Photoresponse-essential oil synergistic antibacterial polymer and application thereof in resisting multiple drug-resistant bacteria

Abstract

The invention discloses a photoresponse-essential oil synergistic antibacterial polymer and an application thereof in resisting multiple drug-resistant bacteria. Citronellol (CT) is subjected to an esterification reaction to prepare a CMA monomer, the CMA monomer and a glycidyl methacrylate (GMA) monomer are copolymerized to prepare a polymer PG9C2, and then TBO is introduced to obtain the antibacterial polymer PG9C2T1. The improvement of the antibacterial activity of the TBO is derived from the improvement of the fat solubility of the TBO, the enhancement of the interaction between the TBO and bacterial cells, the improvement of the accumulation of the TBO in the cells, the penetration of the TBO through cell membranes and the play of roles in the cells, the improvement of the utilization efficiency of ROS, and the sterilization by improving the water solubility of the essential oil through the mechanism of destroying bacterial membranes. According to the invention, high-efficiency and low-toxicity drug-resistant bacteria based on natural products are synthesized, the limitation of a single antibacterial mode is broken through, and technical support is provided for developing a novel low-drug-resistance antibacterial therapy.

Description

Technical Field

[0001] The present invention relates to a light-responsive essential oil synergistic antibacterial polymer and its application in combating multi-drug resistant bacteria, belonging to the technical field of bio-antibacterial polymer materials. Background Art

[0002] Bacterial infections, as an important threat to human health, have pathological impacts throughout the entire history of civilization development. Such infections not only cause local tissue damage, but also lead to systemic pathological reactions, including various clinical complications such as suppurative dermatitis, infective endocarditis, sepsis, and chronic organ failure. The clinical application of antibiotics (especially the discovery of penicillin in the 20th century) has completely changed the treatment mode of infectious diseases, significantly reducing the mortality rate related to infections, and providing safety guarantees for invasive treatments such as modern surgical operations and cancer chemotherapy. Antibacterial drugs represented by β-lactams and polymyxins have achieved remarkable results in controlling Gram-positive and Gram-negative bacterial infections. However, with the widespread use of antibacterial drugs, microorganisms have gradually evolved complex drug resistance mechanisms through genetic variation, selection pressure, and drug abuse. The microbial drug resistance mechanisms are significantly species-specific. Bacteria may have intrinsic resistance to certain antibiotics, or they can acquire drug resistance to antibiotics through chromosomal gene mutations and horizontal gene transfer. The intrinsic resistance of bacterial species to specific antibiotics is the ability to resist the action of the antibiotic due to inherent structural or functional characteristics. The simplest example of the intrinsic drug resistance of a single bacterial species is the lack of sensitive targets for a specific antibiotic; for example, the bactericide triclosan has broad efficacy against Gram-positive bacteria and many Gram-negative bacteria, but it cannot inhibit the Gram-negative bacterium Pseudomonas aeruginosa; Escherichia coli is naturally insensitive to vancomycin; the intrinsic resistance of the genus Pseudomonas to triclosan, etc. Bacteria can also acquire or develop drug resistance to antibiotics. This can be mediated by multiple mechanisms, which are divided into: ① minimizing the intracellular concentration of antibiotics due to poor bacterial penetration or antibiotic efflux; ② modifying antibiotic targets through gene mutations or modifications of the targets; ③ inactivating antibiotics by hydrolysis or modification. For example, Enterobacteriaceae bacteria acquire quinolone resistance genes through conjugative plasmids, resulting in a significant reduction in the clinical efficacy of such drugs. The emergence of vancomycin-resistant Enterococcus highlights the severity of the spread of drug resistance genes. Such multi-drug resistant bacteria have become the key targets for the prevention and control of nosocomial infections.

[0003] As a non-invasive cancer treatment method with broad prospects, photodynamic therapy (PDT) has received extensive attention in recent years. In the past 30 years, various novel photosensitizers (PS), such as chlorine, phthalocyanine, tetrapyrrole, and BODIPY, have been widely used in the clinical treatment of various solid tumors. In addition, hypericin, rose bengal, Nile blue derivatives, and methylene blue have also been used in clinical trials, and some innovative strategies have been proposed to improve the therapeutic effect of PS. PS itself is non-toxic to cells, but under the action of light and oxygen molecules, it will produce reactive oxygen species (ROS), thereby destroying the proteins, nucleic acids, lipids, membranes, and organelles of cells and inducing apoptosis. PDT has many advantages compared with traditional treatment methods (such as surgery, chemotherapy, and radiotherapy). First of all, PDT only plays a role after being activated by specific light, which can effectively reduce systemic toxicity. Secondly, ROS can not only directly damage cancer cells, but also destroy tumor blood vessels, thereby promoting the immune system to recognize and attack cancer cells. In addition, PDT is a local selective treatment method, which can be used in combination with surgery, chemotherapy, or radiotherapy, and can also effectively deal with the problem of multi-drug resistance. And with the extensive application of nanomaterials, the permeability and retention (EPR effect) of PDT have been further enhanced.

