CN110818864B

CN110818864B - Preparation method of high-molecular antibacterial agent for improving antibacterial effect

Info

Publication number: CN110818864B
Application number: CN201911143380.3A
Authority: CN
Inventors: 邵敏; 李阳
Original assignee: Keen Biotechnology Shanghai Co ltd
Current assignee: Keen Biotechnology Shanghai Co ltd
Priority date: 2019-11-20
Filing date: 2019-11-20
Publication date: 2023-01-10
Anticipated expiration: 2039-11-20
Also published as: CN110818864A

Abstract

The invention provides a preparation method of a high-molecular antibacterial agent for improving antibacterial effect, which combines high-efficiency reaction in organic chemistry to prepare a methacrylate-based monomer or an acrylate-based monomer, wherein the small-molecular monomer comprises a carbamate element with hydrogen bond interaction, a tertiary amine element with antibacterial activity and a bacteria microenvironment responsive element. The invention endows the common material with antibacterial performance, and the existence of hydrogen bond interaction elements expands the application range of the macromolecular antibacterial agent.

Description

Preparation method of high-molecular antibacterial agent for improving antibacterial effect

Technical Field

The invention relates to the technical field of biological materials, in particular to a preparation method of a high-molecular antibacterial agent for improving antibacterial effect.

Background

Bacteria are very ancient organisms, one of the main groups of organisms, and the most abundant of all organisms. Bacteria are ubiquitous, and are usually present wherever there is life. They are present in the air breathed, in the water drunk, and in the food eaten by humans. Bacteria can be carried by the airflow from one place to another. The human body is a habitat for a large number of bacteria; can be found on the skin surface, intestinal tract, oral cavity, nose and other body parts.

However, bacteria are an important source of human diseases, and their widespread existence disturbs human lives all the time, and their reproduction and variation threaten human health. The emergence and spread of bacterial resistance to traditional antibiotics has raised a threat to global medical safety. In daily life, people inevitably contact with various materials and become places for bacteria propagation and dissemination; such as various commodities, packaging, appliances, and utilities. Scientific development and improvement of the living standard of human beings lead the health consciousness of human beings to be continuously strengthened, and the application of antibacterial materials gradually draws more attention of people. In order to prevent the spread of bacteria, a plurality of functional materials with bacteriostasis and sterilization performance are produced, and the antibacterial materials can effectively improve the health level of people and benefit human beings. Existing antibacterial agents are roughly divided into three major groups: organic antibacterial agents, inorganic antibacterial agents, and natural antibacterial agents. However, organic antibacterial agents are poor in heat resistance and are easily washed away because of their inherently good water solubility; inorganic antibacterial agents such as nanosilver are release type antibacterial mechanisms, which are poor in stability and easily aggregated to limit antibacterial applications thereof. The natural antibacterial peptide widely existing in the autoimmune system of various cell organisms becomes a fuzz in the antibacterial agent due to its excellent antibacterial property. Thus, natural antimicrobial peptides, which are widely present in the autoimmune system of various cellular organisms, have become targets against which antimicrobial materials compete.

The natural antibacterial peptide is induced in vivo to produce a series of basic polypeptide substances with antibacterial activity, and the natural antibacterial peptide of the autoimmune system widely existing in multicellular organisms has broad-spectrum antibacterial property and also has antibacterial effect on drug-resistant bacteria. Domestic and foreign studies have shown that, in general, natural antimicrobial peptides produce antimicrobial activity by attacking the cytoplasmic membrane of bacteria. The mechanism of action of bacteria towards attacking the cytoplasmic membrane is more difficult to develop resistance than inhibition of bacterial metabolism. Most natural antimicrobial peptides are natural macromolecules with both electropositive and amphiphilic properties, which are critical to their antimicrobial activity. Electropositivity, such that the natural antimicrobial peptide binds to the negatively charged bacterial cell membrane surface by electrostatic complexation; the amphiphilicity endows the hydrophobic groups of the natural antimicrobial peptide with membrane breakage and penetration capabilities that promote subsequent antimicrobial activity.

