A polycationic antibacterial polymer based on imidazole and its preparation and application

Abstract

The present invention belongs to the field of antibacterial polymer synthesis technology, and specifically relates to a polycationic antibacterial polymer based on imidazole and its preparation and application. The present invention utilizes the positive charge carried by the quaternized cationic imidazole salt group to adsorb to the negatively charged bacterial cell membrane, and the hydrophobic segment carried on the polymer can be inserted into the bacterial cell membrane, destroying the bacterial cell membrane, and then achieving an antibacterial effect. At the same time, the imidazole salt cationic polymer designed by the present invention can penetrate into the bacterial biofilm, play a certain killing effect on the bacteria in the bacterial biofilm, thereby obtaining the ability to eradicate the biofilm. It has been found through experiments that the polymer has good antibacterial and bactericidal properties under body temperature incubation conditions. At the same time, the polymer has a certain ability to eradicate biofilm, and its antibacterial and bactericidal properties are concentration-dependent. In addition, the polymer has good biocompatibility and has the potential to be applied to human antimicrobial infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibacterial polymer synthesis, and in particular relates to an imidazole-based polycationic antibacterial polymer and its preparation and application. Background Art

[0002] Due to the increasing incidence of infectious diseases associated with drug-resistant pathogens and the decrease in the number of newly approved antibiotics, researchers have focused their research on the development of antimicrobial materials that can mitigate drug resistance. Based on the different material components, antimicrobial materials can be divided into the following categories: (1) metal-type antimicrobial materials; (2) natural antimicrobial materials; (3) inorganic antimicrobial materials; and (4) organic antimicrobial materials. Among them, organic antimicrobial materials have attracted widespread research interest due to their strong antimicrobial ability, rapid action, and easy dispersion. Organic antimicrobial materials mainly include antimicrobial polymers, antimicrobial peptides, and other organic antimicrobial molecules. Compared with small molecule fungicides, antimicrobial polymers have relatively low toxicity and high antimicrobial efficiency, which has attracted people's attention. With the advancement of polymer science, the regulation of polymer structure has become clearer through the use of advanced polymerization technology, and the relationship between structure and properties can also be more accurately evaluated. With the rapid development of antimicrobial polymer materials, a large number of antimicrobial polymers with different characteristics have been developed. It is generally believed that the mechanism by which antimicrobial polymers exert their bactericidal effect is related to the destructiveness of cell walls and / or cytoplasmic membranes. Antimicrobial polymers are less likely to induce serious bacterial resistance because they have different antimicrobial mechanisms from antibiotics.

[0003] Imidazole salt-based antimicrobial polymers are a new type of antimicrobial cationic polymer. Imidazole is a component of biomolecules, such as amino acids, histidine and related compounds, biotin, and imidazole alkaloids. The imidazole ring has high chemical stability and resistance to hydrogenation. Therefore, imidazole rings and their derivatives can be quaternized to form imidazole salt groups, thereby forming polycationic antimicrobial agents. For example, one study synthesized imidazole-based polymers via in situ photocrosslinking and then anion exchange with L-proline or L-tryptophan. The resulting materials exhibited high antimicrobial properties against Escherichia coli and Staphylococcus aureus, good blood compatibility, low cytotoxicity, and low adsorption to bovine serum albumin. Other studies have also grafted high-density imidazole polymer brushes onto TiO2, and through surface-initiated ring-opening polymerization, the resulting materials exhibited excellent antimicrobial and anti-biofouling properties.

[0004] In general, current research on antimicrobial polymers focuses primarily on side-chain polymers containing pendant cations. However, relatively little research has been conducted on main-chain cationic antimicrobial polymers (polymeric cationic centers) and their antimicrobial activity, likely due to the limited and cumbersome nature of their synthesis methods. However, studies have synthesized small-molecule cationic compounds based on imidazole, quaternary ammonium, and octyldiamine, as well as the corresponding main-chain / side-chain polymers. Comparison revealed that the antimicrobial activity of the three synthesized antimicrobial agents ranked in the order: main-chain cationic polymer > side-chain cationic polymer > small-molecule cationic compound. This suggests that the development of main-chain cationic antimicrobial polymers has the potential to achieve superior antimicrobial efficacy.

