CN107397961B

CN107397961B - Method for improving resistance of polyethyleneimine to enterobacter coli

Info

Publication number: CN107397961B
Application number: CN201710792300.1A
Authority: CN
Inventors: 刘梅; 李娇; 李宣仪
Original assignee: Shaanxi Normal University
Current assignee: Xi'an Tanglong International Hotel Co.,Ltd.
Priority date: 2017-09-05
Filing date: 2017-09-05
Publication date: 2020-10-16
Anticipated expiration: 2037-09-05
Also published as: CN107397961A

Abstract

The invention discloses a method for improving the anti-coliform performance of polyethyleneimine, which is characterized in that mannose is modified on branched polyethyleneimine, the modification method is simple to operate, short in reaction time and low in cost, and the minimum bactericidal concentration of the mannose-modified polyethyleneimine on escherichia coli is 10 mug/mL, which is 22 times lower than that of polyethyleneimine (220 mug/mL); meanwhile, after the HeLa cells are added with 500 mug/mL of antibacterial agents and cultured for 24 hours, the cell activity of the polyethyleneimine modified by adding mannose is kept at 75%, and the cell activity of the polyethyleneimine is reduced to 56%. Therefore, the method of the invention obviously improves the sterilization rate of the polyethyleneimine on the escherichia coli and reduces the cytotoxicity.

Description

Method for improving resistance of polyethyleneimine to enterobacter coli

Technical Field

The invention belongs to the technical field of antibiosis, and particularly relates to a method for improving the coliform resistance of polyethyleneimine.

Background

Escherichia coli (Escherichia coli) is a common pathogenic bacterium in life, often causes diarrhea and septicemia, is a gram-negative short bacillus with blunt ends, circumferential flagella, motion capability and no spores, is widely distributed in nature, so that food has a high probability of being polluted by the Escherichia coli and is easy to cause food poisoning. For example, spinach in the us was contaminated with e.coli in 2006, with disease spreading in half the us; the European "poisonous cucumber" event in 2011 is also an infection caused by the contamination of vegetables with E.coli. Coli has caused numerous large-scale infections worldwide, leading to many deaths. In order to ensure the food safety, consumers can purchase green and healthy food with ease. The research on a novel escherichia coli sterilization method has very important significance.

At present, the common escherichia coli sterilization method mainly adopts antibiotics for sterilization, and as a large amount of antibiotics are used, the escherichia coli generates drug resistance to a plurality of antibiotics, wherein 70% of bacteria have resistance to at least one drug, and the high-concentration antibiotics have certain harm to human bodies. Aiming at the problem of antibiotic resistance of bacteria, novel antibiotics become a research hotspot. The cationic polymer as a novel antibacterial agent has an antibacterial mechanism different from that of conventional antibiotics, so that bacteria hardly develop resistance, and polyethyleneimine is one of typical representatives of cationic antibacterial agents.

Polyethyleneimine (PEI) is a novel polycationic compound with water solubility. According to the difference of molecular structures, the molecular structures of the compounds can be divided into two types, namely linear (L-PEI) and branched (B-PEI), and the molecular structures are as follows:

in the structure of PEI, a large number of amino groups are contained, and it is because the large number of amino groups imparts important characteristics to PEI, i.e., a high positive charge. Therefore, PEI alone (unmodified) can contact negatively charged bacteria via coulombic attraction, disrupting the structure of the bacteria, and thereby inhibiting bacterial growth. Katherine A project group reports in 2012 (Macromolecular Bioscience,2012,12, 1279-one 1289), and PEI alone has selectivity on inhibition of bacteria and has a relatively obvious inhibition effect on staphylococcus aureus (the minimum inhibitory concentration is relatively low); in addition, PEI with different molecular weights has different bacteriostatic effects, and for Escherichia coli, B-PEI₆₀₀The minimum inhibitory concentration is 500 mu g/mL, B-PEI₁₈₀₀The minimum inhibitory concentration is 250 mu g/mL, B-PEI₁₀₀₀₀The minimum inhibitory concentration of (A) is more than 1 g/mL; B-PEI for Staphylococcus aureus₆₀₀(MW:600) minimum inhibitory concentration of 16. mu.g/mL, B-PEI₁₈₀₀The minimum inhibitory concentration is 32 mu g/mL, B-PEI₁₀₀₀₀The minimum inhibitory concentration of (2) is 16 mug/mL. Therefore, the antibacterial effect of the B-PEI on staphylococcus aureus is far better than the antibacterial effect on escherichia coli, and the application of the B-PEI is limited due to the fact that the antibacterial effect of the B-PEI on escherichia coli is not ideal; on the other hand, as the molecular weight of B-PEI increases, the cytotoxicity increases. Therefore, how to reduce the cytotoxicity of PEI and improve the antibacterial effect of PEI on escherichia coli needs to be solved.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for simply and rapidly improving the resistance of polyethyleneimine to enterobacter coli.

