Nanometer antibacterial peptide, preparation method and application thereof

Abstract

The application provides a kind of nano antibacterial peptide and its preparation method and application, belong to biotechnology field, amino acid sequence is as shown in SEQ ID No.1.The application selects arginine to provide positive charge, selects phenylalanine and tryptophan to provide hydrophobicity;Phenylalanine is selected to construct intermolecular pi-pi bond to provide hydrophobic force driven polypeptide self-assembly;Center selects tryptophan zipper to stabilize the overall intramolecular structure, terminal amidation forms hydrogen bond with water molecules, arginine is located at 2, 3, 8 and 9 to form cation-pi bond with tryptophan to promote the process of molecular self-assembly;Symmetrical structure is used to reduce the cytotoxicity of peptide molecules.The application of nano antibacterial peptide in the preparation of drug for treating infectious diseases of gram-negative bacteria or / and gram-positive bacteria.The antibacterial peptide of the application has broad-spectrum inhibitory effect on pathogenic bacteria, and has low hemolytic activity, the therapeutic index reaches 50.79, and has strong stability in physiological environment.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a nano-antimicrobial peptide, its preparation method, and its application. Background Technology

[0002] For over 90 years, antibiotics have been widely used in medicine, food, and animal husbandry. However, the overuse of antibiotics has accelerated the development of drug resistance, posing a significant threat to global public health. The emergence of superbugs has made the search for antibiotic alternatives an urgent priority. Antimicrobial peptides (AMPs) are widely distributed in organisms in nature and possess various biological activities such as antibacterial, antifungal, and anti-inflammatory effects, making them an important component of the host's immune system. AMPs' unique non-specific membrane disruption mechanism makes it difficult for bacteria to develop drug resistance; therefore, AMPs are considered the most promising antibiotic alternatives.

[0003] However, monomeric antimicrobial peptides are susceptible to physiological influences, such as losing their antimicrobial activity in salt ions and simulated serum. Furthermore, their high cytotoxicity continues to limit their clinical potential. Therefore, the focus of monomeric molecule improvement is to enhance their physiological stability and improve biocompatibility at a limited cost. Summary of the Invention

[0004] In view of the above shortcomings, the purpose of this invention is to provide a nano-antimicrobial peptide that has high antimicrobial activity and stable performance in salt ions and serum.

[0005] The technical solution adopted in this invention is as follows: a nano-antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID No. 1, and its C-terminus is amidated with -NH2.

[0006] Furthermore, its self-assembly conditions are: concentration of 10.40-256 μM, incubation at room temperature for 24 hours.

[0007] Another object of the present invention is to provide a method for preparing a nano-antimicrobial peptide as described above, as follows:

[0008] (1) Arginine is chosen to provide positive charge, and phenylalanine and tryptophan are chosen to provide hydrophobicity;

[0009] (2) Phenylalanine was selected to construct intermolecular π-π bonds to provide hydrophobic forces to drive peptide self-assembly; tryptophan was selected at the center to stabilize the overall intramolecular structure, and terminal amidation formed hydrogen bonds with water molecules to further stabilize the peptide. In addition, arginine formed cation-π bonds with tryptophan at positions 2, 3, 8 and 9 to promote the molecular self-assembly process; finally, a centrosymmetric structure was adopted to reduce the cytotoxicity of peptide molecules.

[0010] (3) The polypeptide was synthesized by solid-phase chemical synthesis. Its amino acid sequence is shown in SEQ ID No.1. After the determination of antibacterial activity, hemolytic activity and stability under physiological conditions, it was finally named antimicrobial peptide FRRFW.

[0011] Another object of the present invention is to provide the use of the nano-antimicrobial peptide described above in the preparation of a medicament for treating infectious diseases caused by Gram-negative bacteria and / or Gram-positive bacteria.

