Guanidyl cationic polymer capable of efficiently removing persistent bacteria as well as preparation method and application of guanidyl cationic polymer

Abstract

The invention provides a guanidyl cationic polymer capable of efficiently removing persistent bacteria as well as a preparation method and application of the guanidyl cationic polymer, and belongs to the technical field of biomedical engineering materials. According to the guanidyl cationic polymer, a monomer containing guanidyl at the tail end serves as a cationic block unit, a series of spacer arm front-end atom types with variable side chain spacer arm lengths (ethane-, propane-and pentane-) are designed, x spacer arm lengths and the number m of the cationic block units are 20-50, and therefore the chemical formulas are PN2G, PN3G, PN6G, PO2G, PO3G and PO6G respectively. According to the guanidyl cationic polymer, efficient retention bacteria removal is achieved by directly influencing a bacterial cell membrane mechanism, the effective concentration of the cationic polymer to the retention bacteria can be as low as 4 micrograms per milliliter, and the effect is better than that of current first-line antibiotics and contrast.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering materials, and in particular to a guanidine-based cationic polymer for efficiently removing persister bacteria, and a preparation method and application thereof. Background Art

[0002] Persistence is a survival mechanism that bacteria exhibit in response to a series of adverse environmental conditions during the exponential growth phase. It is a type of microorganism that can spontaneously or passively enter a certain "quiescent" state due to environmental factors during growth, and is a cell subpopulation that has extremely strong tolerance to antimicrobial drugs. When external pressure exists, persisters will maintain long-term survival by regulating metabolic processes and energy metabolism in a slow-growing manner. Currently, persisters are mainly treated with antibiotics. However, large doses and long-term antibiotics are usually prone to drug resistance and are difficult to be effective against persistent bacteria. The main reason is that antibiotics usually require DNA / RNA replication, protein synthesis or cell wall expansion to take effect. However, because bacteria enter a persistent state, the antibiotic target is lost and the antibiotic becomes ineffective.

[0003] Therefore, how to obtain a compound preparation that can effectively eliminate persistent bacteria is a technical problem that needs to be solved at present. Summary of the Invention

[0004] The purpose of the present invention is to provide a guanidine-based cationic polymer for efficiently removing persister bacteria, and its preparation method and application, so as to solve the technical problem that antibiotics produce resistance to the removal of persister bacteria and the removal effect is poor.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a guanidine-based cationic polymer that can effectively remove persistent bacteria. The chemical formula of the guanidine-based cationic polymer is PN x G or PO x G, the structural formula is as follows:

[0007]

[0008] Where x is independently 1 to 6, and m is 20 to 50;

[0009]

[0010] Furthermore, the structural formula of the guanidine-based cationic polymer is selected from one of the following structural formulas:

[0011]

[0012] m=20~50.

[0013] The present invention also provides a method for preparing a guanidine-based cationic polymer that is efficient in removing persistent bacteria, comprising the following steps:

[0014] 1) mixing a polymerization monomer, a chain transfer agent, and an initiator in an organic solvent, and reacting the mixture under heating or blue light to obtain an intermediate polymer;

[0015] 2) mixing the intermediate polymer, 1H-pyrazole-1-carboxamidine hydrochloride, and N,N-diisopropylethylamine in water, and dialyzing and drying the resulting reaction mixture to obtain a dry polymer;

[0016] 3) mixing the dried polymer, water, and hydrogen peroxide solution and reacting them to remove the alkyl chain, and the reaction product is purified by dialysis and freeze-dried to obtain a guanidine-based cationic polymer;

[0017] The polymerizable monomers include 2-aminoethyl methacrylate hydrochloride, N-(3-aminopropyl)methacrylamide hydrochloride, methacrylated 6-(BOC-amino)-1-hexanol, 2-aminoethyl methacrylate hydrochloride, methacrylated tert-butyl N-(3-hydroxypropyl)carbamate, or methacrylated tert-butyl N-(6-aminohexyl)carbamate.

[0018] Further, the chain transfer agent comprises 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid, 4-cyanopentanoic acid dithiobenzoic acid or dithiobenzoic acid cyanoisopropyl ester; the organic solvent comprises N,N-dimethylformamide and / or dimethyl sulfoxide;

[0019] The initiator comprises a thermal initiator azobisisobutyronitrile or a photoinitiator tris(2-phenylpyridine)iridium.

