A low-toxicity cell-penetrating antibacterial peptide, and a preparation method and application thereof

Abstract

This invention discloses a low-toxicity, cell-penetrating antimicrobial peptide, W3L, its preparation method, and its applications. The sequence of this antimicrobial peptide is shown in SEQ ID No. 1, with its C-terminus modified by -NH2 amidation, carrying a net charge of +7. This invention uses arginine to construct a positively charged transmembrane unit, tryptophan to enhance membrane intercalation capability, and a trileucine hydrophobic fragment at the C-terminus to balance toxicity, thus possessing both high-efficiency transmembrane activity and broad-spectrum antimicrobial properties. The antimicrobial peptide is prepared by solid-phase chemical synthesis, purified by reversed-phase liquid chromatography, and identified by mass spectrometry. This antimicrobial peptide has a MIC of only 2-4 μM against various Gram-positive and Gram-negative bacteria, and can eliminate 96.43% of Staphylococcus aureus in macrophages; it shows no significant hemolysis or cytotoxicity at 128 μM, exhibits a high therapeutic index, and demonstrates stable antibacterial activity in various physiological salt ion and serum environments. This invention solves the problems of weak intracellular bactericidal activity and high toxicity of traditional antimicrobial peptides, and can be used to prepare drugs for the prevention and treatment of Gram-related infections, possessing good biosafety and clinical application potential.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a low-toxicity cell-penetrating antimicrobial peptide, its preparation method, and its application. Background Technology

[0002] Antimicrobial peptides (AMPs), as an important component of the innate immune system, possess significant advantages such as broad-spectrum antibacterial activity, low susceptibility to inducing bacterial resistance, good biocompatibility, and easy degradation in vivo with no residue, making them the most promising antibiotic alternative resource for industrialization. However, antimicrobial peptides generally suffer from weak intracellular anti-infection capabilities: most traditional antimicrobial peptides only act on extracellular planktonic bacteria, have difficulty penetrating mammalian cell membrane structures, and cannot eliminate latent pathogens colonizing cells, thus having extremely limited effectiveness against persistent intracellular infections prevalent in livestock farming. Cell-penetrating peptides can efficiently penetrate mammalian cell membranes, but their inherent antibacterial and bactericidal activities are weak, and they cannot effectively eliminate intracellular bacteria when used alone, requiring combination with antibacterial molecules. Currently, there are two methods: the combined use of cell-penetrating peptides and free antimicrobial peptides, and covalent chimerism. In free-peptide combinations, the cell-penetrating peptide and antimicrobial peptide in their free state bind solely through electrostatic interactions and rapidly dissociate in vivo. In the free dipeptide system, only a very small number of antimicrobial peptides can be simultaneously transported into host cells by the cell-penetrating peptide; the vast majority remain extracellularly, significantly reducing the clearance efficiency against intracellular bacteria such as Staphylococcus aureus and Salmonella within macrophages. To improve intracellular efficacy, the concentrations of both peptides must be significantly increased, leading to both hemolytic toxicity and increased raw material costs. In covalently coupled systems, the positively charged transmembrane domain of the cell-penetrating peptide and the bactericidal hydrophobic domain of the antimicrobial peptide present steric hindrance. Splicing these can cause conformational disorder in the peptide folding, weakening the penetration ability of the cell-penetrating peptide or significantly reducing the antimicrobial activity of the antimicrobial peptide, making it difficult to simultaneously maintain the original functions of both peptides. Summary of the Invention

[0003] Based on the above shortcomings, the purpose of this invention is to provide a low-toxicity, cell-penetrating antimicrobial peptide, W3L, thereby solving the problem that existing antimicrobial peptides and cell-penetrating peptides are difficult to kill intracellular bacteria.

[0004] The technical solution adopted in this invention is as follows: a low-toxicity cell-penetrating antimicrobial peptide W3L, the amino acid sequence of which is shown in SEQ ID No.1, and its C-terminus is amidated with -NH2.

