CN111759796A

CN111759796A - Antibiotic hydrogel resisting biofilm and preparation method and application thereof

Info

Publication number: CN111759796A
Application number: CN201910196783.8A
Authority: CN
Inventors: 程义云; 胡婧婧; 胡倩瑜
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2019-03-15
Filing date: 2019-03-15
Publication date: 2020-10-13
Anticipated expiration: 2039-03-15
Also published as: CN111759796B

Abstract

The invention provides an antibiotic hydrogel capable of degrading a biofilm, which is formed by cross-linking oxidized polysaccharide macromolecules, biological enzymes capable of degrading the biofilm and aminoglycoside antibiotics through acid-sensitive Schiff base bonds. The invention also provides the antibiotic hydrogel capable of degrading the biofilm, and the antibiotic hydrogel can be applied to in vitro and in vivo antibiosis. The antibiotic hydrogel capable of degrading the biofilm can release aminoglycoside antibiotics as required on one hand, and simultaneously release biological enzyme components capable of degrading a biofilm matrix on the other hand, so that the biofilm is damaged, the sensitivity of treating the biofilm infection by the aminoglycoside antibiotics is improved, and the antibiotic hydrogel capable of degrading the biofilm is expected to be applied to various clinical anti-infection treatments.

Description

Antibiotic hydrogel resisting biofilm and preparation method and application thereof

Technical Field

The invention belongs to the field of biological materials and antibiosis, and relates to antibiotic hydrogel for an antibacterial biofilm, a preparation method and application thereof.

Background

In recent years, toxic and side effects and drug resistance are caused by abuse and unreasonable use of antibiotics, for example, when the drug dosage is lower than the lowest effective drug concentration, the virulence of bacteria can be increased by inducing gene mutation and the like, and the drug resistance of the bacteria is promoted; high drug concentration can produce various toxic and side effects such as deafness, kidney injury and the like. The bacteria usually exist in the form of a biofilm, and consist of extracellular polysaccharide, extracellular protein, dead bacteria and nucleic acid generated by the lysis of the bacteria, so that the bacteria become barriers for hindering the permeation and diffusion of drugs such as antibiotics and the like, and the bacteria are helped to escape from the immune capture of organisms, the concentration of the drugs required for killing the bacteria in the biofilm is hundreds of times of the drugs required for killing free bacteria, and the bacteria are promoted to generate drug resistance. Since development of novel antibiotics has been progressing slowly due to factors such as economic efficiency and government incentives, the antibacterial efficiency can be improved by administering the antibiotics as needed and destroying the barrier action of the biofilm, and the generation of drug resistance can be resisted from various aspects.

Aiming at reasonable administration of antibiotics, aminoglycoside antibiotic hydrogel capable of being released as required is designed, and aminoglycoside antibiotic is used as a gelling element and is crosslinked with oxidized natural polysaccharide macromolecules into gel through Schiff base bonds. The Schiff base bonds of the gel are broken under an acidic environment generated by bacterial proliferation, so that drugs are released, the release amount of aminoglycoside antibiotics can be automatically adjusted according to the bacterial infection degree, when bacterial infection is serious, the generated acidity is strong, so that the aminoglycoside antibiotics are released more, and when the pH value is restored to a normal physiological pH value after bacteria are killed, the release rate is reduced. Although the gel can reasonably administer aminoglycoside antibiotics to a certain extent, the killing effect on biofilm bacteria is seriously reduced, and the minimum inhibitory concentration on the biofilm bacteria is hundreds of times of that of free bacteria.

The biofilm is mainly composed of extracellular polysaccharide matrix secreted by bacteria, extracellular protein, various nucleic acids generated by bacteria and lysis of dead bacteria, and in recent years, researchers find that the content of the biofilm can be obviously reduced through the corresponding biofilms of the components. Singh proposes that pectinase and amylase can efficiently degrade a biofilm of aspergillus MTCC1323, and alginate can significantly reduce the drug resistance of bacteria in a mucous pseudomonas aeruginosa biofilm to antibiotics. Tetz proves that deoxyribonuclease (DNase) can effectively remove biofilms of various bacteria, so that the curative effect of various antibiotics such as penicillin, cefotaxime and the like on the bacteria in the biofilms is improved. Burrows, however, suggested that proteinase K could disperse the mature bacterial biofilm of listeria. Based on the research bases, the gel can be further introduced with a biological enzyme component for degrading the biological film and prepared into a composite gel, and the biological film component is degraded while the gel is antibacterial so as to improve the sensitivity of aminoglycoside antibiotics.

