An amphiphilic cationic antimicrobial polypeptide and uses thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to an amphiphilic cationic antibacterial polypeptide sequence and application thereof. The present application combines polypeptide fragments with different functions to design a cationic polypeptide TVYV, the polypeptide fragment is shown as SEQ ID NO. 1, the introduction of a targeted amino acid sequence can improve the intracellular penetration efficiency of the polypeptide, the introduction of an enzyme-responsive amino acid sequence can increase the responsive release effect of the polypeptide delivery system to the microenvironment in macrophages, and the introduction of a hydrophobic fragment can enhance the self-assembly driving force of the polypeptide. The antibacterial polypeptide sequence of the present application has good biocompatibility and has activity against Staphylococcus aureus. The antibacterial polypeptide delivery system of the present application can effectively improve the intracellular penetration efficiency of hydrophobic drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an amphiphilic cationic antibacterial polypeptide sequence and application thereof. BACKGROUND

[0002] Staphylococcus aureus is a human pathogen with strong pathogenicity and adaptability, which can secrete leukocidin, alpha-hemolysin and other various virulence factors to destroy the host, infect various host species and tissue sites, cause local pyogenic infection (such as wound infection, furuncle and cellulitis), and induce endocarditis, osteomyelitis and septic arthritis and other serious infectious diseases through systemic spread and transfer. Staphylococcus aureus not only invades extracellular tissues, but also avoids the antibacterial defense mechanism of extracellular hosts to proliferate in mammalian cells (including phagocytes and osteoblasts), causing intracellular infection.

[0003] The discovery and clinical application of antibiotics such as penicillin and vancomycin is one of the greatest achievements in human medicine, which provides a revolutionary development for combating bacterial infection. However, the misuse and abuse of antibiotics accelerate the development of bacterial drug resistance and become one of the great challenges of global public health. In addition, the changing microenvironment of bacterial infection makes antibiotic therapy more difficult and is closely related to the development of antibiotic resistance. Therefore, it is urgent to develop new effective antibacterial agents or improve the therapeutic effect of existing antibiotics.

[0004] Antibacterial peptides (AMPs) are a class of cationic polypeptides with antibacterial, antiviral and antifungal activities, among which natural AMPs are usually endogenous host defense molecules of many organisms, which play a crucial role in the first line of defense against pathogens. The unique membrane-destroying killing mechanism of AMPs not only has the effects of rapid killing kinetics, broad-spectrum activity and low drug resistance on microbial infection, but also can improve the efficiency of treatment by penetrating the cell membrane, promoting intracellular drug accumulation, increasing the sensitivity of cells to drug interference, etc. Therefore, AMPs are expected to become effective antibacterial agents to replace antibiotic therapy, and have good application potential in drug delivery. SUMMARY

[0005] In view of the above technical problems, the purpose of the present application is to provide an amphiphilic cationic antibacterial polypeptide and application thereof, and the specific scheme is as follows:

[0006] An amphiphilic cationic antibacterial polypeptide, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0007] The antibacterial polypeptide is sequentially composed of 23 amino acids, wherein the amino acids at positions 1-4 are an amino acid fragment with targeting effect in TVYV sequence, the amino acids at positions 5-6 are an enzyme-responsive amino acid fragment in TVYV sequence, and the amino acids at positions 18-23 are a hydrophobic fragment

[0008] The hydrophobic fragment comprises a hydrophobic amino acid and / or a hydrophobic fragment composed of an alkyl chain, the hydrophobic amino acid is valine, and the alkyl chain is C15-C18.

[0009] The antibacterial polypeptide is applied to the preparation of an antibacterial infection drug (especially, an antibacterial infection drug against Staphylococcus aureus). The antibacterial polypeptide kills Staphylococcus aureus through membrane damage.

[0010] The antibacterial polypeptide is applied to the delivery of a hydrophobic drug as a drug delivery system. The antibacterial polypeptide can form a nanomicelle for the delivery of a hydrophobic drug, specifically targets phagocytes, stimulates phagocytosis in the phagocytes, and delivers the hydrophobic drug into the cells, so that phagocyte-related diseases can be treated.

