A guanidinium cationic polymer with high efficiency in removing persister bacteria, and a preparation method and application thereof

Abstract

The application provides a guanidyl cationic polymer for efficiently removing retained bacteria, a preparation method and application thereof, and belongs to the technical field of biomedical engineering materials. The guanidyl cationic polymer has a monomer containing a guanidyl group at the end as a cationic block unit, a series of variable side chain spacer arm lengths (ethane-, propane-, and hexane-) and categories of atoms at the front end of the spacer arm are designed, wherein x is the spacer arm length, and the number m of the cationic block unit is 20-50, so the chemical formula is PN2G, PN3G, PN6G, PO2G, PO3G and PO6G, respectively. The guanidyl cationic polymer can efficiently remove retained bacteria by directly affecting the bacterial cell membrane mechanism, and the effective concentration of the cationic polymer on the retained bacteria can be as low as 4 μg / mL, which is better than the current first-line antibiotics and the comparative examples.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical engineering materials, and particularly relates to a guanidyl cationic polymer for efficiently removing persister bacteria and a preparation method and application thereof. BACKGROUND

[0002] Persistence is a survival mechanism that bacteria exhibit during the exponential growth phase to cope with a series of adverse environmental conditions, and it is a subpopulation of microorganisms that can spontaneously enter or passively enter a kind of "static" state due to environmental factors during growth, and has strong resistance to antibacterial drugs. When external pressure exists, persister bacteria can maintain long-term survival in a slow growth mode by regulating metabolic processes and energy metabolism. At present, the main treatment for persister bacteria is through antibiotics, however, large doses of long-term antibiotics usually easily produce drug resistance, and it is difficult to produce effects on persistent bacteria. The main reason is that the action of antibiotics usually needs to be accompanied by DNA / RNA replication, protein synthesis or cell wall expansion, however, due to the entry of bacteria into the persistent state, the loss of antibiotic targets eventually leads to the failure of antibiotics.

[0003] Therefore, how to obtain a compound preparation that can efficiently remove persister bacteria is a technical problem to be solved at present. SUMMARY

[0004] The present application aims to provide a guanidyl cationic polymer for efficiently removing persister bacteria and a preparation method and application thereof, and solve the technical problems of antibiotic resistance and poor removal effect of persister bacteria.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides a guanidyl cationic polymer for efficiently removing persister bacteria, and the chemical formula of the guanidyl cationic polymer is PN x G or PO x G, and the structural formula is as follows:

[0007]

[0008] wherein x is independently 1-6, and m is 20-50;

[0009]

[0010] Further, the structural formula of the guanidyl cationic polymer is selected from one of the following structural formulas:

[0011]

[0012] m = 20-50.

[0013] The application also provides a preparation method of the guanidinium cationic polymer for efficiently removing persister bacteria, comprising the following steps:

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

[0015] 2) mixing the intermediate polymer, 1H-pyrazole-1-carboxamidine hydrochloride and N,N-diisopropylethylamine in water, and performing dialysis and drying on the obtained reaction mixture to obtain a dried polymer;

[0016] 3) mixing the dried polymer, water and hydrogen peroxide solution, and performing reaction to cut off the alkyl chain, and performing dialysis purification and freeze-drying on the reaction product to obtain the guanidinium cationic polymer;

[0017] The polymerization monomer comprises 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; and the organic solvent comprises N,N-dimethylformamide and / or dimethyl sulfoxide.

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

[0020] Further, when the initiator is the thermal initiator azobisisobutyronitrile, the heating temperature is 60-80°C, and the reaction time is 8-20h.

[0021] When the initiator is the photo initiator tris(2-phenylpyridine) iridium, the reaction is performed under blue light condition for 8-20h.

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

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

[0024] Furthermore, the intermediate polymer undergoes a deprotection treatment, which involves dissolving the intermediate polymer in a dichloromethane solution and removing 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; the treatment time is 3–5 h.

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

[0026] In step 2), the mixing is carried out under stirring, the stirring temperature is 40-60℃, and the stirring time is 20-40h.

[0027] Furthermore, in step 3), the volume concentration of the hydrogen peroxide solution is 20-40%, and the 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 the application of a guanidine cationic polymer that efficiently eliminates persistent bacteria in the preparation of formulations that eliminate persistent bacteria.

[0029] The beneficial effects of this invention are:

[0030] 1. This invention is based on the fact that guanidine cationic polymers can ignore the physiological state of persistent bacteria and achieve efficient clearance of persistent bacteria by directly affecting the bacterial cell membrane mechanism. The effective concentration of such cationic polymers against persistent bacteria can be as low as 4 μg / mL, which is superior to current first-line antibiotics and comparative ratios.

