A supramolecular assembly antibacterial peptide preparation and a preparation method thereof

Abstract

The application discloses a kind of supramolecular assembly antibacterial peptide preparation, antibacterial peptide preparation is based on cucurbituril and phenylpropyl host-guest interaction, is formed by cucurbituril supramolecular assembly antibacterial short peptide.The application also discloses the preparation method of the above-mentioned supramolecular assembly antibacterial peptide preparation.Antibacterial peptide preparation of the application is prepared specific sequence antibacterial short peptide by Fmoc solid-phase synthesis method, antibacterial short peptide is added to solvent to obtain short peptide solution, cucurbituril aqueous solution is mixed with short peptide solution, incubation 30 minutes, obtain antibacterial peptide preparation.Antibacterial peptide preparation of the application improves the water solubility of aromatic group-containing antibacterial short peptide, reduces the hemolytic effect of antibacterial peptide preparation on red blood cells, the larger hydrophobic cavity of cucurbituril is bonded with two N-terminal phenylalanine to form host-guest ternary complex, and excellent killing effect is shown to bacteria and fungi.

Description

Technical Field

[0001] This invention relates to a supramolecularly assembled antimicrobial peptide formulation, and also to a method for preparing the above-mentioned supramolecularly assembled antimicrobial peptide formulation. Background Technology

[0002] Cucurbituril, resembling a gourd with open ends, possesses hydrophilic exterior and hydrophobic interior properties, along with strong and highly specific host-guest binding capabilities. It represents a new generation of supramolecular compounds following crown ethers, cyclodextrins, and calixarenes. Cucurbituril exhibits good water solubility and a small cavity size, and can simultaneously bind two guest molecules to form charge-transfer assemblies, making it well-suited for applications in biomedicine, such as molecular self-assembly and drug delivery. Researchers have found that cucurbituril has a strong binding affinity for aromatic amino acids, with the binding affinity to the N-terminal phenylalanine being significantly greater than that to phenylalanine located in the middle of the polypeptide sequence. This is primarily due to the hydrophobic interaction between the benzene ring of phenylalanine and the hydrophobic cavity of cucurbituril, as well as the ionic dipole interaction between the terminal amino group of phenylalanine and the carbonyl group of cucurbituril.

[0003] Drugs assembled via cucurbituril supramolecular assembly exhibit excellent delivery performance. The invention patent "A Targeted Peptide-Cucurbituril Supramolecular Assembly Based on Regulated Microtubule Aggregation and Its Preparation Method and Application" (Application No. 201910292361.0) chemically modifies benzylimidazole onto the peptide backbone, not only preserving the peptide's ability to target microtubule proteins but also providing a target for the non-covalent binding of benzylimidazole and cucurbituril. The invention patent "A Controllable Assembly of Fluorescent Gold Nanoclusters and Its Construction Method" (Application No. 202211342186.X) synthesizes gold nanoclusters containing the FGGGGGKRKC short peptide ligand. Upon addition of cucurbituril, the gold nanoclusters aggregate and assemble into nanoparticles with good specificity, providing a new strategy for drug delivery. The invention patent "A colorimetric method for detecting aminopeptidase N activity" (application number 201711348964.5) uses cucurbituril to recognize and bind to a polypeptide probe to detect aminopeptidase N activity. This supramolecular assembly shows good sensitivity and repeatability.

[0004] Over the past three decades, antimicrobial peptides (AMPs) have received widespread attention in the search for next-generation antibiotics. AMPs are a class of polypeptides encoded by specific genes in plant and animal cells and induced by external conditions, possessing cytotoxic effects against microorganisms such as bacteria, fungi, and viruses. Unlike most antibiotics, AMPs primarily kill or inhibit bacteria through membrane-active mechanisms, without involving site-specific binding or interfering with bacterial metabolism. However, some natural AMPs have poor water solubility, in vivo activity, and safety. Hemolysis can occur at concentrations exceeding a certain threshold, and their cationic and amphiphilic structures can lead to cytotoxicity, severely limiting the clinical application of AMPs. For example, aromatic antimicrobial peptides containing hydrophobic amino acids have low bioavailability due to their poor water solubility. Summary of the Invention

[0005] Objective of the invention: The present invention aims to provide a supramolecularly assembled antimicrobial peptide formulation that can improve the water solubility of aromatic antimicrobial short peptides and enhance their broad-spectrum antimicrobial properties and biocompatibility; another objective of the present invention is to provide a method for preparing the above-mentioned supramolecularly assembled antimicrobial peptide formulation.