Disclosure of Invention

The invention provides a hydrogel capable of releasing aminoglycoside antibiotics as required, and the release amount of the antibiotics can be adjusted according to the degree of bacterial infection. When bacterial infection is serious, the acidity in the hydrogel is stronger, so that the amount of released antibiotics is higher; when the pH value of the hydrogel is normal after bacteria are killed, the release rate is slowed down. And the antibiotic anti-biofilm factor is released while the aminoglycoside antibiotic is released, so that the formed biofilm can be degraded, the protection of the biofilm on bacteria is eliminated, and the sensitivity of the aminoglycoside antibiotic to biofilm infection is improved. The invention is realized by the following technical scheme.

The invention provides a preparation method of antibiotic hydrogel for resisting biofilm, which takes aminoglycoside antibiotic and biological enzyme for degrading biofilm as gelling elements, and directly crosslinks the aminoglycoside antibiotic and oxidized polysaccharide macromolecules into gel through acid-sensitive Schiff base bonds to prepare the antibiotic hydrogel.

In the present invention, the biofilm degrading bio-enzyme includes one or more of a polysaccharide degrading bio-enzyme, a protein degrading bio-enzyme, a nucleic acid degrading bio-enzyme, and the like.

Among them, the biological enzymes that degrade polysaccharides include, but are not limited to, the following species: one or more of pectinase, cellulase, alginase, amylase, etc.

Among them, the protein-degrading biological enzymes include, but are not limited to, the following species: one or more of neutral protease, papain, trypsin, proteinase K, etc.

Among them, the biological enzymes that degrade nucleic acids include, but are not limited to, the following species: one or more of deoxyribonuclease (Dnase), ribonuclease (Rnase), phosphatase, and the like.

In the present invention, the aminoglycoside antibiotics include, but are not limited to, the following species: one or more of netilmicin, isepamicin, capreomycin, ribostamycin, sisomicin, apramycin, amikacin, kanamycin, gentamicin, paromomycin, tobramycin, neomycin, and the like.

In the invention, the polysaccharide polymer is a natural polysaccharide polymer or an artificially synthesized natural polysaccharide polymer.

In the present invention, the polysaccharide polymer includes, but is not limited to, the following materials: one or more of dextran, chitosan, alginic acid, hyaluronic acid, cellulose, lignin, chondroitin, glycosaminoglycan, starch, pectin, mannan, etc.

In the invention, polysaccharide macromolecules are oxidized by an oxidant to obtain aldehyde-containing oxidized polysaccharide macromolecules.

In the invention, the aminoglycoside antibiotics, the biological enzymes for degrading the biofilm and the oxidized polysaccharide macromolecules are crosslinked into gel through acid-sensitive Schiff base bonds.

The aminoglycoside antibiotics, the biological enzyme for degrading the biofilm and the oxidized polysaccharide macromolecule form a colloid system; wherein, the mass percent of the aminoglycoside antibiotics in the gelling system is 0.1-20%, preferably 1%; wherein, the concentration of the biological enzyme in the gelling system is 3-3000u/mL, which is different according to the type of the biological enzyme; wherein, the oxidized polysaccharide polymer accounts for 1 to 10 percent of the mass percent of the gel-forming system, preferably 3 percent; the concentration of the mother solution of the oxidized polysaccharide macromolecules before the preparation of the antibiotic hydrogel is 30mg/mL-200mg/mL, and the hydroformylation ratio is 5% -95%, preferably 50%.

In the invention, the preparation method of the gel is to mix the gel in situ, and the gelation time of the gel is between 1 second and 60 minutes. The gelation time is related to the hydroformylation ratio of the oxidized polysaccharide macromolecules, the dosage and the type of aminoglycoside antibiotics and biological enzymes for degrading the biofilm, and the higher the hydroformylation ratio is, the shorter the gelation time is; the higher the content of antibiotics and biofilm-degrading enzymes or the higher the number of amino groups, the shorter the gelation time.

In the invention, the prepared antibiotic hydrogel resisting the biofilm can simultaneously release aminoglycoside antibiotics reasonably according to requirements and can simultaneously release active ingredients resisting the biofilm.

In the invention, the antibiotic hydrogel for resisting the biofilm can efficiently degrade the biofilm, can inhibit the generation of the biofilm and degrade the mature biofilm, and can obviously improve the antibacterial sensitivity of aminoglycoside antibiotics, especially aiming at the infection of tunica mucosa bacteria.

Wherein the antibiotic hydrogel is used for inhibiting the formation of a bacterial biofilm and degrading a mature bacterial biofilm.

The invention also provides application of the antibiotic hydrogel in preparation of a medicine for inhibiting bacterial infection or environmental bacterial pollution.

