Amphiphilic cationic antibacterial polypeptide and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to an amphiphilic cationic antibacterial polypeptide sequence and application thereof. The cationic polypeptide TVYV is designed by combining polypeptide fragments with different functions, and the polypeptide fragments are as shown in SEQ ID NO.1. The introduction of a targeted amino acid sequence can improve the internalization capability of the polypeptide, and the introduction of an enzyme response amino acid sequence can improve the responsive release effect of a polypeptide delivery system on the microenvironment in macrophages. Hydrophobic fragments are introduced to enhance the self-assembly driving force of the polypeptide. The antibacterial polypeptide sequence disclosed by the invention is good in biocompatibility and has the activity of resisting staphylococcus aureus. The antibacterial polypeptide delivery system provided by the invention can effectively improve the intracellular permeation efficiency of hydrophobic drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an amphiphilic cationic antibacterial polypeptide sequence and application thereof. Background Art

[0002] Staphylococcus aureus is a human pathogen with strong pathogenicity and adaptability. It can secrete a variety of virulence factors that damage the host, such as leukocidin and α-hemolysin, infect a variety of host species and tissue sites, cause local suppurative infections (such as wound infections, furuncles and cellulitis), and induce serious infectious diseases such as endocarditis, osteomyelitis and septic arthritis through systemic diffusion and metastasis. Staphylococcus aureus can not only invade extracellular tissues, but also evade the antibacterial defense mechanism of the extracellular host 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 of human medicine and has provided a revolutionary development in the fight against bacterial infections. However, the misuse and abuse of antibiotics has accelerated the development of bacterial resistance and has become one of the great challenges to global public health. In addition, the ever-changing microenvironment of bacterial infections makes antibiotic treatment more difficult and is closely related to the development of antibiotic resistance. Therefore, there is an urgent need to develop new effective antibacterial agents or improve the therapeutic effects of existing antibiotics.

[0004] Antimicrobial peptides (AMPs) are a class of cationic polypeptides with antibacterial, antiviral and antifungal activities. Natural AMPs are usually endogenous host defense molecules in many organisms and play a vital role in the first line of defense against pathogens. The unique membrane-destructive killing mechanism of AMPs not only enables them to have antimicrobial infection effects with rapid killing kinetics, broad-spectrum activity and low drug resistance, but also improves the efficiency of treatment by penetrating cell membranes, promoting intracellular drug accumulation, and increasing the sensitivity of cells to drug interference. Therefore, AMPs are expected to become effective antibacterial agents to replace antibiotic treatment and have good application potential in drug delivery. Summary of the invention

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

[0006] An amphipathic cationic antibacterial polypeptide, whose amino acid sequence is shown in SEQ ID NO.1.

[0007] The antibacterial peptide is composed of 23 amino acids in sequence, of which amino acids 1-4 are amino acid fragments with targeting effects in the TVYV sequence, amino acids 5-6 are enzyme-responsive amino acid fragments in the TVYV sequence, and amino acids 18-23 are hydrophobic fragments.

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

[0009] The antibacterial polypeptide is used in the preparation of antibacterial infection drugs (especially in the use of anti-Staphylococcus aureus). The antibacterial polypeptide kills Staphylococcus aureus by membrane destruction.

[0010] The antimicrobial polypeptide is used as a drug delivery system to deliver hydrophobic drugs. The antimicrobial polypeptide can form nano-micelles for delivering hydrophobic drugs, and can specifically target phagocytes, stimulate phagocyte endocytosis, deliver hydrophobic drugs into cells, and achieve treatment of phagocyte-related diseases.

[0011] The antimicrobial polypeptide specifically targets macrophages and stimulates macrophage endocytosis, thereby preventing and treating intracellular Staphylococcus aureus infection.

[0012] The present invention combines polypeptide fragments with different functions to design a cationic polypeptide TVYV. The introduction of a targeting amino acid sequence can improve the ability of the polypeptide to be internalized in cells. The introduction of an enzyme-responsive amino acid sequence increases the responsive release effect of the polypeptide delivery system to the microenvironment in macrophages. The introduction of a hydrophobic fragment enhances the self-assembly driving force of the polypeptide. The antibacterial polypeptide sequence of the present invention has good biocompatibility and anti-Staphylococcus aureus activity. The antibacterial polypeptide delivery system of the present invention can effectively improve the intracellular penetration efficiency of hydrophobic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a schematic diagram of the application of the antimicrobial peptide TVYV drug delivery system in treating intracellular infections.

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

[0015] Figure 3 It is the mass spectrum of the antimicrobial peptide TVYV.

[0016] Figure 4 It is the liquid chromatogram of the antimicrobial peptide TVYV.

[0017] Figure 5 This is the concentration-inhibition curve of the antimicrobial peptide TVYV.

[0018] Figure 6It is the hemolytic activity of the antimicrobial peptide TVYV.

[0019] Figure 7 It is the cellular activity of the antimicrobial peptide TVYV.