[0031] 2. This invention uses the RAFT polymerization method to synthesize guanidine-containing polymers, which has a simple preparation process, low cost, can be mass-produced, and has a controllable structure. Attached Figure Description

[0032] Figure 1 This is a synthetic route diagram for the compound PN2G of this invention;

[0033] Figure 2 This is a synthetic route diagram for the compound PN3G of this invention;

[0034] Figure 3 This is a synthetic route diagram for the compound PN6G of this invention;

[0035] Figure 4 This is a synthetic route diagram for the compound PO2G of this invention;

[0036] Figure 5This is a synthetic route diagram for the compound PO3G of this invention;

[0037] Figure 6 This is a synthetic route diagram for the compound PO6G of this invention;

[0038] Figures 7 to 12 The NMR spectrum of the compound of this invention is shown below.

[0039] Figure 13 Synthetic routes for compounds in Comparative Examples 1–3;

[0040] Figure 14 This diagram illustrates the antibacterial effect of the guanidine cationic polymer of the present invention against persistent bacteria.

[0041] Figure 15 This is a morphology diagram of the guanidine cationic polymer after treatment according to the present invention;

[0042] Figure 16 This is a membrane potential diagram of the surface of the guanidine cationic polymer of the present invention. Detailed Implementation

[0043] This invention provides a guanidine-based cationic polymer for efficiently eliminating persistent bacteria, wherein the chemical formula of the guanidine-based cationic polymer is PN. x G or PO x G, with the following structural formula:

[0044]

[0045] Where x is independent, it ranges from 1 to 6, and m ranges from 20 to 50;

[0046]

[0047] In this 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 this invention, the structural formula of the guanidine cationic polymer is preferably one of the following structural formulas:

[0049]

[0050] m = 20~50.

[0051] This invention also provides a method for preparing a guanidine cationic polymer that efficiently eliminates persistent bacteria, comprising the following steps:

[0052] 1) The monomer, chain transfer agent and initiator are mixed in an organic solvent and reacted under heating or blue light conditions to obtain an intermediate polymer;

[0053] 2) The intermediate polymer, 1H-pyrazole-1-formamidinium hydrochloride and N,N-diisopropylethylamine were mixed in water, and the resulting reaction mixture was dialyzed and dried to obtain a dry polymer;

[0054] 3) The dried polymer, water and hydrogen peroxide solution were mixed and reacted to remove the alkyl chain. The reaction product was purified by dialysis and lyophilized to obtain the guanidine cationic polymer.

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

[0056] In this 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 the thermal initiator azobisisobutyronitrile or the photoinitiator tris(2-phenylpyridine)iridium (Ir(ppy)3).

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

[0059] When the initiator is the 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 this invention, the mass ratio of the polymerizing 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 ratio of the polymer monomer to the organic solvent is 100-300 mg: 1-2 mL, preferably 120-200 mg: 1 mL.

[0062] In this invention, the intermediate polymer undergoes a deprotection treatment, which involves dissolving the intermediate polymer in a dichloromethane solution and removing 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 h, preferably 4 h.

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

[0064] In step 2), the mixing is carried out under stirring, the stirring temperature is 40-60℃, preferably 50-55℃, and the stirring time is 20-40h, preferably 24h.

[0065] In this invention, in step 3), the volume concentration of the hydrogen peroxide solution is 20-40%, preferably 25-35%, and more preferably 30%; the 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 the application of a guanidine cationic polymer that efficiently eliminates persistent bacteria in the preparation of formulations that eliminate persistent bacteria.

[0067] The series of guanidino cationic polymers of this invention use monomers with terminal guanidino groups as cationic block units, and are designed with a series of variable side chain spacer arm lengths (ethane-, propane-, pentane-) and spacer arm front atom types, where x is the spacer arm length and m is 20-50, thus the chemical formulas are PN2G, PN3G, PN6G, PO2G, PO3G, and PO6G, respectively. This series of guanidino cationic polymers can completely eliminate persistent bacteria.

[0068] The concept and principle of this invention: Currently, persistent bacteria are mainly treated with antibiotics. However, because bacteria enter a persistent state, the antibiotic target is lost, ultimately leading to antibiotic ineffectiveness. The series of guanidine cationic polymers proposed in this invention directly affect the cell membrane of persistent bacteria, thus ignoring their physiological state and achieving complete eradication. Therefore, directly acting on the cell membrane and ignoring the physiological state of persistent bacteria is the key to designing novel antibacterial polymers.

