CN114315971A

CN114315971A - Preparation of antibacterial lipopeptide LP21 and application thereof in treatment of bacterial infection

Info

Publication number: CN114315971A
Application number: CN202111411195.5A
Authority: CN
Inventors: 王春华; 魏光成; 王浩
Original assignee: Binzhou Medical College
Current assignee: Binzhou Medical College
Priority date: 2021-11-25
Filing date: 2021-11-25
Publication date: 2022-04-12

Abstract

The invention relates to preparation of an artificially synthesized antibacterial lipopeptide LP21 and application thereof in treating bacterial infection, belonging to the field of biological medicine. The sequence of the antibacterial lipopeptide from the N end to the C end is C₈H₁₅O‑WKKLLKWWLKKFKKLD. The antimicrobial lipopeptides are prepared based on key biological characteristics of antimicrobial peptides (positive charge, alpha-helical structure and amphiphilicity). The invention provides a solid-phase synthesis method of the antibacterial lipopeptide. The antibacterial lipopeptide disclosed by the invention has excellent antibacterial activity (the MIC values of escherichia coli ATCC25922 and staphylococcus aureus ATCC29213 are 3 mu M and 0.2 mu M respectively), and has good serum stability and biocompatibility. In vivo antibacterial experiments show that the antibacterial lipopeptide LP21 shows excellent antibacterial efficacy in a mouse in vivo bacterial infection model, is an antibiotic substitute with application potential, and has good application prospects.

Description

Preparation of antibacterial lipopeptide LP21 and application thereof in treatment of bacterial infection

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to preparation of a synthetic cationic amphiphilic antibacterial lipopeptide LP21 with excellent antibacterial activity and application of the synthetic cationic amphiphilic antibacterial lipopeptide LP21 in treatment of bacterial infection.

Background

Since the discovery of penicillin, antibiotics have been widely used globally, and have played an important role in the prevention and treatment of human diseases. However, with the abuse of antibiotics, many bacteria generate drug resistance, which poses a great threat to human health, and the problem of drug resistance becomes a big problem in the current medical field. Therefore, the search for new antibacterial agents is urgent. One of the effective methods for solving the drug resistance of bacteria is to design a novel antibacterial peptide. Antimicrobial peptides are the earliest immunologically active molecules produced by organisms in order to adapt to the environment during long-term evolution, and play an extremely important role in the host's immune defense against pathogen invasion. Natural antimicrobial peptides can exist in almost any life form, where most antimicrobial peptides fold from hydrophilic and hydrophobic residues in the membrane environment into an alpha-helical structure with amphiphilicity and membrane disruption. Traditional antibiotics cause bacterial death by acting on specific bacterial targets, while most of antibacterial peptides exert antibacterial activity through non-receptor-mediated membrane destruction, have unique action mechanism and are not easy to generate drug resistance. Antibiotics are only applicable to bacterial infection, and antibacterial peptides can be used for resisting bacteria, viruses and fungi, so that the antibacterial peptides are used as potential candidate drugs for treating various diseases and have wide application prospects.

Several biological characteristics of natural antimicrobial peptides (e.g., positive charge, alpha-helical structure, and amphiphilicity) are associated with antimicrobial activity. A large number of researches show that the biological activity of the antibacterial peptide is closely related to the number of positive charges, and the proper increase of the number of the positive charges can not only improve the binding capacity of the antibacterial peptide and a bacterial cell membrane, but also effectively reduce the bactericidal concentration of the antibacterial peptide. The alpha-helix secondary structure also plays an important role in maintaining the bioactivity of the antibacterial peptide, the helicity of the antibacterial peptide is beneficial to helping the antibacterial peptide to penetrate the membrane, and when the helical structure is damaged, the membrane penetrating efficiency is obviously reduced. The amphiphilicity of the antimicrobial peptides is also one of the important factors affecting antimicrobial activity. Most antimicrobial peptides exhibit amphiphilicity, i.e., an amphiphilic structure containing hydrophilic and hydrophobic surfaces, which facilitates binding to bacterial membranes. Too low hydrophobicity can cause the affinity of the antibacterial peptide and the bacterial cell membrane to be too low to be combined; too high hydrophobicity tends to cause the antimicrobial peptide to self-aggregate, resulting in not only decreased solubility of the antimicrobial peptide molecule but also increased hemolytic activity thereof. Therefore, designing an antibacterial peptide having a positive charge and an α -helix structure while inserting a hydrophobic amino acid residue to the hydrophilic side or a hydrophilic residue to the hydrophobic side of the α -helix antibacterial peptide can minimize hemolysis and exert optimal antibacterial performance.

Lipopeptides are composed of two parts, hydrophilic peptides and hydrophobic aliphatic hydrocarbon chains, have stronger membrane affinity and broad-spectrum antimicrobial activity, and are considered as very promising antibacterial drugs in the post-antibiotic age. The aliphatic chain modification of the antibacterial peptide is also an important means for solving the problem of poor stability of the antibacterial peptide. Although the antibacterial peptide has more advantages, a plurality of limiting factors exist in the research and development, for example, the antibacterial peptide extracted from natural biological resources has low content and is difficult to separate and purify, so that the production cost is high and the large-scale production is lacked; most of natural antibacterial peptides have longer peptide chains and higher lyso-1 blood page activity, and have lower clinical use value. Therefore, there is a need for molecular design, structural modification and modification of antimicrobial peptides to improve their antimicrobial activity while reducing hemolysis and cytotoxicity. For the design and modification of antibacterial peptide molecules, the substitution or deletion of natural amino acid residues, the combination and cyclization of fragments of the antibacterial peptide and the design of brand new antibacterial peptide from several biological characteristics of the antibacterial peptide are common. Therefore, the present invention has completely new design and artificial synthesis of novel cationic amphiphilic antibacterial lipopeptide LP21 with excellent antibacterial activity based on the biological characteristics (positive charge, alpha-helix structure and amphipathy) of natural antibacterial peptide. The bacterial cell membrane with negative charge is electrostatically attracted by providing positive charge through the introduction of the basic amino acid lysine (Lys), and the formation of bacterial membrane pores is promoted through the hydrophobic interaction between the hydrophobic amino acid leucine (Leu), tryptophan (Trp) and the fatty hydrocarbon chain of fatty acid and bacterial phospholipid, so that the antibacterial activity is generated, and the support is provided for the research on the action mechanism of the antibacterial peptide and the application of the antibacterial peptide. The inventor searches and aligns the full-sequence amino acid structure of the antibacterial lipopeptide of the invention through NCBI protein database, and does not find any identical polypeptide. The antibacterial lipopeptide can provide thought and theoretical basis for the treatment of bacterial infection, and provides an effective way for the design of antibacterial drugs for replacing antibiotics.