[0020] Furthermore, when the initiator is a thermal initiator azobisisobutyronitrile, the heating temperature is 60 to 80° C., and the reaction time is 8 to 20 hours;

[0021] When the initiator is a photoinitiator tris(2-phenylpyridine)iridium, the reaction is carried out under blue light conditions for 8 to 20 hours.

[0022] Furthermore, the mass ratio of the polymerization monomer, chain transfer agent and initiator is 100-300 mg: 3-10 mg: 4 μg-1 mg;

[0023] The usage ratio of the polymerization monomer and the organic solvent is 100-300 mg: 1-2 mL.

[0024] Furthermore, the intermediate polymer is subjected to a deprotection treatment, wherein the deprotection treatment is performed by dissolving the intermediate polymer in a dichloromethane solution and removing the BOC protection under the action of trifluoroacetic acid; the mass ratio of the intermediate polymer, dichloromethane solution and trifluoroacetic acid is 200 mg:1 mL:300-600 μL; and the treatment time is 3-5 h.

[0025] Furthermore, the mass ratio of the intermediate polymer, 1H-pyrazole-1-carboxamidine hydrochloride and N,N-diisopropylethylamine is 60-150:150-200:150-220;

[0026] In the step 2), the mixing is carried out under stirring at a temperature of 40 to 60° C. and a stirring time of 20 to 40 hours.

[0027] Furthermore, in step 3), the volume concentration of the hydrogen peroxide solution is 20-40%, the usage ratio of the intermediate polymer, water and hydrogen peroxide solution is 60-150 mg:2-5 mL:2-5 mL; the reaction temperature is 80-100° C., and the reaction time is 2-5 h.

[0028] The present invention also provides an application of a guanidine-based cationic polymer capable of efficiently removing persister bacteria in the preparation of a preparation for removing persister bacteria.

[0029] Beneficial effects of the present invention:

[0030] 1. The present invention is based on guanidine-based cationic polymers that can ignore the physiological state of persisting bacteria and achieve efficient removal of persisting bacteria by directly affecting the bacterial cell membrane mechanism. The effective concentration of such cationic polymers for persisting bacteria can be as low as 4 μg / mL, which is better than the current first-line antibiotics and comparative examples.

[0031] 2. The present invention adopts RAFT polymerization method to synthesize the polymer containing guanidine group, which has simple preparation process, low cost, can be produced on a large scale and has controllable structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The synthetic route of the compound PN2G of the present invention is shown in FIG.

[0033] Figure 2 The synthetic route of the compound PN3G of the present invention is shown in FIG.

[0034] Figure 3 The synthetic route of the compound PN6G of the present invention is shown in FIG.

[0035] Figure 4 The synthetic route of the compound PO2G of the present invention is shown in FIG.

[0036] Figure 5The synthetic route of the compound PO3G of the present invention is shown in FIG.

[0037] Figure 6 The synthetic route of the compound PO6G of the present invention is shown in FIG.

[0038] Figures 7 to 12 is the nuclear magnetic spectrum NMR diagram of the compound of the present invention;

[0039] Figure 13 The synthetic route of the compounds of Comparative Examples 1 to 3 is shown;

[0040] Figure 14 This is a diagram showing the antibacterial effect of the guanidine-based cationic polymer of the present invention on persistent bacteria;

[0041] Figure 15 This is a morphology diagram of persister bacteria after being treated with the guanidine-based cationic polymer of the present invention;

[0042] Figure 16 This is a diagram of the surface membrane potential of persister bacteria after being treated with the guanidine-based cationic polymer of the present invention. DETAILED DESCRIPTION

[0043] The present invention provides a guanidine-based cationic polymer that can effectively remove persistent bacteria. The chemical formula of the guanidine-based cationic polymer is PN x G or PO x G, the structural formula is as follows:

[0044]

[0045] Where x is independently 1 to 6, and m is 20 to 50;

[0046]

[0047] In the present invention, x is preferably 1, 1.5, 2, 3, 4, 5, or 6; m is preferably 25 to 45, and more preferably 30 to 45.

[0048] In the present invention, the structural formula of the guanidine-based cationic polymer is preferably selected from one of the following structural formulas:

[0049]

[0050] m=20~50.

