Hyperbranched polylysine with high epsilon-linear unit content as well as preparation method and application of hyperbranched polylysine

Abstract

The invention belongs to the field of polymer synthesis and antibacterial materials, and discloses hyperbranched polylysine with high epsilon-linear unit content as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) mixing lysine with a compound of a formula I according to a certain ratio, and carrying out a polymerization reaction to prepare a polymer (II), the molar ratio of lysine being 0-90%, and the molar ratio of the compound of the formula I being 10-100%; (2) removing a borane protecting group to obtain hyperbranched polylysine (III) with high epsilon-linear unit content; according to the present invention, the selective sterilization on fungi / bacteria can be achieved through the structure regulation, the fungi can be resisted / killed only in the specific structure, the sterilization effect on most bacteria is not achieved, and the natural flora of the human body can be effectively protected. The hyperbranched polylysine with the high epsilon-linear unit prepared by the preparation method disclosed by the invention is efficient in fungus killing, low in toxicity and good in biocompatibility.

Description

Technical Field

[0001] The invention belongs to the field of polymer synthesis and antibacterial materials, and specifically relates to a hyperbranched polylysine with a high content of ε-linear units and a preparation method and application thereof. Background Art

[0002] Since lysine contains two amino groups in its structure: α-amino group and ε-amino group, starting from lysine, different polymerization processes can be used to synthesize linear α-polylysine (α-PL) and ε-polylysine (ε-PL), as well as branched polylysine (HPL) with branched structures. In the 1950s, Katchalski et al. discovered that α-PL could inhibit the growth of Gram-positive and Gram-negative bacteria, and copolymers containing lysine could inhibit the growth of Escherichia coli and Mycoplasma pyogenes. However, the antibacterial activity of α-PL is not very high and has certain toxicity. ε-PL synthesized by fermentation has excellent antibacterial effects on both Gram-positive and Gram-negative bacteria and low toxicity, but ε-PL has poor inhibitory effects on fungi, and its chemical synthesis process is immature, the microbial fermentation capacity is limited and the cost is high. In addition, existing antibacterial agents have almost no selectivity for fungi / bacteria.

[0003] Selective sterilization has important applications in many fields. In the medical field, selective bactericidal drugs that only kill specific pathogens can effectively treat infections while minimizing damage to normal flora, thereby reducing side effects and the risk of secondary infections. In addition, reducing the loss of beneficial bacteria can also slow down the development of antibiotic resistance in pathogens to a certain extent. In the food field, it is necessary to selectively kill harmful bacteria and spoilage bacteria without killing probiotics such as lactic acid bacteria, so as to ensure food safety while maintaining its quality and flavor. In the agricultural field, selective killing of harmful microorganisms can protect beneficial microorganisms, which is beneficial to ensuring crop yield and quality and maintaining ecosystem diversity.

[0004] The paper published by Zuzana Kadlecova et al. (Macromolecular Bioscience, 2021, 12, 794-804) reported the use of hyperbranched polylysine (HBPL) as a multifunctional, biodegradable transfection agent for the production of recombinant proteins by transient gene expression and primary cell transfection. HBPL was synthesized by thermal polycondensation reaction using lysine hydrochloride as raw material, which was first neutralized with one equivalent of KOH and then subjected to thermal polycondensation reaction. However, the ratio of α-linear units to ε-linear units of HBPL was not regulated, nor was the antibacterial performance of HBPL reported, especially its selective fungicidal performance. Summary of the invention

[0005] The purpose of the present invention is to provide a hyperbranched polylysine with a high content of ε-linear units and a preparation method and application thereof.

[0006] The technical solution of the present invention is as follows:

[0007] The intermediate (compound of formula I) is obtained by using compounds such as 9-borane bicyclo[3,3,1]-nonane (9-BBN) to form a ring with the α-amino group and carboxyl group of lysine, and then a polymerization reaction is carried out, and then a hyperbranched polylysine with a high content of ε-configuration is obtained by deprotection. The synthesis method of the intermediate has been disclosed in Org. Lett. 2002, 4, 1249-1251. The polymerization reaction includes subjecting the intermediate to a melt polycondensation reaction, or subjecting the intermediate to a solution polycondensation reaction in a solution; or, the intermediate is copolymerized with a lysine monomer in different proportions to a melt polycondensation reaction or a solution polycondensation reaction.