Disclosure of Invention

One of the objectives of the present invention is to provide a cationic amphiphilic antimicrobial lipopeptide LP21 with excellent antimicrobial activity based on the biological characteristics of natural antimicrobial peptides and solid phase peptide synthesis technology. The second purpose of the invention is to apply the antibacterial lipopeptide to the preparation of medicines for treating bacterial infection.

In order to realize the purpose of the invention, the invention provides the following technical scheme:

the amino acid sequence of the cationic amphiphilic antibacterial lipopeptide LP21 is C₈H₁₅O-Trp-Lys-Lys-Leu-Leu-Lys-Trp-Trp-Leu-Lys-Lys-Phe-Lys-Lys-Leu-Asp (abbreviated as C)₈H₁₅O-WKKLLKWWLKKFKKLD), wherein Trp is tryptophan, Lys is lysine, Leu is leucine, Phe is phenylalanine, Asp is aspartic acid, and the fatty acid connected is caprylic acid C₈H₁₆O₂. The design and preparation method comprises the following steps: (1) first, positively charged Lys and hydrophobic amino acids Leu and Trp are designed according to biological characteristics (positive charge, alpha-helix structure and amphipathy) of natural antibacterial peptide, and C is coupled₈H₁₆O₂Obtaining the antibacterial lipopeptide; (2) the solid phase polypeptide synthesis technology has the advantages of simple process, low cost and the like. According to the designed antibacterial lipopeptide sequence, the invention adopts a solid-phase synthesis method to carry out artificial synthesis. The technical scheme is summarized as follows: coupling protected amino acids on 2-chlorotritylechlorideresin one by one from C end to N end according to a pre-designed sequence and deprotecting to obtain polypeptide resin, and then reacting the polypeptide resin with N-caprylic acid C₈H₁₆O₂Coupling to obtain lipopeptide resin, and finally cutting the desired lipopeptide from the resin. The structural formula of the antibacterial lipopeptide is as follows:

carrying out in-vitro antibacterial activity detection on the prepared antibacterial lipopeptide LP21 by adopting a trace broth dilution method; determining the secondary structure of the resulting antimicrobial lipopeptide LP21 using circular dichroism spectroscopy; the serum stability and biocompatibility of the prepared antibacterial lipopeptide LP21 are researched; studying the antibacterial mechanism of the antibacterial lipopeptide LP 21; finally, the antibacterial activity of antibacterial lipopeptide LP21 in vivo was evaluated.

Drawings

Table 1: antibacterial lipopeptide LP21 in vitro antibacterial activity.

FIG. 1: CD spectra of antimicrobial lipopeptide LP21 in pure water and SDS.

Table 2: MIC of antimicrobial lipopeptide LP21 in 12.5% FBS.

FIG. 2: biocompatibility of antimicrobial lipopeptide LP 21. (a) Hemolytic activity of erythrocytes in mice of the NS group. (b) Hemolytic Activity of erythrocytes in mice of the antimicrobial lipopeptide LP21(8mg/kg) group. (c) Relative cell viability of RAW264.7 cells treated with varying concentrations of antimicrobial lipopeptide.

FIG. 3: bacterial biofilm clearance and bacterial membrane permeability assays. (a) The effect of varying concentrations of antimicrobial lipopeptide LP21 on the treatment of mature biofilms of staphylococcus aureus ATCC 29213; (b) coli ATCC25922 was evaluated for outer membrane permeability using an NPN uptake assay; (c) assessing the inner membrane permeability of escherichia coli ATCC25922 using PI; (d) the inner membrane permeability of staphylococcus aureus ATCC29213 was evaluated using PI.

FIG. 4: TEM and SEM images of E.coli ATCC 25922. (a) TEM images of the control group; (b) TEM images of 2h treatment with 2 × MIC antimicrobial lipopeptide LP 21; (c) SEM images of control group; (d) SEM images of 2h treatment with 2 x MIC antimicrobial lipopeptide LP 21.

FIG. 5: coli ATCC25922 cells were treated with 2 xmic antimicrobial lipopeptide LP21 for 0h, 2h, 4h, 6h of membrane damage. Flow cytometry was used to determine the fluorescence intensity of PI (25. mu.g/mL).

FIG. 6: fluorescence images (20X) of E.coli ATCC259220h, 4h, 8h, 12h were treated with antimicrobial lipopeptide LP 21-fluorescein (4 × MIC).