[0005] In summary, in order to further improve the antibacterial properties of functional polymer materials, reduce drug resistance and toxic side effects, and enrich the treatment methods for resisting pathogenic microorganism infections, it is of great significance to prepare imidazole-based polycationic antibacterial polymers with a main chain cationic structure. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the present invention provides an imidazole-based polycationic antibacterial polymer and its preparation method. The positively charged imidazole salt groups in the provided polymer can electrostatically bind to negatively charged bacterial cell membranes, and the hydrophobic segments in the polymer can then intercalate into the bacterial cell membranes, causing bacterial lysis and death. Furthermore, the polymer can penetrate into bacterial biofilms, achieving a certain degree of eradication.

[0007] To achieve the above object, the present invention is implemented through the following technical solutions:

[0008] The first aspect of the present invention provides a polycationic antibacterial polymer compound based on imidazole, the structural formula of the polymer compound is as follows:

[0009]

[0010] R1, R2 are independently selected from C 2-10 、

[0011] Preferably, the polymer compound is selected from any one of the following structural formulas:

[0012]

[0013] The second aspect of the present invention provides a method for preparing the imidazole-based polycationic antibacterial polymer compound according to the first aspect, comprising the following steps:

[0014] S1. Under an inert gas atmosphere, an imidazole monomer and a dithiol alcohol monomer are added to an organic solvent, heated until a distinct yellow color appears, and then the solvent is removed by rotary evaporation. After purification by silica gel column chromatography, a small molecule imidazole monomer (Compound 1) is obtained;

[0015] S2. Dissolve the small molecule imidazole monomer in an organic solvent, cool it to 0°C and then add the dichloroalkane monomer, then heat it to 60-80°C under an inert gas atmosphere and react for 90-100 hours. After the reaction, concentrate and wash to obtain a viscous brown-yellow product, and finally redissolve the product in water, dialyze and dry it to obtain an imidazole-based polycationic antibacterial polymer compound.

[0016] The present invention first obtains a structurally symmetrical small molecule imidazole monomer with imidazole rings exposed at both ends through a click chemistry reaction between an imidazole monomer and a dithiol alcohol monomer using a thiol group and a carbon-carbon double bond; then, a condensation polymerization reaction is carried out with an alkane chain monomer having a terminal disubstituted halogen atom to obtain an AB alternating imidazole-based polycationic antibacterial polymer.

[0017] Preferably, the dimercaptoalcohol monomer is selected from 1,2-dimercaptoethanol, 1,4-dimercaptobutanol, 1,6-dimercaptohexanol, 1,8-dimercaptooctanol, and 2,2'-(ethane-1,2-diylbis(oxy))bis(ethane-1-thiol), and the structural formula of the imidazole monomer is as follows:

[0018]

[0019] Preferably, the dichloroalkane monomer is selected from any one of the following structural formulas:

[0020]

[0021] Preferably, the molar ratio of the imidazole monomer to the dithiol alcohol monomer is 2:1.

[0022] Preferably, the silica gel chromatography column purification is performed using a mixed solvent of dichloromethane and methanol as the mobile phase.

[0023] Preferably, the molar ratio of the small molecule imidazole monomer to the dichloroalkane monomer is 1-2:1-2.

[0024] Preferably, the dialysis is performed for more than two days using a regenerated cellulose dialysis bag with a molecular weight cut-off of 3500 Da.

[0025] The third aspect of the present invention provides the use of the imidazole-based polycationic antibacterial polymer compound described in the first aspect in the preparation of an antibacterial agent or / and a bactericide, wherein the bacteria involved in the antibacterial and / or bactericidal agents include Staphylococcus aureus, Escherichia coli, and Staphylococcus aureus.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The invention discloses a method for preparing a polycationic antibacterial polymer based on imidazole. The method utilizes the positive charge carried by the quaternized cationic imidazole salt group to adsorb to the negatively charged bacterial cell membrane, and the hydrophobic segment carried on the polymer can be inserted into the bacterial cell membrane, destroying the bacterial cell membrane, and then achieving an antibacterial effect. At the same time, the imidazole salt cationic polymer designed by the present invention can penetrate into the bacterial biofilm, play a certain killing effect on the bacteria in the bacterial biofilm, thereby obtaining the ability to eradicate the biofilm. It has been found through experiments that the polymer has good antibacterial and bactericidal properties under body temperature incubation conditions. At the same time, the polymer has a certain ability to eradicate biofilm, and its antibacterial and bactericidal properties are concentration-dependent. In addition, the polymer has good biocompatibility and has the potential to be applied to human antimicrobial infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a synthetic route for imidazole-based polycationic antimicrobial polymers;

