CN113185740B

CN113185740B - Method for improving antibacterial activity and biocompatibility of substrate surface

Info

Publication number: CN113185740B
Application number: CN202110476700.8A
Authority: CN
Inventors: 刘润辉; 武月铭
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2023-09-22
Anticipated expiration: 2041-04-29
Also published as: CN113185740A

Abstract

The invention provides a method for improving the antibacterial activity and biocompatibility of a substrate surface. Specifically, the polymer chain segment of the invention is chemically grafted after the common base material is pretreated to form a functional surface with surface antibacterial activity. The modified functional surface has high antibacterial activity to gram-negative bacteria and/or gram-positive bacteria and/or fungi, and has low hemolytic activity to red blood cells, excellent cell compatibility to mammalian cells and negligible toxicity to long-acting antibacterial activity after in-vivo implantation. Therefore, the modified surface has great application potential in the field of biomedical materials, and provides effective prevention and treatment means for microbial surface infection.

Description

Method for improving antibacterial activity and biocompatibility of substrate surface

Technical Field

The invention belongs to the field of biomedical materials, and relates to a method for improving the antibacterial activity and biocompatibility of a substrate surface, thereby providing an effective prevention and treatment means for microbial surface infection.

Background

With the increasing population aging and the advancing medical technology, the use of biomedical materials (such as catheters, venous indwelling tubes, cardiovascular stents) and medical devices (such as cardiac pacemakers, electrophysiology catheters, hip implants, contact lenses) has increased. However, microbial infection is easily caused during or after the implantation process, and infection sources include bacteria, fungal pathogens, etc., seriously threaten patient health and cause a great economic loss to society. The number of deaths caused by infection worldwide is counted to be tens of millions, accounting for about 20% of the total number of deaths worldwide. Of the number of people dying from infection, 80% of human infections are associated with bacterial infections on the surface of medical materials. More severely, many pathogenic microorganisms exhibit increasingly greater bacterial resistance in the clinic.

Therefore, there is a need in the art to develop a method for providing a substrate surface with excellent antimicrobial properties and biocompatibility.

Disclosure of Invention

The object of the present invention is to provide a method for imparting excellent antibacterial properties and biocompatibility to a substrate surface, and a product produced by the method.

In a first aspect of the present invention, there is provided a method for increasing antimicrobial activity and biocompatibility of a substrate surface, comprising the steps of:

(a) Providing at least a portion of a surface of the substrate,

(b) Pre-activating said at least a portion of the surface to provide said surface with reactive groups selected from the group consisting of: -NH ₂ -Br, -Cl, -OH, -COOH, -CHO, epoxy, oxygen radical, alkenyl, alkynyl, polydopamine complex layer, or combinations thereof, thereby obtaining a pre-activated surface; and

(c) Contacting and reacting a compound or salt thereof with said pre-activated surface to provide a substrate having at least a portion of enhanced surface antimicrobial activity and biocompatibility;

wherein the compound comprises a polymer segment having a structural formula selected from the group consisting of:

in the formula I, R ₁ 、R ₂ And R is ₃ Optionally one of them is-L ₁ R _a Wherein L is ₁ Selected from the group consisting of: a bond, a substituted or unsubstituted C1-C8 alkylene group, a substituted or unsubstituted C2-C8 alkenylene group, a substituted or unsubstituted C2-C8 alkynylene group; r is R _a Selected from the group consisting of: -NR _b R _b Guanidino, biguanide, -N (R) _b ) ₃ ⁺ ；

R ₁ 、R ₂ And R is ₃ In which is not-L ₁ R _a Each of the remaining groups of (a) is independently selected from the group consisting of: H. halogen, -OH, -COOH, C1-C15 alkyl, C2-C15 alkenyl, C2-C15 alkynyl, substituted or unsubstituted C3-C12 cycloalkyl;

R ₄ 、R ₅ and R is ₆ Each independently selected from the group consisting of: H. halogen, -OH, -COOH, substituted or unsubstituted C1-C15 alkyl, substituted or unsubstituted C2-C15 alkenyl, substituted or unsubstituted C2-C15 alkynyl, substituted or unsubstituted C3-C12 cycloalkyl, substituted or unsubstituted benzyl, substituted or unsubstituted phenyl, substituted or unsubstituted 5-10 membered heteroaryl comprising 1-3 heteroatoms selected from O, N, S, substituted or unsubstituted C1-C6 alkyl-Rc, or substituted or unsubstituted C1-C6 alkyl-COO-Rc; each Rc is independently selected from the group consisting of: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted benzyl, substituted or unsubstituted phenyl, substituted or unsubstituted C3-C12 cycloalkyl, and substituted or unsubstituted 5-10 membered heteroaryl including 1-3 heteroatoms selected from O, N, S; or R4 and R5 form with the attached carbon atom a substituted or unsubstituted C3-C12 cycloalkyl group;

x is 5-100; and x+y=100; and is also provided with

n is a positive integer from 1 to 100; or alternatively

In formula II, R ₇ 、R ₈ 、R ₉ 、R ₁₀ 、R ₁₂ 、R ₁₃ And R is ₁₄ Each independently selected from the group consisting of: H. halogen, -OH, -COOH, substituted or unsubstituted C1-C15 alkyl, substituted or unsubstituted C2-C15 alkenyl, substituted or unsubstituted C2-C15 alkynyl, substituted or unsubstituted C3-C12 cycloalkyl; or two adjacent or on the same carbon atomThe substituents form with the attached carbon atom a substituted or unsubstituted C3-C12 cycloalkyl group;

R ₁₁ is-L ₁ R _a Wherein L is ₁ Selected from the group consisting of: a bond, a substituted or unsubstituted C1-C8 alkylene group, a substituted or unsubstituted C2-C8 alkenylene group, a substituted or unsubstituted C2-C8 alkynylene group; r is R _a Selected from the group consisting of: -NR _b R _b Guanidino, biguanide, or-N (R) _b ) ₃ ⁺ The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

R ₁₆ Selected from the group consisting of: H. -OH, substituted or unsubstituted C1-C15 alkyl, substituted or unsubstituted C2-C15 alkenyl, substituted or unsubstituted C2-C15 alkynyl, substituted or unsubstituted C1-C15 alkoxy, substituted or unsubstituted C3-C12 cycloalkyl, substituted or unsubstituted C1-C6 alkyl-C3-C12 cycloalkyl, substituted or unsubstituted benzyl, substituted or unsubstituted phenyl, substituted or unsubstituted 5-10 membered heteroaryl comprising 1-3 heteroatoms selected from O, N, S, substituted or unsubstituted C1-C6 alkyl-Rc, or substituted or unsubstituted C1-C6 alkyl-COO-Rc; each Rc is independently selected from the group consisting of: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted benzyl, substituted or unsubstituted phenyl, substituted or unsubstituted C3-C12 cycloalkyl, and substituted or unsubstituted 5-10 membered heteroaryl including 1-3 heteroatoms selected from O, N, S;

x is 5-100; and x+y=100; and is also provided with

n is a positive integer from 1 to 100;

in formula III, R ₁₇ is-L ₁ R _a Wherein L is ₁ Selected from the group consisting of: a bond, a substituted or unsubstituted C1-C8 alkylene group, a substituted or unsubstituted C2-C8 alkenylene group, a substituted or unsubstituted C2-C8 alkynylene group; r is R _a Selected from the group consisting of: -NR _b R _b Guanidino, biguanide or-N (R) _b ) ₃ ⁺ ；

R ₁₈ Selected from the group consisting of: H. halogen, -OH, -COOH, substituted or unsubstituted C1-C15 alkyl, substituted or unsubstituted C2-C15 alkenyl, substituted or unsubstituted C2-C15 alkynyl, substituted or unsubstituted C3-C12 cycloalkyl, substituted or unsubstituted benzyl, substituted or unsubstituted phenyl, substituted or unsubstitutedA 5-10 membered heteroaryl group comprising 1-3 heteroatoms selected from O, N, S, a substituted or unsubstituted C1-C6 alkyl-Rc, or a substituted or unsubstituted C1-C6 alkyl-COO-Rc; each Rc is independently selected from the group consisting of: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted benzyl, substituted or unsubstituted phenyl, substituted or unsubstituted C3-C12 cycloalkyl, and substituted or unsubstituted 5-10 membered heteroaryl including 1-3 heteroatoms selected from O, N, S;

wherein, in the formula I, the formula II and the formula III, each R _b Each independently selected from the group consisting of substituted or unsubstituted: hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, or two R on the same N atom _b Forming a 4-8 membered heterocyclic group containing one N atom with the attached N atom; and is also provided with

In the formulae, the substitution means that one or more H on each group is independently substituted with a group selected from the group consisting of: deuterium, halogen, -SH, -COOH, -OH, -NH ₂ Benzyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, C2-C6 haloalkenyl, C2-C6 haloalkynyl, C3-C8 cycloalkyl, C6-C10 aryl.

In another preferred embodiment, in each formula, -L ₁ R _a independently-C2-C6 alkyl-R _a Preferably, -C3-C6 alkyl-R _a 。

In another preferred embodiment, R _a Selected from the group consisting of: -NH ₂ Guanidino groupBiguanide, -NH ₃ ⁺ 、-N(CH ₃ ) ₃ ⁺ and-N (C) ₂ H ₅ ) ₃ ⁺ 。

In another preferred embodiment, R ₃ And R is ₆ H.

In another preferred embodiment, R ₁ is-L ₁ R _a ，R ₆ Is H, and R ₄ And R is ₅ Independently selected from the group consisting of: H. substituted or unsubstituted-C1-C6 alkyl, -C3-C8 cycloalkyl, -C1-C6 alkyl-C3-C8 cycloalkyl, -C1-C3 alkyl-COO-benzylThe method comprises the steps of carrying out a first treatment on the surface of the Or R is ₄ And R is ₅ To a linked carbon atom to form a C3-C8 cycloalkyl radical, and R ₄ And R is ₅ At least one is other than H.

In another preferred embodiment, R ₁ And R is ₂ Is H, and R ₃ is-L ₁ R _a 。

In another preferred embodiment, R ₁ 、R ₂ 、R ₄ And R is ₅ H.

In another preferred embodiment, R ₃ is-L ₁ R _a The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

R ₆ Selected from the group consisting of: substituted or unsubstituted C1-C15 alkyl, substituted or unsubstituted C2-C15 alkenyl, substituted or unsubstituted C2-C15 alkynyl, substituted or unsubstituted C3-C12 cycloalkyl, C1-C6 alkyl-Rc or C1-C6 alkyl-COO-Rc; each Rc is independently selected from the group consisting of: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, benzyl, phenyl, C3-C12 cycloalkyl.

In another preferred embodiment, R ₇ 、R ₈ 、R ₉ 、R ₁₀ 、R ₁₂ 、R ₁₃ 、R ₁₄ And R is ₁₅ H.

In another preferred embodiment, R ₁₁ is-L ₁ R _a The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

R ₁₆ Selected from the group consisting of: substituted or unsubstituted C1-C15 alkyl, substituted or unsubstituted C2-C15 alkenyl, substituted or unsubstituted C2-C15 alkynyl, substituted or unsubstituted C3-C12 cycloalkyl, C1-C6 alkyl-Rc or C1-C6 alkyl-COO-Rc; each Rc is independently selected from the group consisting of: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, benzyl, phenyl, C3-C12 cycloalkyl.

In another preferred embodiment, R ₁₆ Selected from the group consisting of: -C1-C6 alkyl, -C3-C8 cycloalkyl, -C1-C6 alkyl-C3-C8 cycloalkyl, -C3-C8 cycloalkyl.

