NL2031339B1

NL2031339B1 - Antibacterial coating formed by copolymerization of chlorhexidine and catecholamine as well as preparation method and application thereof

Info

Publication number: NL2031339B1
Application number: NL2031339A
Authority: NL
Inventors: Li Xiangyang
Original assignee: Univ Anhui Medical
Priority date: 2021-03-23
Filing date: 2022-03-21
Publication date: 2023-01-31
Also published as: CN113244436A; CN113244436B; NL2031339A

Abstract

Disclosed are an antibacterial coating formed by copolymerization of chlorheXidine and catecholamine as well as a preparation method and an application thereof. The antibacterial coating is formed in a process that chlorheXidine and catecholamine are self—assembled to form nanoparticles and the nanoparticles adhere to surfaces of various substrates to form a compact polymerized coating. Compared with chlorhexidine—containing antibacterial materials disclosed in the prior art, the antibacterial coating prepared in the present invention has the same antibacterial function. To solve the technical problem that the antibacterial materials in the prior art are of toxicity and poor biocompatibility, the cytotoxicity is remarkably' improved kn! selecting a. catecholamine substance as a substrate. Meanwhile, the antibacterial coating is high in stability and still has a certain antibacterial effect after being 15 immersed in a simulated body fluid for several days. The antibacterial coating features simple process, easily controlled reaction conditions and low cost.

Description

P1287 /NL

ANTIBACTERIAL COATING FORMED BY COPOLYMERIZATION OF CHLORHEXIDINE

AND CATECHCLAMINE AS WELL AS PREPARATION METHOD AND APPLICATION

THEREOF

TECHNICAL FIELD

The present invention belongs to the technical field of antibacterial coatings, and particularly relates to an antibacterial coating formed by copolymerization of chlorhexidine and catecholamine as well as a preparation method and an application thereof.

BACKGROUND ART

Biologically safe materials capable of being antibacterial stably are one of the most desired materials all the time in the field of biomedical instruments. Realization of being stably antibacterial on the surface of the material can effectively reduce occurrence of infections on the surfaces of medical instruments and the usage amount of antibiotics, so that the service life of the instruments is prolonged greatly. At present, there are certain shortcomings in major modification modes of the antibacterial surface, for example, higher cytotoxicity, narrow antibacterial spectrum, shorter antibacterial time, unstable coating and the like.

Chlorhexidine is a cationic surfactant with considerably high broad spectrum bacteriostatic and bactericidal effects. It is a preferable sterilizing and disinfecting agent with an antibacterial effect to gram positive bacteria and gram negative bacteria. The sterilizing effect of chlorhexidine is mainly originated from its strong electropositivity. The chlorhexidine can be absorbed to osmotic barriers of bacterial serolemma, so that cellular contents are leaked out. The chlorhexidine shows the bacteriostatic action in a low concentration and a sterilizing effect in a high concentration. However, chlorhexidine in the sterilizing concentration has a certain cytotoxicity and a shorter adsorption time, usually about 24 hours, in a material and in vivo, so that in the prior art, its cytocompatibility and sustained stability cannot be improved effectively.

Catecholamine, a class of organic matters with one or more phenolic hydroxyl groups, is characterized by forming a material- independent coating via oxidative polymerization, metal ion coordination and polymerization of amido-containing organic matters. At present, catecholamine has already been one of consti- tuting monomers of material-independent coatings widely applied in the field of material science. Most catecholamines which are electronegative and contain a lot of phenolic hydroxyl groups can be stably bound with chlorhexidine with electropositivity via electric charge effect and covalent effect. Meanwhile, phenols can be adsorbed to various substrates in a material-independent manner and can fix a chlorhexidine and catechol polymer in a material- independent manner, thereby accomplishing surface modification of various materials.

In the present invention, an antibacterial coating is formed via a self-assembling property of chlorhexidine and catecholamine and self-polymerization of catecholamine. It is found by the applicant surprisingly that besides the excellent antibacterial effect, the formed antibacterial coating does not have obvious cytotoxicity, which is because the integral surface level is re- duced as the electronegative catecholamine and the electropositive chlorhexidine are neutralized, so that the integral cytotoxicity is reduced. At the same time, the formed nanoparticles have a certain chlorhexidine releasing ability, thereby playing an in situ antibacterial action.

