Amphiphilic artificial antimicrobial lipopeptide, preparation and application

Abstract

The present invention provides an amphiphilic artificial antibacterial lipopeptide, a preparation and its application in the field of biomedicine. The amphiphilic lipopeptide involved in the present invention is composed of a short peptide containing 5-6 amino acids and a fatty acid chain modified at the N-terminus of the short peptide chain, and the C-terminus of the short peptide chain is terminated by an amide group; the short peptide contains at least 2 consecutive cationic amino acids and 2 consecutive hydrophobic amino acids, and the carbon chain length of the fatty acid chain is between 8 and 16. The lipopeptide has the characteristics of short sequence, broad-spectrum antibacterial activity, anti-resistant bacteria activity, cell membrane targeting, cell selectivity, and secondary structure transformation, and can form a hydrogel through self-assembly. The lipopeptide kills pathogenic bacteria by the mechanism of action of destroying microbial cell membranes, and is less likely to produce resistant strains than traditional antibiotics. The antibacterial lipopeptide of the present invention can be used in a variety of antibacterial, antifungal, and anti-resistant bacteria products.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the design, preparation method and antibacterial application of a class of short lipopeptide molecules. Background Art

[0002] Antimicrobial peptides are a class of antimicrobial materials derived from nature with broad-spectrum antimicrobial activity, and are effective against drug-resistant strains. They are potential candidate drugs for future antimicrobial infections and epidemics (Zasloff, Nature, 2002, 415, 389.). Currently, the most reported antimicrobial peptides have the characteristics of cationic, amphipathic, and α-helical, and can specifically treat related diseases caused by drug-resistant bacteria (Ciumac et al., Journal of Colloid and Interface Science, 2019, 537, 163). Antimicrobial peptides act on the negatively charged bacterial cell membrane, destroy the membrane structure, and cause bacterial cytosol leakage to kill bacteria. Compared with traditional antibiotics, this mechanism of action is not easy to produce drug resistance and can quickly kill bacteria in minutes. However, this type of molecule usually has hemolytic toxicity and protease instability, and therefore usually has the disadvantages of poor biocompatibility, large toxic side effects, short half-life, and low effectiveness during in vivo treatment. Therefore, it is urgent to develop antimicrobial peptide materials and dosage forms that can effectively target drug-resistant bacterial infections.

[0003] At present, the main methods to solve the above defects include artificially designed improved antimicrobial peptides and drug controlled release dosage form design. For example, natural antimicrobial peptides can be improved by genetic engineering and mass-produced by fermentation technology (such as authorized patent CN106674331B). Antimicrobial peptides with artificially designed sequences and prepared by solid / liquid phase chemical synthesis have also gradually occupied more markets with the advancement of peptide synthesis technology (such as authorized patents CN103421090B and CN109320585B). Compared with the sequence length of 20-60 amino acids of most natural antimicrobial peptides, the cost of chemically synthesizing antimicrobial peptides with longer sequences is relatively high, while shortening the sequence of antimicrobial peptides to be synthesized can significantly reduce their preparation costs. On the other hand, peptide chains with too short sequences have the defects of weak antimicrobial activity and poor cell selectivity. For example, when the peptide sequence length is ≤4, the number of positive charges and hydrophobicity of the peptide are difficult to reach the strength that can destroy the bacterial cell membrane and thus kill bacteria; at the same time, such peptides are difficult to form α-helices, and this structure determines the selectivity of antimicrobial peptides for different cells. Therefore, the length of the artificially designed antimicrobial peptides should be reasonably controlled so that they can have secondary structure changes as much as possible on the basis of short sequences. In addition, in order to effectively solve the cytotoxicity and protease instability of antimicrobial peptides, the development of supramolecular self-assembly antimicrobial peptides is also an effective strategy. Through non-covalent cross-linked supramolecular self-assembly, antimicrobial peptide molecules can be bound in the nanostructure formed by molecular self-assembly, and are not released or maintained at a low level in a normal physiological environment, thereby maintaining a low level of cytotoxicity. When in contact with the surface of negatively charged pathogenic microorganisms, antimicrobial peptide molecules will be released from the supramolecular structure and exhibit antibacterial activity. At present, such antimicrobial peptides have been approved to enter the catalog of cosmetic raw materials (such as myristoyl hexapeptide-5), but the research on such short antimicrobial lipopeptides is not comprehensive enough, especially short antimicrobial peptides with a peptide sequence length of 5-6 amino acids.