FIG. 7: in vivo antibacterial activity in mice. (a) Photographs of dorsal wounds of mice treated with antimicrobial lipopeptide LP21 at various times following infection with staphylococcus aureus ATCC 29213. (b) Histological analysis of wound tissue sections stained with H & E after day 10 of antimicrobial treatment. Blue arrow: lymphocytes; red arrow: a blood vessel; gray arrow: a fibroblast. (c) Photographs of mouse eye wounds (obtained from slit lamps) at various times after infection with staphylococcus aureus ATCC29213 were treated with antimicrobial lipopeptide LP 21. (d) Survival of acute peritonitis mice treated with antimicrobial lipopeptide LP21 at a dose of 8 mg/kg. (e) After various times of treatment with the antimicrobial lipopeptides, the mice were subjected to bacterial counts in the peritoneal fluid.

Detailed Description

The following is a description of the present invention for further explanation of its constitution, but the present invention is not to be construed as being limited to the following embodiments.

Example 1: the preparation method of the antibacterial lipopeptide LP21 comprises the following steps:

full sequence of antibacterial lipopeptide: c₈H₁₅O-Trp-Lys-Lys-Leu-Leu-Lys-Trp-Trp-Leu-Lys-Lys-Phe-Lys-Lys-Leu-Asp. This example was synthesized by solid-phase peptide synthesis from C-terminal to N-terminal, wherein the amino acids with protecting groups were Fmoc-Trp (Boc) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Asp (OtBu) -OH. The specific experimental steps are as follows:

(1) synthesis of Fmoc-Trp (Boc) -Lys (Boc) -Leu-Leu-Lys (Boc) -Trp (Boc) -Leu-Lys (Boc) -Phe-Lys (Boc) -Lys- (Boc) Leu-Asp (OtBu) -2-chlorotritylchloride resin polypeptide resin: weighing a certain amount of Fmoc-Asp (OtBu) -2-chlorotritylicloderesin (load: 0.402mmol/g), and swelling with DMF for 30 min; after swelling was complete, the Fmoc protecting group was removed with 20% piperidine in DMF, reacted for 30min, and then washed three times with DMF, DCM and DMF in that order. The indetrione method detects that the product is blue-purple, which indicates that the deprotection reaction is completely carried out. And re-swelling the deprotected resin by using anhydrous DMF for 15min, adding 2 times of molar weight of second amino acid Fmoc-Leu-OH, 2.6 times of molar weight of HOBT, DCC and DIEA, stirring for reacting for 48h, and detecting whether the resin is colorless by an indetrione method, wherein the coupling reaction is complete. Suction filtering the resin, washing with DMF, DCM and DMF several times, filling the resin into a dialysis bag (MW8000-14000), dialyzing and purifying with ethanol, suction filtering, freeze drying to obtain Fmoc-Leu-Asp (OtBu) -2-chlorotritylicloritriederesin, repeating the steps, and coupling one by one from C end to N end until the polypeptide resin is synthesized.

(2)C₈H₁₅Synthesis of O-Trp-Lys-Lys-Leu-Leu-Lys-Trp-Trp-Leu-Lys-Lys-Phe-Lys-Lys-Leu-Asp antimicrobial lipopeptide: removing Fmoc protecting group of the polypeptide resin obtained in (1), and adding 2 times molar amount of C₈H₁₆O₂With 2.6 times the molar amount of EDC and NHS dissolved in DMF to activate C₈H₁₆O₂And (2) adding the polypeptide resin obtained in the step (1) into the DMF solution, stirring for reaction for 24 hours, performing suction filtration, filling the resin into a dialysis bag (MW8000-14000), performing dialysis purification by using ethanol, performing suction filtration, and performing freeze drying to obtain the lipopeptide resin with side chain protecting groups. Cleaving all side chain protecting groups and 2-chlorotriederesin of the lipopeptide resin with cleavage solution trifluoroacetic acid/triisopropylsilane/water (95: 2.5: 2.5, V/V/V), filtering to remove resin, concentrating the cleavage solution, precipitating with glacial ethyl ether and washing multiple times, and finally obtaining the antibacterial lipopeptide by freeze-drying.

Example 2: in vitro antimicrobial activity assay for antimicrobial lipopeptide LP 21:

the Minimum Inhibitory Concentration (MIC) of the antimicrobial lipopeptide was determined by the broth dilution method. Inoculating single colony of purified Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213 into fresh LB culture medium, culturing at 37 deg.C overnight, transferring to new LB culture medium, culturing for 4-6h until growth reaches logarithmic phase, washing with PBS, resuspending bacteria, and making into a concentration of-10⁵CFU/mL of bacterial suspension. Antimicrobial lipopeptides were prepared in sterile water at varying concentrations (final concentration 1. mu.M-128. mu.M) after dilution in multiple proportions and transferred to sterilized 96-well polystyrene plates (50. mu.l/well) and the same volume of bacterial suspension was added to each well to 100. mu.l/well and incubated for 12-16 h. After a prescribed time, the minimum inhibitory concentration was determined as the concentration at which no turbidity was observed at the bottom of the well by visual observation. Experiments were only significant when significant bacterial growth was observed in positive control wells (i.e., containing no antimicrobial lipopeptides).

Example 3: secondary structure of antibacterial lipopeptide LP 21:

the secondary structure of antimicrobial lipopeptide LP21 was determined by circular dichroism (CD spectroscopy). LP21 was dissolved in purified water to prepare a 150. mu.M peptide solution. Meanwhile, LP21 was dissolved in a 30mM Sodium Dodecyl Sulfate (SDS) solution to prepare a peptide solution having a concentration of 150. mu.M. The prepared antimicrobial lipopeptide LP21 solution was scanned at room temperature over the wavelength range of 180-260nm to obtain its CD spectrum. The obtained spectra were then converted to average residue ellipticity using the following equation:

θ_M＝(θobs×1000)/cln

wherein theta is_MIs the average residue ovality (deg.cm)²·dmol^-1) θ obs is the ellipticity (mdeg) observed at a given wavelength after calibration with buffer, c is the concentration of antimicrobial lipopeptide (mM), l is the path length (mM), and n is the number of amino acids of antimicrobial lipopeptide LP 21.

