Preparation method of medical device containing covalently bonded heparin coating

Abstract

Disclosed herein is a method of making a medical device comprising a covalently bonded heparin coating comprising a covalently bonded heparin coating on a substrate wherein the covalently bonded heparin coating is the reaction product of: (a) an isocyanate-containing material on or covalently bonded to the substrate; and (b) a heparin molecule selected from the chemical formula of the claims, said heparin molecule being subjected to a urethane formation reaction or a thiol-isocyanate click reaction with an isocyanate-containing material.

Description

[0001] This application is a divisional application of application number 202080032058.8 filed on March 19, 2020, entitled "Medical Device Containing Covalently Bonded Heparin Coating". Technical Field

[0002] This invention relates to a medical device. In particular, the medical device may include a covalently bonded heparin coating on a substrate (e.g., a polyurethane tube). Background Technology

[0003] Prior disclosures listed or discussed in this specification should not be construed as an admission that such documents are part of the prior art or common general knowledge.

[0004] Heparin is one of the most effective anticoagulants and is widely used as an antithrombotic coating on medical devices, such as synthetic polymer devices. This coating imparts an antithrombotic effect to the device, preventing the formation of blood clots. This antithrombotic effect is necessary when these devices are used as cardiac stents, endovascular stents, or organ stents, or for blood collection and separation applications (Advanced Drug Delivery Reviews 2017, 112, 12-23; US 8,501,212; US 6,953,625; US 8,101,196; US2004 / 0142016; US 2011 / 0274821; WO 2001 / 039814). Typically, this heparin coating must be covalently bonded to the device surface so that it persists in the body for a longer period while avoiding the health risks associated with heparin seeping into the bloodstream from the device surface.

[0005] Existing coating techniques involve generating aldehyde groups (as active sites) on heparin via oxidation reactions (e.g., using nitrous acid or KIO4), followed by Schiff-Base condensation with an amine, reduction with NaCNBH3, and further steps such as thiol-ene click reactions (US 8501212) or the formation of heparin-polyoxyethylene polyamine adducts (US6953625). However, oxidation reactions disrupt part of the heparin structure, inevitably reducing its antithrombotic ability. Furthermore, multi-step coating routes are both time-consuming and expensive.

[0006] In US 8101196, an aqueous solution of benzalkonium chloride is added to an aqueous solution of heparin sodium to prepare a water-insoluble heparin-benzalkonium chloride complex. The heparin-benzalkonium chloride complex is soluble in dichloromethane (DCM), allowing the hydroxyl groups of heparin to react with methacryloyl chloride to attach carbon double bonds to the heparin backbone. Heparin-containing gels are then prepared in medical devices via steps such as polymerization. However, all reactions are carried out in THF or DCM, which is incompatible with some polymer devices, especially polyurethane devices, because the THF or DCM solvents can deform them.

[0007] US2015 / 0352265 discloses a method for preparing a heparin-containing coating on the surface of an oxygenation device [e.g., a hollow fiber membrane (HFM) surface]. In addition to heparin, the coating comprises polyvinylpyrrolidone (PVP) and quaternary aliphatic alkyl ammonium chloride (or bromide), the quaternary aliphatic alkyl ammonium chloride (or bromide) having at least one C7H group. 15 To C 18 H 37 The heparin contains either a common or substituted long-chain aliphatic group. Because alcohols (such as methanol) are used as co-solvents along with other organic solvents (such as tetrahydrofuran), the hydroxyl groups of heparin cannot be covalently bonded to the device. In no case is covalent bonding mentioned in the method of this patent.

[0008] Furthermore, the most common form of heparin is sodium heparin, which is only soluble in water. This limits the application of sodium heparin to medical devices because compounds that react with the hydroxyl group of heparin (such as acryloyl chloride) can also react with water molecules in aqueous solutions of sodium heparin. Therefore, the hydroxyl group of heparin cannot be used as an active site for covalent coating in aqueous solutions.

[0009] Given the above, the choice of solvent is crucial for the covalent coating of heparin onto medical devices. The solvent should completely dissolve the heparin to facilitate the covalent bonding reaction. Furthermore, the solvent should not deform the polymer device, and the chemical reaction should not destroy any portion of the heparin structure.

[0010] Therefore, there is a need to develop a better coating method that allows heparin to be easily and efficiently covalently coated onto medical devices. More importantly, this method must be able to produce a durable heparin coating that does not degrade during the coating process (e.g., by utilizing existing hydroxyl groups in heparin to bond with the device). Furthermore, the method should involve as few steps as possible and be carried out in solvents compatible with all polymer devices, especially polyurethanes, such as acetonitrile. Summary of the Invention

[0011] Various aspects and embodiments of the invention are disclosed with reference to the following numbered documents.

[0012] 1. A medical device comprising a heparin coating covalently bonded to a substrate, wherein the covalently bonded heparin coating is a reaction product of the following substances:

[0013] (a) an isocyanate-containing material on or covalently bonded to a substrate, wherein the isocyanate-containing material contains one or more isocyanate groups; and

[0014] (b) Heparin molecules selected from one or more of the following formulas:

[0015] Ia:(A1) z -heparin;

[0016] Ib: and

[0017] Ic:{([HS(CH2) n ] m -L2-[(CH2) n S(CH2) o O]) z -heparin} p The oxygen atom bonded to heparin is part of the heparin molecule;

[0018] ID: {([HS(CH2)} n' ] m' -L3-[(CH2) n' S(CH2) o' COO]) z -heparin} p' The oxygen atom bonded to heparin is part of the heparin molecule;

[0019] Ie:{([HS(CH2) n" ] m” -L4-[(CH2) n" S(C q H 2q-2 )O]) z -heparin} p" The oxygen atom bonded to heparin is part of the heparin molecule; and

[0020] If: The fixed portion of the heparin molecular network containing Ifi:

[0021]

[0022] The oxygen atom bonded to heparin is part of the heparin molecule; and

[0023] Ifii's network extension:

[0024]

[0025] The oxygen atom bonded to heparin is part of the heparin molecule;

[0026] in:

[0027] Each A1 and A2 is independent of:

[0028] HO-;

[0029] H(OCHR1CH2) x O-, where the terminal oxygen atom is part of the heparin molecule;

[0030] or

[0031] H(OCHR1CHR2) x O-, where R1 and R2 together with the atoms they are attached to form a carbon ring system with 3 to 10 carbon atoms;

[0032] L1 is the cross-linked portion;

[0033] a ranges from 1 to 25;

[0034] Each z is independently between 1 and 50;

[0035] Each n, n', n”, n”', o, and o' is independently 1 to 20;

[0036] Each m, m', m”, m”', p, p', p”, p”' and p”” is independently 1 to 20;

[0037] Each q is independently between 1 and 20;

[0038] Each L2 to L5 is an independent linking group;

[0039] R1 is H or C1 to C 20 alkyl;

[0040] x is between 1 and 20;

[0041] In formula Ib, | represents the covalent bond between the heparin molecule and the cross-linked portion;

[0042] In equation Ifi, each | represents a connection point with another fixed part of equation Ifi or an extension of equation Ifii;

[0043] In equation Ifii, each | represents a connection point with either the fixed part of equation Ifi or another extension of equation Ifii;

[0044] Each heparin molecule is a polyanionic molecule, wherein each negative charge is balanced by a cation, wherein:

[0045] The cation is substantially a quaternary ammonium ion; and

[0046] The isocyanate-containing material is either a coating on a substrate or covalently bonded to the substrate.

[0047] 2. The medical device according to claim 1, wherein in the compound of formula Ib, a is 1 to 10, for example 2 to 5, for example 3 to 4.

[0048] 3. The medical device according to claim 1, wherein in the compounds of formulas Ic to If, each n, n', n”, n"' is independently 1 to 25, for example 2 to 10, for example 3 to 5.

[0049] 4. The medical device according to claim 1 or claim 3, wherein the compound of formula Ic to If contains:

[0050] (a) Each m, m', m”, m”', p, p', p”, p”' and p”” is independently 1 to 10, for example 1 to 3; and / or

[0051] (b) Each q is independently 1 to 15, for example 1 to 10, for example 1 to 6.

[0052] 5. The medical device according to any one of claims 1, 3 to 4, wherein the compounds of formula Ic to If contain:

[0053] (a) Each L2 to L5 is independently either branched or non-branched C 1-10 A linking group in the form of an alkyl chain, wherein the alkyl chain is unsubstituted or is composed of one or more chains selected from C10. 1-6 Substitution of alkyl, O, or N groups, or insertion of heteroatoms (e.g., O, N) into the alkyl chain; and / or

[0054] (b) Each o and o' is independently 1 to 10, for example 1 to 2.

[0055] 6. The medical device according to claim 1, wherein in compounds of formula Ia and Ib, each R1 is H or a C1 to C5 alkyl group.

[0056] 7. The medical device according to claim 1 or claim 6, wherein the compound of formula Ib contains:

[0057] (a) L1 is a crosslinking portion, which is one or more of the following: linear or branched diacyl chloride, linear or branched triacyl chloride, linear or branched diepoxide, linear or branched triepoxide, and linear or branched tetraepoxide, optionally wherein the crosslinking portion is a compound selected from one or more of the following: adipicoyl chloride, dodececanedioyl dichloride, sebacoyl dichloride, suberoyl dichloride, 1,2-butanediol diglycidyl ether, neopentyl glycol diglycidyl ether, and trimethylolpropane triglycidyl ether; and / or

[0058] (b)x is 1 to 10, for example, 1 to 5.