[0006] Technical solution: The supramolecular assembled antimicrobial peptide preparation of the present invention is obtained by assembling cucurbituril and antimicrobial short peptides through host-guest interaction; wherein, both ends of the antimicrobial short peptide sequence are phenylalanine residues, and cucurbituril is included with the phenylpropyl groups at the ends of the antimicrobial short peptide to form a supramolecular structure; the antimicrobial short peptide sequence contains one or more of aspartic acid, glutamic acid, tryptophan, leucine, isoleucine, valine, alanine, or glycine.

[0007] The amino acid residues in the antibacterial short peptide sequence are arranged symmetrically.

[0008] The cucurbita is one or a combination of several of cucurbita[8], cucurbita

[10] , cucurbita

[14] or their derivatives. Large-sized cucurbita have good skeletal flexibility and good solubility in water and organic solvents.

[0009] The preparation method of the above-mentioned supramolecular assembled antimicrobial peptide preparation includes the following steps:

[0010] (1) Prepare antimicrobial short peptides with specific sequences using Fmoc solid-phase synthesis method;

[0011] (2) The prepared antibacterial short peptide is added to a solvent to obtain a short peptide solution;

[0012] (3) Mix cucurbituril aqueous solution with short peptide solution and incubate to obtain antimicrobial peptide preparation.

[0013] In step (2), the solvent is one of water, methanol, ethanol, isopropanol or DMSO.

[0014] In step (2), the short peptide solution contains short peptides with a mass concentration of 0.01 mg / mL to 2 mg / mL.

[0015] In step (3), the mass concentration of cucurbituril in the cucurbituril solution is 0.001 mg / mL; the mass ratio of the short peptide to cucurbituril is 3:1 to 1:5.

[0016] In step (3), the incubation time is no less than 30 minutes.

[0017] The antimicrobial peptide formulation of this invention is prepared by Fmoc solid-phase synthesis to produce antimicrobial short peptides (anionic short peptides) with specific sequences. The antimicrobial short peptides are added to a solvent to obtain a short peptide solution. A cucurbitac aqueous solution is mixed with the short peptide solution and incubated for 30 minutes to obtain the antimicrobial peptide formulation.

[0018] This invention relates to a supramolecularly assembled antimicrobial peptide formulation based on the host-guest interaction between cucurbituril and phenylpropyl groups, which is composed of anionic antimicrobial short peptides assembled from cucurbituril. Cucurbituril molecules exhibit good solubility in water and organic solvents. Cucurbituril has a strong binding affinity for antimicrobial peptides with N-terminal phenylalanine, and can be used to modify aromatic antimicrobial peptides, thereby improving the water solubility of aromatic-containing antimicrobial short peptides. In this supramolecularly assembled antimicrobial peptide formulation, on the one hand, the hydrophobic cavity of the large-sized cucurbituril can bind to two N-terminal phenylalanine groups, forming a host-guest ternary complex, thus enhancing the formulation's antimicrobial activity; on the other hand, the antimicrobial short peptides are anionic, avoiding interactions with cucurbituril molecules (cations interact with the carbonyl group of cucurbituril as an ionic dipole), and reducing the hemolytic effect of the antimicrobial peptide formulation on erythrocytes, thereby improving blood compatibility and cell compatibility.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The antimicrobial peptide preparation formed by the host-guest interaction of cucurbituril and phenylpropyl in the present invention can greatly improve the water solubility of aromatic antimicrobial short peptides. After assembly, the solubility in water is increased by 1 to 10 times compared with the antimicrobial short peptide before assembly, which is conducive to expanding the clinical application prospects of antimicrobial peptides; (2) The present invention uses anionic antimicrobial short peptides, which avoids the interaction with cucurbituril molecules and is conducive to reducing the hemolytic effect of antimicrobial peptide preparations on red blood cells, improving blood compatibility and cell compatibility; (3) The present invention uses the larger hydrophobic cavity bond of cucurbituril to form a host-guest ternary complex, which shows better killing effect on bacteria and fungi and has broad-spectrum antimicrobial properties. Detailed Implementation

[0020] Example 1

[0021] The method for preparing supramolecularly assembled antimicrobial peptide formulations of the present invention includes the following steps:

[0022] (1) The short peptide FLVELLEVLF was synthesized by Fmoc solid-phase synthesis method, and the obtained short peptide was prepared into a short peptide aqueous solution with a mass concentration of 0.01 mg / mL.