[0004] As a natural source of antibacterial agents, plant essential oils usually contain a variety of monomeric components with antibacterial activity, mainly terpene compounds, especially monoterpenes (C10) and sesquiterpenes (C15). In addition, essential oils may also contain diterpene compounds (C20) and other chemical components such as alcohols, aldehydes, phenols, ketones, aliphatic hydrocarbons, and lactone compounds. The unique properties of essential oils make them an indispensable part of traditional medicine, especially widely used in aspects such as antioxidant, antibacterial, anti-inflammatory, antispasmodic, and analgesic. Due to their strong aroma and pharmacological effects, essential oils are widely used in fields such as food preservation, topical medications, and daily health care. Research shows that essential oils and their components can effectively inhibit the growth of bacteria, yeasts, and molds, becoming a natural alternative to traditional chemical antibacterial agents. The mechanism of action of essential oils on bacteria is relatively complex and mainly exerts antibacterial effects through the following several ways: First of all, essential oils can damage the cell walls and cell membranes of bacteria, leading to cell leakage and death; secondly, they can interfere with the energy metabolism system of bacteria, changing the material flow and metabolic processes inside the cells; in addition, essential oils can also affect the protein synthesis, DNA replication, and metabolome of bacteria, changing cell morphology, inhibiting cell division and motility, and even reducing their pathogenicity by destroying the biofilms of bacteria. Some essential oil components can also regulate the signal transduction system of bacteria and affect their toxicity, further enhancing their antibacterial effect.

[0005] However, as a hydrophilic cationic photosensitizer, toluidine blue O (TBO) has insufficient membrane affinity due to its excessive hydrophilicity, and molecular aggregation easily reduces the ROS quantum yield, which reduces the PDT efficiency in these aspects. In addition, essential oils are volatile, have poor water solubility, and have poor stability under environmental conditions such as high temperature and light, which limit their further application in biomedical and other fields. At the same time, during the processing, transportation, storage, and consumption of essential oils, due to the action of factors such as high temperature, humidity, light, and oxygen, they may degrade, resulting in the failure of essential oils and even the production of toxic derivatives.

[0006] The literature (Wang Z., Bai H., Lu C., et al. Light controllable chitosan micelles with ROS generation and essential oil release for the treatment of bacterial biofilm [J]. Carbohydrate Polymers, 2019, 205: 533 - 539.) discloses light - controllable chitosan micelles loaded with thymol for eliminating biofilms. However, it fails to effectively improve the interaction between TBO and bacterial cells to enhance the PDT efficiency, nor does it improve the hydrophilicity of essential oils. Summary of the Invention

[0007] In order to solve the problem that the excessive hydrophilicity of TBO and the hydrophobicity of essential oils lead to low antibacterial activity of both. The object of the present invention is to propose a light - responsive - essential oil synergistic polymer and its application in combating multi - drug - resistant bacteria.

[0008] The present invention discloses a novel light - responsive - essential oil synergistic antibacterial polymer, which utilizes the synergistic effect of toluidine blue O (TBO) and citronellol (CT) to combat multi - drug - resistant bacteria.

[0009] The present invention discloses an application system based on the synergistic effect of the water - soluble cationic photosensitizer molecule toluidine blue O (TBO) and citronellol (CT) to combat multi - drug - resistant bacteria.

[0010] The object of the present invention is achieved through the following technical solutions.

[0011] A light - responsive - essential oil synergistic antibacterial polymer, the polymer includes a main chain and a graft chain. The main chain is a graft - polymerized high - molecular chain of glycidyl methacrylate monomer (GMA) and methyl methacrylate monomer with citronellol group (CMA), and the graft chain is grafted with graft monomer toluidine blue O (TBO) onto the main chain.

[0012] The preparation method of the above - mentioned light - responsive - essential oil synergistic antibacterial polymer includes the following steps:

[0013] (1) Synthesis of citronellyl methacrylate (CMA); in a solvent, citronellol reacts with acyl halide to synthesize CMA through an esterification reaction, and purification is carried out using silica gel column chromatography;

[0014] (2) Synthesis of polymer PG9C2; under nitrogen protection, using methyl 2-bromoisobutyrate as an initiator, glycidyl methacrylate (GMA) and CMA as monomers, CuBr as a catalyst, and 2,2'-bipyridine (BPY) as a ligand, an atom transfer radical polymerization reaction occurs to synthesize polymer PG9C2;

[0015] (3) Synthesis of antibacterial polymer PG9C2T1; under mild conditions, using acid catalysis, the amino group in the TBO molecule undergoes a ring-opening reaction with the epoxy group on the GMA monomer to obtain antibacterial polymer PG9C2T1.