[0059] 8. The medical device according to any one of the preceding claims, wherein the cation is substantially C 1-30 Alkyl quaternary ammonium ions, such as C 1-10 Alkyl quaternary ammonium ions, such as tetrabutylammonium ions.

[0060] 9. The medical device according to any one of the preceding claims, wherein the isocyanate-containing material is covalently bonded to the substrate, optionally wherein the isocyanate-containing material covalently bonded to the substrate is one or more of the following: hexamethylene diisocyanate, poly(hexamethylene diisocyanate), 4,4'-methylenebis(phenyl isocyanate), and trans-1,4-cyclohexyl diisocyanate.

[0061] 10. The medical device according to any one of the preceding claims, wherein the substrate is a polymer, ceramic or metal, optionally wherein the substrate is a polymer selected from one or more of the following: polyurethane, PTFE, polyethylene and PVC (e.g., the substrate is polyurethane).

[0062] 11. A method of forming a medical device as described in any one of claims 1 to 10, the method comprising the following steps:

[0063] (a) Providing an isocyanate-containing material on or covalently bonded to a substrate, said isocyanate-containing material containing one or more isocyanate groups;

[0064] (b) In a polar aprotic solvent, an isocyanate-containing material on or covalently bonded to a substrate reacts with one or more heparin molecules selected from the following formulas:

[0065] Ia:(A1) z -heparin;

[0066] Ib: and

[0067] Ic:{([HS(CH2) n ] m -L2-[(CH2) n S(CH2) o O]) z -heparin} p The oxygen atom bonded to heparin is part of the heparin molecule;

[0068] ID: {([HS(CH2)} n' ] m' -L3-[(CH2) n' S(CH2) o' COO]) z -heparin} p' The oxygen atom bonded to heparin is part of the heparin molecule;

[0069] Ie:{([HS(CH2) n" ] m” -L4-[(CH2) n" S(C q H 2q-2 )O]) z -heparin} p" The oxygen atom bonded to heparin is part of the heparin molecule; and

[0070] If: The fixed portion of the heparin molecular network containing Ifi:

[0071]

[0072] The oxygen atom bonded to heparin is part of the heparin molecule; and

[0073] Ifii's network extension:

[0074]

[0075] The oxygen atom bonded to heparin is part of the heparin molecule;

[0076] To provide the medical device according to any one of claims 1 to 10, wherein:

[0077] Each A1 and A2 is independent of:

[0078] HO-;

[0079] H(OCHR1CH2) x O-, where the terminal oxygen atom is part of the heparin molecule;

[0080] or

[0081] H(OCHR1CHR2) x O-, where R1 and R2 together with the atoms they are attached to form a carbon ring system with 3 to 10 carbon atoms;

[0082] L1 is the cross-linked portion;

[0083] a ranges from 1 to 25;

[0084] Each z is independently between 1 and 50;

[0085] Each n, n', n”, n”', o, and o' is independently 1 to 20;

[0086] Each m, m', m”, m”', p, p', p”, p”' and p”” is independently 1 to 20;

[0087] Each q is independently between 1 and 20;

[0088] Each L2 to L5 is an independent linking group;

[0089] R1 is H or C1 to C 20 alkyl;

[0090] x is between 1 and 20;

[0091] In formula Ib, | represents the covalent bond between the heparin molecule and the cross-linked portion;

[0092] In equation Ifi, each | represents a connection point with another fixed part of equation Ifi or an extension of equation Ifii;

[0093] In equation Ifii, each | represents a connection point with either the fixed part of equation Ifi or another extension of equation Ifii;

[0094] Each heparin molecule is a polyanionic molecule, wherein each negative charge is balanced by a cation, wherein:

[0095] The cation is substantially a quaternary ammonium ion; and

[0096] The isocyanate-containing material is either a coating on a substrate or covalently bonded to the substrate.

[0097] 12. The method according to claim 11, wherein in the compound of formula Ib, a is 1 to 10, for example 2 to 5, for example 3 to 4.

[0098] 13. The method according to claim 11, wherein in the compounds of formulas Ic to If, each n, n', n”, n"' is independently 1 to 25, for example 2 to 10, for example 3 to 5.

[0099] 14. The method according to claim 11 or claim 13, wherein formula Ic is incorporated into the If compound:

[0100] (a) Each m, m', m”, m”', p, p', p”, p”' and p”” is independently 1 to 10, for example 1 to 2; and / or

[0101] (b) Each q is independently 1 to 15, for example 1 to 10, for example 1 to 6.

[0102] 15. The method according to any one of claims 11, 13 to 14, wherein in compounds of formula Ic to If:

[0103] (a) Each L2 to L5 is independently either branched or non-branched C 1-10 A linking group in the form of an alkyl chain, wherein the alkyl chain is unsubstituted or is composed of one or more chains selected from C10. 1-6 Substitution of alkyl, O, or N groups, or insertion of heteroatoms (e.g., O, N) into the alkyl chain; and / or

[0104] (b) Each o and o' is independently 1 to 10, for example 1 to 2.

[0105] 16. The method according to claim 11, wherein in compounds of formula Ia and Ib, each R1 is H or a C1 to C5 alkyl group.

[0106] 17. The method according to claim 11 or claim 16, wherein in the compound of formula Ib:

[0107] (a) L1 is a crosslinking portion, which is one or more of the following: linear or branched diacyl chloride, linear or branched triacyl chloride, linear or branched diepoxide, linear or branched triepoxide, and linear or branched tetraepoxide, optionally wherein the crosslinking portion is a compound selected from one or more of the following: adipicoyl chloride, dodecene diacyl chloride, sebacyl chloride, octanoyl chloride, 1,2-butanediol diglycidyl ether, neopentyl glycol diglycidyl ether, and trimethylolpropane triglycidyl ether; and / or

[0108] (b)x is 1 to 10, for example, 1 to 5.

[0109] 18. The method according to any one of claims 11 to 17, wherein the cation is substantially C 1-30 Alkyl quaternary ammonium ions, such as C 1-10 Alkyl quaternary ammonium ions, such as tetrabutylammonium ions.

[0110] 19. The method according to any one of claims 11 to 18, wherein the isocyanate-containing material is covalently bonded to the substrate, optionally wherein the isocyanate-containing material covalently bonded to the substrate is one or more of the following: hexamethylene diisocyanate, poly(hexamethylene diisocyanate), 4,4'-methylenebis(phenyl isocyanate), and trans-1,4-cyclohexyl diisocyanate.

[0111] 20. The method according to any one of claims 11 to 19, wherein the substrate is a polymer, ceramic or metal, and optionally wherein the substrate is a polymer selected from one or more of the following: polyurethane, PTFE, polyethylene and PVC (e.g., the substrate is polyurethane).

[0112] 21. The method according to any one of claims 11 to 19, wherein the polar aprotic solvent is acetonitrile.

[0113] 22. The method according to claim 11, wherein A1 in compound Ia or A1 and / or A2 in compound Ib is H(OCHR1CH2). x O- or H (OCHR1CHR2) x When O-, the compound of formula Ia or Ib is formed by the reaction of heparin with epoxide.

[0114] 23. The method according to claim 11, wherein the crosslinking between heparin molecules in the compound of formula Ib is provided by (A1). z -Heparin and (A2) z -Heparin, and react these compounds with a crosslinking agent to form, wherein A1, A2 and z are as defined in claim 11, optionally wherein the crosslinking agent is selected from one or more of the following: linear or branched diacyl chloride, linear or branched triacyl chloride, linear or branched diepoxide, linear or branched triepoxide and linear or branched tetraepoxide (e.g., the crosslinking portion is selected from one or more of the following: adipicoyl chloride, dodecene diacyl chloride, sebacyl chloride, octanoyl chloride, 1,2-butanediol diglycidyl ether, neopentyl glycol diglycidyl ether and trimethylolpropane triglycidyl ether).

[0115] 24. The method according to claim 11, wherein the If compound is prepared by reacting a heparin molecule modified to present one or more carbon-carbon double or triple bond functional groups with a compound containing a plurality of thiol groups, wherein optionally the compound containing a plurality of thiol groups is selected from pentaerythritol tetra(3-mercaptopropionate), trimethylolpropane tri(3-mercaptopropionate), and 2,2'-(ethylenedioxy)diethylthiol.

[0116] 25. The method according to claim 24, wherein the heparin molecule modified to present one or more carbon-carbon double bond functional groups or carbon-carbon triple bond functional groups is prepared by reacting heparin with the following substances:

[0117] (a) Compound of formula IIa:

[0118] H2C=C-(CH2) k -XX IIa,

[0119] Where k is 1 to 50 and XX is a halogen (e.g., compound IIa is allyl bromide); or

[0120] (b) Compounds of formula IIb:

[0121] HC≡C-(CH2) k' -XX IIb,

[0122] Where k' is 1 to 50 and XX is a halogen (e.g., compound IIb is propargyl bromide); or

[0123] (c) Compounds of formula IIc:

[0124] XX-C(O)-R2 IIc,

[0125] Where XX is a halogen and R2 is C. 1-50 Alkyl groups (e.g., compounds of formula IIc selected from one or more of the following: 10-undecenoyl chloride, 4-pentenoyl chloride, and acryloyl chloride).