In another preferred embodiment, R ₁₇ is-L ₁ R _a The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

R ₁₈ Selected from the group consisting of: substituted or unsubstituted C1-C15 alkyl, substituted or unsubstituted C2-C15 alkenyl, substituted or unsubstituted C2-C15 alkynyl, substituted or unsubstituted C3-C12 cycloalkyl, C1-C6 alkyl-Rc or C1-C6 alkyl-COO-Rc; each Rc is independently selected from the group consisting of: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, benzyl, phenyl, C3-C12 cycloalkyl.

In another preferred embodiment, in each formula, x is independently 10 to 100, preferably 10 to 90, such as 20, 30, 40, 50, 60, 70 or 80.

In another preferred embodiment, in each, y is independently 5-90, preferably 10-90, such as 20, 30, 40, 50, 60, 70 or 80.

In another preferred embodiment, n is independently a positive integer from 5 to 50, such as 10, 15, 20, 25, 30, 35, or 40.

In another preferred example, the average molecular weight Mn of the compound is in the range 1000-8000, preferably 2000-6000, more preferably 3000-5200, such as about 3500, 4000, 4500 or 5000.

In another preferred embodiment, the compounds have a molecular weight distribution PDI of independently 1 to 1.4, preferably 1.1 to 1.3, more preferably 1.1 to 1.25.

In another preferred embodiment, the compound is a random copolymer or a block copolymer.

In another preferred embodiment, one end of the compound comprises an end group having a reactive group selected from the group consisting of: -SH, -NH ₂ -COOH, -Br, -Cl, -OH, epoxy, alkenyl, alkynyl, -COCl, azido, maleimide, o-dithiopyridyl (OPSS). For example, -C1-C6 alkyl-SH, -NH-C1-C6 alkyl-SH, -CO-C1-C6 alkyl-SH.

In another preferred embodiment, the other end group of the compound is selected from the group consisting of: H. monomer residues, initiator residues. Those skilled in the art will appreciate that the monomer residues, initiator residues, etc. at the end groups are related to the monomer species and initiator species, but do not significantly affect the activity of the polymer segments of the present invention.

In another preferred embodiment, the initiator is selected from the group consisting of: liHMDS, naHMDS, KHMDS trityl mercaptoethylamine, p-tert-butylbenzylamine, triphenylmercapto 3-bromopropyl.

In another preferred embodiment, the monomer residues, initiator residues may be blocked with a blocking agent, preferably selected from the group consisting of: trityl mercaptoethylamine, p-tert-butyl benzylamine.

In another preferred embodiment, the salt of the compound is selected from the group consisting of: hydrochloride, bromate, trifluoroacetate, phosphate, lithium salt, sodium salt, potassium salt.

In another preferred embodiment, the material of the substrate surface is selected from the group consisting of: inorganic nonmetallic biomaterials (such as bioceramics, bioglass, graphene, bone cements and medical carbon materials), biometalline materials (such as stainless steel, cobalt-based and titanium-based alloys, shape memory alloys, noble metals such as silver, platinum, tantalum, niobium, zirconium, palladium, platinum), natural high molecular materials (such as hyaluronic acid, chitosan, alginic acid, cellulose, collagen, gelatin), synthetic high molecular materials (polyetheretherketone, polycaprolactone, polylactic acid, polycarbonate, polyurethane, polyester, polyanhydride, polydimethylsiloxane, polymethyl methacrylate, polyphosphazene, polyamide, polyethylene, polypropylene, polytetrafluoroethylene, polyethylene terephthalate, resins), or combinations thereof (such as composites, assembled materials).

In another preferred embodiment, the material of the substrate surface is selected from the group consisting of: TPU, glass, titanium sheet, stainless steel, gelatin sponge and PEEK.

In another preferred embodiment, the substrate is selected from the group consisting of: orthopedic implants, medical devices, medical catheters, dressings, gels, other antimicrobial medical materials implantable in the body.

In another preferred embodiment, the substrate is selected from the group consisting of: catheters, venous indwelling catheters, cardiovascular stents, cardiac pacemakers, electrophysiological catheters, hip implants or contact lenses.

In another preferred embodiment, the bacteria are selected from the group consisting of: gram positive bacteria, gram negative bacteria, fungi, spores, resting cells.

In another preferred embodiment, the bacteria are selected from the group consisting of: staphylococcus aureus, escherichia coli, acinetobacter, streptococcus suis, nocardia stellate, streptococcus agalactiae, vibrio anguillarum, vibrio alginolyticus, vibrio parahaemolyticus, vibrio harveyi, vibrio cholerae, mannheimia haemolytica, salmonella pullorum, salmonella typhimurium, salmonella cholerae, vibrio fluvialis, aeromonas hydrophila, edwardsiella tarda, pseudomonas fluorescens and salmonella duck, actinobacillus, rhodochrous, bacillus, bacteroides, bifidobacterium, bordetella, brucella, burkholderia, campylobacter, chlamydia, clostridium, corynebacteria, salmonella, and pseudomonas aeruginosa Erickettsia, escherichia, francisella, haemophilus, helicobacter, leptospira, listeria, mannheimia, moraxella, mycobacterium, mycoplasma, neisseria, new rickettsia, xuesiella, porphona, prevotella, pseudomonas, psychrophilia, salmonella, serratia, shewanella, shigella, tanna, treponema, trophera, vibrio, wo Ersi Klebsiella, yersinia, eubacterium, gardnerella, klebsiella, streptococcus, proteus, providencia, or a combination thereof.

In another preferred embodiment, the fungus is selected from the group consisting of: candida, aspergillus, cryptococcus, candida, mucor, penicillium, aspergillus, zygomyces, fusarium, coloring fungi (alternaria), malassezia, chlorella, or combinations thereof. In another preferred embodiment, the antibacterial comprises preventing and/or inhibiting bacterial growth and/or proliferation, reducing and/or killing bacteria.

In another preferred embodiment, the method of preactivation is selected from the group consisting of:

(i) A method of activating a plasma radiator comprising the steps of:

placing a substrate in low-temperature plasma, and exciting and activating to obtain a pre-activated surface with oxygen free radicals, hydroxyl groups, carboxyl groups, epoxy groups and/or aldehyde groups on the surface;

placing the surface pre-activated by the method (i) in a bromoform solution, thereby obtaining a surface brominated pre-activated surface;

(ii) Irradiating the surface of a substrate by ultraviolet ozone, and placing the substrate in a solution of 3-aminopropyl triethoxysilane (APTES) to obtain a pre-activated surface with an aminated surface;

(iii) The substrate is placed in an alkaline solution of dopamine, thereby obtaining a dopamine pre-activated surface.

In another preferred embodiment, in method (iii) one or more features selected from the group consisting of:

(a) The solvent of the alkaline solution is tris (hydroxymethyl) aminomethane hydrochloride;

(b) The alkaline solution has a pH of 8.5-10, such as 9 or 9.5.

In another preferred embodiment, the preactivation temperature of each preactivation process is independently from 0 to 120 ℃, such as from 10 to 100 ℃, from 20 to 60 ℃, or from 30 to 40 ℃.

In another preferred embodiment, the preactivation time of each preactivation process is independently from 5min to 48h, such as 10min, 30min, 1h, 2h, 6h, 12h or 24h.

In another preferred embodiment, in step (c), the reaction is carried out by immersing the pre-activated surface in a solution of the compound or salt thereof, preferably at a concentration of 0.1-10mg/mL, preferably 0.5-5mg/mL, such as 1mg/mL, 2mg/mL, 3mg/mL or 4mg/mL.

In another preferred embodiment, in step (c), the temperature of the reaction is from 0 to 120 ℃, such as from 10 to 100 ℃, from 30 to 80 ℃ or from 40 to 60 ℃.

In another preferred embodiment, in step (c), the reaction is carried out for a period of time ranging from 1h to 48h, such as 2h, 4h, 6h, 12h or 24h.

In another preferred embodiment, the reaction further comprises one or more of the following steps:

(d) Blocking the reaction site; and/or

(e) Washing and drying.

In another preferred embodiment, the compound comprises a segment selected from the group consisting of:

In the formulae, x is 5-100; and x+y=100; n is a positive integer from 1 to 100.

In another preferred embodiment, the compound is selected from the group consisting of:

In a second aspect of the invention there is provided a substrate having an increased antimicrobial activity and biocompatibility of said at least a portion of the surface as prepared by the method of the first aspect of the invention, and a product comprising or prepared from said substrate, and said product comprising at least a portion of the surface having an increased antimicrobial activity and biocompatibility.

In another preferred embodiment, the average grafting density of the compounds on at least a portion of the surface of the substrate is not less than 0.1chain/nm ² Preferably, 0.2chain/nm or more ² Or is greater than or equal to 0.23chain/nm ² The average grafting density of the compound is 0.1-2chain/nm on at least one part of the surface of the substrate ² ，0.15-1chain/nm ² 、0.2-0.5chain/nm ² Or e.g. 0.25chain/nm ² 、0.3chain/nm ² 、0.35chain/nm ² Or 0.4chain/nm ² 。

In another preferred embodiment, the surface contact angle of at least a portion of the surface of the substrate is reduced by 20-100 °, more preferably by 30-80 °, by 40 °, 45 °, 50 °, 55 °, 60 °, 65 °, or 75 °, compared to the untreated substrate surface.

In a third aspect of the invention there is provided a substrate as described in the second aspect of the invention for use in the preparation of a product having improved antibacterial activity and biocompatibility.

In another preferred embodiment, the product is selected from the group consisting of: orthopedic implants, medical devices, medical catheters, dressings, gels, other antimicrobial medical materials implantable in the body.

In another preferred embodiment, the product is selected from the group consisting of: catheters, venous indwelling catheters, cardiovascular stents, cardiac pacemakers, electrophysiological catheters, hip implants or contact lenses.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 is a graph of percent hemolysis and scanning electron microscopy morphology of a polyurethane modified polymer surface (TPU-1) and a TPU unmodified surface versus a negative positive control;

FIG. 2 shows the results of a substrate surface cytotoxicity test; wherein A and C are two cell contact toxicity experiments of observing the surface of the substrate by a microscope; b and D are the percent cell viability of the substrate surface extract;

FIG. 3 is a comparison of the number of antibacterial colonies in vivo for polymer modified and unmodified surfaces;

FIG. 4 is HE staining of polymer modified and unmodified surfaces (arrows labeled as inflammatory cells);

FIG. 5 is a gram bacterial stain (labeled with an arrow as a bacterium or colony) for polymer modified and unmodified surfaces;

FIG. 6 is a scanning electron microscope image of polymer modified and unmodified surface bacteria with polymer modified surface contact for bacterial membrane breakage;

FIG. 7 shows cytoplasmic membrane permeabilities of polymer modified and unmodified surfaces, bacterial cytoplasmic membrane permeabilities of polymer modified surface contacts;

FIG. 8 shows the electrical conductivity of polymer modified and unmodified surfaces, with increased electrical conductivity of bacterial cells contacted by the polymer modified surfaces.

FIG. 9 shows the surface cell compatibility of example 2.

FIG. 10 AFM characterization of polymer modified and unmodified surfaces.

FIG. 11 contact angle characterization of polymer modified and unmodified surfaces.

Detailed Description

The present inventors have conducted extensive and intensive studies and, through a large number of screening and testing, have provided a method for improving antibacterial activity and biocompatibility of a substrate surface. Specifically, the method includes attaching the polymer segment to at least a portion of the surface of the substrate via a chemical bond, thereby improving the antimicrobial activity and biocompatibility of the substrate surface. The present inventors have found that the above polymer segment, a compound containing the same or a salt thereof has strong cytotoxicity at a bactericidal concentration in a solution state, but surprisingly, at 90% bactericidal rate when attached to a substrate surface, cytotoxicity of the modified substrate is rather lower than that of the substrate being modified, thereby being very suitable for improving antibacterial activity and biocompatibility of the substrate surface. The present invention has been completed on the basis of this finding.

Terminology

Unless defined otherwise, 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 invention belongs.