SUMMARY

The present invention is intended to provide an antibacterial coating formed by copolymerization of chlorhexidine and catecholamine as well as a preparation method and an application thereof. The antibacterial coating can be used for surface modification of biomedical materials, and solves the problems that existing modified antibacterial surface cannot be continuously and stably antibacterial and is poor in biocompatibility, thereby providing a new choice for an antibacterial material on the surface of a material.

In order to solve the above-mentioned technical problems, the present invention is realized via the following technical solution:

The present invention is an antibacterial coating formed by copolymerization of chleorhexidine and catecholamine, wherein the antibacterial coating is formed in a process that chlorhexidine and catecholamine are self-assembled to form nanoparticles and the nanoparticles adhere to surfaces of various substrates to form a compact polymerized coating.

Further, a preparation method of the antibacterial coating includes the following steps:

Sl: preparing a catecholamine aqueous solution of a certain concentration and a chlorhexidine solution of a certain concentration;

S2: slowly dropping the chlorhexidine solution prepared in S1 into the catecholamine aqueous solution, and allowing catecholamine and chlorhexidine to be fully polymerized; and

S3: leaving the mixture still for 24 hours to deposit a chlorhexidine and catecholamine polymerized coating on a substrate.

Further, the catecholamine is dopamine, noradrenaline, tannic acid, gallic acid, brown algae polyphenol or epigallocatechin gallate.

Further, the catecholamine is preferably brown algae polyphenol.

Further, the concentration of catecholamine is crucial for polymerization of the nanoparticles and has a crucial impact on forming of the coating. Catecholamines of different concentrations have different electric properties and contents of phenolic hydroxyl groups, so that catecholamines will affect polymerization of the nanoparticles and the coating. A concentration range of catecholamine is 0.05-10 mg/mL.

Further, screened by a gradient concentration experiment, the concentration of catecholamine is preferably 0.05-2 mg/mL.

Further, the concentration of chlorhexidine is crucial for polymerization of the nanoparticles and further has a crucial im-

pact on forming of the coating. Chlorhexidine of different concentrations is different in antibacterial ability, quantity of electric charges and quantity of functional groups, so that chlorhexidine will affect function and polymerization of the nanoparticles and the coating. A concentration range of chlorhexidine is 0.05-10 mg/mL.

Further, screened by a gradient concentration experiment, the concentration of chlorhexidine is preferably 0.05-1.6 mg/mL.

Further, in S1, a concentration of the brown algae polyphenol aqueous solution is 0.25 mg/mL; and a concentration of the chlorhexidine solution is 0.8 mg/mL.

Further, a pH value of a polymerization process in S2 is 8.5.

Further, the antibacterial coating is applied to prepare antibacterial applications, antibacterial bone materials and antibacterial dental materials.

Further, the antibacterial coating can be deposited on the surface of a bone filler or a bone implant, so that its biocompatibility is improved. Moreover, the antibacterial coating of the application has good stability, and is still kept intact without exposing the substrate when the bone filler or the bone implant deforms.

Further, the nanoparticles can be obtained by freeze drying of the reaction solution in S1, and can further be blended into various hydrogels to prepare hydrogel products with an antibacterial effect.,

Further, the method for preparing the antibacterial coating can be further adjusted as follows: placing a sample in the reaction solution, and volatilizing the solution by means of drying to obtain the antibacterial material with the surface adsorbing the antibacterial nanoparticles.

Further, the hydrogel product can be used for an antibacterial application or a contact lens lining.

The present invention has the following beneficial effects: 1. Compared with chlorhexidine-containing antibacterial materials disclosed in the prior art, the antibacterial coating prepared in the present invention has the same antibacterial function. In addition, to solve the technical problem that the antibacterial materials in the prior art are of toxicity and poor biocompatibility, the cytotoxicity is remarkably improved by selecting a catecholamine substance as a substrate, in particular for a coating formed by polymerization of brown algae polyphenol 5 and chlorhexidine. It has a certain promoting effect on cell proliferation. Meanwhile, the antibacterial coating is high in stability and still has a certain antibacterial effect after being immersed in a simulated body fluid for several days. 2. The antibacterial coating is material-independent, namely, the antibacterial coating can be adhered to and polymerized on the surfaces of different materials including titanium, stainless steel, silicon and glass. 3. The method for preparing the antibacterial coating provided by the present invention features simple process, easily controlled reaction conditions and low cost.

