Self-assembled nanofiber antibacterial peptide based on coiled spiral structure as well as preparation method and application of self-assembled nanofiber antibacterial peptide
By designing a self-assembled nanofiber antimicrobial peptide KLI4 with a coiled helical structure, the problems of high biotoxicity and poor stability of antimicrobial peptides were solved, achieving effective inhibition of Gram bacteria and good biocompatibility, and showing potential for application as a novel antimicrobial drug.
Patent Information
- Application Number
- CN202511544110.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-28
- Publication Date
- 2026-02-17
AI Technical Summary
Existing antimicrobial peptides suffer from high biotoxicity and poor in vivo stability in clinical applications, which limits their widespread use.
A self-assembled nanofiber antimicrobial peptide, KLI4, based on a coiled helical structure, was designed and prepared by solid-phase chemical synthesis. Under specific conditions, it self-assembled into a nanostructure with the amino acid sequence shown in SEQ ID No. 1. It exhibits strong antibacterial activity and good biocompatibility.
It achieved significant inhibitory effects against Gram-positive and Gram-negative bacteria, and showed good biocompatibility with human erythrocytes and porcine jejunal epithelial cells, demonstrating its potential as a novel antibacterial drug.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a self-assembled nanofiber antimicrobial peptide based on a coiled helical structure, its preparation method, and its application. Background Technology
[0002] Antimicrobial peptides, as key components of the innate immune system, have become an important research direction in combating bacterial infections due to their broad-spectrum antimicrobial activity and low drug resistance. However, their clinical application is limited by potential biotoxicity and unstable antimicrobial activity in the in vivo environment. The supramolecular structure of antimicrobial peptides can enhance their membrane-disrupting ability through synergistic effects, thereby improving antimicrobial efficacy, desensitizing biotoxicity, and enhancing stability in complex physiological environments. α-helices are the most common secondary structure type of antimicrobial peptides. The coiled-coil structure is a supercoiled bundle formed by at least two α-helices intertwined through hydrophobic interactions, thus providing a structural basis for the assembly of supramolecular antimicrobial peptides. By altering the amino acid sequence and oligomeric state, coiled-coil structures can be regulated to form different supramolecular structures (nanofibers, nanoparticles, nanocages, etc.), providing flexible regulatory means and broad application prospects for antimicrobial peptides and their applications. In light of this, developing a self-assembled nanofiber antimicrobial peptide based on a coiled helical structure is expected to overcome the technical bottlenecks of high biotoxicity and poor in vivo stability of traditional antimicrobial peptides, providing key technical support for the development of novel and highly effective antimicrobial drugs. This is of great significance for solving the problem of drug-resistant bacterial infections and ensuring public health security. Summary of the Invention
[0003] In view of the above shortcomings, the purpose of this invention is to provide a self-assembled nanofiber antimicrobial peptide based on a coiled helical structure, which has strong antibacterial activity and good biocompatibility.
[0004] The technical solution adopted in this invention is as follows: a self-assembled nanofiber antimicrobial peptide KLI4 based on a coiled helical structure, the amino acid sequence of which is shown in SEQ ID No. 1.
[0005] Furthermore, its molecular formula is shown in formula (I):
[0006] , formula (I).
[0007] The present invention also provides a method for preparing the antimicrobial peptide KLI4 based on a self-assembled nanofiber with a coiled helical structure as described above, the steps of which are as follows:
[0008] Step S1: Using the α-helical heptapeptide repeat sequence (abcdefg)4 template, lysine residues are selected at positions b and c to provide positive charges;
[0009] Step S2: Fill leucine and isoleucine into positions a and d respectively to provide intermolecular hydrophobic forces, and select lysine and glutamic acid to fill positions e and g respectively to provide intermolecular electrostatic forces;
[0010] Step S3: Fill the center f position of the hydrophilic surface with tryptophan to optimize the hydrophobicity of the peptide. Based on this, repeat the sequence four times to obtain the peptide sequence shown in SEQ ID No. 1.