[0126] 26. The medical device according to any one of claims 1 to 10 is used as a blood-compatible cardiac stent or intravascular stent to prevent the formation of blood clots.

[0127] 27. The medical device according to any one of claims 1 to 10 is used as a blood-compatible organ support to prevent the formation of blood clots.

[0128] 28. The medical device according to any one of claims 1 to 10 is used for blood collection and separation.

[0129] 29. The medical device according to any one of claims 1 to 10 is used for the prevention of blood clots. Attached Figure Description

[0130] Figure 1The present invention illustrates the preparation of a heparin-coated medical device by means of an isocyanate-coated polyurethane device (15) and modified or unmodified heparin-tetrabutylammonium (TBA) (20 or 30) through the following reactions: (a) a urethane formation reaction in acetonitrile (25); or (b) a thiol-isocyanate click reaction in acetonitrile (35).

[0131] Figure 2 A schematic diagram is shown of coating heparin-TBA (40) onto a PHMDI or HDI-coated PU tube (15) via a urethane formation reaction (25) in acetonitrile to obtain the heparin-coated PU tube (50) of the present invention.

[0132] Figure 3 This demonstrates the process of converting heparin-O (CHR) via a carbamate formation reaction (25). 1 CHR 2 O) n A schematic diagram of the heparin-coated PU tube (65) of the present invention, in which H-TBA (60) is coated on PHMDI or HDI coated PU tube (15).

[0133] Figure 4 A schematic diagram is shown of crosslinking 40 or 60 with a binder (42) to form APC crosslinked heparin (70), and then coating it onto a PHMDI or HDI coated PU tube (15) by a carbamate formation reaction (25) to obtain the heparin-coated PU tube (75) of the present invention.

[0134] Figure 5 The following schematic diagram is shown: (a) preparation of modified heparin-TBA (80) having olefin functional groups; (b) preparation of thiol-functionalized heparin-TBA (95) to be coated onto PHMDI or HDI coated PU tube (15) by thiol-isocyanate click reaction (35) to obtain the heparin-coated PU tube (100) of the present invention.

[0135] Figure 6 A schematic diagram is shown showing the reaction of ethynyl-functionalized heparin-TBA (82) with a linker (85) to form thiol-functionalized heparin-TBA (97 and 98).

[0136] Figure 7 The following photographs are shown: (a) Samples 4-10 of the present invention after in vitro testing with goat blood and immersion in deionized water; (b) untreated PU tubes (encased in blood clots) after in vitro testing with goat blood and immersion in deionized water.

[0137] Figure 8 The following SEM images are shown: (a) Sample 5 (using heparin-O (CHR) 1 CHR2 O) n (a) H-TBA(60) coated PHMDI coated PU tube; (b) control sample after in vitro blood test (uncoated PU tube). Detailed Implementation

[0138] Surprisingly, it has been found that a more efficient heparin coating can be obtained by covalently bonding heparin to a substrate via a reaction in which isocyanates form covalent bonds between heparin itself or functionalized heparin molecules. A key feature of this process (and the resulting product) is the use of heparin molecules, where the countercation to the negative charge on heparin is a quaternary ammonium cation. Surprisingly, the use of these substances allows for the use of a wider range of solvents that are compatible with polymeric materials that may be particularly suitable as substrates. Therefore, a medical device is disclosed comprising a covalently bonded heparin coating on a substrate, wherein the covalently bonded heparin coating is a reaction product of the following substances:

[0139] (a) an isocyanate-containing material on or covalently bonded to a substrate, the isocyanate-containing material containing one or more isocyanate groups; and

[0140] (b) Heparin molecules selected from one or more of the following formulas:

[0141] Ia:(A1) z -heparin;

[0142] Ib: and

[0143] Ic:{([HS(CH2) n ] m -L2-[(CH2) n S(CH2) o O]) z -heparin} p The oxygen atom bonded to heparin is part of the heparin molecule;

[0144] ID: {([HS(CH2)} n' ] m' -L3-[(CH2) n' S(CH2) o' COO]) z -heparin} p' The oxygen atom bonded to heparin is part of the heparin molecule;

[0145] Ie:{([HS(CH2) n" ] m” -L4-[(CH2) n" S(C q H 2q-2 )O])z -heparin} p" The oxygen atom bonded to heparin is part of the heparin molecule; and

[0146] If: The fixed portion of the heparin molecular network containing Ifi:

[0147]

[0148] The oxygen atom bonded to heparin is part of the heparin molecule; and

[0149] Ifii's network extension:

[0150]

[0151] The oxygen atom bonded to heparin is part of the heparin molecule;

[0152] in:

[0153] Each A1 and A2 is independent of:

[0154] HO-;

[0155] H(OCHR1CH2) x O-, where the terminal oxygen atom is part of the heparin molecule;

[0156] or

[0157] H(OCHR1CHR2) x O-, where R1 and R2 together with the atoms they are attached to form a carbon ring system with 3 to 10 carbon atoms;

[0158] L1 is the cross-linked portion;

[0159] a ranges from 1 to 25;

[0160] Each z is independently between 1 and 50;

[0161] Each n, n', n”, n”', o, and o' is independently 1 to 20;

[0162] Each m, m', m”, m”', p, p', p”, p”' and p”” is independently 1 to 20;

[0163] Each q is independently between 1 and 20;

[0164] Each L2 to L5 is an independent linking group;

[0165] R1 is H or C1 to C 20 alkyl;

[0166] x is between 1 and 20;

[0167] In formula Ib, | represents the covalent bond between the heparin molecule and the cross-linked portion;

[0168] In equation Ifi, each | represents a connection point with another fixed part of equation Ifi or an extension of equation Ifii;

[0169] In equation Ifii, each | represents a connection point with either the fixed part of equation Ifi or another extension of equation Ifii;

[0170] Each heparin molecule is a polyanionic molecule, wherein each negative charge is balanced by a cation, wherein:

[0171] The cation is substantially a quaternary ammonium ion; and

[0172] The isocyanate-containing material is either a coating on a substrate or covalently bonded to the substrate.

[0173] In the embodiments described herein, the expression "comprising" can be interpreted as the features that need to be mentioned, but does not limit the presence of other features. Alternatively, the expression "comprising" can also refer to the situation where only the listed components / features are intended to be present (e.g., the expression "comprising" can be replaced by the phrases "consists of" or "consists essentially of"). It is explicitly contemplated that both broad and narrow interpretations can be applied to all aspects and embodiments of the invention. In other words, the expression "comprising" and its synonyms can be replaced by the phrases "consists of" or "essentially of" or their synonyms, and vice versa.

[0174] Heparin is a sulfated polysaccharide primarily composed of alternating sequences of hexuronic acid and 2-amino-2-deoxy-D-glucose. The repeating units of heparin are as follows: Figure 2 As shown. Heparin is readily available in an unfractionated form, which can comprise molecules with a wide range of molecular weights. Commercially available heparin typically contains heparin chains with molecular weights ranging from 1,000 Daltons or less to 50,000 Daltons or more. Examples of heparin used in this invention may have a molecular weight of 3,000 to 30,000 Daltons. The heparin used in this invention is heparin in which the anion (-COO) on the heparin backbone... - and -SO3 - The counteracting cations are primarily quaternary ammonium cations, and more specifically, quaternary alkylammonium cations.

[0175] Figure 1A general overview of the process for producing a coated heparin medical device is provided. In a first case, substrate 10 is reacted with or coated with material 12 (not shown) to provide an isocyanate-containing substrate 15. It should be understood that, for clarity, only one isocyanate-containing group is shown, but multiple isocyanate-containing groups may be present on the surface of the substrate. The isocyanate molecules may be covalently bonded to the surface of the substrate (e.g., using diisocyanate molecules that react with the surface of the substrate) or they may be coated thereon. The isocyanate-containing substrate 15 may then be reacted with hydroxyl-containing heparin 20 (the hydroxyl group may be a hydroxyl group directly located on the heparin backbone, or a hydroxyl group spaced apart from the heparin backbone by a linking group) or thiol-containing heparin 30, wherein the thiol group is spaced apart from the heparin backbone by a linking group.

[0176] The polymer can be synthesized using any suitable substrate material, such as ceramic, metal, or more specifically, synthetic polymers. Suitable synthetic polymers that may be mentioned herein include, but are not limited to, polyurethane, PTFE, polyethylene, or PVC. In a particular embodiment of the invention, the substrate may be polyurethane. In embodiments of the invention that may be mentioned herein, isocyanate-containing materials may be covalently bonded to the substrate. In embodiments where isocyanate-containing materials are covalently bonded to the substrate, the resulting covalently bonded isocyanate may be one or more of, but not limited to, hexamethylene diisocyanate, poly(hexamethylene diisocyanate), 4,4'-methylenebis(phenyl isocyanate), and trans-1,4-cyclohexylene diisocyanate.

[0177] Examples of products obtained when using compounds of formula Ia are in Figure 2 and 3 As shown in the image. Figure 2 The connection between the hydroxyl group directly on the backbone of a partial heparin molecule 40 and the isocyanate-containing substrate 15 is shown. It should be noted that in this example, the cation counteracting the anion in heparin is a tetrabutylammonium ion. However, it should be understood that any suitable quaternary ammonium cation can be used. For example, in the aspects and embodiments of the invention disclosed herein, the quaternary ammonium cation can be C... 1-30 Alkyl quaternary ammonium ions, such as C 1-10 Alkyl quaternary ammonium ions, such as tetrabutylammonium ions.