[0069] The technical solutions provided by the present invention will be 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] Weigh 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), and dissolve them in 1 mL of N,N-dimethylformamide. Then, degas with N2 for 30 min, react in a sand bath at 70 °C for 12 hours, dialyze against a methanol solution at 1000 Da for 48 h, collect the solution, and dry it to obtain polymer PN2. Based on this, weigh 80 mg of PN2, 160 mg of 1H-pyrazole-1-formamidinium 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 the polymer was purified by dialyzing with 1000 Da in methanol for 24 hours. The dried polymer was dissolved in 3 mL of deionized water, and 3 mL of 30% hydrogen peroxide solution was added. The mixture was reacted at 90°C for 3 hours to remove the alkyl chain. Finally, the polymer was purified by dialyzing with 1000 Da in water for 24 hours, 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 was weighed as a 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 min, reacted in a sand bath at 70 °C for 12 hours, dialyzed against a methanol solution at 1000 Da for 48 h, collected, and dried. The polymer PN3 was obtained. Based on this, 80 mg of PN3, 160 mg of 1H-pyrazole-1-formamidinium hydrochloride, and 190 mg of N,N-diisopropylethylamine (DIEA) were weighed and dissolved in 1 mL of deionized water. The reaction mixture was stirred at 55°C for 24 hours, and the polymer was purified by dialyzing with 1000 Da in methanol for 24 hours. The dried polymer was dissolved in 3 mL of deionized water, and 3 mL of 30% hydrogen peroxide solution was added. The mixture was reacted at 90°C for 3 hours to remove the alkyl chain. Finally, the polymer was purified by dialyzing with 1000 Da in water for 24 hours, 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 methacrylylated 6-(BOC-amino)-1-hexanol, 4.5 mg of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid (DDMAT), and 4 μg of Ir(ppy)3 were dissolved in 1 mL of dimethyl sulfoxide and reacted under blue light for 8 h. The mixture was then dialyzed against methanol solution at 1000 Da for 48 h, collected, and dried to synthesize polymer PN6. Next, 200 mg of PN6 was added to 1 mL of dichlorosol and 500 μL of trifluoroacetic acid and stirred at room temperature for 4 h to remove BOC protection. Based on this, 80 mg of PN6, 160 mg of 1H-pyrazole-1-formamidinium 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 the polymer was purified by dialyzing with 1000 Da in methanol for 24 hours. The dried polymer was dissolved in 3 mL of deionized water, and 3 mL of 30% hydrogen peroxide solution was added. The mixture was reacted at 90°C for 3 hours to remove the alkyl chain. Finally, the polymer was purified by dialyzing with 1000 Da in water for 24 hours. The final lyophilized product was the guanidine-based 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-methylpropionic acid (DDMAT), and 4 μg of Ir(ppy)3 were weighed and dissolved in 1 mL of dimethyl sulfoxide. The mixture was reacted under blue light for 8 h, then dialyzed against methanol solution at 1000 Da for 48 h. The solution was collected and dried to synthesize polymer PO2. Next, 80 mg of PO2, 160 mg of 1H-pyrazole-1-formamidinium hydrochloride, and 190 mg of N,N-diisopropylethylamine (DIEA) were weighed and dissolved in 1 mL of deionized water. The reaction mixture was stirred at 55°C for 24 hours, and the polymer was purified by dialyzing with 1000 Da in methanol for 24 hours. The dried polymer was dissolved in 3 mL of deionized water, and 3 mL of 30% hydrogen peroxide solution was added. The mixture was reacted at 90°C for 3 hours to remove the alkyl chain. Finally, the polymer was purified by dialyzing with 1000 Da in water for 24 hours, and finally lyophilized to obtain the guanidine 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 methacrylamide-modified N-(3-hydroxypropyl)carbamate tert-butyl, 4.5 mg of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid (DDMAT), and 4 μg of Ir(ppy)3 were dissolved in 1 mL of dimethyl sulfoxide and reacted under blue light for 8 h. The mixture was then dialyzed against methanol solution at 1000 Da for 48 h, collected, and dried to synthesize polymer PO3. Next, 200 mg of PO3 was added to 1 mL of dichloroisocyanurate solution and 500 μL of trifluoroacetic acid, and stirred at room temperature for 4 h to remove BOC protection. Based on this, 80 mg of PO3, 160 mg of 1H-pyrazole-1-formamidinium 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 the polymer was purified by dialyzing with 1000 Da in methanol for 24 hours. The dried polymer was dissolved in 3 mL of deionized water, and 3 mL of 30% hydrogen peroxide solution was added. The mixture was reacted at 90°C for 3 hours to remove the alkyl chain. Finally, the polymer was purified by dialyzing with 1000 Da in water for 24 hours, 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 methacryloyl-N-(6-aminohexyl)carbamate tert-butyl ester, 4.5 mg of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid (DDMAT), and 4 μg of Ir(ppy)3 were weighed and dissolved in 1 mL of dimethyl sulfoxide. The mixture was reacted under blue light for 8 h, then dialyzed against methanol solution at 1000 Da for 48 h, collected, and dried. Polymer PO6 was synthesized. Next, 200 mg of PO6 was added to 1 mL of dichlorosol and 500 μL of trifluoroacetic acid, and stirred at room temperature for 4 h to remove BOC protection. Based on this, 80 mg of PO6, 160 mg of 1H-pyrazole-1-formamidinium hydrochloride, and 190 mg of N,N-diisopropylethylamine (DIEA) were weighed and dissolved in 1 mL of deionized water. The reaction mixture was stirred at 55°C for 24 hours, and the polymer was purified by dialyzing with 1000 Da in methanol for 24 hours. The dried polymer was dissolved in 3 mL of deionized water, and 3 mL of 30% hydrogen peroxide solution was added. The mixture was reacted at 90°C for 3 hours to remove the alkyl chain. Finally, the polymer was purified by dialyzing with 1000 Da in water for 24 hours, 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 with 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 with 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 with 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 with 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 with 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 with 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 with isopropyl dithiobenzoate, 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 with isopropyl dithiobenzoate, 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 with isopropyl dithiobenzoate, 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 with isopropyl dithiobenzoate, 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 with isopropyl dithiobenzoate, 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 with isopropyl dithiobenzoate, and R1 is... m = 45.