[0016] The synthesis route of the monomer citronellyl methacrylate (CMA) is as follows:

[0017]

[0018] The synthesis routes of the polymers PG9C2 and PG9C2T1 are as follows:

[0019]

[0020] Furthermore, in step (1), the solvent is dichloromethane and the acyl halide is methacryloyl chloride.

[0021] Furthermore, in step (1), the molar ratio of citronellol to acyl halide is 1:1 - 1:1.2.

[0022] Furthermore, in step (1), the temperature of the esterification reaction is 0 - 30 °C and the reaction time is 4 - 8 h.

[0023] Furthermore, in step (2), the molar ratio of GMA to CMA is 5:1 - 5.5:1.

[0024] Furthermore, in step (2), the temperature of the polymerization reaction is 50 - 70 °C and the reaction time is 12 - 18 h.

[0025] Furthermore, in step (3), the acid is one of hydrochloric acid, sulfuric acid, and boric acid.

[0026] Furthermore, in step (3), the molar ratio of polymer PG9C2 to toluidine blue O is 1:15 - 1:20.

[0027] Furthermore, in step (3), the temperature of the ring-opening reaction is 25 - 35 °C and the reaction time is 8 - 12 h.

[0028] The application of the above-mentioned light-responsive essential oil synergistic antibacterial polymer against multi-drug resistant bacteria, especially Gram-positive bacteria such as Staphylococcus aureus and methicillin-resistant Staphylococcus aureus, has high antibacterial activity (the MIC values against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus are 62.5 μg / mL -1 , and the MIC value against Escherichia coli is 125 μg / mL -1 ).

[0029] The present invention proposes a novel light-responsive essential oil synergistic antibacterial polymer for combating multi-drug resistant bacteria. As a proof of concept, first, the hydrophobic citronellol was esterified with methacryloyl chloride to obtain the citronellyl methacrylate (CMA) monomer. Then, using methyl 2-bromoisobutyrate as the initiator, glycidyl methacrylate (GMA) and CMA as monomers, CuBr as the catalyst, and 2,2'-bipyridine (BPY) as the ligand, the polymer PG9C2 was synthesized by atom transfer radical polymerization (ATRP). Finally, the antibacterial polymer PG9C2T1 was obtained by the ring-opening reaction of the amino group in the TBO molecule with the epoxy group on the GMA monomer. The structures, degrees of polymerization, and dispersities of the polymers PG9C2 and PG9C2T1 were characterized by nuclear magnetic resonance hydrogen spectroscopy ( 1 1H NMR) and gel permeation chromatography (GPC). The in vitro photodynamic antibacterial experiment of the polymer PGCT was carried out to evaluate its antibacterial activity, and its singlet oxygen kinetics, biocompatibility, and accumulation in bacterial cells were characterized. A possible synergistic antibacterial mechanism was proposed through live-dead bacterial staining and changes in the morphology of bacterial cells.

[0030] The present invention enhances the antibacterial efficacy of PDT and optimizes the therapeutic effect of plant essential oils by improving the interaction between PS and bacterial cells, and constructs an antibacterial polymer based on the synergistic effect of the water-soluble cationic photosensitizer molecule toluidine blue O (TBO) and citronellol (CT) against multi-drug resistant bacteria. The polymer PG9C2 containing a citronellol structural unit was synthesized by atom transfer radical polymerization (ATRP). Subsequently, the antibacterial polymer PG9C2T1 was prepared by the directional modification of the TBO photosensitive group on the polymer side chain under mild conditions. This system innovatively integrates the cationic characteristics, singlet oxygen generation ability of TBO, and lipophilic characteristics of citronellol, and significantly improves the antibacterial performance through molecular-level synergistic effects.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] (1) An antibacterial polymer based on the synergistic effect of the water-soluble cationic photosensitizer molecule toluidine blue O (TBO) and citronellol (CT) against multidrug-resistant bacteria provided by the present invention. In vitro antibacterial experiments show that the polymer exhibits higher antibacterial activity than its single components. The improvement of its antibacterial activity is due to the fact that the polymer increases the lipophilicity of TBO, enhances its interaction with bacterial cells, increases its accumulation in cells, enables it to penetrate the cell membrane and play a role inside the cell, and improves the utilization efficiency of ROS. At the same time, it realizes sterilization by improving the water solubility of the essential oil and thus destroying the bacterial membrane.