[0178] While the aim is to remove all other cations from the heparin molecule, leaving only quaternary ammonium cations, this may not be entirely achievable. Therefore, as stated above, quaternary ammonium cations constitute the major cation composition associated with the heparin molecule used in the methods and products disclosed herein. When used herein, the term “substantially” is intended to mean that at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99%, for example, at least 99.999% of the cations are quaternary ammonium ions.

[0179] Figure 3 The diagram illustrates the use of a linker to functionalize the heparin backbone, with the linker terminally having a hydroxyl group, where the hydroxyl group is covalently bonded to an isocyanate. This is achieved by employing a heparin molecule 40 and reacting it with a suitable epoxide 41 to generate a hydroxyl group separate from the heparin backbone, as shown in diagram 60. When only one epoxide is needed to react with the hydroxyl group of the backbone to provide the linker, a ring-opening oligomerization reaction may occur, producing an oligomer 41 formed from up to 20 epoxides. The resulting functionalized material 60 can be reacted with an isocyanate-functionalized substrate 15 to provide a covalently bonded product 65. Two epoxides can be used. The first epoxide can be an epoxide in a straight-chain alkyl chain, or the epoxide can be formed as part of a carbide ring.

[0180] It should be understood that Figure 2 and Figure 3 The above-described embodiment is a specific example of the reaction product of compound Ia, wherein A1 is:

[0181] HO-( Figure 2 );or

[0182] H(OCHR1CH2) x O-, where the terminal oxygen atom is part of the heparin molecule; or H(OCHR1CHR2). x O-, where R1 and R2, together with the atoms they are attached to, form a carbon ring system with 3 to 10 carbon atoms. Figure 3 In embodiments of the invention, x can be any suitable value, such as 1 to 20, 1 to 10, or 1 to 5.

[0183] To avoid any doubt, it should be noted that more than one Al group can be used in compounds of formula Ia to form covalent bonds with each heparin molecule. For example, 1 to 50 Al groups can be used per heparin molecule to form covalent bonds with the isocyanate in the substrate.

[0184] Figure 4 The process of bonding a compound of formula Ib to an isocyanate-functionalized substrate is illustrated in general. A linker 42 first reacts to crosslink the heparin chains together. The heparin chains described herein can be 40 or 60. These crosslinked heparin chains can then react with the isocyanate-functionalized substrate 15 to produce the desired product 75.

[0185] In the compound of formula Ib:

[0186]

[0187] A value of 1 to 25, such as 1 to 10, 2 to 5, or 3 to 4, indicates that multiple heparin compounds are cross-linked together via a cross-linking agent. For the avoidance of doubt, the cross-linking will occur in a manner that provides a cross-linked heparin molecular network, and Formula Ib is intended to represent a cross-linked heparin molecular network. It should be understood that A1 and A2 in Formula Ib above may take equivalent values ​​to A1 as described above with respect to compounds of Formula Ia. Finally, each z may independently be 1 to 50, representing the connection point between each heparin molecule and the isocyanate-functionalized substrate.

[0188] In embodiments of the present invention, the crosslinking portion L1 can be one or more of the following: straight-chain or branched diacyl chloride, straight-chain or branched triacyl chloride, straight-chain or branched diepoxide, straight-chain or branched triepoxide, and straight-chain or branched tetraepoxide. Examples of such groups include, but are not limited to, adipicoyl chloride, dodecyl diacyl chloride, sebacyl chloride, octanoyl chloride, 1,2-butanediol diglycidyl ether, neopentyl glycol diglycidyl ether, and trimethylolpropane triglycidyl ether.

[0189] In some embodiments of the invention relating to compounds of formulas Ia and Ib, each R1 may be H or a C1 to C5 alkyl group.

[0190] Examples of products obtained by reacting isocyanate-functionalized substrates with compounds of formulas Ic to If are shown in Figure 5 (b) and Figure 6 In embodiments of the invention using such compounds, one or more of the following may be used:

[0191] (i) Each n, n', n”, n”' is independently 1 to 25, for example 2 to 10, for example 3 to 5;

[0192] (ii) Each m, m', m”, m”', p, p', p”, p”' and p”” is independently 1 to 10, for example 1 to 3;

[0193] (iii) Each q is independently 1 to 15, for example 1 to 10, for example 1 to 6;

[0194] (iv) Each L2 to L5 is independently either branched or non-branched C 1-10 A linking group in the form of an alkyl chain, wherein the alkyl chain is unsubstituted or is composed of one or more chains selected from C10. 1-6 Substitution of alkyl, O, or N groups, or insertion of heteroatoms (such as O or N) into the alkyl chain; and / or

[0195] (v) Each o and o' is independently 1 to 10, for example 1 to 2.

[0196] In compounds of formula Ic-lf, one or more thiol groups are grafted onto the heparin molecule. This is achieved by first functionalizing heparin with an alkene- or alkyne-containing group, which can be done using a suitable alkylene halide (e.g., allyl bromide), such as... Figure 5 As shown in a. Subsequently, olefin-functionalized heparin can react with a compound containing at least two thiol groups in a thiol-olefin click reaction to generate thiol-functionalized heparin, which can be used to participate in the covalent bond formation reaction with the isocyanate-functionalized substrate in a thiol-isocyanate click reaction, such as... Figure 5 b and Figure 6 As shown. Figure 5 b and Figure 6 As shown, compounds of formula Ic-lf can contain various different network structures depending on the reaction of heparin with a thiol-containing compound, and can have more than two thiol groups (e.g., 2, 3, 4, 5, 6, 7, or 8 thiol groups). These multiple thiol groups can crosslink multiple heparin molecules together to create a heparin molecular network, which is then bonded to an isocyanate-functionalized substrate via free thiol groups. Further details regarding methods for producing the disclosed medical device are provided below.

[0197] It is believed that compounds of formula Ib-lf, which can form a heparin molecular network, can provide products with excellent stability—even comparable to products using formula Ia compounds (substantial stability).

[0198] For example, Figure 6 This demonstrates that thiol groups can be introduced into alkynyl-functionalized heparin-TBA (82), which is prepared by reacting 40 with an alkynyl halide (instead of an allyl halide). Figure 6 As shown, alkyne-functionalized heparin-TBA (82) can be crosslinked via a thiol-alkyne click reaction via PETMP 85 in the presence of a tertiary amine catalyst (e.g., DMAP) (92). Typically, one alkynyl group on 82 reacts with two thiol groups, which may result from: (1) a single PETMP molecule forming 97; or (2) two PETMP molecules forming 98. It should be understood that it is important in these reactions that the thiol group is in molar excess compared to the alkynyl (or alkenyl) group in the thiol-alkyne (or thiol-alkene) click reaction, thus the product contains free thiol groups.

[0199] The Examples section below provides specific examples of compounds of formulas Ia to If.

[0200] This article also provides a method for forming the medical device as described above, the method comprising the following steps:

[0201] (a) Providing an isocyanate-containing material on or covalently bonded to a substrate, wherein the isocyanate-containing material contains one or more isocyanate groups;

[0202] (b) In a polar aprotic solvent, an isocyanate-containing material, either on or covalently bonded to a substrate, reacts with one or more heparin molecules selected from the following formulas:

[0203] Ia:(A1) z -heparin;

[0204] Ib: and

[0205] Ic:{([HS(CH2) n ] m -L2-[(CH2) n S(CH2) o O]) z -heparin} p The oxygen atom bonded to heparin is part of the heparin molecule;

[0206] ID: {([HS(CH2)} n' ] m' -L3-[(CH2) n' S(CH2) o' COO]) z -heparin} p' The oxygen atom bonded to heparin is part of the heparin molecule;

[0207] Ie:{([HS(CH2) n" ] m” -L4-[(CH2) n" S(C q H 2q-2 )O]) z -heparin} p" The oxygen atom bonded to heparin is part of the heparin molecule; and

[0208] If: The fixed portion of the heparin molecular network containing Ifi:

[0209]

[0210] The oxygen atom bonded to heparin is part of the heparin molecule; and

[0211] Ifii's network extension:

[0212]

[0213] The oxygen atom bonded to heparin is part of the heparin molecule;

[0214] To provide a medical device according to any one of claims 1 to 10, wherein:

[0215] Each A1 and A2 is independent of:

[0216] HO-;

[0217] H(OCHR1CH2) x O-, where the terminal oxygen atom is part of the heparin molecule;

[0218] or

[0219] H(OCHR1CHR2) x R1 and R2, together with the atoms they are attached to, form a carbon ring system with 3 to 10 carbon atoms;

[0220] L1 is the cross-linked portion;

[0221] a ranges from 1 to 25;

[0222] Each z is independently between 1 and 50;

[0223] Each n, n', n”, n”', o, and o' is independently 1 to 20;

[0224] Each m, m', m”, m”', p, p', p”, p”' and p”” is independently 1 to 20;

[0225] Each q is independently between 1 and 20;

[0226] Each L2 to L5 is an independent linking group;

[0227] R1 is H or C1 to C 20 alkyl;

[0228] x is between 1 and 20;

[0229] In formula Ib, | represents the covalent bond between the heparin molecule and the cross-linked portion;

[0230] In equation Ifi, each | represents a connection point with another fixed part of equation Ifi or an extension of equation Ifii;

[0231] In equation Ifii, each | represents a connection point with either the fixed part of equation Ifi or another extension of equation Ifii;

[0232] Each heparin molecule is a polyanionic molecule, wherein each negative charge is balanced by a cation, wherein:

[0233] The cation is substantially a quaternary ammonium ion; and

[0234] The isocyanate-containing material is a coating on the substrate or covalently bonded to the substrate.

