阅读说明：本技术 花菁类染料ir780的抗菌新用途 (Novel antibacterial application of cyanine dye IR780 ) 是由 陈刚 范雪莲 宗荣玲 于 2020-11-24 设计创作，主要内容包括：本发明属于生物医药领域,具体涉及花菁类染料IR780或其可接受的盐作为抗菌药物的新应用。具体表现为IR780对革兰氏阳性菌金黄色葡萄球菌及革兰氏阴性菌大肠杆菌、铜绿假单胞杆菌、鲍曼不动杆菌的敏感株及耐药株均具有明显的抑制作用,IR780能破坏菌体的细胞膜、增加其通透性导致DNA外渗,对菌体DNA及可溶性蛋白具有降解作用。本发明为目前花菁类染料IR780的应用提供了一种新的选择,具有新的临床应用前景。(The invention belongs to the field of biomedicine, and particularly relates to a novel application of a cyanine dye IR780 or an acceptable salt thereof as an antibacterial drug. The specific expression is that IR780 has obvious inhibition effect on sensitive strains and drug-resistant strains of gram-positive bacteria staphylococcus aureus, gram-negative bacteria escherichia coli, pseudomonas aeruginosa and acinetobacter baumannii, IR780 can destroy cell membranes of thalli and increase permeability of the thalli to cause DNA extravasation, and has degradation effect on thalli DNA and soluble protein. The invention provides a new choice for the application of the existing cyanine dye IR780 and has a new clinical application prospect.)

1. Use of a cyanine-type dye IR780, or an acceptable salt thereof, in the manufacture of an agent or medicament for the inhibition and/or prevention and/or treatment of bacterial infections.

2. Use according to claim 1, wherein the bacteria are gram-positive or gram-negative bacteria.

3. The use according to claim 2, wherein the bacteria are one or more of sensitive strains or resistant strains of staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, acinetobacter baumannii.

4. The use according to claim 2, wherein the bacteria is one or more of methicillin-resistant staphylococcus aureus ATCC 44300, methicillin-resistant staphylococcus aureus T144, staphylococcus aureus-resistant strain BW15, staphylococcus aureus-resistant strain BWMR26, staphylococcus aureus-sensitive strain ATCC 29213, escherichia coli-sensitive strain ATCC 25922, escherichia coli-sensitive strain cmcc (b)44102, pseudomonas aeruginosa-sensitive strain ATCC 27853, acinetobacter baumannii-sensitive strain ATCC 19606, acinetobacter baumannii-resistant strain AB14 and acinetobacter baumannii-resistant strain AB 16.

5. The use according to claim 1, wherein the agent or medicament is an injection, a tablet, a capsule, a granule, a liposome, a nanoparticle, a microsphere or an emulsion.

6. The use according to claim 1, characterized in that the effective dose of the cyanine dye IR780 is between 1 and 32 μ g/mL.

7. An antibacterial agent, characterized in that it comprises a cyanine-type dye IR780 or an acceptable salt thereof.

Technical Field

The invention belongs to the technical field of biological medicines, relates to a new application of a cyanine dye IR780, and particularly relates to an application of IR780 or acceptable salts thereof in inhibiting bacterial infection.

Background

Currently, the abuse of antibiotics causes the drug resistance of bacteria to become a worldwide problem, and therefore, the search for new high-efficiency antibacterial drugs is imperative.

IR780 is a lipophilic cationic micromolecular cyanine dye, belongs to an indole tricarbocyanine dye, and has a maximum absorption peak at an absorption wavelength of 780 nm. The molecular formula of IR780 is C₃₆H₄₄ClIN₂Molecular weight 667Da, its structural formula is as follows:

currently, IR780 is gaining wide attention by virtue of its superior properties-it can accumulate to the tumor site without the need to couple to tumor targeting ligands; secondly, the fluorescent probe is considered to be a good in vivo imaging fluorescent probe due to strong fluorescence; IR780 has stronger absorption characteristic in a near-infrared region with good tissue penetrability and higher photothermal conversion efficiency, and is often used as a photothermal therapeutic agent for tumors under the laser irradiation condition; and fourthly, the medicine is fast to clear in vivo, cannot be phagocytized by a reticuloendothelial system, and has higher safety. At present, IR780 is mainly used as a fluorescent probe for in vivo and in vitro imaging and laser-assisted optical treatment, but the direct antibacterial action of the probe is not reported at home and abroad.