[0033] (2) An antibacterial polymer based on the synergistic effect of the water-soluble cationic photosensitizer molecule toluidine blue O (TBO) and citronellol (CT) against multidrug-resistant bacteria provided by the present invention improves the water solubility of the essential oil by combining with the water-soluble photosensitizer molecule, and at the same time enhances the hydrophobicity of TBO, improving the PDT efficiency and the efficacy of the essential oil. The present invention provides ideas for designing highly efficient and low-toxic antibacterial materials based on natural products, breaks through the limitations of a single antibacterial mode, and provides a theoretical basis and technical path for the development of new antibacterial therapies with low drug resistance. Description of the Drawings

[0034] Figure 1 Schematic diagram of the mechanism of action of the photo-responsive - essential oil synergistic antibacterial polymer PG9C2T1 against multidrug-resistant bacteria.

[0035] Figure 2 For CT and CMA 1 1H NMR spectrum of the nuclear magnetic resonance hydrogen spectrum.

[0036] Figure 3 1H NMR spectrum of the polymers PG9C2 and PG9C2T1.

[0037] Figure 4 Kinetic spectrum of singlet oxygen generation of the polymer PG9C2T1, where (a) and (b) are the ultraviolet absorption changes under irradiation at different times; (c) is the ultraviolet absorption decay at 411 nm; (d) is the pseudo-first-order fitting curve.

[0038] Figure 5 Minimum inhibitory concentration (MIC) of the polymer PG9C2T1.

[0039] Figure 6 In vitro photodynamic antibacterial plate count of the polymer PG9C2T1.

[0040] Figure 7 Bioaccumulation of the polymer PG9C2T1 inside bacterial cells.

[0041] Figure 8 Diagram of the morphological changes of bacteria induced by the polymer PG9C2T1.

[0042] Figure 9 Effect diagram of live / dead bacteria staining induced by polymer PG9C2T1

[0043] Figure 10 Hemolysis test diagram of polymer PG9C2T1, where (a) is the hemolysis test result; (b) is the hemolysis test photo Detailed implementation manners

[0044] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some representative embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the present invention

[0045] Figure 1 Schematic diagram of the mechanism of action of the photo-responsive essential oil synergistic antibacterial polymer against multi-drug resistant bacteria, including the following information: First, the cationic property of the TBO structural unit mediates its electrostatic binding to the negatively charged bacterial cell surface; Second, the citronellol component promotes the adsorption and enrichment of the polymer in the bacterial lipid bilayer through hydrophobic interaction; Finally, light triggers the targeted generation of reactive oxygen species (ROS) in the membrane microenvironment, driving an efficient synergistic bactericidal process

[0046] Example 1:

[0047] Synthesize the monomer citronellyl methacrylate CMA

[0048] In a reaction system of a three-necked flask under nitrogen protection, citronellol (CT, 9.1 mL, 0.05 mol) and triethylamine (8.3 mL, 0.06 mol) were successively added and dissolved in 100 mL of dichloromethane solvent. After configuring the device with a condensing reflux system, the system was placed in an ice-water bath environment, the mechanical stirring was started and maintained at a constant speed. Methacryloyl chloride (5.3 mL, 0.06 mol) was slowly added through a constant-pressure dropping funnel, and the dropping rate was controlled to keep the reaction temperature within the range of 0 - 5 °C. The dropping process lasted for 30 min to ensure the homogenization of the reaction. Subsequently, the low-temperature environment was removed, and the reaction system was continuously stirred at room temperature for 8 h to complete the esterification process. After the reaction, the precipitated triethylamine hydrochloride was removed by vacuum filtration, and the obtained filtrate was concentrated to 1 / 3 of the original volume by a rotary evaporator. After the concentrated solution was transferred to a separatory funnel, liquid-liquid extraction was carried out three times (3×50 mL) with ether as the extractant. The organic phases were combined and washed once with 0.1 mol / L dilute hydrochloric acid and saturated sodium bicarbonate solution respectively to remove unreacted acyl chloride and basic by-products. The washed organic layer was added with excessive anhydrous magnesium sulfate and dried for 12 h to completely remove water, and then the desiccant was removed by filtration. After the ether was removed from the filtrate by vacuum distillation, purification was carried out by silica gel column chromatography, and a mixed solvent of n-hexane / ethyl acetate (volume ratio 9:1) was used as the eluent. The target component was collected and the solvent was removed by rotary evaporation to finally obtain a high-purity CMA product, which was stored in a -20 °C refrigerator for standby.

[0049] As Figure 2 shown, by comparing the 1 1H NMR spectra of CT and CMA, significant structural changes can be observed: Taking the olefinic proton (=CH-, 5.1 ppm, labeled as a) in the CT molecule as a reference, the chemical shift of the characteristic peak of C-CH2-O after the esterification reaction migrated from 3.7 ppm (labeled as b) to 4.2 ppm (labeled as b'). In addition, characteristic peaks of the methacryloyl group (CH2=, 5.6 ppm and 6.1 ppm, labeled as c') appeared in the CMA spectrum, and the integral area had a quantitative relationship with the corresponding number of protons. The displacement changes of these characteristic peaks and the appearance of new characteristic peaks, combined with the integral area analysis, fully confirmed the successful synthesis of high-purity CMA monomers.