[0004] The short antimicrobial lipopeptide designed by the present invention maintains strong antimicrobial activity and is effective against drug-resistant bacteria on the basis of having a short sequence; and still has α-helical characteristics, so that it has good charge selectivity and cell selectivity; due to its supramolecular self-assembly dosage form, its biocompatibility and stability are also good. Summary of the invention

[0005] In order to solve the problems of long sequences, high costs and poor stability of existing antimicrobial peptides, the present invention designs a series of short-sequence antimicrobial lipopeptides. On the one hand, the present invention discloses a series of amphiphilic antimicrobial lipopeptides with antibacterial and antifungal activities, and a method for preparing a self-assembly formulation thereof, wherein the antimicrobial lipopeptides are composed of 5-6 amino acid sequences, and the cost of chemical synthesis is significantly reduced compared to antimicrobial peptides generally composed of 20-60 amino acids; and the N-terminus of the antimicrobial lipopeptide is connected to a fatty acid chain, which can promote the self-assembly of the antimicrobial peptide and enhance the membrane-breaking and antimicrobial activity of the antimicrobial peptide.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] In the first aspect, the short peptide sequence in the amphiphilic antimicrobial lipopeptide proposed by the present invention consists of 5-6 amino acids, and has at least 2 consecutive cationic amino acids and 2 consecutive hydrophobic amino acids, and is modified with a C8-C16 fatty acid chain at the N-terminus of the short peptide chain.

[0008] Among them, cationic amino acids include arginine (Arg, R), lysine (Lys, K), histidine (His, H); hydrophobic amino acids include phenylalanine (Phe, F), alanine (Ala, A), leucine (Leu, L), isoleucine (Ile, I), tryptophan (Trp, W), proline (Pro, P), valine (Val, V), glycine (Gly, G); the amino acid used to connect the fatty acid chain and the N-terminus of the short peptide chain is glycine (Gly, G) or a cationic amino acid. Preferably, the cationic amino acid is selected from one or both of arginine and lysine; the hydrophobic amino acid is selected from one or more of phenylalanine, isoleucine, valine, and glycine.

[0009] Preferably, the lipopeptide has the following structure: C8-KKIII-NH2, C8-GKKIII-NH2, C8-KKIIVV-NH2, C 12 -GIIKKI-NH2, C 12 -GKKIII-NH2, C 12 -KKIII-NH2, C 12 -KKIIF-NH2, C 12 -KKIFF-NH2, C 12 -KKFFF-NH2, C 16 -RRRVVV-NH2, C 16 -KRRFF-NH2, C 16 -RRKII-NH2, C 16 -KKKII-NH2, C 16 -GKKKII-NH2; wherein CH3(CH2) n-2 CO-, abbreviated as C n -, C8, C 12 , C 16 They are caprylic acid chain, lauric acid chain and palmitic acid chain connected to the N-terminus, and NH2 is the amide group capping the C-terminus of the short peptide chain.

[0010] In a second aspect, the present invention provides an antibacterial drug preparation, which is prepared by the above-mentioned amphiphilic antibacterial lipopeptide or a derivative sequence containing any of the above-mentioned sequences and the following common excipients, wherein the excipient concentration is 10 -3~1g / L; the preparation has a dosage form with a nanofiber structure, such as gel, sol, emulsion, and microneedle patch.

[0011] The auxiliary materials include: phosphate, and / or tromethamine buffer salt (Tris), and / or tris(hydroxymethyl)glycine buffer salt, and / or borate, and / or adenosine triphosphate (ATP), and / or chitosan, and / or hyaluronic acid, and / or poly(N-isopropylacrylamide) (PNIPAM), and / or poly(lactic acid-glycolic acid copolymer), and / or sodium alginate, and / or polyvinyl alcohol, and / or cellulose, and / or genipin, and / or polyvinyl pyrrolidone, and / or one or more metal element ions (Na, Mg, Al, K, Ca, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ag, Au, U).