[0008] A method for preparing hyperbranched polylysine with a high content of ε-linear units comprises the following steps:

[0009] (1) mixing lysine and a compound of formula I in a certain ratio and performing a polymerization reaction to obtain a polymer (II), wherein the molar proportion of lysine is 0-90% and the molar proportion of the compound of formula I is 10-100%;

[0010] (2) removing the borane protecting group to obtain a hyperbranched polylysine (III) with a high content of ε-linear units;

[0011]

[0012]

[0013] Among them, m, n, x and y are the degree of polymerization, 1≤m, n, x, y≤500, the structure is an ε-linear unit, the structure is an α-linear unit.

[0014] Preferably, the degree of polymerization of the hyperbranched polylysine is 1≤m, n, x, y≤200, 10≤m+n+x+y≤200, and the molecular weight distribution is 1.10-3.00; more preferably, the degree of polymerization of the hyperbranched polylysine is 10≤m, n, x, y≤60, 20≤m+n+x+y≤200, and the molecular weight distribution is 1.30-2.20.

[0015] Preferably, the molar proportion of lysine is 0%-50%, more preferably, the molar proportion of lysine is 0%-20%.

[0016] Preferably, the polymerization reaction includes a melt polycondensation reaction or a solution polycondensation reaction, and the solvent of the solution polymerization reaction is one or more of ethylene glycol, propylene glycol, ethylene glycol monomethyl ether, diethylene glycol monomethyl ether, anisole, dioxane, N,N-dimethylformamide, NMP, N-methylpyrrolidone, dimethyl sulfoxide, and cyclopentane. In the melt polycondensation reaction, the polymerization process may or may not use a catalyst. The catalyst may be selected from one or more of metal salts; metal organic compounds or metal organic complexes; tertiary amines or quaternary ammonium salts; boric acid and its derivatives, phosphoric acid or phosphonic acid and its derivatives; and carboxylates.

[0017] Preferably, the polymerization reaction temperature in step 1) is 100° C.-220° C., and the reaction time is 30 minutes-48 hours; and the reaction temperature in step 2) is controlled at 20-90° C., and the reaction time is 30 minutes-24 hours.

[0018] Preferably, the polymerization reaction temperature in step 1) is 140° C.-200° C., and the time is 1 hour-24 hours; the compound of formula I is prepared by reacting lysine hydrochloride with 9-BBN.

[0019] Preferably, the removal of the borane protecting group is achieved by reacting the polymer (II) with a 25%-38% hydrochloric acid solution.

[0020] Application of hyperbranched polylysine with high content of ε-linear units prepared by the above method in selective killing of fungi.

[0021] Preferably, the molar content of the ε-linear unit is more than 50% of the total molar content of the ε-linear unit and the α-linear unit.

[0022] Preferably, the molar content of the ε-linear units is 55% to 95%, more preferably, the molar content of the ε-linear units is 70% to 90%.

[0023] The principle of the present invention is that α-polylysine can only kill bacteria, and ε-polylysine has a certain degree of bactericidal effect on fungi, but the effect is worse than that of hyperbranched polylysine, which may be because compared with ε-polylysine, hyperbranched polylysine has a higher charge density while containing ε-linear units. Therefore, by regulating the proportion of each structural unit of hyperbranched polylysine and increasing the proportion of ε-linear units, selective fungicide can be achieved.

[0024] 9-BBN can selectively protect the α-NH2 of lysine, thereby reducing the reactivity of α-NH2 during the polymerization process and increasing the reactivity of ε-NH2. Compared with the hyperbranched polylysine obtained by polymerization of lysine hydrochloride monomer, the hyperbranched polylysine obtained by polymerization of 9-BBN-lysine intermediate (compound of formula I) has a higher content of ε-linear units. By copolymerizing the compound of formula I with lysine and adjusting the feed ratio of the intermediate to the lysine monomer, the ratio of ε- / α-configuration in the hyperbranched polylysine can be regulated, thereby achieving selective killing of bacteria / fungi.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention can effectively adjust the antibacterial properties and toxicity of HPL by adjusting the branching degree and the ratio of α-linear units to ε-linear units. In particular, the ratio of α-linear units to ε-linear units in HPL can be controlled and adjusted through a special reaction pathway, thereby adjusting the antibacterial properties and toxicity.