As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40 ℃, preferably 25±5 ℃.

Terminology

In the present invention, unless otherwise indicated, terms used have the ordinary meanings known to those skilled in the art.

When substituents are described by conventional formulas written from left to right, the substituents are also includedChemically equivalent substituents are obtained when writing structural formulae from right to left. For example, -CH ₂ O-is equivalent to-OCH ₂ -。

Throughout the specification, the terms "optionally substituted" or "may be substituted" and the like mean that the group may or may not be further substituted or fused with one or more non-hydrogen substituents (to form a polycyclic ring system). Substituents for suitable chemically suitable specific functional groups will be apparent to those skilled in the art.

As used herein, the term "alkyl" refers to a straight or branched chain alkyl group containing several carbon atoms, wherein "C ₁ -C ₁₅ Alkyl "means a straight or branched chain alkyl having 1 to 15 carbon atoms, including alkyl having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbon atoms, alkyl preferably being, for example, C ₁ -C ₂ 、C ₁ -C ₃ 、C ₁ -C ₄ 、C ₁ -C ₅ 、C ₁ -C ₆ 、C ₁ -C ₇ 、C ₁ -C ₈ 、C ₁ -C ₉ 、C ₁ -C ₁₀ 、C ₂ -C ₃ 、C ₂ -C ₄ 、C ₂ -C ₅ 、C ₂ -C ₆ 、C ₃ -C ₄ 、C ₃ -C ₅ 、C ₃ -C ₆ 、C ₃ -C ₇ 、C ₃ -C ₈ 、C ₄ -C ₅ 、C ₄ -C ₆ Or C _5-6 . Typical "alkyl" groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, and,Pentyl, isopentyl, heptyl, 4-dimethylpentyl, octyl, 2, 4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl and the like. In the present invention, alkyl groups also include substituted alkyl groups. "substituted alkyl" means that one or more positions in the alkyl group are substituted, especially 1 to 4 substituents, and may be substituted at any position.

As used herein, the term "C ₁ -C ₁₅ Alkoxy "means a straight or branched chain alkoxy group having 1 to 15 carbon atoms, having C ₁ -C ₁₅ alkyl-O-structure, C ₁ -C ₁₅ Alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy, and the like, with ethoxy being preferred. C (C) ₁ -C ₁₅ Alkoxy groups also include substituted C ₁ -C ₁₅ An alkoxy group.

As used herein, the term "C ₁ -C ₁₅ Alkyl hydroxy "means-C ₁ -C ₁₅ alkylene-OH, -C ₁ -C ₁₅ Alkylene has the definition as above, C ₁ -C ₁₅ Alkyl hydroxy groups include, but are not limited to, -CH ₂ OH、-CH ₂ CH ₂ OH。C ₁ -C ₁₅ The alkyl hydroxy groups also include substituted C ₁ -C ₁₅ Alkyl hydroxy.

As used herein, the term "C ₁ -C ₁₅ Alkylsulfonyl "means C ₁ -C ₁₅ Alkyl S (=O) ₂ -。

As used herein, the term "C ₁ -C ₁₅ alkyl-C ₆ -C ₁₅ Aryl "means-C ₁ -C ₁₅ alkyl-C ₆ -C ₁₅ Aryl radicals, e.g. -CH ₂ CH ₂ CH ₂ Ph、-Bn。

As used herein, the term "C ₁ -C ₁₅ Alkyl ester group "means C ₁ -C ₁₅ Alkyl C (=O) -O-or-C (=O) -O-C ₁ -C ₁₅ An alkyl group.

As used herein, the term "thio C1-C15 alkyl ester group" refers to C ₁ -C ₁₅ Alkyl C (=S) -O-or-C (=S) -O-C ₁ -C ₁₅ An alkyl group.

As used herein, the term "guanidino" refers to NHC (=nh) NH-, and the term "biguanide" refers to-NH-C (CH) -NH ₂ 。

As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon having at least 2 carbon atoms and at least one double bond. Alkenyl group can be packaged Includes any number of carbon atoms, where "C2-C15 alkenyl" refers to a straight or branched hydrocarbon having 2 to 15 carbon atoms and at least one double bond, e.g., C ₂ 、C _2- C ₃ 、C _2- C ₄ 、C _2- C ₅ 、C _2- C ₆ 、C _2- C ₇ 、C _2- C ₈ 、C _2- C ₉ 、C _2- C ₁₀ 、C ₃ 、C _3- C ₄ 、C _3- C ₅ 、C _3- C ₆ 、C ₄ 、C _4- C ₅ 、C _4- C ₆ 、C ₅ 、C _5- C ₆ And C ₆ . Alkenyl groups may have any suitable number of double bonds including, but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include, but are not limited to, vinyl (vinyl group)), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1, 3-pentadienyl, 1, 4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1, 3-hexadienyl, 1, 4-hexadienyl, 1, 5-hexadienyl, 2, 4-hexadienyl, or 1,3, 5-hexatrienyl. As with the alkyl groups described above, alkenyl groups may be substituted or unsubstituted.

As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon having at least 2 carbon atoms and at least one triple bond. Alkynyl groups may include any number of carbon atoms, "C2-C15 alkynyl" refers to straight or branched chain hydrocarbons having 2 to 15 carbon atoms and at least one triple bond, e.g., C ₂ 、C _2- C ₃ 、C _2- C ₄ 、C _2- C ₅ 、C _2- C ₆ 、C _2- C ₇ 、C _2- C ₈ 、C _2- C ₉ 、C _2- C ₁₀ 、C ₃ 、C _3- C ₄ 、C _3- C ₅ 、C _3- C ₆ 、C ₄ 、C _4- C ₅ 、C _4- C ₆ 、C ₅ 、C _5- C ₆ And C ₆ . Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl Alkynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1, 3-pentadiynyl, 1, 4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1, 3-hexadiynyl, 1, 4-hexadiynyl, 1, 5-hexadiynyl, 2, 4-hexadiynyl, or 1,3, 5-hexatriynyl. As with the alkyl groups described above, alkynyl groups may be substituted or unsubstituted.

The term "aryl" refers to aromatic cyclic hydrocarbon compounds wherein "C6-C12 aryl" refers to aromatic cyclic hydrocarbon compounds containing 6, 7, 8, 9, 10, 11 or 12 ring carbon atoms, having 1 to 3 rings, especially mono-and bi-cyclic groups such as phenyl, biphenyl or naphthyl. The aromatic ring of the aryl group may be linked by a single bond (e.g., biphenyl), or condensed (e.g., naphthalene, anthracene, etc.), where the aromatic ring contains two or more aromatic rings (bicyclic, etc.). "substituted aryl" means that one or more positions in the aryl group are substituted, especially 1 to 3 substituents, and can be substituted at any position.

As used herein, the term "heteroaryl" refers to a heteroaromatic system containing 1 to 3 atoms selected from N, O, S, wherein "5-12 membered heteroaryl" refers to a 5-12 membered heteroaromatic system containing 1 to 3 atoms selected from N, O, S. Heteroaryl is preferably a 5 to 10 membered ring, more preferably 5 or 6 membered ring, and heteroaryl includes, but is not limited to, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazolyl, tetrazolyl, and the like. "heteroaryl" may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more groups independently selected from alkyl, deuteroalkyl, haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, alkylthio, alkylamino, halogen, amino, nitro, hydroxy, mercapto, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylthio, oxo (-oxo), carboxyl, and carboxylate.

As used herein, the term "cycloalkyl" refers to a fully saturated cyclic hydrocarbon group having several carbon atoms, wherein "C ₃ -C ₁₂ Cycloalkyl "means a fully saturated cyclic hydrocarbon group having 3 to 12 carbon atoms, preferably C ₃ -C ₄ 、C ₃ -C ₅ 、C ₃ -C ₆ 、C ₃ -C ₇ 、C ₃ -C ₈ 、C ₃ -C ₉ 、C ₃ -C ₁₀ . "substitution C ₃ -C ₁₂ Cycloalkyl "means that one or more positions in the cycloalkyl group, especially 1-4 substituents, may be substituted at any position, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. In the present invention, "cycloalkyl" is intended to include "substituted cycloalkyl".

As used herein, the term "cycloalkenyl" refers to an unsaturated cyclic hydrocarbon group having 1-3 double bonds with several carbon atoms, where "C ₄ -C ₁₂ Cycloalkenyl "means an unsaturated cyclic hydrocarbon group having from 4 to 12 carbon atoms with 1 to 3 double bonds, preferably C ₆ -C ₁₀ Cycloalkenyl, C ₄ -C ₆ Cycloalkenyl groups, cycloalkenyl groups include, but are not limited to, cyclobutenyl, cyclopentenyl, cyclohexenyl.

As used herein, the term "heterocyclyl" refers to a fully saturated or partially unsaturated cyclic group having several (3 or more) ring atoms and having 1-3 heteroatoms, wherein "5-12 membered heterocyclyl" refers to a fully saturated or partially unsaturated cyclic group having 5-12 ring atoms and having 1-3 heteroatoms (including but not limited to, e.g., 3-7 membered monocyclic, 6-11 membered bicyclic, or 8-12 membered tricyclic ring systems). Wherein the nitrogen or sulfur atom may be oxidized and the nitrogen atom may be quaternized. The heterocyclic group may be attached to any heteroatom or carbon atom residue of a ring or ring system molecule. Typical monocyclic heterocycles include, but are not limited to, azetidinyl, pyrrolidinyl, oxetanyl, pyrazolinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, hexahydroazepinyl, 4-piperidonyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1, 3-dioxanyl, and tetrahydro-1, 1-dioxythiophene, and the like. Polycyclic heterocyclyl groups include spiro, fused and bridged heterocyclic groups; wherein the heterocyclic groups of the spiro ring, the condensed ring and the bridged ring are optionally connected with other groups through single bonds, or are further connected with other cycloalkyl groups, heterocyclic groups, aryl groups and heteroaryl groups through any two or more atoms on the ring in a parallel ring manner; the heterocyclic group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more groups independently selected from alkyl, deuteroalkyl, haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, alkylthio, alkylamino, halogen, amino, nitro, hydroxy, mercapto, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylthio, oxo, carboxyl, and carboxylate. Heterocyclyl groups include, but are not limited to: tetrahydropyrrolyl, tetrahydrofuranyl, piperidinyl, piperazinyl, and the like.

When a substituent is a non-terminal substituent or a related group, it is a subunit of the corresponding group, typically a divalent group, the definition of the above group applies equally to divalent, trivalent, etc., multivalent groups thereof. For example, alkyl groups having one H atom removed are alkylene groups (e.g., methylene, ethylene, propylene, isopropylene (e.g.)) Butylene (e.g.)>) Pentylene (e.g.)>) Hexyl ene (e.g.)>) Heptyl (e.g.)>) Etc.), cycloalkyl corresponds to cycloalkylene (e.g.: />Etc.), the heterocyclyl corresponds to the heterocyclylene group (e.g., as:) Cycloalkyl corresponds to a heterocyclyl group (e.g.: /> Etc.), alkoxy corresponds to alkyleneoxy (-CH ₂ O-、-CH ₂ CH ₂ -O-CH ₂ -、-CH ₂ OCH ₂ CH ₂ CH ₂ (-), etc.

As used herein, the term "plurality" refers to two or more, such as 2, 3, 4, 5 or 6.

As described herein, the compounds of the present invention may be substituted with any number of substituents or functional groups to extend their inclusion. In general, the term "substituted", whether appearing before or after the term "optional", in the context of the present invention includes the general formula of substituents, means that the specified structural substituent is used in place of the hydrogen radical. When multiple of a particular structure are substituted at a position with multiple particular substituents, the substituents may be the same or different at each position. The term "substitution" as used herein includes all permissible organic compound substitutions. In a broad sense, permissible substituents include acyclic, cyclic, branched, unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic organic compounds. In the present invention, the heteroatom nitrogen may have a hydrogen substituent or any of the permissible organic compounds described hereinabove to supplement the valence state thereof. Furthermore, the present invention is not intended to be limited in any way to allow substitution of organic compounds.

