Self-assembled nano-capture antibacterial peptide KIL4 as well as preparation method and application thereof
By designing and assembling self-contained nanoparticles to capture the antimicrobial peptide KIL4, the issues of biotoxicity and environmental sensitivity of antimicrobial peptides in clinical applications have been resolved. This has enabled the effective inhibition and capture of Gram-positive and Gram-negative bacteria, providing technical support for novel antimicrobial drugs.
Patent Information
- Application Number
- CN202511526587.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-24
- Publication Date
- 2026-02-06
AI Technical Summary
Existing antimicrobial peptides face issues of biotoxicity and environmental sensitivity in clinical applications, making it difficult to fully realize their antimicrobial efficacy.
A self-assembled nanoparticle-capturing antimicrobial peptide, KIL4, was designed. By forming a nanostructure through specific amino acid sequences and self-assembly conditions, its stability and targeted binding ability were enhanced. It was prepared and purified by solid-phase chemical synthesis, and its nanomorphology and biocompatibility were tested.
It achieves significant inhibition of both Gram-positive and Gram-negative bacteria, exhibits good biocompatibility and bacterial capture ability, and provides technical support for novel antibacterial drugs.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a self-assembled nano-capture antimicrobial peptide KIL4, 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, they face two major challenges in practical applications: first, potential biotoxicity may damage normal host cells; second, their activity is easily affected by the environment, making it difficult to fully exert their antimicrobial efficacy. These two problems restrict their translation into clinical drugs. Therefore, it is necessary to optimize the performance of antimicrobial peptides in multiple dimensions: on the one hand, nanostructures can provide protection for antimicrobial peptides, reducing their degradation in vivo and significantly improving stability and bioavailability; on the other hand, the spatial effects and surface properties of nanostructures can enhance the targeted binding ability of antimicrobial peptides to bacteria, thereby improving antimicrobial activity. Summary of the Invention
[0003] Based on the above shortcomings, the purpose of this invention is to provide a self-assembled nano-capture antimicrobial peptide KIL4, which has dual effects of antibacterial and bacterial capture, and has excellent biocompatibility.
[0004] The technical solution adopted in this invention is as follows: a self-assembled nano-capture antimicrobial peptide KIL4, 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 self-assembled nano-capture antimicrobial peptide KIL4 as described above, the steps of which are as follows:
[0008] Step S1: Select the α-helical heptapeptide repeat sequence (abcdefg)4 template and select lysine residues at positions b and c to provide positive charges;
[0009] Step S2: Isoleucine and leucine are filled into positions a and d respectively to provide intermolecular hydrophobic forces, and lysine and glutamic acid are filled into 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, biocompatibility detection, and bacterial capture ability detection, and is finally named antimicrobial peptide KIL4.
[0012] The present invention also provides a self-assembly method for the self-assembly of the antimicrobial peptide KIL4 captured by nanoparticles as described above, characterized in that the self-assembly conditions are: a concentration of 65.16-256 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 nano-capture antimicrobial peptide KIL4 as described above in the preparation of medicaments 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 nano-capture antimicrobial peptide KIL4 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 a nanofiber network structure, 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 KIL4 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 KIL4;
[0019] Figure 2 The chromatogram of the antimicrobial peptide KIL4 is shown.
[0020] Figure 3 The graph shows the critical aggregation concentration of the antimicrobial peptide KIL4 and the linear fitting value of the critical aggregation concentration. In the graph, a is the ANS fluorescence spectrum of the nano-captured antimicrobial peptide KIL4, and b is the critical aggregation concentration.
[0021] Figure 4 This is a negative staining image of the antimicrobial peptide KIL4;
[0022] Figure 5 The graph shows the determination of the hemolytic activity of the antimicrobial peptide KIL4.
[0023] Figure 6 This is a graph showing the assay of the cytotoxicity of the antimicrobial peptide KIL4.
[0024] Figure 7 This is a graph showing the bacterial capture ability of the antimicrobial peptide KIL4.
[0025] Figure 8 This is a graph showing the binding capacity of the antimicrobial peptide KIL4 to LPS.
[0026] Figure 9 This figure shows the effect of the antimicrobial peptide KIL4 on the outer membrane permeability of Escherichia coli. Detailed Implementation
[0027] 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.
[0028] Example 1
[0029] Design of the antimicrobial peptide KIL4: The α-helical heptapeptide repeat sequence (abcdefg)4 was selected as the template. Lysine was selected to provide positive charge at positions b and c. Isoleucine and leucine were filled into positions a and d, respectively, to provide intermolecular hydrophobic forces. Lysine and glutamic acid were selected to fill positions e and g, respectively, to provide intermolecular electrostatic forces. Tryptophan was filled into position f at 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 KIL4 is shown in Table 1.
[0030] Table 1 KIL4 gene sequence
[0031] peptides amino acid sequence molecular weight Net charge KIL4 IKKLKWEIKKLKWEIKKLKWEIKKLKWE 3722.69 +8
[0032] The molecular structure of this antimicrobial peptide is shown in formula (I):
[0033]
[0034] Formula (I).
[0035] Example 2
[0036] Synthesis of antimicrobial peptide KIL4 using solid-phase chemical synthesis:
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 7. After completing the connection of all amino acids, wash the resin and dry it.
[0044] 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.
[0045] 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.