[0027] (2) The present invention can achieve selective disinfecting of fungi / bacteria through structural regulation. In a specific structure, it can only resist / kill fungi, but has no disinfecting effect on most bacteria, which can effectively protect the natural flora of the human body.

[0028] (3) The high-ε-linear unit hyperbranched polylysine prepared by the present invention has high fungicidal efficiency, low toxicity and good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the H-NMR spectrum of the hyperbranched polylysine prepared in Example 1.

[0030] Figure 2 This is the NMR carbon spectrum of the hyperbranched polylysine prepared in Example 1.

[0031] Figure 3 It is a bar graph of the antibacterial properties of hyperbranched polylysine in Example 2.

[0032] Figure 4 These are photos of the antibacterial performance test results of Example 2. The upper picture is the test result of the microbroth dilution method, and the lower picture is the test result of the plate dilution method, Escherichia coli (left), Staphylococcus aureus (middle), and Candida albicans (right).

[0033] Figure 5 SEM images of Escherichia coli (top), Staphylococcus aureus (middle), and Candida albicans (bottom) after treatment with hyperbranched poly-lysine. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with specific embodiments, but the embodiments of the present invention are not limited thereto. For process parameters not particularly noted, conventional techniques may be used.

[0035] Example 1

[0036] Synthesis of Hyperbranched Polylysine

[0037] (1) Melt polycondensation

[0038] 9-BBN-lysine (compound of formula I), lysine hydrochloride and KOH were added to a 100 mL round-bottom flask, connected to a water separator, heated to 180°C in an oxygen-free environment, stirred for reaction for 6 hours, and then stopped heating. The product was dissolved in methanol and precipitated in ether. 2 g of the polymer intermediate was dissolved in 5 mL of methanol, 25 mL of concentrated hydrochloric acid was added, stirred at room temperature for 24 hours, precipitated in tetrahydrofuran, precipitated repeatedly for 3 times, and freeze-dried at -50 degrees for 24 hours to obtain hyperbranched polylysine.

[0039] (2) Solution polycondensation

[0040] The compound of formula I, lysine hydrochloride and KOH were dissolved in DMSO, added to a 100 mL round-bottom flask, connected to a water separator, heated to 180°C in an oxygen-free environment, stirred for 6 hours, and the heating was stopped, and the product was precipitated into ether. 2 g of the polymer intermediate was dissolved in 5 mL of methanol, 25 mL of concentrated hydrochloric acid was added, stirred at room temperature for 24 hours, precipitated into tetrahydrofuran, repeatedly precipitated 3 times, and freeze-dried to obtain hyperbranched polylysine.

[0041] By adjusting the ratio of the compound of formula I to lysine hydrochloride, hyperbranched polylysine with different ε-configuration contents can be obtained. The H NMR spectrum of the obtained hyperbranched polylysine is as follows: Figure 1 As shown, the NMR carbon spectrum is Figure 2 The specific feed ratio and ε-configuration content are shown in Table 1, wherein HPL-Bn is a hyperbranched polylysine prepared by melt polycondensation, and S-HPL-Bn is a hyperbranched polylysine prepared by solution polymerization.

[0042] Table 1

[0043]

[0044]

[0045] Comparative Example 1 91.32 g of lysine hydrochloride and 28.05 g of KOH were added to a 500 mL round-bottom flask, a water separator was connected, and the mixture was stirred at 180 ° C for 4 h under a nitrogen environment. The heating was stopped, the reaction system was cooled to room temperature, and the polymer was dissolved in methanol and precipitated in ether to obtain 75.2 g of hyperbranched polylysine. The ε-configuration content of the product in Comparative Example 1 was 30%.

[0046] From the above comparison, it can be seen that by controlling the molar ratio of the compound of formula I to lysine hydrochloride, the structure of the hyperbranched polylysine can be effectively regulated, and a hyperbranched polylysine with a high ε-linear unit content can be obtained.