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

[0021] Fig. 9 This is the TEM morphology characterization and particle size distribution diagram of the composite micelles after the antibacterial peptide TVYV is loaded with the hydrophobic drug rifampicin PFP.

[0022] Fig.10 These are the ultraviolet absorption spectra and circular dichroism spectra of the antibacterial peptide TVYV before and after loading the hydrophobic drug rifampicin PFP.

[0023] Fig.11 This is an intuitive morphological image and SEM morphological characterization image of the composite hydrogel after the antibacterial peptide TVYV is loaded with the hydrophobic drug rifampicin PFP.

[0024] Fig.12 This is the macrophage uptake of the antimicrobial peptide TVYV before and after it is loaded with the hydrophobic drug rifampicin PFP. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0027] 1. Design of the antimicrobial peptide TVYV: The peptide delivery system with cell membrane targeting function can effectively promote cell penetration, control intracellular drug release, and improve drug utilization. It is a drug delivery system with great development prospects. To this end, we designed a new amphiphilic peptide nanoplatform (TKPR-V(Cit)-YGRKKRRQRRR-VVVVVV, TVYV) with enzyme responsiveness and specific targeting to macrophages for the treatment of local infections caused by Staphylococcus aureus, while achieving antibiotic delivery and clearance of intracellular bacteria. Schematic diagram of the intracellular Staphylococcus aureus clearance model, as shown in Figure 1 shown.

[0028] The polypeptide is mainly composed of four parts, among which the TKPR sequence has the ability to specifically target macrophages and enhance their phagocytic ability; YGRKKRRQRRR (TAT, a cell-penetrating peptide from HIV-1TAT protein) serves as a carrier for targeting and destroying Staphylococcus aureus in macrophages; V(Cit) (Val-Cit) serves as a connector with cathepsin B responsiveness and is located in the middle of the two domains of TKPR and TAT; 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, chemical structure as Figure 2 shown.

[0030] Synthesis of antimicrobial peptides: The peptides were synthesized by solid phase synthesis. The mass spectrometry and chromatographic results of the peptides were as follows: Figure 3 and Figure 4 shown.

[0031] 2. Antibacterial activity test of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antibacterial peptides

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

[0033] The revived 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 incubated in LB liquid medium at 37°C overnight, and diluted to 10 6 CFU / mL. In a 96-well plate, antimicrobial peptides of different concentrations (200μg / mL, 150μg / mL, 100μg / mL, 50μg / mL, 25μg / mL, 12.5μg / mL) were mixed with bacterial solution in a ratio of 1:1. After incubation at 37°C for 24 hours, the absorbance value OD of each well at 600nm was measured with a multifunctional microplate reader. The antimicrobial peptide concentration corresponding to the group with no obvious bacterial growth was the minimum inhibitory concentration MIC. After determining the MIC value, a small amount of bacterial solution with no obvious bacterial growth was aspirated and applied to LB solid culture medium, incubated at 37°C for 18-24 hours, and the lowest concentration without bacterial growth was the minimum bactericidal concentration MBC. Each group of experiments was repeated three times.

[0034] The results are as follows Figure 5 As shown, it can be seen that the antibacterial peptide TVYV has a concentration-dependent antibacterial activity against Staphylococcus aureus

[0035] 3. In vitro hemolytic activity test of antimicrobial peptides

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

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

[0038] Among them, OD 待测 is the experimental group data, OD 阴性 For the negative control PBS data, OD 阳性 The data are for positive control ultrapure water.

[0039] Figure 6 This is the result of the hemolytic activity experiment. It can be seen from the results that the antimicrobial peptide TVYV exhibits very low blood cell toxicity, indicating that the linear structure of antimicrobial peptides is very safe and has a very high drugability.

[0040] IV. Cytotoxicity test of antimicrobial peptide TVYV, measured by CCK-8 method

[0041] The CCK-8 cytotoxicity detection kit (Biyuntian Biotechnology) was used to evaluate the toxic effects of antimicrobial peptides on cells. Mouse normal fibroblast L929 cells and human umbilical vein endothelial cells HUVEC cells were used as cell lines. HUVEC and L929 cells frozen in a liquid nitrogen tank were taken out, quickly thawed in a 37°C water bath, and inoculated in 3mL DMEM cell culture medium containing 10% fetal bovine serum and 1% double antibody (penicillin and streptomycin), and placed in a cell culture incubator with 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 2mL of trypsin digestion solution was added to digest for about 1min; then, the trypsin digestion solution was removed, and 3mL of cell culture medium was added to carefully blow off the adherent cells, and transferred to a 15mL centrifuge tube and centrifuged at 800rpm for 3min; the centrifuged cells were resuspended in a certain amount of cell culture medium and blown evenly, and after cell counting, 1×104 The cells were inoculated at a density of 100 cells / well in a 96-well plate and cultured overnight at 37°C and 5% CO2. After the cells adhered to the plate, the original culture medium was removed and the cells were washed with PBS.