In the invention, the aminoglycoside antibiotic gel resisting the biofilm is applied to the infection treatment of clinical various wounds, intubation, device implantation and the like, and can also be applied to the treatment of bacterial pollution caused by other biofilms.

The invention is mainly characterized in that:

(1) the hydrogel is formed by directly mixing aminoglycoside antibiotics and biological enzyme for degrading biofilm as cross-linking agents with oxidized polysaccharide macromolecules. The preparation method is simple and the used materials are safe.

(2) The component of the anti-biofilm is enzyme corresponding to the component of the biomembrane, and the anti-biofilm has high removal efficiency on the biomembrane, short action time and wide universality on strains. Not only can inhibit the formation of biofilm, but also can degrade mature bacterial biofilm.

(3) The hydrogel can reasonably release biological enzyme resisting biofilm and aminoglycoside antibiotic according to the infection degree of bacteria.

(4) The hydrogel improves the antibacterial sensitivity of aminoglycoside antibiotics by destroying the barrier of a biological membrane, and has the advantages of simple operation method, simple action mechanism and obvious biological membrane resisting effect. Can be applied to biofilm infection of various bacteria.

Drawings

FIG. 1 is a graph showing the effect of pectinase on minimum inhibitory concentration of amikacin in example 1.

FIG. 2 is a graph showing the effect of the neutral protease on the minimum inhibitory concentration of amikacin in example 2.

FIG. 3 is a graph showing the effect of nuclease on minimum inhibitory concentration of amikacin in example 3.

FIG. 4 is a photograph of a complex gel of amikacin with pectinase, neutral protease and nuclease in examples 4-6.

FIG. 5 is a photograph of a composite gel of the pectinase in example 7 with isepamicin, netilmicin, kanamycin, gentamicin, tobramycin, and paromomycin, respectively.

FIG. 6 is a graph of the effect of pectinase on Pseudomonas aeruginosa biofilm formation as analyzed by crystal violet staining in example 8.

FIG. 7 is a graph of the plate colony counting assay of example 9 analyzing the effect of pectinase on mature Pseudomonas aeruginosa biofilms.

FIG. 8 is a graph of the effect of neutral protease on Pseudomonas aeruginosa biofilm formation as analyzed by crystal violet staining in example 10.

FIG. 9 is a graph of the plate colony counting assay of example 11 to analyze the effect of neutral protease on mature Pseudomonas aeruginosa biofilms.

FIG. 10 is a graph of the effect of DNase on the formation of a biofilm of Pseudomonas aeruginosa in example 12, as analyzed by crystal violet staining.

FIG. 11 is a graph of the plate colony counting assay of example 13 analyzing the effect of DNase on mature Pseudomonas aeruginosa biofilms.

FIG. 12 shows the inhibition zones of the amikacin/pectinase/oxidized dextran complex gel against Pseudomonas aeruginosa and Staphylococcus aureus in example 14.

FIG. 13 is a graph showing the toxicity of amikacin/pectinase/oxidized dextran complex gel on cells analyzed by the MTT method in example 15.

FIG. 14 shows the hemolysis behavior of the amikacin/pectinase/oxidized dextran complex gel of example 16.

FIG. 15 is a statistical chart of the number of colonies of Kunming mice infected with Pseudomonas aeruginosa tunica for three days without any treatment and infected portions of the skin in example 17.

FIG. 16 is a statistical chart of the number of colonies of Kunming mice treated with amikacin/oxidized dextran gel and infected with Pseudomonas aeruginosa tunica for three days and infected portions of the skin in example 18.

FIG. 17 is a statistical chart of the number of colonies of Kunming mice treated with pectinase/oxidized dextran gel and infected with Pseudomonas aeruginosa tunica for three days and infected parts of the skin in example 19.

FIG. 18 is a statistical chart of the number of colonies of Kunming mice treated with amikacin/pectinase/oxidized dextran complex gel and infected with Pseudomonas aeruginosa tunica for three days and infected portions of the skin in example 20.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, test methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art, except for those specifically mentioned below, and the present invention is not particularly limited thereto.

First, the effect of the anti-biofilm component on the bactericidal effect of aminoglycoside antibiotics:

example 1: influence of pectinase on sensitivity of aminoglycoside antibiotics in killing free bacteria and tunica capsularis

Amikacin was selected as a model representative drug for aminoglycoside antibiotics and pectinase was selected as a representative for the degradation of polysaccharide components in the anti-biofilm preparation. Pseudomonas aeruginosa was inoculated into 96-well plates at an initial concentration of 10 bacteria at 100. mu.L per well⁴CFU/mL, incubated in an incubator at 37 ℃ for three days, during which the medium was changed once a day to culture mature biofilms. On the fourth day, the medium was removed, fresh medium was added, and amikacin was added at different concentrations, respectively, and its minimum inhibitory concentration against bacteria in the mature biofilm was determined. Meanwhile, pectinase (200u/mL) with a certain concentration is introduced into amikacin solutions with different concentrations, and the minimum inhibitory concentration of the mixture of the pectinase and the amikacin on bacteria in a mature biofilm is detected.