[0011] Step S4: The peptide is synthesized using a solid-phase chemical synthesis method, and then purified by reversed-phase high-performance liquid chromatography and identified by mass spectrometry to complete the preparation of the peptide. The peptide is then subjected to nano-morphological characterization, antibacterial activity detection, and biocompatibility detection, and finally named antimicrobial peptide KLI4.
[0012] The present invention also provides a self-assembly method for the antimicrobial peptide KLI4 based on the self-assembled nanofiber of the coiled helical structure as described above, characterized in that the self-assembly conditions are: a concentration of 9.95-256 μM, incubation at 37°C for 24 hours to achieve self-assembly into a nanostructure.
[0013] The present invention also provides the use of the self-assembled nanofiber antimicrobial peptide KLI4 based on a coiled helical structure as described above in the preparation of a medicament for treating infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria.
[0014] Furthermore, the Gram-positive bacteria are Staphylococcus aureus, Staphylococcus epidermidis, or Enterococcus faecalis.
[0015] Furthermore, the Gram-negative bacteria mentioned are Escherichia coli, Pseudomonas aeruginosa, or Salmonella typhimurium.
[0016] The present invention also provides a medicament for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections as described above, said medicament containing a self-assembled nanofiber antimicrobial peptide KLI4 based on a coiled helical structure as described above.
[0017] The beneficial effects and advantages of this invention are as follows: The antimicrobial peptides of this invention can self-assemble into nanofibers, exhibiting the ability to capture *Escherichia coli*. It shows significant inhibitory effects on Gram-negative bacteria (*Escherichia coli*, *Salmonella typhimurium*) and Gram-positive bacteria (*Staphylococcus aureus*). Tests have shown that high concentrations of KLI4 exhibit good biocompatibility with human erythrocytes and porcine jejunal epithelial cells (IPEC-J2), providing effective technical support for the development of novel antimicrobial drugs. Attached Figure Description
[0018] Figure 1 This is the mass spectrum of the antimicrobial peptide KLI4;
[0019] Figure 2 The chromatogram of the antimicrobial peptide KLI4 is shown.
[0020] Figure 3 The graph shows the critical aggregation concentration and linear fitting value of the critical aggregation concentration of the antimicrobial peptide KLI4, where a is the ANS fluorescence spectrum of the nano-captured antimicrobial peptide KLI4, and b is the critical aggregation concentration.
[0021] Figure 4 This is a negative staining image of the antimicrobial peptide KLI4;
[0022] Figure 5 The graph shows the determination of the hemolytic activity of the antimicrobial peptide KLI4.
[0023] Figure 6 This is a graph showing the assay of the cytotoxicity of the antimicrobial peptide KLI4.
[0024] Figure 7 This is a graph showing the binding capacity of the antimicrobial peptide KLI4 to LPS.
[0025] Figure 8 This figure shows the effect of the antimicrobial peptide KLI4 on the outer membrane permeability of Escherichia coli. Detailed Implementation
[0026] 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.
[0027] Example 1
[0028] Design of antimicrobial peptide KLI4: The α-helical heptapeptide repeat sequence (abcdefg)4 template was selected, and lysine was selected at positions b and c to provide positive charge; leucine and isoleucine were filled at positions a and d respectively to provide intermolecular hydrophobic interaction, and lysine and glutamic acid were selected at positions e and g respectively to provide intermolecular electrostatic interaction; tryptophan was filled at position f in the center of the hydrophilic side to optimize the hydrophobicity of the peptide; the sequence was repeated 4 times to achieve a sufficient number of positive charges. The amino acid sequence of the resulting antimicrobial peptide KLI4 is shown in Table 1.
[0029] Table 1 KLI4 gene sequence
[0030] peptides amino acid sequence molecular weight Net charge KLI4 LKKIKWE LKKIKWE LKKIKWE LKKIKWE 3722.69 +8
[0031] The molecular structure of this antimicrobial peptide is shown in formula (I):
[0032]
[0033] Formula (I).