[0023] (2) Glycourea and formaldehyde were polymerized under acidic conditions. The type of acid, reaction temperature and raw material ratio were adjusted to synthesize cucurbita[8]. Then, based on its different solubility in hydrochloric acid solutions of different concentrations, it was separated by recrystallization multiple times to obtain cucurbita[8]. (In the example, cucurbita was synthesized using the following method reported in the literature: Day A, Arnold AP, Blanch RJ.J.Org.Chem.2001,66,8094-8100.Aav R,Kaabel S, M.ComprehensiveSupramolecular Chemistry II.2017,3,203-220.);

[0024] (3) Prepare a cucurbituril[8] solution with a mass concentration of 0.001 mg / mL; mix the cucurbituril solution with the short peptide solution, the mass ratio of the short peptide to cucurbituril[8] is 3:1, incubate for 30 minutes to obtain the assembled antimicrobial peptide preparation.

[0025] Using rabbit blood cells as a model, the hemolytic performance of supramolecularly assembled antimicrobial peptide preparations was evaluated. The hemolytic rate of antimicrobial short peptides before and after assembly by cucurbituril[8] was recorded. The results are shown in Table 1. Unassembled antimicrobial short peptides were used as the control group.

[0026] Table 1 Hemolysis rate of antimicrobial short peptides before and after cucurbituril[8] assembly

[0027]

[0028] As shown in Table 1, compared with unassembled short peptides, the antimicrobial peptide preparation assembled with cucurbituril [8] of this invention has a hemolysis rate of less than 5% on red blood cells, and is judged to be non-hemolytic. This is because cucurbituril [8] has good water solubility and cavity size, which reduces the hemolytic effect on blood cells and improves the biocompatibility of antimicrobial short peptides.

[0029] The cell viability of the antimicrobial peptide preparation was evaluated by co-culturing it with mouse fibroblasts L929 before and after assembly with cucurbitacin [8]. The results are shown in Table 2, with the unassembled antimicrobial peptide as the control group.

[0030] Table 2 Cell survival rates of antimicrobial short peptides before and after cucurbituril[8] assembly

[0031]

[0032] As shown in Table 2, compared with antimicrobial short peptides, the cell survival rate of L929 cells treated with the antimicrobial peptide preparation assembled by cucurbituril[8] of this invention was significantly improved. This is because cucurbituril[8] assembly not only improved the water solubility of the antimicrobial peptide, but also reduced the toxicity of the antimicrobial peptide to normal cells and improved the biocompatibility of the antimicrobial peptide.

[0033] The antibacterial properties of the antimicrobial peptide preparation assembled with cucurbituril [8] were determined by a quantitative bacterial killing method. The results showed that the antimicrobial properties were significantly improved compared with those of a concentration of 6*10. 7 ±0.01*10 7 After 20 min of treatment with E. coli at CFU / mL, the antimicrobial peptide preparation assembled from cucurbituril [8] prepared in Example 1 decreased to 1.02*10. 2 ±0.02*10 2 The concentration of CFU / mL was 6.73*10⁻⁶ CFU / mL, while the control group of unassembled antimicrobial short peptides had a concentration of 6.73*10⁻⁶ CFU / mL. 5 ±0.03*10 5 CFU / mL; with a concentration of 5.84*10 7 ±0.03*10 7 After 20 min of treatment with Staphylococcus aureus at CFU / mL, the antimicrobial peptide preparation assembled with cucurbituril [8] decreased to 1.42*10. 2 ±0.01*10 2 The concentration of CFU / mL was 8.44*10⁻⁶ CFU / mL, while the control group of unassembled antimicrobial short peptides had a concentration of 8.44*10⁻⁶ CFU / mL. 5 ±0.02*10 5 CFU / mL; with a concentration of 6.2*10 6 ±0.03*10 6 After co-culturing Candida albicans with CFU / mL for 20 min, the antimicrobial peptide preparation assembled with cucurbituril[8] decreased to 2.01*10. 2 ±0.02*10 2 CFU / mL, while the control group of unassembled antimicrobial short peptides had a concentration of 5.38*10. 4 ±0.03*10 4 CFU / mL. Cucurbita[8] showed strong bactericidal effects against Gram-positive, Gram-negative bacteria and fungi.

