A lipopolysaccharide-responsive nano antimicrobial peptide and its preparation method and application

Abstract

The present invention discloses a lipopolysaccharide-responsive nano antimicrobial peptide and its preparation method and application, which belongs to the field of biotechnology. The amino acid sequence of the antimicrobial peptide is as shown in SEQ ID No. 1. Also disclosed is the application of the antimicrobial peptide of the present invention in the preparation of a drug for treating diseases caused by Gram-negative bacteria infections. The antimicrobial peptide of the present invention can target and bind to lipopolysaccharide to form nanofibers for capture, and then present them in agglomerates to phagocytic cells for phagocytosis, fully avoiding the killing of beneficial bacteria in the host. In summary, the antimicrobial peptide of the present invention can target Gram-negative bacteria to break the membrane and kill bacteria, fully avoiding the accidental killing of beneficial bacteria in the host, and effectively reducing the generation of drug resistance. Therefore, it is a nano-capture peptide with immune regulation function that has great development potential and high application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a lipopolysaccharide-responsive nano antimicrobial peptide and a preparation method and application thereof. Background Art

[0002] Antimicrobial peptides (AMPs), also known as host defense peptides, are crucial components of the innate immune system. Their membrane-permeating and bactericidal mechanisms, which are less likely to induce bacterial resistance, make them considered promising alternatives to antibiotics. Despite their unparalleled advantages, AMPs still face challenges in clinical application and animal husbandry. On the one hand, traditional AMPs, due to their broad-spectrum antimicrobial activity, often inadvertently kill beneficial bacteria in the host and also have a damaging effect on normal intestinal microflora, disrupting the balance of the intestinal microecological environment and inducing severe secondary infections. On the other hand, after antibiotic-induced bacterial death, Gram-negative bacteria release membrane-bound lipopolysaccharide (LPS) into the bloodstream. Endotoxemia triggers an uncontrolled systemic inflammatory response, leading to collateral damage to host tissues and disruption of physiological processes, potentially resulting in septic shock and high mortality. Therefore, mobilizing the body's immune system to collaboratively resist and capture bacterial infection is an effective strategy that can further reduce the inflammatory response induced by LPS. Therefore, the new design of LPS-responsive self-assembling capture peptides is of great significance for solving the problem of drug resistance and finding alternatives to antibiotics. Summary of the Invention

[0003] Based on the above problems, the purpose of the present invention is to provide a lipopolysaccharide-responsive nano antimicrobial peptide, which can assemble into a nano-network structure in the presence of lipopolysaccharide, has a good killing effect on Gram-negative bacteria, and has good biocompatibility. At the same time, it can promote the phagocytosis of cell clusters by macrophages, and reduce the possibility of bacteria developing drug resistance.

[0004] The technical solution adopted by the present invention is as follows: a lipopolysaccharide-responsive nano antimicrobial peptide FI2, whose amino acid sequence is shown in SEQ ID No.1.

[0005] Furthermore, the molecular formula of the lipopolysaccharide-responsive nano antimicrobial peptide FI2 is shown in formula (I):

[0006]

[0007] Furthermore, the self-assembly method of the lipopolysaccharide-responsive nano antimicrobial peptide FI2 described above has the following self-assembly conditions: placing the antimicrobial peptide FI2 with a concentration of 8-256 μM in deionized water containing lipopolysaccharide with a concentration of 20 μg / ml and incubating at 37°C for 24 hours.

[0008] Another object of the present invention is to provide a use of the lipopolysaccharide-responsive nano antimicrobial peptide FI2 as described above in the preparation of a drug for treating diseases caused by Gram-negative bacteria infections.

[0009] Furthermore, the Gram-negative bacteria are Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli or Salmonella typhimurium.

[0010] Another object of the present invention is to provide a drug suitable for treating and / or preventing Gram-negative bacterial infection as described above, wherein the drug contains the lipopolysaccharide-responsive nano antimicrobial peptide FI2 as described above.