[0034] Example 2
[0035] Synthesis of antimicrobial peptide KLI4 using solid-phase chemical synthesis:
[0036] 1. Weigh an appropriate amount of 2-CTC resin and place it in dichloromethane (DCM) for swelling treatment, soaking for 1 hour. Subsequently, use dimethylformamide (DMF) to clean the resin three times to remove surface impurities.
[0037] 2. Accurately weigh 1 equivalent (eq) of the first protected amino acid and 1.5 equivalents of diisopropylethylamine (DIEA), and use DMF as a solvent to mix with the resin and react for 2 hours.
[0038] 3. After the reaction is complete, the resin is dried and then washed three times with DMF. Subsequently, a mixed solution of methanol and DIEA is added to carry out the end-capping reaction for 1 hour.
[0039] 4. After the end-capping reaction is complete, clean the resin and use a 20% piperidine DMF solution for deprotection treatment to remove the Fmoc protecting group. The reaction time is 10 minutes, and the operation is repeated twice.
[0040] 5. After washing the resin, add 3 equivalents of the second amino acid, 3 equivalents of 1-hydroxybenzotriazole (HOBT) and 3 equivalents of dicyclohexylcarbodiimide (DIC), and use DMF as solvent to carry out the coupling reaction for 1.5 hours.
[0041] 6. Repeat steps 4 and 5 to sequentially connect the following amino acid residues until the last amino acid residue at the N-terminus is connected, and remove the Fmoc protecting group at the N-terminus.
[0042] 7. After completing the connection of all amino acids, wash the resin and dry it.
[0043] 8. The resin was cleaved using a mixed solution of 95% trifluoroacetic acid (TFA), 2% triisopropylsilane (Tis), 2% ethylenedithiol (EDT) and 1% water, while the protecting groups of the peptide side chains were removed. The reaction time was 2 hours.
[0044] 9. After the reaction is complete, filter the resin, wash the filtrate with ice-cold ether, then centrifuge to collect the precipitate and obtain the crude polypeptide.
[0045] 10. Finally, the crude polypeptide was purified by liquid chromatography and the final polypeptide KLI4 was obtained by freeze drying.
[0046] The final mass spectrum of the antimicrobial peptide KLI4 is as follows: Figure 1 As shown; the chromatogram of the antimicrobial peptide KLI4 is as follows. Figure 2 As shown.
[0047] Example 3
[0048] Nanoscale characterization of antimicrobial peptide KLI4:
[0049] Critical aggregation concentration (CAC) determination: The critical aggregation concentration (CAC) was determined using a 1-aniline-8-naphthalenesulfonic acid (ANS) fluorescent probe. 1 µL of ANS (final concentration 1 mM, dissolved in 100% DMF) was added to different concentrations of the antimicrobial peptide KLI4 dissolved in phosphate buffer and incubated at 37°C for 24 h. The mixed samples were then transferred to 96-well plates, and fluorescence spectroscopy was performed using a microplate reader with excitation wavelength of 369 nm and emission wavelengths of 440 nm–550 nm. The CAC value of the peptide was then calculated using Origin software. The results are shown below. Figure 3 ,from Figure 3 (A) It can be seen that with the increase of concentration, the fluorescence intensity of the antimicrobial peptide KLI4 increases sharply in the range of 440 nm-550 nm, indicating the presence of nanomolecules in the solution and preliminarily determining the formation of nanostructures. Subsequent fitting analysis using Origin software, such as... Figure 3 As shown in (B), the CAC value of the antimicrobial peptide KLI4 is 9.95 μM.
[0050] Example 4
[0051] Negative staining: After incubation at 128 μM for 12 h, 20 μL of the peptide solution was placed on a copper grid plate for 2 min, and stained with 0.1% phosphotungstic acid for 10 s. Finally, the morphology of the air-dried peptides was examined using a Hitachi H-7650 transmission electron microscope (Hitachi H-7650, Japan). The results are shown below. Figure 4 Clear nanofiber structures were observed to form in the solution of the antimicrobial peptide KLI4.