[0012] The above preparation is preferably in the form of gel, sol or microneedle patch.

[0013] The present invention is not limited to the above description, and various dosage forms containing antimicrobial lipopeptides can also be prepared by replacing lipopeptide molecules and / or excipients, and mixing single and / or multiple dosage forms.

[0014] In a fifth aspect, the present invention provides the use of the above-mentioned amphiphilic antimicrobial lipopeptide in the preparation of a drug that can simultaneously kill Gram-positive bacteria, Gram-negative bacteria and fungi. The drug can be administered by in situ injection, in situ smearing or in situ application to kill Gram-positive bacteria, Gram-negative bacteria and fungi, thereby achieving anti-infection and improving the quality of life.

[0015] Compared with the existing antimicrobial lipopeptides, the antimicrobial lipopeptides and preparations disclosed in the present invention have the following advantages: (1) The lipopeptide molecular sequence of the present invention is designed based on the secondary structure transformation characteristics and the peptide chain is relatively short. The α-helical structure transformed after interacting with the negatively charged cell membrane can destroy the bacterial and fungal envelope structure, thereby killing the bacteria. This secondary structure transformation makes the antimicrobial lipopeptide cell-selective, so that it can kill pathogens while maintaining low cytotoxicity. (2) Compared with most of the disclosed non-assembly host antimicrobial lipopeptide molecules and preparations, the antimicrobial lipopeptide and preparation materials of the present invention have good biocompatibility, low toxic side effects, and resistance to degradation. (3) It has a broad-spectrum antimicrobial activity and can kill Gram-positive bacteria, Gram-negative bacteria and fungi at the same time, and can effectively kill drug-resistant bacteria. (4) Compared with the antimicrobial peptide molecules extracted from nature, the artificially designed molecules of the present invention have the advantages of a single component and a clear biochemical activity mechanism, and can be mass-produced by solid-phase or liquid-phase peptide synthesis methods, with low production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described in detail below in conjunction with the accompanying drawings:

[0017] Figure 1 is a representative antimicrobial lipopeptide molecule of the present invention;

[0018] Figure 2 Gels, sols, and microneedle patches prepared from representative antimicrobial lipopeptide molecules of the present invention;

[0019] Figure 3 Figure 2 is a nanofiber diagram formed by a representative antimicrobial lipopeptide of the present invention;

[0020] Figure 4 The antibacterial activity of the representative antibacterial lipopeptides of the present invention against different drug-resistant strains is determined;

[0021] Figure 5 The fluorescence leakage data caused by the representative antimicrobial lipopeptides of the present invention to negatively charged and uncharged liposome nanovesicles encapsulating fluorescent dyes, respectively;

[0022] Figure 6 The secondary structure changes of the representative antimicrobial lipopeptide of the present invention in a normal solution environment and a negatively charged environment. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is described in detail below through preferred implementation modes and examples, but the present invention is not limited to the following implementation modes and examples.

[0024] If specific techniques or conditions are not specified in the implementation methods and examples, reference may be made to relevant literature in the art or the instructions for related products and instruments.

[0025] Example 1: The chemical synthesis method of the peptide molecules provided by the present invention is described in detail below in conjunction with specific examples:

[0026] Representative antibacterial lipopeptide molecules provided by the present invention include C8-KKIII-NH2, C8-GKKIII-NH2, C8-KKIIVV-NH2, C 12 -GIIKKI-NH2, C 12 -GKKIII-NH2, C 12 -KKIII-NH2, C 12 -KKIIF-NH2, C 12 -KKIFF-NH2, C 12 -KKFFF-NH2, C 16 -RRRVVV-NH2, C 16 -KRRFF-NH2, C 16 -RRKII-NH2, C 16 -KKKII-NH2, C 16 -GKKKII-NH2(partial molecular structure such as Figure 1), can be synthesized by solid phase peptide synthesis, and the C-terminus of the short peptide chain is amino-treated. 12 , C 16 They are octanoic acid chain, lauric acid chain, palmitic acid chain connected to the N-terminus, G is glycine, V is valine, I isoleucine, F is phenylalanine, K is lysine, and R is arginine; NH2 is the amide group capping the C-terminus of the short peptide chain, which reduces one negatively charged carboxylic acid group in the peptide chain.

