Use of polysialic acid or a derivative thereof in an anti-foreign body reaction

Abstract

The present application relates to the use of polysialic acid or its derivatives in anti-foreign body reaction, in particular, to a polysialic acid anti-foreign body reaction material for resisting biological contamination, for regulating macrophage phenotype to reduce inflammatory response, and for resisting foreign body reaction. The polysialic acid material includes a bulk material prepared from polysialic acid and its derivatives, or a coating material for surface modification of the material, which can resist protein adsorption, cell adhesion and adhesion of various microorganisms, can act on immune cells, such as regulating the phenotype of macrophages, thereby reducing acute and chronic inflammatory response, and has excellent anti-foreign body reaction effect as an implant material.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomaterials, and in particular to the use of polysialic acid or its derivatives in resisting foreign body reaction. BACKGROUND

[0002] Biomaterials, as the core components of implanted devices and tissue engineering in modern medicine, have long been challenged by foreign body response (FBR) in vivo. FBR is a cascade reaction mediated by the immune system, which usually starts when the implant comes into contact with body fluids. Proteins, platelets and other biological molecules are rapidly adsorbed on the surface within seconds, which in turn activates immune cells such as macrophages, leading to local chronic inflammation and the formation of fibrotic capsules. This capsule not only limits the normal interaction between the implant and the surrounding tissue, but also can cause functional failure, increased risk of infection or thrombosis and other complications. For example, in cardiovascular stent implantation, protein adsorption recruits platelets and triggers the coagulation cascade, further exacerbating inflammation and thrombosis; in brain electrode implantation, blood protein adsorption on the electrode surface activates microglia and further activates astrocytes to form a package, leading to signal attenuation or even electrode failure.

[0003] Current strategies to address FBR mainly revolve around material surface modification and bulk design. The most commonly used strategy is to modify the surface of the implanted material with a hydrophilic coating, such as PEG chains, hydrophilic polypeptide polymers or zwitterionic polymers, etc. The hydrophilic properties of these coatings can weaken the non-polar interaction between proteins and hydrophobic surfaces, while also resisting protein adsorption through surface hydration layers and steric effects, thereby reducing non-specific protein adsorption. Another strategy focuses on immune regulation, such as using nanomedicine carriers combined with anti-inflammatory drugs (such as curcumin, interferon, etc.) and immobilized on the material surface, or coating the surface with polymers with immune regulation function, thereby constructing a coating with long-term immune regulation function to inhibit inflammatory response at different stages after implantation. However, these strategies still have limitations: the long-term stability and processing adaptability of surface coatings are insufficient, the optimization of material bulk design lacks universality for physical properties, and drug strategies may face challenges in toxicity or drug release control.

[0004] Therefore, developing anti-FBR materials with anti-protein adsorption, immune regulation functions and suitable for a variety of substrates is still a technical bottleneck that needs to be broken through in the field of biomedical engineering. SUMMARY

[0005] The purpose of the present application is to provide the use of polysialic acid or its derivatives for antibacterial, protein adsorption inhibition, macrophage phenotype regulation and inflammation reduction, and for resisting foreign body reaction.

[0006] In a first aspect of the application, there is provided the use of a polysialic acid or a derivative thereof, or a material prepared from a polysialic acid or a derivative thereof, for the manufacture of a medical material for use in combating a foreign body reaction.

[0007] In another preferred embodiment, the polysialic acid is of the formula I

[0008] I

[0009] n is an integer from 2 to 400.

[0010] In another preferred embodiment, n is an integer from 10 to 400, for example 20, 50, 100, 200, 300, 400.

[0011] In another preferred embodiment, the polysialic acid has a molecular weight of from 600 Da to 100,000 Da, preferably from 5 kDa to 100 kDa, for example 10 kDa, 30 kDa, 50 kDa, 100 kDa or more than 30 kDa.

[0012] In another preferred embodiment, the polysialic acid derivative is a compound of the formula II or III, or a pharmaceutically acceptable salt thereof,

[0013]

[0014] wherein,

[0015] each R1is independently selected from -OR a , -NR b R c ;

[0016] each R2is independently selected from C1-C12 alkyl, C1-C12 haloalkyl, 2-12 membered heteroalkyl, C1-C12 alkylsulfonyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C12 cycloalkyl, C6-C12 aryl, 5-12 membered heteroaryl, 3-12 membered heterocyclyl, C1-C6 alkylguanidinyl, C1-C6 alkylester, propenyl, methacryl, -(CH2) m -OR a , -(CH2) m -NR b R c ;

[0017] each R3is independently selected from hydrogen, C1-C12 alkyl, C1-C12 alkylaldehyde, -R’-O-R”, -COR a ’, -CH(OH)-R b ’, -CH2-CH(OH)-R c ’;

[0018] R4, R a , R b , R c each independently selected from the group consisting of hydrogen, -(CH2CH2O) p -CH2CH2-N3, -(CH2CH2) p -N3, C1-C15 alkyl, C1-C15 alkylamino, C2-C15 alkenyl, C2-C15 alkynyl, C1-C15 alkylhydroxy, C1-C15 alkylformyl, C1-C15 alkylsulfonyl, -R'-COOR", -R'-CO-R", -R'-O-R"-, -R'-S-R", C3-C12 cycloalkyl, C4-C12 cycloalkenyl, 5-12 membered heterocyclyl, C6-C12 aryl, 5-12 membered heteroaryl, benzyl (Bn), or R a and R b form, together with the N atom to which they are attached, a 3-8 membered substituted or unsubstituted heterocyclyl;

[0019] R a , R b , R c each independently selected from the group consisting of hydrogen, C1-C15 alkyl, C2-C15 alkenyl, C2-C15 alkynyl, C1-C15 alkylformyl, C1-C15 alkylsulfonyl, -R'-COOR", -R'-CO-R", -R'-O-R"-, -R'-S-R", C3-C12 cycloalkyl, C4-C12 cycloalkenyl, 5-12 membered heterocyclyl, C6-C12 aryl, 5-12 membered heteroaryl;

[0020] R' is independently selected from the group consisting of null, C1-C15 alkylene, C2-C15 alkenylene, C2-C15 alkynylene, C3-C12 cycloalkylene, C4-C12 cycloalkenylene, 3-12 membered heterocyclylene, C6-C12 arylene, 5-12 membered heteroarylene;

[0021] R" is independently selected from the group consisting of C1-C15 alkyl, C1-C15 alkylamino, C2-C15 alkenyl, C2-C15 alkynyl, C3-C12 cycloalkyl, C4-C12 cycloalkenyl, 3-12 membered heterocyclyl, C6-C12 aryl, 5-12 membered heteroaryl;

[0022] each m is independently an integer from 1 to 12;

[0023] each n is independently an integer from 2 to 400;

[0024] each p is independently an integer from 1 to 5.

[0025] In another preferred embodiment, the polysialic acid derivative is a homopolymer or a copolymer.

[0026] In another preferred embodiment, the polysialic acid derivative copolymer is a random copolymer, a block copolymer or an alternating copolymer.

[0027] In another preferred embodiment, the polysialic acid derivative is a compound according to formula II-1, II-2, II-3 or formula III-1, or a pharmaceutically acceptable salt thereof,

[0028]

[0029] wherein R1, R2, R3, R4, n are each independently as described herein.

[0030] In another preferred embodiment, R1is -NHR b .