[0029] Figure 2 A route for synthesizing imidazole-based polycationic antimicrobial polymers using 1,4-dimercaptobutanol and 1,4-dichlorobutane monomers;

[0030] Figure 3 H NMR spectrum of small molecule imidazole monomer synthesized with 1-vinylimidazole and dimercaptobutanol as reactants ( 1 H NMR);

[0031] Figure 4 The H NMR spectrum of the mixed imidazole-based polycationic antibacterial polymer condensation polymerization monomers ( 1 HNMR);

[0032] Figure 5 The nuclear magnetic resonance 1H spectrum of the imidazole-based polycationic antibacterial polymer ( 1 H NMR);

[0033] Figure 6 The antibacterial growth curves of the imidazole-based polycationic antibacterial polymer after sequentially interacting with three model bacteria (Staphylococcus aureus ATCC 43300, Escherichia coli ATCC 25922, and Staphylococcus aureus ATCC 6538);

[0034] Figure 7 This is a laser confocal image of live-dead staining after the interaction of imidazole-based polycationic antimicrobial polymer with bacteria (ATCC 6538);

[0035] Figure 8This is a laser confocal image of live-dead staining after the interaction of imidazole-based polycationic antimicrobial polymer with bacteria (ATCC 25922);

[0036] Figure 9 This is a characterization diagram of the eradication ability of imidazole-based polycationic antimicrobial polymers against bacterial (ATCC 6538) biofilms;

[0037] Figure 10 Figure 2 is a diagram showing the cytotoxicity evaluation of imidazole-based polycationic antimicrobial polymers. DETAILED DESCRIPTION

[0038] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0039] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0040] Example 1 Synthesis of Imidazole-Based Polycationic Antibacterial Polymer (Compound 2)

[0041] according to Figure 1 、 Figure 2 The synthetic route of compound 2 includes the following steps:

[0042] (1) Synthesis of Compound 1

[0043] Under argon protection, imidazole monomer and dithiol alcohol monomer (see Table 1 for details) were added to a flask containing 25 mL of anhydrous acetonitrile at a 2:1 molar ratio. The system was then heated to 70°C under argon protection and reacted overnight, resulting in a distinct yellow color. After the reaction, the solvent was removed by rotary evaporation under reduced pressure, and silica gel column chromatography was performed using a mixed solvent of dichloromethane and methanol (v / v = 1 / 10) as the mobile phase. Purification by silica gel column chromatography yielded a pale yellow-green oily liquid, Compound 1 (see Table 1 for details).

[0044] Taking R1=C4H8 corresponding to 1,4-dimercaptobutanol as an example, 5 mg of the synthesized compound 1 (small molecule imidazole monomer) was dissolved in 0.5 mL of deuterated chloroform and characterized by 400 MHz nuclear magnetic resonance (NMR) hydrogen spectrum. The NMR structure diagram was analyzed, indicating that the small molecule imidazole monomer structure was correct and could be used for the next reaction ( Figure 3 ).

[0045] (2) Synthesis of Compound 2

[0046] In a 25 mL reaction tube, compound 1 (6.44 mmol) obtained in step (1) was dissolved in 5 mL DMSO. The system was cooled to 0°C in an ice bath, and then dichloroalkane monomer (see Table 1 for details) (6.46 mmol) was added dropwise. Then, under argon protection, the reaction was heated to 70°C for 96 h. The reaction was stopped in an ice bath, and cold ether (100 times the volume of the reaction system) was added dropwise to the reaction system. The reaction was washed twice with cold ether, and then a vacuum pump was connected to remove the remaining small amount of ether to obtain a viscous brown-yellow product. 10 mL of deionized water was then added to the product to dissolve it, and it was dialyzed for two days using a regenerated cellulose dialysis bag with a molecular weight cutoff of 3500 Da. After freeze-drying, a yellow solid was obtained. The product was a very viscous yellow liquid at room temperature, namely compound 2 (see Table 1 for details), and finally an imidazole-based polycationic antibacterial polymer was obtained.