[0011] The beneficial effects and advantages of the present invention are as follows: The antimicrobial peptide FI2 of the present invention has a significant inhibitory effect on Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium, but has no significant effect on Gram-positive bacteria such as Staphylococcus aureus. However, under physiological conditions, it has a significant capture effect on Escherichia coli and promotes the phagocytic ability of macrophages to bacterial clusters. This can not only capture bacteria and trigger the innate immune system to achieve antibacterial purposes, but also target Gram-negative bacteria to break the membrane and kill bacteria, fully avoiding the accidental killing of beneficial bacteria in the host and effectively reducing the development of drug resistance. In addition, the antimicrobial peptide FI2 of the present invention does not show hemolysis at the maximum tested concentration in the presence or absence of lipopolysaccharide. In summary, the antimicrobial peptide FI2 of the present invention is a lipopolysaccharide-responsive self-assembling antimicrobial peptide with high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the mass spectrum of antimicrobial peptide FI2;

[0013] Figure 2 is the chromatogram of antimicrobial peptide FI2;

[0014] Figure 3 The critical aggregation concentration of antimicrobial peptide FI2 and the linear fitting value of the critical aggregation concentration;

[0015] Figure 4 Negative staining images of antimicrobial peptide FI2, including (a) control group LPS, (b) control group FI2, (c) control group FI2 and LPS co-incubated for 12 h;

[0016] Figure 5 Negative staining images of antimicrobial peptide FI2 co-incubated with E. coli; (a) control group, (b, c, d) control group FI2 co-incubated with LPS for 2 h;

[0017] Figure 6 This is a graph showing the determination of the hemolytic activity of the antimicrobial peptide FI2;

[0018] Figure 7The figure shows the determination of cytotoxicity of antimicrobial peptide FI2;

[0019] Figure 8 Antimicrobial peptide FI2 promotes macrophage phagocytosis of E. coli 25922;

[0020] Figure 9 To promote the phagocytosis of Escherichia coli by macrophages, (a) DAPI staining of the control group, (b) EGFP staining of the control group, (c) merge of the control group, (d) DAPI staining of the FI2-treated group; (e) EGFP staining of the FI2-treated group, (f) merge of the FI2-treated group. DETAILED DESCRIPTION

[0021] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0022] Example 1

[0023] Design of the antimicrobial peptide FI2: In coiled-coil peptides, the seven-residue pattern typically described by the heptad abcdefg is repeated multiple times along the central axis of the helix. Residues at positions a and d, or at any positions i and i+3, are arranged along the same ridge of the helix. Here, the FI zipper is placed at positions a and d of the α-helical heptad, serving as the hydrophobic group for the overall tether. Furthermore, lipopolysaccharide (LPS) on the outer membrane of Gram-negative bacteria and teichoic and uronic acid teichoic acids on the cell wall of Gram-positive bacteria are both anionic species, attracting antimicrobial peptides through electrostatic interactions. Net positive charge is a key factor influencing bacterial capture by antimicrobial peptides. To prevent repulsion between positive charges and hinder nanofiber formation and further enhance their potential, positively charged arginine residues are added to positions b and e of the heptad. Alanine separates the two segments, creating an alternating hydrophobic and hydrophilic structure, forming the FRAIRAX sequence template. This allows for further nanostructure formation through cationic and anionic complementarity between peptides, where X represents arginine and aspartic acid. The peptide sequence template, FRAIRAX, is repeated twice, forming a discrete ridge of repeating residues along the helical axis and exhibiting a one-to-one central symmetry in the overall structure. Ultimately, the peptide sequence (FRAIRAD)2(RARFARI)2 was designed; its amino acid sequence is shown in Table 1.

[0024] Table 1 Amino acid sequence of lipopolysaccharide-responsive nanoantimicrobial peptide FI2

[0025]

[0026] Its molecular structure is shown in formula (I).

[0027]

[0028] Example 2

[0029] Synthesis of lipopolysaccharide-responsive nanoantimicrobial peptide FI2 by solid-phase chemical synthesis

[0030] 1. The preparation of antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus, and is completed by a peptide synthesizer. Weigh 3g of RINK resin (substitution degree 0.3mmol / g) into a 150ml reactor and soak it with 50ml of dichloromethane (DCM). After 2 hours, wash the resin with nitrogen-dimethylformamide (DMF) 3 times the volume of the resin, and then drain it. Repeat this four times, and drain the resin for use. Add a certain amount of 20% piperidine (piperidine / DMF) to the reactor and shake it on a decolorization shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with 3 times the volume of the resin in DMF, and then drain it;

[0031] 2. Take a small amount of resin and test it with the ninhydrin (Nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100℃ for 1 minute). If the resin is colored, it means that the deprotection is successful. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, add 20ml of DMF to dissolve it, then add 3ml of N, N-diisopropylcarbodiimide (DIC) and shake for 1min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30℃ for reaction;

[0032] 3. After 2 hours, cap the resin with a certain amount of acetic anhydride (acetic anhydride: DIEA: DCM = 1:1:2) for half an hour, then wash four times with 3 times the volume of DMF and drain. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake on a decolorization shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash four times with DMF and then drain.