[0005] Its molecular formula is shown in formula (I):

[0006]

[0007] Formula (I)

[0008] The present invention also provides a method for preparing a low-toxicity, cell-penetrating antimicrobial peptide W3L as described above, the steps of which are as follows:

[0009] Step 1: Insert tryptophan between two arginine residues and repeat three times to form a peptide chain with the sequence RWRRWRRWR. Then, link three consecutive leucine residues to the C-terminus of the peptide chain to obtain peptide W3L, whose sequence is shown in SEQ ID No.1. Finally, amidate the C-terminus of peptide W3L with -NH2, and finally carry a net charge of +7.

[0010] Step 2: The polypeptide W3L was synthesized using a solid-phase chemical synthesis method. After purification by reversed-phase high-performance liquid chromatography and identification by mass spectrometry, the polypeptide was subjected to antibacterial activity testing, intracellular antibacterial activity testing, hemolytic activity testing, cytotoxicity testing, and salt ion stability testing. Finally, it was named antimicrobial peptide W3L.

[0011] Another object of the present invention is to provide the use of the low-toxicity, cell-penetrating antimicrobial peptide W3L as described above in the preparation of a medicament for treating infectious diseases caused by Gram-positive and / or Gram-negative bacteria.

[0012] Furthermore, the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis, or Staphylococcus epidermidis.

[0013] Furthermore, the Gram-negative bacteria mentioned are Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, or Salmonella pullorum.

[0014] Another object of the present invention is to provide a medicament suitable for treating and / or preventing infections caused by Gram-positive and / or Gram-negative bacteria, said medicament containing the low-toxicity, cell-penetrating antimicrobial peptide W3L as described above.

[0015] This invention has the following advantages and beneficial effects: The antimicrobial peptide W3L of this invention exhibits good low-toxicity activity and intracellular antimicrobial activity, which is expected to solve the shortcoming of weak intracellular anti-infection ability in existing antimicrobial peptides. Antimicrobial activity, intracellular antimicrobial activity, hemolytic activity, cytotoxicity, and salt ion stability tests were performed on the antimicrobial peptide W3L of this invention. It was found that the antimicrobial peptide W3L has high inhibitory activity against several tested Gram-negative and Gram-positive bacteria, including *Escherichia coli*, *Pseudomonas aeruginosa*, *Salmonella typhimurium*, *Staphylococcus aureus*, *Enterococcus faecalis*, and *Staphylococcus epidermidis*. Simultaneously, the antimicrobial peptide W3L has a significant scavenging effect on *S. aureus* 43300 in RAW264.7, clearing 96.43% of the intracellular *S. aureus* 43300. The antimicrobial peptide W3L also has low toxicity and good biocompatibility, without causing significant hemolysis at 128 μM and without exhibiting significant cytotoxicity at 128 μM. In addition, the antimicrobial peptide W3L maintains a low MIC under different physiological concentrations of salt ions and serum environments, exhibiting high salt ion stability and serum stability. In summary, the antimicrobial peptide W3L of this invention has the potential to become a broad-spectrum antimicrobial agent for treating infections caused by Gram-positive and Gram-negative bacteria. Attached Figure Description

[0016] Figure 1 The reversed-phase high-performance liquid chromatogram of W3L, a low-toxicity cell-penetrating antimicrobial peptide mediated by arginine and anchored at the leucine terminus.

[0017] Figure 2 This is the mass spectrum of W3L, a low-toxicity, cell-penetrating antimicrobial peptide mediated by arginine and anchored at the leucine terminus.

[0018] Figure 3 The diagram shows the intracellular antimicrobial activity of the cell-penetrating antimicrobial peptide W3L.

[0019] Figure 4 The hemolytic activity of the cell-penetrating antimicrobial peptide W3L is shown in the graph.

[0020] Figure 5 This is a cytotoxicity diagram of the cell-penetrating antimicrobial peptide W3L. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings.