Example 4: serum stability of antibacterial lipopeptide LP 21:

varying concentrations of antimicrobial lipopeptide LP21 were mixed in equal volumes with an initial concentration of 50% serum (FBS) and incubated at 37 ℃ for 0.5 h. After these pretreatments, the MIC values of the antimicrobial lipopeptides were determined (MIC determination was the same as in example 2), to determine their serum stability.

Example 5: biocompatibility of antimicrobial lipopeptide LP 21:

biocompatibility was studied by measuring hemolysis and cytotoxicity of antimicrobial lipopeptide LP21 on mammalian cells.

Two male mice (23-25g) were selected in the hemolytic assay and the antimicrobial lipopeptide (8mg/kg) was injected into the mice via the tail vein. Two drops of blood were collected after 2 hours by orbital bleeding of mice, then the blood was diluted with physiological saline (NS) and the red blood cell morphology was observed under a fluorescent microscope.

Cytotoxicity of antimicrobial lipopeptide LP21 on human embryonic kidney cells RAW264.7 cells was determined by using the CCK-8 method. Cells (1X 10) in DMEM medium (supplemented with 10% fetal bovine serum)⁴Individual cells/well) were seeded into 96-well plates, then contained 5% CO at 37 ℃₂IncubatorAnd incubated overnight. The antibacterial lipopeptide is diluted in multiple proportions, added to a cell culture at a final concentration of 1-128 mu M, and incubated for 24 hours. After the specified time had been reached, the cell culture was incubated with CCK-8 (10. mu.L) for 2 hours at 37 ℃ and the absorbance was measured at 450nm using a microplate reader to calculate the cell viability of RAW264.7 cells and thereby determine the cytotoxicity of antimicrobial lipopeptide LP 21.

Example 6: membrane disruption of antimicrobial lipopeptide LP 21:

the inhibitory effect of antimicrobial lipopeptide LP21 on mature bacterial biofilms was determined by crystal violet staining. A single colony of Staphylococcus aureus ATCC29213 was picked from the dish and added to LB medium for overnight incubation. The bacterial solution (100. mu.l/well) was transferred to a 96-well plate and incubated for 72 hours, and planktonic bacteria were removed and washed twice with PBS. LP21 was diluted in multiple ratios, added to a 96-well plate (100. mu.l/well) at a final concentration of 1-64. mu.M as a test group, containing neither bacterial solution nor antimicrobial lipopeptide as a negative control, containing bacterial solution but not antimicrobial lipopeptide as a positive control, and incubated at 37 ℃ for 24 hours. The supernatant was removed and the biofilm was washed twice with PBS. Methanol (100 μ l) was added to each well and fixed for 15 min. The methanol was removed, air dried, stained with crystal violet (0.1%, 100 μ l) for 10min, washed 5 times with PBS and dried at room temperature. Ethanol (95%, 100 μ l) was added to each well and shaken slightly several times to allow the biofilm to completely release crystal violet. The absorbance was measured at 595nm with a multifunctional microplate reader and the bacterial biofilm eradication rate was calculated using the following formula.

Biofilm eradication rate (%) - (a)₁₀₀―A)/(A₁₀₀―A₀)]×100％

A represents the absorbance of antimicrobial lipopeptide LP21 at a given concentration; a. the₀Represents the absorbance of the negative control; a. the₁₀₀The absorbance of the positive control group is shown.

The outer membrane permeability of antimicrobial lipopeptide LP21 was determined using the fluorescent dye 1-N-phenylnaphthylamine (NPN). Coli was cultured in LB at 37 ℃ overnight, cultured in fresh LB for 3-6h, and bacterial cells were centrifuged at 3600rpm, washed 3 times with PBS, and then resuspended in PBS to obtain OD₆₀₀The strain was 0.5. NPN (final concentration of 10. mu.M) was added to the quartz ratioBackground fluorescence was recorded in the bacterial suspension in the cuvette using a fluorescence spectrophotometer. Different concentrations of antimicrobial lipopeptides were added to the cuvettes and changes in fluorescence intensity were recorded until no further increase in fluorescence intensity was observed (excitation λ 350nm, emission λ 420 nm). After the antibacterial lipopeptide is added, NPN is doped into a bacterial membrane to increase the fluorescence intensity. Data collected over 10 minutes were averaged. The percentage of NPN uptake was calculated by the following equation:

NPNuptake(％)＝(Fobs-F₀)/(F₁₀₀-F₀)×100％

wherein Fobs is the fluorescence observed at a given peptide concentration, F₀Representing the initial fluorescence in the absence of peptide, F₁₀₀Represents the fluorescence upon addition of polymyxin B (10. mu.g/mL) and was used as a positive control.

The ability of antimicrobial lipopeptide LP21 to penetrate the inner membrane was evaluated using the propidium iodide uptake assay. Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213 were cultured in LB at 37 ℃ overnight, and then re-cultured in fresh LB to obtain OD₆₀₀Strain 0.35. Bacterial cells were centrifuged at 3600rpm, washed 3 times with PBS, and resuspended in PBS. Respectively incubating the bacterial suspension with antibacterial lipopeptide solutions (1-64 mu M) with different concentrations after dilution in multiple proportions at 37 ℃ for 60min, then adding Propidium Iodide (PI) into the suspension, and respectively measuring the fluorescence intensity at the excitation wavelength of 520nm and the emission wavelength of 620nm by using a multifunctional microplate reader.

