Cationic polymer and its preparation method and application

Abstract

This invention discloses a cationic polymer, its preparation method, and its applications, belonging to the field of polymer materials technology. The structural formula is shown in any one of formulas (I)-(IV). The preparation method includes: catalyzing the reaction of 2-acetylphenothiazine or 2-acetylphenotoxazine with 1-bromo-4-chlorobutane in an organic solvent using a catalyst to obtain a first intermediate; catalyzing the reaction of the first intermediate with malononitrile in an organic solvent using a catalyst to obtain a second intermediate; subjecting the monomers to free radical polymerization under the action of an initiator to obtain a third intermediate; and reacting the second and third intermediates in an organic solvent to obtain the cationic polymer. This cationic polymer exhibits good biocompatibility, has inhibitory effects on various bacteria, and can act as a sensitizer, significantly reducing the concentration of antibiotics used and achieving antibacterial treatment under low-concentration drug action.

Description

Technical Field

[0001] This invention relates to the field of polymer materials technology, specifically to a cationic polymer, its preparation method, and its applications. Background Technology

[0002] Phototherapy, capable of in-situ treatment of lesions under light-induced conditions, has opened new avenues for disease treatment research and attracted considerable interest. Photodynamic therapy (PDT), as a type of phototherapy, offers advantages such as controllable light, non-invasiveness, and effectiveness. Bacterial infectious diseases seriously threaten human health and even life, posing a significant global challenge. Increasing evidence suggests that bacteria can indirectly contribute to the development of other diseases. The overuse of antibiotics has led to the emergence of multidrug-resistant bacteria, making antibiotic resistance a major threat to global healthcare. The World Health Organization's "Priority Pathogens List" specifically emphasizes the severity of antibiotic-resistant Gram-negative bacteria, with carbapenem-resistant Gram-negative bacteria listed as the most important pathogens.

[0003] Developing novel antibacterial drugs is one of the effective ways to treat bacterial infectious diseases, aiming to combat bacterial resistance and improve treatment efficacy. Chinese patent document CN109705262A discloses a cationic antibacterial polymer, its preparation method, and its application. This cationic antibacterial polymer is methyl methacrylate-co-dimethylaminoethyl methacrylate bromoethane, abbreviated as P(MMA). n -co-DMAEMA m + C2H5Br -(n) represents the molar content of methyl methacrylate in the cationic polymer, and m represents the molar content of poly(dimethylaminoethyl methacrylate) bromoethane. The polymer synthesized by this invention has good antibacterial properties, which can be adjusted by modifying the polymer chain segments. Chinese patent document CN113416309A discloses a sulfonium salt cationic polymer with antibacterial properties. This invention designs a sulfonium salt cationic polymer with a sulfonium salt structure in the main chain. It utilizes the interaction between the sulfonium salt cation in the molecular structure and the electronegative structure on the surface of the bacterial cell membrane, while coordinating with the remaining hydrophilic and hydrophobic components in the molecular structure. The group acts on the bacterial cell membrane, causing the bacterial cell membrane to disintegrate and thus killing the bacteria; Chinese patent document with publication number CN113929892A discloses a metal-containing high-efficiency cationic antibacterial polymer, which is composed of a macromolecular polyester backbone and side groups, wherein the macromolecular polyester backbone is generated by the condensation of trimethylolpropane allyl ether and diacyl chloride, and the side groups are dimethylaminopyridinium amine-metal complexes; because the dimethylaminopyridinium amine metal complexes have a strong coordination interaction with phosphatidylserine on the surface of the bacterial membrane, the metal cationic polymer of this invention can interact strongly with the bacterial membrane.

[0004] However, there are few reports of antibacterial cationic polymers that combine AIE properties and ROS generation capabilities in the current technology. Summary of the Invention

[0005] This invention provides a cationic polymer with an alkyl chain as the main chain and quaternary ammonium salts modified with phenothiazine / phenotoxazine and malononitrile derivatives as side chains. It can be used to generate reactive oxygen species (ROS) in response to light to inhibit the growth and proliferation of bacteria, and can enhance the antibacterial effect in synergy with traditional antibiotics.

[0006] The specific technical solution adopted is as follows:

[0007] A cationic polymer, with a structural formula as shown in any one of formulas (I)-(IV):

[0008]

[0009] In equations (I)-(IV), 10 > n > 2, and X is a halogen.