[0034] The antimicrobial activity of the cucurbitacin[8]-assembled antimicrobial peptide preparation was detected by determining the MIC value using the micro-broth dilution method. *Escherichia coli*, *Pseudomonas aeruginosa*, and *Acinetobacter baumannii* were cultured in Luria-Bertani broth, while *Staphylococcus aureus* was cultured in tryptone soybean broth. The results showed that the minimum inhibitory concentration (MIC) of the cucurbitacin[8]-assembled antimicrobial peptide preparation prepared in Example 1 was 2 μg / mL against *Escherichia coli*, 4 μg / mL against *Pseudomonas aeruginosa*, 4 μg / mL against *Acinetobacter baumannii*, and 2 μg / mL against *Staphylococcus aureus*. In contrast, the MIC of the unassembled antimicrobial short peptide control group against all four bacteria was 256 μg / mL. This indicates that the cucurbitacin[8]-assembled antimicrobial peptide preparation prepared in Example 1 has significant antimicrobial effects against *Escherichia coli*, *Pseudomonas aeruginosa*, *Acinetobacter baumannii*, and *Staphylococcus aureus*.

[0035] The antimicrobial activity of the cucurbitacin[8]-assembled antimicrobial peptide preparation prepared in Example 1 against drug-resistant bacteria was tested by double dilution. Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) were cultured in tryptone soybean broth. The results showed that the minimum inhibitory concentration (MIC) of the cucurbitacin[8]-assembled antimicrobial peptide preparation against MRSA was 8 μg / mL, and the MIC against MRSE was 8 μg / mL. The MIC of the unassembled antimicrobial peptide control group against both drug-resistant bacteria was 256 μg / mL. The results showed that the antimicrobial effect of the cucurbitacin[8]-assembled antimicrobial peptide preparation against drug-resistant bacteria was 32 times that of the unassembled antimicrobial peptide, indicating that the antimicrobial activity of the antimicrobial peptide was significantly enhanced.

[0036] Example 2

[0037] The method for preparing supramolecularly assembled antimicrobial peptide formulations of the present invention includes the following steps:

[0038] (1) The short peptide FVVDIWIDVVF was synthesized by Fmoc solid-phase synthesis method, and the obtained short peptide was prepared into a short peptide aqueous solution with a mass concentration of 0.1 mg / mL.

[0039] (2) Glycourea and formaldehyde were polymerized under acidic conditions. The type of acid, reaction temperature and raw material ratio were adjusted to synthesize cucurbita

[10] . Cucurbita was separated by recrystallization multiple times according to its different solubility in hydrochloric acid solutions of different concentrations

[10] .

[0040] (3) Prepare a cucurbituril

[10] solution with a mass concentration of 0.001 mg / mL; mix the cucurbituril solution with the short peptide solution, the mass ratio of the short peptide to cucurbituril

[10] is 1:1, incubate for 30 minutes to obtain the assembled antimicrobial peptide preparation.

[0041] The hemolysis rate of antimicrobial short peptides before and after assembly by cucurbituril

[10] was recorded, and the hemolytic performance of supramolecularly assembled antimicrobial peptide preparations was evaluated. The results are shown in Table 3, with unassembled antimicrobial short peptides as the control group.

[0042] Table 3 Hemolysis rate of antimicrobial short peptides before and after cucurbituril

[10] assembly

[0043]

[0044]

[0045] As shown in Table 3, compared with unassembled short peptides, the antimicrobial peptide preparation assembled with cucurbituril

[10] of the present invention has a hemolysis rate of less than 5% on red blood cells, and is judged to be non-hemolytic. This is because cucurbituril

[10] has good water solubility and cavity size, which improves the biocompatibility of the antimicrobial short peptide.

[0046] The cell viability of the antimicrobial peptide preparation was evaluated by co-culturing it with mouse fibroblasts L929 before and after assembly of the antimicrobial short peptide with cucurbituril

[10] . The results are shown in Table 4, with the unassembled antimicrobial short peptide as the control group.

[0047] Table 4 Cell viability of antimicrobial short peptides before and after cucurbituril

[10] assembly

[0048]

[0049] As shown in Table 4, compared with antimicrobial short peptides, the cell survival rate of L929 cells treated with the antimicrobial peptide preparation assembled by cucurbituril

[10] of this invention was significantly improved. This indicates that the assembly of cucurbituril

[10] not only improved the water solubility of the antimicrobial peptide, but also reduced the toxicity of the antimicrobial peptide to normal cells.

