Pseudomonas aeruginosa dimer-resistant nano antibacterial peptide with pH (Potential of Hydrogen) triggered and regulated as well as preparation method and application of nano antibacterial peptide

Abstract

The invention discloses preparation and application of a pH-triggered and regulated anti-pseudomonas aeruginosa dimer nano antibacterial peptide C2HF, and belongs to the technical field of biology. Comprising a branched chain A and a branched chain B, the amino acid sequence of the branched chain A is HHCFFF, the amino acid sequence of the branched chain B is FFFCHHH, and the branched chain A and the branched chain B are connected through a disulfide bond formed between thiol groups in two cysteine residues. The antibacterial peptide C2HF is self-assembled into a nanofiber structure in a PB (pH = 6.0) solution, the C2HF has strong anti-pseudomonas aeruginosa activity under the acidic condition, the hemolytic toxicity of the antibacterial peptide C2HF is almost zero, and the antibacterial peptide C2HF has extremely high application potential under the acidic condition for treating pseudomonas aeruginosa infection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a pH-triggered and regulated anti-Pseudomonas aeruginosa dimer nanopeptide, its preparation method, and its application. Background Technology

[0002] Antimicrobial peptides (AMPs) are a class of highly promising novel antimicrobial molecules, offering a new solution for combating multidrug-resistant bacterial infections. Unlike traditional antibiotics that act on limited, fixed targets, antimicrobial peptides primarily kill bacteria by physically disrupting their membrane integrity. This antimicrobial mechanism reduces the probability of bacteria developing resistance to antimicrobial peptides. The ordered nanostructures of self-assembled antimicrobial peptides contribute to improved activity and stability, thereby enhancing their activity against drug-resistant bacteria. Furthermore, self-assembled nanomedicine delivery systems can mitigate the pharmacokinetic / pharmacodynamic defects of antimicrobial peptides, improve their shelf life, stability, and bioavailability, and prolong their half-life. Therefore, self-assembled antimicrobial peptides hold great potential in treating bacterial infections, especially those caused by drug-resistant bacteria.

[0003] The integration of pH-triggered regulation technology provides a key solution for the targeted optimization of antimicrobial peptides. Bacterial infection sites often exhibit specific pH microenvironments that differ significantly from the pH of the normal physiological environment. pH-triggered antimicrobial peptides can specifically sense changes in environmental pH, activating antimicrobial activity in the acidic or alkaline microenvironment where the target bacteria reside. They selectively kill pathogens while having minimal impact on beneficial bacteria and human cells in the normal physiological environment. This helps maintain the homeostasis of the in vivo microbial community while effectively fighting infection, reducing treatment-related side effects. Therefore, there is a need for antimicrobial peptides with self-assembly technology and pH-triggered regulation mechanisms. Summary of the Invention

[0004] Based on the above shortcomings, the purpose of this invention is to provide a pH-triggered and regulated dimeric antimicrobial peptide C2HF against Pseudomonas aeruginosa, which can spontaneously assemble into a nanofiber structure under acidic conditions using cation-π interactions. It has a good killing effect on Pseudomonas aeruginosa in an acidic environment of pH 6.0 and good biocompatibility, while its antimicrobial activity is significantly reduced at pH 7.4.

[0005] The technical solution adopted in this invention is as follows: a pH-triggered and regulated anti-Pseudomonas aeruginosa dimeric nanopeptide C2HF, comprising branch A and branch B, wherein the amino acid sequence of branch A is HHHCFFF, the amino acid sequence of branch B is FFFCHHH, and the Cys of branch A and branch B are connected by disulfide bonds.

[0006] Furthermore, the molecular formula of the antimicrobial peptide C2HF, as described above, is shown in formula (I):

[0007]

[0008] Formula (I)

[0009] Furthermore, as described above, the antimicrobial peptide C2HF self-assembles into a nanofiber structure under acidic conditions through cation-π interactions between branch A and branch B.

[0010] Furthermore, the self-assembly conditions of the antimicrobial peptide C2HF as described above are as follows: the antimicrobial peptide C2HF is dissolved in a 10 mM PB buffer solution at pH 6.0, the concentration of the antimicrobial peptide C2HF is 32-256 μM, and incubated at room temperature for 12 hours to self-assemble into a nanofiber structure.

[0011] Furthermore, the preparation method of the antimicrobial peptide C2HF as described above is as follows: Two branches A and B are designed, with the sequence of branch A being HHHCFFF and the sequence of branch B being FFFCHHH; branch A and branch B are synthesized separately using solid-phase chemical synthesis, wherein the Cys groups of branch A and branch B are connected by disulfide bonds, and then identified by mass spectrometry and purified by reversed-phase high-performance liquid chromatography to obtain the antimicrobial peptide C2HF.