[0051] The present invention also provides a method for preparing a guanidine-based cationic polymer that is efficient in removing persistent bacteria, comprising the following steps:

[0052] 1) mixing a polymerization monomer, a chain transfer agent, and an initiator in an organic solvent, and reacting the mixture under heating or blue light to obtain an intermediate polymer;

[0053] 2) mixing the intermediate polymer, 1H-pyrazole-1-carboxamidine hydrochloride, and N,N-diisopropylethylamine in water, and dialyzing and drying the resulting reaction mixture to obtain a dry polymer;

[0054] 3) mixing the dried polymer, water, and hydrogen peroxide solution and reacting them to remove the alkyl chain, and the reaction product is purified by dialysis and freeze-dried to obtain a guanidine-based cationic polymer;

[0055] The polymerizable monomers include 2-aminoethyl methacrylate hydrochloride, N-(3-aminopropyl)methacrylamide hydrochloride, methacrylated 6-(BOC-amino)-1-hexanol, 2-aminoethyl methacrylate hydrochloride, methacrylated tert-butyl N-(3-hydroxypropyl)carbamate, or methacrylated tert-butyl N-(6-aminohexyl)carbamate.

[0056] In the present invention, the chain transfer agent comprises 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid, 4-cyanopentanoic acid dithiobenzoic acid or dithiobenzoic acid cyanoisopropyl ester, preferably 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid; the organic solvent is preferably N,N-dimethylformamide and / or dimethyl sulfoxide;

[0057] The initiator comprises a thermal initiator azobisisobutyronitrile or a photoinitiator tris(2-phenylpyridine)iridium (Ir(ppy)3).

[0058] In the present invention, when the initiator is a thermal initiator azobisisobutyronitrile, the heating temperature is 60 to 80° C., preferably 70° C.; the reaction is preferably carried out in a sand bath, and the reaction time is 8 to 20 hours, preferably 12 hours;

[0059] When the initiator is a photoinitiator tris(2-phenylpyridine)iridium, the reaction is carried out under blue light for 8 to 20 hours, preferably 8 to 10 hours.

[0060] In the present invention, the mass ratio of the polymerization monomer, chain transfer agent and initiator is 100-300 mg: 3-10 mg: 4 μg-1 mg, preferably 120-200 mg: 5-8 mg: 4 μg-0.45 mg;

[0061] The usage ratio of the polymerization monomer and the organic solvent is 100-300 mg:1-2 mL, preferably 120-200 mg:1 mL.

[0062] In the present invention, the intermediate polymer is subjected to a deprotection treatment, and the deprotection treatment is to dissolve the intermediate polymer in a dichloromethane solution and remove the BOC protection under the action of trifluoroacetic acid; the mass ratio of the intermediate polymer, dichloromethane solution and trifluoroacetic acid is 200 mg:1 mL:300-600 μL, preferably 200 mg:1 mL:400-500 μL; the treatment time is 3-5 hours, preferably 4 hours.

[0063] In the present invention, the mass ratio of the intermediate polymer, 1H-pyrazole-1-carboxamidine hydrochloride and N,N-diisopropylethylamine is 60-150:150-200:150-220, preferably 80-100:160-200:190-220;

[0064] In the step 2), the mixing is carried out under stirring at a temperature of 40 to 60° C., preferably 50 to 55° C.; and the stirring time is 20 to 40 hours, preferably 24 hours.

[0065] In the present invention, in step 3), the volume concentration of the hydrogen peroxide solution is 20-40%, preferably 25-35%, and more preferably 30%; the usage ratio of the intermediate polymer, water, and hydrogen peroxide solution is 60-150 mg:2-5 mL:2-5 mL, preferably 80-120 mg:3-5 mL:3-5 mL; the reaction temperature is 80-100° C., preferably 90-95° C.; and the reaction time is 2-5 h, preferably 3-5 h.

[0066] The present invention also provides an application of a guanidine-based cationic polymer capable of efficiently removing persister bacteria in the preparation of a preparation for removing persister bacteria.

[0067] The present invention's series of guanidine-based cationic polymers utilize terminal guanidine-containing monomers as cationic block units. These polymers are designed with variable side chain spacer arm lengths (e.g., ethane-, propane-, and pentane-) and spacer tip atom types. Where x represents the spacer arm length and m represents the number of cationic block units, the chemical formulas are PN2G, PN3G, PN6G, PO2G, PO3G, and PO6G, respectively. These guanidine-based cationic polymers are capable of thoroughly eliminating persistent bacteria.