[0235] It should be understood that the compound of formula la-lf and the substrate material are the same as those defined above.

[0236] The polar aprotic solvent can be any suitable polar aprotic solvent. However, when the substrate is a polymer material, the polar aprotic solvent is preferably a material that does not dissolve or otherwise significantly degrade the selected polymer. In specific embodiments of the invention that may be mentioned herein, the polar aprotic solvent can be acetonitrile. It should be understood that the use of quaternary ammonium ions enables the use of polar aprotic solvents in the reactions described herein.

[0237] A1 is H(OCHR1CH2) x O- or H (OCHR1CHR2) x Compounds of formula Ia with the O- group can be formed by reacting heparin with epoxides. A1 and / or A2 are H (OCHR1CH2). x O- or H (OCHR1CHR2) x O-type Ib compounds can be formed by the reaction of heparin with epoxides.

[0238] In compounds of formula Ib, the crosslinking between heparin molecules in the formula Ib compound provides (A1). z -Heparin and (A2) z -Heparin (where A1, A2, and z are as defined above), and react these compounds with a crosslinking agent to form the crosslinking agent. The crosslinking agent may be selected from one or more of the following: straight-chain or branched diacyl chlorides, straight-chain or branched triacyl chlorides, straight-chain or branched diepoxides, straight-chain or branched triepoxides, and straight-chain or branched tetraepoxides. Examples of such crosslinking agents include, but are not limited to, adipicoyl chloride, dodecyl diacyl chloride, sebacyl chloride, octanoyl chloride, 1,2-butanediol diglycidyl ether, neopentyl glycol diglycidyl ether, and trimethylolpropane triglycidyl ether.

[0239] Compounds of formula Ic-lf can be prepared by reacting heparin molecules modified to exhibit one or more carbon-carbon double or triple bond functional groups with a compound containing multiple thiol groups, optionally selected from pentaerythritol tetra(3-mercaptopropionate), trimethylolpropane tri(3-mercaptopropionate), and 2,2'-(ethylenedioxy)diethylthiol. Heparin molecules modified to exhibit one or more carbon-carbon double or triple bond functional groups can be prepared by reacting heparin with the following substances:

[0240] (a) Compound of formula IIa:

[0241] H2C=C-(CH2) k-XX IIa,

[0242] Where k is 1 to 50 and XX is a halogen (e.g., compound IIa is allyl bromide); or

[0243] (b) Compounds of formula IIb:

[0244] HC≡C-(CH2) k' -XX IIb,

[0245] Where k' is 1 to 50 and XX is a halogen (e.g., compound IIb is propargyl bromide); or

[0246] (c) Compounds of formula IIc:

[0247] XX-C(O)-R2 IIc,

[0248] Where XX is a halogen and R2 is C. 1-50 Alkyl groups (e.g., compounds of formula IIc selected from one or more of the following: 10-undecanoyl chloride, 4-pentenoyl chloride and acryloyl chloride).

[0249] In reactions (a) through (c) listed above, additional reactants and / or reagents may be present as needed. For example, in reactions (a) and (b) above, heparin may be pretreated with a base (e.g., NaH) before reacting with the compound of formula IIa or IIb. Full details of the experimental methods can be obtained from the considerations in the experimental section below.

[0250] It should be understood that the resulting heparin-coated substrate can be used to treat or prevent conditions affecting the subject. Therefore, the device disclosed herein can be provided as a medical device:

[0251] (a) Used as a blood-compatible cardiac stent or intravascular stent to prevent the formation of blood clots;

[0252] (b) Used as a blood-compatible organ support to prevent the formation of blood clots;

[0253] (c) Used for blood collection and separation; and

[0254] (d) Used to prevent blood clots.

[0255] These medical devices can be used in suitable treatment methods, wherein the method includes the step of embedding the medical device in a subject in need of it to provide the desired effect described above. It should be understood that at least a portion of these medical devices can be consumed when used by a subject in need of treatment, and the use of these medical devices in drug preparation as described above is also provided.

[0256] (a) Used as a blood-compatible cardiac stent or intravascular stent to prevent the formation of blood clots;

[0257] (b) Used as a blood-compatible organ support to prevent blood clot formation;

[0258] (c) Used for blood collection and separation; and

[0259] (d) Used to prevent blood clots.

[0260] Other aspects and embodiments of the invention are provided in the following non-limiting examples.

[0261] Example

[0262] This invention relates to a medical device comprising a covalently bonded heparin coating on a substrate, which can be used as follows: Figure 1 The schematic diagram illustrates the process. First, an isocyanate-coated device 15 is prepared by coating a polyurethane (PU) medical device or tube (10) with 12[poly(hexamethylene diisocyanate)] (PHMDI) or hexamethylene diisocyanate (HDI). Subsequently, device 15 can be coated with modified or unmodified heparin-tetrabutylammonium (heparin-TBA) via the following reactions: (a) a urethane formation reaction in acetonitrile (25); or (b) a thiol-isocyanate click reaction in acetonitrile (35).

[0263] For the urethane formation reaction (25), a coating reaction occurs between the hydroxyl groups of 20 (heparin-TBA or epoxide-functionalized heparin-TBA) and the isocyanate groups on the surface of 15. For reaction 35, a covalent reaction occurs between the thiol groups of thiol-functionalized heparin-TBA (30) and the isocyanate groups on the surface of 15.

[0264] Materials and methods

[0265] The materials were purchased from the sources provided below.

[0266] Acetonitrile (Sigma-Aldrich, anhydrous, 99.8%)

[0267] Hexane (Sigma-Aldrich, anhydrous, 95%)

[0268] Tetrabutylammonium chloride (Sigma-Aldrich, ≥97%)

[0269] Triethylamine (Tokyo Chemical Industry Co., Ltd., >99.0%)

[0270] Calcium chloride (Sigma-Aldrich, ≥97%)

[0271] Goat blood (Quad Five),

[0272] Sodium citrate dihydrate (Sigma-Aldrich, ≥99%)

[0273] Glutaraldehyde solution (Sigma-Aldrich, Grade I, 25% aqueous solution).

[0274] Poly(hexamethylene diisocyanate) (Sigma-Aldrich, viscosity 1300-2200 cP (25℃)),

[0275] Hexamethylene diisocyanate (Sigma-Aldrich, ≥98%)

[0276] Adipic acid chloride (Sigma-Aldrich, 98%)

[0277] Pentaerythritol tetra(3-mercaptopropionate) (Sigma-Aldrich, >95%)

[0278] Allyl bromide (Sigma-Aldrich, 99%)

[0279] Sodium hydride (Strem Chemicals, 60% mineral oil dispersion),

[0280] Sodium chloride (Sigma-Aldrich, ≥99.5%)

[0281] Heparin (Yantai Dongcheng Biochemical Co., Ltd.)

[0282] 4-(Dimethylamino)pyridine (Sigma-Aldrich, ≥99%)

[0283] Cyclohexene oxide (Sigma-Aldrich, 98%), and

[0284] Dialysis membrane (Spectrum Laboratories, molecular weight cutoff (MWCO) 3,500).

[0285] The substrate is a double-lumen polyurethane conduit (or polyurethane tube). The conduit has a diameter of 2.67 mm and a surface area of ​​1.87 cm². 2 / length of catheter per cm.

[0286] Moisture-sensitive compounds are handled in an argon-protected MBRAUN glove box. Deionized (DI) water is supplied from the ELGA ultrapure water treatment system (PURELAB Option).

[0287] Recorded using a JEOL 500MHz spectrometer 1 H NMR and 13 C NMR spectrum.

[0288] SEM imaging was performed using a JEOL JSM 6700F.

[0289] Infrared (IR) spectra were recorded using a Bruker Vertex 80V vacuum FTIR spectrometer. Note the FTIR absorption intensities: s = strong; vs = very strong; m = moderate; w = weak; and vw = very weak.

[0290] General Step 1—Preparation of Heparin-TBA (40)

[0291] According to the reported method, heparin-TBA was prepared by ion exchange of sodium heparin with tetrabutylammonium chloride (TBACL) in water (40) (ACS Appl. Mater. Interfaces 2016, 8, 8376-8385).

[0292] Typically, tetrabutylammonium chloride solution (TBACL, 1.6 g in 4 mL deionized water) is slowly added to a 50 mL flask containing heparin sodium solution (1 g in 6 mL deionized water) with stirring at room temperature. The resulting mixture is stirred at room temperature for 2 days to allow the Na+ to settle. + The ions (of sodium heparin) were completely exchanged with the TBA ions (of TBACL). The solution was then dialyzed with deionized water for 16 h using a dialysis membrane (molecular weight cutoff (MWCO) of 3500) to remove NaCl and excess TBACL. The water in the solution was removed by rotary evaporation (35°C water bath) to obtain a white solid product, which was further dried overnight under high vacuum at room temperature using an oil pump.