[0027] The specific lipopeptide synthesis method is as follows:

[0028] (1) Weigh appropriate amounts of fatty acids (octanoic acid chain, lauric acid chain, or palmitic acid chain), amide MHBA resin, Fmoc-protected side chain uncharged amino acids (glycine, and / or valine, and / or isoleucine, and / or phenylalanine), and C-terminal Fmoc-protected and side chain Boc-protected positively charged amino acids (lysine and / or arginine), and dissolve them in dimethylformamide (DMF) solvent;

[0029] (2) According to the requirements of CEM's Liberty blue microwave solid phase peptide synthesizer, the sample in the above step (1) is placed in a designated container according to the lipopeptide sequence to start synthesizing lipopeptide molecules;

[0030] (3) After the peptide synthesis is completed, the product is added with a cleavage agent prepared from trifluoroacetic acid (TFA), triisopropylsilane (TIS), and water to remove the resin, stirred at room temperature for 3 h, and filtered to collect the filtrate;

[0031] (4) adding a small amount of iced ether and collecting the precipitate by centrifugation, repeating the process three times, and then purifying the sample by preparative reverse HPLC, and finally freeze-drying the purified sample in vacuum to obtain the final product powder.

[0032] (5) The purity and molecular weight of the lipopeptide were tested by high performance liquid chromatography and mass spectrometry, confirming that the synthesized molecular sequence was correct and the purity was above 95%.

[0033] Example 2: The following provides the preparation method and material characterization of the antibacterial drugs in the form of gel, sol, and microneedle patch of the present invention. Taking the selected representative antibacterial lipopeptide molecules as an example, the remaining materials can be prepared according to this method through fine-tuning of the formula and conditions:

[0034] 2.1 Gel dosage form:

[0035] a. Weigh a certain amount of antimicrobial lipopeptide powder, such as C 16 -RRRVVV-NH2, dissolved in ultrapure water, the concentration range of lipopeptide molecules is 10 -3 ~10 2g / L, preferably, between 1 and 20 g / L (mass content is 0.1 to 2 wt%);

[0036] b. Accelerate the dissolution of peptide powder by stirring, ultrasound (5-30s), and heating (40-100°C, 5-30min);

[0037] c. Weigh a certain amount of phosphate and sodium chloride powder and dissolve them in ultrapure water. The concentration range of phosphate is 10 -3 ~1g / L, preferably, between 0.1~1g / L (mass content is 0.01~0.1wt%); the concentration range of sodium chloride is 10 -3 ~1g / L, preferably, between 0.1 and 1g / L (mass content is 0.01 to 0.1wt%);

[0038] d. Completely dissolve the phosphate and sodium chloride powders by stirring and ultrasonication (5 to 30 seconds);

[0039] e. The lipopeptide solution and the excipient solution are mixed in a volume ratio of 1:1, and stirred evenly, and the pH range is adjusted to 3 to 11 by adding 1M hydrochloric acid or sodium hydroxide, preferably, the pH is between 6 and 8;

[0040] f. After aging for 6 to 72 hours, preferably 24 to 72 hours, an invertible hydrogel dosage form can be obtained ( Figure 2 a);

[0041] g. Take a small amount of the hydrogel obtained in step f and dilute it with ultrapure water at a volume ratio of 1:10, and apply it on a carbon-supported copper grid. Using transmission electron microscopy technology, it can be observed that the antimicrobial lipopeptide self-assembles into a nanofiber structure ( Figure 3 ).

[0042] 2.2 Sol dosage form:

[0043] a. Weigh a certain amount of C8-KKIIVV-NH2 powder and dissolve it in ultrapure water. The concentration range of lipopeptide molecules is 10 -3 ~10 2 g / L, preferably, between 0.1 and 10 g / L (mass content is 0.01 to 1 wt%);

[0044] b. Accelerate the dissolution of peptide powder by stirring, ultrasound (5-30s), and heating (40-100°C, 5-30min);

[0045] c. Weigh a certain amount of tromethamine buffer salt and silver nitrate powder as auxiliary materials and dissolve them in ultrapure water. The concentration range of tromethamine buffer salt is 10 -3 ~10g / L, preferably, between 0.1 and 1g / L (mass content is 0.01 to 0.1wt%); the concentration range of silver nitrate is 10-3 ~1g / L, preferably, between 0.01 and 0.1g / L (mass content is 0.001 to 0.01wt%);