As shown in figure 1, the minimum inhibitory concentration of amikacin to Pseudomonas aeruginosa in mature biofilm is 640ug/mL and 320 times of the minimum inhibitory concentration of free Pseudomonas aeruginosa of 2ug/mL, however, under the action of pectinase, the minimum inhibitory concentration of amikacin to Pseudomonas aeruginosa in biofilm is 32ug/mL, which indicates that the sensitivity of amikacin to tunica bacteria is significantly improved with the assistance of pectinase.

Example 2: influence of neutral protease on sensitivity of aminoglycoside antibiotics in killing free bacteria and tunica capsularis

The mature biofilm was prepared according to the method of example 1, and treated by adding neutral protease in the same way, as shown in fig. 2, it was observed that the killing performance of amikacin against enveloped bacteria could be significantly improved when the amount of neutral protease used was 3u/mL, and the MIC value of 640ug/mL was reduced to 8 ug/mL.

Example 3: influence of phosphatase on sensitivity of aminoglycoside antibiotics in killing free bacteria and tunica capsularis

Mature biofilms were prepared according to the method of example 1, and were treated by the same method with phosphatase, as shown in fig. 3, it was observed that the killing performance of amikacin against tunica bacteria was significantly improved when the phosphatase was 12.5u/mL, and the MIC value of 640ug/mL was reduced to 16 ug/mL.

Secondly, preparing aminoglycoside/anti-biofilm agent/oxidized polysaccharide polymer composite gel:

example 4: preparation of aminoglycoside/anti-biofilm agent/oxidized natural polysaccharide molecule composite gel 1

Amikacin is selected as a model representative drug of aminoglycoside antibiotics, pectinase is selected as a representative for degrading polysaccharide components in the anti-biofilm preparation, and dextran is taken as an example of oxidized polysaccharide macromolecules. Wherein 10uL amikacin (mother liquor is 100mg/mL), 15 uL pectinase (final concentration is 200u/mL), and 75 uL oxidized dextran (mother liquor is 50mg/mL) are mixed for about 2 minutes to form gel.

Example 5: preparation of aminoglycoside/anti-biofilm agent/oxidized polysaccharide polymer composite gel 2

Amikacin is selected as a model representative drug of aminoglycoside antibiotics, neutral protease is a representative of degraded protein components in the anti-biofilm preparation, and dextran is taken as an example of oxidized polysaccharide molecules. Wherein 10 mu L of amikacin (mother liquor is 100mg/mL), 15 mu L of neutral protease (mother liquor is 3u/mL), and 75 mu L of oxidized dextran (mother liquor is 50mg/mL) are mixed and gelatinized within about 2 minutes.

Example 6: preparation of aminoglycoside/anti-biofilm agent/oxidized polysaccharide polymer composite gel 3

Amikacin was selected as a model representative drug for aminoglycoside antibiotics and phosphatase was selected as a representative for the degradation of nucleic acid components in anti-biofilm formulations. Wherein 10 mu L of amikacin (mother liquor is 100mg/mL), 15 mu L of 5' -phosphodiesterase (final concentration is 12.5u/mL), and 75 mu L of oxidized dextran (mother liquor is 50mg/mL) are mixed and colloid is formed in about 2 minutes.

As shown in FIG. 4, amikacin can form stable complex gel with pectinase, neutral protease and DNase.

Example 7: preparation of aminoglycoside/anti-biofilm agent/oxidized polysaccharide polymer composite gel

Selecting other aminoglycoside antibiotic drugs, such as isepamicin, netilmicin, kanamycin, gentamicin, tobramycin, and paromomycin. The antibiotic component is represented by pectinase, and is used for preparing composite gel with different aminoglycoside antibiotic drugs. Wherein, 10 mul of various aminoglycoside antibiotic medicine solution (mother solution is 100mg/mL), 15 mul of 5' -phosphodiesterase (final concentration is 12.5u/mL), and 75 mul of oxidized dextran (mother solution is 50mg/mL) are mixed and can be gelled, and the gelling time is between 1min and 5 min.

As shown in FIG. 5, pectinase can be used to form a complex gel with various aminoglycoside antibiotics and oxidized dextran.