Morphological and intracellular changes of bacterial cells treated with antimicrobial lipopeptide LP21 were observed using Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). Coli ATCC25922 was cultured to logarithmic growth phase, then suspended in PBS and adjusted to about 5X 10⁷CFU/mL, incubated with 2 × MIC of antimicrobial lipopeptide for 120 min, and negative control with no antimicrobial lipopeptide. After the prescribed time, E.coli was centrifuged at 3500rpm for 5min, and bacterial cell pellets were collected and washed 3 times with PBS. The bacterial cell particles were fixed with 2.5% (v/v) glutaraldehyde overnight at 4 ℃, washed 5 times with PBS, fixed 90min with 1% osmic acid and washed 3 times with PBS. In fractionated ethanol (50%, 70%, 80%, 95%, 100%) each 15m was dehydratedin, treatment with acetone 2 times, then treatment with acetone and penetrant 3h, and soaking with pure penetrant overnight. Bacterial samples were embedded and placed at 37 ℃ for 12h, 45 ℃ for 12h, and 60 ℃ for 24 h. After the specified time, bacterial sections were prepared using a microtome, placed on a copper mesh with a carbon support film, stained with uranyl acetate and lead citrate, and finally observed by TEM. After the observation by TEM, the copper mesh was sprayed with gold for SEM observation.

The uptake of antimicrobial lipopeptide LP21 by E.coli ATCC25922 was quantitatively assessed by Flow Cytometry (FCM). Coli ATCC25922 was incubated with 4 × MIC final concentration of antimicrobial lipopeptide at 37 ℃ for 2h, 4h, 6h, centrifuged, washed three times with PBS, incubated with PI (final concentration 25 μ g/mL) for 20min at room temperature, washed with PBS to remove unbound dye, and resuspended in 400uL of PBS. FCM was used for detection at 488nm excitation wavelength and the results were recorded.

To further investigate the distribution of antimicrobial lipopeptide LP21 in bacterial cells, E.coli ATCC25922 was incubated with antimicrobial lipopeptide-fluorescein and observed by laser scanning confocal microscopy (CLSM). Escherichia coli (OD)₆₀₀0.5) was incubated with antimicrobial lipopeptide-fluorescein (4 × MIC) at 37 ℃. After incubation for 2h, 4h, 6h, the bacterial cells were centrifuged at 3600rpm for 5min, and the cell pellet was collected and washed three times with PBS. Propidium iodide PI (final concentration 25ug/mL) was then added and incubated for 20min, washed in PBS to remove unbound dye and resuspended. Smear, images were captured with CLSM and fluorescence signal distribution was observed.

Example 7: in vivo antibacterial experiments

Normal male mice with an average body weight of 25g were used in vivo experiments.

Mouse back infection model: the in vivo antimicrobial efficacy of antimicrobial lipopeptide LP21 was evaluated using a mouse back wound model infected with staphylococcus aureus ATCC 29213. Mice were anesthetized with an intraperitoneal injection of 4% chloral hydrate (0.01 mL/g). Wounds were first created on the dorsal surface of mice with a 6mm biopsy punch and infected with Staphylococcus aureus ATCC29213 (1X 10)⁸CFU/wound), an infection model is established. After 24 hours of modeling, the antimicrobial lipopeptide solution (20 μ L,0.004g/ml) was dropped onto the wound site once a day and the wound healing was recorded with a camera. 10 days after administration, the skin at the wound site was fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned, and finally stained with Hematoxylin and Eosin (HE). The pathological sections were observed with a microscope.

Mouse eye infection model: culturing Staphylococcus aureus ATCC29213 on agar plate at 37 deg.C for 24h, separating single colony, and adjusting bacterial liquid concentration to-10 with PBS⁷CFU/mL, for corneal infection in mice. All mice received slit lamp examination prior to infection to ensure that the cornea was normal. Mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.01mL/g), followed by a drop of 1% lidocaine hydrochloride as local anesthesia and corneal irrigation with sterile NS. A suspension (15. mu.L) of Staphylococcus aureus ATCC29213 was applied to the corneal surface by scratching the cornea with a sterile blade. 12h after infection, antimicrobial lipopeptide (15uL, 0.003g/mL) was dropped onto the corneal surface once a day for 3 consecutive days. Mice were observed daily for ocular infection and recovery with slit lamps.

Mouse peritonitis model: by suspending E.coli ATCC25922 (200. mu.L, 10. mu.L)⁸CFU/mL) was injected into the abdominal cavity of a mouse to establish a mouse peritonitis model. If the model is successfully constructed, the mouse will expel mucous stool, mental depression, rolling, and messy fur. Following successful modeling, antimicrobial lipopeptide LP21 (200. mu.L, 0.001g/mL, 8mg/kg) was injected intraperitoneally into mice in the experimental group, while the control group was injected with the same volume of NS. Mice were euthanized at different time points (12h, 24h, 48h), and then the abdominal cavity of the mice was rinsed with 1mL of sterile NS and 200 μ L of peritoneal fluid was withdrawn. After dilution, the plates were spread on agar plates and incubated at 37 ℃ for 24 h. After the specified time, the number of colonies in each plate was counted, and then the total number of CFUs per ml was calculated to evaluate the antibacterial activity in vivo.

The antibacterial lipopeptide LP21 prepared by the invention has good biomedical performance:

(1) the antibacterial lipopeptide LP21 has excellent in vitro antibacterial activity

In vitro antibacterial activity was determined by determining the MIC values of antimicrobial lipopeptide LP21 against e.coli ATCC25922 and s.aureus ATCC 29213. As shown in Table 1, the MIC values for the antimicrobial lipopeptides of the present invention were small for both bacteria, indicating that the antimicrobial lipopeptide LP21 has strong in vitro antimicrobial activity.