[0046] 10. Finally, the crude polypeptide was purified by liquid chromatography and the final polypeptide KIL4 was obtained by freeze drying.
[0047] The final mass spectrum of the antimicrobial peptide KIL4 is as follows: Figure 1 As shown; the chromatogram of the antimicrobial peptide KIL4 is as follows. Figure 2 As shown.
[0048] Example 3
[0049] Nanoscale characterization of antimicrobial peptide KIL4:
[0050] 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 peptides of different concentrations dissolved in phosphate buffer and incubated at 37°C for 24 h. The mixed samples were 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 peptides was then calculated using Origin software. The results are shown below. Figure 3 .
[0051] from Figure 3 (A) It can be seen that as the peptide concentration increases, the fluorescence intensity of the antimicrobial peptide KIL4 increases sharply in the range of 440 nm to 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 KIL4 is 65.16 μM.
[0052] Example 4
[0053] 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 A dense and clear nanofiber network structure can be observed in the antimicrobial peptide KIL4 solution.
[0054] Example 5
[0055] In vitro hemolytic activity, cytotoxicity, and bactericidal activity assays of the antimicrobial peptide KIL4:
[0056] 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 KIL4 did not cause hemolytic toxicity even at a high concentration (128 μM), which indicates that the antimicrobial peptide KIL4 has good biocompatibility.
[0057] 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 .
[0058] from Figure 6 It can be seen that after treatment with a high concentration of antimicrobial peptide KIL4 (64 μM), the survival rate of IPEC-J2 cells was 85.85%, indicating that antimicrobial peptide KIL4 has good biocompatibility and has the potential to become an antibiotic alternative.
[0059] 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 KIL4 exhibits significant antibacterial activity against both common Gram-negative and Gram-positive bacteria.
[0060] Table 2. Minimum inhibitory concentrations of antimicrobial peptide KIL4 against common pathogens.
[0061] strain Minimum inhibitory concentration (μM) Gram-negative bacteria E. coli 25922 2 E.coli K88 2 E. coli K99 4 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 4 E.faecalis 29212 8 S.epidermidis 12228 2
[0062] Example 6
[0063] Antimicrobial peptide KIL4 bacterial agglutination test
[0064] E. coli ATCC 25922 cells grown to mid-log phase were centrifuged (5000 g, 5 min) and washed 2-3 times with HEPES buffer (pH=7.0). The bacterial culture was then resuspended in HEPES buffer, and the bacterial concentration was adjusted to OD600=0.2. 2 mL of bacterial culture was pipetteed into sterilized, clear, capped cuvettes. Different concentrations of the antimicrobial peptide were then added to the cuvettes containing the bacterial culture. The cuvettes were incubated horizontally at room temperature for 6 h, and the bacterial aggregation and sedimentation were observed. Cuvettes containing only the bacterial culture and not treated with the antimicrobial peptide served as the control group. Figure 7 It is evident that the antimicrobial peptide KIL4 induces rapid aggregation and sedimentation of Escherichia coli.
[0065] Example 7
[0066] Determination of the LPS binding capacity of the antimicrobial peptide KIL4 and its effect on bacterial outer membrane permeability:
[0067] 1. The LPS binding ability testing method is as follows:
[0068] 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 8 As shown, the antimicrobial peptide KIL4 has excellent LPS binding ability.
[0069] 2. The method for detecting bacterial outer membrane permeability is as follows:
[0070] 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 9 As shown, the antimicrobial peptide KIL4 disrupts the outer membrane of E. coli ATCC 25922 in a dose-dependent manner.
Claims
1. A self-assembled nano-capture antimicrobial peptide KIL4, characterized in that, Its amino acid sequence is shown in SEQ ID No.
1.
2. The self-assembled nano-capture antimicrobial peptide KIL4 according to claim 1, characterized in that, Its molecular formula is shown in formula (I): , formula (I).
3. The method for preparing the self-assembled nano-capture antimicrobial peptide KIL4 according to claim 1, characterized in that, The steps are as follows: Step S1: Select the α-helical heptapeptide repeat sequence (abcdefg)4 template and select lysine residues at positions b and c to provide positive charges; Step S2: Isoleucine and leucine are filled into positions a and d respectively to provide intermolecular hydrophobic forces, and lysine and glutamic acid are filled into positions e and g respectively to provide intermolecular electrostatic forces; 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. 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, biocompatibility detection, and bacterial capture ability detection, and is finally named antimicrobial peptide KIL4.
4. The self-assembly method for self-assembling nano-captured antimicrobial peptide KIL4 according to claim 1, characterized in that, Its self-assembly conditions are: concentration of 65.16-256 μM, incubation at 37℃ for 24 hours can self-assemble into nanostructures.
5. The use of the self-assembled nano-capture antimicrobial peptide KIL4 according to claim 1 in the preparation of a medicament for treating infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria.
6. The application according to claim 5, characterized in that: The Gram-positive bacteria mentioned are Staphylococcus aureus, Staphylococcus epidermidis, or Enterococcus faecalis.
7. The application according to claim 5, characterized in that: The Gram-negative bacteria mentioned are Escherichia coli, Pseudomonas aeruginosa, or Salmonella typhimurium.
8. A drug suitable for treating and / or preventing infections caused by Gram-positive and / or Gram-negative bacteria, characterized in that, The drug contains a self-assembled nano-capture antimicrobial peptide KIL4 as described in claim 1.