However, the difficulty of extracting and separating the natural antibacterial peptide greatly limits the application of the natural antibacterial peptide to the antibacterial material. Therefore, there are many reports in the literature that polycationic natural-like antibacterial peptides such as poly-N-substituted lysine, poly-N-substituted aspartic acid, oligomer, and polymer have the same antibacterial activity as natural antibacterial peptides. To date, partially synthetic mimetic and antibacterialopeptides have entered clinical trials as antimicrobials or metabolic modulators. However, these antibacterial-mimetic peptides and antibacterial-like peptides are difficult to maintain their long-lasting antibacterial function because they are easily washed away by water due to their inherent high water solubility. In addition, the high water solubility also limits the affinity with common materials, and is difficult to blend with some of the most widely used high molecular materials such as polyurethane, polyamide and the like or form an effective antibacterial coating.

On the other hand, the antibacterial peptide mimics and antibacterial peptide analogues have high cytotoxicity because of high water solubility, so that the antibacterial peptide mimics and the antibacterial peptide analogues can be dissolved in a large amount in the presence of water. Therefore, its biocompatibility also limits its application in the field of antibacterial materials

Therefore, how to realize the controllable release and even the responsive release of the polymeric antibacterial agent is still a serious challenge and a problem to be solved under the condition of enhancing the material affinity and biocompatibility of the polymeric antibacterial agent and simultaneously ensuring the antibacterial activity of the material. Designing such an optimized antimicrobial system requires the introduction of new motifs and the application of a straightforward and efficient chemical structure design. .

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a preparation method of a high-molecular antibacterial agent for improving antibacterial efficacy, wherein hydrogen bond-containing elements are introduced into the high-molecular antibacterial agent, and the antibacterial property, the application range and the biocompatibility of the polymer are improved by adjusting the length of alkyl chains on electropositive tertiary amine elements, so that the antibacterial efficacy of the high-molecular antibacterial agent is improved.

In order to achieve the purpose, the invention adopts the technical scheme that:

a preparation method of a high-molecular antibacterial agent for improving antibacterial effect is used for preparing a methacrylate-based monomer or an acrylate-based monomer by combining with high-efficiency reaction in organic chemistry, wherein a small-molecular monomer comprises a carbamate element with hydrogen bond interaction, a tertiary amine element with antibacterial activity and a bacterial microenvironment responsive element, and has the following structure:

wherein R has 7 structures as above, and m, n =1 to 11.

The invention is further described by using polyethylene glycol (PEG-CTA) of the terminal group modified trithioester as a chain transfer agent, polymerizing the monomers by reversible addition-fragmentation chain transfer polymerization (RAFT) to obtain a block copolymer with controllable polymerization degree and polymerization degree distribution of the hydrophobic chain segment, and polymerizing the monomers by using micromolecular trithioester as a chain transfer agent to obtain a homopolymer.

As further described herein, the hydrophilic segment uses polyacrylic acid (PAA), poly (N-isopropylacrylamide) (PNIPAM), poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC) or poly (N, N-dimethylaminoethyl methacrylate (PDMAEMA)) to obtain the corresponding amphiphilic block copolymer, also by RAFT polymerization.

The invention is further described, after the amphiphilic block polymer with proper hydrophilic and hydrophobic chain segment length obtained after optimization is dissolved by adopting a corresponding solvent, the polymer is formed into a film on the surfaces of other materials containing hydrogen bonds in a coating mode, so that the polymer and the materials are closely connected through hydrogen bond interaction.

The invention is further described by the following specific processes: the polymer is fully dissolved in the cosolvent, and then is uniformly sprayed on the surface of other materials or is mixed with organic solutions of other materials, and the polymer is obtained after the organic solvents are evaporated.

For further description of the present invention, the hydrogen bonding material is polyamide or polyurethane or cotton.