Thirdly, evaluating the performance of aminoglycoside/anti-biofilm agent/oxidized polysaccharide polymer composite gel for inhibiting and degrading biofilm

Example 8: evaluation of performance of amikacin/pectinase/oxidized dextran composite gel 1 in inhibiting biofilm

Putting a cover glass with the diameter of 2.0cm into a 24-hole plate as a carrier, adding pseudomonas aeruginosa bacterial liquid for culturing, constructing an in-vitro biomembrane model of pseudomonas aeruginosa, and adding an antibacterial material at the same time. Totally divided into 4 groups, wherein the first group is added with amikacin/pectinase/oxidized dextran composite gel 1, the second group is added with amikacin/oxidized dextran gel, the third group is added with pectinase/oxidized dextran gel, and the fourth group is not treated. After 48h of incubation, biofilm formation on the slides was observed using crystal violet staining and the stained pictures were photographed using a microscope.

As shown in FIG. 6, very dense biofilms were present on slides that were not treated, and the biofilm content was relatively low on slides treated with amikacin/oxidized polysaccharide gel or pectinase/oxidized dextran gel, whereas no significant biofilm was present on slides treated with amikacin/pectinase/oxidized dextran complex gel. The combination of pectinase and amikacin can obviously inhibit the generation of the pseudomonas aeruginosa biofilm.

Example 9: evaluation of performance of amikacin/pectinase/oxidized dextran composite gel 1 in degrading biofilm

Placing cover glass with diameter of 2.0cm as carrier into 24-well plate, adding Pseudomonas aeruginosa bacterial liquid (initial concentration of 10)⁶CFU/mL) for 3 days, and constructing an in vitro mature biofilm model of Pseudomonas aeruginosa. On the fourth day, the antimicrobial material was added. Totally divided into 4 groups, wherein the first group is added with amikacin/pectinase/oxidized dextran composite gel 1, the second group is added with amikacin/oxidized dextran gel, the third group is added with pectinase/oxidized dextran gel, and the fourth group is not treated. After 24h of culture, the number of viable bacteria in the mature biofilm was counted by plate colony counting.

As shown in FIG. 7, the number of viable bacteria in mature biofilm of Pseudomonas aeruginosa was 5.0 × 10 without any antibacterial treatment¹⁰CFU/mL, the number of viable bacteria treated by amikacin/oxidized dextran gel or pectinase/oxidized dextran gel is 7.0 × 10⁹CFU/mL and 3.67 × 10¹⁰After the amikacin/pectinase/oxidized dextran composite gel is treated, the number of the living bacteria in the biological envelope is obviously reduced to 2.67 × 10⁷CFU/mL. The polysaccharide matrix of the mature biofilm can be destroyed by degrading the polysaccharide by pectinase, and the amikacin can be assisted to efficiently kill bacteria in the biofilm. Therefore, the pectinase can obviously improve the sensitivity of the amikacin to bacteria in a mature biological membrane.

Example 10: evaluation of biofilm inhibition performance of amikacin/neutral protease/oxidized polysaccharide polymer composite gel 2

Placing cover glass with diameter of 2.0cm as carrier into 24-well plate, adding Pseudomonas aeruginosa bacterial liquid (initial concentration of 10)⁶CFU/mL), constructing an in vitro biofilm model of Pseudomonas aeruginosa, and adding antibacterial agentA material. Totally divided into 4 groups, wherein the first group is added with amikacin/neutral protease/oxidized polysaccharide composite gel 2, the second group is added with amikacin/oxidized polysaccharide gel, the third group is added with neutral protease/oxidized polysaccharide gel, and the fourth group is not subjected to any treatment. After 48h of incubation, biofilm formation on the slides was observed using crystal violet staining and the stained pictures were photographed using a microscope.

As shown in fig. 8, very dense biofilms were present on slides that were not treated, the biofilm content on slides treated with amikacin/oxidized dextran gel or neutral protease/oxidized dextran gel was relatively low, and no significant biofilm was present on slides treated with amikacin/neutral protease/oxidized dextran complex gel. The combination of neutral protease and amikacin can obviously inhibit the generation of the pseudomonas aeruginosa biofilm.

Example 11: amikacin/neutral protease/oxidized dextran composite gel 2 evaluation of performance of degrading biofilm

Placing cover glass with diameter of 2.0cm in 24-well plate, adding Pseudomonas aeruginosa bacterial liquid (initial concentration of 10)⁶CFU/mL) for 3 days, and constructing an in vitro mature biofilm model of Pseudomonas aeruginosa. On the fourth day, the antimicrobial material was added. Totally divided into 4 groups, wherein the first group is added with amikacin/neutral protease/oxidized dextran composite gel 2, the second group is added with amikacin/oxidized dextran gel, the third group is added with neutral protease/oxidized dextran gel, and the fourth group is not treated. After 24h of culture, the number of bacteria surviving in the mature biofilms was counted by plate colony counting.