(2) The antibacterial lipopeptide LP21 has an alpha-helical structure in a membrane simulation environment

The alpha-helix secondary structure plays an important role in maintaining the bioactivity of the antibacterial peptide. Here, the secondary structure of antimicrobial lipopeptides in aqueous solution and in a membrane-simulated environment SDS was analyzed using CD spectroscopy (fig. 1). The results showed that the antimicrobial lipopeptides showed a negative band at about 200nm in purified water, indicating that their structure in purified water is random. However, the CD spectrum of the antimicrobial lipopeptide in 30mM DS solution showed two strong negative bands at approximately 208nm and 220nm and a positive band near 195nm, indicating that the antimicrobial lipopeptide exhibits an alpha-helical structure under membrane mimetic conditions. These results suggest that the alpha-helical structure of the antimicrobial lipopeptides may play a key role in their antimicrobial activity.

(3) The antibacterial lipopeptide LP21 has good serum stability and biocompatibility

We investigated the serum stability of the antimicrobial lipopeptides by measuring their antimicrobial activity after treatment with serum. As can be seen from Table 2, the MIC of the antimicrobial lipopeptide to Escherichia coli ATCC25922 after the antimicrobial lipopeptide is incubated with 50% FBS in equal volume is 8 mu M, which shows that the antimicrobial lipopeptide can still maintain good antimicrobial activity in serum, and thus the antimicrobial lipopeptide has good serum stability.

Hemolytic activity and cytotoxicity are important indicators for verifying drug safety and cell selectivity. We analyzed the hemolytic activity by the morphology of erythrocytes under a fluorescence microscope (fig. 2a, b). FIG. 2a shows the morphology of erythrocytes in NS group and FIG. 2b shows the morphology of erythrocytes in antibacterial lipopeptide group, and it can be seen that the morphology of erythrocytes in NS group and antibacterial lipopeptide group is normal, and no hemolysis is observed. This indicates that the antimicrobial lipopeptides are not hemolytic to normal cells.

The CCK-8 method is adopted to detect the cytotoxicity of the antibacterial lipopeptide on human embryonic kidney cells RAW264.7 cells. As can be seen from FIG. 2c, the relative cell viability of RAW264.7 cells was above 80% at concentrations of antimicrobial lipopeptide below 32. mu.M, indicating that the toxicity of antimicrobial lipopeptide on RAW264.7 cells was very low below 32. mu.M. However, when the concentration is higher than 64 μ M, cytotoxicity is drastically increased, and the relative survival rate of RAW264.7 cells is less than 30%. This indicates that the antimicrobial lipopeptides have low cytotoxicity to mammalian cells, i.e., selectivity for bacterial cells, over a range of concentrations. The above results show that antimicrobial lipopeptides at concentrations below 32. mu.M are highly biocompatible with mammalian cells.

(4) The antibacterial lipopeptide LP21 has membrane breaking mechanism and high antibacterial efficiency

The ability of the antimicrobial lipopeptides to disrupt the mature biofilm formed by staphylococcus aureus ATCC29213 was studied by crystal violet staining. First, mature biofilms formed after four days of culture from staphylococcus aureus ATCC29213 were established in advance to evaluate the ability of antimicrobial lipopeptides to disrupt biofilms. Figure 3a shows that after 24h treatment with antimicrobial lipopeptide, biofilm was greatly reduced in a concentration dependent manner and significant destruction (> 85%) of the biofilm was observed at 16 μ M antimicrobial lipopeptide. The dose-dependent effect of bacterial biofilms on antimicrobial lipopeptides suggests that the lipopeptides are able to penetrate extracellular polymeric matrix barriers, disrupting mature biofilms.

The outer membrane is important for gram-negative bacteria and plays an important role in protecting the bacterial cells without affecting the exchange of substances required to sustain the life of the bacteria. The fluorescence intensity of NPN is very weak in aqueous environment and strong in hydrophobic environment such as extracellular membrane. NPN was excluded from the intact outer membrane of the bacteria, but if the outer membrane was destroyed, it could enter the membrane phospholipid layer, resulting in significant fluorescence. Here, NPN uptake experiments were used to study the ability of antimicrobial lipopeptides to induce outer membrane penetration of E.coli ATCC 25922. As can be seen from fig. 3B, the addition of the antimicrobial lipopeptide significantly increased the NPN uptake of escherichia coli ATCC25922, and the NPN uptake exhibited a concentration-dependent effect on the antimicrobial lipopeptide, indicating that the antimicrobial lipopeptide had the ability to strongly disrupt the outer membrane of escherichia coli ATCC25922, and was comparable to polymyxin B, which is likely due to the increased ability of the antimicrobial lipopeptide to bind to the bacterial cell membrane due to the conjugation of fatty acid, thereby increasing the antimicrobial activity. When the concentration of antimicrobial lipopeptide was greater than 4 μ M, NPN uptake did not increase further, indicating that the available surface area of the bacterial outer membrane may have been destroyed by this concentration of antimicrobial lipopeptide.

To achieve sufficient membrane permeability, the antimicrobial lipopeptides must reach and penetrate the inner membrane. PI uptake experiments were used to assess the ability of antimicrobial lipopeptide LP21 to induce bacterial inner membrane penetration. When PI binds to DNA, PI emits intense fluorescence indicating impaired intimal integrity. As can be seen from FIG. 3c, in the E.coli ATCC25922 group, PI fluorescence intensity increased with increasing concentration of antimicrobial lipopeptide, reaching saturation at 16. mu.M. However, in the S.aureus ATCC29213 group, the fluorescence intensity was saturated at 4. mu.M (FIG. 3d), indicating that the antimicrobial lipopeptides were able to disrupt the inner membrane of these bacteria, leading to the diffusion of PI into the bacterial cells, giving off fluorescence, and also indicating that the antimicrobial lipopeptides were more effective in penetrating or lysing the inner membrane of S.aureus ATCC29213 than the inner membrane of E.coli ATCC 25922. As demonstrated above, the antimicrobial lipopeptides have excellent ability to penetrate or lyse membranes and exert antimicrobial effects through membrane disruption mechanisms.