[0011] The antibacterial polypeptide specifically targets macrophages, stimulates phagocytosis in the macrophages, and realizes the prevention and treatment of intracellular Staphylococcus aureus infection.

[0012] The cationic polypeptide TVYV is designed by combining polypeptide fragments with different functions. The introduction of a targeting amino acid sequence can improve the intracellularization ability of the polypeptide, the introduction of an enzyme-responsive amino acid sequence can increase the responsive release effect of the polypeptide delivery system on the microenvironment in macrophages, and the introduction of a hydrophobic fragment can enhance the self-assembly driving force of the polypeptide. The antibacterial polypeptide sequence has good biocompatibility and has antibacterial activity against Staphylococcus aureus. The antibacterial polypeptide delivery system can effectively improve the intracellular penetration efficiency of a hydrophobic drug. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is an application schematic diagram of the antibacterial polypeptide TVYV drug delivery system for treating intracellular infection.

[0014] Figure 2 is a chemical molecular formula structure diagram of the antibacterial polypeptide TVYV.

[0015] Figure 3 is a mass spectrum diagram of the antibacterial polypeptide TVYV.

[0016] Figure 4 is a liquid chromatogram of the antibacterial polypeptide TVYV.

[0017] Figure 5 is a concentration-bacteriostatic curve of the antibacterial polypeptide TVYV.

[0018] Figure 6Hemolytic activity of antibacterial peptide TVYV.

[0019] Figure 7 Cell activity of antibacterial peptide TVYV.

[0020] Figure 8 TEM morphology characterization diagram and particle size distribution diagram of antibacterial peptide TVYV nanomicelles.

[0021] Figure 9 TEM morphology characterization diagram and particle size distribution diagram of antibacterial peptide TVYV loaded with hydrophobic drug rifampicin PFP after complex micelles.

[0022] Figure 10 UV absorption spectrum and circular dichroism spectrum of antibacterial peptide TVYV before and after loading hydrophobic drug rifampicin PFP.

[0023] Figure 11 Direct visual morphology diagram and SEM morphology characterization diagram of antibacterial peptide TVYV loaded with hydrophobic drug rifampicin PFP after complex hydrogel.

[0024] Figure 12 Macrophage uptake of antibacterial peptide TVYV before and after loading hydrophobic drug rifampicin PFP. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0026] The amino acid sequence of the amphiphilic cationic antibacterial peptide TVYV is shown in SEQ ID NO. 1.

[0027] I. Design of antibacterial peptide TVYV: The polypeptide delivery system with cell membrane targeting function can effectively promote cell penetration, control intracellular drug release, and improve drug utilization, and is a very promising drug delivery system. Therefore, we designed a new type of amphiphilic peptide nanoplatform (TKPR-V(Cit)-YGRKKRRQRRR-VVVVVV, TVYV) with enzyme responsiveness and specific targeting of macrophages, for the treatment of local infections caused by Staphylococcus aureus, while achieving antibiotic delivery and intracellular bacterial clearance. The intracellular Staphylococcus aureus clearance model is shown in Figure 1 .

[0028] The polypeptide is mainly composed of four parts, wherein the TKPR sequence has the ability to specifically target macrophages and enhance phagocytosis; YGRKKRRQRRR (TAT, a cell-penetrating peptide from HIV-1 TAT protein) serves as a carrier for targeting and destroying Staphylococcus aureus in macrophages; V(Cit) (Val-Cit) serves as a linker responsive to cathepsin B, located between the TKPR and TAT domains; the incorporation of VVVVVV (six valine residues) is to provide hydrophobicity and driving force for self-assembly.

[0029] TVYV: Thr-Lys-Pro-Arg-Val-Cit-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Val-Val-Val-Val-Val-Val, the chemical structure is shown as Figure 2 .

[0030] Synthesis of antibacterial polypeptide: the polypeptide was synthesized by solid-phase synthesis, and the mass spectrum and chromatogram results of the polypeptide are shown in Figure 3 and Figure 4 .