As a further description of the invention, acetone or dioxane or tetrahydrofuran is used as the co-solvent.

Compared with the prior art, the invention has the advantages that:

a hydrogen bond interaction element carbamate is introduced to a repeating structural unit of the structure of the high-molecular antibacterial agent, and alkyl chains with different lengths and structures are introduced to nitrogen of tertiary amine to regulate the pKa value of the tertiary amine and the water solubility of the whole molecule on the tertiary amine element playing an antibacterial role in the repeating structural unit. Hydrogen bond interaction elements such as a urethane bond, an amide bond and a urea bond are widely applied to the field of high polymer materials, and the hydrogen bond interaction elements endow high polymer materials such as nylon-66, polyurethane, protein, liquid crystal and the like with excellent performance.

Therefore, the introduction of hydrogen bond interaction elements can effectively improve the affinity of the antibacterial polymer and the commonly applied polymer material. The high molecular material can be dispersed and fused with other hydrogen bond-containing materials in modes of blending, surface coating and the like, so that the antibacterial property of the common material is endowed. The existence of hydrogen bond interaction elements expands the application range of the macromolecular antibacterial agent.

The alkyl chain length is regulated and controlled on the tertiary amine acting element on the high molecular antibacterial agent, the water solubility of the high molecular antibacterial agent is reduced along with the increase of the alkyl chain length, the reduction of the water solubility relatively slows down the release of the high molecular antibacterial agent, and on the contrary, the alkyl chain length on the tertiary amine acting element is reduced, the water solubility and the release of the high molecular antibacterial agent are also increased, so that the long-acting and durable antibacterial effect of the material is realized;

the polymer antibacterial agent which can responsively release the tertiary amine element with antibacterial activity is prepared, and the polymer antibacterial agent does not release the tertiary amine element with antibacterial function in an aseptic environment, so that the polymer antibacterial agent has good biocompatibility, but can responsively release the tertiary amine element with antibacterial activity in a bacterial microenvironment to kill bacteria; that is, only in the bacterial environment, the tertiary amine element with antibacterial activity on the polymer can be released to kill bacteria.

Drawings

FIG. 1 is the nuclear magnetic hydrogen spectrum of N, N-dimethylamino monomer, N-dimethylaminoethoxycarbonylaminoethyl methacrylate; nuclear magnetic hydrogen spectrum of N, N-diethylamino monomer, N-diethylaminoethoxycarbonylaminoethyl methacrylate; and the nuclear magnetic hydrogen spectrum of N, N-di-N-propylamino monomer, namely N, N-di-N-propylamine ethoxy carbonyl aminoethyl methacrylate.

FIG. 2 is a nuclear magnetic hydrogen spectrum of a monomer of N, N-diisopropylamino, namely N, N-diisopropylamino ethoxycarbonyl aminoethyl methacrylate; the nuclear magnetic hydrogen spectrum of the N-monosubstituted isobutylamino monomer, namely N-monosubstituted isobutylamino ethoxycarbonyl aminoethyl methacrylate; and nuclear magnetic hydrogen spectra of N, N-methylethylamino monomer, N-methylethylaminoethoxycarbonyl aminoethyl methacrylate.

FIG. 3 is a nuclear magnetic hydrogen spectrum of a hydrogen peroxide activated antimicrobial active monomer.

FIG. 4 shows an acid-base titration curve of a series of monomers such as N, N-dimethylamino monomer (N, N-dimethylaminoethoxycarbonylaminoethylmethacrylate), N-diethylamino monomer (N, N-diethylaminoethoxyoxycarbonylaminoethylmethacrylate), N-di-N-propylamino monomer (N, N-di-N-propylaminoethoxycarbonylaminoethylmethacrylate), N-diisopropylamino monomer (N, N-diisopropylaminoethoxyoxycarbonylaminoethylmethacrylate), N-monosubstituted isobutylamino monomer (N-monosubstituted isobutylaminoethoxycarbonylaminoethyl methacrylate), and N, N-methylethylamino monomer (N, N-methylethylaminoethoxycarbonylaminoethyl methacrylate).