[0047] Taking the polymer (P4) of 1,4-dimercaptobutanol and 1,4-dichlorobutane with R1=C4H8 and R2=C4H8 as an example, 10 mg of the mixed sample (mixed sample after adding dichloroalkane monomer) was dissolved in 0.5 mL of deuterated chloroform and characterized by 400 MHz nuclear magnetic hydrogen spectrum. The nuclear magnetic structure diagram was analyzed, indicating that the ratio of compound 1 and dichloroalkane monomer in the imidazole-based polycationic antibacterial polymer condensation polymerization was appropriate ( Figure 4 At the same time, 10 mg of the sample (compound 2) was dissolved in 0.5 mL of deuterated DMSO and characterized by 400 MHz nuclear magnetic hydrogen spectroscopy, indicating that the structure of the imidazole-based polycationic antibacterial polymer was correct ( Figure 5 In addition, the cLogP values of the main structural unit segments of the obtained polycationic antibacterial polymer were calculated using ChemDraw 19.0, and the results are shown in Table 2. As shown in Table 2, the cLogP values corresponding to R1=C4H8 and R2=C4H8 are 2.810 and 3.106, respectively, indicating that both segments have a certain degree of hydrophobicity.

[0048] Table 1 Structural formulas of dithiol monomer, compound 1, dichloroalkane monomer and compound 2 in the synthesis of imidazole-based polycationic antibacterial polymers

[0049]

[0050]

[0051] Table 2 cLogP values of the main structural units of imidazole-based polycationic antibacterial polymers

[0052]

[0053] Example 2 Analysis of antibacterial properties of imidazole-based polycationic antibacterial polymers

[0054] The subsequent performance analysis is illustrated by taking the polymer (P4) of 1,4-dimercaptobutanol and 1,4-dichlorobutane with R1=C4H8 and R2=C4H8 as an example.

[0055] Bacterial cells (ATCC 43300, ATCC 25922, ATCC 6538) were cultured in TSB medium at 37°C overnight to mid-logarithmic phase and resuspended in TSB to an OD of 600 =0.1. In addition, the imidazole-based polycationic antibacterial polymer was dissolved in sterile PBS buffer (pH=7.4) and diluted to different concentrations with PBS buffer. After UV irradiation for 15 minutes, it was mixed with the bacterial cell suspension in a 96-well plate at a volume ratio of 1:1. At the same time, TSB / PBS medium (1:1) inoculated with each strain was used as a positive control, and TSB / PBS medium (1:1) without bacteria inoculation was used as a negative control. All samples and control groups were incubated at 37°C, and the OD was measured intermittently using a microplate reader. 600 , drawn as a curve.

[0056] From the inhibition growth curve ( Figure 6 ) It can be seen that the imidazole-based polycationic antibacterial polymers acted on three model bacteria (ATCC 43300, ATCC 25922, and ATCC 6538) in sequence, and had a good inhibitory effect at 32 μg / mL, 16 μg / mL, and 32 μg / mL, respectively.

[0057] At the same time, after obtaining the 24-h mixed bacterial solution according to the above method, 50 μL of bacterial suspension was extracted from the final mixture in each well, transferred to a TSB agar culture dish, and incubated at 37°C for 24 h. Each experiment was repeated three times in parallel.

[0058] The MIC is defined as the lowest polymer concentration that inhibits bacterial growth (% inhibition <10%); the MBC value is determined as the concentration of the imidazole-based polycationic antimicrobial polymer that reduces the number of viable bacterial cells by greater than 9 log compared to the control without polymer. As shown in Table 3, the average molecular weight (Mn) of the imidazole-based polycationic antimicrobial polymer, as measured by nuclear magnetic resonance (NMR), demonstrates good antibacterial and bactericidal effects against methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus (S. aureus), Streptococcus faecalis (S. faecalis), and Escherichia coli (E. coli).

[0059] Furthermore, in order to observe the bacterial activity after polymer treatment, the bacteria in the logarithmic phase were collected by centrifugation (4000 rpm, 5 min), washed once with PBS, and diluted to OD 600 = 0.2. The bacterial suspension was then incubated with the polymer at the MIC concentration for 10 h. After incubation, the cells were washed twice with PBS and the cells were labeled with a live / dead fluorescent dye (SYTO9 / PI) and the bacterial viability was observed using a laser confocal microscope.

[0060] from Figure 7 、 8 As can be seen in the PI channel, a large amount of red fluorescence signal was detected in the experimental group, indicating the death of a large number of Staphylococcus aureus and Escherichia coli. This is because the presence of a large number of high-density cations on the imidazole-based polycationic antimicrobial polymer can induce bacterial aggregation, causing bacteria to form bacterial clusters. Simultaneously, the negatively charged microenvironment induced by the bacterial clusters promotes further aggregation of the polycationic antimicrobial polymer to the bacterial clusters, thereby inducing localized mass bacterial death.