Of course, all the above-mentioned advantages are not necessarily achieved at the same time to implement any product of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

In order to describe the technical solution of the embodiments of the present invention more clearly, the accompany- ing drawings required to describe the embodiments will be intro- duced briefly below. It is apparent that the accompanying drawings described below are merely some embodiments of the present invention, and those of ordinary skill in the art further can obtain other drawings according to those accompanying drawings without making creative efforts.

FIG. 1 is a statistical graph of varying results of a surface contact angle before and after modification of a titanium surface by the antibacterial coating.

FIG. 2 is a flat pattern of SEM results of a titanium surface before and after modification of the titanium surface by the antibacterial coating.

FIG. 3 is x-ray photoelectron spectra before and after polymerization of chlorhexidine and catecholamine.

FIG. 4 is a flat pattern of an inhibition zone of staphylococcus aureus around a chlorhexidine and catecholamine modified sample.

FIG. 5 is a flat pattern of an inhibition zone around after compounding of various phenols and chlorhexidine.

FIG. 6 is a statistical graph of absorbance value results of a bacterial solution obtained by co-culture of chlorhexidine and catecholamine (different concentrations) modified samples and staphylococcus aureus.

FIG. 7 is a statistical graph of cytotoxicity assay results of different chlorhexidine and catecholamine modified samples.

FIG. 8 is a flat pattern of results of dying cytoskeletons and cell nuclei (phalloidin/DAPI) after surface cells of different chlorhexidine and catecholamine modified samples are cultured for one day.

FIG. 9 is a flatten pattern of Rhodamine 123 dyeing results after surface cells of different chlorhexidine and catecholamine modified samples are co-cultured for one day and three days.

In the drawings, control is titanium, and numbers such as 0, 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 are the concentrations (mg/mL) of chlorhexidine and catecholamine.

According to the drawings, the following conclusions can be drawn:

It can be known from FIG. 1 that the contact angle of the titanium surface changes remarkably after the titanium surface is modified, so that success of modification is proved to a certain extent.

It is shown by the results in FIG. 2 that the chlorhexidine and catecholamine polymer can be polymerized in form of nanoparticles, and the chlorhexidine and catecholamine coating can be formed as a lot of nanoparticles are gathered.

It is shown by the results in FIG. 3 that a characteristic peak of the titanium element of the substrate is covered after chlorhexidine is introduced, which indicates that chlorhexidine and catecholamine can be polymerized on the titanium surface to form an intact coating.

It is shown in FIG. 4 that the chlorhexidine and catecholamine coating has the antibacterial action, and its antibacterial action is enhanced increasingly with increase of a feeding concentration of chlorhexidine. Appearance of the inhibition zone further shows that chlorhexidine on the surface of the coating has a better diffusing ability.

It is shown by the results in FIG. 5 that various phenols can be polymerized with chlorhexidine on the titanium surface to form the coating, and have effective diffusible antibacterial abilities.

It is shown by the results in FIG. 6 that the chlorhexidine and catecholamine (different concentrations) coatings have higher in situ antibacterial abilities.

It is shown in FIG. 7 that via MTT detection, all the coatings do not have cytotoxicity, wherein the low-concentration chlorhexidine modified sample has a certain cell promoting effect.

It is shown by the results in FIG. 8 that the chlorhexidine and catecholamine coating has no obvious impact on proliferation of cells and spreading of cytoskeletons, without obvious cytotoxicity.

It is shown by the results in FIG. 9 that in a cellular bacterial co-culture environment, the chlorhexidine and catecholamine modified sample can inhibit bacteria effectively and does not have cytotoxicity.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the accompanying drawings in the embodiments of the present invention. The described embodiments are merely a part of, rather than all of, the embodiments of the present invention.

Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

Example 1 (Preparation of a brown algae polyphenol and chlorhexidine composite antibacterial coating) 1. A matrix cleaning step: a polished matrix was fed into an ultrasonic cleaner first, the matrix was respectively cleaned with

15-30 kHz ultrasonic waves in acetone and anhydrous alcohol in sequence for 5-10 min, and dried. 2. Brown algae polyphenol and chlorhexidine were respectively dissolved in a Tris solution with pH=8.5, a final concentration of the brown algae polyphenol in the solution was kept at 0.5 mg/mL, and then a chlorhexidine solution was added, wherein the chlorhexidine concentrations were respectively 0 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.4 mg/mL, 0.8 mg/mL and 1.6 mg/mL.