[0033] 4. Take a small amount of resin and test it using the ninhydrin method (two drops each of Test A and Test B, react at 100°C for 1 minute). If the resin shows color, deprotection is successful. Weigh an appropriate amount of the second amino acid and HOBT into a 50ml centrifuge tube, dissolve them in 25ml of DMF, then add 2.5ml of DIC and shake well for 1 minute. Once the solution is clear, add it to the reactor and place the reactor on a shaker at 30°C for reaction. After 1 hour, take a small amount of resin and test it using the ninhydrin method (two drops each of Test A and Test B, react at 100°C for 1 minute). If the resin is colorless, the reaction is complete; if the resin shows color, condensation is incomplete and the reaction should be continued. After the reaction is complete, wash the resin four times with DMF, drain it, and add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor. Shake on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group from the resin. After deprotection, wash with DMF four times and then drain to check whether the protection is removed;

[0034] 5. After the last amino acid is attached, remove the protection, wash four times with DMF, and then drain the resin with methanol. Then, use 95% cutting solution (trifluoroacetic acid: 1,2-ethanedithiol: 3, isopropylsilane: water = 95:2:2:1) to cut the peptide from the resin (add 10 ml of cutting solution per gram of resin) and centrifuge four times with icy ether (cutting solution: ether = 1:9). Finally, use HPLC to separate and purify, and then freeze-dry to obtain a peptide of a certain purity;

[0035] 6. Identification of polypeptides: The polypeptides obtained above were analyzed by electrospray mass spectrometry, such as Figure 1 The molecular weight shown in the mass spectrum is basically consistent with the theoretical molecular weight in Table 1, and the purity of the polypeptide is greater than 95%.

[0036] Example 3

[0037] Nanoscale characterization of antimicrobial peptide FI2:

[0038] Critical aggregation concentration determination: In order to detect the ability of the antimicrobial peptide FI2 to form nanostructures at a lipopolysaccharide concentration of 20 μg / ml, the critical aggregation concentration (CAC) was determined using a 1-aniline-8-naphthalenesulfonic acid (ANS) fluorescent probe. 1 μL of ANS (final concentration of 1 mM, dissolved in 100% DMF) was added to different concentrations of peptides (dissolved in deionized water with an LPS concentration of 20 μg / ml) and incubated at 37°C for 24 hours. The mixed sample was transferred to a 96-well plate and the fluorescence spectrum was scanned using a fluorescence microplate reader with an excitation wavelength of 369 nm and an emission wavelength of 440 nm-550 nm. The CAC value of the peptide was then calculated using Origin software. The test results are shown in Figure 3 .

[0039] from Figure 3 (a) shows that under the condition of LPS concentration of 20 μg / ml, as the concentration increases, the fluorescence intensity of antimicrobial peptide FI2 in the range of 440nm-550nm increases sharply, indicating the presence of nano-macromolecules in the solution. The formation of nanostructures was preliminarily determined. Subsequently, Origin software was used for fitting analysis, as shown in Figure 2. Figure 3 As shown in (b), the CAC value of antimicrobial peptide FI2 was 28.4 μM.