[0031] In another preferred embodiment, R b is selected from -(CH2CH2O) p -CH2CH2-N3, -(CH2CH2) p -N3.

[0032] In another preferred embodiment, R2is selected from C1-C6alkyl, C1-C6haloalkyl, 2-6 membered heteroalkyl, C2-C6alkenyl, C2-C6alkynyl.

[0033] In another preferred embodiment, R2is selected from C1-C6alkyl, C2-C6alkenyl, preferably C2-C4alkenyl.

[0034] In another preferred embodiment, R3is selected from -COR a .

[0035] In another preferred embodiment, R a is selected from hydrogen, C1-C15alkyl, C2-C15alkenyl, C2-C15alkynyl.

[0036] In another preferred embodiment, R a is C2-C6alkenyl, preferably C2-C4alkenyl.

[0037] In another preferred embodiment, R4is selected from -(CH2CH2O) p -CH2CH2-N3, -(CH2CH2) p -N3.

[0038] In another preferred embodiment, p is 1, 2, 3, 4 or 5.

[0039] In another preferred embodiment, the term "heteroalkyl" refers to an alkyl group containing 1, 2, or 3 heteroatoms selected from N, O, S, including alkoxy, alkyl- amino, alkyl-S-alkyl, alkylmercapto, alkyl-O-alkyl, and the like.

[0040] In another preferred embodiment, the poly sialic acid derivative is selected from the following compounds or pharmaceutically acceptable salts thereof:

[0041] ,

[0042] wherein n is an integer from 20 to 400.

[0043] In another preferred embodiment, the poly sialic acid derivative has a molecular weight of 600 Da to 100,000 Da, preferably 5 kDa to 100 kDa, for example 10 kDa, 30 kDa, 50 kDa, 100 kDa, or more than 30 kDa.

[0044] In another preferred embodiment, the pharmaceutically acceptable salt is a pharmaceutically acceptable salt of a carboxyl group in the poly sialic acid derivative with a base.

[0045] In another preferred embodiment, the pharmaceutically acceptable salt is selected from the group consisting of sodium salt, potassium salt, magnesium salt, calcium salt, ammonium salt.

[0046] In another preferred embodiment, the material is a hydrogel material or a coating material.

[0047] In another preferred embodiment, the hydrogel material is obtained by crosslinking poly sialic acid or a derivative thereof with an optional macromolecule.

[0048] In another preferred embodiment, the macromolecule is selected from the group consisting of gelatin, hyaluronic acid, starch, chitosan, sodium alginate, sodium carboxymethyl cellulose, carboxymethyl cellulose, or a combination thereof.

[0049] In another preferred embodiment, the crosslinking comprises crosslinking by a crosslinking agent or photocrosslinking by a photoinitiator.

[0050] In another preferred embodiment, the crosslinking agent is selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), glutaraldehyde, divinyl sulfone (DVS), or a combination thereof.

[0051] In another preferred embodiment, the photoinitiator is selected from the group consisting of Irgacure 2959, Irgacure 1173, LAP, Eosin Y, or a combination thereof.

[0052] In another preferred embodiment, the hydrogel material is obtained by crosslinking poly sialic acid with an optional macromolecule by a crosslinking agent.

[0053] In another preferred embodiment, the hydrogel material is prepared by photo-crosslinking the poly-sialic acid derivative containing C=C double bond and optional macromolecule by a photo-crosslinking agent.

[0054] In another preferred embodiment, the hydrogel material is prepared by a method selected from Method I or Method II:

[0055] The Method I comprises the following steps:

[0056] A1, blending the hydrogel raw material components with a base and a crosslinking agent to obtain a pre-gel solution, wherein the hydrogel raw material components are poly-sialic acid and optional macromolecule;

[0057] A2, heating and crosslinking the pre-gel solution, and post-processing to obtain the hydrogel material;

[0058] The Method II comprises the following steps:

[0059] B1, blending the poly-sialic acid derivative containing C=C double bond with a photo-initiator, and then performing photo-crosslinking, and after post-processing, obtaining the hydrogel material.

[0060] In another preferred embodiment, in step A1, the base is selected from the group consisting of sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, ammonia, potassium carbonate, or a combination thereof.

[0061] In another preferred embodiment, in step A1, the mass ratio of the hydrogel raw material components to the base is 5-200:1, preferably 10-100:1.

[0062] In another preferred embodiment, in step A1, the mass ratio of the hydrogel raw material components to the crosslinking agent is 1-200:1, preferably 1-50:1.

[0063] In another preferred embodiment, in step A1, in the hydrogel raw material components, when the macromolecule is added, the mass ratio of poly-sialic acid to the macromolecule is (0.5-1.5):(0.5-1.5), preferably (0.8-1.2):(0.8-1.2), for example 1:1.

[0064] In another preferred embodiment, in step A2, the heating and crosslinking refers to crosslinking at 25-65℃, preferably 35-50℃ for 1-48h, preferably 2-12h.

[0065] In another preferred embodiment, in step A2, the post-processing comprises swelling the obtained hydrogel to equilibrium in a buffer solution, and then performing dialysis to obtain the hydrogel material, wherein the buffer solution is selected from PBS and physiological saline.

[0066] In another preferred embodiment, in step B1, the mass ratio of the poly sialic acid derivative containing C=C double bond to the photoinitiator is 1-200:1, preferably 10-100:1 or 1-50:1.

[0067] In another preferred embodiment, in step B1, the light irradiation refers to the use of ultraviolet light with a wavelength of 350-370 nm.

[0068] In another preferred embodiment, in step B1, the intensity of the light irradiation is 300-700 mW / cm 2 light intensity.

[0069] In another preferred embodiment, in step B1, the time of the light irradiation is 1-30 min, preferably 2-10 min.

[0070] In another preferred embodiment, in step B1, the post-treatment comprises soaking the obtained hydrogel in water to obtain the hydrogel material.

[0071] In another preferred embodiment, the coating material is a coating of poly sialic acid or its derivative on a substrate, and the substrate is selected from the group consisting of medical inorganic non-metallic materials, medical inorganic metallic materials, and medical polymer materials.

[0072] In another preferred embodiment, in the coating material, the poly sialic acid or its derivative is covalently grafted to the surface of the substrate by a chemical method.

[0073] In another preferred embodiment, the medical inorganic non-metallic material is selected from the group consisting of glass, conductive glass, silicon, and ceramic.

[0074] In another preferred embodiment, the medical inorganic metallic material is selected from the group consisting of gold, tungsten, titanium, titanium alloy, nickel-titanium alloy, and titanium-chromium alloy.

[0075] In another preferred embodiment, the medical polymer material is selected from the group consisting of polyurethane, polydimethylsiloxane, polystyrene, poly-epsilon-caprolactone, polyacrylamide, polymethyl methacrylate, polypropylene, polycarbonate, PBS resin, polytetrafluoroethylene, polylactic acid, and polyamide.

[0076] In another preferred embodiment, the substrate is selected from the group consisting of nickel-titanium sheet, glass, and PDMS.

[0077] In another preferred embodiment, the coating material is prepared by a method comprising:

[0078] C1, pretreating the substrate with dopamine to graft hydroxyl groups on the surface;

[0079] C2, mixing poly sialic acid or its derivative with the pretreated substrate and activating with an activating agent to obtain the coating.