[0050] Example 2:

[0051] Synthesis of GMA, CMA copolymer PG9C2

[0052] In a Schlenk reactor under nitrogen protection, bromoisobutyryl bromide (0.5 mL, 3.9 mmol), methyl methacrylate of citronellol obtained in Example 1 (CMA, 1.7 g, 7.5 mmol), glycidyl methacrylate (GMA, 5 mL, 38 mmol), and 2,2-bipyridine (BPY, 0.61 g, 3.9 mmol) were successively introduced and dispersed in a 15 mL tetrahydrofuran (THF) solvent system. After deoxygenating the system through three freeze-pump-thaw cycles, copper(I) bromide (CuBr, 0.56 g, 4.2 mmol) was added, and two more deoxygenation treatments were carried out to ensure an inert atmosphere. After sealing the reactor, it was continuously stirred in an oil bath at 55 °C for 12 h to complete atom transfer radical polymerization (ATRP). In the reaction termination stage, air was introduced into the system and it was quickly cooled to -196 °C (liquid nitrogen bath), and then the reaction mixture was diluted with THF. The copper catalyst was removed by column chromatography on neutral alumina, and the eluate was collected, concentrated by rotary evaporation, and then purified by precipitation twice with cold n-hexane (0 °C). The resulting white solid product was dried in a vacuum oven at 40 °C for 24 h to obtain the target polymer PG9C2.

[0053] Example 3:

[0054] Synthesis of photo-responsive - essential oil antibacterial polymer PG9C2T1

[0055] The polymer PG9C2 obtained in Example 2 (0.1 g, 0.05 mmol) and toluidine blue O (TBO, 0.3 g, 1 mmol) were placed in a single-necked reactor, 5 mL of dimethyl sulfoxide (DMSO) was added as a solvent, and hydrochloric acid (catalytic amount, 10% mmol) was introduced to promote the ring-opening of the epoxy group. After the system was deoxygenated by nitrogen bubbling for 10 min, the reaction was stirred at room temperature (25 °C) for 8 h. The reaction solution was transferred to a dialysis bag with a molecular weight cut-off of 500 Da and dialyzed against deionized water for 48 h (changing the medium every 12 h) to remove unreacted TBO and small molecule by-products. Finally, the dialysis solution was freeze-dried to obtain the ring-opening product PG9C2T1 as a dark blue powder.

[0056] Figure 3 1H NMR spectra of polymers PG9C2 and PG9C2T1. The spectrum of PG9C2 clearly shows the characteristic peaks (e and f) of the CMA monomer and the characteristic peaks (b, c, d) of the GMA monomer. Specifically, the signal at 3.63 ppm corresponds to the H a proton in the initiator, while the signals at 2.63 ppm, 2.83 ppm, 3.22 ppm, 3.78 ppm, and 4.3 ppm are attributed to the H c 、H b 、H dProtons. In addition, the signal at 5.1 ppm corresponds to the H in the CMA monomer e protons, while the H f proton signal of the H in the CMA monomer is shifted from 4.2 ppm to 3.98 ppm, further confirming the successful synthesis of PG9C2. In the spectrum of PG9C2T x , the signals appearing in the range of 7.27 ppm to 7.98 ppm correspond to the four hydrogen protons on the phenothiazine ring in the TBO molecule, indicating that TBO has been successfully grafted onto the polymer backbone through the ring-opening reaction. This result confirms the successful synthesis of PG9C2T x .

[0057] Example 4:

[0058] Determination of the singlet oxygen generation kinetics of polymer PG9C2T1

[0059] As a widely recognized 1 O2 detection molecular probe, 1,3-diphenylisobenzofuran (DPBF) exhibits fast reaction kinetics and high sensitivity to trace singlet oxygen. The irreversible oxidation of DPBF will cause a characteristic concentration-dependent attenuation of its ultraviolet-visible absorption spectrum, especially the decrease in absorbance at 411 nm.

[0060] A test solution was prepared by uniformly mixing 5 μM of polymer PG9C2T1 with the DPBF solution in DMSO. Subsequently, the reaction system was irradiated with a 660 nm laser source at 25 °C. Real-time spectrophotometric monitoring was carried out at 30-second intervals using a UV spectrophotometer and the equipped quartz cuvette. The absorbance measurement at 411 nm was baseline-corrected with the solvent blank. The generation kinetics of singlet oxygen was quantified by analyzing the change in absorbance with time through first-order derivative, and the 1O2 generation rate constant (k) was calculated using non-linear regression of the pseudo-first-order kinetic model: ln(A / A0) = kt, where A0 and A represent the initial absorbance and the time-dependent absorbance value, respectively.