[0012] Another object of the present invention is to provide the use of the antimicrobial peptide C2HF as described above in the preparation of a medicament for treating infectious diseases caused by Pseudomonas aeruginosa in an acidic environment.

[0013] This invention has the following advantages and beneficial effects: Testing of its antibacterial activity and biosafety revealed that it exhibits good antibacterial effects against Pseudomonas aeruginosa in an acidic environment (pH=6.0) and demonstrates excellent biocompatibility when co-cultured with cells. In summary, the antimicrobial peptide C2HF has extremely high application potential for diseases caused by Pseudomonas aeruginosa infection under acidic conditions. Attached Figure Description

[0014] Figure 1 The mass spectrum of the nano-antimicrobial peptide C2HF;

[0015] Figure 2 The high-performance liquid chromatogram of the nano-antimicrobial peptide C2HF;

[0016] Figure 3 The diagram shows the minimum aggregation concentration (CAC) of the nano-antimicrobial peptide C2HF.

[0017] Figure 4 The image shows the hemolytic activity of the nano-antimicrobial peptide C2HF. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0019] Example 1

[0020] Design of antimicrobial peptides

[0021] Design Principle: To obtain an antimicrobial peptide that can effectively kill Pseudomonas aeruginosa and has good biocompatibility, a supramolecular nanostructure was formed by the non-covalent self-assembly of a single peptide molecule. The antimicrobial activity and biocompatibility of this structure were then tested, resulting in a self-assembled antimicrobial peptide with high application value. Amino acids, due to their different side chains, can provide different interaction forces for stabilizing the three-dimensional structure of proteins, such as hydrophobic interactions, electrostatic interactions, and hydrogen bonds, to achieve the chemical and physicochemical stability of the protein's stereostructure and realize the minimum free energy for pairing. The imidazole group of His (isoelectric point 6.5) is protonated and positively charged under weakly acidic conditions, forming a cation-π interaction with Phe. It can also stabilize the supramolecular structure through π-π interactions and hydrogen bonds, playing an important role in protein interactions and chemical catalysis. The isoelectric point of the imidazole group is 6.5. Under weakly acidic conditions, the protonation of the His side chain leads to cation-π interactions with Phe, thereby promoting supramolecular self-assembly in an acidic environment. Meanwhile, Phe-rich antimicrobial peptides can interact strongly with the phospholipid bilayer and promote peptide insertion into the phospholipid bilayer to achieve permeability or form pores.

[0022] The amino acid sequence of the antimicrobial peptide C2HF is as follows:

[0023]

[0024] Table 1 shows the C2HF amino acid sequence of the antimicrobial peptide.

[0025]

[0026] The molecular formula of the antimicrobial peptide C2HF is shown in formula (I):

[0027]

[0028] Formula (I)

[0029] Example 2

[0030] Synthesis of antimicrobial peptides by solid-phase chemical synthesis

[0031] The peptide was synthesized using the Fmoc solid-phase synthesis method and then lyophilized. The purity of the product was determined by electrospray ionization mass spectrometry (ESI-MS) and reversed-phase high-performance liquid chromatography (RP-HPLC), and the purity of the antimicrobial peptide was greater than 95%.

[0032] Example 3

[0033] The critical aggregation concentration (CAC) of nanopeptides was determined using a sodium 1-aniline-8-naphthalenesulfonate (ANS) fluorescent probe. Dye preparation: ANS powder was dissolved in N,N-dimethylformamide (DMF) to a concentration of 40 mM and stored at low temperature in the dark for later use. Different concentrations of antimicrobial peptide C2HF (1-256 μM) were dissolved in PB (10 mM, pH 6.0) buffer and incubated at room temperature for 12 hours. Subsequently, different concentrations of antimicrobial peptide C2HF were mixed with ANS (diluted 500-fold). The fluorescence intensity was monitored using a microplate reader at an excitation wavelength of 360 nm and an emission wavelength of 420-670 nm, with a slit width of 2 nm. Figure 3 As shown, significant fluctuations in fluorescence intensity can be observed, indicating that the antimicrobial peptide C2HF has self-assembly capabilities.

[0034] Example 4

[0035] Assay of antimicrobial activity of antimicrobial peptide C2HF

[0036] The minimum inhibitory concentration (MIC) of peptides was determined using the standard microbroth dilution method. Logarithmic-phase bacteria were diluted to ~2 × 10⁻⁶. 5 CFU / mL. 50 μL of antimicrobial peptide C2HF at different concentrations (final concentration 1-128 μM) and an equal volume of bacterial suspension were added to each well of a 96-well plate. A negative control (culture medium only) and a positive control (bacteria and culture medium) were also included. The 96-well plates were then incubated at 37 ℃ for 18-20 hours. The absorbance at 492 nm was measured using a microplate reader. Each experiment was performed in duplicate, and these tests were repeated at least three times. The results are shown in Table 2.