[0068] The concept and principle of this invention: Currently, persister bacteria are primarily treated with antibiotics. However, as bacteria enter a persistent state, the antibiotics lose their target and ultimately become ineffective. The series of guanidine-based cationic polymers proposed in this invention directly affect the cell membranes of persister bacteria, disregarding their physiological state and thereby achieving complete elimination. Therefore, direct action on the cell membrane and disregarding the physiological state of persisters are key to the design of new antimicrobial polymers.

[0069] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0070] Example 1

[0071] The synthesis of PN2G includes the following steps:

[0072] 120 mg of 2-aminoethyl methacrylate hydrochloride, 5 mg of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid (DDMAT), and 0.45 mg of azobisisobutyronitrile (AIBN) were dissolved in 1 mL of N,N-dimethylformamide. The mixture was then degassed with N2 for 30 minutes, reacted in a 70°C sand bath for 12 hours, and dialyzed against methanol using a 1000 Da solution for 48 hours. The resulting product was collected and dried. Polymer PN2 was obtained. On this basis, 80 mg of PN2, 160 mg of 1H-pyrazole-1-carboximidamide hydrochloride, and 190 mg of N,N-diisopropylethylamine (DIEA) were dissolved in 1 mL of deionized water. The reaction mixture was stirred at 55°C for 24 hours and dialyzed against methanol at 1000 Da for 24 hours to purify the polymer. The dried polymer was dissolved in 3 mL of deionized water and 3 mL of 30% hydrogen peroxide solution was added and reacted at 90°C for 3 hours to remove the alkyl chain. Finally, the polymer was dialyzed against water at 1000 Da for 24 hours to purify the polymer and finally lyophilized to obtain the guanidine cationic polymer PN2G. The synthesis steps are as follows: Figure 1 , R1 is m=45.

[0073] Example 2

[0074] The synthesis of PN3G includes the following steps:

[0075] 120 mg of N-(3-aminopropyl)methacrylamide hydrochloride as the monomer, 5 mg of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid (DDMAT), and 0.45 mg of azobisisobutyronitrile (AIBN) were dissolved in 1 mL of N,N-dimethylformamide. The mixture was then degassed with N2 for 30 minutes, reacted in a 70°C sand bath for 12 hours, and dialyzed against methanol using a 1000 Da solution for 48 hours. The resulting product was collected and dried. Polymer PN3 was obtained. Based on this, 80 mg of PN3, 160 mg of 1H-pyrazole-1-carboximidamide hydrochloride, and 190 mg of N,N-diisopropylethylamine (DIEA) were dissolved in 1 mL of deionized water. The reaction mixture was stirred at 55°C for 24 hours and dialyzed against methanol at 1000 Da for 24 hours to purify the polymer. The dried polymer was dissolved in 3 mL of deionized water and 3 mL of 30% hydrogen peroxide solution was added and reacted at 90°C for 3 hours to remove the alkyl chain. Finally, the polymer was dialyzed against water at 1000 Da for 24 hours to purify the polymer and finally lyophilized to obtain the guanidine cationic polymer PN3G. The synthesis steps are as follows: Figure 2 , R1 is m=45.

[0076] Example 3

[0077] The synthesis of PN6G includes the following steps:

[0078] 200 mg of methacryloylated 6-(BOC-amino)-1-hexanol, 4.5 mg of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropanoic acid (DDMAT), and 4 μg of Ir(ppy)3 were dissolved in 1 mL of dimethyl sulfoxide and reacted under blue light for 8 hours. The mixture was then dialyzed against methanol using a 1000 Da filter for 48 hours, collected, and dried to synthesize polymer PN6. Next, 200 mg of PN6 was added to 1 mL of dichloromethane solution and 500 μL of trifluoroacetic acid and stirred at room temperature for 4 hours to remove the BOC protection. On this basis, 80 mg of PN6, 160 mg of 1H-pyrazole-1-carboximidamide hydrochloride, and 190 mg of N,N-diisopropylethylamine (DIEA) were dissolved in 1 mL of deionized water. The reaction mixture was stirred at 55°C for 24 hours and dialyzed against methanol at 1000 Da for 24 hours to purify the polymer. The dried polymer was dissolved in 3 mL of deionized water and 3 mL of 30% hydrogen peroxide solution was added and reacted at 90°C for 3 hours to remove the alkyl chain. Finally, the polymer was dialyzed against water at 1000 Da for 24 hours to purify the polymer. The polymer was finally lyophilized to obtain the guanidino cationic polymer PN6G. The synthesis steps are as follows: Figure 3 , R1 is m=45.