[0080] In another preferred embodiment, in step C1, the dopamine pretreatment refers to immersing the substrate into a mixed solution of dopamine hydrochloride and Tris HCl buffer.

[0081] In another preferred embodiment, the mixed solution is a dopamine hydrochloride solution with a concentration of 0.5-5 mg / mL configured using Tris HCl buffer with a pH of 8-9.

[0082] In another preferred embodiment, in step C2, the activation reagent is EDCI / NHS.

[0083] In another preferred embodiment, the source of biofouling is a protein, a nucleic acid, a serum, a plasma, a platelet, a cell, or a microorganism.

[0084] In another preferred embodiment, the anti-foreign-body response comprises:

[0085] (1) resisting adhesion of proteins, cells, and / or microorganisms;

[0086] (2) resisting biofouling; and / or

[0087] (3) reducing acute and / or chronic inflammatory responses.

[0088] In another preferred embodiment, the cell comprises a fibroblast.

[0089] In another preferred embodiment, the microorganism comprises Staphylococcus aureus and / or Acinetobacter baumannii.

[0090] In another preferred embodiment, the resisting adhesion of proteins, cells, and / or microorganisms comprises:

[0091] (1) resisting adhesion of fibroblasts;

[0092] (2) resisting adhesion of Staphylococcus aureus;

[0093] (3) resisting adhesion of Acinetobacter baumannii; and / or

[0094] (4) resisting adhesion of immunoglobulins.

[0095] In another preferred embodiment, the anti-foreign-body response comprises one or more selected from the group consisting of:

[0096] (1) modulating macrophage phenotype to reduce inflammatory responses;

[0097] (2) resisting protein adsorption, cell adhesion, and adhesion of various microorganisms.

[0098] In another preferred embodiment, the macrophage is selected from the group consisting of RAW264.7, THP-1, BV2, or BMDM, or a combination thereof.

[0099] In another preferred embodiment, the anti-foreign body reaction has one or more characteristics selected from the group consisting of:

[0100] (1) the adsorption effect of the material prepared from polysialic acid or its derivative on protein, cell and / or microorganism is reduced by at least 50%, preferably at least 60%, more preferably at least 70% compared with that of untreated material;

[0101] (2) the material prepared from polysialic acid or its derivative reduces M1 type macrophage polarization, which is manifested by that the surface of the material prepared from polysialic acid or its derivative can reduce the expression of M type macrophage marker iNOS by at least 50%, preferably at least 60%, more preferably at least 70%;

[0102] (3) after the material prepared from polysialic acid or its derivative is implanted into the body, the inflammatory cell layer around the material is thin, the inflammatory reaction is low, and the density of fibrous deposition around the tissue is small, close to that of normal tissue.

[0103] In another preferred embodiment, the medical material is an implant material.

[0104] In another preferred embodiment, the medical material is an implant material or a surface coating of a medical device.

[0105] In another preferred embodiment, the implant material is selected from the group consisting of a medical catheter, a medical stent, an implanted prosthesis, an implanted electrode, and an implanted biosensor.

[0106] In another preferred embodiment, the material of the medical device is selected from the group consisting of a medical inorganic non-metallic material, a medical inorganic metallic material, and a medical polymeric material.

[0107] In another preferred embodiment, the medical device is a medical device that needs to be implanted into the human body.

[0108] In another preferred embodiment, the medical material functions by being implanted into the human body or by contacting with human tissues.

[0109] In another preferred embodiment, the medical material further comprises a drug and / or a cell having a therapeutic and / or repairing effect.

[0110] In another preferred embodiment, the drug is selected from the group consisting of a small molecule chemical drug, a protein drug, an antibody drug, and an RNA drug.

[0111] In another preferred embodiment, the medical material is in the form of a hydrogel.

[0112] In another preferred embodiment, the medical material hydrogel can be used as a carrier of drugs, for loading drugs and / or cells with therapeutic and / or repairing effects, for drug modification, drug delivery, cell carrier, tissue repair, wound dressing.

[0113] In a second aspect of the present application, a polysialic acid derivative is provided, as described in the first aspect of the present application.

[0114] In a third aspect of the present application, a method for preparing the polysialic acid derivative as described in the second aspect of the present application is provided, and the method is selected from the following methods:

[0115] Method I:

[0116]

[0117] The polysialic acid is mixed with the raw material R3-O-R3, and the pH is adjusted to 8-8.5, and the reaction is carried out at 0-4℃ for 18-48h to obtain the compound II-1;

[0118] Method II:

[0119]

[0120] The polysialic acid is mixed with the raw material R1-H under the activation system of EDC / NHS, and the reaction is carried out at 0-4℃ for 18-48h to obtain the compound II-2;

[0121] Method III:

[0122]

[0123] (2-1) The polysialic acid is mixed with a base, heated to reflux for 18-48h, and then the pH is adjusted to neutral with an acid to obtain an intermediate;

[0124] (2-2) The intermediate is mixed with the raw material R2-CO-O-CO-R2, and the pH is adjusted to 8-8.5, and the reaction is carried out at 0-4℃ for 18-48h to obtain the compound II-3;

[0125] Method IV:

[0126]

[0127] (4-1) The polysialic acid is mixed with sodium periodate, and the reaction is carried out in the dark to obtain an intermediate;

[0128] (4-2) The intermediate is mixed with R4-NH2 under the action of sodium cyanoborohydride, and the reaction is carried out at 20-45℃ for 2-5 days to obtain the compound IV-1;

[0129] wherein R1, R2, R3, R4, n are each independently as described in the present application.

[0130] In a fourth aspect of the present application, a medical material is provided, which is a medical material implant or a surface coating of a medical device, and which contains (a) a polysialic acid or a derivative thereof, or (b) a material prepared from a polysialic acid or a derivative thereof as a raw material; and which is a hydrogel material or a coating material.

[0131] In another preferred embodiment, the polysialic acid, the polysialic acid derivative, the material prepared from a polysialic acid or a derivative thereof as a raw material, the medical material, the hydrogel material, the coating material are each independently as described in the first aspect of the present application.

[0132] In a fifth aspect of the present application, a hydrogel material is provided, which is obtained by cross-linking a polysialic acid or a derivative thereof with an optional macromolecule selected from the group consisting of gelatin, hyaluronic acid, starch, chitosan, sodium alginate, sodium carboxymethyl cellulose, carboxymethyl cellulose, or a combination thereof.

[0133] In a sixth aspect of the present application, a coating material is provided, in which a polysialic acid or a derivative thereof is covalently grafted onto the surface of a substrate by a chemical method, and which is a coating of a polysialic acid or a derivative thereof on a substrate, and the substrate is selected from the group consisting of a medical inorganic non-metallic material, a medical inorganic metallic material, and a medical polymeric material.

[0134] It should be understood that, within the scope of the present application, each of the technical features described above and in the following (e.g., in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0135] Figure 1 A polysialic acid derivative prepared in Example 1 is shown.

[0136] Figure 2 A polysialic acid derivative prepared in Example 2 is shown.

[0137] Figure 3 A polysialic acid derivative prepared in Example 3 is shown.

[0138] Figure 4 A polysialic acid derivative prepared in Example 4 is shown.

[0139] Figure 5 Protein adsorption results on different material surfaces modified with polysialic acid in Example 8 are shown.