[0124] Comparative Example 1

[0125] The synthesis of Comparative Example 1 includes 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 against methanol solution at 1000 Da for 48 h, collect the solution, and dry it. Based on this, weigh 80 mg of the dried sample, 160 mg of 1H-pyrazole-1-formamidinium hydrochloride, and 190 mg of N,N-diisopropylethylamine (DIEA), and dissolve them in 1 mL of deionized water. Stir the reaction mixture at 55 °C for 24 h, and dialyze against methanol at 1000 Da for 24 h to purify the polymer. Collect the solution, dry it, and obtain Comparative Example 1. The synthesis steps are as follows: Figure 13 .

[0127] Comparative Example 2

[0128] The synthesis of Comparative Example 2 includes 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 weighed and dissolved in 2 mL of N,N-dimethylformamide. The mixture was then degassed with N2 for 30 min, reacted in a sand bath at 70 °C for 12 hours, dialyzed against methanol solution at 1000 Da for 48 h, collected, and dried. Based on this, 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, thoroughly mixed, and dissolved in 10 mL of DMSO. The mixture was stirred and reacted at room temperature for 24 h, then dialyzed against methanol at 1000 Da for 24 h to purify the polymer, collected, and dried. 80 mg of dried polymer, 160 mg of 1H-pyrazole-1-formamidinium 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 the polymer was purified by dialyzing in methanol at 1000 Da for 24 hours. The purified polymer was then collected and dried to obtain Comparative Example 2. The synthesis steps are as follows. Figure 13 .

[0130] Comparative Example 3

[0131] The synthesis of Comparative Example 3 includes the following steps:

[0132] 200 mg of 4-vinylbenzyl chloride, 5 mg of 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid (DDMAT), and 0.9 mg of azobisisobutyronitrile (AIBN) were weighed and dissolved in 2 mL of N,N-dimethylformamide. The mixture was then degassed with N2 for 30 min, reacted in a sand bath at 70 °C for 12 hours, dialyzed against methanol solution at 1000 Da for 48 h, and collected and dried. Based on this, 80 mg of the dried sample, 160 mg of 1H-pyrazole-1-formamidinium hydrochloride, and 190 mg of N,N-diisopropylethylamine (DIEA) were weighed and dissolved in 1 mL of deionized water. The reaction mixture was stirred at 55 °C for 24 h, and dialyzed against methanol at 1000 Da for 24 h to purify the polymer. The collected and dried sample yielded Comparative Example 3. The synthesis steps are as follows. Figure 13 .

[0133] This invention evaluates the effectiveness of guanidine cationic polymers in eliminating persistent bacteria. For example... Figure 14The results showed that 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 could completely eliminate persistent bacteria. However, the control group, using vancomycin at a concentration of 50x MIC, showed 100% survival of persistent bacteria. Furthermore, as shown in Table 1, the therapeutic effects of the series of cationic polymers designed in this invention on persistent bacteria were significantly better than those in the control group.