Then the sclution was added onto the surface of the sample and left still for 24 hours. 3. A sample cleaning step: the sample was placed in RO water, subjected to ultrasonic treatment separated from water, respectively cleaned with 15-30 kHz ultrasonic waves for 5-10 min and then dried in cold air.

Example 2 (Preparation of brown algae polyphenol and chlorhexidine composite nanoparticles) 1. Brown algae polyphenol and chlorhexidine were respectively dissolved in a Tris solution with pH=8.5, a final concentration of the brown algae polyphenol in the solution was kept at 0.5 mg/mL, and then a chlorhexidine solution was added, wherein the chlorhexidine concentrations were respectively 0 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.4 mg/mL, 0.8 mg/mL and 1.6 mg/mL.

Then the solution was added onto the surface of the sample and left still for 24 hours. 2. The solution was centrifugalized by a 3,000g centrifugal force for 10 min to obtain the nancparticles suspended in the liquid.

Example 3 (Preparation of a dopamine and chlorhexidine composite antibacterial coating) 1. A matrix cleaning step: a polished matrix was fed into an ultrasonic cleaner first, the matrix was respectively cleaned with 15-30 kHz ultrasonic waves in acetone and anhydrous alcohol in sequence for 5-10 min, and dried. 2. Dopamine and chlorhexidine were respectively dissolved in a Tris solution with pH=8.5, a final concentration of the dopamine in the solution was kept at 0.5 mg/mL, and then a chlorhexidine solution was added, wherein the chlorhexidine concentrations were respectively 0 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.4 mg/mL, 0.8 mg/mL and 1.6 mg/mL. Then the solution was added onto the surface of the sample and left still for 24 hours. 3. A sample cleaning step: the sample was placed in RO water, subjected to ultrasonic treatment separated from water, respectively cleaned with 15-30 kHz ultrasonic waves for 5-10 min and then dried in cold air.

Example 4 (Preparation of a norepinephrine and chlorhexidine composite antibacterial coating) 1. A matrix cleaning step: a polished matrix was fed into an ultrasonic cleaner first, the matrix was respectively cleaned with 15-30 kHz ultrasonic waves in acetone and anhydrous alcohol in sequence for 5-10 min, and dried. 2. Norepinephrine and chlorhexidine were respectively dissolved in a Tris solution with pH=8.5, a final concentration of the norepinephrine in the solution was kept at 0.5 mg/mL, and then a chlorhexidine solution was added, wherein the chlorhexidine concentrations were respectively 0 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.4 mg/mL, 0.8 mg/mL and 1.6 mg/mL. Then the solution was added onto the surface of the sample and left still for 24 hours. 3. A sample cleaning step: the sample was placed in RO water, subjected to ultrasonic treatment separated from water, respectively cleaned with 15-30 kHz ultrasonic waves for 5-10 min and then dried in cold air.

Example 5 (Preparation of a tannic acid and chlorhexidine composite antibacterial coating) 1. A matrix cleaning step: a polished matrix was fed into an ultrasonic cleaner first, the matrix was respectively cleaned with 15-30 kHz ultrasonic waves in acetone and anhydrous alcohol in sequence for 5-10 min, and dried. 2. Tannic acid and chlorhexidine were respectively dissolved in a Tris solution with pH=8.5, a final concentration of the tannic acid in the solution was kept at 0.5 mg/mL, and then a chlorhexidine solution was added, wherein the chlorhexidine concentrations were respectively 0 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.4 mg/mL, 0.8 mg/mL and 1.6 mg/mL. Then the solution was added onto the surface of the sample and left still for 24 hours. 3. A sample cleaning step: the sample was placed in RO water, subjected to ultrasonic treatment separated from water, respectively cleaned with 15-30 kHz ultrasonic waves for 5-10 min and then dried in cold air.

In the description of the specification, the description with reference to the terms “an embodiment”, “examples”, “specific examples” and the like means that specific features, structures, materials, or features described in connection with the embodiments or examples are included in at least one embodiment or example of the present invention. In the description, schematic expressions of the terms may not refer to the same embodiments or examples. Furthermore, specific features, structures, materials or characteristics described can be combined in any one or more embodiments or exemplary embodiments in a proper manner.