The effect of antimicrobial lipopeptide LP21 on the surface morphology and intracellular changes of E.coli ATCC25922 was visualized using TEM and SEM. The control group of E.coli partially had rod-like, circular or oval shapes, the membranes were intact and smooth, and the membrane boundaries were very distinct (FIGS. 4a and 4 c). As can be seen from FIGS. 4b and 4d, after 2 hours of treatment with 2 × MIC antimicrobial lipopeptide LP21, the bacterial membrane showed wrinkles, vesicles and even rupture, and the flux of the bacterial content was observed, as compared with the control group, and it was found that antimicrobial lipopeptide LP21 caused serious damage to the bacterial membrane, indicating that it had very excellent membrane penetration ability and lysis activity. The antibacterial action of conventional antibiotics often involves key intracellular targets in the bacterial biosynthetic pathway, but the antibacterial lipopeptides can rapidly disrupt bacterial membranes by mechanical means, which helps overcome bacterial resistance to conventional antibiotics.

The membrane rupture effect of the antibacterial lipopeptide is researched by quantitatively detecting the uptake condition of escherichia coli ATCC25922 to the antibacterial lipopeptide at different times through flow cytometry. As can be seen from FIG. 5, when the antibacterial lipopeptide and the Escherichia coli cells act together for 2h, 4h and 6h, the ratio of the fluorescent bacteria with destroyed membrane is 96.0%, 87.3% and 65.6%, respectively, which indicates that the antibacterial lipopeptide has strong membrane destruction capability and destruction efficiency.

The distribution of antimicrobial lipopeptide LP21 in E.coli ATCC25922 cells was further analyzed by laser confocal. LP 21-fluorescein was obtained by labeling LP21 with fluorescein. The fluorescence image (FIG. 6) shows that 0h, i.e., no bacteria were effectively stained with PI without the addition of LP 21-fluorescein, and that no green fluorescence of LP 21-fluorescein, which could cause lysis of the bacterial membrane, was found in the bacteria. After the treatment for 4h, 8h and 12h by adding LP 21-fluorescein, the red fluorescence emitted by PI and the green fluorescence intensity of LP 21-fluorescein are gradually enhanced and reach the maximum intensity at 12h, which shows that the antibacterial lipopeptide LP21 can gradually destroy the integrity of the bacterial membrane to kill the bacteria with the increase of the action time.

(5) The antibacterial lipopeptide LP21 has obvious in-vivo antibacterial effect

The in vivo antimicrobial efficacy of antimicrobial lipopeptides was evaluated using a dorsal wound model of mice infected with staphylococcus aureus ATCC 29213. Wound healing was observed at different time points, as shown in fig. 7a, the antibacterial lipopeptide group healed faster than the NS group. Although NS-treated wounds also achieved similar closure rates to the antibacterial lipopeptide group on day 11, NS group wounds had more exudate in the early stages of healing, while the antibacterial lipopeptide group had slight exudate in the early stages of healing, indicating that antibacterial lipopeptides had significant antibacterial effects on the wounds. Furthermore, analysis of dorsal wound tissue sections of mice 11 days after infection showed that both NS group and antimicrobial lipopeptide LP21 group had lymphocytic inflammatory infiltrates (fig. 7b, blue arrows) compared to the control group, but more lymphocytic inflammatory infiltrates were found in NS group compared to LP21 group. While tissue damage and even necrosis was found in the NS group, only slight lymphocytic infiltration, no tissue damage or necrosis, was found in the wounds treated with antimicrobial lipopeptide LP21, suggesting that antimicrobial lipopeptide LP21 may reduce inflammatory responses and improve immune activity. More neovasculoid was found in group LP21 compared to the NS group (fig. 7b, red arrow), indicating that antimicrobial lipopeptide LP21 has the function of promoting vascular growth. The LP21 group found more fibroblast formation than the NS group (fig. 7b, grey arrows), which also indicates that the antimicrobial lipopeptide LP21 can promote fibroblast formation to aid in wound healing. In conclusion, the antimicrobial lipopeptide LP21 of the invention can reduce inflammatory infiltration of lymphocytes, and promote more neovascularization and fibroblast formation while exerting excellent antimicrobial effect, which indicates that the antimicrobial lipopeptide is a very promising antimicrobial agent.

Staphylococcus aureus is one of the major pathogens of bacterial keratitis. The in vivo antibacterial efficacy of antibacterial lipopeptides was assessed by using a model of bacterial keratitis in the eyes of mice infected with staphylococcus aureus ATCC 29213. 12h after infection of mouse cornea with Staphylococcus aureus ATCC29213, mice were treated with PBS and antimicrobial lipopeptide, respectively, and then observed for recovery of corneal infection at different times by slit lamp microscopy, as shown in FIG. 7 c. After 48h, the PBS group found corneal defects and significant plaques, while the antimicrobial lipopeptide LP21 group had clear corneas with no signs of corneal defects. These results indicate that antimicrobial lipopeptides can effectively inhibit corneal infection in mice caused by staphylococcus aureus ATCC 29213.