[0050] The antibacterial properties of the antimicrobial peptide preparation assembled with cucurbituril

[10] were determined by a quantitative bacterial killing method. The results showed that the antimicrobial properties were significantly improved compared with those of a concentration of 6.15*10. 7 ±0.02*10 7 After 20 min of treatment with E. coli at CFU / mL, the antimicrobial peptide preparation assembled from cucurbituril

[10] prepared in Example 2 decreased to 1.7*10. 2 ±0.01*10 2 CFU / mL, while the control group of unassembled antimicrobial short peptides had a concentration of 6.38*10. 5 ±0.02*10 5 CFU / mL; with a concentration of 4.67*10 7 ±0.03*10 7 After 20 min of treatment with Staphylococcus aureus at CFU / mL, the antimicrobial peptide preparation assembled with cucurbituril

[10] decreased to 1.3*10. 2 ±0.04*102 CFU / mL, while the control group of unassembled antimicrobial short peptides had a concentration of 6.38*10. 5 ±0.03*10 5 CFU / mL; with a concentration of 8.21*10 6 ±0.04*10 6 After co-culturing Candida albicans with CFU / mL for 20 min, the antimicrobial peptide preparation assembled with cucurbituril

[10] decreased to 3.83*10. 2 ±0.02*10 2 CFU / mL, while the control group of unassembled antimicrobial short peptides had a concentration of 5.63*10. 4 ±0.03*10 4 CFU / mL. Cucurbita

[10] showed strong bactericidal effects against Gram-positive, Gram-negative bacteria and fungi.

[0051] The antimicrobial activity of the cucurbitacin

[10] -assembled antimicrobial peptide preparation was detected by determining the MIC value using the micro-broth dilution method. *Escherichia coli*, *Pseudomonas aeruginosa*, and *Acinetobacter baumannii* were cultured in Luria-Bertani broth, while *Staphylococcus aureus* was cultured in tryptone soybean broth. The results showed that the minimum inhibitory concentration (MIC) of the cucurbitacin

[10] -assembled antimicrobial peptide preparation prepared in Example 2 was 1 μg / mL against *Escherichia coli*, 4 μg / mL against *Pseudomonas aeruginosa*, 8 μg / mL against *Acinetobacter baumannii*, and 4 μg / mL against *Staphylococcus aureus*. In contrast, the MIC of the unassembled antimicrobial short peptide control group against all four bacteria was 256 μg / mL. This indicates that the cucurbitacin

[10] -assembled antimicrobial peptide preparation prepared in Example 2 has significant antimicrobial effects against *Escherichia coli*, *Pseudomonas aeruginosa*, *Acinetobacter baumannii*, and *Staphylococcus aureus*.

[0052] The antimicrobial activity of the cucurbitacin

[10] -assembled antimicrobial peptide preparation prepared in Example 2 against drug-resistant bacteria was tested by double dilution. Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) were cultured in tryptone soybean broth. The results showed that the minimum inhibitory concentration (MIC) of the cucurbitacin

[10] -assembled antimicrobial peptide preparation against MRSA was 8 μg / mL, and against MRSE was 4 μg / mL. In contrast, the MIC of the unassembled antimicrobial peptide control group against both drug-resistant bacteria was 256 μg / mL. These results demonstrate that the antimicrobial activity of the cucurbitacin

[10] -assembled antimicrobial peptide preparation against drug-resistant bacteria was significantly enhanced.

[0053] Example 3

[0054] The method for preparing supramolecularly assembled antimicrobial peptide formulations of the present invention includes the following steps:

[0055] (1) The short peptide FLALEIIELALF was synthesized by Fmoc solid-phase synthesis method, and the obtained short peptide was prepared into a short peptide aqueous solution with a mass concentration of 1 mg / mL.

[0056] (2) Glycourea and formaldehyde were polymerized under acidic conditions. The type of acid, reaction temperature and raw material ratio were adjusted to synthesize cucurbita

[14] . Cucurbita was separated by recrystallization multiple times according to its different solubility in hydrochloric acid solutions of different concentrations

[14] .

[0057] (3) The obtained cucurbituril

[14] was prepared into a cucurbituril aqueous solution with a mass concentration of 0.001 mg / mL; the cucurbituril aqueous solution was mixed with the short peptide aqueous solution, the mass ratio of the short peptide to cucurbituril

[14] was 1:3, and incubated for 30 minutes to obtain the assembled antimicrobial peptide preparation.

[0058] The hemolysis rate of the antimicrobial short peptide before and after assembly by cucurbituril

[14] was recorded, and the hemolytic performance of the supramolecularly assembled antimicrobial peptide preparation was evaluated. The results are shown in Table 5, with the unassembled antimicrobial short peptide as the control group.