[0052] Example 5
[0053] In vitro hemolytic activity, cytotoxicity, and bactericidal activity assays of antimicrobial peptide KLI4:
[0054] 1. Hemolytic Activity Assay: 1 mL of fresh blood from healthy individuals was collected and centrifuged (4 ℃, 1000×g, 5 min). The blood was washed three times with PBS (10 mM) buffer, the supernatant was discarded, and the precipitated blood cells were collected and resuspended in PBS (10 mM) buffer to a final volume of 10 mL. An equal volume of the diluted red blood cell suspension was then placed in a 96-well plate and mixed with peptide solutions of different concentrations. After incubation at 37 ℃ for 1 h, the plate was centrifuged (1000×g, 10 min), and the supernatant was transferred to a new 96-well plate. Wells treated with 0.1% Triton X-100 served as positive controls, and wells without peptide treatment served as negative controls. The absorbance was measured using a microplate reader at a wavelength of 570 nm. A peptide concentration that caused 50% hemolytic activity was defined as cytotoxic. The test results are attached. Figure 5As can be seen, the antimicrobial peptide KLI4 did not cause hemolytic toxicity even at a high concentration (128 μM), which indicates that the antimicrobial peptide KLI4 has good biocompatibility.
[0055] 2. Cytotoxicity assay: IPEC-J2 porcine jejunal epithelial cells, revived from liquid nitrogen, were inoculated into a culture medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibodies, and passaged at 37°C and 5% CO2. The cultured cells were then digested with 0.25% trypsin and adjusted to a culture medium volume of 2–4 × 10⁻⁶ cells / mL. 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 16–18 h. Then, 50 µL of LMTT (5 mg / mL) was added to each well, and incubation continued for 4 h. After incubation, the supernatant was discarded, and the crystals at the bottom of the wells were dissolved in 100 µL of DMSO. The absorbance of each well was measured at 570 nm using a microplate reader. Culture medium wells served as blank controls. Results are shown below. Figure 6 .
[0056] from Figure 6 It can be seen that after treatment with a high concentration of antimicrobial peptide KLI4 (64 μM), the survival rate of IPEC-J2 cells was 99.69%, indicating that antimicrobial peptide KLI4 has good biocompatibility and has the potential to become an antibiotic alternative.
[0057] 3. Bactericidal activity assay: The minimum inhibitory concentration (MIC) of the antimicrobial peptide was determined using the microdilution method. Different concentrations of peptide were added to BSA solution in 96-well plates, followed by the addition of an equal volume of solution with a final concentration of 1×10⁻⁶. 5 CFUmL -1 The final peptide concentration in the 96-well plate ranged from 0.5 to 128 μM. After incubation for 24 hours, the absorbance was measured using a microplate reader at a wavelength of 600 nm. The results are shown in Table 2. It can be seen that the antimicrobial peptide KLI4 exhibits significant antibacterial activity against both common Gram-negative and Gram-positive bacteria.
[0058] Table 2. Minimum inhibitory concentrations of antimicrobial peptide KLI4 against common pathogens.
[0059] strains Minimum inhibitory concentration (μM) Gram-negative bacteria E. coli 25922 2 E.coli K88 4 E. coli K99 2 S. typhimurium 14028 4 S. typhimurium C7731 4 P. aeruginosa 27853 4 Gram-positive bacteria S. aureus 29213 4 S.aureus 25923 4 S. aureus 43300 8 E.faecalis 29212 4 S.epidermidis 12228 8
[0060] Example 6
[0061] Determination of the LPS binding capacity of antimicrobial peptide KLI4 and its effect on bacterial outer membrane permeability:
[0062] 1. The LPS binding ability testing method is as follows:
[0063] The binding affinity of the two parents to LPS was detected using the BODIPY-TR-cadaverine probe. E. coli O111:B4 LPS (50 μg / mL) and BC (5 μg / mL) were incubated in the dark for 4 h. The peptides (50 μL) were serially diluted with Tris buffer (pH 7.4) in 96-well plates, and an LPS-BC mixture was added to each well. Fluorescence intensity was measured using a fluorescence spectrophotometer at excitation wavelength of 580 nm and emission wavelength of 620 nm. Results are shown below. Figure 7 As shown, the antimicrobial peptide KLI4 binds to LPS in a dose-dependent manner.