[0140] Figure 6Protein adsorption results of poly sialic acid or its derivative modified PDMS surface in Example 9 are shown.

[0141] Figure 7 Microscope photos of fibroblast adhesion on poly sialic acid modified nickel titanium surface in Example 10 are shown.

[0142] Figure 8 Fluorescence area quantification analysis results of fibroblast adhesion on poly sialic acid modified nickel titanium surface in Example 10 are shown.

[0143] Figure 9 Results of poly sialic acid hydrogel reducing inflammatory phenotype of macrophage RAW264.7 in Example 11 are shown.

[0144] Figure 10 Results of poly sialic acid modified glass surface reducing inflammatory phenotype of macrophage RAW264.7 in Example 12 are shown.

[0145] Figure 11 Optical photos of poly sialic acid hydrogel mouse in vivo foreign body reaction test in Example 13 are shown.

[0146] Figure 12 HE staining and Masson staining analysis photos of poly sialic acid hydrogel mouse in vivo foreign body reaction test in Example 13 are shown.

[0147] Figure 13 Optical photos of poly sialic acid and hyaluronic acid blended hydrogel mouse in vivo foreign body reaction test in Example 14 are shown.

[0148] Figure 14 Optical photos of surface poly sialic acid modified polydimethylsiloxane (PDMS) mouse in vivo foreign body reaction test in Example 15 are shown.

[0149] Figure 15 Microscope photos results of poly sialic acid modified nickel titanium surface Staphylococcus aureus adhesion in Example 16 are shown.

[0150] Figure 16 Fluorescence area quantification analysis results of poly sialic acid modified nickel titanium surface Staphylococcus aureus adhesion in Example 16 are shown.

[0151] Figure 17 Microscope photos results of poly sialic acid modified glass surface Acinetobacter baumannii adhesion in Example 17 are shown.

[0152] Figure 18 Fluorescence area quantification analysis results of poly sialic acid modified glass surface Acinetobacter baumannii adhesion in Example 17 are shown. DETAILED DESCRIPTION

[0153] The present inventors have made extensive and in-depth research and for the first time found that a polysialic acid or a derivative thereof can be used for resisting foreign body reaction.

[0154] The present application relates to a polysialic acid anti-foreign body reaction material for resisting biological contamination, for regulating macrophage phenotype to reduce inflammatory response, and for resisting foreign body reaction. The polysialic acid material includes a bulk material prepared from polysialic acid and its derivatives, such as a hydrogel, or a coating material for surface modification of a material, which can resist protein adsorption, cell adhesion and adhesion of various microorganisms, can act on immune cells, such as regulating the phenotype of macrophages, thereby reducing acute and / or chronic inflammatory response, and has excellent anti-foreign body reaction effect as an implant material.

[0155] On this basis, the present application is completed.

[0156] Terms

[0157] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0158] As used herein, the terms "comprising", "including", "containing", are interchangeable and include both open- and closed-ended definitions. In other words, the terms include "consisting of" and "consisting essentially of".

[0159] As used herein, the term "about", used in reference to a particular recited numerical value, means that the value can vary from the recited value by not more than 1%. For example, as used herein the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0160] Polysialic acid (PSA) is a linear polysaccharide composed of N-acetylneuraminic acid monomers connected by α-2,8- or α-2,9-glycosidic bonds, which is non-immunogenic and has a large number of negative charges. The molecular chain can form a stable hydration layer with water molecules, and at the same time, electrostatic repulsion occurs between PSA and negatively charged plasma proteins (such as fibrinogen and albumin), which physically isolates the material surface from direct contact with proteins, thereby inhibiting non-specific protein adsorption and subsequent immune response. On the other hand, PSA can bind to Siglec receptors on the surface of innate immune cells such as macrophages and neutrophils, transmitting inhibitory signals, thereby relieving inflammatory response in the subacute and chronic inflammation stages. Therefore, PSA has both anti-protein adsorption and immune regulation functions, and contains a large number of modifiable sites, which is suitable for surface modification of various materials or preparation of bulk materials, and is used for preparing anti-FBR materials.

[0161] The anti-foreign body reaction function of the polysialic acid material comes from the anti-protein adsorption function and the immune regulation function brought by its negative charge.

[0162] The specific polysialic acid or its derivative of the present application, or the hydrogel or surface modified coating material prepared therefrom has a more excellent protein, cell and / or microorganism adsorption effect and a reducing effect of M1 macrophage polarization and inflammation, and thus has a more excellent anti-foreign body reaction effect.

[0163] There are also some researches on the use of polysialic acid derivatives for anti-foreign body reaction in the prior art. However, the effect of the prior art, for example, on non-specific protein adsorption is poor, which is only reduced by about 25%, and for example, the M1 macrophage marker iNOS is still highly expressed, while the use of the polysialic acid or its derivative of the present application can reduce the expression of iNOS to a level comparable to that of the negative control.

[0164] The main advantages of the present application include:

[0165] The present application finds that polysialic acid or its derivative can be used to inhibit the adhesion of proteins, bacteria, etc., can act on immune cells, reduce acute and chronic inflammatory reactions, and has excellent anti-foreign body reaction effect as an implant material.

[0166] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples are not specified, which are usually carried out according to the conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0167] Materials

[0168] Unless otherwise specified, the molecular weight of the polysialic acid used in the examples is 30-100 kDa.

[0169] Example 1: Synthesis of hydroxyl-modified acrylate polysialic acid derivative

[0170]

[0171] Step (1), accurately weigh 30 mg of polysialic acid in a 10 mL reaction bottle, dissolve in 1 mL of pure water, and put in a stirring bar to start stirring. Slowly add 200 μL of acrylic anhydride to the reaction bottle under ice bath conditions. Prepare a 1M concentration of sodium hydroxide solution, and adjust the pH of the above reaction system to 8 ~ 8.5. Place the reaction bottle in a 4 °C reaction for 24 hours;

[0172] Step (2), transfer the reaction solution to a dialysis bag and dialyze with pure water at 4 °C for 2 days;

[0173] Step (3), freeze-drying the product after dialysis to obtain the product.

[0174] The nuclear magnetic resonance results are shown in Figure 1 .

[0175] Example 2: Synthesis of polysialic acid derivative with carboxyl-modified azido group

[0176]

[0177] Step (1), accurately weigh 20 mg of polysialic acid in a 10 mL reaction bottle, dissolve in 3 mL of MES buffer, and put in a stirring bar to start stirring. Add 14.7 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 7.3 mg of N-hydroxysuccinimide (NHS) to the reaction bottle under ice bath conditions, and react for 30 minutes. Add 13.9 mg of 1-amino-11-azido-3,6,9-trioxaundecane to the above reaction system. Place the reaction bottle in a 4 °C incubator for 24 hours to end the reaction; N

[0178] Step (2), transfer the reaction solution to a dialysis bag and dialyze against pure water at 4 °C for 2 days;

[0179] Step (3), freeze-drying the product after dialysis to obtain the product.

[0180] The nuclear magnetic resonance results are shown in Figure 2 .