The in vivo antibacterial effect of antimicrobial lipopeptide LP21 was evaluated by intraperitoneal injection of escherichia coli ATCC25922 to establish a mouse acute peritonitis model. After 2h of successful modeling, mice developed symptoms of cramping, listlessness, slow response, etc., while excreting viscous stools. NS and antibacterial lipopeptides were then injected into the abdominal cavity of mice and the bacteria were counted by drawing abdominal fluid separately at different time points. Since all mice in the NS group died within 48h, bacterial counts were only performed within 48 h. The results showed that none of the mice survived within 48h for the NS group and 67% survival within 48h for the LP21 group (fig. 7 d). With increasing treatment time, the number of bacteria in the NS group was almost the same as the initial amount within 24h, while the number of e.coli ATCC25922 in the LP21 group gradually decreased and significantly decreased within the initial 12h (fig. 7 e). It was shown that antimicrobial lipopeptide LP21 was effective in inhibiting bacterial growth in mice. The in vivo experiments prove that the antibacterial lipopeptide LP21 has effective in vivo antibacterial activity and good function of promoting wound healing.

Sequence listing

<110> Binzhou medical college

<120> preparation of antibacterial lipopeptide LP21 and application thereof in treatment of bacterial infection

<141> 2021-11-25

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Trp Lys Lys Leu Leu Lys Trp Trp Leu Lys Lys Phe Lys Lys Leu Asp

1 5 10 15

Claims

1. An antimicrobial lipopeptide LP21, wherein the antimicrobial lipopeptide LP21 has the sequence C from N-terminus to C-terminus₈H₁₅O-Trp-Lys-Lys-Leu-Leu-Lys-Trp-Trp-Leu-Lys-Lys-Phe-Lys-Lys-Leu-Asp (abbreviated as C)₈H₁₅O-WKKLLKWWLKKFKKLD), wherein Trp is tryptophan, Lys is lysine, Leu is leucine, Phe is phenylalanine, Asp is aspartic acid, and the fatty acid connected is caprylic acid C₈H₁₆O₂. The structural formula of the antibacterial lipopeptide is shown as the following figure:

2. an antimicrobial lipopeptide LP21 according to claim 1, prepared by the process comprising:

(1) synthesis of Fmoc-Trp (Boc) -Lys (Boc) -Leu-Leu-Lys (Boc) -Trp (Boc) -Leu-Lys (Boc) -Phe-Lys (Boc) -Lys- (Boc) Leu-Asp (OtBu) -2-chlorotrityl chloride resin: adopting a solid-phase synthesis method, sequentially coupling amino acids from the C end to the N end of the antibacterial lipopeptide, weighing a certain amount of Fmoc-Asp (OtBu) -2-chlorotrityl chloride resin (load: 0.402mmol/g) and mixing with DMF to swell for 30min, wherein Fmoc is an amino protecting group of Asp, OtBu is a carboxyl protecting group on an Asp side chain, and the 2-chlorotrityl chloride resin is connected on the other carboxyl of the Asp; after swelling, removing the Fmoc protecting group by using a DMF solution containing 20% piperidine, and then sequentially washing with DMF, DCM and DMF three times, wherein if the indetrione method detects that the product is blue black, the Fmoc protection removal reaction is completely performed; swelling the resin without Fmoc protection again by using anhydrous DMF for 15 minutes, then adding 2 times of molar weight of second amino acid Fmoc-Leu-OH, 2.6 times of molar weight of HOBT, DCC and DIEA, stirring for reacting for 48 hours, and if the indetrione method detects no color, indicating that the coupling reaction is complete; and (2) performing suction filtration on the resin, washing the resin with DMF, DCM and DMF for multiple times in sequence, filling the resin into a dialysis bag (MW 8000-.

(2)C₈H₁₅Synthesis of O-Trp-Lys-Lys-Leu-Leu-Lys-Trp-Trp-Leu-Lys-Lys-Phe-Lys-Lys-Leu-Asp antimicrobial lipopeptide: removing Fmoc protecting group of the polypeptide resin obtained in (1), and adding 2 times molar amount of C₈H₁₆O₂With 2.6 times the molar amount of EDC and NHS dissolved in DMF to activate C₈H₁₆O₂Adding the polypeptide resin obtained in the step (1) into the DMF solution, stirring for 24h, performing suction filtration, filling the resin into a dialysis bag (MW 8000-; cleavage solution (TFA: Tis: H) was used₂O95: 2.5: 2.5) cleaving all side chain protecting groups of the lipopeptide resin and the resin for 1.5h, filtering to remove the resin, concentrating the cleavage solution, precipitating with glacial ethyl ether and washing several times, and freeze-drying to obtain antibacterial lipopeptide C₈H₁₅O-Trp-Lys-Lys-Leu-Leu-Lys-Trp-Trp-Leu-Lys-Lys-Phe-Lys-Lys-Leu-Asp。

3. The antimicrobial lipopeptide LP21 according to claim 1, wherein the antimicrobial lipopeptide prepared by the method of broth dilution in trace amounts is tested for in vitro antimicrobial activity, and the results show that the antimicrobial lipopeptide of the present invention has excellent in vitro antimicrobial activity; the secondary structure of the antibacterial lipopeptide is determined by circular dichroism spectroscopy, and the result shows that the antibacterial lipopeptide shows an alpha-helical structure in a membrane simulation environment; the serum stability and biocompatibility of the prepared antibacterial lipopeptide are researched, and the result shows that the antibacterial lipopeptide has good stability and biocompatibility; the antibacterial mechanism of the antibacterial lipopeptide is researched, and the result shows that the antibacterial lipopeptide has a strong membrane rupture mechanism; in vivo experiments show that the antibacterial lipopeptide has good in vivo antibacterial activity and can be applied to the preparation of medicines for treating bacterial infection.