Disclosure of Invention

The purpose of the invention is as follows: in order to solve the problems of the existing antibacterial drugs, the invention discovers that the widely applied cyanine dye IR780 has an effective antibacterial effect, and provides a new application of the IR780 in the field of biomedicine.

The technical scheme is as follows: in order to solve the technical problem, the invention provides application of a phthalocyanine dye IR780 or an acceptable salt thereof in preparing an agent or a medicament for inhibiting and/or preventing and/or treating bacterial infection.

Wherein the bacteria are gram-positive bacteria or gram-negative bacteria.

Wherein, the bacteria include but not limited to one or more of sensitive strains or drug-resistant strains of staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and acinetobacter baumannii.

Wherein, the bacteria include but not limited to one or more of methicillin-resistant staphylococcus aureus ATCC 44300, methicillin-resistant staphylococcus aureus T144, staphylococcus aureus resistant strain BW15, staphylococcus aureus resistant strain BWMR26, staphylococcus aureus sensitive strain ATCC 29213, escherichia coli sensitive strain ATCC 25922, escherichia coli sensitive strain CMCC (B)44102, pseudomonas aeruginosa sensitive strain ATCC 27853, acinetobacter baumannii sensitive strain ATCC 19606, acinetobacter baumannii resistant strain AB14 and acinetobacter baumannii resistant strain AB 16.

Wherein, the reagent or the medicament comprises but not limited to injection, tablets, capsules, granules, liposomes, nanoparticles, microspheres or emulsion.

Wherein, the effective dosage of the phthalocyanine dye IR780 is preferably 1-32 mug/mL.

The present disclosure also includes an antimicrobial agent comprising the cyanine-type dye IR780 or an acceptable salt thereof

The product of the invention comprises a product for inhibiting the growth of bacteria, a product for treating and/or preventing diseases caused by bacteria and a product for preventing and treating putrefaction caused by bacteria.

Has the advantages that: compared with the prior art, the invention has the advantages that: at present, IR780 is mainly used as a fluorescent probe for in vivo and in vitro imaging and laser-assisted optical treatment, but the direct antibacterial action of the probe is not reported at home and abroad. The invention discovers the direct antibacterial action of the cyanine dye IR780 for the first time, has obvious inhibition effect on sensitive strains and drug-resistant strains of gram-positive bacteria and gram-negative bacteria, belongs to the new application of the IR780, and is beneficial to developing a series of antibacterial products and relieving the problem of bacterial infection.

Drawings

FIG. 1, antibacterial activity of IR780 against Staphylococcus aureus resistant strain MRSA 44300;

a: time sterilization curve of IR780 against staphylococcus aureus MRSA 44300;

b: the effect of IR780 on changes in the DNA content of s.aureus MRSA 44300 extravasation;

c: degradation of the DNA of staphylococcus aureus MRSA 44300 by IR 780;

d: degradation of the protein of staphylococcus aureus MRSA 44300 by IR 780;

FIG. 2, antibacterial activity of IR780 against Staphylococcus aureus resistant strain MRSA T144;

a: time sterilization curve of IR780 against staphylococcus aureus resistant strain MRSA T144;

b: the influence of IR780 on the change of the DNA content of the staphylococcus aureus drug-resistant strain MRSA T144 extravasation;

c: IR780 degradation of Staphylococcus aureus resistant strain MRSA T144 DNA;

d: IR780 degradation of protein of Staphylococcus aureus resistant strain MRSA T144;