[0181] Example 3: Synthesis of polysialic acid derivative with acetyl-hydrolyzed and acrylamide-modified groups

[0182]

[0183] Step (1), accurately weigh 50 mg of polysialic acid in a 10 mL reaction bottle. Prepare a 1 M concentration of sodium hydroxide in water and ethanol mixed solution, and add it to the above reaction bottle. Heat to reflux for 24 hours to end the reaction;

[0184] Step (2), remove the ethanol in the reaction solution by rotary evaporation;

[0185] Step (3), dilute with pure water and adjust the pH of the reaction system to neutral with 0.5 M hydrochloric acid;

[0186] Step (4), transfer the reaction solution to a dialysis bag and dialyze against pure water at 4 °C for 2 days;

[0187] Step (5), freeze-drying the product after dialysis to obtain the deacetylated polysialic acid derivative; ​

[0188] Step (6), 30 mg of the above deacetylated polysialic acid derivative was accurately weighed in a 10 mL reaction bottle, dissolved in 1 mL of pure water, and placed in a stirring bar to start stirring. 200 μL of acrylic anhydride was slowly added dropwise to the reaction bottle under ice bath conditions. A 1 M concentration of sodium hydroxide solution was prepared, and the pH of the above reaction system was adjusted to 8 ~ 8.5. The reaction bottle was placed in a 4 °C reaction for 24 hours to end;

[0189] Step (7), the reaction solution was transferred to a dialysis bag and dialyzed with pure water at 4 °C for 2 days;

[0190] Step (8), the product after dialysis was freeze-dried to obtain the product.

[0191] The nuclear magnetic resonance results are shown in Figure 3 .

[0192] Example 4: Synthesis of polysialic acid derivative with azide group modified end group

[0193]

[0194] Step (1), 50 mg of polysialic acid was accurately weighed in a 10 mL reaction bottle, dissolved in 1 mL of pure water, and placed in a stirring bar to start stirring. 3.7 mg of sodium periodate was added to the above reaction bottle, and the reaction was carried out in the dark for 3 hours;

[0195] Step (2), 500 μL of ethylene glycol was added to the above reaction system, and the stirring was continued for 2 hours to terminate the reaction;

[0196] Step (3), the reaction solution was transferred to a dialysis bag and dialyzed with a 0.05% NH4HCO3 solution at 4 °C for 24 hours, and then dialyzed with pure water for 6 hours;

[0197] Step (4), the product after dialysis was freeze-dried to obtain an intermediate with an aldehyde group as the end group;

[0198] Step (5), 30 mg of the above intermediate was accurately weighed in a 10 mL reaction bottle, dissolved in 1 mL of 0.5 M concentration K2HPO4 solution, and placed in a stirring bar to start stirring. 0.33 mg of 1-amino-11-azido-3,6,9-trioxaundecane and 0.31 mg of sodium cyanoborohydride were added to the above reaction bottle, and the reaction was carried out at 35 °C for 3 days;

[0199] Step (6), the reaction solution was transferred to a dialysis bag and dialyzed with a 0.05% NH4HCO3 solution at 4 °C for 24 hours, and then dialyzed with pure water for 6 hours;

[0200] Step (7), the product after dialysis was freeze-dried to obtain a polysialic acid derivative with azide group modified end group.

[0201] NMR results are shown as Figure 4

[0202] Example 5: Preparation of 1,4-butanediol diglycidyl ether cross-linked polysialic acid hydrogel

[0203] Step (1), 40 mg of polysialic acid (molecular weight of 10 kDa, 30 kDa, 50 kDa, 100 kDa or more than 30 kDa) was accurately weighed in a 1.5 mL reaction bottle, 200 μL of 0.5 M concentration of sodium hydroxide solution was added, and ultrasonic was applied for 5 minutes until the solution was clear. 22 μL of 1,4-butanediol diglycidyl ether (BDDE) was added to the above solution and mixed uniformly to obtain a pre-gel solution;

[0204] Step (2), the above solution was added to a mold, and cross-linked at 40 °C for 4 hours;

[0205] Step (3), the shaped polysialic acid hydrogel was placed in PBS for swelling to equilibrium, then transferred to a dialysis bag and dialyzed with PBS for 24 hours to obtain a BDDE cross-linked polysialic acid hydrogel.

[0206] Example 6: Preparation of polysialic acid and hyaluronic acid blended hydrogel

[0207] Step (1), 20 mg of polysialic acid and 20 mg of hyaluronic acid (molecular weight of 46 kDa and 289 kDa, respectively) were accurately weighed in a 1.5 mL reaction bottle, 200 μL of 0.5 M concentration of sodium hydroxide solution was added, and ultrasonic was applied for 5 minutes until the solution was clear.

[0208] Step (2), 22 μL of 1,4-butanediol diglycidyl ether (BDDE) was added to the above solution and mixed uniformly;

[0209] Step (3), the above solution was added to a mold, and cross-linked at 40 °C for 4 hours.

[0210] Step (4), the shaped polysialic acid hydrogel was placed in PBS for swelling to equilibrium, then transferred to a dialysis bag and dialyzed with PBS for 24 hours to obtain a BDDE cross-linked polysialic acid hydrogel.

[0211] Example 7: Preparation of photo-cross-linked polysialic acid acrylate derivative hydrogel

[0212] ​Step (1), 40 mg of the modified polyacrylate derivative of polysialic acid prepared in Example 1 was accurately weighed in a 1.5 mL reaction bottle, dissolved in an aqueous solution containing 0.1% of a photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), and ultrasonicated for 5 minutes until the solution was clear;

[0213] Step (2), the above solution was added to a round hole silicone rubber mold, and irradiated with ultraviolet light of a wavelength of 365 nm at a light intensity of 500 mW / cm2for 3-10 minutes to crosslink the solution. 2 Step (3), the formed polysialic acid hydrogel was immersed in pure water for 24 hours to obtain a photo-crosslinked polysialic acid acrylate derivative hydrogel.

[0214] Step (3), the formed polysialic acid hydrogel was immersed in pure water for 24 hours to obtain a photo-crosslinked polysialic acid acrylate derivative hydrogel.

[0215] Example 8: Protein adsorption on polysialic acid modified surface proteins

[0216] Step (1), a smooth nickel-titanium sheet, a glass sheet, and a PDMS sheet were cleaned with ethanol and water alternately and dried with nitrogen;

[0217] Step (2), a dopamine hydrochloride solution with a concentration of 1 mg / mL was prepared using a Tris HCl buffer with a pH of 8.5, the pretreated sheet of step (1) was immersed in the solution for 24 hours, and then taken out and cleaned with ethanol and water alternately and dried with nitrogen;

[0218] Step (3), a polysialic acid solution with a concentration of 5 mg / mL was prepared using pure water, EDCI and NHS were added and activated in an ice bath for 30 minutes, the pretreated sheet of step (2) was immersed in the solution for 24 hours, and then taken out and cleaned with ethanol and water alternately and dried with nitrogen;

[0219] Step (4), a protein adsorption test was performed using a horseradish peroxidase labeled goat anti-human immunoglobulin as a model protein, 35 μL of the protein was added to the surface of the modified and unmodified sheet (limited to a 6 mm diameter hole by a silicone rubber mold) and incubated for 20 minutes, after PBS cleaning, the nickel-titanium sheet was placed in a 24-well plate, an o-phenylenediamine solution (1 mL, 1 mg / mL) was added, and the enzyme catalytic reaction was started, after 15 minutes, the reaction was terminated with an equal volume of 2 M sulfuric acid, and the optical density (OD) value was read by an enzyme marker at a test wavelength of 492 nm, and the protein adsorption amount after background subtraction was proportional to the OD value.