[0022] Example 1

[0023] Design of antimicrobial peptides

[0024] 1. First, using arginine as the core positively charged unit, and leveraging the high-density positive charge from its side-chain guanidinium group, the peptide binds to anionic phospholipids on the surface of bacterial and macrophage membranes via electrostatic interactions, triggering endocytosis and allowing the peptide to smoothly enter macrophages to clear intracellular Staphylococcus aureus. Second, tryptophan is interspersed between arginine residues. Utilizing the strong affinity of the tryptophan indole ring for the lipid bilayer, the peptide embeds into the membrane structure, synergistically enhancing the electrostatic adsorption effect of arginine, thus doubly improving the efficiency of the peptide in penetrating the host cell membrane and bacterial biofilm. Simultaneously, the hydrophobic ratio of the peptide is precisely controlled between 40% and 60%, and three consecutive leucine hydrophobic anchoring fragments are placed at the C-terminus of the peptide chain. Due to the mild embedding characteristics of leucine into the lipid bilayer, this approach balances potent intracellular antibacterial effects with low toxicity and excellent biocompatibility. Finally, the C-terminus of the peptide is modified with -NH2 amidation to increase the overall net positive charge of the peptide, enhancing its targeting selectivity for bacterial cell membranes and further strengthening its overall antibacterial activity, resulting in the antimicrobial peptide W3L. Its amino acid sequence is shown in SEQ ID No. 1. The sequence, molecular weight, and charge number of the antimicrobial peptide W3L are shown in Table 1.

[0025] Table 1. Sequence, molecular weight, and charge number of polypeptide W3L

[0026]

[0027] Example 2

[0028] Synthesis and Identification of Antimicrobial Peptides

[0029] The designed antimicrobial peptide was synthesized by Jier Biochemical (Shanghai) Co., Ltd. via solid-phase synthesis and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain the target compound, which was used for subsequent determination of antibacterial activity, intracellular antibacterial activity, hemolytic activity, cytotoxicity, and salt ion stability.

[0030] The reversed-phase high-performance liquid chromatogram of the antimicrobial peptide W3L is attached. Figure 1 .

[0031] The mass spectrum of the antimicrobial peptide W3L is attached. Figure 2 .

[0032] Example 3

[0033] Antimicrobial activity of antimicrobial peptides

[0034] The minimum inhibitory concentration (MIC) of the antimicrobial peptide was determined using the micro-broth dilution method. The designed and successfully synthesized antimicrobial peptide was dissolved in sterile ultrapure water in a sterile operating room to a concentration of 2.56 mM, obtaining a stock solution for subsequent bioactivity assays. Bacteria were cultured overnight at 37°C and 220 rpm on a shaker, then transferred to a new MHB culture medium until the logarithmic growth phase. The bacteria cultured to the logarithmic phase were then adjusted to OD using a UV-Vis spectrophotometer.600 nm =0.38 ~ 0.40 for later use. Dilute the prepared bacteria 1000 times, and add 50 μL to 50 μL of sterile 96-well plates containing different concentrations of antimicrobial peptides in BSA. Seal the plates tightly with sealing film to prevent bacterial contamination, and incubate at 37°C for 16-18 h. After incubation, the negative control wells remained clear and transparent, indicating that the experiment was uncontaminated. The turbidity of each well was observed visually and measured using a microplate reader at a wavelength of 492 nm. The lowest peptide concentration at which the turbidity did not increase compared to the negative control was defined as the MIC of the antimicrobial peptide. Three independent replicates were performed, with each replicate in triplicate. The minimum inhibitory concentration of the antimicrobial peptide is shown in Table 2.

[0035] Table 2 Antimicrobial activity of antimicrobial peptide W3L (μM)

[0036]

[0037] As can be seen from the table above, the MIC values ​​of the antimicrobial peptide W3L against all bacteria were 2 to 4 μM, demonstrating good broad-spectrum antimicrobial activity.

[0038] Example 4

[0039] Intracellular antimicrobial activity of antimicrobial peptides

[0040] The activity of antimicrobial peptides against intracellular bacteria was evaluated by constructing a *S. aureus* 43300 infected cell model. Cell culture medium was prepared at a ratio of high-glucose DMEM:fetal bovine serum:penicillin-streptomycin = 89:10:1. RAW264.7 cells were cultured in a 37°C incubator containing 5% CO2 until adherent growth was achieved, and then the cell density was adjusted to approximately 2 × 10⁶ cells / year. 5 Add 1 mL of cell suspension to each well of a 24-well plate and incubate overnight at 37°C in a 5% CO2 incubator. Then, culture the bacteria overnight at 37°C with shaking at 220 rpm, and transfer to fresh MHB. Culture until the logarithmic growth phase, then adjust the bacteria to OD. 600 nm = 0.38 ~ 0.40 for later use. Mix the prepared cells and bacteria at a volume ratio of 1:10, and add the sample to a 24-well plate. Incubate at 37°C in a 5% CO2 incubator for 1 h, then add gentamicin to a final concentration of 100 μg / mL and continue incubating for 1 h. Add antimicrobial peptide or vancomycin to the 24-well plate to a final concentration of 8-32 μM, and incubate at 37°C in a 5% CO2 incubator for 4 h. Discard the culture medium in the 24-well plate, wash 2-3 times with PBS, resuspend the cells in 1 mL of PBS, and then add 0.025% Triton X-100 to release intracellular bacteria from the cells. Dilute the above solution and spread it evenly on MHA plates. Incubate at 37°C for 18 h and count the colonies. Perform three independent replicate experiments.