FIG. 3, antibacterial activity of IR780 against Staphylococcus aureus resistant strain BW 15;

a: time sterilization curve of IR780 against staphylococcus aureus resistant strain BW 15;

b: the effect of IR780 on the change in the exosmotic DNA content of staphylococcus aureus resistant strain BW 15;

c: degradation of IR780 on the DNA of a staphylococcus aureus drug-resistant strain BW 15;

d: the degradation effect of IR780 on the protein of a staphylococcus aureus drug-resistant strain BW 15;

FIG. 4, antibacterial activity of IR780 against Staphylococcus aureus resistant strain BWMR 26;

a: time sterilization curve of IR780 against Staphylococcus aureus resistant strain BWMR 26;

b: the effect of IR780 on the change of the DNA content of the Staphylococcus aureus resistant strain BWMR26 extravasation;

c: degradation of IR780 on the DNA of a staphylococcus aureus drug-resistant strain BWMR 26;

d: the degradation effect of IR780 on the protein of a staphylococcus aureus drug-resistant strain BWMR 26;

FIG. 5, antibacterial activity of IR780 against S.aureus-susceptible strain S.29213;

a: time sterilization curve of IR780 against S.29213, a sensitive strain of Staphylococcus aureus;

b: the effect of IR780 on the change in the content of extravasated DNA of S.29213, a susceptible strain of Staphylococcus aureus;

c: IR780 degradation of DNA of staphylococcus aureus sensitive strain S.29213;

d: IR780 degradation of S.29213 protein of staphylococcus aureus sensitive strain;

FIG. 6, antibacterial activity of IR780 against E.coli susceptible strain ATCC 25922;

a: time sterilization curve of IR780 against E.coli sensitive strain ATCC 25922;

b: effect of IR780 on changes in the exosmotic DNA content of escherichia coli susceptible strain ATCC 25922;

c: degradation of IR780 DNA of Escherichia coli sensitive strain ATCC 25922;

d: degradation of proteins of an Escherichia coli sensitive strain ATCC 25922 by IR 780;

FIG. 7, antibacterial activity of IR780 against E.coli-susceptible strain CMCC (B) 44102;

a: time sterilization curve of IR780 against E.coli sensitive strain CMCC (B) 44102;

b: effect of IR780 on changes in the content of extravasated DNA of escherichia coli susceptible strain cmcc (b) 44102;

c: degradation of IR780 DNA of sensitive strain CMCC (B)44102 of Escherichia coli;

d: degradation of IR780 protein of sensitive strain CMCC (B)44102 of Escherichia coli;

FIG. 8, antibacterial activity of IR780 against P.aeruginosa susceptible strain ATCC 27853;

a: time sterilization curve of IR780 against pseudomonas aeruginosa susceptible strain ATCC 27853;

b: the effect of IR780 on the variation in the exosmotic DNA content of the susceptible strain of Pseudomonas aeruginosa ATCC 27853;

c: degradation of IR780 DNA of a pseudomonas aeruginosa sensitive strain ATCC 27853;

d: degradation of proteins of a pseudomonas aeruginosa sensitive strain ATCC 27853 by IR 780;

FIG. 9, antibacterial activity of IR780 against Acinetobacter baumannii sensitive strain ATCC 19606;

a: time sterilization curve of IR780 against acinetobacter baumannii sensitive strain ATCC 19606;

b: the effect of IR780 on changes in the amount of extravasated DNA of the Acinetobacter baumannii susceptible strain ATCC 19606;

c: IR780 degradation of DNA of Acinetobacter baumannii sensitive strain ATCC 19606;

d: degradation of proteins of an acinetobacter baumannii sensitive strain ATCC 19606 by IR 780;