[0079] Example 4

[0080] The synthesis of PO2G includes the following steps:

[0081] 200 mg of 2-aminoethyl methacrylate hydrochloride, 4.5 mg of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropanoic acid (DDMAT), and 4 μg of Ir(ppy)3 were dissolved in 1 mL of dimethyl sulfoxide and reacted under blue light for 8 hours. The mixture was then dialyzed against methanol using a 1000 Da filter for 48 hours, collected, and dried. Polymer PO2 was synthesized. Next, 80 mg of PO2, 160 mg of 1H-pyrazole-1-carboxamidine hydrochloride, and 190 mg of N,N-diisopropylethylamine (DIEA) were dissolved in 1 mL of deionized water. The reaction mixture was stirred at 55°C for 24 hours and dialyzed against methanol at 1000 Da for 24 hours to purify the polymer. The dried polymer was dissolved in 3 mL of deionized water and 3 mL of 30% hydrogen peroxide solution was added and reacted at 90°C for 3 hours to remove the alkyl chain. Finally, the polymer was dialyzed against water at 1000 Da for 24 hours to purify the polymer and finally lyophilized to obtain the guanidine-based cationic polymer PO2G. The synthesis steps are as follows: Figure 4 , R1 is m=45.

[0082] Example 5

[0083] The synthesis of PO3G includes the following steps:

[0084] 200 mg of methacryloylated tert-butyl N-(3-hydroxypropyl)carbamate, 4.5 mg of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropanoic acid (DDMAT), and 4 μg of Ir(ppy)3 were dissolved in 1 mL of dimethyl sulfoxide and reacted under blue light for 8 hours. The mixture was then dialyzed against methanol using a 1000 Da filter for 48 hours, collected, and dried to synthesize the polymer PO3. Next, 200 mg of PO3 was added to 1 mL of dichloromethane solution and 500 μL of trifluoroacetic acid and stirred at room temperature for 4 hours to remove the BOC protection. On this basis, 80 mg of PO3, 160 mg of 1H-pyrazole-1-carboximidamide hydrochloride, and 190 mg of N,N-diisopropylethylamine (DIEA) were dissolved in 1 mL of deionized water. The reaction mixture was stirred at 55°C for 24 hours and dialyzed against methanol at 1000 Da for 24 hours to purify the polymer. The dried polymer was dissolved in 3 mL of deionized water and 3 mL of 30% hydrogen peroxide solution was added and reacted at 90°C for 3 hours to remove the alkyl chain. Finally, the polymer was dialyzed against water at 1000 Da for 24 hours to purify the polymer and finally lyophilized to obtain the guanidine cationic polymer PO3G. The synthesis steps are as follows: Figure 5 , R1 is m=45.

[0085] Example 6

[0086] The synthesis of PO6G includes the following steps:

[0087] 200 mg of methacryloylated tert-butyl N-(6-aminohexyl)carbamate, 4.5 mg of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropanoic acid (DDMAT), and 4 μg of Ir(ppy)3 were dissolved in 1 mL of dimethyl sulfoxide and reacted under blue light for 8 hours. The mixture was then dialyzed against methanol using a 1000 Da filter for 48 hours, collected, and dried. Polymer PO6 was synthesized. Next, 200 mg of PO6 was added to 1 mL of dichloromethane solution and 500 μL of trifluoroacetic acid and stirred at room temperature for 4 hours to remove the BOC protection. On this basis, 80 mg of PO6, 160 mg of 1H-pyrazole-1-carboximidamide hydrochloride, and 190 mg of N,N-diisopropylethylamine (DIEA) were dissolved in 1 mL of deionized water. The reaction mixture was stirred at 55°C for 24 hours and dialyzed against methanol at 1000 Da for 24 hours to purify the polymer. The dried polymer was dissolved in 3 mL of deionized water and 3 mL of 30% hydrogen peroxide solution was added and reacted at 90°C for 3 hours to remove the alkyl chain. Finally, the polymer was dialyzed against water at 1000 Da for 24 hours to purify the polymer and finally lyophilized to obtain the guanidine cationic polymer PO6G. The synthesis steps are as follows: Figure 6 , R1 is m=45.