[0047] Example 2 Antibacterial Performance Test

[0048] Antibacterial: 36 mg of the hyperbranched polylysine of Example 1 and Comparative Example 1 were dissolved in 3 mL of sterile PBS to obtain a 12 mg / mL mother solution for antibacterial performance testing. The testing method is as follows:

[0049] The antibacterial properties of hyperbranched poly-lysine were tested by the microbroth dilution method. A small amount of bacterial colonies were picked up with an inoculation loop and placed in MH broth medium. The culture was cultured at 37°C overnight to revive the bacterial strain and grow to the mid-logarithmic growth stage. The bacterial solution concentration was adjusted to ~10 6 CFU / mL. Take 175 μL of bacterial suspension and add it to a 96-well plate, then add 25 μL of gradient diluted hyperbranched poly-lysine solution, place the 96-well plate in a 37°C incubator for 24 hours, and use an enzyme reader to detect OD 600 To determine the bacterial growth, the turbidity of each well was observed with the naked eye. The minimum inhibitory concentration (MIC) was defined as the concentration of the antibacterial agent required to inhibit 90% bacterial growth compared with the control group.

[0050] Antifungal: 36 mg of the hyperbranched polylysine of Example 1 and Comparative Example 1 were dissolved in 3 mL of sterile MOPS to obtain a 12 mg / mL mother solution for antifungal performance testing. The testing method is as follows:

[0051] The antifungal properties of hyperbranched poly-lysine were tested by the microbroth dilution method. A small number of fungal colonies were picked up with an inoculation loop and placed in SDB medium. The culture was cultured at 35°C for 24 hours to revive the activity of the fungus and grow to the mid-logarithmic growth stage. The concentration of the bacterial solution was adjusted to 10~10 with RPMI1640-MOPS medium. 3 CFU / mL. Take 175 μL of bacterial suspension and add it to a 96-well plate, then add 25 μL of gradient diluted hyperbranched poly-lysine solution, place the 96-well plate in a 35°C incubator for 48 hours, and use an enzyme reader to detect OD 600To determine the fungal growth, the turbidity of each well was observed with the naked eye. The minimum inhibitory concentration (MIC) was defined as the concentration of the antibacterial agent required to inhibit 90% fungal growth compared with the control group.

[0052] Table 2 Antibacterial performance test results (MIC value, unit μg / mL)

[0053]

[0054] It can be seen from Table 2 that, compared with Comparative Example 1, the hyperbranched polylysine of the present invention can kill only fungi but not bacteria, thereby achieving a selective bactericidal effect.

[0055] Example 3 Blood compatibility test

[0056] 36 mg of the hyperbranched polylysine obtained in Example 1 was dissolved in 3 mL of sterile PBS solution to obtain a 12 mg / mL mother solution for in vitro hemolysis test. The test results are shown in Table 3. The test method is as follows:

[0057] Take 1 mL of fresh rabbit blood, collect red blood cells after full centrifugation, and dilute with sterile PBS to obtain a 2% (v / v) red blood cell suspension. Add 175 μL of red blood cell suspension to a 1.5 mL sterile centrifuge tube, then add 25 μL of gradient diluted hyperbranched polylysine solution, add 25 μL of 0.5% TX-100 solution to the positive control, and add 25 μL of sterile PBS to the negative control, incubate at 37°C for 1 hour, centrifuge, and draw 150 μL of supernatant into a 96-well plate, and use an enzyme reader to detect OD 540 , HC 10 It is defined as the polymer concentration that causes 10% hemolysis of erythrocytes.

[0058] Table 3 Hemolysis test results (HC 10 Value, unit: μg / mL)

[0059] <![CDATA[HC 10 ]]> HPL-B100 >1500 HPL-B95 >1500 HPL-B90 >1500 HPL-B80 >1500 HPL-B70 >1500 HPL-B60 >1500 HPL-B50 >1500 HPL-B40 >1500 HPL-B30 >1500 HPL-B20 >1500 HPL-B10 >1500

[0060] Example 4 Cytotoxicity Test

[0061] 36 mg of the hyperbranched polylysine obtained in Example 1 was dissolved in 3 mL of sterile PBS solution to obtain a 12 mg / mL mother solution for cytotoxicity testing. The test results are shown in Table 4. The test method is as follows:

[0062] After digestion and elution of mouse embryonic fibroblast NIH3T3 / L929 cells in good growth condition, a cell suspension of 40,000 cells / mL was prepared. 200 μL of the cell suspension was added to a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 12 hours until the cells adhered to the wall. Then 25 μL of gradient diluted hyperbranched poly-lysine solution was added and the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours. The cytotoxicity was tested by MTT method. IC 50 It was defined as the polymer concentration that inhibited cell growth by 50%.