[0010] The present invention also provides a method for preparing the aforementioned cationic polymer, specifically comprising the following steps:

[0011] (1) Under the protection of an inert gas, 2-acetylphenthiazide or 2-acetylphenoxazine and 1-bromo-4-chlorobutane are reacted in an organic solvent by a catalyst to obtain the first intermediate;

[0012] (2) Under the protection of an inert gas, the first intermediate and malononitrile are reacted in an organic solvent by a catalyst to obtain the second intermediate;

[0013] (3) Under the action of an initiator, the monomer undergoes free radical polymerization to obtain a third intermediate; the second and third intermediates are reacted in an organic solvent to obtain the cationic polymer.

[0014] This invention synthesizes a polymer with an alkyl chain as the main chain and tertiary amine groups on the side chains via free radical polymerization. By quaternizing the tertiary amines on the side chains and introducing cations, aggregation-induced emission (AIE) units, and photosensitizer units into the side chains, an AIE cationic polymer with ROS-generating capability is obtained. Since bacteria have a negatively charged surface, this cationic polymer can interact with bacteria to inhibit their growth and proliferation.

[0015] Preferably, in step (1), the catalyst is sodium hydride.

[0016] Preferably, in step (1), the reaction conditions for 2-acetylphenothiazine or 2-acetylphenotoxazine and 1-bromo-4-chlorobutane are ice bath, 2h-10h.

[0017] Preferably, in step (2), the catalyst is amine acetate-acetic acid.

[0018] Preferably, in step (2), the reaction conditions for the first intermediate and malononitrile are 80-150℃ for 2-10h.

[0019] In step (3), the polymerization monomer includes propyl 3-(dimethylamino)methacrylate or dimethylallylamine; the initiator includes, but is not limited to, azobisisobutyronitrile, etc.

[0020] Preferably, in step (3), the reaction conditions for the second and third intermediates are 40-90°C for 12-96 hours.

[0021] This invention also provides the application of the aforementioned cationic polymer in the preparation of antibacterial agents; the cationic polymer has good biocompatibility and acts as an antibacterial photosensitizer to inhibit various bacteria (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus MRSA, Escherichia coli, carbapenem-resistant multidrug-resistant Escherichia coli CREc, etc.).

[0022] By controlling the side chain length of this cationic polymer, its solubility, reactive oxygen species (ROS) generation performance, and antibacterial effect can be controlled.

[0023] The present invention also provides an antibacterial drug comprising the aforementioned cationic polymer and an antibiotic, wherein the antibiotic includes, but is not limited to, levofloxacin (LVFX), moxifloxacin, imipenem, meropenem, vancomycin, etc.

[0024] Studies have found that this cationic polymer has an inhibitory effect on a variety of bacteria. When it works synergistically with antibiotics such as levofloxacin, it can reduce the antibacterial concentration while improving the antibacterial effect.

[0025] The aforementioned antibacterial drug is a topical preparation, used in conjunction with light treatment. The cationic polymer, upon light exposure, generates reactive oxygen species (ROS) in a photoresponsive manner, achieving energy conversion. Furthermore, it synergistically enhances the antibacterial effect with traditional antibiotics, enabling bacterial inhibition at low antibiotic concentrations.

[0026] More preferably, the conditions for the light treatment are: light density 0.01~200mW / cm². 2 Irradiation time: 0.5–40 min.

[0027] Preferably, in the antibacterial drug, the mass ratio of cationic polymer to antibiotic is 10-80:1.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] (1) This invention synthesizes a polymer with an alkyl chain as the main chain and tertiary amine groups on the side chain by free radical polymerization reaction. By quaternizing the tertiary amines on the side chain, cations, aggregation-induced emission (AIE) motifs and photosensitizer motifs are introduced into the side chain to obtain an AIE cationic polymer with ROS generation capability.

[0030] (2) The cationic polymer provided by the present invention has good biocompatibility and causes little or no damage to normal cells. At the same time, as a photosensitizer, it has an inhibitory effect on a variety of bacteria, especially the growth and proliferation of carbapenem-resistant multidrug-resistant Escherichia coli and other drug-resistant bacteria.

[0031] (3) The present invention uses the cationic polymer and antibiotics such as levofloxacin in combination, and with the aid of light conditions, the MIC value (minimum inhibitory concentration) of the antibiotic can be greatly reduced. That is, the cationic polymer can act as a sensitizer, greatly reducing the concentration of antibiotics used, and achieving antibacterial treatment under the action of low concentration of drugs. Attached Figure Description

[0032] Figure 1 This is a schematic diagram illustrating the synergistic antibacterial effect of the cationic polymer and levofloxacin.