[0046] d. Completely dissolve the auxiliary material powder by stirring and ultrasonication (5 to 30 seconds);

[0047] e. The lipopeptide solution and the excipient solution are mixed in a volume ratio of 1:1, and stirred evenly, and the pH range is adjusted to 3 to 11 by adding 1M nitric acid solution, preferably, the pH is between 6 and 8;

[0048] f. After stirring and aging for 6 to 12 hours, a sol formulation can be obtained ( Figure 2 b).

[0049] 2.3 Microneedle patch:

[0050] a. Weigh a certain amount of C 16 -RRKII-NH2 powder, dissolved in ultrapure water, the concentration range of lipopeptide molecules is 10 -3 ~10 2 g / L, preferably, between 1 and 20 g / L (mass content is 0.1 to 2 wt%);

[0051] b. Accelerate the dissolution of peptide powder by stirring, ultrasound (5-30s), and heating (40-100°C, 5-30min);

[0052] c. Weigh a certain amount of hyaluronic acid powder and dissolve it in ultrapure water, wherein the concentration of hyaluronic acid is in the range of 1 to 200 g / L, preferably, between 20 and 200 g / L (mass content is 2 to 20 wt %);

[0053] d. Completely dissolve the hyaluronic acid powder by stirring and ultrasonication (5 to 30 seconds);

[0054] e. The lipopeptide solution and the hyaluronic acid solution are mixed in a volume ratio of 1:1, and stirred evenly, and the pH range is adjusted to 3 to 11 by adding 1M hydrochloric acid or sodium hydroxide, preferably, the pH is between 6 and 8;

[0055] f. Select conventional microneedle molds and related modules in the art; the selected microneedle mold is generally a square with a side length of 0.5-5 cm, or a circle with a diameter of 0.5-5 cm, or various patterns within the range of 0.5-5 cm, including 100-900 microneedle forming holes, a height of 50-1000 μm, a bottom diameter of 100-600 μm, a top of 1-10 μm, and the microneedle forming holes are cones or polygonal pyramids. Preferably, the selected microneedle mold is a square with a side length of 2 cm, or a circle with a diameter of 2 cm, including 400 microneedle forming holes, a height of 300 μm, a bottom diameter of 300 μm, a top of 10 μm, and the microneedle forming holes are cones.

[0056] g. Pour the solution obtained in step e into the microneedle mold, remove the bubbles in the mold, cure it after ultraviolet light irradiation, and finally peel off the microneedle patch from the microneedle mold (such as Figure 2 c).

[0057] Example 3: Determination of hydrophilicity of antimicrobial lipopeptides, taking a representative antimicrobial lipopeptide molecule as an example:

[0058] The antibacterial lipopeptide was dissolved in ultrapure water, and the peptide with a concentration range of 0.01-1 mg / mL was injected into the HPLC instrument, and the mobile phase A was water containing 0.1% trifluoroacetic acid, and the mobile phase B was acetonitrile containing 0.1% trifluoroacetic acid. The gradient elution parameters were: 0-1 minute, 95% A+5% B; 1-40 minutes, 95% A+5% B to 5% A+95% B; 40-45 minutes, 5% A+95% B to 95% A+5% B. The HPLC retention time is the peak time of the lipopeptide after testing by this method. The results are shown in Table 1 in Implementation 4. The longer the retention time, the stronger the hydrophobicity of the lipopeptide. The retention time of the lipopeptide of the n-octanoic acid group is short (~19 minutes), the retention time of the lipopeptide of the lauric acid group is medium (~22-25 minutes), and the retention time of the lipopeptide of the palmitic acid group is long (~25-26 minutes).