[0293] After drying the product overnight to remove all water, heparin-TBA was observed to be soluble in water, THF, dichloromethane, and acetonitrile. The good solubility of heparin-TBA in acetonitrile is particularly useful because it allows for the coating of heparin-TBA (40) onto polymer devices using more chemically compatible solvents. The synthesized product was obtained through… 1 H, 13 The sample was characterized by C NMR and FTIR spectra and is referred to as "Sample 1".

[0294] 1 ¹H NMR (500MHz, D₂O, δ = ppm): 0.94 (triple peak, J) C-H =7.5Hz), 1.35 (sextile peak, J C-H =7.4Hz), 1.64 (quintet, J C-H =7.9Hz), 3.19 (triple peak, J C-H =8.3Hz), 3.62-5.44 (broad multipeak).

[0295] 13C NMR (125Hz, D2O, δ=ppm): 12.91, 19.19, 23.16, 58.14, 69.37 (broad peak), 76.00 (broad peak).

[0296] FTIR absorption frequency of sample 1 (heparin-TBA particles, cm⁻¹) -1 ): 2959(m), 2934(w), 2874(m), 1616(s), 1464(m), 1381(m), 1216(vs), 1150(w), 1107(w), 1012(vs), 935 (w), 879(s), 801(w), 739(s), 682(vw), 621(vw), 608(vw), 576(s), 548(vw), 530(vw), 487v(w), 414(vw).

[0297] General Step 2—Preparation of PHMDI-coated medical PU tubing

[0298] In a glove box (argon-protected), PHMDI (1.0 g) and anhydrous acetonitrile (20 mL) were added to a 50 mL Schlenk flask and gently shaken for approximately 5 min to form a solution. Six medical PU tube samples (2.67 mm in diameter, 2 cm in length each) were immersed in the PHMDI-acetonitrile solution. The Schlenk flask was then removed from the glove box, connected to a Schlenk tubing line (nitrogen-protected), and heated to 60 °C (in an oil bath) for 5 h. During the 5 h coating time, the Schlenk flask was gently shaken for approximately 1 min every 30 min of reaction time to prevent the PU tubes from sticking together or adhering to the inner surface of the Schlenk flask. After 5 h of incubation, the polyurethane tubes were removed and dried in an oven at 60 °C for 1 h. The PHMDI-coated sample is designated "Sample 2" and characterized by FTIR spectroscopy.

[0299] FTIR absorption frequency (cm) of uncoated PU tube -1 ): 3316(s), 2922(s), 2851(s), 2797(w), 1688(vs), 1527(vs), 1447(m), 1413(vw), 1366(m), 1319(m), 1228(s), 1188(s), 1098 / 1081(vs, double acromion), 1046(w, acromion), 981(m), 899(w), 779(m), 636(m), 610(s), 453(w).

[0300] Sample 2 (PHMDI coated PU tube, cm) -1The FTIR absorption frequencies of the sample are: 3307 (s, broad peak), 2931 (s), 2857 (m), 2263 (vs), 1766 (m), 1684 (vs), 1639 (w, shoulder peak), 1515 (vs), 1352 (m), 1211 (s), 1095 / 1081 (m, double shoulder peak), 1041 (w), 981 (w), 897 / 876 (w, double shoulder peak), 771 (m), 729 (w), 636 (m), 609 (s), and 582 (w, shoulder peak).

[0301] General Step 3—Preparation of HDI-coated Medical PU Tubes

[0302] In a glove box (argon protection), HDI (1.0 g), dibutyltin dilaurate (DBTDL, 120 mg), and anhydrous hexane (20 mL) were added to a 50 mL Schlenk flask and gently shaken for approximately 5 min to obtain a solution. Six medical PU tube samples (approximately 2.67 mm in diameter, 2 cm in length each) were then immersed in the HDI solution. The Schlenk flask was then removed from the glove box, connected to a Schlenk tubing line (under nitrogen protection), and heated to 60 °C (in an oil bath) for 2 h. The PU tubes were then removed, washed with anhydrous hexane (10 mL × 4) and anhydrous acetonitrile (10 mL × 4), and vacuum dried for 1 h. The HDI-coated sample is designated "Sample 3" and characterized by FTIR spectroscopy.

[0303] Sample 3 (HDI-coated PU tube, cm) -1 The FTIR absorption frequencies of the sample are: 3320(s), 2927(s), 2852(s), 2797(w), 2268(m), 1977(vw), 1689(vs), 1662(w, acromion), 1526(s), 1447(m), 1365(w), 1320(m), 1228(m), 1188(m), 1099 / 1081(vs, double acromion), 1045(m, acromion), 982(m), 899(vw), 779(m), 636(m), 610(s), 451(w).

[0304] Example 1

[0305] Heparin-TBA (40) is coated onto a PHMDI-coated polyurethane tube (15) via a urethane formation reaction (25).

[0306] To prepare the heparin-coated device (50) of the present invention, heparin-TBA (40) can be directly used in the urethane formation reaction 25 (in acetonitrile), because the hydroxyl groups of heparin can directly react with the isocyanate groups of the PHMDI-coated tube 15. Figure 2 ).

[0307] In the usual reaction, under argon protection, heparin-TBA40 (500 mg) and anhydrous acetonitrile (10 mL) are added to a 50 mL Schlenk flask and gently shaken for 5 min to obtain a solution. The Schlenk flask is then removed from the glove box, connected to a Schlenk line (under nitrogen protection), and heated to 60 °C in an oil bath.

[0308] Two freshly prepared PHMDI-coated PU tubes (prepared according to general step 2) were immersed in the solution and incubated at 60°C for 6 hours. During the 6-hour coating period, the Schlenk flask was gently shaken for approximately 1 minute every 30 minutes of reaction time to prevent the PU tubes from sticking together or adhering to the inner surface of the Schlenk flask. After 6 hours of incubation, the PU tubes were removed and dried in an oven at 60°C for 2 hours. The synthesized heparin-TBA-coated tube 50 is designated as "Sample 4" and characterized by FTIR spectroscopy.

[0309] Sample 4 (PHMDI-coated PU tube coated with heparin-TBA, cm) -1 The FTIR absorption frequencies of the sample are: 3419 (s, broad peak), 2962 (m), 2937 (w, shoulder peak), 2876 (m), 1615 (s), 1486 (w), 1464 (m), 1413 (w), 1382 (w), 1346 (vw), 1214 (vs), 1149 (w), 1109 (vw), 1053 (vw, shoulder peak), 1024 (vw, shoulder peak), 1009 (vs), 937 (w), 880 (m), 806 (m), 739 (m), 577 (m), 531 (w), 488 (w), and 416 (w).

[0310] Example 2

[0311] Heparin-O (CHR) is coated onto a PHMDI or HDI-coated polyurethane tube (15) via a urethane formation reaction (25). 1 CHR 2 O) n H-TBA(60)

[0312] In addition to directly coating heparin-TBA onto isocyanate-coated medical devices, other modified forms of heparin-TBA (40) can also be used. In one embodiment of the invention, heparin-TBA (40) can be modified with an epoxide (41) before being coated onto a PHMDI or HDI-coated PU tube (15) via a urethane formation reaction (25). Figure 3 ).

[0313] The ring-opening reaction of epoxides with the hydroxyl groups of heparin-TBA allows some hydroxyl groups to be repositioned away from the heparin carbon skeleton to promote the formation of urethane with isocyanates (25). This means that epoxide-modified heparin can be coated onto PU tubes more effectively or more easily than heparin-TBA.

[0314] Typically, the reaction can be carried out at elevated temperatures with or without a catalyst to promote the reaction. Examples of catalysts include triethylamine, dimethylaminopyridine (DMAP), and 1,1,3,3-tetramethylurea. The general formula for epoxide-modified heparin (60) can be represented as heparin-O (CHR). 1 CHR 2 O) n H-TBA, where R 1 and R 2 The substituents are epoxides, and n is the number of epoxide molecules reacted. Epoxide-modified heparin-TBA (60) can be coated onto PU tubes in the same manner as heparin-TBA coating, such as... Figure 3 As shown. One example of the epoxide used is cyclohexene oxide.

[0315] Heparin-O (CHR) was coated onto a PHMDI-coated polyurethane tube (15). 1 CHR 2 O) n H-TBA(60)

[0316] In the usual reaction, heparin-TBA (40) (1.0 g), cyclohexene oxide (256 mg), and anhydrous acetonitrile (10 mL) were added to a 50 mL Schlenk flask in a glove box (under argon protection) and gently shaken for 5 min to form a solution. The Schlenk flask was then removed from the glove box, connected to a Schlenk line (under nitrogen protection), and heated to 80 °C in an oil bath with stirring for 8 h to obtain the corresponding heparin-O (CHR) solution. 1 CHR 2 O) n H-TBA (60). The reaction mixture was cooled to room temperature, and then two PHMDI-coated polyurethane tubes (prepared according to general step 2) were immersed in the solution at room temperature for 1 min. The coated tubes were then removed and dried in an oven at 60 °C for 30 min. This coating step was repeated once, followed by drying in an oven for 1 h. The obtained sample is designated "Sample 5" and characterized by FTIR spectroscopy.