[0012] The present invention has the following advantages and beneficial effects: the experimental route for preparing the high-efficiency nano-antimicrobial peptide by this method is simple; the nano-characterization, antibacterial activity, hemolytic activity, physiological salt and serum stability determination of the prepared nano-antimicrobial peptide revealed that the antimicrobial peptide FRRFW has a strong inhibitory effect on pathogens, almost no hemolysis of red blood cells, a therapeutic index of 50.79, and strong resistance under physiological conditions, thus having high application value, bactericidal activity, good biocompatibility and stability, and the potential to become an antibiotic alternative. Attached Figure Description

[0013] Figure 1 Reversed-phase high-performance liquid chromatography chromatogram of the nano-antimicrobial peptide FRRFW;

[0014] Figure 2 Mass spectrum of the nano-antimicrobial peptide FRRFW;

[0015] Figure 3 Fluorescence spectrum of nano-antimicrobial peptides;

[0016] Figure 4 Critical aggregation concentration determination graph of nano-antimicrobial peptides;

[0017] Figure 5 Nanomorphology of antimicrobial peptides. 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 nano-antimicrobial peptides

[0021] (1) Arginine is selected to provide positive charge, and phenylalanine and tryptophan are selected to provide hydrophobicity to meet the basic characteristics of antimicrobial peptides; (2) Phenylalanine is selected to construct intermolecular π-π bonds to provide hydrophobic force to drive peptide self-assembly; tryptophan is selected at the center to stabilize the overall intramolecular structure, and terminal amidation forms hydrogen bonds with water molecules to further stabilize the peptide segment; arginine forms cation-π bonds with tryptophan at positions 2, 3, 8 and 9 to promote the molecular self-assembly process; finally, a centrosymmetric structure is adopted to reduce the cytotoxicity of peptide molecules.

[0022] (3) The template for the nano-antimicrobial peptide was found to be: FRRFWWFRRF-NH2, with the amino acid sequence shown in SEQ ID No.1, and named as antimicrobial peptide FRRFW.

[0023] The amino acid sequence of the nano-antimicrobial peptide FRRFW is as follows:

[0024]

[0025] Table 1. Amino acid sequences of peptides

[0026]

[0027] Example 2

[0028] Solid-phase chemical synthesis of antimicrobial peptide sequence: N-terminus -A1A2A3……A n -C-end

[0029] a. Resin swelling: Weigh Fmoc-Phe-Resin resin and pour it into the reaction column. Add DCM and soak for 30 minutes, then dry.

[0030] b. Deprotection: Add an appropriate amount of deprotection solution to the reaction column, purge with nitrogen and stir for 30 minutes, then dry under vacuum.

[0031] c. Weighing: Within 30 minutes of deprotection, calculate the required amounts of amino acids, condensing agent, and NMM reaction agents for each step based on the prepared quantities, and then weigh the amino acid Fmoc-A for the next step. n-1 .

[0032] d. Deprotection washing: Add an appropriate amount of DMF to the reaction column, agitate with nitrogen for 2 minutes, then dry. Repeat this process 6 times. e. Feeding: After washing and deprotection, and after detection, add the weighed feed material in the order of each peptide, then add a small amount of reaction solution, then add alkali and NMM. Adjust the gas mixture evenly, flush off the resin adhering to the inner wall of the reaction column with DCM, and then record the reaction time. The reaction time is 30 minutes.

[0033] f. Washing after reaction: Drain the solution in the reaction column, add an appropriate amount of DMF to wash, agitate with nitrogen for 2 minutes, drain, and repeat the operation 3 times.

[0034] g. Detection: Take an appropriate amount (10-20 beads) of resin in a small test tube and add two drops each of solutions A, B, and C. Heat in a dry heater for 3 minutes (110 degrees Celsius). If the solution turns blue and the resin is discolored and opaque after removal, the reaction is incomplete and needs to be repeated. If the solution is slightly yellow and the resin is colorless and transparent, the reaction is complete and the next amino acid can be ligated. Repeat steps bf above until the last amino acid is ligated.

[0035] h. Washing and drying after synthesis: The above peptides were dried under vacuum. An appropriate amount of methanol was added to the reaction column, and the column was agitated with nitrogen for 2 minutes. The mixture was then dried under vacuum. An appropriate amount of DCM was added, and the column was agitated with nitrogen for 2 minutes. This process was repeated 3 times. Finally, an appropriate amount of methanol was added to the reaction vessel, and the column was agitated with nitrogen for 2 minutes. This process was repeated twice. The resin was then placed in a suitable container and vacuum dried in a vacuum desiccator for 12 hours before cutting.

[0036] Cutting: Put the dried resin into a suitable round-bottom flask, add an appropriate amount of the prepared cutting solution (1g / 10ml), and place it in a constant temperature shaker at 25℃ for 2 hours.

[0037] Filtration: Filter out the resin particles using a 50ml sintered glass funnel, then pour the filtrate into a 100ml centrifuge tube, add 6-8 times the volume of anhydrous diethyl ether while stirring, and the precipitated white solid is the desired crude polypeptide.