[0220] The results are shown in Table 1. Figure 5 As can be seen, the surface modified by polysialic acid can resist 50-80% of protein adsorption, and has an anti-foreign body reaction effect.

[0221] Example 9: Protein adsorption on polysialic acid derivative modified PDMS surface

[0222] Step (1): Clean the PDMS sheet with ethanol and water alternately, and then dry it with nitrogen.

[0223] Step (2): Prepare a dopamine hydrochloride solution with a concentration of 1 mg / mL using Tris HCl buffer at pH 8.5. Immerse the pretreated tablets from step (1) in the solution for 24 hours, remove them and wash them alternately with ethanol and water, and then dry them with nitrogen.

[0224] Step (3): The polysialic acid derivatives obtained in Examples 1, 2, 3 and 4 were respectively prepared into solutions with pure water at a concentration of 5 mg / mL. EDCI and NHS were added and activated in an ice bath for 30 minutes. The pretreated tablets in step (2) were immersed in the solution for 24 hours. They were then taken out and washed alternately with ethanol and water and dried with nitrogen.

[0225] Step (4): The protein adsorption test used horseradish peroxidase-labeled goat anti-human immunoglobulin as the model protein. 35 μL of protein was added to the surface of the slide before and after modification (with a silicone rubber mold to restrict the diameter of the circular wells to 6 mm) and incubated for 20 minutes. After washing with PBS, the nickel-titanium slide was placed in a 24-well plate and o-phenylenediamine solution (1 mL, 1 mg / mL) was added to start the enzyme-catalyzed reaction. After 15 minutes, the reaction was terminated with an equal volume of 2 M sulfuric acid. The optical density (OD) value was read at a test wavelength of 492 nm using an ELISA reader. After background subtraction, the amount of protein adsorbed was proportional to the OD value.

[0226] The results are as follows Figure 6 As shown, the surfaces modified with polysialic acid derivatives can resist protein adsorption and have an anti-foreign substance reaction effect. Moreover, the effect of resisting protein adsorption is excellent, and it can reduce it by more than 60%.

[0227] Example 10: Polysialic acid-modified nickel-titanium surface fibroblast adhesion

[0228] Step (1): Clean the polished nickel-titanium sheet with ethanol and water alternately, and then dry it with nitrogen.

[0229] Step (2): Prepare a dopamine hydrochloride solution with a concentration of 1 mg / mL using Tris HCl buffer at pH 8.5. Immerse the pretreated nickel-titanium sheet in the solution for 24 hours. Remove the nickel-titanium sheet and wash it alternately with ethanol and water, and then dry it with nitrogen.

[0230] Step (3): Prepare a polysialic acid solution with a concentration of 5 mg / mL using pure water, add EDCI and NHS and activate in an ice bath for 30 minutes. Immerse the pretreated nickel-titanium sheet in the solution for 24 hours, remove the nickel-titanium sheet and wash it alternately with ethanol and water, and dry it with nitrogen.

[0231] Step (4), the modified and unmodified nickel titanium sheet obtained in step (3) was respectively placed in a 24-well plate, 1 mL, 10 5 fibroblasts were added, and incubated in a carbon dioxide incubator for 3 days. The culture solution was DMEM. The nickel titanium sheet was taken out and gently washed with PBS to remove the unadhered cells. The amount of cells was observed under a microscope.

[0232] The results are shown in Figures 7-8 The surface modified by polysialic acid can resist 78% of fibroblast adhesion.

[0233] Example 11: Phenotype regulation of macrophage RAW264.7 by polysialic acid hydrogel

[0234] Step (1), RAW264.7 cells were plated in a 96-well plate at 2×10 4 per well, and incubated in a carbon dioxide incubator for 24 hours;

[0235] Step (2), the polysialic acid hydrogel prepared in Example 5 was cut into a disc with a diameter of 5 mm. One hydrogel and 1 μg / mL LPS were added to each well, and incubated with the cells for 24 hours. The LPS-only group was used as a positive control, and the group without hydrogel and LPS was used as a negative control.

[0236] Step (3), the hydrogel and supernatant were discarded, and washed twice with PBS. 100 μL of 4% paraformaldehyde was added to each well at 4°C for 30 minutes. After the fixing solution was discarded, the wells were washed twice with PBS. 100 μL of 0.1% TX-100 solution was added for permeabilization for 15 minutes. After the supernatant was discarded, the wells were washed twice with PBS. 100 μL of 5% skim milk powder solution was added for blocking for 1 hour. After the skim milk powder solution was discarded, the wells were washed twice with PBS.

[0237] Step (4), M1 macrophage marker iNOS was diluted with 5% bovine serum albumin in PBS at a dilution of 1:300. 30 μL of the corresponding antibody was added to each well, and incubated at 4°C for 14-16 hours. After the antibody was discarded, the wells were washed twice with PBS.

[0238] Step (5), the fluorescently labeled secondary antibody was diluted with PBS at a dilution of 1:300. 30 μL of the secondary antibody was added to each well, and incubated at room temperature for 1-2 hours in the dark. After the antibody was discarded, the wells were washed twice with PBS.

[0239] Step (6), propidium iodide (PI) was diluted with PBS to a concentration of 5 μg / mL, 30 μL of PI solution was added to each well, and incubated at room temperature for 15 minutes in the dark. After the antibody was discarded, the wells were washed twice with PBS. Finally, 100 μL of PBS was added, and the wells were observed and photographed using a confocal microscope.

[0240] The results, as shown in Figure 9 Figure 6, showed that polysialic acid hydrogel can regulate the phenotype of macrophages, reduce M1 polarization, and inhibit inflammatory response, and the effect is excellent, which can reach a level basically comparable to that of the negative control.

[0241] Example 12: Regulation of the phenotype of macrophages RAW264.7 by polysialic acid modified glass surface

[0242] Step (1), the glass pieces were cleaned with ethanol and water alternately, dried with nitrogen, and attached to a silicone rubber mold, each hole having a diameter of 7 mm;

[0243] Step (2), a dopamine hydrochloride solution with a concentration of 1 mg / mL was prepared using Tris HCl buffer with a pH of 8.5. The nickel-titanium piece glass piece pretreated in step (1) was immersed in the solution for 24 hours. The glass piece was then cleaned with ethanol and water alternately and dried with nitrogen;

[0244] Step (3), a polysialic acid solution with a concentration of 5 mg / mL was prepared using pure water, and EDCI and NHS were added and activated in an ice bath for 30 minutes. The glass piece pretreated in step (2) was immersed in the solution for 24 hours. The glass piece was then cleaned with ethanol and water alternately and dried with nitrogen;

[0245] Step (4), RAW264.7 cells were inoculated on the surface of the modified or unmodified glass pieces at a concentration of 5×10 4 cells per hole, and incubated in a carbon dioxide incubator for 24 hours;

[0246] Step (5), 1 μg / mL LPS was added to the polysialic acid modified holes, and 1 μg / mL LPS was added to the unmodified holes as a positive control. No LPS was added to the unmodified holes as a negative control.

[0247] Step (6), the culture medium was discarded, and the wells were washed twice with PBS. 30 μL of 4% paraformaldehyde was added to each well and incubated at 4 °C for 30 minutes. After the fixing solution was discarded, the wells were washed twice with PBS. 30 μL of 0.1% TX-100 solution was added to each well and incubated for 15 minutes. After the supernatant was discarded, the wells were washed twice with PBS. 30 μL of 5% skimmed milk powder solution was added to each well and incubated for 1 hour. After the skimmed milk powder solution was discarded, the wells were washed twice with PBS.