[0317] Sample 5 (coated with heparin-O (CHR) 1 CHR 2 O) nH-TBA (60) PHMDI coated polyurethane tube (15), cm -1 The FTIR absorption frequencies of the sample are: 3402 (s, broad peak), 2961 (s), 2936 (w, shoulder), 2875 (m), 1620 (s), 1514 (vw), 1486 (vw, shoulder), 1461 (m), 1421 (w), 1381 (w), 1345 (vw), 1215 (vs), 1151 (w, shoulder), 1108 (vw), 1054 (vw, shoulder), 1025 / 1012 (vs, double shoulder), 937 (w), 882 (m), 807 (w), 761 / 741 (m, double shoulder), 624 (w, shoulder), 624 (vw), 606 (vw), 577 (m), 534 (vw), 425 (w).

[0318] Heparin-O (CHR) was coated onto the HDI-coated polyurethane tube (15). 1 CHR 2 O) n H-TBA(60)

[0319] Using the above-mentioned method, heparin-O (CHR) is coated onto a PHMDI-coated polyurethane tube (15). 1 CHR 2 O) n The same steps were followed to coat the HDI-coated PU tubes with heparin-O (CHR)

[60] . 1 CHR 2 O) n H-TBA(60). The obtained sample is designated as "Sample 6" and characterized by FTIR spectroscopy.

[0320] Sample 6 (coated with heparin-O (CHR) 1 CHR 2 O) n H-TBA (60) HDI-coated polyurethane tube (15), cm -1 The FTIR absorption frequencies of the sample are: 3423 (s, broad peak), 2962 (s), 2937 (w), 2877 (m), 1684 (w, shoulder), 1658 (w, shoulder), 1617 (s), 1526 (w), 1487 (w), 1464 (w), 1412 (w), 1381 (w), 1320 (w), 1219 (vs), 1071 (vw, shoulder), 1054 (vw, shoulder), 1027 / 1012 (vs, double shoulder), 984 (vw, shoulder), 886 (m), 742 (w), 635 (w), 609 (s).

[0321] Example 3

[0322] On a PHMDI-coated polyurethane tube (15), APC crosslinked heparin-TBA (40) or APC crosslinked heparin-O (CHR) is coated via a urethane formation reaction 25. 1 CHR 2 O) n H-TBA(60)

[0323] Heparin-TBA (40) or epoxide-modified heparin-O (CHR) 1 CHR 2 O) n H-TBA (60) can be further modified to form intermolecular crosslinks with its respective adjacent heparin molecules. In one specific embodiment of the invention, crosslinking can be achieved by reacting the hydroxyl groups of 40 or 60 with the linker 42 (i.e., adipic acid chloride (APC)) in the presence of a base (triethylamine) to obtain the crosslinking product 70. Figure 4 Alternatively, intermolecular crosslinking can be achieved by reacting 40 or 60 with a linker containing multiple epoxy groups. Intermolecular crosslinking increases the bonding density of heparin on isocyanate-coated medical devices, thereby reducing heparin leakage from the medical device. This improves the lifespan of the heparin coating obtained on the device.

[0324] APC crosslinked heparin-TBA (40) was coated onto a PHMDI-coated polyurethane tube (15).

[0325] In the usual reaction, heparin-TBA (40) (1.0 g) and anhydrous acetonitrile (10 mL) were added to a 50 mL Schlenk flask in a glove box (protected by argon) and gently shaken for 5 min to form a solution. Triethylamine (192 mg) was then added to the solution. With stirring, APC solution (120 mg in 1.5 mL ACN) was added dropwise to this solution over 5 min, and the reaction mixture was stirred at room temperature for 30 min. Two PHMDI-coated PU tubes (prepared according to general step 2) were immersed in the solution at room temperature for 1 min, then removed and dried in an oven at 60 °C for 10 min. The coating step was repeated twice, followed by drying in an oven at 60 °C for 2 h. The obtained sample is designated "Sample 7" and characterized by FTIR spectroscopy.

[0326] Sample 7 (PMHDI-coated PU tube coated with APC crosslinked heparin-TBA (40), cm) -1The FTIR absorption frequencies of the sample are: 3317 (s, broad peak), 2960 (s), 2935 (m), 2875 (m), 1715 (w), 1687 (w), 1660 (w), 1625 (w), 1525 (m), 1465 (m), 1381 (w), 1319 (vw), 1224 (vs), 1151 (vw), 1106 (vw, shoulder), 1055 (vw, shoulder), 1027 / 1013 (vs, double shoulder), 938 (w), 884 (m), 805 (w), 779 (w), 741 (w), 686 (w), 610 (m), 577 (w), 492 (vw).

[0327] APC crosslinked heparin-O (CHR) was coated onto a PHMDI-coated polyurethane tube (15). 1 CHR 2 O) n H-TBA(60)

[0328] Heparin-O (CHR) was prepared following the same steps as in Example 2. 1 CHR 2 O) n H-TBA (60) solution. After cooling to room temperature, APC solution (60 mg dissolved in 1 mL ACN) was added dropwise to solution 60 over 5 min with stirring, and then the reaction mixture was stirred at room temperature for 30 min. Two PHMDI-coated PU tubes (prepared according to general step 2) were immersed in the solution at room temperature for 1 min, then removed and dried in an oven at 60 °C for 10 min. The coating step was repeated twice, and then dried in an oven at 60 °C for 2 h. The obtained sample is designated "Sample 8" and characterized by FTIR spectroscopy.

[0329] Sample 8 (coated with APC crosslinked heparin-O (CHR) 1 CHR 2 O) n H-TBA(60) PMHDI coated PU tube, cm -1 The FTIR absorption frequencies of the sample are: 3313 (s, broad peak), 2928 (s), 2852 (s), 2796 (w), 1689 (s), 1661 (w, shoulder peak), 1525 (s), 1447 (m), 1412 (vw), 1366 (w), 1320 (w), 1227 (s), 1188 (s), 1098 (w, shoulder peak), 1080 (vs), 1041 (m, shoulder peak), 982 (m), 898 (w), 808 (vw), 779 (m), 636 (m), 609 (s), 443 (w).

[0330] Example 4

[0331] PETMP crosslinked heparin-TBA (95) was coated onto polyurethane tubes via a thiol-isocyanate click reaction at 35°C.

[0332] In addition to the carbamate formation reaction 25, the isocyanate-coating device (15) can also be coated with thiol-isocyanate via a thiol-isocyanate click reaction in acetonitrile (35) using thiol-functionalized heparin TBA (95, 97, and 98). Figure 5 b and Figure 6 The reaction occurs between the thiol groups on the thiol-functionalized heparin-TBA and the isocyanate groups on the 15 surface.

[0333] In one embodiment of the invention, a thiol group can be introduced into heparin-TBA (40) in two steps. First, a carbon double or triple bond is introduced into heparin-TBA. In a conventional step, heparin-TBA (40) introduces a carbon double bond by alkylation with NaH / allyl bromide or by reaction with an acyl chloride to provide a modified heparin-TBA (80) having an olefinic functional group. Figure 5 a).

[0334] Subsequently, thiol groups are introduced via a thiol-ene click reaction (a reaction between a thiol group and a double bond) or a thiol-alkynyl click reaction (a reaction between a thiol group and a triple bond). The thiol-ene click reaction involves crosslinking 80 with an excess of a polythiol compound (e.g., pentaerythritol tetra(3-mercaptopropionate) (PETMP, 85)) in the presence of a tertiary amine catalyst (e.g., DMAP) (90) to obtain PETMP-crosslinked heparin (95) with an excess of thiol groups (95). Figure 5 b). Then, PETMP crosslinked heparin 95 is covalently coated onto the isocyanate coating device (15) by a thiol-isocyanate click reaction (35) to obtain a coated tube 100.

[0335] PETMP crosslinked heparin-TBA (95) was coated onto a PHMDI-coated polyurethane tube (15) via a thiol-isocyanate click reaction 35.

[0336] In a glove box (protected by argon), heparin-TBA40 (1.0 g) and anhydrous acetonitrile (10 mL) were added to a 50 mL Schlenk flask and gently shaken for 5 min to obtain a solution. Under stirring at room temperature, a slurry of NaH (26 mg) in 2 mL of anhydrous acetonitrile was added dropwise over 5 min (generating gas), and stirring was continued for 20 min. Then, a solution of allyl bromide (79 mg) in 2 mL of anhydrous acetonitrile was added dropwise over 5 min, and stirring was continued for 3 h. 4-Dimethylaminopyridine (DMAP, 40 mg) was added, and stirring was continued for 2 h. Under stirring at room temperature, the resulting solution was added dropwise to a solution of pentaerythritol tetra(3-mercaptopropionate) (PETMP, 160 mg; 85) in 10 mL of anhydrous acetonitrile, and stirring was continued for 6 h to obtain a PETMP crosslinked heparin-TBA solution (95) containing excess thiol groups.

[0337] Two PHMDI-coated polyurethane tubes (prepared according to general step 2) were immersed in PETMP crosslinked heparin-TBA solution 95 for 1 min at room temperature, then removed and dried in an oven at 60°C for 30 min. This coating step was repeated twice, and the tubes were then dried in an oven at 60°C for 1 h. The obtained sample is designated "Sample 9" and characterized by FTIR spectroscopy.