[0059] Table 5 Hemolysis rate of antimicrobial short peptides before and after cucurbituril

[14] assembly

[0060]

[0061] As shown in Table 5, compared with unassembled short peptides, the antimicrobial peptide preparation assembled with cucurbituril

[14] of the present invention has a hemolysis rate of less than 5% on red blood cells, and is judged to be non-hemolytic. This is because cucurbituril

[14] has good water solubility and cavity size, which reduces the hemolytic effect on blood cells.

[0062] The cell viability of the antimicrobial peptide preparation was evaluated by co-culturing it with mouse fibroblasts L929 before and after assembly of the antimicrobial short peptide with cucurbituril

[14] . The results are shown in Table 6, with the unassembled antimicrobial short peptide as the control group.

[0063] Table 6 Cell viability of antimicrobial short peptides before and after cucurbituril

[14] assembly

[0064]

[0065] As shown in Table 6, compared with antimicrobial short peptides, the cell survival rate of L929 cells treated with the antimicrobial peptide preparation assembled by cucurbituril

[14] of this invention was significantly improved. This is because cucurbituril

[14] assembly not only improved the water solubility of the antimicrobial peptide, but also enhanced its biocompatibility.

[0066] The antibacterial properties of the antimicrobial peptide preparation assembled from cucurbituril

[14] were determined by a quantitative bacterial killing method. The results showed that the antimicrobial properties were significantly improved compared with those of a concentration of 4.5 × 10⁻⁶. 7 ±0.03*10 7After 20 min of treatment with E. coli at CFU / mL, the antimicrobial peptide preparation assembled from cucurbituril

[14] prepared in Example 3 decreased to 1.26*10. 2 ±0.02*10 2 CFU / mL, while the control group of unassembled antimicrobial short peptides had a concentration of 5.1*10. 5 ±0.02*10 5 CFU / mL; with a concentration of 6.3*10 7 ±0.04*10 7 After 20 min of treatment with Staphylococcus aureus at CFU / mL, the antimicrobial peptide preparation assembled with cucurbituril

[14] decreased to 1.5*10. 2 ±0.03*10 2 The concentration of CFU / mL was higher than that of the unassembled antimicrobial short peptide control group, which was 7.22*10. 5 ±0.04*10 5 CFU / mL; with a concentration of 7.22*10 6 ±0.05*10 6 After co-culturing Candida albicans with CFU / mL for 20 min, the antimicrobial peptide preparation assembled with cucurbituril

[14] decreased to 2.6*10. 2 ±0.01*10 2 CFU / mL, while the control group of unassembled antimicrobial short peptides had a concentration of 4.89*10. 4 ±0.03*10 4 CFU / mL. Cucurbita

[14] showed strong bactericidal effects against Gram-positive, Gram-negative bacteria and fungi.

[0067] The antimicrobial activity of the cucurbitacin

[14] -assembled antimicrobial peptide preparation was detected by determining the MIC value using the micro-broth dilution method. *Escherichia coli*, *Pseudomonas aeruginosa*, and *Acinetobacter baumannii* were cultured in Luria-Bertani broth, while *Staphylococcus aureus* was cultured in tryptone soybean broth. The results showed that the minimum inhibitory concentration (MIC) of the cucurbitacin

[14] -assembled antimicrobial peptide preparation prepared in Example 3 was 2 μg / mL against *Escherichia coli*, 2 μg / mL against *Pseudomonas aeruginosa*, 4 μg / mL against *Acinetobacter baumannii*, and 4 μg / mL against *Staphylococcus aureus*. In contrast, the MIC of the unassembled antimicrobial short peptide control group against all four bacteria was 256 μg / mL. This indicates that the cucurbitacin

[14] -assembled antimicrobial peptide preparation prepared in Example 3 has significant antimicrobial effects against *Escherichia coli*, *Pseudomonas aeruginosa*, *Acinetobacter baumannii*, and *Staphylococcus aureus*.

[0068] The antimicrobial activity of the cucurbitacin

[14] -assembled antimicrobial peptide preparation prepared in Example 3 against drug-resistant bacteria was tested by double dilution. Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) were cultured in tryptone soybean broth. The results showed that the minimum inhibitory concentration (MIC) of the cucurbitacin

[14] -assembled antimicrobial peptide preparation against MRSA was 4 μg / mL, and the MIC against MRSE was also 4 μg / mL. In contrast, the MIC of the unassembled antimicrobial short peptide control group against both drug-resistant bacteria was 256 μg / mL. These results demonstrate that the antimicrobial effect of the cucurbitacin

[14] -assembled antimicrobial peptide preparation against drug-resistant bacteria is 64 times that of the unassembled antimicrobial short peptide.