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

[0135]

[0136] To visualize the damage effect on persistent bacteria, Figure 15 The figures show the morphology of bacteria treated with guanidine cationic polymers. As shown, after treatment with the 1x MIC guanidine cationic polymer, varying degrees of wrinkling, depressions, and even perforations appeared on the surface of each bacterium. Some bacteria were so broken that their morphology was no longer observable. In contrast, the control group, treated with vancomycin at a concentration of 50x MIC, showed normal morphology, clear boundaries, and good membrane integrity. Furthermore, the bacterial membrane integrity of the bacteria treated in the comparative experiment was better, showing a significant difference compared to the cationic polymer designed in this invention. Therefore, the above results further demonstrate the highly efficient removal ability of guanidine cationic polymers for persistent bacteria.

[0137] Figure 16 The figure shows the membrane potential of the surviving bacteria after treatment with guanidino cationic polymer. As shown, after treatment with 1x MIC guanidino cationic polymer, the membrane potential of the surviving bacteria changed from approximately -26mV to +20mV. Therefore, this large-scale change in the membrane potential of the surviving bacteria can disregard the physiological state of the surviving bacteria and ultimately lead to complete elimination of the surviving bacteria.

[0138] As shown in the above embodiments, this invention provides a highly efficient guanidine-based cationic polymer for eliminating persistent bacteria, its preparation method, and its applications. This invention is based on the fact that guanidine-based cationic polymers can eliminate persistent bacteria efficiently by directly affecting the bacterial cell membrane mechanism, regardless of the physiological state of the bacteria. The effective concentration of such cationic polymers against persistent bacteria can be as low as 4 μg / mL, which is superior to current first-line antibiotics and comparative studies. This invention uses the RAFT polymerization method to synthesize guanidine-containing polymers, which has a simple preparation process, low cost, can be mass-produced, and has a controllable structure.

[0139] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A highly efficient guanidine cationic polymer for eliminating persistent bacteria, characterized in that, The chemical formula of the guanidine cationic polymer is PN. x G or PO x G, with the following structural formula: or, , Where x is independent and ranges from 1 to 6, and m ranges from 20 to 50; 。 2. The guanidine cationic polymer for efficiently eliminating persistent bacteria according to claim 1, characterized in that, The structural formula of the guanidino cationic polymer is selected from one of the following structural formulas: ； ，m=20~50。 3. The method for preparing the highly efficient guanidine cationic polymer for eliminating persistent bacteria as described in claim 1 or 2, characterized in that, Includes the following steps: 1) The monomer, chain transfer agent and initiator are mixed in an organic solvent and reacted under heating or blue light to obtain an intermediate polymer; 2) The intermediate polymer, 1H-pyrazole-1-formamidinium hydrochloride and N,N-diisopropylethylamine were mixed in water, and the resulting reaction mixture was dialyzed and dried to obtain a dry polymer; 3) The dried polymer, water and hydrogen peroxide solution were mixed and reacted to remove the alkyl chain. The reaction product was purified by dialysis and lyophilized to obtain the guanidine cationic polymer. The polymer monomer is 2-aminoethyl methacrylate hydrochloride, N-(3-aminopropyl)methacrylamide hydrochloride, methacrylated 6-(BOC-amino)-1-hexanol, 2-aminoethyl methacrylate hydrochloride, methacrylated N-(3-hydroxypropyl)carbamate tert-butyl ester or methacrylated N-(6-aminohexyl)carbamate tert-butyl ester.

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

5. The method for preparing the highly efficient guanidine cationic polymer for eliminating persistent bacteria according to claim 4, characterized in that, When the initiator is a thermal initiator azobisisobutyronitrile, the heating temperature is 60~80℃ and the reaction time is 8~20h; When the initiator is the photoinitiator tris(2-phenylpyridine)iridium, the reaction is carried out under blue light for 8-20 hours.

6. The method for preparing the highly efficient guanidine cationic polymer for eliminating persistent bacteria according to any one of claims 3 to 5, characterized in that, The mass ratio of the polymerizing monomer, chain transfer agent and initiator is 100~300mg:3~10mg:4μg~1mg; The ratio of the polymer monomer to the organic solvent is 100~300mg:1~2mL.

7. The method for preparing the highly efficient guanidine cationic polymer for eliminating persistent bacteria according to claim 6, characterized in that, The intermediate polymer undergoes a deprotection treatment, which involves dissolving the intermediate polymer in a dichloromethane solution and removing 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.

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

9. The method for preparing the highly efficient guanidine cationic polymer for eliminating 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 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℃, and the reaction time is 2-5 h.

10. The use of the guanidino cationic polymer of claim 1 or 2, which is highly efficient at eliminating persistent bacteria, in the preparation of formulations for eliminating persistent bacteria.

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