[0033] Figure 2The graph shows the ROS generation efficiency of the cationic polymer and the control small molecule detected using the fluorescent probe DCFH. In the graph, A is PON-CN+DCFH, B is PN-CN+DCFH, C is the second intermediate CN+DCFH, D is rose red RB+DCFH, E is DCFH, and F is the reactive oxygen species ratio curve.

[0034] Figure 3 The graphs show the inhibition rates of cationic polymers against Staphylococcus aureus (S. aureus) and methicillin-resistant Staphylococcus aureus (MRSA) under dark and light conditions. In graph A, PON-CN inhibits S. aureus and MRSA, while graph B shows the inhibition rates of PN-CN against these bacteria.

[0035] Figure 4 The graphs show the inhibition rates of cationic polymers against Escherichia coli (E. coli) and carbapenem-resistant multidrug-resistant E. coli (CREc) under dark and light conditions. A represents the inhibition rate of PON-CN against E. coli and carbapenem-resistant multidrug-resistant E. coli (CREc), and B represents the inhibition rate of PN-CN against E. coli and carbapenem-resistant multidrug-resistant E. coli (CREc).

[0036] Figure 5 The graph shows the synergistic inhibition rate of cationic polymers and LVFX against carbapenem-resistant multidrug-resistant Escherichia coli (CREc) under dark and light conditions. A represents the synergistic effect of PON-CN and LVFX under dark conditions, B represents the synergistic effect of PON-CN and LVFX under dark conditions, C represents the synergistic effect of PON-CN and LVFX under light conditions, and D represents the synergistic effect of PON-CN and LVFX under light conditions.

[0037] Figure 6 The graphs show the zeta potential statistics. A represents the zeta potential statistics for PON-CN, PON-CN+LVFX, and PON-CN+LVFX and LVFX after 30 minutes. B represents the zeta potential statistics for PN-CN, PN-CN+LVFX, and PN-CN+LVFX and LVFX after 30 minutes. C represents the zeta potential statistics for CREc, CREc+PON-CN, CREc+PON-CN+LVFX, and CREc+LVFX. D represents the zeta potential statistics for CREc, CREc+PN-CN, CREc+PN-CN+LVFX, and CREc+LVFX.

[0038] Figure 7This is a schematic diagram illustrating the synergistic effect of cationic polymers and levofloxacin, combined with white light irradiation, in treating skin inflammation in mice. Detailed Implementation

[0039] The present invention will be further described below with reference to the following examples and accompanying drawings. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0040] Example 1: Synthesis of cationic polymer PON-CN

[0041] (1) The structural formula of the cationic polymer PON-CN is shown below:

[0042]

[0043] (2) The synthetic route of the second intermediate CN is shown in the following formula:

[0044]

[0045] 2-Acetylphenothiazine (2 g, 8.30 mmol), 1-bromo-4-chlorobutane (2.82 g, 16.59 mmol), and sodium hydride (0.96 g, 40 mmol) were added to a 250 mL two-necked flask equipped with a condenser. The nitrogen gas was purged three times. N,N-dimethylformamide (30 mL) was injected under nitrogen protection. After the injection was completed, the mixture was reacted in an ice bath for 4 h. The reaction was quenched with water, and the precipitate was rapidly stirred in a saturated sodium chloride solution. The precipitate was then filtered, filtered through a column, and the DCM:PE ratio was 1:1 to obtain the first intermediate. The intermediate was then dried overnight in a vacuum oven.

[0046] The first intermediate (0.3 g, 0.83 mmol) and malononitrile (0.165 g, 2.49 mmol) were added to a 250 mL two-necked flask with a condenser and a water separator. The nitrogen gas was purged three times. Ammonium acetate (0.218 g, 2.83 mmol) was dissolved in acetic acid (10 mL). Toluene (30 mL) and an acetic acid-ammonium acetate solution were injected under nitrogen protection. After the injection was completed, the mixture was heated to reflux at 110 °C for 2 h to obtain the second intermediate CN in 34% yield.

[0047] The 1H NMR spectrum data of the second intermediate CN are as follows: 1 H NMR(400MHz,Chloroform-d)δ7.22–7.17(m,2H),7.14–7.10(m,1H),7.07–7.03(m,2H),6.97(t d,J=7.5,1.2Hz,1H),6.89(dd,1H),3.93(t,2H),3.56(t,2H),2.61(s,3H),2.08–1.85(m,2H).