[0088] Example 7

[0089] The synthesis of PN2G includes the following steps:

[0090] Same as Example 1, except that the chain transfer agent 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid is replaced by 4-cyanopentanoic acid dithiobenzoic acid, and R1 is m=45.

[0091] Example 8

[0092] The synthesis of PN3G includes the following steps:

[0093] Same as Example 2, except that the chain transfer agent 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid is replaced by 4-cyanopentanoic acid dithiobenzoic acid, and R1 is m=45.

[0094] Example 9

[0095] The synthesis of PN6G includes the following steps:

[0096] Same as Example 3, except that the chain transfer agent 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid is replaced by 4-cyanopentanoic acid dithiobenzoic acid, and R1 is m=45.

[0097] Example 10

[0098] The synthesis of PO2G includes the following steps:

[0099] Same as Example 4, except that the chain transfer agent 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid is replaced by 4-cyanopentanoic acid dithiobenzoic acid, and R1 is m=45.

[0100] Example 11

[0101] The synthesis of PO3G includes the following steps:

[0102] Same as Example 5, except that the chain transfer agent 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid is replaced by 4-cyanopentanoic acid dithiobenzoic acid, and R1 is m=45.

[0103] Example 12

[0104] The synthesis of PO6G includes the following steps:

[0105] Same as Example 6, except that the chain transfer agent 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid is replaced by 4-cyanopentanoic acid dithiobenzoic acid, and R1 is m=45.

[0106] Example 13

[0107] The synthesis of PN2G includes the following steps:

[0108] Same as Example 1, except that the chain transfer agent 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid is replaced by dithiobenzoic acid cyanoisopropyl ester, and R1 is m=45.

[0109] Example 14

[0110] The synthesis of PN3G includes the following steps:

[0111] Same as Example 2, except that the chain transfer agent 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid is replaced by dithiobenzoic acid cyanoisopropyl ester, and R1 is m=45.

[0112] Example 15

[0113] The synthesis of PN6G includes the following steps:

[0114] Same as Example 3, except that the chain transfer agent 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid is replaced by dithiobenzoic acid cyanoisopropyl ester, and R1 is m=45.

[0115] Example 16

[0116] The synthesis of PO2G includes the following steps:

[0117] Same as Example 4, except that the chain transfer agent 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid is replaced by dithiobenzoic acid cyanoisopropyl ester, and R1 is m=45.

[0118] Example 17

[0119] The synthesis of PO3G includes the following steps:

[0120] Same as Example 5, except that the chain transfer agent 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid is replaced by dithiobenzoic acid cyanoisopropyl ester, and R1 is m=45.

[0121] Example 18

[0122] The synthesis of PO6G includes the following steps:

[0123] Same as Example 6, except that the chain transfer agent 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid is replaced by dithiobenzoic acid cyanoisopropyl ester, R1 is m=45.

[0124] Comparative Example 1

[0125] Comparative Example 1 synthesis comprises the following steps:

[0126] Weigh 200 mg of methacrylamide, 5 mg of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid (DDMAT), and 0.9 mg of azobisisobutyronitrile (AIBN) and dissolve them in 2 mL of N,N-dimethylformamide. Then degas with N2 for 30 min, react in a sand bath at 70 ° C for 12 hours, dialyze with 1000 Da in methanol solution for 48 hours, collect and dry. On this basis, weigh 80 mg of the above-mentioned dried sample, 160 mg of 1H-pyrazole-1-carboximidamide hydrochloride and 190 mg of N,N-diisopropylethylamine (DIEA) and dissolve them in 1 mL of deionized water. The reaction mixture was stirred at 55 ° C for 24 hours, and dialyzed in methanol with 1000 Da for 24 hours to purify the polymer, and then collected and dried to obtain Comparative Example 1. The synthesis steps are as follows Figure 13 .