[0063] Table 4 Cytotoxicity test results (IC 50 Value, unit: μg / mL)

[0064] <h2 style=";text-align:left;direction:ltr"><![CDATA[IC <h2 style=";text-align:left;direction:ltr"> 50 <h2 style=";text-align:left;direction:ltr"> (NIH3T3)]]><h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"><![CDATA[IC <h2 style=";text-align:left;direction:ltr"> 50 <h2 style=";text-align:left;direction:ltr"> (L929)]]><h2 style=";text-align:left;direction:ltr"> HPL-B100 >1500 >1500 HPL-B95 >1500 >1500 HPL-B90 >1500 >1500 HPL-B80 >1500 >1500 HPL-B70 >1500 >1500 HPL-B60 >1500 >1500 HPL-B50 >1500 >1500 HPL-B40 >1500 >1500 HPL-B30 >1500 >1500 HPL-B20 >1500 >1500 HPL-B10 >1500 >1500

[0065] It can be seen from the above embodiments and comparative examples that the high-ε-linear unit hyperbranched polylysine prepared by the present invention can selectively and efficiently kill fungi without killing bacteria, and has low toxicity and good biocompatibility.

[0066] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing hyperbranched polylysine with a high content of ε-linear units, characterized in that: The steps include: (1) mixing lysine and a compound of formula I in a certain ratio and performing a polymerization reaction to obtain a polymer (II), wherein the molar proportion of lysine is 0-90% and the molar proportion of the compound of formula I is 10-100%; (2) removing the borane protecting group to obtain a hyperbranched polylysine (III) with a high content of ε-linear units; Among them, m, n, x and y are the degree of polymerization, 1≤m, n, x, y≤500, the structure is an ε-linear unit, the structure is an α-linear unit.

2. The preparation method according to claim 1, characterized in that: The degree of polymerization of the hyperbranched polylysine is 1≤m, n, x, y≤200, 10≤m+n+x+y≤200, and the molecular weight distribution is 1.10-3.00; more preferably, the degree of polymerization of the hyperbranched polylysine is 10≤m, n, x, y≤60, 20≤m+n+x+y≤200, and the molecular weight distribution is 1.30-2.

20.

3. The preparation method according to claim 2, characterized in that: The molar proportion of the lysine is 0%-50%, more preferably, the molar proportion of the lysine is 0%-20%.

4. The preparation method according to claim 1, 2 or 3, characterized in that: The polymerization reaction includes a melt polycondensation reaction or a solution polycondensation reaction. The solvent of the solution polymerization reaction is one or more of ethylene glycol, propylene glycol, ethylene glycol monomethyl ether, diethylene glycol monomethyl ether, anisole, dioxane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, and sulfolane.

5. The preparation method according to claim 4, characterized in that: In step 1), the polymerization reaction temperature is 100° C.-220° C., and the reaction time is 30 minutes-48 hours; in step 2), the reaction temperature is controlled at 20-90° C., and the reaction time is 30 minutes-24 hours.

6. The preparation method according to claim 5, characterized in that: The polymerization reaction temperature in step 1) is 140°C-200°C, and the time is 1 hour-24 hours; the compound of formula I is prepared by reacting lysine hydrochloride with 9-BBN; and the removal of the borane protecting group is achieved by reacting the polymer (II) with concentrated hydrochloric acid.

7. Hyperbranched polylysine with a high content of ε-linear units obtained by the method according to any one of claims 1 to 6.

8. Application of hyperbranched polylysine with high content of ε-linear units in selective killing of fungi.

9. The use according to claim 8, characterized in that: The molar content of the ε-linear unit accounts for more than 50% of the total molar number of the ε-linear unit and the α-linear unit.

10. The use according to claim 9, characterized in that: The molar content of the ε-linear units is 55% to 95%, and more preferably, the molar content of the ε-linear units is 70% to 90%.

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