[0037] Table 2. Minimum inhibitory concentration (μM) of antimicrobial peptide C2HF

[0038]

[0039] As shown in Table 2, the nanopeptide C2HF exhibits excellent activity against four types of Pseudomonas aeruginosa, including spectinomycin-resistant Pseudomonas aeruginosa 109004, with a minimum inhibitory concentration of 2-4 μM.

[0040] Example 5

[0041] Determination of the hemolytic activity of antimicrobial peptide C2HF

[0042] Blood preparation: Fresh human blood was drawn and centrifuged at 3000-3500 rpm for 10 minutes at 4°C. The supernatant was aspirated, and phosphate-buffered saline (PBS) pH=6.0 was filtered through a 0.22 μm aqueous filter membrane. The PBS was then added to red blood cells, and the red blood cells were washed three times by centrifugation. Finally, the red blood cells were resuspended in 10 volumes of PBS. Dilution of antimicrobial peptide C2HF: PBS was added to columns 1-12 of a 96-well plate, with 90 µL added to the first column and 50 µL added to the other columns. 10 µL of the dissolved nanopeptide solution was added to column 1 of the 96-well plate, serially diluted to column 10. Red blood cell addition: 50 µL of resuspended red blood cells was added to columns 1-12 of the 96-well plate per well. 0.1% Triton X-100 was added to column 12 as a positive control (100% hemolysis), and column 11 as a negative control. Incubate the 96-well plate at 37 °C for 1 hour, then centrifuge at 1000 × g for 5 minutes at 4 °C. Transfer 50 µL of the supernatant from each well to a new 96-well plate. Result determination: Measure the absorbance at 570 nm using a microplate reader. Perform each experiment in duplicate, and repeat the tests at least three times.

[0043] like Figure 4 As shown, the antimicrobial peptide C2HF exhibited negligible hemolytic activity at all tested concentrations, indicating that the antimicrobial peptide C2HF has good biocompatibility.

[0044] In summary, the antimicrobial peptide C2HF exhibits excellent activity against four types of Pseudomonas aeruginosa (including spectinomycin-resistant Pseudomonas aeruginosa 109004), with a minimum inhibitory concentration of 2-4 μM, and shows almost no toxicity to human erythrocytes, demonstrating extremely high application potential.

Claims

1. A pH-triggered, regulated dimeric antimicrobial nanopeptide C2HF against Pseudomonas aeruginosa, characterized in that: It includes branch A and branch B, wherein the amino acid sequence of branch A is: HHHCFFF, and the amino acid sequence of branch B is: FFFCHHH, and the Cys of branch A and branch B are connected by disulfide bonds.

2. The pH-triggered, regulated anti-Pseudomonas aeruginosa dimeric nanopeptide C2HF according to claim 1, characterized in that, Its molecular formula is shown in formula (I): Formula (I).

3. The pH-triggered, regulated anti-Pseudomonas aeruginosa dimeric nanopeptide C2HF according to claim 1, characterized in that: Under acidic conditions, branch A and branch B self-assemble into nanofiber structures through cation-π interactions.

4. The self-assembly method of pH-triggered regulated anti-Pseudomonas aeruginosa dimeric nanopeptide C2HF according to claim 1, characterized in that, The self-assembly conditions are as follows: the antimicrobial peptide C2HF is dissolved in 10 mM PB buffer at pH 6.0, and the concentration of the antimicrobial peptide C2HF is 32-256 μM. The mixture is incubated at room temperature for 12 hours to self-assemble into a nanofiber structure.

5. The method for preparing a pH-triggered, regulated anti-Pseudomonas aeruginosa dimeric nanopeptide C2HF according to claim 1, characterized in that, The method is as follows: Two branches, A and B, are designed. The sequence of branch A is HHHCFFF, and the sequence of branch B is FFFCHHH. Branch A and branch B are synthesized separately using solid-phase chemical synthesis. The Cys groups of branch A and branch B are then linked by disulfide bonds. After identification by mass spectrometry and purification by reversed-phase high-performance liquid chromatography, the antimicrobial peptide C2HF is obtained.

6. The application of the pH-triggered and regulated anti-Pseudomonas aeruginosa dimeric nanopeptide C2HF according to claim 1 in the preparation of a drug for treating infectious diseases caused by Pseudomonas aeruginosa in acidic environments.

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