[0127] Comparative Example 2

[0128] Comparative Example 2 synthesis comprises the following steps:

[0129] 191 mg of acrylic acid, 5 mg of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid (DDMAT), and 0.9 mg of azobisisobutyronitrile (AIBN) were dissolved in 2 mL of N,N-dimethylformamide. The mixture was then degassed with N for 30 minutes, reacted in a 70°C sand bath for 12 hours, and dialyzed against methanol at 1000 Da for 48 hours, collected, and dried. Next, 100 mg of the dried polymer, 95 mg of trans-4-aminocyclohexanol hydrochloride, 227 mg of N,N-dicyclohexylcarbodiimide (DCC), and 11 mg of 4-dimethylaminopyridine (DMAP) were weighed and thoroughly mixed. The mixture was then dissolved in 10 mL of DMSO and stirred at room temperature for 24 hours. The polymer was then purified by dialysis against methanol at 1000 Da for 24 hours, collected, and dried. 80 mg of the dried polymer, 160 mg of 1H-pyrazole-1-carboxamidine hydrochloride, and 190 mg of N,N-diisopropylethylamine (DIEA) were dissolved in 1 mL of deionized water. The reaction mixture was stirred at 55°C for 24 hours, and dialyzed against methanol at 1000 Da for 24 hours to purify the polymer. The polymer was then collected and dried to obtain Comparative Example 2. The synthesis steps are as follows: Figure 13 .

[0130] Comparative Example 3

[0131] Comparative Example 3 synthesis comprises the following steps:

[0132] Weigh 200 mg of 4-vinylbenzyl chloride, 5 mg of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid (DDMAT), and 0.9 mg of azobisisobutyronitrile (AIBN) and dissolve them in 2 mL of N,N-dimethylformamide. Then degas with N2 for 30 min, react in a sand bath at 70 ° C for 12 hours, dialyze with 1000 Da in methanol solution for 48 hours, collect and dry. On this basis, weigh 80 mg of the above-mentioned dried sample, 160 mg of 1H-pyrazole-1-carboxamidine hydrochloride and 190 mg of N,N-diisopropylethylamine (DIEA) and dissolve them in 1 mL of deionized water. The reaction mixture was stirred at 55 ° C for 24 hours, and dialyzed in methanol with 1000 Da for 24 hours to purify the polymer, and then collected and dried to obtain Comparative Example 3. The synthesis steps are as follows Figure 13 .

[0133] The present invention evaluates the effect of guanidine-based cationic polymers on the removal of persistent bacteria. Figure 14As shown, PN2G at 32 μg / mL, PN3G at 16 μg / mL, PN6G at 16 μg / mL, PO2G at 8 μg / mL, PO3G at 4 μg / mL, and PO6G at 16 μg / mL completely eliminated persister bacteria. However, a control group using vancomycin at 50x the MIC concentration showed 100% survival of persister bacteria. Furthermore, as shown in Table 1, the cationic polymers designed in this invention demonstrated significantly superior therapeutic efficacy against persister bacteria compared to the control group.

[0134] Table 1: Antibacterial effects of guanidine-based cationic polymers and comparative examples on persistent bacteria

[0135]

[0136] To visualize the damaging effects on persister bacteria, Figure 15 Figure 3 shows the morphology of bacteria treated with a guanidine-based cationic polymer. As shown, after treatment with 1x the MIC of a guanidine-based cationic polymer, the surfaces of individual bacteria exhibited varying degrees of wrinkling, depressions, and even perforations. Some bacteria were so fragmented that their morphology could no longer be discerned. However, the control group, treated with vancomycin at 50x the MIC, exhibited normal appearance, clear boundaries, and excellent membrane integrity. Furthermore, the membrane integrity of the bacteria treated with the comparative example was significantly better, significantly different from that of the cationic polymer designed in this invention. These results further demonstrate the effective removal of persister bacteria by guanidine-based cationic polymers.

[0137] Figure 16 This graph shows the surface membrane potential of persister bacteria after treatment with a guanidine-based cationic polymer. As shown, after treatment with 1x the MIC of a guanidine-based cationic polymer, the membrane potential of the persister bacteria shifted from an initial -26 mV to approximately +20 mV. This significant shift in membrane potential can be achieved regardless of the physiological state of the persister bacteria, ultimately leading to complete elimination of the persister bacteria.

[0138] As can be seen from the above examples, the present invention provides a guanidine-based cationic polymer that is highly effective in removing persister bacteria, as well as its preparation method and application. Based on the ability of guanidine-based cationic polymers to disregard the physiological state of persister bacteria, the present invention achieves efficient removal of persister bacteria by directly affecting the bacterial cell membrane mechanism. The effective concentration of such cationic polymers against persister bacteria can be as low as 4 μg / mL, which is superior to current first-line antibiotics and comparative examples. The present invention uses RAFT polymerization to synthesize guanidine-containing polymers, which has a simple preparation process, low cost, large-scale production, and controllable structure.