As used herein, the term "substituted" refers to a compound in which any one or more hydrogens on the designated atom are replaced with a selection from the group consisting of the indicated substituents, provided that the normal valency of the designated atom is not exceeded, and the resulting compound from the substitution is stable, i.e., can be isolated, characterized, and subjected to a biological activity test.

The substitution, unless specifically stated, refers to substitution of one or more H on each group independently with a group selected from the group consisting of: deuterium, halogen, -SH, -OH, -NH ₂ Benzyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, C2-C6 haloalkenyl, C2-C6 haloalkynyl, C3-C8 cycloalkyl, C6-C10 aryl.

Unless otherwise indicated, it is assumed that any heteroatom in an underfilling state has sufficient hydrogen atoms to complement its valence.

As used herein, the term "blend (statistic coplymer)" or "random" refers to polymers formed by the random linkage of two or more monomers polymerized simultaneously.

As used herein, the term "block-type" refers to a polymer formed by the sequential polymerization of two or more monomers, at least one of which is of a pendant amino or hydroxyl structure, formed from the attachment of different segments.

The structural formula or name of the monomer given in the present invention may only give a specific configuration or not give a specific configuration, and the monomer may also include all other configurations corresponding to the given configuration.

Active ingredient

In the present invention, the active ingredient is a polymer segment, a compound containing the same or a salt thereof.

As used herein, the terms "compound of the invention" and "polymer of the invention" are used interchangeably to refer to a compound comprising a polymer segment of the invention.

In the present invention, the polymer segment is selected from the group consisting of:

wherein R is ₁ 、R ₂ 、R ₃ R ₄ 、R ₅ 、R ₆ 、R ₇ 、R ₈ 、R ₉ 、R ₁₀ 、R ₁₂ 、R ₁₃ 、R ₁₄ 、R ₁₅ R ₁₆ 、R ₁₇ 、R ₁₈ And each x, y, n is as defined above.

Unless otherwise indicated, all compounds present in the present invention are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (i.e., racemates). Among all the compounds of the invention, each chiral carbon atom may optionally be in the R configuration or in the S configuration, or in a mixture of R and S configurations.

Certain compounds of the invention possess an asymmetric carbon atom (optical center) or double bond; racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., isolated enantiomers) are all intended to be included within the scope of the present invention. When compounds provided herein have a defined stereochemistry (denoted R or S, or indicated with dashed or wedge-shaped bonds), those compounds will be understood by those skilled in the art to be substantially free of other isomers (e.g., at least 80%,90%,95%,98%,99% and up to 100% free of other isomers).

As used herein, the term "salt of a compound" means that the compounds of the present invention can be used in the form of salts derived from pharmaceutically or physiologically acceptable acids or bases. These salts include, but are not limited to, salts formed with acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid. Other salts include: salts with alkali or alkaline earth metals (such as sodium, potassium, calcium or magnesium), and in the form of esters, carbamates or other conventional "prodrugs".

In general, the polymer segments of the present invention have a proportion of structural unit side chains (e.g. -L ₁ R _a Part) contains-NR _b R _b Guanidino, biguanide, -N (R) _b ) ₃ ⁺ Basic groups (R) capable of generating positive charges _a A group) which is in accordance with the present inventionThe compounds of (2) are closely related to the surface antibacterial activity and preferably contain-L ₁ R _a The proportion of structural units of the side chains is 10-100% or 50-80%.

More preferably, when R is _a L with groups bound to the skeleton ₁ When the linking group has 2 or more carbon atoms, the compound and the surface exhibit further antibacterial activity, preferably L ₁ Is a C2-C6 alkylene group, more preferably a C2-C4 alkylene group.

Most preferably, the compound is selected from the group consisting of:

The present inventors have found that the above polymer segment, a compound containing the same or a salt thereof has strong cytotoxicity at a bactericidal concentration in a solution state, but surprisingly, when attached to a substrate surface, the cytotoxicity of the modified substrate is rather lower in the case of 90% bactericidal rate than in the case of the modified substrate. Accordingly, the present invention provides the use of the above polymer segment, a compound comprising the same, or a salt thereof for improving the antibacterial activity and biocompatibility of a substrate surface.

The invention provides a method for improving the antibacterial activity and biocompatibility of a substrate surface. Specifically, the method includes the step of chemically bonding the polymer segment to at least a portion of the surface of the substrate, thereby improving the antimicrobial activity and biocompatibility of the surface of the substrate.

The polymer segments may be modified onto the substrate surface using surface chemistry commonly used in the art.

More specifically, the steps may be included:

(a) Providing at least a portion of a surface of the substrate,

(b) Pre-activating the at least a portion of the surface,providing the surface with a reactive group selected from the group consisting of: -NH ₂ -Br, -Cl, -OH, -COOH, -CHO, epoxy, oxygen radical, alkenyl, alkynyl, polydopamine complex layer, or combinations thereof, thereby obtaining a pre-activated surface; and

wherein the compound comprises the polymer segment described above.

Typically, the compounds also include end groups having reactive groups selected from the group consisting of: -SH, -NH ₂ -COOH, -Br, -Cl, -OH, epoxy, alkenyl, alkynyl, -COCl, azido, maleimide, o-dithiopyridyl (OPSS). The reactive group may be attached to the polymer segment via a linking group such as C1-C6 alkylene, -CO-C1-C6 alkylene, -NH-C1-C6 alkylene, or the like. The reactive groups are used to react with reactive groups of the pre-activated substrate surface to attach the polymer segments of the present invention to the substrate surface. The selection of the reactive group of the compound according to the preactivation method or the preactivation method according to the reactive group of the compound is known to the person skilled in the art and may be selected, for example, from the group consisting of nucleophilic substitution reactions based on mercapto-halogen atoms, addition reactions of mercapto-epoxy groups, addition reactions of mercapto-alkenyl/alkynyl or azido-alkynyl groups, and the like.

Those skilled in the art will appreciate that the monomer residues on the end groups, initiator residues, end groups having reactive groups as described above, and the like, do not or should not significantly affect the activity of the polymer segments of the present invention.

The present invention has no particular requirement on the basic activation method, and can be selected according to the type of the substrate. Commonly used, preactivation methods include, but are not limited to: a plasma radiator activation method, an ultraviolet ozone irradiation method, a dopamine activation method, and the like. In particular, when the preactivation method is a dopamine activation method, there is no particular requirement for the end groups of the compound, and there is no need to have a reactive group, and it may be only H, a monomer residue, an initiator residue, or the like.

After the grafting reaction is completed, the reaction sites may also be blocked with a blocking reagent. The blocking reagent may be routinely selected according to the pre-activation method. The blocking reagent also has groups that react with reactive groups on the pre-activated surface, blocking other moieties being generally biologically inert. Such as the blocking of surface groups reactive with sulfhydryl groups with mercaptoglycerol.

Preferably, the material of the substrate surface is selected from the following group: inorganic nonmetallic biomaterials (such as bioceramics, bioglass, graphene, bone cements and medical carbon materials), biometalline materials (such as stainless steel, cobalt-based and titanium-based alloys, shape memory alloys, noble metals such as silver, platinum, tantalum, niobium, zirconium, palladium, platinum), natural high molecular materials (such as hyaluronic acid, chitosan, alginic acid, cellulose, collagen, gelatin), synthetic high molecular materials (polyetheretherketone, polycaprolactone, polylactic acid, polycarbonate, polyurethane, polyester, polyanhydride, polydimethylsiloxane, polymethyl methacrylate, polyphosphazene, polyamide, polyethylene, polypropylene, polytetrafluoroethylene, polyethylene terephthalate, resins), or combinations thereof (such as composites, assembled materials).

The substrate may be, but is not limited to, orthopedic implants, medical devices, medical catheters, dressings, gels, other antimicrobial medical materials implantable into the body.

In another preferred embodiment, the bacteria include, but are not limited to: gram positive bacteria, gram negative bacteria, fungi, spores or resting cells.

In another preferred embodiment, the antibacterial comprises preventing and/or inhibiting bacterial growth and/or proliferation, reducing and/or killing bacteria.

As used herein, the terms "untreated", "pre-treatment", "unmodified" are used interchangeably to refer to substrates that have not been treated with or prior to performing the methods of the present invention, also referred to herein as blank substrates.

In another preferred example, "an increased antimicrobial activity" means an increase in antimicrobial activity of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, or 200% or more in the treated substrate surface compared to the substrate surface prior to treatment by the method of the present invention; alternatively, the bacterial viability of the treated substrate surface is reduced by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, or 200% or more, as compared to the surface of the substrate prior to treatment by the method of the present invention.

In another preferred example, "increased biocompatibility" means that the hemolytic activity and/or cytotoxicity of the treated substrate surface is reduced, e.g. by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more or 200% or more, with respect to the hemolytic activity of human red blood cells, compared to the surface of the substrate prior to treatment by the method of the invention. The surface treated by the method has no obvious hemolytic activity on human red blood cells, murine red blood cells and the like; has no obvious cytotoxicity to mammalian cells such as mouse embryo fibroblasts, african monkey kidney cells, human umbilical vein endothelial cells, canine kidney cells, arterial smooth muscle cells and the like.

Product and use

The invention also provides a substrate having at least a portion of the surface having improved antimicrobial activity and biocompatibility, prepared by the method of the first aspect of the invention, and a product comprising or prepared from the substrate, and comprising at least a portion of the surface having improved antimicrobial activity and biocompatibility.

The invention also provides the application of the substrate in preparing a product with antibacterial activity and improved biocompatibility.

Typically, the products include, but are not limited to: orthopedic implants, medical devices, medical catheters, dressings, gels, other antimicrobial medical materials implantable in the body. Such as catheters, venous indwelling catheters, cardiovascular stents, cardiac pacemakers, electrophysiological catheters, hip implants or contact lenses, etc.

The main advantages of the invention include:

1. the surface of the substrate obtained by the method can obtain excellent microbial resistance, has no toxicity to normal cells, low inflammatory response and good biocompatibility, and is very suitable for surface modification of biomedical materials.

2. The compound of the present invention can play a role in resisting microorganisms by destroying the microbial membrane structure, and thus has a broad-spectrum and long-lasting performance in inhibiting microorganisms.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Examples

Preparation of alpha-amino acid polymers

Example 1: preparation of random alpha-amino acid copolymer by initiating N-epsilon-t-butoxycarbonyl-DL-lysine-N-carboxycyclic anhydride and L-glutamic acid-5-benzyl ester-N-carboxycyclic anhydride with hexamethyldisilazane lithium amide (LiHMDS)

Lithium hexamethyldisilazide (33.4 mg,0.2 mmol) was accurately weighed and prepared as a 0.1M solution in tetrahydrofuran (2 mL) for further use. N-epsilon-t-butoxycarbonyl-DL-lysine-N-carboxyl cyclic anhydride and L-glutamic acid-5-benzyl ester-N-carboxyl cyclic anhydride are weighed, and tetrahydrofuran is used as a solvent. 1.8mL of N- ε -t-butoxycarbonyl-DL-lysine-N-carboxycyclic anhydride (0.2M) and 0.2mL of 5-benzyl glutamate-N-carboxycyclic anhydride (0.2M) were mixed and stirred with a magnet (monomer ratio x: y=9:1, for example). To a stirred reaction flask, 0.8mL of a lithium hexamethyldisilazide solution having a concentration of 0.1M was added. The mixture was stirred at room temperature in a glove box for 5 minutes. After the polymerization reaction was completed, the reaction mixture was blocked overnight with triphenylmercaptoethylamine. Cold petroleum ether (45 mL) was poured into the reaction mixture, and the white floc which precipitated was collected by centrifugation, dried in air flow, redissolved in tetrahydrofuran (1.5 mL), and precipitated by adding a large amount of cold petroleum ether. This dissolution-precipitation process was repeated three times in total to obtain a purified copolymer. The molecular weight mn=6200 g/mol and the molecular weight distribution Mw/mn=1.16 of the polymer obtained were identified by Gel Permeation Chromatography (GPC).