[0038] Washing: Seal the centrifuge tubes and centrifuge at 4000 rpm for 3 minutes. Remove the tubes, discard the supernatant, add ether, stir well with a glass rod, and centrifuge again. Repeat this washing process 5 times.

[0039] Drying: The polypeptide, after being washed 5 times, was placed in a vacuum desiccator and dried under vacuum for 24 hours. The resulting white powder is the crude polypeptide, which was weighed and prepared for purification.

[0040] Purification: The column was equilibrated for 30 min using 0.2 mol / L sodium sulfate (phosphate pH = 7.4). The peptide was dissolved in 90% acetonitrile aqueous solution, filtered, and added to a reversed-phase atmospheric pressure column. Gradient elution was performed (eluting solvent was a mixture of methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30) at a flow rate of 1 mL / min and a detection wavelength of 220 nm. The main peak was collected and lyophilized. Further purification was performed using a reversed-phase C18 column with elution buffer A being 0.1% TFA / water solution and elution buffer B being 0.1% TFA / acetonitrile solution at a flow rate of 1 mL / min. The main peak was collected again and lyophilized.

[0041] Identification: The antimicrobial peptides obtained above were analyzed by electrospray ionization mass spectrometry (e.g., Figure 1As shown), the purity of the antimicrobial peptide is greater than 95% (e.g., Figure 2 (As shown).

[0042] Example 3

[0043] Biological activity assay of nano-antimicrobial peptides

[0044] 1. Determination of antibacterial activity: The minimum inhibitory concentration (MIC) of the peptide was determined using the standard microbroth dilution method. Logarithmic-phase bacteria were diluted to 10⁻⁶. 5 CFU / ml. Add 50 μL of peptides at different concentrations (final peptide concentration 1-128 μM) and an equal volume of bacterial suspension to each well of a 96-well plate. Include a negative control (culture medium only) and a positive control (bacteria and culture medium). Incubate the 96-well plate at 37°C for 18-20 hours. Read the microplate at 492 nm (OD500). 492 The absorbance value was measured at ( ) to determine the minimum inhibitory concentration. Three independent replicates were performed, with each replicate in duplicate. The results are shown in Table 2.

[0045] Table 2. Antimicrobial activity of antimicrobial peptides (μM)

[0046]

[0047] As shown in Table 2, FRRFW exhibits high antibacterial activity against common pathogens.

[0048] 2. Determination of hemolytic activity: Fresh human erythrocyte suspension was collected and diluted 10-fold with PBS (pH=7.4). 50 μL of peptides of different concentrations (final peptide concentration 1-512 μM) and an equal volume of erythrocyte suspension were placed in each well of a 96-well plate. Human erythrocyte suspensions treated with 0.1% Triton X-100 were used as positive controls, and untreated human erythrocyte suspensions were used as negative controls. The 96-well plates were incubated at 37°C for 1 hour. After centrifugation (1000g, 5 min, 4°C), 50 μL of supernatant was collected from the mixture and transferred to a new 96-well plate. The plate was then read from 570 nm (OD200) using a microplate reader. 570 The absorbance value was measured at ( ). The hemolysis rate was calculated using the following formula:

[0049] Hemolysis rate (%) = [(sample OD)] 570 —Negative control OD 570 ) / (Positive control OD 570 —Negative control OD 570 The minimum hemolytic concentration (MCC) is the concentration at which the antimicrobial peptide causes a 15% hemolysis rate. The test results are shown in Table 3.

[0050] Table 3 Hemolytic Activity and Therapeutic Index of Nanopeptides

[0051]

[0052]

[0053] The nano-antimicrobial peptide FRRFW did not exhibit hemolytic activity within the detection range. Its therapeutic index was calculated using the geometric mean of the minimum hemolytic concentration and the minimum inhibitory concentration, and the therapeutic index reached 50.79.

[0054] Example 4

[0055] Nanoscale characterization of antimicrobial peptides

[0056] 1. Critical Aggregation Concentration Determination: To detect the ability of antimicrobial peptides to form nanostructures, the critical aggregation concentration (CAC) was first determined using a 1-aniline-8-naphthalenesulfonic acid (ANS) fluorescent probe. 1 μL of ANS (1 mM, dissolved in DMF) was added to different concentrations of peptides (dissolved in deionized water), and incubated at 37°C for 15 min. The mixed samples were transferred to 96-well plates, and fluorescence spectra were monitored from 440 nm to 550 nm using an F-4500 fluorescence spectrophotometer (Hitachi, Japan) with an excitation wavelength of 369 nm. The CAC values ​​of the peptides were then calculated using Origin software. The results are shown below. Figure 3-4 .