FIG. 5 shows gel permeation chromatography spectra of a series of polymers such as N, N-dimethylamino polymer (i.e., N-dimethylaminoethoxyethyl methacrylate), N-diethylamino polymer (N, N-diethylaminoethoxycarbonyl aminoethyl methacrylate), N-methylethylamino polymer (N, N-methylethylaminoethoxycarbonyl aminoethyl methacrylate), N-diisopropylamino polymer (N, N-diisopropylaminoethoxycarbonyl aminoethyl methacrylate), and N-monosubstituted isobutylamino polymer (N-monosubstituted isobutylaminoethoxycarbonyl aminoethyl methacrylate)).

FIG. 6 is a transmission electron micrograph of an assembly formed after titration of a polymer of N, N-diisopropylamino group by acid base.

FIG. 7 is a graph depicting the antimicrobial activity of a series of nitrogen-substituted urethane polymers.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

In a first step, the following nitrogen-substituted urethane monomer M is prepared:

the tertiary amine element with antibacterial activity, the substitution of the tertiary amine is shown as the following figure:

the preparation method comprises the following steps: 20.0mmol of N-substituted tertiary aminoethanol was dissolved in 60mL of dry methylene chloride, followed by the addition of dibutyltin dilaurate (DBTL, 50-1000. Mu.L) as a condensation catalyst, and 22.0mmol of isocyanoethyl methacrylate was slowly added with stirring at 0 ℃ to 100 ℃. After 6 hours of reaction, the organic solvent was removed by rotary evaporation under reduced pressure (rotary evaporation), and the crude product was dissolved in methylene chloride, washed three times with saturated brine and dried over anhydrous magnesium sulfate. The crude product was then concentrated by rotary evaporation and purified by column chromatography (silica gel as the stationary phase packing and ethyl acetate as the mobile phase eluting solvent), then the organic solvent was removed by rotary evaporation and dried under vacuum to give pure white M (18.0 mmol, 90%).

In the second step, the following nitrogen-substituted urethane polymers were further prepared by RAFT polymerization:

a PEG (polyethylene glycol) chain segment is used as a hydrophilic chain segment, and the length of the chain segment can be randomly selected, such as x = 4-1000; the polymerization degree of the hydrophobic segment can be effectively changed by changing polymerization parameters and conditions, and specifically, y =2 to 250 is obtained, and the polymer (P1) in which x =60 and y =40 is selected is exemplified.

The preparation method comprises the following steps: 8mmol M monomer, 0.6g (0.26 mmol) PEG-CTA chain transfer agent and 10.6mg (0.064 mmol) Azobisisobutyronitrile (AIBN) initiator are dissolved in 1-100mL dioxane, added into a polymerization tube containing a magnetic stirrer, and placed in an oil bath at 0-100 ℃ after freeze-pumping circulation. The polymerization reaction is terminated after 12 hours by using a liquid nitrogen cooling mode, and after the room temperature is recovered, the reaction product is precipitated in anhydrous ether and repeatedly precipitated for three times. The product was dried in a vacuum oven to give polymer P1,2.56g (81.2%).

The third step: preparation of aqueous assemblies of nitrogen-substituted urethane polymers:

1) P1 was dissolved in 1 part by volume of an acidic aqueous solution (for example, an aqueous hydrochloric acid solution having a pH = 2), 9 parts of a weakly alkaline water was slowly added under stirring, and the pH of the system was adjusted to about 7.4, which is a normal physiological pH. The resulting whitish emulsion was placed in a dialysis bag and dialyzed against water until the acid-base neutralized salts were removed. This gave vesicles (V1) with a diameter of about 450nm, as visualized by TEM in FIG. 5.