As shown in FIG. 9, the number of viable bacteria in mature biofilm of Pseudomonas aeruginosa without any antibacterial treatment was 6.67 × 10⁸CFU/mL, after treatment with amikacin/oxidized dextran gel or neutral protease/oxidized dextran gel, the number of viable bacteria in the biofilm was 7 × 10⁶CFU/mL and 2.2 × 10⁸CFU/mL. After the amikacin/neutral protease/oxidized dextran composite gel treatment, the number of the surviving bacteria in the biological envelope is only2.6×10⁵CFU/mL. It can be seen that the neutral protease can destroy the mature biofilm by degrading the extracellular protein matrix, and can assist amikacin in efficiently killing bacteria in the biofilm. Therefore, it can be concluded that neutral protease can significantly improve the sensitivity of amikacin to bacteria in mature biofilms.

Example 12: evaluation of performance of amikacin/5' -phosphodiesterase/oxidized dextran composite gel 3 in inhibiting biofilm

Placing cover glass with diameter of 2.0cm in 24-well plate, adding Pseudomonas aeruginosa bacterial liquid (initial concentration of 10)⁶CFU/mL), constructing an in vitro slide biofilm model of the pseudomonas aeruginosa, and adding an antibacterial material at the same time. Totally divided into 4 groups, wherein the first group is added with amikacin/5 '-phosphodiesterase/oxidized dextran composite gel 1, the second group is added with amikacin/oxidized dextran gel, the third group is added with 5' -phosphodiesterase/oxidized dextran gel, and the fourth group is not treated at all. After 48h of culture, biofilm formation on the slides was observed by crystal violet staining.

As shown in fig. 10, very dense biofilms were present on slides that were not treated, and the biofilm content was relatively low on slides treated with amikacin/oxidized dextran gel or 5 '-phosphodiesterase/oxidized dextran gel, while no significant biofilm was present on slides treated with amikacin/5' -phosphodiesterase/oxidized dextran complex gel. The combination of 5' -phosphodiesterase and amikacin can obviously inhibit the generation of the pseudomonas aeruginosa biofilm.

Example 13: amikacin/5' -phosphodiesterase/oxidized dextran composite gel 3 performance evaluation of degraded biofilm

Putting a cover glass with the diameter of 2.0cm into a 24-hole plate as a carrier, adding pseudomonas aeruginosa bacterial liquid, culturing for 3 days, and constructing an in-vitro mature biomembrane model of pseudomonas aeruginosa. On the fourth day, the antimicrobial material was added. Totally divided into 4 groups, wherein the first group is added with amikacin/5 '-phosphodiesterase/oxidized dextran composite gel 3, the second group is added with amikacin/oxidized dextran gel, the third group is added with 5' -phosphodiesterase/oxidized dextran gel, and the fourth group is not treated at all. After 24h of culture, the number of bacteria surviving in the mature biofilms was counted by plate colony counting.

As shown in FIG. 11, the number of bacteria in the mature biofilm of Pseudomonas aeruginosa was 4.67 × 10 without any antibacterial treatment¹⁰CFU/mL, after treatment with amikacin/oxidized dextran gel or 5' -phosphodiesterase/oxidized dextran gel, the number of bacteria surviving in the biofilm was 5.67 × 10⁸CFU/mL and 2.33 × 10¹⁰CFU/mL. after amikacin/5' -phosphodiesterase/oxidized dextran complex gel treatment, the bacteria surviving in the biomembrane is only 1.63 × 10⁷CFU/mL. It can be seen that 5' -phosphodiesterase can destroy mature biofilm by degrading nucleic acid, and can efficiently kill bacteria in the biofilm after being combined with amikacin. Thus, it can be concluded that 5' -phosphodiesterase can significantly improve the sensitivity of amikacin to bacteria in mature biofilms.

Antibacterial sensitivity evaluation of tetra, aminoglycoside/anti-biofilm agent/oxidized polysaccharide polymer composite gel

Example 14: evaluation of antibacterial Activity of aminoglycoside/envelope enzyme/oxidized polysaccharide Complex gel by agar punch method

The method comprises the steps of inoculating pseudomonas aeruginosa and staphylococcus aureus in a 6-well plate, incubating for three days to construct a mature biofilm, removing an upper culture medium, and preparing the mature biofilm at the bottom into a bacterial suspension by using ultrasound. LB solid plates were prepared while adding the mature biofilm bacteria suspension to the medium to a final concentration of 10⁵CFU/mL, using 50 u L gun head to set on the agar plate hole 3, in the hole to add 20L amikacin/pectinase/oxidized dextran gel, amikacin/oxidized dextran gel and pectinase/oxidized dextran gel. And (5) incubating for 24h, and detecting the difference of the inhibition zones.