The technical scheme for solving the technical problems is as follows: mannose is modified on branched polyethyleneimine.

The method for modifying mannose on branched polyethyleneimine comprises the following steps: adding branched polyethyleneimine and mannose into a PBS buffer solution with pH of 7.4, and stirring at 80-100 ℃ for 30-60 minutes.

The method for modifying mannose on branched polyethyleneimine is preferably: the branched polyethyleneimine and mannose were added to a PBS buffer solution having a pH of 7.4, and stirred at 90 ℃ for 40 minutes.

In the method, the mass ratio of the branched polyethyleneimine to the mannose is 1: 0.09-0.72, and preferably the mass ratio of the branched polyethyleneimine to the mannose is 1: 0.24-0.36.

In the synthesis process of modifying mannose on branched polyethyleneimine, the aldehyde group on the mannose and the amino group on the branched polyethyleneimine can react as follows:

R₂C＝O+R'NH₂——R₂C＝NR'+H₂O

that is, the aldehyde group and primary amine generate imine (Schiff base structure) containing carbon-nitrogen double bond, thereby forming polymer particles with fluorescence, namely mannose modified polyethyleneimine (Man-PEI CPs).

According to the invention, mannose can be quickly and effectively modified on polyethyleneimine, and on one hand, the Man-PEI CPs synthesized by the method increase the binding opportunity of the polyethyleneimine and escherichia coli through the specific binding of the mannose and flagellin fimH on the surface of the escherichia coli; on the other hand, the Schiff base structure formed between the mannose and the polyethyleneimine is also helpful for improving the bactericidal capability of the polyethyleneimine on escherichia coli. The method has the advantages of simple operation, short reaction time, low cost, no need of excessive chemical modification and complex large-scale instruments, and the Man-PEI CPs synthesized by the method have excellent antibacterial performance and lower cytotoxicity.

Drawings

FIG. 1 is a chart of the UV-VISIBLE absorption spectra of mannose (Man), B-PEI (MW:600), Man-PEI CPs of example 3, and the fluorescence emission spectra of Man-PEI CPs.

FIG. 2 is an SEM image of B-PEI (MW: 600).

FIG. 3 is an SEM image of the Man-PEI CPs of example 3.

FIG. 4 is a graph of B-PEI (MW:600) and Man-PEI CPs from example 3¹HNRM Spectrum (D)₂O，600MHz)。

FIG. 5 is a photograph of the total number of Colonies (CFU) on LB broth agar plates after sterilization of Man-PEI CPs (10. mu.g/mL) synthesized at different ratios (B-PEI: Man) in examples 1-4 for 3 h.

FIG. 6 is a graph showing the bactericidal effect of B-PEI and Man-PEI CPs (10. mu.g/mL) of different molecular weights on E.coli in examples 3, 5 and 6.

FIG. 7 shows the bactericidal activity of B-PEI (MW:600) on E.coli at different concentrations.

FIG. 8 is the bactericidal ratio of the Man-PEI CPs of example 3 against E.coli at different concentrations.

FIG. 9 shows the viability of HeLa cells at different concentrations of B-PEI (MW:600) and Man-PEI CPs from example 3.

Detailed Description

The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.

Example 1

1g of B-PEI with a weight-average molecular weight of 600 was added to 7mL of 10mM PBS buffer solution with pH of 7.4, followed by stirring, 0.09g of mannose was added thereto, followed by stirring at 90 ℃ for 40min, cooling, dialysis in a dialysis bag for 24 hours to obtain Man-PEICPs, and the Man-PEICPs were stored in a refrigerator at 4 ℃.

Example 2

1g of B-PEI with a weight-average molecular weight of 600 was added to 7mL of 10mM PBS buffer solution with pH of 7.4, followed by stirring, 0.18g of mannose was then added, followed by stirring at 90 ℃ for 40min, cooling, dialysis in a dialysis bag for 24 hours to obtain Man-PEICPs, and the Man-PEICPs were stored in a refrigerator at 4 ℃.