[0059] Example 4: Determination of antibacterial activity of antimicrobial lipopeptides, taking representative antimicrobial lipopeptide molecules as an example:

[0060] Prepare hydrolyzed casein peptone broth (Mueller-Hinton Broth, MHB) routinely. Dilute the antimicrobial lipopeptide sample in a 96-well plate with MHB by a 2-fold dilution method to prepare an antimicrobial lipopeptide solution with a concentration gradient. The bacteria tested include Escherichia coli (E. coli, ATCC 25922), Staphylococcus aureus (S. aureus, ATCC 6538), and clinically extracted drug-resistant bacteria such as extended-spectrum β-lactamase Escherichia coli (ESBL E. coli) and methicillin-resistant Staphylococcus aureus (MRSA). Take a single colony in MHB and culture it overnight at 37°C. Dilute the bacteria cultured overnight with MHB, and the final bacterial concentration is 106 CFU / mL, and mixed with the antimicrobial lipopeptide solution prepared by the 2-fold dilution method at a volume ratio of 1:1. After 24 hours of incubation, the absorbance at 600 nm (OD 600 ), the minimum inhibitory concentration is the minimum antimicrobial lipopeptide concentration that inhibits bacterial growth. Each sample was repeated 3 times.

[0061] Prepare RPMI-1640 medium routinely. Dilute the antimicrobial lipopeptide sample in 96-well plates with RPMI-1640 medium by 2-fold dilution to prepare an antimicrobial lipopeptide solution with a concentration gradient. Take a single colony of Candida albicans (C.albicans, SC5314) in RPMI-1640 medium and culture it overnight at 37°C. Dilute the overnight cultured fungus with RPMI-1640 medium to a final fungal concentration of 10 5 CFU / mL, and mixed with the antimicrobial lipopeptide solution prepared by the 2-fold dilution method at a volume ratio of 1:1. After 48 hours of incubation, the absorbance at 600 nm (OD 600 ), the minimum inhibitory concentration is the minimum antimicrobial lipopeptide concentration that inhibits fungal growth. Each sample was repeated 3 times.

[0062] The sequences, hydrophilicity, and antibacterial activity of representative antimicrobial lipopeptides are shown in Table 1. The longer the HPLC retention time, the stronger the hydrophobicity of the lipopeptide, and the smaller the minimum inhibitory concentration, the stronger the antibacterial activity of the antimicrobial lipopeptide. The results showed that the representative antimicrobial lipopeptides had antibacterial activity against Gram-negative bacteria, Gram-positive bacteria, and fungi, and some antimicrobial lipopeptides with moderate hydrophilicity had better antibacterial activity.

[0063] Table 1: HPLC retention time and minimum inhibitory concentration of representative antimicrobial lipopeptides

[0064]

[0065] Representative antimicrobial lipopeptides have antimicrobial activity against multidrug-resistant bacteria. Figure 4 As shown, it can be seen that the antimicrobial lipopeptides can kill drug-resistant bacteria at the 10 μM level, and the minimum inhibitory concentration for drug-resistant bacteria is lower than that for non-resistant bacteria, indicating that they have better anti-resistant bacteria activity. It should be pointed out that the antimicrobial lipopeptides designed by the present invention have antimicrobial activity against drug-resistant bacteria, and only some antimicrobial lipopeptides are shown in the figure.

[0066] Example 5: Detection of membrane-breaking activity of antimicrobial lipopeptides, taking representative antimicrobial lipopeptide molecules as an example:

[0067] 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC) and 1-Palmitoyl-2-oleoylphosphatidylglycerol (POPG) were selected to represent the uncharged and negatively charged phospholipid models, respectively. Uncharged cholesterol (Cholesterol) was selected to represent the components of the human cell membrane model. The POPG:POPC phospholipid powders with a molar ratio of 3:7 were mixed to prepare unilamellar liposome nanovesicles simulating the negatively charged bacterial membrane model. The Cholesterol:POPC liposome powders with a molar ratio of 5:5 were mixed to prepare unilamellar liposome nanovesicles simulating the uncharged human cell model. 40mM carboxyfluorescein (CF) was added during the preparation of nanovesicles, and the carboxyfluorescein outside the nanovesicles was filtered out through a 60g / L Sephadex G50 chromatography column to prepare nanovesicles encapsulating fluorescein dye.