[0338] Sample 9 (PHMDI-coated PU tube coated with PETMP crosslinked heparin-TBA, cm) -1 The FTIR absorption frequencies of the sample are: 3318 (s, broad peak), 2960 (w, shoulder peak), 2932 (s), 2858 (m), 2270 (w), 1683 (s), 1659 (m), 1521 (s), 1466 (m), 1348 (vw), 1217 (vs), 1097 (vw), 1074 (w), 1034 (m), 1013 (vw, shoulder peak), 984 (vw, shoulder peak), 887 (w), 805 (vw), 775 (w), 735 (vw), 635 (w), and 609 (s).

[0339] On an HDI-coated polyurethane tube (15), PETMP crosslinked heparin-TBA (95) was coated via a thiol-isocyanate click reaction 35.

[0340] Two HDI-coated polyurethane tubes 15, prepared according to general step 3, were immersed in PETMP crosslinked heparin-TBA solution 95 (prepared according to the above steps in the current embodiment) for 1 min. The samples were then dried in an oven at 60°C for 1 h. The obtained sample is designated "Sample 10" and characterized by FTIR spectroscopy.

[0341] Sample 10 (HDI-coated PU tube coated with PETMP crosslinked heparin-TBA, cm)-1 The FTIR absorption frequencies of the sample are: 3327 (s, broad peak), 2962 (s), 2936 (m), 2876 (m), 1737 (m), 1687 (w), 1647 (w), 1620 (m), 1526 (w), 1487 (vw), 1465 (w), 1414 (vw), 1382 (w), 1354 (vw), 1320 (vw), 1226 (vs), 1097 (vw, shoulder), 1070 (vw, shoulder), 1029 (vs), 1014 (vw, shoulder), 886 (m), 806 (vw), 779 (m), 741 (w), 635 (w), 609 (s), 538 (vw), 465 (vw), 452 (vw), 425 (vw).

[0342] Example 5

[0343] In vitro blood tests on heparin-coated PU tubing.

[0344] The antithrombotic effects of heparin-coated samples 4-10, uncoated PU tubes (or “control samples” here) were tested using goat blood. PHMDI-coated PU tubes (from general step 2) and HDI-coated PU tubes (from general step 3) were also tested.

[0345] Sample preparation

[0346] Cut the sample or PU tube / catheter into 1 cm segments. Before testing, incubate 4 to 10 samples in ultrapure water for 24 hours to release unbound heparin-TBA.

[0347] Test steps

[0348] The test was conducted at room temperature. Each 1 cm sample segment was placed into a 15 mL polypropylene (PP) tube containing 1 mL of goat blood. Table 1 lists the details of the goat blood used. Table 2 lists the test setup.

[0349] Table 1. Goat Blood Information

[0350]

[0351] Table 2. Test setup for samples 4-10

[0352]

[0353] To begin the test, a volume of CaCl2 aqueous solution (0.68% w / v) was added to a PP tube to achieve a 1:4 molar ratio (CaCl2 to Na3-citrate). The resulting mixture was vigorously shaken to achieve rapid and homogeneous mixing. After adding CaCl2, the time required for coagulation of the control sample was measured using a stopwatch. The measurement was stopped when coagulation of the control sample was visually observed. All coagulation tests for samples 4-10 were immediately terminated by removing the samples from the blood, and the coagulation status of the samples was observed. All samples were washed with 1×PBS, and the adsorbed blood cells were fixed by immersion in 1×PBS with 2.5% glutaraldehyde overnight at 4°C, followed by drying. After cell fixation, all samples were rapidly rinsed with water and vacuum dried, and the surface morphology of the samples was observed using a JOEL JSM-6700 scanning electron microscope.

[0354] result

[0355] Samples 4 through 10 all showed antithrombotic activity. No blood clots were observed on the surface of the heparin-coated PU tubes. Figure 7 a). Conversely, in the original PU tube ( Figure 7 b) Numerous blood clots formed on the surfaces of PHMDI-coated PU tubing and HDI-coated PU tubing (results not provided here).

[0356] Compare SEM images with control samples ( Figure 8 Compared to sample 5, (b) Figure 8 a) Contains very little platelet adhesion. The porous surface of PU is still clearly visible on the coated sample 5, while the surface of the control sample is covered with platelets and fibrinogen.

Claims

1. A method for preparing a medical device comprising a covalently bonded heparin coating, the method comprising the following steps: (a) Providing an isocyanate-containing material on or covalently bonded to a substrate, said isocyanate-containing material containing one or more isocyanate groups; (b) In a polar aprotic solvent, an isocyanate-containing material on or covalently bonded to a substrate reacts with one or more heparin molecules selected from the following formulas: Ia:(A1) z -heparin; Ib: and Ic:{([HS(CH2) n ] m -L2-[(CH2) n S(CH2) o O]) z -heparin} p The oxygen atom bonded to heparin is part of the heparin molecule; ID: {([HS(CH2)} n' ] m' -L3-[(CH2) n' S(CH2) o' COO]) z -heparin} p' The oxygen atom bonded to heparin is part of the heparin molecule; Ie:{([HS(CH2) n" ] m” -L4-[(CH2) n" S(C q H 2q-2 )O]) z -heparin} p" The oxygen atom bonded to heparin is part of the heparin molecule; and If: The fixed portion of the heparin molecular network containing Ifi: The oxygen atom bonded to heparin is part of the heparin molecule; and Ifii's network extension: The oxygen atom bonded to heparin is part of the heparin molecule; To provide a medical device according to any one of claims 1 to 10, wherein: Each A1 and A2 is independent of: HO-; H(OCHR1CH2) x O-, where the terminal oxygen atom is part of the heparin molecule; or H(OCHR1CHR2) x O-, where R1 and R2 together with the atoms they are attached to form a carbon ring system with 3 to 10 carbon atoms; L1 is the cross-linked portion; a ranges from 1 to 25; Each z is independently between 1 and 50; Each n, n', n”, n”', o, and o' is independently 1 to 20; Each m, m', m”, m”', p, p', p”, p”' and p”” is independently 1 to 20; Each q is independently between 1 and 20; Each L2 to L5 is an independent linking group; R1 is H or C1 to C 20 alkyl; x is between 1 and 20; In formula Ib, | represents the covalent bond between the heparin molecule and the cross-linked portion; In equation Ifi, each | represents a connection point with another fixed part of equation Ifi or an extension of equation Ifii; In equation Ifii, each | represents a connection point with either the fixed part of equation Ifi or another extension of equation Ifii; Each heparin molecule is a polyanionic molecule, wherein each negative charge is balanced by a cation, wherein: The cation is substantially a quaternary ammonium ion; and Isocyanate-containing materials are either coated onto a substrate or covalently bonded to the substrate. The heparin molecules react with isocyanate-containing materials to form urethane or to undergo a thiol-isocyanate click reaction.

2. The preparation method according to claim 1, characterized in that, The quaternary ammonium ion is tetrabutylammonium ion.

3. The preparation method according to claim 2, characterized in that, The polar aprotic solvent is acetonitrile.

4. The preparation method according to claim 1, characterized in that, The isocyanate-containing material of the substrate is one or more of the following: hexamethylene diisocyanate, poly(hexamethylene diisocyanate), 4,4'-methylene bis(phenyl isocyanate), and trans-1,4-cyclohexyl diisocyanate.

5. The preparation method according to claim 1, characterized in that, Where A1 in compound Ia or A1 and / or A2 in compound Ib is H(OCHR1CH2) x O- or H (OCHR1CHR2) x When O-, the compound of formula Ia or Ib is formed by the reaction of heparin with epoxide.

6. The preparation method according to claim 5, characterized in that, In the preparation process of the Ic-If compound, before heparin reacts with the epoxide, heparin first reacts with tetrabutylammonium chloride to form heparin-TBA.

7. The preparation method according to claim 1, characterized in that, Preparation process of Ic-If compound: First, heparin is functionalized with olefin or alkyne groups. Then, olefin-functionalized heparin reacts with a compound containing at least two thiol groups in a thiol-olefin click reaction to generate thiol-functionalized heparin.

8. The preparation method according to claim 7, characterized in that, In the preparation process of the Ic-If compound, heparin first reacts with tetrabutylammonium chloride to form heparin-TBA, and then heparin is prepared by thiol functionalization.

9. The preparation method according to claim 1, characterized in that, In the compound of formula Ib, the crosslinking between heparin molecules provides (A1). z -Heparin and (A2) z -Heparin, and react these compounds with a crosslinking agent to form a crosslinking agent selected from one or more of the following: linear or branched diacyl chloride, linear or branched triacyl chloride, linear or branched diepoxide, linear or branched triepoxide, and linear or branched tetraepoxide (e.g., the crosslinking portion is selected from one or more of the following: adipicoyl chloride, dodecene diacyl chloride, sebacyl chloride, octanoyl chloride, 1,2-butanediol diglycidyl ether, neopentyl glycol diglycidyl ether, and trimethylolpropane triglycidyl ether).

10. The preparation method according to claim 1, characterized in that, The If compound is prepared by reacting a heparin molecule modified to present one or more carbon-carbon double or triple bond functional groups with a compound containing multiple thiol groups, wherein the compound containing multiple thiol groups is optionally selected from pentaerythritol tetra(3-mercaptopropionate), trimethylolpropane tri(3-mercaptopropionate), and 2,2'-(ethylenedioxy)diethylthiol.

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