[0040] Example 4

[0041] Negative staining: After incubating the antimicrobial peptide FI2 with 20 μg / ml lipopolysaccharide for 12 hours, 20 μL of the bacterial solution was placed on a copper mesh plate for 2 minutes and stained with 0.1% phosphotungstic acid for 10 seconds. Finally, the peptide morphology was examined under a Hitachi H-7650 transmission electron microscope (Hitachi H-7650, Japan) after air drying. The test results are shown in Figure 4 .from Figure 4 (b, c) It can be seen that the peptide sample alone is in a dispersed state, and in the presence of lipopolysaccharide, a dense nanofiber structure is formed. Negative staining bacterial TEM: Escherichia coli cells in the logarithmic growth phase were washed by centrifugation with PBS (10mM) buffer and resuspended to OD 600 nm =0.4. E. coli cells were combined with peptide nanofibers (32 μM) and incubated at 37°C for 2 h. 20 μL of the bacterial solution was placed on a copper mesh plate for 2 min, and the E. coli cells were stained with 0.1% phosphotungstic acid for 3 s. Finally, the air-dried bacterial morphology was examined under a Hitachi H-7650 transmission electron microscope (Hitachi H-7650, Japan). The test results are shown in Figure 5 .and Figure 5 (a) Compared with Figure 5 (b) It can be observed that the antimicrobial peptide FI2 is attached to the surface of E. coli, which further verifies that the antimicrobial peptide FI2 targets the cell membrane surface of Gram-negative bacteria. Figure 5 (c, d) The antimicrobial peptide FI2 was incubated with E. coli to assemble into nanofibers, which further captured the E. coli.

[0042] Example 5

[0043] Determination of in vitro hemolytic activity, cytotoxicity and bactericidal activity of antimicrobial peptide FI2:

[0044] 1. Hemolytic activity assay: Collect 1 mL of fresh healthy human blood and centrifuge it (4°C, 1000×g, 5 min). Wash it three times with PBS (10 mM) buffer, discard the supernatant, collect the precipitated blood cells and resuspend them to 10 mL with PBS (10 mM) buffer solution. Subsequently, an equal volume of diluted red blood cell suspension was placed in a 96-well plate and mixed with polypeptide solutions of different concentrations. After incubation at 37°C for 1 hour, centrifuge it (1000×g, 10 min), and aspirate the supernatant and transfer it to a new 96-well plate. The wells treated with 0.1% Triton X-100 served as positive controls, and the wells without polypeptide treatment served as negative controls. The absorbance value was determined using an enzyme-linked microplate reader with a wavelength of 570 nm. The polypeptide concentration that caused 50% hemolytic activity was defined as cytotoxic. See attached for test results. Figure 6 .

[0045] pass Figure 6 It can be seen that the antimicrobial peptide FI2 (128 μM) still did not cause hemolytic toxicity at high concentrations, which indicates that the antimicrobial peptide FI2 has good biocompatibility.

[0046] 2. Cytotoxicity assay: Cells frozen in liquid nitrogen were revived and inoculated into a culture medium containing 10% fetal bovine serum and 1% double-antibody, and subcultured at 37°C and 5% CO2. The cultured cells were digested with 0.25% trypsin and the cell density was adjusted to 2-4×10 5 cells / mL. 50 μL of cell suspension was mixed with 50 μL of peptides of different concentrations in a 96-well plate and incubated at 37°C and 5% CO2 for 24 h. 50 μL of MTT (5 mg / mL) was then added to each well and incubated for another 4 h. After incubation, the supernatant was discarded and the crystals at the bottom of the well were dissolved with 100 μL of DMSO. The absorbance of each well was measured at 570 nm using a microplate reader. The culture medium wells served as blank controls. The test results are shown in Figure 7 .

[0047] from Figure 7 It can be seen that even after treatment with high concentrations of antimicrobial peptide FI2, the cell survival rates of HEK 293T and Caco-2 were greater than 50%, and the cell survival rate of RAW264.7 was greater than 50% at a concentration of 32 μM, indicating that antimicrobial peptide FI2 has good biocompatibility and has the potential to become an antibiotic alternative.

[0048] 3. Bactericidal activity determination: The minimum inhibitory concentration of antimicrobial peptides was determined by microdilution method. Different concentrations of peptides were added to BSA solution in a 96-well plate, and then an equal volume of BSA with a final concentration of 1×10 5 CFUmL -1The final peptide concentration in the 96-well plate ranged from 0.5 to 128 μM. After 24 hours of incubation, the absorbance was measured using a microplate reader at a wavelength of 600 nm. The results are shown in Table 2.

[0049] Table 2 Antibacterial activity against 8 Gram-negative bacterial strains (μM)

[0050]

[0051] GM a : Geometric mean MIC value of Gram-negative bacteria

[0052] It can be seen from Table 2 that under physiological conditions, the antimicrobial peptide FI2 has bactericidal ability and exhibits strong antibacterial activity against Gram-negative bacteria.