[0064] 2. The method for detecting bacterial outer membrane permeability is as follows:
[0065] Determination of bacterial outer membrane permeability: The effect of antimicrobial peptides on the outer membrane of *E. coli* ATCC25922 was detected using the cell membrane-sensitive fluorescent dye NPN. *E. coli* ATCC 25922 was cultured to the logarithmic growth phase, centrifuged (5000g, 5 min) to collect the cells, washed three times with 5 mM HEPES buffer (containing 5 mM glucose, pH 7.2), and reselected until OD600nm = 0.2. A final concentration of 10 μM NPN was added, and the cells were incubated at 37°C in the dark for 30 min. The bacterial culture was then added to 100 μL per well of a 96-well plate, and fluorescence intensity was immediately measured after adding various concentrations of antimicrobial peptides until fluorescence release stabilized. The excitation wavelength was set at 350 nm and the emission wavelength at 420 nm. Results are as follows: Figure 8 As shown, the antimicrobial peptide KLI4 has excellent ability to disrupt the outer membrane.
Claims
1. A self-assembling nanofiber antimicrobial peptide based on a coiled coil structure KLI4, characterized in that, The amino acid sequence of which is shown as SEQ ID No.
1.
2. The self-assembled nanofiber antibacterial peptide KLI4 based on the coiled coil structure according to claim 1, characterized in that, The molecular formula of which is shown as formula (I): , formula (I).
3. The preparation method of the self-assembled nanofiber antibacterial peptide KLI4 based on the coiled coil structure according to claim 1, characterized in that, The steps are as follows: Step S1: using alpha-helix heptad repeat sequence (abcdefg)4template, selecting lysine at b, c position to provide positive charge; Step S2: filling leucine and isoleucine to a, d position to provide intermolecular hydrophobic interaction, selecting lysine and glutamic acid to fill e, g position to provide intermolecular electrostatic interaction; Step S3: filling tryptophan at the center of the hydrophilic surface f position to optimize the hydrophobicity of the polypeptide, on this basis, the sequence is repeated four times, and the obtained polypeptide sequence is shown as SEQ ID No. 1; Step S4: the polypeptide is synthesized by solid-phase chemical synthesis method, and after further reversed-phase high performance liquid chromatography purification and mass spectrometry identification, the preparation of the polypeptide is completed, and the polypeptide is subjected to nano-morphology characterization detection, antibacterial capacity detection and biocompatibility detection, and finally named as antibacterial peptide KLI4.
4. The self-assembly method of the self-assembled nanofiber antimicrobial peptide KLI4 based on the coiled coil structure according to claim 1, characterized in that, The self-assembly conditions thereof are: concentration of 9.95-256 μM, incubation at 37℃ for 24 hours to self-assemble into nanostructure.
5. The application of a self-assembled nanofiber antibacterial peptide KLI4 based on a coiled coil structure according to claim 1 in the preparation of a drug for treating infectious diseases caused by gram-positive bacteria or / and gram-negative bacteria.
6. Use according to claim 5, characterized in that: The gram-positive bacteria are Staphylococcus aureus, Staphylococcus epidermidis or Enterococcus faecalis.
7. Use according to claim 5, characterized in that: The gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa or Salmonella typhimurium.
8. A medicament suitable for the treatment and / or prophylaxis of Gram-positive and / or Gram-negative bacterial infections, characterized in that, The drug contains the self-assembled nanofiber antibacterial peptide KLI4 based on a coiled coil structure according to claim 1.