The preferred embodiments of the present invention disclosed above are merely used to help elaborating the present invention.

The preferred embodiments neither elaborate all details nor limit the present invention to the specific implementation modes. It is apparent that many modifications and variations can be made according to the contents of the description. These embodiments are selected and described specifically in the description to better explain the principle and actual application of the present invention, so that those skilled in the art can better understand and utilize the present invention. The present invention is merely subject to the claims and the full coverage and equivalents thereof.

Claims

CONCLUSIESCONCLUSIONS

1. Antibacteriële coating gevormd door copolymerisatie van chloor- hexidine en catecholamine, waarbij de antibacteriële coating wordt gevormd in een proces waarbij chloorhexidine en catecholamine wor- den onderworpen aan zelf-assemblage om nanodeeltjes te vormen en de nanodeeltjes hechten aan oppervlakken van een verscheidenheid aan substraten om een compacte gepolymeriseerde coating te vormen.1. Antibacterial coating formed by copolymerization of chlorhexidine and catecholamine, the antibacterial coating being formed in a process in which chlorhexidine and catecholamine are self-assembled to form nanoparticles and the nanoparticles adhere to surfaces of a variety of substrates to form a compact polymerized coating.

2. Werkwijze voor het bereiden van de antibacteriële coating vol- gens conclusie 1, omvattende de volgende stappen: Sl: het bereiden van een waterige catecholamine-oplossing van een bepaalde concentratie en een chloorhexidine-oplossing van een be- paalde concentratie; S2: het langzaam druppelen van de chloorhexidine-oplossing die is bereid in S1 in de waterige catecholamine-oplossing, en toestaan dat catecholamine en chloorhexidine volledig worden gepolymeri- seerd; en S3: het mengsel 24 uur stil laten staan om een met chloorhexidine en catecholamine gepolymeriseerde coating op een substraat af te zetten.A method for preparing the antibacterial coating according to claim 1, comprising the steps of: S1: preparing an aqueous catecholamine solution of a certain concentration and a chlorhexidine solution of a certain concentration; S2: slowly dripping the chlorhexidine solution prepared in S1 into the aqueous catecholamine solution, and allowing catecholamine and chlorhexidine to polymerize completely; and S3: allowing the mixture to stand still for 24 hours to deposit a chlorhexidine and catecholamine polymerized coating on a substrate.

3. Werkwijze voor het bereiden van de antibacteriële coating vol- gens conclusie 2, waarbij het catecholamine dopamine, noradrenali- ne, looizuur, galluszuur, bruine algenpolyfenol of epigallocate- chinegallaat is.The method for preparing the antibacterial coating according to claim 2, wherein the catecholamine is dopamine, norepinephrine, tannic acid, gallic acid, brown algae polyphenol or epigallocatechin gallate.

4. Werkwijze voor het bereiden van de antibacteriële coating vol- gens conclusie 3, waarbij in S1 een concentratie van de waterige polyfenoloplossing van bruine algen 0,25 mg/ml is en een concen- tratie van de chloorhexidine-oplossing 0,8 mg/ml is.The method for preparing the antibacterial coating according to claim 3, wherein in S1, a concentration of the aqueous polyphenol solution of brown algae is 0.25 mg/ml and a concentration of the chlorhexidine solution is 0.8 mg/ml ml is.

5. Werkwijze voor het bereiden van de antibacteriële coating vol- gens een van de conclusies 2 tot 4, waarbij een pH-waarde van een polymerisatieproces in S2 8,5 is.The method for preparing the antibacterial coating according to any one of claims 2 to 4, wherein a pH value of a polymerization process in S2 is 8.5.

6. Aanbrengen van de antibacteriële coating gevormd door copolyme- risatie van chloorhexidine en catecholamine volgens conclusie 1, waarbij de antibacteriële coating wordt aangebracht om antibacte- riële toepassingen, antibacteriële botmaterialen en antibacteriële tandheelkundige materialen te bereiden.Application of the antibacterial coating formed by copolymerization of chlorhexidine and catecholamine according to claim 1, wherein the antibacterial coating is applied to prepare antibacterial applications, antibacterial bone materials and antibacterial dental materials.