[0053] Example 6

[0054] Determination of the immunomodulatory ability of antimicrobial peptide FI2:

[0055] 1. Gentamicin protection test: To evaluate whether the antimicrobial peptide FI2 promotes the phagocytosis of bacterial clusters by macrophages under physiological conditions, the bacterial content in macrophages was detected by gentamicin protection test. E. coli 25922 culture in the logarithmic growth phase was collected by centrifugation at 3000 rpm for 5 minutes, washed three times with PBS buffer, and resuspended in PBS buffer. The bacterial concentration was adjusted to OD 600nm =0.4. 16μM and 32μM antimicrobial peptides FI2 and FF2 were added to the bacterial suspension and co-cultured at 37°C for 2 hours. The bacterial particles pretreated with peptides were added to the cell culture medium of RAW 264.7 and cultured at 37°C for 2 hours. The cell culture medium was aspirated and washed 3 times. A solution containing 50μg / mL of gentamicin was added to eliminate extracellular bacteria. 0.1% Triton X-100 was used for lysis for 10 minutes. 50μL of lysate was aspirated and 450μL of high-temperature sterilized PBS was added for gradient dilution. 100μL of the dilution was evenly inoculated on the MHA plate culture medium. Cultured overnight in a 37°C incubator, the colony count (CFU) of each sample was calculated, the experiment was repeated three times, and the test results are shown in the table. Figure 8 .

[0056] from Figure 8 As can be seen, compared with the control group, pretreatment with 16μM and 32μM antimicrobial peptide FI2 significantly increased the internalization of E. coli particles in RAW 264.7 cells in a dose-dependent manner. This suggests that antimicrobial peptide FI2 can capture bacteria and further enhance the phagocytic ability of phagocytes.

[0057] 2. Macrophage phagocytosis of bacteria test: In order to visually observe the effect of antimicrobial peptide FI2 on macrophage phagocytosis of bacterial clusters, the phagocytosis of Escherichia coli by RAW 264.7 was observed using a fluorescent inverted microscope. Prepare E. coli particles containing green fluorescent protein, collect E. coli cells by centrifugation at 3000rpm for 5 minutes, and resuspend in PBS buffer. Add a peptide solution with a final concentration of 16μM and co-culture with the bacterial suspension at 37°C for 2 hours. Add the bacterial particles pretreated with the peptide to the cell culture medium of RAW264.7, culture at 37°C for 2 hours, aspirate the cell culture medium and wash 3 times, add Triton X-100 to fix for 10 minutes, aspirate Triton X-100 and wash 3 times, and add an anti-fade agent containing DAPI. Observe with a fluorescent inverted microscope. See the test results. Figure 9 .

[0058] The results are as follows Figure 9 As shown in the figure, compared with the control, E. coli particles treated with the antimicrobial peptide FI2 aggregated, and the phagocytosis of aggregated E. coli particles by RAW 264.7 macrophages was enhanced. This indicates that the nanocapture peptide can capture bacteria and further promote the uptake of phagocytes.

[0059] Therefore, the antimicrobial peptide FI2 of the present invention can target and bind to lipopolysaccharide to form nanofibers for capture, and then present them in agglomerates to phagocytic cells for phagocytosis, thereby fully avoiding the killing of beneficial bacteria in the host.

Claims

1. A lipopolysaccharide-responsive nano antimicrobial peptide FI2, characterized in that: Its amino acid sequence is shown in SEQ ID No. 1, and its molecular formula is shown in formula (I):

2. The self-assembly method of a lipopolysaccharide-responsive nano antimicrobial peptide FI2 according to claim 1, characterized in that: The self-assembly conditions were as follows: antimicrobial peptide FI2 at a concentration of 28.4-256 μM was placed in deionized water containing lipopolysaccharide at a concentration of 20 μg / ml and incubated at 37° C. for 24 hours.

3. Use of the lipopolysaccharide-responsive nano antimicrobial peptide FI2 according to claim 1 in the preparation of a medicament for treating diseases caused by Gram-negative bacteria, wherein the Gram-negative bacteria are Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli or Salmonella typhimurium.

4. A drug suitable for treating and / or preventing Gram-negative bacterial infection, characterized in that: The medicine contains the lipopolysaccharide-responsive nano antimicrobial peptide FI2 as claimed in claim 1.

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