As shown in fig. 12, it can be seen that the inhibition zone of amikacin against biofilm bacteria is significantly increased in the presence of pectinase, and it can be seen that pectinase has no antibacterial effect, so it can be seen that the sensitivity of amikacin treatment on biofilm bacteria is significantly enhanced with the aid of pectinase.

Biological safety of penta-aminoglycoside/anti-biofilm agent/oxidized polysaccharide polymer composite gel

Example 15: cytotoxicity of aminoglycoside/envelope enzyme/oxidized polysaccharide polymer composite gel

NIH 3T3 cells were seeded in 96-well plates at 5000 cells/well, 37 ℃ and 5% CO₂And incubating for 24 hours under the complete culture condition, and carrying out an experiment when the cell density reaches more than 80%. The cytotoxicity test takes amikacin/pectinase/oxidized dextran composite gel 1 as an example, and 200 mu L of the composite gel 1 is placed in a culture medium without bovine serum albumin, soaked for 24h, and then the leachate is taken out and supplemented with bovine serum albumin to make the final concentration 10%. Preparing the leachate into different concentrations, respectively adding the leachate into micropores inoculated with NIH 3T3 cells, wherein each pore is 50 mu L, and observing the survival rate of the cells by adopting an MTT method after treating for 4 hours.

As shown in FIG. 13, the survival rate of NIH 3T3 cells in the leachate of 10mg/mL amikacin/pectinase/oxidized dextran complex gel can still reach 92.6%, and it can be seen that the amikacin/pectinase/oxidized dextran complex gel has no obvious toxicity to normal tissue cells.

Example 16 haemolysis of aminoglycoside/antibiofilm agent/oxidized polysaccharide Polymer composite gel

The hemolysis test takes amikacin/pectinase/oxidized dextran composite gel 1 as an example. 1mL of mouse blood was collected by an eyeball bleeding method to prepare a 2% blood cell suspension. Separately, 20. mu.L of complex gel 1, 20. mu.L of Triton X-100 (0.5%), and PBS (20. mu.L) having pH 7.4 were prepared, and 1mL of blood cell suspension was added to each sample, treated at 37 ℃ for 1 hour, and photographed for comparison. The mixture was centrifuged at 2000r/min for 5 minutes, the supernatant was examined for absorbance at 540nm and the absorbance of the Triton X-100 treated supernatant was set to 100% reference. The relationship between the obtained treated serum absorbance and the material is shown in FIG. 14.

As shown in fig. 14, the absorbance of the serum treated by the amikacin/pectinase/oxidized dextran complex gel 1 was 2.62%, while the absorbance of the serum treated by PBS was 3.14%, and it was also seen from the photograph that no significant hemolysis occurred, indicating a good blood safety performance.

Hexa, evaluation of in vivo antibacterial Activity of aminoglycoside/anti-biofilm agent/oxidized polysaccharide Polymer composite gel

Example 17 evaluation of skin infection of Kunming mouse by Epilobium aeruginosa

The Kunming mouse group is anesthetized, the back is shaved, no antibacterial material is injected in the body, and 70 mu L (10 mu L) is directly injected into the back⁸CFU/mL) of Episella aeruginosa. After three days, the infection of the site of the Kunming mouse injected with bacteria was observed, and the skin of the infected site was removed to count the bacterial colonies by the spot plate method.

As shown in FIG. 15, skin of the infected part of Kunming mouse which had not been treated with any antibacterial material was severely ulcerated, and the number of colonies per unit volume was 1.5 × 10¹⁰CFU/mL, the appearance of the skin indicates the presence of a severe infection.

Example 18: evaluation of in vivo antibacterial effect of amikacin/oxidized dextran hydrogel on pseudomonas aeruginosa

The antibacterial effect of the amikacin/oxidized dextran hydrogel (with the drug content) in vivo is researched. The specific method comprises the following steps: after 8 weeks of Kunming mice were anesthetized, the back was shaved, and the mice were injected subcutaneously with amikacin/oxidized dextran hydrogel (100 μ L) in the back, followed by 70 μ L (10 μ L)⁸CFU/mL) of Episella aeruginosa. After three days, the infection of the site of the Kunming mouse injected with bacteria was observed, and the skin of the infected site was removed to count the bacterial colonies by the spot plate method.