[0061] As Figure 4 shown in (a) and (b) of 1 , the absorbance decay behavior of DPBF treated by light irradiation of polymer PG9C2T1 is highly similar to that of free TBO. The characteristic absorption peak at 411 nm in the experimental group shows a sharp downward trend, fully confirming that the polymer PG9C2T1 has significant photoinduced Figure 4 O2 generation ability. To further clarify the mechanism of the photodynamic reaction, Figure 4 in (c) shows the dynamic decay curve of the absorbance of DPBF at 411 nm, and the data was fitted and analyzed using the quasi-first-order kinetic model ( 1The rate constant for O2 formation can be determined by calculating the slope of the first-order kinetic equation shown in the figure. After calculation, the 1 O2 generation rate constants of TBO and PG9C2T1 are 0.0141 s -1 and 0.0148 s -1 respectively. The kinetic parameters of PG9C2T1 and TBO are highly close, indicating that the polymerization process has not significantly affected the photodynamic activity of the photosensitive group.

[0062] Example 5:

[0063] Determination of the minimum inhibitory concentration of polymer PG9C2T1, plate counting method

[0064] The MIC is defined as the lowest concentration of an antimicrobial agent that completely inhibits microbial growth in vitro. The microbroth dilution method was used in the experiment: The polymer PG9C2T1 (4 mg / mL) and LB medium were added to the first well of a 96-well plate at a ratio of 100 μL:80 μL, and after two-fold serial dilution, the final volume in each well was 180 μL. Bacterial suspensions of E. coli, S. aureus, and MRSA in the logarithmic growth phase (OD 600 = 0.1, approximately 10 8 CFU / mL) were diluted to 10 6 CFU / mL, and 20 μL of the bacterial suspension was added to each well. The experimental group was set with a light-irradiated group and a dark group. The light-irradiated group was irradiated under a 660 nm LED light source for 60 min. At the same time, a positive control (LB + bacterial suspension) and a negative control (pure LB) were set. After the well plate was sealed, it was cultured at 37 °C for 24 h. The MIC value was determined as the lowest concentration without turbidity observed with the naked eye, and three parallel samples were set for each experiment. Figure 5 The MIC values of the polymer against three multi-drug resistant bacteria are shown. For the E. coli strain, the polymer PG9C2T1 showed significantly enhanced antibacterial activity (125 μg mL -1 ) under 660 nm light irradiation, and compared with free TBO (500 μg mL -1 ), the activity was increased by 4-fold. This 4-fold enhancement indicates a synergistic effect between the terpene essential oil component and the photoactivated TBO moiety. The polymer PG9C2T1 also showed a relatively low MIC value (500 μg mL -1 ) under dark conditions, compared with TBO (2000 μg mL -1 ), which can be attributed to the enhanced membrane permeability due to the amphiphilic polymer-lipid interaction. Similar antibacterial enhancement patterns were observed for the S. aureus and MRSA strains.

[0065] Plate counting method: E. coli, S. aureus, and MRSA were all inoculated into LB broth medium and cultured with shaking at 37 °C to obtain a concentration of 10 7Bacterial suspension with a concentration of CFU / ml. To ensure sufficient absorption of bacteria by the sample, 100 μL of the bacterial suspension was taken and mixed with 500, 250, 125, 62.5, 31.25, and 15.625 μg mL -1 of the polymer PG9C2T1 and incubated in a gas bath thermostatic oscillator at 37 °C in the dark for 60 min. Then, 100 μL of the mixture was spread on an LB broth agar solid medium. After irradiation with a 660 nm LED light source for 60 min, it was incubated at 37 °C in the dark for 24 h, and the bacterial growth was observed and counted. As Figure 6 shown, under the condition of the same mass concentration, the number of colony-forming units on the plates of the three test strains after light treatment with the polymer PG9C2T1 was significantly less than that of the TBO group, and the bactericidal effect showed an obvious concentration dependence, indicating that the photo-responsive - essential oil synergistic antibacterial polymer PG9C2T1 has more excellent photodynamic antibacterial activity.

[0066] Example 6:

[0067] Bioaccumulation of polymer PG9C2T1

[0068] Keep the bacterial concentrations of E. coli, S. aureus, and MRSA at 10 8 CFU / mL. The experimental protocol included incubating each bacterial suspension with 100 μg mL -1 of the polymer PG9C2T1 in a gas bath thermostatic oscillator at 37 °C in the dark for 60 min. The incubated samples were centrifuged (3000 rpm, 5 min) to precipitate the bacterial cells, and then washed three times consecutively with PBS to remove the residual extracellular compounds. Fluorescence analysis was performed using a confocal laser scanning microscope (CLSM) with an excitation wavelength of 488 nm and the emission filter set to 5070 - 550 nm. Under the same magnification (100× oil immersion lens) and photomultiplier tube gain settings, bright-field and fluorescence images were captured to compare the intracellular fluorescence intensities of the TBO control group and the polymer group.