Example 3

1g of B-PEI with a weight-average molecular weight of 600 was added to 7mL of 10mM PBS buffer solution with pH of 7.4, followed by stirring, 0.36g of mannose was added thereto, followed by stirring at 90 ℃ for 40min, cooling, dialysis in a dialysis bag for 24 hours to obtain Man-PEICPs, and the Man-PEICPs were stored in a refrigerator at 4 ℃.

Example 4

1g of B-PEI with a weight-average molecular weight of 600 was added to 7mL of 10mM PBS buffer solution with pH of 7.4, followed by stirring, 0.72g of mannose was then added, followed by stirring at 90 ℃ for 40min, cooling, dialysis in a dialysis bag for 24 hours to obtain Man-PEICPs, and the Man-PEICPs were stored in a refrigerator at 4 ℃.

As can be seen from FIG. 1, the Man-PEI CPs show a new absorption peak at 352nm, while B-PEI and mannose show no absorption peak above 250 nm. On the other hand, solutions of Man-PEI CPs at lambda_em＝460_nmIt showed strong fluorescence. As can be seen from FIGS. 2 and 3, M is compared with B-PEI_an-PEI CP_sAre monodisperse and present as coarse spheres of about 15 μm in diameter. These results all demonstrate that mannose is successfully modified on polyethyleneimine, forming new polymer particles. As can be seen in FIG. 4, M_an-PEICP_sIs/are as follows¹The H NMR spectrum showed a new peak at 8.40ppm, whereas B-PEI did not have a peak at this position, which is attributed to H₂This result further confirms that mannose is modified to polyethyleneimine by the schiff base structure.

Example 5

Adding 1g of B-PEI with the weight-average molecular weight of 1800 to 7mL of 10mM PBS buffer solution with the pH value of 7.4, stirring uniformly, and adding 0.36g of mannitolSugar, stirring at 90 deg.C for 40min, cooling, dialyzing in dialysis bag for 24 hr to obtain M_an-PEICP_sAnd then stored in a refrigerator at 4 ℃.

Example 6

Adding 1g of B-PEI with weight average molecular weight of 10000 into 7mL of 10mM PBS buffer solution with pH value of 7.4, stirring uniformly, adding 0.36g of mannose, stirring at 90 ℃ for 40min, cooling, and dialyzing in a dialysis bag for 24h to obtain M_anPEICPs, which were subsequently stored in a refrigerator at 4 ℃.

To prove the beneficial effects of the invention, the inventors conducted comparative tests on the performances of the B-PEI and the corresponding Man-PEICPs in examples 1-6, and the specific test conditions were as follows:

1. test for Escherichia coli resistance

The antimicrobial properties of an antimicrobial agent are generally characterized by a Minimum Bactericidal Concentration (MBC). MBC refers to the minimum concentration of antimicrobial agent required to completely kill a particular test bacterium, with smaller MBC values indicating greater ability of the material to kill the growth of that microorganism. The determination of the minimum bactericidal concentration consists of the following steps:

(1) weighing 25.0g of LB broth, adding the LB broth into 1L of distilled water, boiling to fully dissolve the LB broth, adjusting the pH value to 7.2-7.3, adding 15g of agar powder, boiling to dissolve the agar powder, and autoclaving at 121 ℃ for 20 min. After sterilization, the plates were poured on a sterile table for use. Taking out the Escherichia coli strain cryopreservation tube from a refrigerator at-80 deg.C, diluting the bacteria solution with sterile water by a proper time after the bacteria solution is dissolved, spreading 100 μ L of the bacteria solution on a plate, and culturing at 37 deg.C for 18 h.