[0248] Step (7), M1 macrophage marker iNOS and M2 macrophage marker antibody Mrc1 were diluted with PBS containing 5% bovine serum albumin at 1:300, 30 μL of corresponding antibody was added to each well, and incubated at 4 °C for 14-16 hours. After the antibody was discarded, it was rinsed twice with PBS;

[0249] Step (8), the secondary antibody was diluted with PBS at 1:300, 30 μL of the secondary antibody was added to each well, and incubated at room temperature for 1-2 hours in the dark. After the antibody was discarded, it was rinsed twice with PBS;

[0250] Step (9), propidium iodide (PI) was diluted with PBS to a concentration of 5 μM, 30 μL of PI solution was added to each well, and incubated at room temperature for 15 minutes in the dark. After the antibody was discarded, it was rinsed twice with PBS, and finally 100 μL of PBS was added. Photographs were taken with a confocal microscope for observation.

[0251] The results are shown in Figure 10 It can be seen that the polysialic acid modified surface can regulate the phenotype of macrophages, reduce M1 polarization and induce M2 polarization.

[0252] Example 13: Polysialic acid hydrogel foreign body reaction test in mice

[0253] The hydrogel obtained in Example 5 was subjected to ultraviolet sterilization for 30 minutes. 6-8 week old male C57BL / 6J mice were selected, the mice were anesthetized with isoflurane gas, and the mice were depilated on the back. An 8 mm long vertical incision was made on both sides of the mouse back, and the hydrogel was implanted subcutaneously below the fascia. After implantation, the incision was sutured. The mice were euthanized under anesthesia at 1 week and 4 weeks after implantation. The same method was used to implant polyethylene glycol hydrogel into mice and handle them.

[0254] The skin tissue of the implanted material was fixed with 4% paraformaldehyde tissue fixative for 12 hours, and sections were made for H&E staining and Masson staining analysis.

[0255] The results are shown in Figures 11-12 It can be seen that the polysialic acid hydrogel has a very thin layer of inflammatory cells around it, and the inflammatory response is low; the density of fibrous deposition in the surrounding tissue is small, close to normal tissue, and has a very good effect of reducing foreign body reaction.

[0256] Example 14: Polysialic acid and hyaluronic acid blended hydrogel foreign body reaction test in mice

[0257] The hydrogel obtained in Example 6 was sterilized by ultraviolet for 30 minutes. 6-8 weeks old male C57BL / 6J mice were selected, the mice were anesthetized by isoflurane gas, and the back of the mice was depilated. An 8 mm long incision was made on both sides of the back of the mice, and the hydrogel was implanted under the subcutaneous fascia. After implantation, the incision was sutured. After 1 week of implantation, the mice were euthanized under anesthesia, and the skin tissue of the implanted material was collected to observe the tissue inflammation.

[0258] As shown in Figure 13 Example 6, the hydrogel prepared by blending polysialic acid and hyaluronic acid with different molecular weights had a very good effect of reducing foreign body reaction.

[0259] Example 15: In vivo foreign body reaction test of surface polysialic acid modified polydimethylsiloxane (PDMS)

[0260] Step (1), cut PDMS into a disc with a diameter of 5 mm, and clean it with ethanol and water alternately, and dry it with nitrogen;

[0261] Step (2), prepare a dopamine hydrochloride solution with a concentration of 1 mg / mL using Tris HCl buffer with a pH of 8.5, immerse the PDMS sheet pretreated in step (1) in the solution for 24 hours, take out the PDMS sheet and clean it with ethanol and water alternately, and dry it with nitrogen;

[0262] Step (3), prepare a polysialic acid solution with a concentration of 5 mg / mL using pure water, add EDCI and NHS, and activate it in an ice bath for 30 minutes. Immerse the PDMS sheet pretreated in step (2) in the solution for 24 hours, take out the PDMS sheet and clean it with ethanol and water alternately, and dry it with nitrogen;

[0263] Step (4), the PDMS sheet obtained in step (3) was sterilized by ultraviolet for 30 minutes. 6-8 weeks old male C57BL / 6J mice were selected, the mice were anesthetized by isoflurane gas, and the back of the mice was depilated. An 8 mm long incision was made on both sides of the back of the mice, and the hydrogel was implanted under the subcutaneous fascia. After implantation, the incision was sutured. After 1 week of implantation, the mice were euthanized under anesthesia. The same method was used to implant unmodified PDMS into the mice and process them. The skin tissue of the implanted material was collected to observe the tissue inflammation.

[0264] As shown in Figure 14 Example 6, the hydrogel prepared by blending polysialic acid and hyaluronic acid with different molecular weights had a very good effect of reducing foreign body reaction.

[0265] Example 16: Staphylococcus aureus adhesion on a nickel-titanium surface modified with polysialic acid

[0266] Step (1): Clean the polished nickel-titanium sheet with ethanol and water alternately, and then dry it with nitrogen.

[0267] Step (2): Prepare a dopamine hydrochloride solution with a concentration of 1 mg / mL using Tris HCl buffer at pH 8.5. Immerse the nickel-titanium sheet pretreated in step (1) in the solution for 24 hours. Remove the nickel-titanium sheet and wash it alternately with ethanol and water, and then dry it with nitrogen.

[0268] Step (3): Prepare a polysialic acid solution with a concentration of 5 mg / mL using pure water, add EDCI and NHS and activate in an ice bath for 30 minutes. Immerse the pretreated nickel-titanium sheet in the solution for 24 hours, remove the nickel-titanium sheet and wash it alternately with ethanol and water, and dry it with nitrogen.

[0269] Step (4): Place the modified and unmodified nickel-titanium sheets obtained in step (3) into 24-well plates respectively, and add 1 mL of 10 6 Staphylococcus aureus at a concentration of 1 / mL was incubated at 37 °C for 3 days in LB broth medium. The nickel-titanium slide was removed and the unadhered cells were gently washed away with PBS. The bacterial count was observed by staining and photographing.

[0270] The results are as follows Figures 15-16 As shown, surfaces modified with polysialic acid can resist 99% bacterial adhesion.

[0271] Example 17: Polysialic acid-modified glass surface adhesion of Acinetobacter baumannii

[0272] Step (1): Clean the glass slide with ethanol and water alternately, and then dry it with nitrogen gas;

[0273] Step (2): Prepare a dopamine hydrochloride solution with a concentration of 1 mg / mL using Tris HCl buffer at pH 8.5. Immerse the glass slides pretreated in step (1) in the solution for 24 hours. Remove the glass slides and wash them alternately with ethanol and water, and then dry them with nitrogen.

[0274] Step (3): Prepare a polysialic acid solution with a concentration of 5 mg / mL using pure water, add EDCI and NHS and activate in an ice bath for 30 minutes. Immerse the glass slide after the pretreatment in step (2) in the solution for 24 hours. Take out the glass slide and wash it alternately with ethanol and water, and dry it with nitrogen.

[0275] Step (4): Place the modified and unmodified glass slides obtained in step (3) into 24-well plates respectively, and add 1 mL of 10 6The glass pieces were incubated with 105CFU / mL of S. aureus in a 37 °C incubator for 3 days, the culture medium was LB broth medium, the glass pieces were gently washed with PBS to remove the unattached cells, and the number of bacteria was observed by staining and taking pictures.