The polymer which was drawn dry was added with 2mL of trifluoroacetic acid and 5% (v/v) triethylsilane, after shaking gently at room temperature overnight, excess trifluoroacetic acid was blown off, the resulting viscous liquid was dissolved in 0.5mL of methanol, 45mL of ice-methyl t-butyl ether was added to precipitate a white precipitate, and the dissolution-precipitation process was repeated three times, to thereby obtain a random polymer with deprotected side chain amino groups. The subunit ratio of the polymer is obtained by nuclear magnetic resonance hydrogen spectrum characterization, and x in the final polymer: y=9.26: 1. the deprotected polymer was again dissolved in 5mL of ultrapure water and filtered and lyophilized for subsequent bioactivity testing.

Example 2: preparation of random alpha-amino acid copolymer by initiating N-epsilon-t-butoxycarbonyl-DL-lysine-N-carboxyl cyclic anhydride and DL-norleucine-N-carboxyl cyclic anhydride with hexamethyldisilazane lithium amide (LiHMDS)

The experimental procedure was the same as in example 1, except that DL-norleucine-N-carboxyanhydride was substituted for L-glutamic acid-5-benzyl ester-N-carboxyanhydride; 0.4mL of a lithium hexamethyldisilazide solution having a concentration of 0.1M was added instead of 0.8mL of a lithium hexamethyldisilazide solution having a concentration of 0.1M. The molecular weight mn=7500 g/mol and the molecular weight distribution Mw/mn=1.19 of the polymer obtained. The subunit ratio of the polymer is obtained by nuclear magnetic resonance hydrogen spectrum characterization, and x in the final polymer: y=6.12: 4.

Example 3: preparation of random alpha-amino acid copolymer by initiating N-epsilon-t-butoxycarbonyl-DL-lysine-N-carboxyl cyclic anhydride and DL-norvaline-N-carboxyl cyclic anhydride with hexamethyldisilazane lithium amide (LiHMDS)

The experimental method was the same as in example 1, except that DL-norvaline-N-carboxylmethylanhydride was used instead of L-glutamic acid-5-benzyl ester-N-carboxylmethylanhydride; 0.4mL of a lithium hexamethyldisilazide solution having a concentration of 0.1M was added instead of 0.8mL of a lithium hexamethyldisilazide solution having a concentration of 0.1M. The molecular weight mn=7100 g/mol and the molecular weight distribution Mw/mn=1.19 of the polymer obtained. The subunit ratio of the polymer is obtained by nuclear magnetic resonance hydrogen spectrum characterization, and x in the final polymer: y=6: 4.

example 4: preparation of random alpha-amino acid copolymer by initiating N-epsilon-t-butoxycarbonyl-DL-lysine-N-carboxycyclic anhydride and DL-2-aminobutyric acid-N-carboxycyclic anhydride with hexamethyldisilazane lithium amide (LiHMDS)

The experimental method was the same as in example 1, except that DL-2-aminobutyric acid-N-carboxycyclic anhydride was substituted for L-glutamic acid-5-benzyl ester-N-carboxycyclic anhydride; 0.4mL of a lithium hexamethyldisilazide solution having a concentration of 0.1M was added instead of 0.8mL of a lithium hexamethyldisilazide solution having a concentration of 0.1M. The molecular weight mn=6800 g/mol and the molecular weight distribution Mw/mn=1.19 of the polymer obtained. The subunit ratio of the polymer is obtained by nuclear magnetic resonance hydrogen spectrum characterization, and x in the final polymer: y=6: 4.

Example 5: preparation of random alpha-amino acid copolymer by initiating N-epsilon-t-butoxycarbonyl-DL-lysine-N-carboxyl cyclic anhydride and DL-alanine-N-carboxyl cyclic anhydride with hexamethyldisilazane lithium amide (LiHMDS)

The experimental procedure was the same as in example 1, except that DL-alanine-N-carboxyanhydride was substituted for L-glutamic acid-5-benzyl ester-N-carboxyanhydride; 0.4mL of a lithium hexamethyldisilazide solution having a concentration of 0.1M was added instead of 0.8mL of a lithium hexamethyldisilazide solution having a concentration of 0.1M. The molecular weight mn=5900 g/mol and the molecular weight distribution Mw/mn=1.19 of the polymer obtained. The subunit ratio of the polymer is obtained by nuclear magnetic resonance hydrogen spectrum characterization, and x in the final polymer: y=4: 6.

example 6: hexamethyldisilyl lithium amide (LiHMDS) initiates homo-polymerization of N-epsilon-t-butoxycarbonyl-DL-lysine-N-carboxycyclic anhydride and post-polymerization modification

Lithium hexamethyldisilazide (33.4 mg,0.2 mmol) was accurately weighed and prepared as a 0.1M solution in tetrahydrofuran (2 mL) for further use. Weighing N-epsilon-t-butoxycarbonyl-DL-lysine-N-carboxyl cyclic anhydride and using tetrahydrofuran as a solvent. To a stirred reaction flask, 0.8mL of a lithium hexamethyldisilazide solution having a concentration of 0.1M was added. The mixture was stirred at room temperature in a glove box for 5 minutes. After the polymerization reaction was completed, the reaction mixture was blocked overnight with triphenylmercaptoethylamine. Cold petroleum ether (45 mL) was poured into the reaction mixture, and the white floc which precipitated was collected by centrifugation, dried in air flow, redissolved in tetrahydrofuran (1.5 mL), and precipitated by adding a large amount of cold petroleum ether. This dissolution-precipitation process was repeated three times in total to obtain a purified copolymer. The molecular weight mn=6000 g/mol and the molecular weight distribution Mw/mn=1.16 of the polymer obtained were identified by Gel Permeation Chromatography (GPC). The polymer which was drawn dry was added with 2mL of trifluoroacetic acid and 5% (v/v) triethylsilane, after shaking gently at room temperature overnight, excess trifluoroacetic acid was blown off, the resulting viscous liquid was dissolved in 0.5mL of methanol, 45mL of ice-methyl t-butyl ether was added to precipitate a white precipitate, and the dissolution-precipitation process was repeated three times, to thereby obtain a random polymer with deprotected side chain amino groups. The deprotected polymer was again dissolved in 5mL of ultrapure water and lyophilized for post-polymer modification by filtration.

The deprotected polymer (10 mg) was weighed out and dissolved in anhydrous methanol, and 3 equivalents of 1H-pyrazole-1-carboxamide hydrochloride and 4 equivalents of N, N diisopropylethylamine were added to the amine molar mass of the polymer chain. The resulting mixture was heated at 55deg.C under nitrogen atmosphereAbout 24 hours. The solvent was then removed in a rotary evaporator and the polymer was purified by three precipitation with methanol-acetone to give a white powder. By using ¹ HNMR, FT-IR demonstrated a degree of guanylation approaching 100%. Again dissolved in 5mL of ultrapure water, filtered and lyophilized for antifungal bioactivity test.

Example 7: preparation of random alpha-amino acid copolymer by initiating N-epsilon-t-butoxycarbonyl-DL-ornithine-N-carboxyl cyclic anhydride and DL-norleucine-N-carboxyl cyclic anhydride with hexamethyldisilazane lithium amide (LiHMDS)

The experimental procedure was the same as in example 6, except that N- ε -t-butoxycarbonyl-DL-ornithine-N-carboxyanhydride and DL-norleucine-N-carboxyanhydride were substituted for N- ε -t-butoxycarbonyl-DL-lysine-N-carboxyanhydride. The molecular weight mn=6100 g/mol and the molecular weight distribution Mw/mn=1.16 of the polymer obtained. The subunit ratio of the polymer is obtained by nuclear magnetic resonance hydrogen spectrum characterization, and x in the final polymer: y=4: 6.

Preparation of beta-amino acid polymers

Example 8: preparation of random beta-amino acid copolymer by triggering beta-3-amino-N-benzyloxycarbonyl-L-alanine-N-carboxythiocarbonyl cyclic anhydride and beta-3-aminoheptanoic acid-N-carboxythiocarbonyl cyclic anhydride with triphenylmercaptoethylamine

In a glove box protected by nitrogen, beta-3-amino-N-benzyloxycarbonyl-L-alanine-N-carboxythiocarbonyl anhydride and beta-3-aminoheptanoic acid-N-carboxythiocarbonyl anhydride were weighed, 1.2mL of beta-3-amino-N-benzyloxycarbonyl-L-alanine-N-carboxythiocarbonyl anhydride (0.2M) and 0.8mL of beta-3-aminoheptanoic acid-N-carboxythiocarbonyl anhydride (0.2M) were mixed in a reaction flask using dry N, N-dimethylformamide as a solvent, and then stirred by adding a magnet. The initiator triphenylmercaptoethylamine was weighed and prepared into a solution (0.2M), 100. Mu.L of the solution was rapidly added into a reaction flask, the reaction was stirred at room temperature in a glove box for 3 days, the reaction solution was taken out of the glove box, cold petroleum ether (45 mL) was added, after white flocculent precipitate was separated out, the solution was collected by centrifugation, then tetrahydrofuran (1 mL) was used for dissolution, and the solution was precipitated with cold petroleum ether, and the polymer with a protecting group on the side chain amino group was obtained after repeating the above steps three times. The molecular weight mn=3300, the molecular weight distribution pdi=1.14 of the polymer having a protecting group in the side chain was identified by Gel Permeation Chromatography (GPC). The polymer was drawn down, 2mL of a mixture of hydrobromic acid, acetic acid and trifluoroacetic acid (33:67:100) and 5% (v/v) triethylsilane were added, and the viscous liquid obtained by blowing off the excess liquid after shaking slightly at room temperature overnight was dissolved in 0.5mL of methanol, and 45mL of glacial methyl t-butyl ether was added to precipitate out a white precipitate, and the dissolution-precipitation process was repeated three times, to thereby obtain a random polymer with deprotected side chain amino groups. The subunit ratio of the polymer is obtained by nuclear magnetic resonance hydrogen spectrum characterization, and x in the final polymer: y=6: 4. the deprotected polymer was again dissolved in 5mL of ultrapure water and filtered and lyophilized for surface antimicrobial testing.

Example 9: preparation of random peptoid copolymer by triggering N-carbobenzoxy-amino ethyl-carboxyl cyclic anhydride and N-cyclohexyl-carboxyl cyclic anhydride with triphenylmercapto ethylamine

In a glove box protected by nitrogen, N-benzyloxycarbonyl-aminoethyl-carboxyanhydride and N-cyclohexyl-carboxyanhydride were weighed, 1.8mL of N-benzyloxycarbonyl-aminoethyl-carboxyanhydride (0.2M) and 0.2mL of N-cyclohexyl-carboxyanhydride (0.2M) were mixed in a reaction flask using dry N, N-dimethylformamide as a solvent, and then magnetons were added thereto and stirred. The initiator triphenylmercaptoethylamine was weighed and prepared into a solution (0.2M), 100. Mu.L of the solution was rapidly added into a reaction flask, the reaction was stirred at room temperature in a glove box for 3 days, the reaction solution was taken out of the glove box, cold petroleum ether (45 mL) was added, after white flocculent precipitate was separated out, the solution was collected by centrifugation, then tetrahydrofuran (1 mL) was used for dissolution, and the solution was precipitated with cold petroleum ether, and the polymer with a protecting group on the side chain amino group was obtained after repeating the above steps three times. The molecular weight mn=3100 and the molecular weight distribution pdi=1.14 of the polymer having a protecting group in the side chain were identified by Gel Permeation Chromatography (GPC). The polymer was drawn down, added with 2mL of a mixture of hydrobromic acid, acetic acid and trifluoroacetic acid (33:67:100) and 5% (v/v) triethylsilane, gently shaken at room temperature overnight and then excess trifluoroacetic acid was blown off, the resulting viscous liquid was dissolved in 0.5mL of methanol, and 45mL of ice-methyl t-butyl ether was added to precipitate a white precipitate, and the dissolution-precipitation process was repeated three times, thereby obtaining a random polymer with deprotected side chain amino groups. The subunit ratio of the polymer is obtained by nuclear magnetic resonance hydrogen spectrum characterization, and x in the final polymer: y=9: 1. the deprotected polymer was again dissolved in 5mL of ultrapure water and filtered and lyophilized for surface antimicrobial testing.