As shown in FIG. 16, the skin of the infected site of the mice injected with the amikacin/oxidized dextran hydrogel was partially ulcerated, and the number of colonies per unit volume of the skin was 8.9 × 10⁹CFU/mL, the appearance of the skin also indicates the presence of a degree of infection.

Example 19: evaluation of in vivo bacteriostatic effect of pectinase/oxidized dextran gel

The prepared pectinase/oxidized dextran gel is researched to have an in-vivo antibacterial effect.

The specific method comprises the following steps: after 8 weeks of Kunming mice were anesthetized, the back was shaved, and the mice were injected subcutaneously with pectinase/oxidized dextran gel, followed by 70 μ L (10)⁸CFU/mL) of Episella aeruginosa. After three days, the infection of the Kunming mouse at the bacterial injection site was observed, and the skin at the infected site was removed and the number of bacteria in the skin was counted by a plate colony counting method.

As shown in FIG. 17, the skin of the infected site of the mice injected with the pectinase/oxidized dextran gel was substantially intact, and the colony count per unit volume of the skin was 1.3 × 10¹⁰CFU/mL, the appearance of the skin also showed that no serious infection was present.

Example 20: evaluation of in vivo antibacterial effect of amikacin/pectinase/oxidized dextran composite gel

The prepared amikacin/pectinase/oxidized dextran composite gel 1 is researched for the in vivo antibacterial effect.

The specific method comprises the following steps: after 8 weeks of Kunming mice were anesthetized, the mice were shaved, and the mice were injected subcutaneously with amikacin/pectinase/oxidized dextran complex gel 1, followed by 70 μ L (10)⁸CFU/mL) of Episella aeruginosa. After three days, the infection of the Kunming mouse at the bacterial injection site was observed, and the skin at the infected site was removed and the number of bacteria in the skin was counted by a plate colony counting method.

As shown in FIG. 18, the skin of the infected site of the mice injected with amikacin/pectinase/oxidized dextran complex gel 1 was substantially intact, and the number of colonies per unit volume of the skin was 2.6 × 10⁸CFU/mL, the appearance of the skin also showed that no serious infection was present.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.

Claims

1. The preparation method of the antibiotic hydrogel for resisting the biofilm is characterized in that aminoglycoside antibiotics and biological enzymes for degrading the biofilm are used as cross-linking elements, and the aminoglycoside antibiotics and the biological enzymes are cross-linked with oxidized polysaccharide macromolecules into gel through acid-sensitive Schiff base bonds, so that the antibiotic hydrogel is prepared.

2. The method of claim 1, wherein the biofilm degrading biological enzymes comprise one or more of polysaccharide degrading biological enzymes, nucleic acid degrading biological enzymes and protein degrading biological enzymes.

3. The method of claim 2, wherein the biological enzyme degrading the polysaccharide is selected from one or more of pectinase, alginate, cellulase, and amylase; the protein degrading biological enzyme is selected from one or more of neutral protease, papain, trypsin and proteinase K; the biological enzyme for degrading nucleic acid is selected from one or more of deoxyribonuclease Dnase, ribonuclease RNase and phosphatase.

4. The method of claim 1, wherein the aminoglycoside antibiotic is selected from the group consisting of one or more of netilmicin, isepamicin, capreomycin, ribostamycin, sisomicin, apramycin, amikacin, kanamycin, gentamicin, paromomycin, tobramycin, and neomycin.

5. The method of claim 1, wherein the polysaccharide macromolecules are selected from one or more of dextran, chitosan, alginic acid, hyaluronic acid, cellulose, lignin, chondroitin, glycosaminoglycans, starch, pectin, mannan; oxidizing the polysaccharide macromolecules by an oxidant to obtain the oxidized polysaccharide macromolecules containing aldehyde groups.

6. The method of claim 1, wherein the aminoglycoside antibiotic, the biofilm degrading enzyme, and the oxidized polysaccharide macromolecule comprise a gel-forming system; wherein, the aminoglycoside antibiotics accounts for 0.1 to 20 percent of the mass of the colloid system; the concentration of the biological enzyme in the gelling system is 3-3000 u/mL; the oxidized polysaccharide polymer accounts for 1-10% of the gel-forming system by mass, and the hydroformylation ratio is 5-95%.

7. The method of claim 1, wherein the temperature of said forming is room temperature; the gelling time is between 1 second and 60 minutes.

8. An antibiotic hydrogel prepared by the method of any one of claims 1 to 7.

9. Use of the antibiotic hydrogel of claim 8 to inhibit biofilm formation and degrade mature biofilms.

10. Use of the antibiotic hydrogel of claim 8 in the manufacture of a medicament for inhibiting bacterial infection or bacterial contamination of an environment.