[0041] like Figure 3 As shown, the antimicrobial peptide W3L has a significant scavenging effect on S. aureus 43300 in RAW264.7, and can remove 96.43% of S. aureus 43300 in the cells.

[0042] Example 5

[0043] Hemolytic activity of antimicrobial peptides

[0044] To assess the safety of antimicrobial peptides, the destructive behavior of peptides at concentrations ranging from 2 to 128 μM on human erythrocytes (hRBCs) was investigated. One mL of fresh blood was collected from healthy volunteers and stored in heparin sodium anticoagulant tubes. The blood was centrifuged at 3000 rpm for 10 min at 4 °C, the supernatant was discarded, and the blood was washed 2–3 times with PBS. The blood was then resuspended in 10 mL of PBS. 50 μL of the erythrocyte suspension was mixed with 50 μL of antimicrobial peptides at different concentrations, incubated at 37 °C for 1 h, and then centrifuged at 1000 rpm for 10 min at 4 °C. 70 µL of the supernatant was transferred to a new 96-well plate, and the absorbance was measured at 570 nm using a microplate reader. The average values ​​for each group were taken and compared. 50 μL of erythrocytes with 50 μL of PBS served as a negative control; 50 μL of erythrocytes with 50 μL of 0.1% Tritonx-100 served as a positive control. This experiment was repeated three times. Minimum hemolytic concentration (MHC) refers to the lowest peptide concentration that can cause 10% hemolysis of red blood cells. Test results are available in the appendix to the product manual. Figure 5 Biocompatibility of antimicrobial peptides was evaluated by the minimum concentration (MHC) at which they caused 10% hemolysis of human erythrocytes. The therapeutic potential was further evaluated by calculating the antimicrobial peptide therapeutic index (SI). The results are shown in Table 3.

[0045] Table 3 Hemolytic activity of antimicrobial peptide W3L

[0046]

[0047] a The geometric mean (GM) of the minimum inhibitory concentration of antimicrobial peptides against bacteria was used to calculate the therapeutic index when no detectable antimicrobial activity was observed at 64 µM.

[0048] b MHC is the lowest concentration of antimicrobial peptide that causes 10% hemolysis of human red blood cells (hRBCs). When no detectable hemolytic activity is observed at 128 μM, 256 μM is used to calculate the therapeutic index.

[0049] c SI is calculated as MHC / GM. The higher the therapeutic value, the greater the therapeutic potential.

[0050] Included in the instruction manual Figure 4 It can be seen that antimicrobial peptide W3L did not cause hemolysis at the highest concentration tested (hemolysis rate less than 10%). The minimum hemolytic concentration of antimicrobial peptide W3L is much higher than its minimum inhibitory concentration, indicating that antimicrobial peptide W3L has high biocompatibility while exerting antimicrobial activity.

[0051] Example 6

[0052] Cytotoxicity of antimicrobial peptides

[0053] The cytotoxicity of antimicrobial peptides was determined using the MTT assay, with RAW264.7 macrophages selected for the assay.