Example 10: preparation of beta-peptoid copolymer by initiating beta-N-p-fluorophenyl-N-carboxythiocarbonyl cyclic anhydride and beta-N-benzyloxycarbonyl-amino ethyl-N-carboxythiocarbonyl cyclic anhydride by p-tert-butylbenzylamine

beta-N-p-fluorophenyl-N-carboxythiocarbonyl anhydride and beta-N-benzyloxycarbonyl-aminoethyl-N-carboxythiocarbonyl anhydride were weighed, 1.6mL of beta-N-benzyloxycarbonyl-aminoethyl-N-carboxythiocarbonyl anhydride (0.2M) and 0.4mL of beta-N-p-fluorophenyl-N-carboxythiocarbonyl anhydride (0.2M) were mixed in a reaction flask using N, N-dimethylformamide as a solvent, and then a magneton was added thereto and stirred. The initiator p-tert-butyl benzylamine is weighed and prepared into a solution (0.2M), 100 mu L of the solution is quickly added into a reaction bottle, the reaction is stirred at room temperature in a glove box for 3 days, the reaction solution is taken out of the glove box, cold petroleum ether (45 mL) is added, centrifugal collection is carried out after white flocculent precipitate is separated out, tetrahydrofuran (1 mL) is used for dissolution, cold petroleum ether is used for precipitation, and the polymer with a side chain amino protecting group is obtained after repeating the steps for three times. The molecular weight mn=3500, the molecular weight distribution pdi=1.12 of the polymer having a protecting group in the side chain was identified by Gel Permeation Chromatography (GPC). The polymer which is pumped out is added with 2mL of mixed solution of hydrobromic acid, acetic acid and trifluoroacetic acid (33:67:100), after the mixed solution is slightly shaken for 2 hours at room temperature, the excessive trifluoroacetic acid is blown off, the obtained viscous liquid is dissolved in 0.5mL of methanol, 45mL of methyl tert-butyl ether with ice is added to separate out white precipitate, and the dissolving-precipitating process is repeated for three times, so that the random polymer with deprotected side chain amino is obtained. The subunit ratio of the polymer is obtained by nuclear magnetic resonance hydrogen spectrum characterization, and x in the final polymer: y=8: 2. the deprotected polymer was again dissolved in 5mL of ultrapure water and filtered and lyophilized for surface antimicrobial testing.

Preparation of oxazoline polymers

Example 11: preparation of oxazoline copolymer by triphenylmercapto 3-bromopropyl initiated N-epsilon-t-butoxycarbonyl-2- (aminopropyl) oxazoline and cyclohexyloxazoline

In a glove box protected by nitrogen, N-epsilon-t-butoxycarbonyl-2- (aminopropyl) oxazoline and cyclohexyloxazoline are weighed and dried N, N-dimethylacetamide is used as a solvent. 1.2mL of N- ε -t-butoxycarbonyl-2- (aminopropyl) oxazoline (0.2M) and 0.8mL of cyclohexyloxazoline (0.2M) were mixed in a reaction flask and stirred with a magnet. The triphenylmercapto 3-bromopropyl initiator is weighed to prepare a solution (0.2M), 0.1mL of the solution is quickly added into a reaction bottle, the reaction is stirred at 120 ℃ for 6 hours, cooled to room temperature, cold petroleum ether (45 mL) is added, centrifugal collection is carried out after white flocculent precipitate is separated out, tetrahydrofuran (1 mL) is used for dissolution, cold petroleum ether is used for precipitation, and the polymer with a side chain amino protecting group is obtained after three times of repetition. The molecular weight mn=3170, and the molecular weight distribution pdi=1.18 of the polymer was identified by Gel Permeation Chromatography (GPC). Then 2mL of trifluoroacetic acid and 5% (v/v) of triethylsilane are shaken overnight to remove protective groups in the polymer, most of the trifluoroacetic acid is blown off, white precipitate is separated out by adding ice methyl tertiary butyl ether (50 mL), the white precipitate is collected by centrifugation, methanol (1 mL) is used for dissolving, the ice ethyl ether (50 mL) is used for precipitating, after repeating the steps for three times, residual solvent is pumped out by an oil pump, the subunit proportion of the polymer is obtained by nuclear magnetic resonance hydrogen spectrum characterization, and finally x in the polymer is obtained: y=6: 4. and respectively dissolving the sample with ultrapure water (5 mL), and finally freeze-drying to obtain the deprotected oxazoline copolymer for surface antibacterial test.

Example 12: modification of polyurethane TPU substrate surface

The purchased polyurethane TPU was cut into flakes (circular, d=1 cm), and the TPU flakes were washed under ultrasound with 2% tween-20 (98% deionized water) for 15 minutes, followed by two washes with deionized water/absolute ethanol for 15 minutes each. Blow-drying with nitrogen. The surface of the cleaned TPU sheet is then activated using a plasma radiator. Specifically, the sheet was placed in a chamber and irradiated with a plasma irradiator at 70W on each side for 5 minutes at an oxygen flow rate (0.3-0.4 MPa). After irradiation, the pieces were immersed in a solution containing 1:10 bromoform: toluene (v/v) was placed in a functionalizing agent for 7-8 hours to functionalize the activated TPU surface with brominated groups. The bromoform-modified TPU flakes were washed with toluene, methylene chloride and ethanol. They were then stored in a vacuum vessel and dried overnight. The polymer prepared in example 11 (1 mg/mL, dissolved in 10% (V/V) glycerophosphate buffer solution) from example 1, example 2, example 3, example 4, example 5, example 6, example 7, example 8, example 9, example 7 was added dropwise to the dried bromoform-modified TPU surface, incubated for 9-10 hours, followed by 10. Mu.L of 10% (V/V) mercaptoglycerol phosphate buffer solution to block the excess reaction sites, and then the whole mixture was incubated at room temperature for 3-4 hours. And finally, flushing the surface of the substrate with deionized water and drying with nitrogen. The functional surfaces formed are named in table 1 below:

TABLE 1

The polymers are obtained from the examples	The polyurethane surface obtained
		Example 1	TPU-1
Example 2	TPU-2
		Example 3	TPU-3
Example 4	TPU-4
		Example 5	TPU-5
Example 6	TPU-6
		Example 7	TPU-7
Example 8	TPU-8
		Example 9	TPU-9
Example 11	TPU-10

Example 13: modification of glass surfaces

After the surface of the glass is alternately ultrasonically cleaned by the water and ethanol, the glass is respectively placed in 24 pore plates for standby after being dried by nitrogen. The dopamine hydrochloride was then dissolved in tris hydrochloride (ph=8.5, tris-HCl) solution to give 1mg/mL dopamine solution. 1mL of the solution was added to each well plate, the material surface was immersed, and the reaction was slowly shaken for 24 hours. After the reaction is finished, the base material is taken out, the surface of the material is cleaned for 3 times by ultra-pure water ultrasonic for 1min, and the material is dried for standby.

The substrate was then placed in a 5mL centrifuge tube, 1.2mL of polymer solution (prepared from example 1, example 2, example 10, example 11 (1 mg/mL) was immersed in the surface and heated at 60℃for 10 hours, the substrate was then removed, alternately rinsed with aqueous ethanol, and dried for use, the functional surface formed was named as Table 2 below:

TABLE 2

The polymers are obtained from the examples	The obtained glass surface
		Example 1	Glass-1
Example 2	Glass-2
		Example 10	Glass-3
Example 11	Glass-4

Example 14: modification of metal surfaces

After the surface of the titanium sheet or 316 stainless steel material is alternately ultrasonically cleaned by using water and ethanol, the titanium sheet or 316 stainless steel material is respectively placed in 24 pore plates for standby after being dried by nitrogen. The dopamine hydrochloride was then dissolved in tris hydrochloride (ph=8.5, tris-HCl) solution to give 1mg/mL dopamine solution. 1mL of the solution was added to each well plate, the material surface was immersed, and the reaction was slowly shaken for 24 hours. After the reaction is finished, the base material is taken out, the surface of the material is cleaned for 3 times by ultra-pure water ultrasonic for 1min, and the material is dried for standby.

The substrate was then placed in a 5mL centrifuge tube, 1.2mL of polymer solution (1 mg/mL) was added to submerge the surface, and heated at 60℃for 10 hours. Taking out the base material, alternately flushing with water and ethanol, and drying for later use.

The functional surfaces formed are named in table 3 below:

TABLE 3 Table 3

Example 15: modification of gelatin sponge surface

After the gelatin sponge is ultrasonically cleaned by ultrapure water and internal moisture is extruded, the gelatin sponge is respectively placed in 24 pore plates for standby after being dried by nitrogen. Dopamine hydrochloride was dissolved in tris hydrochloride (ph=8.5, tris-HCl) solution to give 1mg/mL dopamine solution. 1mL of the solution was added to each well plate, and a gelatin sponge holder (5 mm. Times.5 mm) was immersed in the above mixed solution, and the reaction was slowly shaken for 24 hours. After the reaction is finished, the base material is taken out, the surface of the material is cleaned by ultra-pure water for 1min, and the surface is extruded by forceps for multiple times and dried for standby.

The substrate was then placed in a 5mL centrifuge tube, 1.2mL of polymer solution (1 mg/mL) was added to submerge the surface, and heated at 60℃for 10 hours. The substrate was removed, rinsed alternately with aqueous ethanol and lyophilized overnight to give the desired surface as shown in table 4.

TABLE 4 Table 4

The polymers are obtained from the examples	The obtained sponge surface
		Example 2	Gelatin sponge-1

Example 16: polyether ether ketone (PEEK) surface modification

After the surface of the polyether-ether-ketone (PEEK) material is alternately ultrasonically cleaned by using water and ethanol, respectively placing the materials in 24 pore plates for standby after nitrogen blow-drying. The dopamine hydrochloride was then dissolved in tris hydrochloride (ph=8.5, tris-HCl) solution to give 1mg/mL dopamine solution. 1mL of the solution was added to each well plate, the material surface was immersed, and the reaction was slowly shaken for 24 hours. After the reaction is finished, the base material is taken out, the surface of the material is cleaned for 3 times by ultra-pure water ultrasonic for 1min, and the material is dried for standby.

The functional surfaces formed are named in table 5 below:

TABLE 5

The polymers are obtained from the examples	The PEEK surface obtained
		Example 1	PEEK-1
Example 2	PEEK-2
		Example 11	PEEK-3

Gold surface modification

The gold surface was prepared by dropping a polymer (1 mg/mL, compound of example 1) onto the surface of a gold sheet to form a thiol-gold bond, and after overnight, alternately rinsing the excess compound with deionized water and ethanol to obtain gold-1.

Example 17: graft characterization of modified substrates

The TPU-1 surface was characterized using ellipsometry, atomic Force Microscopy (AFM) and surface contact to demonstrate successful grafting of the polymer to the TPU surface.