[0069] Example 4

[0070] The method for preparing supramolecularly assembled antimicrobial peptide formulations of the present invention includes the following steps:

[0071] (1) The short peptide FGGDWDGGF was synthesized by Fmoc solid-phase synthesis method, and the obtained short peptide was prepared into a short peptide aqueous solution with a mass concentration of 2 mg / mL.

[0072] (2) Glycourea and formaldehyde were polymerized under acidic conditions. The type of acid, reaction temperature and raw material ratio were adjusted to synthesize cucurbita[8]. Cucurbita was separated by recrystallization multiple times according to its different solubility in hydrochloric acid solutions of different concentrations[8].

[0073] (3) Prepare a cucurbituril[8] solution with a mass concentration of 0.001 mg / mL; mix the cucurbituril solution with the short peptide solution, the mass ratio of the short peptide to cucurbituril[8] is 1:5, incubate for 30 minutes to obtain the assembled antimicrobial peptide preparation.

[0074] The hemolysis rate of the antimicrobial short peptide before and after assembly by cucurbituril [8] was recorded, and the hemolytic performance of the supramolecularly assembled antimicrobial peptide preparation was evaluated. The results are shown in Table 7, with the unassembled antimicrobial short peptide as the control group.

[0075] Table 7 Hemolysis rate of antimicrobial short peptides before and after cucurbituril [8] assembly

[0076]

[0077] As shown in Table 7, compared with unassembled short peptides, the antimicrobial peptide preparation assembled with cucurbituril [8] of this invention has a hemolysis rate of less than 5% on red blood cells, and is therefore determined to be non-hemolytic. This is because cucurbituril [8] has good water solubility and cavity size, which reduces the hemolytic effect on blood cells.

[0078] The cytotoxicity of antimicrobial peptide preparations was evaluated by detecting the cell viability of antimicrobial short peptides before and after assembly with cucurbituril[8] and co-cultured with mouse fibroblasts L929. The results are shown in Table 8, with unassembled antimicrobial short peptides as the control group.

[0079] Table 8 Cell survival rates of antimicrobial short peptides before and after cucurbituril [8] assembly

[0080]

[0081] As shown in Table 8, compared with antimicrobial short peptides, the cell survival rate of L929 cells treated with the antimicrobial peptide preparation assembled by cucurbituril[8] of this invention was significantly improved. This is because cucurbituril[8] assembly not only improved the water solubility of the antimicrobial peptide, but also enhanced its biocompatibility.

[0082] The antibacterial properties of the antimicrobial peptide preparation assembled with cucurbituril [8] were determined by a quantitative bacterial killing method. The results showed that the antimicrobial properties were significantly improved compared with those of a concentration of 7.23*10. 7 ±0.02*10 7 After 20 min of treatment with E. coli at CFU / mL, the antimicrobial peptide preparation assembled with cucurbituril [8] prepared in Example 4 decreased to 1.05*10. 2 ±0.02*10 2 The concentration of CFU / mL was higher than that of the unassembled antimicrobial short peptide control group, which was 7.72*10. 5 ±0.03*10 5 CFU / mL; with a concentration of 7*10 7 ±0.03*10 7 After 20 min of treatment with Staphylococcus aureus at CFU / mL, the antimicrobial peptide preparation assembled with cucurbituril[8] decreased to 1.28*10. 2 ±0.02*10 2 CFU / mL, while the control group of unassembled antimicrobial short peptides had a concentration of 6.28*10. 5 ±0.03*10 5 CFU / mL; with a concentration of 6.8*10 6 ±0.04*10 6 After co-culturing Candida albicans with CFU / mL for 20 min, the antimicrobial peptide preparation assembled with cucurbituril[8] decreased to 3.56*10. 2 ±0.02*10 2 CFU / mL, while the control group of unassembled antimicrobial short peptides had a concentration of 5.56*10. 4 ±0.02*10 4 CFU / mL. Cucurbita[8] showed strong bactericidal effects against Gram-positive, Gram-negative bacteria and fungi.