2) P1 was dissolved in 1 part by volume of an organic solution (as exemplified by dioxane), and 9 parts of water were slowly added with stirring. The obtained whitish emulsion is placed in a dialysis bag and dialyzed in water until the organic solvent in the system is removed. This gave vesicles (V1) with a diameter of about 450nm, as visualized by TEM in FIG. 1.

Application example 1 antimicrobial activity characterization of nitrogen-substituted urethane polymers:

100 units of nitrogen-substituted polyurethane polymer and 200 parts of polyurethane are melted and blended, a film of 5 x 5cm is prepared after extrusion and granulation, and a series of antibacterial activity characterizations of the nitrogen-substituted polyurethane polymer are obtained after antibacterial tests are carried out according to relevant standards (the blank control is polyethylene glycol monomethyl ether, the molecular weight is 2000). The results show that: compared with a blank control, the nitrogen-substituted polyurethane polymer and the polyurethane material can be effectively blended, and the carbamate and the polyurethane material of the nitrogen-substituted polyurethane polymer can improve the affinity through the hydrogen bond-hydrogen bond interaction, so that the antibacterial durability is improved, which is consistent with the design expectation of the invention.

In the present embodiment, all the components are general standard components or components known to those skilled in the art, and the structure and principle thereof can be known to those skilled in the art through technical manuals or through routine experiments.

In the present invention, unless otherwise expressly stated or limited, "above" or "below" a first feature means that the first and second features are in direct contact, or that the first and second features are not in direct contact but are in contact with each other via another feature. Also, a first feature "above," "over," and "on" a second feature includes that the first feature is directly above and obliquely above the second feature, or merely means that the first feature is at a higher level than the second feature, and a first feature "below," "under," and "under" the second feature includes that the first feature is directly above and obliquely above the second feature, or merely means that the first feature is at a lower level than the first feature.

In the description of the present specification, reference to the description of the terms "one embodiment," "some embodiments," "an example," "a specific example" or "some examples" or the like means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention, and schematic representations of the terms in this specification do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments without departing from the principles and spirit of the present invention.

Claims

1. A preparation method of a high-molecular antibacterial agent capable of improving antibacterial effect is used for preparing a methacrylate-based monomer or an acrylate-based monomer by combining with high-efficiency reaction in organic chemistry, wherein the methacrylate-based monomer or the acrylate-based monomer comprises a carbamate unit with hydrogen bond interaction, a tertiary amine unit with antibacterial activity and a bacterial microenvironment responsive unit, and is characterized by having the following structure:

wherein, R is tertiary amine element with antibacterial activity, has 6 structures as above, m, n = 1-11, polyethylene glycol with terminal group modified trithioester is used as chain transfer agent, reversible addition-fragmentation chain transfer polymerization (RAFT) is adopted to polymerize the monomer, and block copolymer with controllable polymerization degree of hydrophobic chain segment and polymerization degree distribution is obtained.

2. The preparation method of the polymeric antibacterial agent with the antibacterial effect improved according to claim 1, wherein after the obtained amphiphilic block polymer with the length of the hydrophilic and hydrophobic chain segments is dissolved by a corresponding solvent, the polymer is formed into a film on the surface of other materials containing hydrogen bonds in a coating mode, so that the polymer is tightly connected with the materials through the interaction of the hydrogen bonds.

3. The preparation method of the polymeric antibacterial agent with improved antibacterial effect according to claim 2 is characterized by comprising the following specific steps: the polymer is fully dissolved in the cosolvent, and then is uniformly sprayed on the surface of other materials or is mixed with organic solutions of other materials, and the polymer is obtained after the organic solvents are evaporated.

4. The method for preparing a polymeric antimicrobial agent with enhanced antimicrobial activity as claimed in claim 2, wherein the hydrogen bonding material is selected from polyamide, polyurethane or cotton.

5. The method as claimed in claim 3, wherein the co-solvent is acetone, dioxane or tetrahydrofuran.