[0042] Use DMEM cell culture medium (containing 10% fetal bovine serum and 1% double antibody) to dilute the peptide to different concentrations (the final concentration gradient is consistent with the MIC determination), add it to the 96-well plate with cells, and culture it at 37°C and 5% CO2. Among them, 100 μL of cell culture medium without peptide was added to the positive control, and 100 μL of cell culture medium was added to the negative control. After 24 hours of culture, 10 μL of CCK-8 reagent was added to each well, and then placed in the incubator for incubation for 1 hour, then taken out and the absorbance at 450nm was detected using a multifunctional microplate reader. This experiment was repeated 3 times.

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

[0044] Among them, A 待测 is the experimental group data, A 阴性 is the negative control cell culture medium data, A 阳性 The data are for positive control cell fluid.

[0045] The results are as follows Figure 7 Compared with the control group, the cell activity of the antimicrobial polypeptide TVYV was maintained above 60%, and no obvious cytotoxicity was shown within the MIC range, which indicates that the antimicrobial polypeptide provided by the present invention has good cell biocompatibility, no negative effect on cell proliferation and cell activity, and no toxic threat to normal cells, and can be further studied and developed as a biological antimicrobial polypeptide.

[0046] 5. Preparation and characterization of antimicrobial peptide delivery system

[0047] Antimicrobial peptide TVYV nanomicelles: TVYV lyophilized powder (10 mg) was dissolved in ultrapure water to obtain 10 mg / mL TVYV / UP mother solution. 100 μL of TVYV mother solution was slowly added with 100 μL of sodium hydroxide (NaOH) (pH=13) and 300 μL of ultrapure water in sequence, and stirred (300 rpm) for several hours. The TEM morphology and particle size distribution of the antimicrobial peptide TVYV nanomicelles are shown in Figure 2. Figure 8 shown.

[0048] Composite micelles after the antibacterial peptide TVYV is loaded with the hydrophobic drug rifampicin RFP: Weigh a small amount of RFP powder and dissolve it in a certain amount of dimethyl sulfoxide (DMSO) to obtain a 20mg / mL RFP / DMSO mother solution. Take 50μL RFP / DMSO mother solution and slowly add 500μL 10mg / mL TVYV / UP and 1.9mL UP in sequence, stir thoroughly and slowly add 50μL NaOH (pH=13), and stir at 300rpm for about 20h. Place the above-mentioned mixed solution in a dialysis bag (2000Da) and dialyze at room temperature for 24h to remove DMSO in the solution. The results of the TEM morphology and particle size distribution of the composite micelles are shown in Fig. 9 The relevant UV absorption spectrum and circular dichroism spectrum results are shown in Fig.10 shown.

[0049] Composite hydrogel formed by antimicrobial peptide TVYV and hydrophobic drug rifampicin RFP: Take 10μL 20mg / mL RFP / DMSO in a 2mL sample bottle, and slowly add 100μL 10mg / mL TVYV / UP and 90μL UP in sequence, stir thoroughly, slowly add 50μL NaOH (pH=13), mix well and incubate at 37℃ overnight. The intuitive morphology and SEM morphology characterization of the composite hydrogel are shown in the figure. Fig.11 shown.

[0050] VI. Macrophage uptake of the antimicrobial peptide TVYV before and after loading the hydrophobic drug rifampicin PFP

[0051] According to the above steps, mouse mononuclear macrophage RAW 264.7 cells were inoculated into 6-well plates, cultured for 12 hours, and then treated with LPS for 16 hours; 2) the cells were washed with PBS, and 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) was added to the experimental group, and only 1 mL of DMEM (without FBS) was added to the control group; after incubation at 37°C for 1 hour, the cells were washed twice and resuspended in 1 mL of PBS, and the FITC fluorescence intensity was detected using a flow cytometer. Fig.12 The results of cellular uptake showed that the antimicrobial peptide TVYV can effectively enhance the uptake ability of hydrophobic drugs in macrophages.

[0052] The present invention is not limited to the above-mentioned embodiments. Those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and purpose of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

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

1.

2. An amphiphilic cationic antimicrobial polypeptide according to claim 1, characterized in that: The amino acids at positions 1-4 are amino acid fragments with targeting effects in the sequence, the amino acids at positions 5-6 are enzyme-responsive amino acid fragments in the sequence, and the amino acids at positions 18-23 are hydrophobic fragments.

3. An amphiphilic cationic antimicrobial polypeptide according to claim 2, characterized in that: The hydrophobic segment includes a hydrophobic segment composed of a hydrophobic amino acid and / or an alkyl chain, the hydrophobic amino acid is valine, and the alkyl chain is C15-C18.

4. Use of the amphiphilic cationic antibacterial polypeptide according to claim 1 in the preparation of antibacterial infection drugs.

5. Use of the amphiphilic cationic antimicrobial polypeptide as claimed in claim 1 as a drug delivery system for delivering hydrophobic drugs.

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