1. Ellipsometry is used for characterizing the thickness of the polymer layer on the TPU surface, and the characterization result proves that the thickness of the polymer layer is 2.35+/-0.02 nm. The grafting density of the TPU-1 surface polymer is indirectly obtained to be 0.23chain/nm through the calculation of a formula sigma= (hpNA)/Mn ² 。

AFM was used to characterize TPU-1 surface roughness and surface morphology, as shown in FIG. 10, after polymer modification, TPU-1 surface roughness was 3.79nm, while unmodified TPU surface roughness was 3.23nm, the overall surface roughness did not change much, but from AFM images the surface morphology was found to change significantly, and the surface was seen to be uniform in morphology of the grafted polymer. This phenomenon can prove that the polymer has been successfully grafted onto the TPU surface.

3. The surface contact instrument was used to characterize the hydrophilicity and hydrophobicity of the TPU surface before and after modification. As shown in FIG. 11, the results show that the TPU surface contact angle before unmodified is 98℃and exhibits hydrophobic character, while the TPU-1 surface contact angle after polymer modification is greatly reduced, being only 36.9 ℃. This phenomenon demonstrates that the hydrophilicity of the TPU surface increases after the polymer grafting is successful.

Example 18: antibacterial ability test of substrate surface

Methicillin-resistant staphylococcus aureus s.aausa 300 and escherichia coli e.coli JM109 were represented as gram-positive and gram-negative strains, respectively. Candida albicans and cryptococcus neoformans are used as fungi.

The strain was cultured overnight in LB liquid medium at 37℃and 150rpm. Centrifuging the cultured microorganism at 4000r/min for 5min, and pouringThe supernatant was removed and the strain was diluted with calcium-magnesium ion free phosphate buffer solution at ph=7.0-7.4, the procedure was repeated three times, finally scaled with a microplate reader, and cell density was 5×10 according to OD value ⁵ The CFU/mL bacterial suspension was used as the working solution. The modified substrate surfaces were placed in 24 well plates, each surface being uniformly covered with 80 μl of bacterial working solution. After incubation at 37 ℃ for 2.5 hours, 1000 μl PBS was slowly added to each well, and the plate was sonicated for 3 minutes and vortexed for 2 minutes to ensure detachment of bacteria from the surface. The sterilization rate of the surface was calculated using the following formula. To obtain the colony count on the surface, 30. Mu.L of bacterial suspension was transferred from each well to an agar plate. (Prior to testing of the gelatin sponge group, the gelatin sponge was cut into small pieces with scissors, sonicated for 3 minutes, and homogenized with a homogenizer for 2 minutes

The plates were incubated overnight at 37 ℃. According to colony count (C) _{Experimental sample} ) The antibacterial ability of the substrate was judged, and bacterial suspensions that were not incubated with any surface were used as negative controls (C _{Negative control} ). At least three replicates were performed per experiment.

The antibacterial ability test was performed according to the above method on the blank substrate (the blank substrate was previously cleaned), the pretreated substrate, and the surfaces of the materials prepared in examples 12 to 17, respectively. The results of the antibacterial ability are shown in tables 6 to 7:

TABLE 6

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TABLE 7

Note that: the antibacterial rate of the surface of the material is required to be more than 90 percent, and the antibacterial effect can be reported only according with the national standard (JISZ 2901-2000).

Example 19: substrate surface hemolysis experiment

Taking the TPU-1 surface as an example, substrate surface hemolysis was verified. Fresh human blood was centrifuged at 1000rpm for 15 minutes and then washed 3 times with TBS buffer to obtain human red blood cells (hRBC). The hRBCs obtained were diluted to a 5% (v/v) suspension with TBS for use. mu.L of TBS was dropped onto the polymer-modified surface, and then 50. Mu.L of the above-mentioned 5% concentration red blood cell suspension was added to the surface. After incubation at 37℃for 1 hour, the suspension on the surface was collected and centrifuged at 3700rpm for 5 minutes. Then, 80. Mu.L of the suspension was added to each well of a 96-well plate, and OD values were collected at 405nm by a microplate reader. The negative control was 4 wells with 50. Mu.L TBS buffer and 50. Mu.L red blood cell suspension, and the positive control was 4 wells with Triton X-100 and red blood cell suspension. Wherein the polymer surface is designated A _sample The negative control is designated A _negative Positive control was designated A _positive . The percent hemolysis in each well was calculated using the following formula:

after hemolysis analysis, the morphology of hRBC on the TPU-1 surface was characterized using scanning electron microscope SEM. The surface was immersed in 2.5wt% glutaraldehyde at 4℃overnight. The sides were then gently rinsed 3 times with PBS and dehydrated using gradient ethanol (30%, 50%,70%,80%,90%,95%, 100%) for 5 minutes at each gradient. Finally, the samples were dried under nitrogen for SEM characterization.

As shown in FIG. 1, the polymer modified surface has low hemolytic activity, and the erythrocyte morphology is well ensured.

Example 20: substrate surface cytotoxicity experiment

Taking TPU-1 surface as an example, the cytotoxicity of the substrate surface was verifiedSex. Two types of mammalian cells, human umbilical vein endothelial cells (HUVEC ATCC PCS-100-010) and NIH-3T3 fibroblasts (3T 3 ATCC CRL-1658) were cultured in TCPS dishes using DMEM (10% FBS,100U/mL penicillin, 100. Mu.g/mL streptomycin and 2mM L-glutamine) at 37℃with 5% CO ₂ Culturing under atmosphere. When the coverage of cells in the dishes reached about 80-90%, the cells were isolated from the dishes, collected and diluted in DMEM to a final concentration of 8×10 ⁴ cells/mL. The TPU-1 surface was placed in a 24-well plate and each well was thoroughly submerged with 1mL of cell suspension. The unmodified TPU surface was used for comparison in the same panel. After incubation of the plates for 24 hours at 37 ℃, the cells were incubated with 20 μl 1:1 (Acridine Orange/Ethidium Bromide staining kit, AO/EB) and incubated for a further 10 minutes in the absence of light, the living cells fluoresce green and the dead cells fluoresce red. After removing the staining solution and washing the surface with 500 μlpbs, the stained cells were observed using a fluorescence microscope.

The toxicity of the extract obtained from the TPU-1 surface was also evaluated using the elution test method. The TPU-1 surface was immersed in 1mL of DMEM medium at 37℃for 24 hours to obtain an extract. The extract was added to a 96-well plate of HUVEC and NIH-3T3 cells at 37℃at 8X 10 ⁴ Cell density of cells/well was pre-cultured for 24h. Then 10 μLMTT solution (5 mg/mL) was added to each well and the plates incubated for 4 hours in the absence of light. After removal of the solution in each well, 150 μldmso was added and the plate incubated at 37 ℃ for an additional 15 minutes. Finally, the plate was shaken for 10 minutes to dissolve the purple MTT-carboxamide crystals and the OD was recorded at 570nm using a microplate reader. The following formula was used to calculate cell viability. OD with polymer surface overflow therein ₅₇₀ Is denoted as A _test Only DMEM medium was added to 4 wells of the same 96-well plate as a blank, designated A _blank DMEM medium and cells (without polymer surface overflow) were added to 4 wells as positive controls, designated a _control . The percent cell survival per well was calculated using the following formula:

as a result, as shown in FIG. 2, cells were normally adhered and spread on the surface of the polymer-modified TPU, and no significant cytotoxicity was found on the surface. In addition, no significant cytotoxicity was seen on the surface of the other modified materials of the present invention.

Example 21: in vivo antibacterial Activity test

The experimental animals all follow the ethical standard of Shanghai public health clinical center animals in the feeding and experimental operation process. 1% sodium pentobarbital (50 mg/kg) was used preoperatively to anesthetize disease-free Sprague-Dawley (SD) rats (female, 200-250g,8-10 weeks) by intraperitoneal injection. Excess hair on the back of the rat was shaved off with a pet razor and the back of the rat was applied with depilatory cream to remove residual hair. The back was then rubbed with saline and finally sterilized with alcohol. Both TPU-1 and bare TPU pieces were incubated with MRSA (S.aureusiUSA300 LAC) 5X 105CFU/mL for 2.5h at 37℃and then implanted into the left and right sides of the back of the same rat, respectively. TPU-1 with bacterial liquid on the surface is implanted along the opening on the back side of the rat along the openings on two sides of the midline on the back of the rat, and then the opening on the back of the rat is sutured. After 1, 3 and 7 days of implantation, taking out TPU and TPU-1 along the back opening, placing the TPU and TPU-1 into a centrifuge tube containing normal saline, shearing an upper layer tissue contacted with TPU and TPU-1 bacterial liquid into the centrifuge tube, adding dissociation liquid into one part to perform homogenization and centrifugation treatment on the tissue, and performing plating culture on the supernatant to count bacterial plates; another aliquot of the bacterial solution contacted upper tissue was cut and placed in paraformaldehyde for staining with HE (matrixin-eosin staining) and gram bacteria for histological section staining analysis.

Colony count comparison results as shown in fig. 3-5, the polymer modified antimicrobial surface was able to effectively and consistently sterilize. HE staining found that the polymer modified surface had good in vivo compatibility with low inflammatory response. Gram bacteria staining found that the surrounding tissue bacteria of the polymer modified surface were reduced.

Example 22: surface antibacterial mechanism test

When the polymer is immobilized on the surface, the polymer molecules no longer have free ability and the antimicrobial pattern/mechanism of the polymer may be changed accordingly. It is therefore necessary to perform certain tests on antimicrobial surfaces to elucidate the possible bactericidal modes/mechanisms of polymer modification of the surface.

1. And observing the morphology of the bacteria after the bacteria react with the surface by using a scanning electron microscope. And (3) similar to the observation of red blood cells after bacteria are inoculated on the surface, paraformaldehyde fixing is carried out, and the appearance of bacteria is observed after gradual dehydration.

As a result, in the SEM of FIG. 6, the surface of the cell membrane of the gram-negative bacteria E.coli and the surface of the cell membrane of the gram-positive bacteria MRSA after 2.5 hours of surface culture of the unmodified TPU are smooth and complete, and the cell membrane surface of the gram-negative bacteria E.coli and the cell membrane of the gram-positive bacteria MRSA after the TPU-P culture are wrinkled and damaged, and obvious abnormal bacterial morphology is shown. This indicates that contact sterilization is performed after the TPU-P surface is contacted with E.coli and MRSA.

Plasma membrane permeability assay of TPU-1 surface bacterial suspensions were diluted to a density of 108CFU/mL using the following protocol, and then 10. Mu.L of 1-naphthylaminobenzene fluorescent probe (NPN, 2.1929mg/mL in DMSO) was added to 10mL of bacterial suspension to obtain working solution. The TPU-1 surfaces were placed in a 24-well plate and each surface was uniformly covered with 80. Mu.L of working solution. After incubation at 37 ℃ for 0.5, 1.5 and 2.5 hours, 200 μl of N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid (HEPES) buffer was slowly added to each well, and the plate was then sonicated for 3 minutes and vortexed for 2 minutes to ensure detachment of the bacteria from the surface. mu.L of the solution was transferred into a 96-well plate to record the fluorescence intensity of each well at 350nm excitation and 420nm emission.

NPN is a hydrophobic fluorescent dye, and when the permeability of the cell outer membrane is damaged, the NPN dye can be combined at the damage position of the cell outer membrane to emit fluorescence. As a result, as shown in fig. 7, the polymer modified surface showed a significant increase in fluorescence intensity after sterilization by contact with e.coli, and continued to maintain this trend until the end of the experiment, while the unmodified surface and the control (viable bacteria) did not have a significant change in fluorescence intensity. The results indicate that the outer membrane permeability of the bacteria is changed after surface contact sterilization.