[0031] II. Antibacterial activity test of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antibacterial polypeptide

[0032] Standard strain: Staphylococcus aureus (CICC 10384)

[0033] The recovered bacterial solution was inoculated on LB solid medium by streaking method and incubated at 37°C overnight. Then, a single colony on the plate was picked and inoculated in LB liquid medium at 37°C overnight, and then diluted with LB liquid medium to 10 6 CFU / mL. In the 96-well plate, different concentrations (200 μg / mL, 150 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL) of antibacterial polypeptide were mixed with the bacterial solution at 1:1, and after incubation at 37°C for 24 h, the absorbance value OD of each well at 600 nm was measured by multifunctional enzyme label instrument. The antibacterial polypeptide concentration corresponding to the group without obvious bacterial growth was the minimum inhibitory concentration MIC. After determining the MIC value, a small amount of bacterial solution without obvious bacterial growth was inoculated on LB solid medium and incubated at 37°C for 18-24 h, and the minimum concentration without bacterial growth was the minimum bactericidal concentration MBC. Each group of experiments was repeated three times.

[0034] The results are shown in Figure 5 It can be seen that the antibacterial polypeptide TVYV has concentration-dependent antibacterial activity

[0035] III. In vitro hemolytic activity test of antibacterial polypeptide

[0036] Fresh rabbit whole blood containing sodium citrate was selected, centrifuged (15 min, 1500 rpm) to remove plasma, washed with PBS for 3 times, and the obtained red blood cells were diluted in PBS solution at 1:25 (4% v / v). The above diluted red blood cell suspension (500 μL) was placed in 500 μL PBS solution and 500 μL ultrapure water (UP) as positive and negative controls, respectively, and 500 μL cell suspension and 500 μL polypeptide / PBS solution of different concentrations were mixed in a 2 mL centrifuge tube. After incubation at 37°C for 1 h, centrifugation was performed at 1500 rpm for 5 min, and 100 μL supernatant was transferred to a 96-well plate, and the absorbance value at 540 nm was detected on an enzyme marker. Each group of experiments was repeated three times, and the average value of the obtained data was taken.

[0037] Hemolytic activity = (OD 待测 - OD 阴性 ) / (OD 阳性 - OD 阴性 ) x 100%

[0038] Wherein, OD 待测 is the experimental group data, OD 阴性 is the negative control PBS data, and OD 阳性 is the positive control ultrapure water data.

[0039] Figure 6 The hemolytic activity experiment results show that the antibacterial polypeptide TVYV has very low blood cell toxicity, indicating that the linear structure of the antibacterial polypeptide is very safe and has a high drug development potential.

[0040] IV. Cytotoxicity test of antibacterial polypeptide TVYV, determined by CCK-8 method

[0041] The CCK-8 cytotoxicity detection kit (Bi Yun Tian Biotechnology) was used to evaluate the toxic effect of the antibacterial polypeptide on cells. The HUVEC and L929 cells stored in liquid nitrogen tank were taken out, quickly thawed in a 37°C water bath, and inoculated in 3 mL DMEM cell culture medium containing 10% fetal bovine serum and 1% double-antibiotic (penicillin and streptomycin), and placed in a cell incubator at a constant temperature of 37°C and 5% CO2. When the cell density reached more than 80%, the original culture medium in the cell culture dish was removed, and the cells were washed with PBS, and 2 mL trypsin digestion solution was added for digestion for about 1 min. Then, the trypsin digestion solution was removed, and 3 mL cell culture medium was added to carefully blow off the adherent cells, which were transferred to a 15 mL centrifuge tube and centrifuged at 800 rpm for 3 min. The centrifuged cells were resuspended in a certain amount of cell culture medium and blown evenly, counted by cell counting, and inoculated in 96-well plates at a concentration of 1 x 104 The cells were seeded in 96-well plates at a density of cells / well, and cultured overnight at 37°C in a 5% CO2 atmosphere. After the cells adhered, the original culture solution was removed, and the cells were washed with PBS.

[0042] The polypeptide was diluted with DMEM cell culture solution (containing 10% fetal bovine serum and 1% double-antibiotic) to different concentrations (the final concentration gradient was consistent with the MIC determination), and added to the 96-well plate with cells, and incubated at 37°C in a 5% CO2 atmosphere. Among them, 100 μL of cell culture solution without polypeptide was added as a positive control, and 100 μL of cell culture solution was added as a negative control. After 24 h of culture, 10 μL of CCK-8 reagent was added to each well, and then incubated in the incubator for 1 h before being taken out. The absorbance value at 450 nm was detected using a multifunctional enzyme label instrument. This experiment was repeated three times.