(2) In a clean bench, 20mL of sterilized liquid medium was transferred to a 100mL conical flask, a 10. mu.L pipette was used to aspirate a complete colony from the plate, the pipette tip was driven into the conical flask, and the plate was incubated in a shaker (37 ℃ C., 260r/min) for 14h to allow OD of the bacterial suspension₆₀₀＝1。

(3) Taking out the bacteria liquid incubated for 14h in the shaking table, sucking 5mL into a 10mL centrifuge tube, centrifuging (6000r/min,2min), removing supernatant, adding 5mL physiological saline, mixing uniformly, centrifuging (6000r/min,2min), repeating twice, adding 5mL physiological saline into the thallus washed out of the culture medium, mixing uniformly until the thallus is ready to be culturedThe bacterial suspension was diluted to 1 × 10 with PBS buffer at pH 7.4⁵After doubling, adding B-PEI or Man-PEICPs solutions with different concentrations, putting the solutions into a shaker (37 ℃, 260r/min) for incubation for 3h, taking 100 mu L of the solution, coating the solution on a flat plate, performing inverted culture at 37 ℃ for 18h, and observing the growth condition of colonies; a blank control experiment is carried out by replacing B-PEI and Man-PEI CPs with PBS buffer solution, and experiments are carried out in parallel for 3 times.

The calculation method of the sterilization rate comprises the following steps:

the bactericidal rate was (1-number of colonies in experimental group/number of colonies in blank group) × 100%

As can be seen from fig. 5, in examples 1 to 4, the antibacterial effect of Man-PEI CPs was significantly improved with the increasing amount of mannose, and the bactericidal rate against escherichia coli was 100% when B-PEI: Man was 1:0.36 (example 3). Not only the B-PEI with the molecular weight of 600, but also the B-PEI with other molecular weights are connected with mannose with the same proportion, so that the sterilizing effect is obviously improved in fig. 6.

FIGS. 7 and 8 further confirm that the minimum bactericidal concentration of B-PEI is 220. mu.g/mL when the molecular weight is 600, and the 99.9% bactericidal rate can be achieved when the Man-PEI CPs are 10. mu.g/mL, so that compared with B-PEI, the Man-PEI CPs reduce the minimum bactericidal concentration of B-PEI by 22 times, and effectively improve the bactericidal rate of B-PEI on Escherichia coli.

2. Cytotoxicity assays

Cytotoxicity to HeLa cells was evaluated according to the 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide (MTT) method. The culture medium containing 10% fetal calf serum was used to prepare a single cell suspension, which was seeded into a 96-well plate at 1000 cells per well, 200. mu.L per well volume, and 5% CO at 37 ℃ in a medium containing 5% fetal calf serum₂And (4) performing medium incubation for 12 h. Equal concentration gradients of B-PEI and Man-PEI CPs were diluted with culture medium to give final concentrations of 10, 50, 100, 250, 500. mu.g/mL to 96-well plates, each repeated 3 times. After 24h incubation, the cells were washed with PBS buffer, and then 20. mu.L aliquots of MTT were added to each well to remove B-PEI and Man-PEI CPs and incubation continued for 4 h. Finally, 150 μ L of dimethyl sulfoxide was added to each well, and the mixture was shaken on a shaker at a low speed for 15min to dissolve the crystals sufficiently. The absorbance of each well was measured at 490nm of an enzyme linked immunosorbent assay.

As shown in FIG. 9, the Man-PEI CPs were not cytotoxic at MBC (10. mu.g/mL), whereas B-PEI showed some cytotoxicity at MBC (220. mu.g/mL). When the concentration of the antibacterial drug is increased to 500 mug/mL, after the CPs are added with the Man-PEI and cultured for 24 hours, the cell viability is still maintained at 75%, and the cell viability is reduced to 56% when the B-PEI is added. Therefore, compared with B-PEI, the Man-PEI CPs improve the sterilization effect of the B-PEI on Escherichia coli, and simultaneously reduce the cytotoxicity of the B-PEI, which further proves that the B-PEI CPs have good application prospects as antibacterial agents.

Claims

1. The application of mannose-modified polyethyleneimine in preparing anti-escherichia coli medicines is as follows: adding branched polyethyleneimine and mannose into a PBS (phosphate buffer solution) with the pH =7.4, wherein the mass ratio of the branched polyethyleneimine to the mannose is 1: 0.09-0.72, and stirring for 30-60 minutes at 80-100 ℃.

2. The use of mannose-modified polyethyleneimine according to claim 1 for the preparation of an anti-escherichia coli medicament, wherein: the branched polyethyleneimine and mannose were added to a PBS buffer of pH =7.4, and stirred at 90 ℃ for 40 minutes.

3. The use of mannose-modified polyethyleneimine according to claim 1 or 2 for the preparation of an anti-escherichia coli medicament, wherein: the mass ratio of the branched polyethyleneimine to the mannose is 1: 0.24-0.36.