3. Conductivity testing of the surface the change in conductivity in the solution caused by release of bacterial cytoplasmic material during the surface contact sterilization of TPU-1 was evaluated. Coli and MRSA were diluted to working solutions with cell densities of 5X 105CFU/mL, respectively. The TPU-1 surfaces were placed in a 24-well plate and each surface was uniformly covered with 80. Mu.L of working solution. After incubation at 37 ℃ for 0.5, 1, 1.5, 2 and 2.5h, 2420 μl of PBS was slowly added to each well, and the plate was sonicated for 3 min and vortexed for 2 min. Then, 2500. Mu.L of the solution per well was transferred to a centrifuge tube (15 mL) and the conductivity of each tube was measured using a conductivity meter.

As a result, as shown in FIG. 8, for the gram-negative bacteria E.coli, the conductivity of the TPU-1 surface contact sterilized bacterial suspension increased with time, reached the highest value at 2h, and stabilized around 2.34. Mu.s/cm thereafter; the conductivity of the TPU bacterial suspension is always maintained at about 1.36 mu s/cm, and the TPU bacterial suspension shows lower conductivity. For gram positive bacteria MRSA, the conductivity of the bacteria suspension after the surface contact sterilization of TPU-1 is increased continuously along with the increase of time, reaches the highest value at 1.5h, and is stabilized at about 2.44 mu s/cm after that; the conductivity of the TPU bacterial suspension was maintained at about 1.43 mu s/cm at all times, exhibiting lower conductivity. This suggests that the TPU-P surface contact sterilization mechanism is sterilization by disrupting the cell membrane.

Taken together, the results indicate that the polymer surface kills bacteria by disrupting the bacterial membrane after contact.

Example 23: solution antibacterial ability and solution toxicity of polymer

The polymer obtained in example 2 was tested for antimicrobial activity and solution toxicity:

minimum Inhibitory Concentration (MIC) in solution was tested by diluting the bacterial solution to 2X 10 with MH medium ⁵ cfu/mL is reserved. The polymer was diluted in 96-well plates with MH medium at a concentration ranging from 400 to 3.13. Mu.g/mL. Then, 50. Mu.L of diluted bacterial liquid was added to each well to make the total volume of the bacterial liquid and the polymer 100. Mu.L, and the mixture was gently shaken for 10 seconds and allowed to stand for 9 hours in a mold incubator at 37 ℃. Then the OD600 is read by an enzyme-labeled instrument, only MH culture medium is added into 4 holes in the same 96-well plate to serve as negative control, and the MH culture medium and bacteria are added into 4 holesThe liquid (without polymer) served as a positive control. Two replicates were tested at a time and repeated twice at different times. The percentage of bacterial growth per well uses the formula:and (5) calculating. The calculated data is then plotted and the MIC value is the lowest concentration of polymer that inhibits bacterial growth.

The result was that the MIC of the polymer of example 2 for Staphylococcus aureus was 50. Mu.g/mL in the solution state.

And (5) testing hemolytic toxicity. The polymer was diluted stepwise with twice the TBS buffer in 96-well plates to give a polymer concentration in the range of 400 to 3.13. Mu.g/mL. Then 50. Mu.L of the diluted red blood cell suspension was added to each well, and the mixture was gently shaken for 10 seconds and allowed to stand for 1 hour in a mold incubator at 37 ℃. The wells were placed in a centrifuge for centrifugation (3700 rpm,5 min) and 80 μl of supernatant from each well was transferred to another 96 well plate (each time the gun head was replaced). If bubbles appear after transfer, the bubbles are required to be pricked by using toothpick to be sticky to isooctanol, then an enzyme-labeled instrument is used for reading OD405, 4 holes in the same 96-well plate are added with TBS buffer solution and red blood cell suspension liquid to serve as negative controlTriton X-100 and red blood cell suspension were added to 4 wells as positive controls +.>Two replicates were tested at a time and repeated twice at different times. The percent hemolysis per well was calculated using the formula:and (5) calculating. The calculated data is then plotted as the lowest concentration of polymer to lyse 50% of the red blood cells.

The result is that the polymer of example 2 is a lysotoxic HC in solution ₅₀ 6.25 μg/mL.

However, according to the results of TPU-2 in Table 6, the polymer of example 2 has a surface sterilization rate of 90% or more for both gram-positive and gram-negative bacteria. In addition, the thermoplastic polyurethane has excellent compatibility to blood cells, and the blood cells have no adverse reaction on the surface of TPU-2; and has no adverse reaction to human umbilical vein endothelial cells (HUVEC ATCC PCS-100-010) and NIH-3T3 fibroblasts (3T 3 ATCC CRL-1658) (see FIG. 9).

In summary, the solution sterilization selectivity (HC 50/MIC) of the polymer of example 2 is 0.125, which has strong toxicity, that is, the compound of the present application can cause serious damage to normal cells in the concentration of killing bacteria in the solution state, which severely limits the application of the compound in vivo sterilization, and the inventors surprisingly found that when the compound is used as a modification on the surface of a substrate to improve the antibacterial capability of the surface, the biocompatibility of the surface of the obtained substrate is significantly improved (not only no extra cytotoxicity is brought, but also the cytotoxicity is lower than that of a blank substrate before modification) under the condition that the sterilization rate of more than 90% is achieved, so that the compound is very suitable for being used as a surface modification material for surface modification of a biomedical material, and the application of the compound is significantly expanded.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. A method for improving antimicrobial activity and biocompatibility of a substrate surface, comprising the steps of:

(a) Providing at least a portion of a surface of the substrate,

in the formula I, R ₁ is-L ₁ R _a Wherein L is ₁ Selected from substituted or unsubstituted C1-C8 alkylene; r is R _a Selected from the group consisting of: -NR _b R _b Guanidino, biguanide, -N (R) _b ) ₃ ⁺ ；

R ₂ 、R ₃ And R is ₆ Each independently is H;

and R is ₄ And R is ₅ Independently selected from the group consisting of: H. substituted or unsubstituted-C1-C6 alkyl, substituted or unsubstituted-C3-C8 cycloalkyl, substituted or unsubstituted-C1-C3 alkyl-COO-benzyl, substituted or unsubstituted phenyl, and R ₄ And R is ₅ At least one is other than H;

or alternatively, the process may be performed,

In the formula I, R ₃ is-L ₁ R _a Wherein L is ₁ Selected from substituted or unsubstituted C1-C8 alkylene; r is R _a Selected from the group consisting of: -NR _b R _b Guanidino, biguanide, -N (R) _b ) ₃ ⁺ The method comprises the steps of carrying out a first treatment on the surface of the And R is ₆ Selected from C3-C12 cycloalkyl;

R ₁ and R is ₂ Each independently is H;

R ₄ and R is ₅ Each independently selected from the group consisting of: H. a substituted or unsubstituted C1-C15 alkyl group;

x is 5-100; and x+y=100; and is also provided with

n is a positive integer from 1 to 100; or alternatively

In formula II, R ₇ 、R ₈ 、R ₉ 、R ₁₀ 、R ₁₂ 、R ₁₃ 、R ₁₄ And R is ₁₅ Each independently of the otherGround is selected from H;

R ₁₁ is-L ₁ R _a Wherein L is ₁ Selected from substituted or unsubstituted C1-C8 alkylene; r is R _a Selected from the group consisting of: -NR _b R _b Guanidino, biguanide, or-N (R) _b ) ₃ ⁺ The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

R ₁₆ Selected from the group consisting of: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted-C3-C8 cycloalkyl, substituted or unsubstituted-C1-C6 alkyl-C3-C8 cycloalkyl;

x is 5-100; and x+y=100; and is also provided with

n is a positive integer from 1 to 100;

in formula III, R ₁₇ is-L ₁ R _a Wherein L is ₁ Selected from substituted or unsubstituted C1-C8 alkylene; r is R _a Selected from the group consisting of: -NR _b R _b Guanidino, biguanide or-N (R) _b ) ₃ ⁺ ；

R ₁₈ Selected from substituted or unsubstituted C1-C15 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, substituted or unsubstituted benzyl or substituted or unsubstituted phenyl;

x is 5-100; and x+y=100; and is also provided with

n is a positive integer from 1 to 100;

wherein, in the formula I, the formula II and the formula III, each R _b Each independently selected from hydrogen; and is also provided with

In the formulae, the substitution means that one or more H on each group is independently substituted with a group selected from the group consisting of: deuterium, halogen, -SH, -COOH, -OH, -NH ₂ ；

And the end of the compound attached to the surface comprises an end group having a reactive group selected from the group consisting of: -SH, -NH ₂ -COOH, -Br, -Cl, -OH, epoxy, alkynyl, -COCl, azido, maleimide, o-dithiopyridyl (OPSS); wherein the reactive groups are configured to react with the reactive groups of the pre-activated substrate surface to thereby attach the polymer segment to the substrate surface;

the other end of the compound is H;

the bacteria are selected from the group consisting of: gram positive bacteria, gram negative bacteria, fungi, spores, resting cells;

the average grafting density of the compound on at least one part of the surface of the substrate is more than or equal to 0.2chain/nm ² 。

2. The method of claim 1, wherein in each formula, -L ₁ R _a independently-C2-C6 alkyl-R _a 。

3. The method of claim 1, wherein the compound has a polymer segment of formula I:

in the formula I, R ₁ is-L ₁ R _a Wherein L is ₁ Selected from C1-C6 alkylene; r is R _a Selected from the group consisting of: -NR _b R _b Guanidino, biguanide, -N (R) _b ) ₃ ⁺ ；

R ₂ 、R ₃ And R is ₆ Each independently is H;

and R is ₄ And R is ₅ Independently selected from the group consisting of: H. -C1-C6 alkyl, -C3-C8 cycloalkyl, -C1-C3 alkyl-COO-benzyl, and R ₄ And R is ₅ At least one is other than H;

or alternatively, the process may be performed,

in the formula I, R ₃ is-L ₁ R _a Wherein L is ₁ Selected from C1-C6 alkylene; r is R _a Selected from the group consisting of: -NR _b R _b Guanidino, biguanide, -N (R) _b ) ₃ ⁺ The method comprises the steps of carrying out a first treatment on the surface of the And R is ₆ Selected from C3-C12 cycloalkyl;

R ₁ 、R ₂ 、R ₄ and R is ₅ Each independently is H.

4. The method of claim 1, wherein R _a Selected from the group consisting of: -NH ₂ Guanidino groupBiguanide, -NH ₃ ⁺ 。

5. The method of claim 1, wherein x is independently 10 to 100 in each formula.

6. The method of claim 1, wherein the substrate surface is selected from the group consisting of: bioceramics, bioglass, graphene, bone cement and medical carbon materials, stainless steel, cobalt-based and titanium-based alloys, shape memory alloys, silver, tantalum, niobium, zirconium, palladium, platinum, hyaluronic acid, chitosan, alginic acid, cellulose, collagen, gelatin, resins, or combinations thereof.

7. The method of claim 6, wherein the resin is selected from the group consisting of: polyether ether ketone, polycaprolactone, polylactic acid, polycarbonate, polyurethane, polyester, polyanhydride, polydimethylsiloxane, polymethyl methacrylate, polyphosphazene, polyamide, polyethylene, polypropylene, polytetrafluoroethylene, or combinations thereof.

8. The method of claim 7 wherein the polyester is polyethylene terephthalate.

9. The method of claim 1, wherein the compound comprises a segment selected from the group consisting of:

10. The method of claim 1, wherein one end of the compound comprises an end group having a reactive group selected from the group consisting of: -SH, -NH ₂ 、-COOH。

11. The method of claim 1, wherein the compound comprises a segment selected from the group consisting of:

12. The method of claim 1, wherein said compound is selected from the group consisting of:

13. A substrate having improved antimicrobial activity and biocompatibility over at least a portion of the surface prepared by the method of claim 1.

14. Use of a substrate according to claim 13 for the preparation of a product with improved antibacterial activity and biocompatibility.