[0048] (3) The synthetic route of the cationic polymer PON-CN is shown in the following formula:

[0049]

[0050] 3-(dimethylamino)methacrylate (0.342 g, 2 mmol) and azobisisobutyronitrile (0.012 mg, 0.12 mmol) were added to a 25 mL polymerization tube. The nitrogen gas was purged three times. Ethanol (1 mL) was injected under nitrogen protection. After the injection was completed, the reaction was carried out at 70 °C for 24 h. After the reaction was completed, 0.5 mL of dichloromethane was added to aid dissolution. The reaction solution was slowly added dropwise to n-hexane under rapid stirring to precipitate the third intermediate, which was then dried overnight in a vacuum oven.

[0051] The third intermediate (0.03 g, 0.175 mmol) and the second intermediate CN (0.143 g, 1.75 mmol) were added to a 25 mL polymerization tube, and tetrahydrofuran (2 mL) was injected. After the injection was complete, the reaction was carried out at 70 °C for 48 h. After the reaction was completed, 0.5 mL of dichloromethane was added to aid dissolution. The reaction solution was slowly added dropwise to n-hexane under rapid stirring to precipitate the cationic polymer PON-CN. The polymer was dried overnight in a vacuum oven. The yield of cationic polymer PON-CN was 40%.

[0052] Molecular weight test results show that the cationic polymer PON-CN has 10 > n > 2, where n is a non-integer.

[0053] The 1H NMR data for the cationic polymer PON-CN are as follows: 1 H NMR(400MHz,Chloroform-d)δ7.22–7.17(m,2H),7.14–7.10(m,1H),7.07–7.03(m,2H),6.97(t d,J=7.5,1.2Hz,1H),6.89(dd,1H),3.93(t,2H),3.56(t,2H),2.61(s,3H),2.08–1.85(m,2H).

[0054] Example 2 Synthesis of cationic polymer PN-CN

[0055] The only difference between this embodiment and Example 1 is that the polymerizing monomer is replaced with dimethylallylamine; the second intermediate CN prepared in Example 1 is reacted with the third intermediate formed by the free radical polymerization of dimethylallylamine to obtain the cationic polymer PN-CN. The synthetic route is shown in the following formula:

[0056]

[0057] The 1H NMR spectrum data of the cationic polymer PN-CN are as follows: 1 ¹H NMR (400MHz, Chloroform-d) δ 7.21–7.10 (m, 3H), 7.04–6.78 (m, 4H), 4.05–3.79 (m, 2H), 3.64 (s, 2H), 3.56 (q, J = 5.8, 5.4 Hz, 3H), 2.16–1.68 (m, 9H), 1.26 (s, 4H). Sample analysis

[0058] The Staphylococcus aureus and Escherichia coli used in the experiment were purchased, while methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant multidrug-resistant Escherichia coli (CREc) were clinically isolated from the Third Affiliated Hospital of Guangzhou Medical University.

[0059] The method for testing the antibacterial rate was as follows: 10 mL of fresh LB medium was poured into a 50 mL centrifuge tube, and 10 μL of bacterial suspension was injected into the corresponding centrifuge tube. The tubes were then incubated overnight on a shaker (37℃, 1700 r / min). The bacterial suspension was washed three times with PBS and mixed with the sample to be tested to prepare mixtures of different concentrations. The bacterial suspension was then diluted to 5.0 × 10⁻⁶. 5 The bacterial count was set at 50,000 cells / mL. 100 μL of this mixture was then added to a 96-well plate (50,000 bacteria per well). The plate was shaken for 10 seconds, and the absorbance at 600 nm was measured using a microplate reader. The plate was then incubated on a shaker for 16 hours (37°C, 1700 rpm). If illumination was required, the plate was incubated on a shaker for 1 hour, then placed under a white light lamp (40 mW / cm²). 2 After 20 minutes, place the 96-well plate on a shaker and continue culturing for 15 hours (37℃, 1700 r / min). After culturing, place the plate on a microplate reader, shake for 10 seconds to disperse the bacteria, and measure the absorbance at 600 nm to calculate the inhibition rate.

[0060] The method for testing the efficacy of antibacterial drugs is as follows: a 1.5×1.5cm incision is made on the back. 2 A circular wound was created on the mouse. CREc (50 μL, 5 × 10⁻⁶ ml) was applied to the wound. 8 / each), wait 4 days for the wound to suppurate, then apply PON-CN (50μL, 150μM), LVFX (50μL, 3.5μg / mL), or PON-CN+LVFX (50μL, 150μM+3.5μg / mL) to the wound for 1 hour, and then irradiate the wound with a white light lamp (150mW / cm²). 2 (20 min) Both the PBS and PON-CN+LVFX groups had one group requiring illumination and one group not requiring illumination. The PON-CN group only required illumination, while the LVFX group did not require illumination.