[0057] As the concentration increased, the fluorescence intensity of the nano-antimicrobial peptide FRRFW gradually increased within the detection range, indicating the presence of nano-macromolecules in the solution and preliminarily determining the formation of nanostructures. Subsequently, the CAC value of FRRFW was determined to be 10.40 μM using Origin software fitting analysis.

[0058] 2. Morphology Analysis: To further analyze the nanomorphology of the antimicrobial peptide, the peptide (2.56 mM) was diluted to a concentration of 4-256 μM in deionized water and incubated at room temperature for 24 hours. The sample was deposited on a carbon-coated mesh and observed using a Hitachi h-7800TEM (Hitachi, Japan) at 100 kV with 1% phosphotungstic acid negative staining for 30 seconds. The results are shown in […]. Figure 5 .

[0059] The antimicrobial peptide FRRFW forms loose nanosheets at 4 μM, transforms into nanowires at a CAC value, and transforms into more compact spherical micelles and vesicle structures when the concentration is further increased to 256 μM.

[0060] Determination of the physiological stability of nano-antimicrobial peptides

[0061] To assess the stability of peptides under physiological conditions, these assays used *E. coli* ATCC 25922 as a Gram-negative bacterial model. In the salt stability assay, the folding changes of the peptide's MIC value were evaluated after dissolving salt powder in a 0.2% BSA solution. The final salt concentrations were: NaCl, 150 mM; KCl, 4.5 mM; NH4Cl, 6 mM; CaCl2, 2 mM; ZnCl2, 8 mM; and MgCl2, 1 mM, FeCl3, 4 mM. For serum sensitivity assays, equal volumes of FRRFW were incubated with 25%, 50%, and 100% serum dissolved in 0.2% BSA.

[0062] Table 4. Minimum inhibitory concentration (μM) of FRRFW against Escherichia coli ATCC 25922 under physiological conditions.

[0063]

[0064] As shown in Table 4, only 50% serum concentration and NaCl and CaCl2 slightly affected the antibacterial activity of FRFW, and the MIC change was within 4-fold, indicating that the antibacterial short peptide FRFW has strong salt ion stability and serum stability.

Claims

1. A nano-antimicrobial peptide, characterized in that, Its amino acid sequence is shown in SEQ ID No.1, and its C-terminus is amidated with -NH2.

2. The self-assembly method of a nano-antimicrobial peptide according to claim 1, characterized in that, Its self-assembly conditions are: concentration of 10.40-256 μM, incubation at room temperature for 24 hours.

3. The method for preparing nano-antimicrobial peptides according to claim 1, characterized in that, The preparation method steps are as follows: S1: Arginine is chosen to provide a positive charge, and phenylalanine and tryptophan are chosen to provide hydrophobicity; S2: Phenylalanine is selected to construct intermolecular π-π bonds, providing hydrophobic forces to drive peptide self-assembly; tryptophan is selected at the center to stabilize the overall intramolecular structure, and terminal amidation forms hydrogen bonds with water molecules to further stabilize the peptide; arginine forms cation-π bonds with tryptophan at positions 2, 3, 8, and 9 to promote the molecular self-assembly process; finally, a centrosymmetric structure is adopted to reduce the cytotoxicity of peptide molecules; S3: The polypeptide was synthesized using solid-phase chemical synthesis. Its amino acid sequence is shown in SEQ ID No.

1. After testing for antibacterial activity, hemolytic activity, and stability under physiological conditions, it was finally named the antimicrobial peptide FRRFW.

4. The use of the nano-antimicrobial peptide according to claim 1 in the preparation of a medicament for treating infectious diseases caused by Gram-negative bacteria and / or Gram-positive bacteria, wherein the Gram-negative bacteria is *Escherichia coli* (…). Escherichiacoli Salmonella typhimurium ( Salmonella typhimurium ) or Pseudomonas aeruginosa ( Pseudomonasaeruginosa The Gram-positive bacteria mentioned are Staphylococcus epidermidis (Staphylococcus epidermidis). Staphylococcus epidermidis Staphylococcus aureus ( Staphylococcus aureus ) or Enterococcus faecalis ( Enterococcus faecalis ).

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