[0069] The results are as Figure 7As shown, fluorescence images of the accumulation and adhesion distribution of TBO and polymer PG9C2T1 in bacterial cells captured using laser scanning confocal microscopy (CLSM) technology. Significantly enhanced red fluorescence intensity was detected in the experimental groups of the three tested strains treated with polymer PG9C2T1, visually confirming that the polymer can effectively adhere to bacteria and has better photosensitizer enrichment ability. However, no corresponding red fluorescence was observed in the bacteria treated with the TBO control group. At the same time, the high consistency of the fluorescence signal with the bacterial localization in bright-field imaging further verified the effective accumulation of the polymer in bacterial cells. This is attributed to the lipophilic characteristics of the polymer, the tendency of molecular aggregation, and the strong interaction between essential oil molecules and bacterial cells, which improve the accumulation of TBO in bacterial cells. These physicochemical properties are beneficial for it to penetrate the cell membrane barrier and achieve intracellular penetration.

[0070] Example 7:

[0071] Morphological changes of bacteria induced by polymer PG9C2T1

[0072] Keep the bacterial concentrations of E. coli, S. aureus and MRSA at 10 8 CFU / mL, add 5 mL of the bacterial suspension and 100 μg / mL -1 of polymer PG9C2T1, incubate in a gas bath thermostatic oscillator at 37 °C in the dark for 60 min, and then irradiate with a 660 nm light source for 60 min. Collect 1 mL of the mixture, centrifuge the sample (5000 rpm, 5 min) to precipitate the bacterial cells and collect the bacterial cells, and then wash three times continuously with PBS. The obtained precipitate was fixed in 2.5% glutaraldehyde at 4 °C for 12 h. After completion, wash three times continuously with PBS, and then dehydrate the sample with a series of graded ethanol solutions (30, 50, 70, 85, 95 and 100%). The dehydrated sample was vacuum dried and used for SEM characterization.

[0073] The results are as Figure 8 shown. The ultrastructural changes of the bacterial cells before and after treatment with PG9C2T1 under light illumination were observed using a scanning electron microscope (SEM) system. It can be seen that the bacteria in the control group showed typical complete morphological characteristics: the cell outline was clear, a small amount of vesicle structures and localized depressions were visible on the membrane surface, but no membrane lysis phenomenon was observed. In contrast, large-area membrane structure collapse and disintegration occurred in the bacterial cells treated with PG9C2T1, confirming that the polymer can achieve bactericidal effects by deforming bacterial cells through destroying the bacterial cell structure.

[0074] Example 8:

[0075] Live and dead bacteria staining experiment

[0076] Using the LIVE / DEAD BacLight Bacterial Viability Kit, the integrity of the bacterial membrane was detected by CLSM. E. coli, S. aureus and MRSA bacterial suspensions (10 8 CFU / mL) were incubated with 100 μg / mL -1 of the polymer PG9C2T1 in the dark at 37 °C for 30 min and then irradiated with a 660 nm light source for 60 min. The suspension (1 mL) was collected, centrifuged at 5000 rpm for 5 min, and then the supernatant was removed and washed three times with PBS. The bacteria were resuspended in PBS (1 mL), and the dyes SYTO9 (1.5 μL) and PI (1.5 μL) were added. The mixture was incubated in the dark for 15 min, and 10 μL was dropped onto a glass slide. Fluorescence was observed using a dual-channel CLSM. The excitation wavelengths of SYTO 9 and PI were 488 nm and 535 nm, respectively. The emission wavelengths of SYTO 9 and PI were 500 - 540 nm and 583 - 688 nm, respectively.

[0077] The results are as Figure 9 shown. The changes in bacterial membrane permeability before and after polymer treatment were evaluated by the dual-fluorescence labeling method. In the experiment, the specific membrane integrity probe propidium iodide (PI) was used to label bacteria with damaged membranes (red fluorescence, emission wavelength 525 nm), and at the same time, SYTO 9 (green fluorescence) was used to stain live bacteria to accurately distinguish the survival status of the bacterial population. Under light illumination, all three experimental bacteria in the PG9C2T1 treatment group showed significant characteristics of membrane damage: the red fluorescence signal of PI was significantly enhanced, while the intensity of the green fluorescence of SYTO 9 decreased sharply. This result indicates that the photodynamic action induced by the polymer can lead to the loss of bacterial membrane integrity and permeability changes, thereby inducing a lethal effect. It is worth noting that weak red fluorescence was also detected under dark conditions, suggesting that PG9C2T1 may have inherent antibacterial activity independent of light illumination.