[0043] Cell activity = (A 待测 -A 阴性 ) / (A 阳性 -A 阴性 ) x 100%

[0044] Among them, A 待测 is the experimental group data, A 阴性 is the negative control cell culture medium data, and A 阳性 is the positive control cell solution data.

[0045] The results are shown in Figure 7 . Compared with the control group, the cell activity of the antibacterial polypeptide TVYV was maintained at more than 60%, and no obvious cytotoxicity was shown within the MIC range, which can indicate that the antibacterial polypeptide provided by the application has good cell biocompatibility, and has no negative effect on cell proliferation and cell activity, and will not pose a toxic threat to normal cells, and can be used as a biological antibacterial polypeptide for further research and development.

[0046] V. Preparation and characterization results of antibacterial polypeptide delivery system

[0047] Antibacterial polypeptide TVYV nanomicelles: TVYV freeze-dried powder (10 mg) was dissolved in ultrapure water to obtain a 10 mg / mL TVYV / UP mother liquor. 100 μL of TVYV mother liquor was slowly added to 100 μL of sodium hydroxide (NaOH) (pH = 13) and 300 μL of ultrapure water, respectively, and stirred (300 rpm) for several hours. The results of the TEM morphology and particle size distribution of the antibacterial polypeptide TVYV nanomicelles are shown in Figure 8 .

[0048] Complex micelles of antibacterial polypeptide TVYV loaded with hydrophobic drug rifampicin RFP: A small amount of RFP powder was dissolved in a certain amount of dimethyl sulfoxide (DMSO) to obtain a 20 mg / mL RFP / DMSO mother liquor. 50 μL of the RFP / DMSO mother liquor was slowly added to 500 μL of 10 mg / mL TVYV / UP and 1.9 mL of UP, respectively, and stirred at 300 rpm for about 20 h. The mixed solution was placed in a dialysis bag (2000 Da) and dialyzed at room temperature for 24 h to remove DMSO in the solution. The TEM morphology and particle size distribution of the complex micelles are shown in Figure 9 The related ultraviolet absorption spectrum and circular dichroism spectrum are shown in Figure 10

[0049] Complex hydrogel of antibacterial polypeptide TVYV and hydrophobic drug rifampicin RFP: 10 μL of 20 mg / mL RFP / DMSO was added to a 2 mL sample bottle, and 100 μL of 10 mg / mL TVYV / UP and 90 μL of UP were slowly added in sequence, and 50 μL of NaOH (pH = 13) was slowly added after stirring. The mixed solution was incubated at 37°C overnight. The direct visual morphology and SEM morphology characterization of the complex hydrogel are shown in Figure 11

[0050] Six. Macrophage uptake of antibacterial polypeptide TVYV before and after loading with hydrophobic drug rifampicin RFP

[0051] According to the above steps, mouse monocyte macrophage RAW 264.7 cells were inoculated in a 6-well plate, cultured for 12 h, and then treated with LPS for 16 h; 2) The cells were washed with PBS, and the experimental groups were added with 1 mL of DMEM (without FBS) containing FITC-TVYV (50 μg / mL), FITC-RFP@TVYV (50 μg / mL, expressed as the concentration of TVYV), and FITC-RFP (0.05 μg / mL), respectively. The control group was only added with 1 mL of DMEM (without FBS). After incubation at 37°C for 1 h, the cells were washed twice and resuspended in 1 mL of PBS, and the FITC fluorescence intensity was detected using a flow cytometer. Figure 12 The results of cell uptake show that the antibacterial polypeptide TVYV can effectively enhance the uptake of hydrophobic drugs in macrophages.

[0052] The present application is not limited to the above embodiments, and those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents.​​

Claims

1. An amphiphilic cationic antibacterial polypeptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. The use of the amphiphilic cationic antimicrobial polypeptide as described in claim 1 in the preparation of drugs against Staphylococcus aureus infection.

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