[0276] The results are shown in Figures 17-18 The surface modified by polysialic acid can resist 99% of bacterial adhesion.

[0277] It is understood that various modifications or changes in the application can be made by those skilled in the art after reading the above description of the application, and these equivalent forms also fall within the scope defined by the claims appended to this application.

Claims

1. The use of a material prepared from polysialic acid or its derivatives, characterized in that, This is used to prepare a medical material for resisting foreign body reactions; The polysialic acid is shown in Formula I below. I n is an integer between 20 and 400; The molecular weight of the polysialic acid is 10 kDa to 100 kDa; The polysialic acid derivative is a compound of formula II-1, II-2, II-3 or III-1, or a pharmaceutically acceptable salt thereof. Where R1 is -NHR b R b Selected from -(CH2CH2O) p -CH2CH2-N3、-(CH2CH2) p -N3; R2 is a C2-C6 alkenyl group; R3 is -COR a ';R a 'It is a C2-C6 alkenyl group; R4 is selected from -(CH2CH2O) p -CH2CH2-N3、-(CH2CH2) p -N3; Each p is independently 1, 2, 3, 4 or 5; n is an integer between 20 and 400; The molecular weight of the polysialic acid derivative is 10 kDa to 100 kDa; The medical material is a medical implant or medical device with a surface coating material; the coating material is a coating of polysialic acid or its derivatives on a substrate, and the substrate is selected from the group consisting of: medical inorganic non-metallic materials, medical inorganic metallic materials, and medical polymer materials. The medical inorganic non-metallic material is glass; the medical inorganic metallic material is selected from the group consisting of: gold, tungsten, titanium, titanium alloy, nickel-titanium alloy, and titanium-chromium alloy; the medical polymer material is selected from the group consisting of: polydimethylsiloxane and polytetrafluoroethylene. The coating material is prepared by the following method, the method comprising: C1. Pre-treat the substrate with dopamine and graft amino groups onto the surface. The dopamine pre-treatment refers to immersing the substrate in a mixed solution of dopamine hydrochloride and Tris HCl buffer for pretreatment. C2. Polysialic acid or its derivatives are mixed with a pretreated substrate and activated with an activating agent to obtain the coating, wherein the activating agent is EDCI / NHS; Furthermore, the anti-foreign body response includes resisting the adhesion of proteins, cells and microorganisms, and the resistance to the adhesion of proteins, cells and microorganisms includes: (1) resisting fibroblast adhesion; (2) resisting Staphylococcus aureus adhesion; (3) resisting Acinetobacter baumannii adhesion; and (4) resisting immunoglobulin adhesion.

2. The use as described in claim 1, characterized in that, The polysialic acid derivative is a compound selected from the following structures or a pharmaceutically acceptable salt thereof: ， Where n is an integer between 20 and 400.

3. The use as described in claim 1, characterized in that, The molecular weight of the polysialic acid is 30 kDa to 100 kDa; The polysialic acid derivative is a compound selected from the following structures: ， Where n is an integer between 20 and 400; The molecular weight of the polysialic acid derivative is 30 kDa to 100 kDa; The medical inorganic non-metallic material is glass; the medical inorganic metallic material is nickel-titanium alloy; the medical polymer material is selected from the following group: polydimethylsiloxane and polytetrafluoroethylene.

4. The use as described in claim 1, characterized in that, The mixed solution is a dopamine hydrochloride solution with a concentration of 0.5-5 mg / mL prepared using Tris HCl buffer at pH 8-9.

5. The use as described in claim 1, characterized in that, The substrate is selected from the group consisting of: nickel-titanium sheets, glass, and PDMS.

6. The use as described in claim 1, characterized in that, The medical implant material is selected from the following group: medical catheter, medical stent, implantable prosthesis, implantable electrode, and implantable biosensor.

7. The use as described in claim 1, characterized in that, The medical material works by being implanted into the human body or by coming into contact with human tissue.

8. The use as described in claim 1, characterized in that, The polysialic acid or its derivative is polysialic acid, wherein the polysialic acid is shown in Formula I below. I n is an integer between 20 and 400; The molecular weight of the polysialic acid is 30 kDa to 100 kDa; The medical material is a medical implant or medical device with a surface coating material; the coating material is a coating of polysialic acid on a substrate, and the substrate is selected from the group consisting of: nickel-titanium sheet, glass, and PDMS; The coating material is prepared by the following method, the method comprising: C1. Pre-treat the substrate with dopamine and graft amino groups onto the surface. The dopamine pre-treatment refers to immersing the substrate in a mixed solution of dopamine hydrochloride and Tris HCl buffer for pre-treatment. The mixed solution is a dopamine hydrochloride solution with a concentration of 0.5-5 mg / mL prepared using Tris HCl buffer with a pH of 8-9. C2. Polysialic acid or its derivatives are mixed with a pretreated substrate and activated with an activating agent to obtain the coating, wherein the activating agent is EDCI / NHS; Furthermore, the anti-foreign body response includes resisting the adhesion of proteins, cells and microorganisms, and the resistance to the adhesion of proteins, cells and microorganisms includes: (1) resisting fibroblast adhesion; (2) resisting Staphylococcus aureus adhesion; (3) resisting Acinetobacter baumannii adhesion; and (4) resisting immunoglobulin adhesion.

9. A medical material, characterized in that, The medical material is a medical implant or medical device with a surface coating material, and the medical material contains a material prepared from polysialic acid or its derivatives; The polysialic acid is shown in Formula I below. I n is an integer between 20 and 400; The molecular weight of the polysialic acid is 10 kDa to 100 kDa; The polysialic acid derivative is a compound of formula II-1, II-2, II-3 or III-1, or a pharmaceutically acceptable salt thereof. Where R1 is -NHR b R b Selected from -(CH2CH2O) p -CH2CH2-N3、-(CH2CH2) p -N3; R2 is a C2-C6 alkenyl group; R3 is -COR a ';R a 'It is a C2-C6 alkenyl group; R4 is selected from -(CH2CH2O) p -CH2CH2-N3、-(CH2CH2) p -N3; Each p is independently 1, 2, 3, 4 or 5; n is an integer between 20 and 400; The molecular weight of the polysialic acid derivative is 10 kDa to 100 kDa; The coating material is a coating made by coating polysialic acid or its derivatives onto a substrate, wherein the substrate is selected from the group consisting of: medical inorganic non-metallic materials, medical inorganic metallic materials, and medical polymer materials; The medical inorganic non-metallic material is glass; the medical inorganic metallic material is selected from the group consisting of: gold, tungsten, titanium, titanium alloy, nickel-titanium alloy, and titanium-chromium alloy; the medical polymer material is selected from the group consisting of: polydimethylsiloxane and polytetrafluoroethylene. The coating material is prepared by the following method, the method comprising: C1. Pre-treat the substrate with dopamine and graft amino groups onto the surface. The dopamine pre-treatment refers to immersing the substrate in a mixed solution of dopamine hydrochloride and Tris HCl buffer for pretreatment. C2. Polysialic acid or its derivatives are mixed with a pretreated substrate and activated with an activating agent to obtain the coating, wherein the activating agent is EDCI / NHS.

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