[0078] Example 9:

[0079] Hemolysis test of the polymer PG9C2T1

[0080] The hemolysis of the polymer was evaluated by the rabbit red blood cell hemolysis test. Fresh rabbit blood red blood cells were slowly aspirated into a centrifuge tube, and then a certain amount of PBS was added for centrifugal washing. The supernatant was discarded to remove the broken blood cells and plasma in the blood, and the process was repeated 2-3 times until the supernatant after centrifugation remained transparent. After the last centrifugation to remove the supernatant, the mass of the remaining red blood cells was weighed, and the red blood cells were diluted with PBS buffer to a content of 5% (V / V). The sample was prepared with PBS buffer, and PBS and triton were used as negative and positive controls respectively. The polymer PG9C2T1 and the red blood cell solution were incubated in the dark at 37 °C in a gas bath constant temperature oscillator for 2 h as the dark group, and at the same time, a light group was set to incubate for 1 h and then irradiated for 30 min. After the culture was completed, the culture solution was centrifuged, 100 μL of the supernatant was taken and placed in a 96-well plate, and the absorbance value A of OD540 was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The calculation formula for the hemolysis rate is as follows:

[0081] Hemolysis rate (%) = (A - A n ) / (A p - A n ) × 100%

[0082] where A, A n , A p are the absorbance values of the experimental group, negative control group and positive control group respectively.

[0083] The results are as Figure 10 shown. The potential application value of the polymer was evaluated by testing its hemolytic activity. At the equivalent bactericidal concentration, the hemolysis rate of the polymer PG9C2T1 showed a positive correlation trend with the increase in concentration, which was due to the non-specific cell action caused by the overall cationic characteristics conferred by the high loading of TBO in the polymer. It is worth noting that the hemolysis rate increased significantly after 1 h of light treatment, but the overall value still remained at a low level, which was related to the cytotoxic effect of 1 O2 generated under photodynamic action. The experimental data comprehensively showed that PG9C2T1 had good biosafety under dark conditions, and its photoactivated state could produce a controllable cell damage effect, which was consistent with the design concept of selective cytotoxicity required for its use as a targeted antibacterial agent.

Claims

1. A light-responsive-essential oil synergistic antibacterial polymer, characterized in that: The polymer comprises a main chain and a graft chain. The main chain is a polymer chain grafted with glycidyl methacrylate monomer GMA and citronellol methyl methacrylate monomer CMA. The graft chain is grafted on the main chain with graft monomer TBO.

2. The method for preparing a light-responsive-essential oil synergistic antibacterial polymer according to claim 1, characterized in that: The following steps are involved: (1) Synthesizing citronellol methyl methacrylate (CMA); in a solvent, citronellol and an acyl halide undergo an esterification reaction to synthesize CMA, and purify the CMA using a silica gel column chromatography; (2) Synthesis of polymer PG9C2; under nitrogen protection, methyl 2-bromoisobutyrate was used as an initiator, glycidyl methacrylate GMA and citronellol methyl methacrylate CMA were used as monomers, CuBr was used as a catalyst, and 2,2-bipyridine (BPY) was used as a ligand to synthesize polymer PG9C2 by atom transfer radical polymerization; (3) Synthesis of the antibacterial polymer PG9C2T1; Under mild conditions, the amino group in the TBO molecule and the epoxy group on the GMA monomer were catalyzed by acid to undergo a ring-opening reaction to obtain the antibacterial polymer PG9C2T1.

3. The preparation method according to claim 2, characterized in that: In step (1), the solvent is dichloromethane and the acyl halide is methacryloyl chloride.

4. The preparation method according to claim 2, characterized in that: In step (1), the molar ratio of citronellol to acyl halide is 1:1-1:1.

2.

5. The preparation method according to claim 2, characterized in that: In step (1), the temperature of the esterification reaction is 50-70° C., and the reaction time is 4-8 hours.

6. The preparation method according to claim 2, characterized in that: In step (2), the molar ratio of GMA to CMA is 5:1-5.5:

1.

7. The preparation method according to claim 2, characterized in that: The polymerization reaction temperature is 50-70° C., and the reaction time is 12-18 hours.

8. The preparation method according to claim 2, characterized in that: In step (3), the molar ratio of polymer PG9C2 to toluidine blue O is 1:15-1:

20.

9. The preparation method according to claim 2, characterized in that: In step (3), the temperature of the ring-opening reaction is 25-35° C., and the reaction time is 8-12 h.

10. Use of a light-responsive-essential oil synergistic antibacterial polymer according to claim 1 in combating multi-drug resistant bacteria, characterized in that: The multi-drug resistant bacteria include Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus.

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