[0054] Prepare high-glucose DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-antibiotics, and store at 4°C for later use. Before use, place the medium in a 37°C water bath to bring it to room temperature. Seed cells frozen in liquid nitrogen into the appropriate complete medium after a water bath for thawing, and then incubate in a CO2 incubator. Observe under a microscope; when the cells cover more than 80% of the bottom of the cell culture flask, the next passage can be performed. After RAW264.7 cells are cultured in high-glucose DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-antibiotics until they cover 80% of the bottom of the culture flask, discard the medium and wash the cells 2-3 times with PBS. Add fresh complete medium and gently tap until the cells are completely detached to prepare a cell suspension. Add 50 µL of the cell suspension to each well in columns 1-11 of a 96-well plate, 3-5 × 10⁶ cells per well. 5 Cells were incubated overnight at 37°C in a 5% CO2 incubator. A new sterile 96-well plate was used, and the antimicrobial peptide was serially diluted with complete culture medium to the 10th column. 50 µL of each concentration of antimicrobial peptide was extracted and added to columns 1-10 of the 96-well plate containing cell suspension. 50 µL of complete culture medium was added to column 11 as a positive control, and 100 µL of complete culture medium was added to column 12 as a negative control. The 96-well plate was incubated for another 4 h in a 5% CO2 incubator. 50 µL of 5 mg / mL MTT solution was added to each well of the 96-well plate, and the plate was incubated for another 3 h in a 5% CO2 incubator. All liquid in each well was aspirated and discarded. 100 µL of DMSO was added to each well to fully dissolve the purple crystals, and the absorbance was measured at 570 nm. This experiment was independently repeated three times. The results are shown below. Figure 5 .

[0055] Included in the instruction manual Figure 5 It can be seen that the cell survival rate of the antimicrobial peptide W3L was higher than 80% in the concentration range of 1 ~ 128 μM, and it did not show obvious cytotoxicity.

[0056] Example 7

[0057] Salt ion stability of antimicrobial peptides

[0058] The MIC values ​​of antimicrobial peptides under different physiological concentrations of salt ions were determined using a *S. aureus* 43300 spectrometer. Different concentrations of salt ions were dissolved in 0.2% BSA (containing 0.01% glacial acetic acid) solution: NaCl 300 mM, KCl 9 mM, CaCl2 5 mM, MgCl2 2 mM, NH4Cl 12 μM, ZnCl2 16 μM, and FeCl3 8 μM. The MICs of the antimicrobial peptides in different salt ions were determined using the microbroth dilution method. This experiment was independently repeated three times. The results are shown in Table 4.

[0059] Table 4 Salt ion stability of antimicrobial peptide W3L

[0060]

[0061] As can be seen from Table 4, the MIC values ​​of antimicrobial peptide W3L against S. aureus 43300 under physiological saline conditions range from 2 µM to 4 µM, exhibiting strong salt ion stability.

Claims

1. A low-toxicity, cell-penetrating antimicrobial peptide, W3L, characterized in that: Its amino acid sequence is shown in SEQ ID No.1, and its C-terminus is amidated with -NH2.

2. The low-toxicity, cell-penetrating antimicrobial peptide W3L as described in claim 1, characterized in that, Its molecular formula is shown in formula (I). Formula (I).

3. The method for preparing a low-toxicity, cell-penetrating antimicrobial peptide W3L as described in claim 1, characterized in that, The steps are as follows: Step 1: Insert tryptophan between two arginine residues and repeat three times to form a peptide chain with the sequence RWRRWRRWR. Then, link three consecutive leucine residues to the C-terminus of the peptide chain to obtain polypeptide W3L, whose sequence is shown in SEQ ID No.

1. Finally, amidation is performed on the C-terminus of polypeptide W3L with -NH2, resulting in a net charge of +7. Step 2: The polypeptide W3L was synthesized using a solid-phase chemical synthesis method. After purification by reversed-phase high-performance liquid chromatography and identification by mass spectrometry, the polypeptide was subjected to antibacterial activity testing, intracellular antibacterial activity testing, hemolytic activity testing, cytotoxicity testing, and salt ion stability testing. Finally, it was named antimicrobial peptide W3L.

4. The use of the low-toxicity, cell-penetrating antimicrobial peptide W3L as described in claim 1 in the preparation of a medicament for treating infectious diseases caused by Gram-positive and / or Gram-negative bacteria.

5. The application according to claim 4, characterized in that: The Gram-positive bacteria mentioned are Staphylococcus aureus, Enterococcus faecalis, or Staphylococcus epidermidis.

6. The application according to claim 4, characterized in that: The Gram-negative bacteria mentioned are Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, or Salmonella pullorum.

7. 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 low-toxicity, cell-penetrating antimicrobial peptide W3L as described in claim 1.

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