FIG. 10, antibacterial activity of IR780 against Acinetobacter baumannii drug-resistant strain AB 14;

a: time sterilization curve of IR780 against Acinetobacter baumannii drug-resistant strain AB 14;

b: the influence of IR780 on the change of the DNA content of the acinetobacter baumannii drug-resistant strain AB14 extravasation;

c: IR780 degradation of Acinetobacter baumannii drug-resistant strain AB14 DNA;

d: IR780 degradation of Acinetobacter baumannii drug-resistant strain AB16 protein;

FIG. 11, antibacterial activity of IR780 against Acinetobacter baumannii drug-resistant strain AB 16;

a: time sterilization curve of IR780 against Acinetobacter baumannii drug-resistant strain AB 16;

b: the influence of IR780 on the change of the DNA content of the acinetobacter baumannii drug-resistant strain AB16 extravasation;

c: IR780 degradation of Acinetobacter baumannii drug-resistant strain AB16 DNA;

d: IR780 degradation of Acinetobacter baumannii drug-resistant strain AB16 protein.

Detailed Description

The present invention is further illustrated by the following specific examples, it should be noted that, for those skilled in the art, variations and modifications can be made without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Materials and equipment:

(1) IR780 iodide was obtained from Sigma, USA;

(2) the bacterial culture medium is purchased from Haibo biotechnology limited company of Qingdao high-tech industrial garden;

(3) the bacterial genome DNA extraction kit is purchased from Tiangen Biotechnology limited;

(4) the electrophoresis related material is purchased from Jiangsu Kai-based biotechnology, Inc.;

(5) all strains were stored in the laboratory.

Example 1: MIC value determination of IR780 against bacteria

MIC determination of standard strains and clinical isolates was performed on IR780 with reference to the minimal broth dilution method recommended by the american Clinical Laboratory Standards Institute (CLSI), and MIC results were judged to be performed mainly with reference to CLSI M100-S25(2015) standard. Diluting the medicine at multiple ratios of 512, 256, 128, 64, 32, 16, 8, 4, 2 and 1 μ g/mL by using MH culture medium, respectively adding into the front 10 wells of a U-shaped plate with 96 wells, and diluting the detected strain to 10 by using MH culture medium⁵-10⁶CFU/mL, 100. mu.L of each well was added to each well, 100. mu.L of culture medium was added to the 11 th well as a negative control, and 100. mu.L of bacterial suspension was added to the 12 th well as a positive control. And (5) culturing in an incubator at 37 ℃ for 12-16h, reading the result, and taking the minimum concentration for inhibiting the growth of bacteria as the MIC value. To ensure the accuracy of the experimental results, the experiment was repeated three times.

As shown in Table 1, the results show that IR780 has broad-spectrum antibacterial activity on gram-positive bacteria and gram-negative bacteria including sensitive bacteria and drug-resistant bacteria of staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and acinetobacter baumannii, particularly has better inhibitory effect on staphylococcus aureus and even MRSA strains, and the minimum inhibitory concentration range is 1-4 mug/mL.

TABLE 1

Example 2: time-kill-curve determination of IR780 against bacteria

The strains in Table 1 were grown to logarithmic phase of bacterial suspension (OD)₆₀₀Diluting the strain by 1:1000 times (0.8-1), and adding 1mL of diluted bacterial liquid into a bacteria shaking tube. According to the experimental result of MIC, IR780 (0-32 mu g/mL) with different concentrations is incubated with a bacterial solution, the temperature is 37 ℃ and 250rmp is carried out, and no liquid medicine is added into a control group. Take 30. mu.L of coated plate at 0, 3, 6, 9, 12, 24h, respectively. Placing the plate at 37 deg.C, culturing for 12-16h, and counting single colonyDrawing a growth curve chart and observing the growth trend of the bacteria.

As shown in A of FIGS. 1 to 11, the bacteria in each control group rapidly grew and propagated within 0 to 12 hours, and the bacteria grew in a stationary phase within 12 to 24 hours. After adding different concentrations of IR780, the growth of each bacterium was significantly inhibited and there was a significant activity-dose relationship, and in combination with the MIC results, it was determined that IR780 was a strong antibacterial agent.