[0061] The ROS generation efficiency of the cationic polymer and the control small molecule was detected using the fluorescent probe DCFH. The results are as follows: Figure 2 As shown in Figure AE, the reactive oxygen species ratio curve is as follows: Figure 2 As shown in F, the results indicate that the cationic polymer PON-CN has the highest ROS yield, followed by the cationic polymer PN-CN.

[0062] The statistical graphs showing the inhibition rates of cationic polymers against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) under dark and light conditions are as follows: Figure 3 As shown in A and B, the results indicate that the cationic polymers PON-CN and PN-CN can significantly inhibit the growth and proliferation of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) at low concentrations under light conditions.

[0063] The statistical graphs of the inhibition rates of cationic polymers against *Escherichia coli* and carbapenem-resistant multidrug-resistant *Escherichia coli* CREc under dark and light conditions are shown below. Figure 4 As shown in A and B, the cationic polymers PON-CN and PN-CN exhibit excellent antibacterial effects against Escherichia coli (E. coli) under light conditions, and their antibacterial effect against carbapenem-resistant multidrug-resistant E. coli (CREc) increases with increasing concentration.

[0064] Figure AD shows the statistical graph of the inhibition rate of cationic polymer and LVFX synergistically against carbapenem-resistant multidrug-resistant Escherichia coli CREc under dark and light conditions. It indicates that the antibacterial effect of cationic polymer and LVFX synergistically is better under light conditions.

[0065] Figure 6 The zeta potential statistics show that the cationic polymer interacted with carbapenem-resistant multidrug-resistant Escherichia coli (CREc), and the cationic polymer + levofloxacin also interacted with carbapenem-resistant multidrug-resistant Escherichia coli (CREc).

[0066] The schematic diagram of the synergistic antibacterial effect of the cationic polymer and levofloxacin is shown below. Figure 1 As shown in the diagram, the synergistic effect of cationic polymers and levofloxacin, combined with white light irradiation, cures skin inflammation in mice. Figure 7 As shown in the results of the antibacterial drug efficacy test, the skin of mice healed after 10 days of treatment with cationic polymer and levofloxacin combined with white light irradiation.

[0067] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cationic polymer, characterized in that, The structural formula is shown in any one of formulas (Ⅰ)-(Ⅱ): In equations (Ⅰ)-(Ⅱ), 10 > n > 2, and X is a halogen.

2. The method for preparing the cationic polymer according to claim 1, characterized in that, Includes the following steps: (1) Under the protection of an inert gas, 2-acetylphenthiazide and 1-bromo-4-chlorobutane are reacted in an organic solvent by a catalyst to obtain the first intermediate; (2) Under the protection of an inert gas, the first intermediate and malononitrile are reacted in an organic solvent by a catalyst to obtain the second intermediate; (3) Under the action of an initiator, the monomer undergoes free radical polymerization to obtain a third intermediate; the second and third intermediates are reacted in an organic solvent to obtain the cationic polymer.

3. The method for preparing the cationic polymer according to claim 2, characterized in that, In step (1), the catalyst is sodium hydride; in step (2), the catalyst is amine acetate-acetic acid.

4. The method for preparing the cationic polymer according to claim 2, characterized in that, In step (1), the reaction conditions for 2-acetylphenthiazide and 1-bromo-4-chlorobutane are ice bath, 2h-10h.

5. The method for preparing the cationic polymer according to claim 2, characterized in that, In step (2), the reaction conditions for the first intermediate and malononitrile are 80-150℃ for 2-10h.

6. The method for preparing the cationic polymer according to claim 2, characterized in that, In step (3), the polymer monomers include propyl 3-(dimethylamino)methacrylate or dimethylallylamine.

7. The method for preparing the cationic polymer according to claim 2, characterized in that, In step (3), the reaction conditions for the second and third intermediates are 40-90℃ for 12-96h.

8. The use of the cationic polymer according to claim 1 in the preparation of antibacterial agents.

9. An antibacterial drug, characterized in that, The invention includes the cationic polymer of claim 1 and the antibiotic, wherein the antibiotic includes levofloxacin, moxifloxacin, imipenem, meropenem or vancomycin.

10. The antibacterial drug according to claim 9, characterized in that, The antibacterial drug is a topical preparation, used in conjunction with light treatment.

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