[0061] To further determine the polymer's ability to clear biofilms, an imidazole-based polycationic antimicrobial polymer was dissolved in culture medium to create polymer solutions of varying concentrations. These solutions were then incubated for 24 hours with Staphylococcus aureus biofilms pre-cultured using conventional plate culture. Biofilm biomass was measured using the crystal violet method.

[0062] from Figure 9 It can be seen that at a higher concentration of 256 μg / mL, the imidazole-based polycationic antimicrobial polymer had a certain effect on clearing the biofilm, and the biomass of the treatment group was reduced by about 50%.

[0063] In addition, the cytotoxicity of the polymer was evaluated using 3T3 cells: cells were incubated with a solution of an imidazole-based polycationic antimicrobial polymer for 12 hours. After removing the supernatant, 15 μL of MTT (5 mg / mL) was added. Four hours later, 150 μL of DMSO was added to dissolve the MTT. Finally, relative cell viability (fluorescence absorbance at 450 nm) was measured using a microplate reader.

[0064] from Figure 10 It can be seen that the cell compatibility of the imidazole-based polycationic antibacterial polymer is concentration-dependent, and it has good cell compatibility at a concentration of 8 μg / mL.

[0065] Table 3 Statistics of MIC and MBC results of imidazole-based polycationic antibacterial polymers against different bacteria

[0066]

[0067] In addition, the other polymers in Table 1 (P1-3, P5-15) also have good antibacterial or bactericidal effects like P4.

[0068] In summary, the imidazole-based polycationic antimicrobial polymer compound provided by the present invention exhibits excellent antibacterial and bactericidal properties under body temperature incubation conditions. Furthermore, the polymer has a certain ability to eradicate biofilms, and its antibacterial and bactericidal properties are concentration-dependent. Furthermore, the polymer has good biocompatibility and has the potential to be used in the fight against microbial infections in humans.

[0069] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. A polycationic antibacterial polymer compound based on imidazole, characterized in that: The structural formula of the polymer compound is shown below: R1, R2 are independently selected from C 2-10 .

2. The imidazole-based polycationic antibacterial polymer compound according to claim 1, characterized in that: The polymer compound is selected from any one of the following structural formulas:

3. The method for preparing the imidazole-based polycationic antibacterial polymer compound according to any one of claims 1 or 2, characterized in that: The following steps are involved: S1. Under an inert gas atmosphere, an imidazole monomer and a dimercaptoalcohol monomer are added to an organic solvent, heated until a distinct yellow color appears, and then the solvent is removed by rotary evaporation. After purification by silica gel chromatography, a small molecule imidazole monomer is obtained; the dimercaptoalcohol monomer is selected from 1,2-dimercaptoethanol, 1,4-dimercaptobutanol, 1,6-dimercaptohexanol, and 1,8-dimercaptooctanol; the structural formula of the imidazole monomer is as follows: S2. Dissolving a small molecule imidazole monomer in an organic solvent, cooling the mixture to 0°C and then adding a dichloroalkane monomer, followed by heating to 60-80°C under an inert gas atmosphere for a reaction of 90-100 hours. After the reaction, the mixture is concentrated and washed to obtain a viscous brown-yellow product, which is then redissolved in water, dialyzed, and dried to obtain an imidazole-based polycationic antibacterial polymer compound; the dichloroalkane monomer is selected from any one of the following structural formulas: 。 4. The method for preparing the imidazole-based polycationic antibacterial polymer compound according to claim 3, wherein: The molar ratio of the imidazole monomer to the dithiol alcohol monomer is 2:

1.

5. The method for preparing the polycationic antibacterial polymer compound based on imidazole according to claim 3, characterized in that: The product was purified by silica gel column chromatography using a mixed solvent of dichloromethane and methanol as the mobile phase.

6. The method for preparing the imidazole-based polycationic antibacterial polymer compound according to claim 3, characterized in that: The molar ratio of the small molecule imidazole monomer to the dichloroalkane monomer is 1-2:1-2.

7. The method for preparing the imidazole-based polycationic antibacterial polymer compound according to claim 3, characterized in that: The samples were dialyzed for more than two days using a regenerated cellulose dialysis tubing with a molecular weight cut-off of 3500 Da.

8. Use of the imidazole-based polycationic antibacterial polymer compound according to any one of claims 1 or 2 in the preparation of an antibacterial agent or / and a bactericide, characterized in that: The bacteria involved in the bacteriostatic agent and / or bactericide include Staphylococcus aureus and Escherichia coli.

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