Example 3: effect of IR780 on bacterial DNA extravasation

The strains in Table 1 were grown to logarithmic phase of bacterial suspension (OD)₆₀₀0.8-1) diluted with MH broth at a ratio of 1:1000, and 1mL of the diluted bacterial solution was added to a shake tube. According to the MIC experiment result, 1/4MIC, 1/2MIC and MIC drug concentration are selected to be incubated with the bacterial liquid, the temperature is 37 ℃ and the rpm is 250, and no liquid medicine is added into a control group. After incubation for 12h, centrifugation was carried out at 12000 rmp for 2min, and the supernatant was collected and the DNA content of the supernatant was determined by a microspectrophotometer.

The results are shown in B diagrams of FIGS. 1-11, in order to detect the influence of IR780 on the permeability of bacterial cell membranes, the extravasation amount of macromolecular substances (DNA) in cells after being added with drugs is further determined, and after incubation for 12h, the DNA content of 1/4MIC, 1/2MIC and drug treatment groups of MIC are obviously increased compared with those of a control group, and the drug treatment groups have dose dependence. It was demonstrated that IR780 did alter the cell membrane permeability of the various bacteria tested.

Example 4: degradation of bacterial DNA by IR780

The strains in Table 1 were grown to logarithmic phase of bacterial suspension (OD)₆₀₀0.8-1) diluted with MH broth at a ratio of 1:1000, and 1mL of the diluted bacterial solution was added to a shake tube. According to the MIC experiment result, 1/4MIC, 1/2MIC and MIC drug concentration are selected to be incubated with the bacterial liquid, the temperature is 37 ℃ and the rpm is 250, and no liquid medicine is added into a control group. After incubation for 12h, bacterial genomic DNA was extracted according to the kit instructions, and the degradation of bacterial DNA with increasing drug concentration was detected by agarose gel electrophoresis.

As shown in the C diagrams of FIGS. 1-11, in order to investigate whether the IR780 antibiotic degrades released biomass, we further investigated the effect of IR780 on released nucleic acids, and showed that IR780 can degrade DNA released by various bacteria to varying degrees with increasing dose. Thus, IR780 is not only effective in killing bacteria, but also successfully degrades biomass released by the bacteria.

Example 5: effect of IR780 on bacterial soluble protein content

The strains in Table 1 were grown to logarithmic phase of bacterial suspension (OD)₆₀₀0.8-1) diluted with MH broth at a ratio of 1:1000, and 1mL of the diluted bacterial solution was added to a shake tube. According to the MIC experiment result, 1/4MIC, 1/2MIC and MIC drug concentration are selected to be incubated with the bacterial liquid, the temperature is 37 ℃ and the rpm is 250, and no liquid medicine is added into a control group. After incubation for 12h, centrifugation is carried out at 12000 rmp for 2min, the supernatant is discarded, the bacterial pellet is washed by PBS, 200 mu L of PBS is added after washing twice, and the bacterial pellet is fully shaken and resuspended. And (3) placing the turbid liquid on ice to prevent local high heat, cracking bacteria by using a bacteria ultrasonic crusher, performing ultrasonic treatment for 3s at a power of 200W for 6s at an interval, and acting for 3min to ensure that the turbid liquid becomes clear and transparent and is not adhered. Changes in bacterial proteins were detected by SDS-PAGE.

As shown in the D diagrams of FIGS. 1-11, clear and bright protein bands can be seen in the control groups of various bacteria, after the treatment with IR780, the total amount of soluble protein in the thalli of the drug-adding group is obviously reduced along with the increase of the dosage, and part of the soluble protein has no protein band at the MIC dosage, which indicates that IR780 has obvious influence on the content of the soluble protein in various bacteria tested, and probably causes the outflow of the soluble protein in the thalli or has degradation effect on the protein by increasing the cell membrane permeability.