[0068] The antimicrobial lipopeptide sample was diluted in a 96-well plate with PBS buffer by a 2-fold concentration dilution method to prepare an antimicrobial lipopeptide solution with a concentration gradient, and the liquid volume in each well was 100 μL. Subsequently, 0.1 mg / mL of nanovesicles loaded with fluorescein dye were added to each well of the 96-well plate. After 30 minutes of reaction, the fluorescein dye leaked from the nanovesicles was read out using a fluorescence spectrometer, with an excitation wavelength of 490 nm and an emission wavelength of 515 nm. Among them, the fluorescence emission signal of the untreated nanovesicles represents 0% cytosol leakage, and the fluorescence emission signal of the nanovesicles treated with 1% Triton X-100 represents 100% cytosol leakage.

[0069] like Figure 5 As shown, the representative antimicrobial lipopeptides all showed selective destruction of different cell models, that is, the destruction of negatively charged POPG / POPC nanovesicles simulating bacteria and fungi was greater than the destruction of uncharged Cholesterol / POPC nanovesicles simulating human cell membranes. This shows that the designed antimicrobial lipopeptides can selectively kill negatively charged bacteria and fungi, while being less toxic to uncharged host cells. In addition, a series of palmitoyl-modified antimicrobial lipopeptides that are easier to self-assemble cause almost no damage to host cells, indicating their superior cell selectivity.

[0070] Example 6: Detection of the secondary structure of antimicrobial lipopeptides, taking a representative antimicrobial lipopeptide molecule as an example:

[0071] The secondary structure of lipopeptides was detected using Chirascan series circular dichroism spectrometers. Lipopeptide samples were prepared in phosphate buffer to allow self-assembly. They were then diluted to a concentration of 0.1-0.5 mg / mL in phosphate buffer and 25 mM sodium dodecyl sulfate, respectively, to detect changes in the secondary structure of antimicrobial lipopeptides in an uncharged physiological environment and in a negatively charged bacterial and fungal environment. The optical path of the circular dichroism spectrum was 0.1-1 mm, and the scanning range was 190-260 nm.

[0072] like Figure 6 As shown, representative antimicrobial lipopeptides exhibit random structures (peak at 200nm) or β-folds (peak at 220nm) when self-assembled. When in a negatively charged bacterial environment, they transform into α-helices (double peaks at 205nm and 220nm) to varying degrees. In the random structure and β-fold, antimicrobial lipopeptides are not easy to interact with uncharged cell membranes, while the α-helical structure transformed after interacting with negatively charged cell membranes can destroy the bacterial and fungal envelope structure, thereby killing pathogens. This secondary structure transformation makes the antimicrobial lipopeptide cell-selective, so that it can kill pathogens while maintaining low cytotoxicity.

[0073] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned implementation method and embodiments, but the present invention is not limited to the above-mentioned method, especially the specific molecule, dosage form and application, that is, it does not mean that the present invention can only be implemented by relying entirely on the above-mentioned specific method. The technicians in the relevant technical field should understand that any transformation or improvement of the present invention, the replacement, combination and modification of the raw materials and auxiliary materials of the product of the present invention, the selection of specific method steps, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An antibacterial drug preparation, characterized in that: Comprising an amphiphilic antimicrobial lipopeptide and one or more pharmaceutically acceptable excipients; The amphiphilic antimicrobial lipopeptide is composed of a short peptide containing 6 amino acids and a fatty acid chain modified at the N-terminus of the short peptide chain, and the C-terminus of the short peptide chain is capped by an amide group; The short peptide sequence is KKIIVV, wherein I is isoleucine, K is lysine, and V is valine; The fatty acid modified at the N-terminus of the short peptide chain KKIIVV is n-octanoic acid; The auxiliary material is selected from one or more of phosphate, tromethamine buffer salt, tris(hydroxymethyl)methylglycine buffer salt, borate, adenosine triphosphate, chitosan, hyaluronic acid, poly(N-isopropylacrylamide), poly(lactic acid-glycolic acid copolymer), sodium alginate, polyvinyl alcohol, cellulose, polyvinyl pyrrolidone, and metal element ions.

2. Use of the antibacterial drug preparation according to claim 1 in the preparation of a broad-spectrum antibacterial drug for treating infections caused by Escherichia coli, Staphylococcus aureus, and Candida albicans.

3. The use according to claim 2, characterized in that: The broad-spectrum antibacterial drug is in the form of gel, sol or microneedle patch.

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