[0139] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A guanidine-based cationic polymer for efficiently removing persistent bacteria, characterized in that: The chemical formula of the guanidine-based cationic polymer is PN x G or PO x G, the structural formula is as follows: Where x is independently 1 to 6, and m is 20 to 50; 2. The guanidine-based cationic polymer for efficiently removing persistent bacteria according to claim 1, characterized in that: The structural formula of the guanidine-based cationic polymer is selected from one of the following structural formulas: m＝20～50。 3. The method for preparing the guanidine-based cationic polymer for efficiently removing persistent bacteria according to claim 1 or 2, characterized in that: The following steps are involved: 1) mixing a polymerization monomer, a chain transfer agent, and an initiator in an organic solvent, and reacting the mixture under heating or blue light to obtain an intermediate polymer; 2) mixing the intermediate polymer, 1H-pyrazole-1-carboxamidine hydrochloride, and N,N-diisopropylethylamine in water, and dialyzing and drying the resulting reaction mixture to obtain a dry polymer; 3) mixing the dried polymer, water, and hydrogen peroxide solution and reacting them to remove the alkyl chain, and the reaction product is purified by dialysis and freeze-dried to obtain a guanidine-based cationic polymer; The polymerizable monomers include 2-aminoethyl methacrylate hydrochloride, N-(3-aminopropyl)methacrylamide hydrochloride, methacrylated 6-(BOC-amino)-1-hexanol, 2-aminoethyl methacrylate hydrochloride, methacrylated tert-butyl N-(3-hydroxypropyl)carbamate, or methacrylated tert-butyl N-(6-aminohexyl)carbamate.

4. The method for preparing a guanidine-based cationic polymer for efficiently removing persistent bacteria according to claim 3, characterized in that: The chain transfer agent comprises 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid, 4-cyanopentanoic acid dithiobenzoic acid or dithiobenzoic acid cyanoisopropyl ester; the organic solvent comprises N,N-dimethylformamide and / or dimethyl sulfoxide; The initiator comprises a thermal initiator azobisisobutyronitrile or a photoinitiator tris(2-phenylpyridine)iridium.

5. The method for preparing a guanidine-based cationic polymer for efficiently removing persistent bacteria according to claim 4, characterized in that: When the initiator is a thermal initiator azobisisobutyronitrile, the heating temperature is 60 to 80° C. and the reaction time is 8 to 20 hours; When the initiator is a photoinitiator tris(2-phenylpyridine)iridium, the reaction is carried out under blue light conditions for 8 to 20 hours.

6. The method for preparing a guanidine-based cationic polymer for efficiently removing persistent bacteria according to any one of claims 3 to 5, characterized in that: The mass ratio of the polymerization monomer, chain transfer agent and initiator is 100-300 mg: 3-10 mg: 4 μg-1 mg; The usage ratio of the polymerization monomer and the organic solvent is 100-300 mg: 1-2 mL.

7. The method for preparing a guanidine-based cationic polymer for efficiently removing persistent bacteria according to claim 1, characterized in that: The intermediate polymer is subjected to a deprotection treatment, wherein the deprotection treatment comprises dissolving the intermediate polymer in a dichloromethane solution and removing the BOC protection under the action of trifluoroacetic acid; the mass ratio of the intermediate polymer, the dichloromethane solution and the trifluoroacetic acid is 200 mg:1 mL:300-600 μL; and the treatment time is 3-5 hours.

8. The method for preparing a guanidine-based cationic polymer for efficiently removing persistent bacteria according to claim 1, 5 or 7, characterized in that: The mass ratio of the intermediate polymer, 1H-pyrazole-1-carboxamidine hydrochloride and N,N-diisopropylethylamine is 60-150:150-200:150-220; In the step 2), the mixing is carried out under stirring at a temperature of 40 to 60° C. and a stirring time of 20 to 40 hours.

9. The method for preparing a guanidine-based cationic polymer for efficiently removing persistent bacteria according to claim 8, characterized in that: In step 3), the volume concentration of the hydrogen peroxide solution is 20-40%, and the usage ratio of the intermediate polymer, water, and hydrogen peroxide solution is 60-150 mg:2-5 mL:2-5 mL; the reaction temperature is 80-100° C., and the reaction time is 2-5 hours.

10. Use of the guanidine-based cationic polymer for efficiently eliminating persister bacteria according to claim 1 or 2 in the preparation of a preparation for eliminating persister bacteria.

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