layers. Including; The method according to any one of claims 1 to 5, wherein the first boric acid buffer solution has a pH higher 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.

7. The method according to claim 6, wherein the rinsing step is optionally carried out after steps iii), iv), v) and / or vi), optionally with an aqueous solution.

8. The method according to claim 6 or 7, wherein the incubation of the surface in the heparin conjugate solution is carried out for at least 15 minutes in each step.

9. The method according to any one of claims 1 to 8, wherein the first boric acid buffer solution and the second boric acid buffer solution contain compounds selected from the group consisting of boric acid esters, boric acid and / or its variants, sodium tetraborate decahydrate, 1,3-dioxa-5-aza-2,4,6-triborinan and / or any combination thereof.

10. The method according to any one of claims 6 to 9, wherein the concentration of the polyallylamine (PAA) primer in the first boric acid 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 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.

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

5.

13. A heparin-functionalized surface comprising a coating obtained by the method described in any one of claims 1 to 12.

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, stent, ventricular assist device, extracorporeal membrane oxygenation device, 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 anticomplement 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 claim 14 or 15, and ii) The process of embedding the device into an object that requires it. Methods that include...

20. A method for providing an improved 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 process 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, wherein the first boric acid buffer solution forms a primer layer. iv) Optionally, a step of rinsing the primer layer. v) Incubating the primer layer obtained in the previous step in a heparin conjugate solution that forms a heparin conjugate layer. vi) Optionally, a step of rinsing the surface of step v), vii) Optionally, incubate the heparin conjugate layer obtained in the previous step in a first boric acid buffer solution to form an additional primer layer. viii) Optionally, a step of rinsing the surface of step vii), ix) Optionally, incubate the additional primer layer in a heparin conjugate solution to form an additional heparin conjugate layer. x) Optionally, a step of rinsing the surface in step ix. xi) optionally repeat steps vii) to x) to produce any third or more layers that form a coating. xii) A step of incubating the coating in a second boric acid buffer solution, wherein the second boric acid buffer solution has a pH greater than 7, preferably 8 to 12, preferably 9 to 11.5, more preferably 10 to 11, and more preferably about 10.5, and xiii) A step of incubating the coating from step xii) by treating the surface with an acylation solution containing an acylation agent, Includes, The first borate buffer solution has a pH 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. The acylation solution contains an acylation agent at a concentration of at least 0.2 vol%, and The method wherein the incubation of the coating in the second boric acid buffer solution in step viiii) is carried out for at least 10 minutes.

Images