[0083] The antimicrobial activity of the cucurbitacin[8]-assembled antimicrobial peptide preparation was detected by determining the MIC value using the micro-broth dilution method. *Escherichia coli*, *Pseudomonas aeruginosa*, and *Acinetobacter baumannii* were cultured in Luria-Bertani broth, while *Staphylococcus aureus* was cultured in tryptone soybean broth. The results showed that the minimum inhibitory concentration (MIC) of the cucurbitacin[8]-assembled antimicrobial peptide preparation prepared in Example 4 was 1 μg / mL against *Escherichia coli*, 2 μg / mL against *Pseudomonas aeruginosa*, 4 μg / mL against *Acinetobacter baumannii*, and 2 μg / mL against *Staphylococcus aureus*. In contrast, the MIC of the unassembled antimicrobial short peptide control group against all four bacteria was 256 μg / mL. This indicates that the cucurbitacin[8]-assembled antimicrobial peptide preparation prepared in Example 4 has significant antimicrobial effects against *Escherichia coli*, *Pseudomonas aeruginosa*, *Acinetobacter baumannii*, and *Staphylococcus aureus*.

[0084] The antimicrobial activity of the cucurbitacin[8]-assembled antimicrobial peptide preparation prepared in Example 4 against drug-resistant bacteria was tested by double dilution. Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) were cultured in tryptone soybean broth. The results showed that the minimum inhibitory concentration (MIC) of the cucurbitacin[8]-assembled antimicrobial peptide preparation against MRSA was 8 μg / mL, and the MIC against MRSE was 8 μg / mL. In contrast, the MIC of the unassembled antimicrobial short peptide control group against both drug-resistant bacteria was 256 μg / mL. The results demonstrate that the antimicrobial effect of the cucurbitacin[8]-assembled antimicrobial peptide preparation against drug-resistant bacteria was 32 times that of the unassembled antimicrobial short peptide, and the antimicrobial activity of the antimicrobial short peptide was significantly enhanced.

[0085] Example 5

[0086] The method for preparing supramolecularly assembled antimicrobial peptide formulations of the present invention includes the following steps:

[0087] (1) The short peptide FELLELLEF was synthesized by Fmoc solid-phase synthesis method, and the obtained short peptide was prepared into a short peptide aqueous solution with a mass concentration of 0.15 mg / mL.

[0088] (2) Glycourea and formaldehyde were polymerized under acidic conditions. The type of acid, reaction temperature and raw material ratio were adjusted to synthesize cucurbita

[10] . Cucurbita was separated by recrystallization multiple times according to its different solubility in hydrochloric acid solutions of different concentrations

[10] .

[0089] (3) Prepare a cucurbituril

[10] solution with a mass concentration of 0.001 mg / mL; mix the cucurbituril solution with the short peptide solution, the mass ratio of the short peptide to cucurbituril

[10] is 2:1, incubate for 30 minutes to obtain the assembled antimicrobial peptide preparation.

[0090] The hemolysis rate of antimicrobial short peptides before and after assembly by cucurbituril

[10] was recorded, and the hemolytic performance of supramolecularly assembled antimicrobial peptide preparations was evaluated. The results are shown in Table 9, with unassembled antimicrobial short peptides as the control group.

[0091] Table 9 Hemolysis rate of antimicrobial short peptides before and after cucurbituril

[10] assembly

[0092]

[0093] As shown in Table 9, compared with unassembled short peptides, the antimicrobial peptide preparation assembled with cucurbituril

[10] of the present invention has a hemolysis rate of less than 5% on red blood cells, and is judged to be non-hemolytic. This is because cucurbituril

[10] has good water solubility and cavity size, which reduces the hemolytic effect on blood cells and improves the biocompatibility of antimicrobial short peptides.

[0094] The cell viability of the antimicrobial peptide preparation was evaluated by co-culturing it with mouse fibroblasts L929 before and after assembly of the antimicrobial short peptide with cucurbituril

[10] . The results are shown in Table 10, with the unassembled antimicrobial short peptide as the control group.

[0095] Table 10 Cell viability of antimicrobial short peptides before and after cucurbituril

[10] assembly

[0096]

[0097]

[0098] As shown in Table 10, compared with antimicrobial short peptides, the cell survival rate of L929 cells treated with the antimicrobial peptide preparation assembled by cucurbituril

[10] of this invention was significantly improved. This is because cucurbituril

[10] assembly not only improved the water solubility of the antimicrobial peptide, but also reduced the toxicity of the antimicrobial peptide to normal cells.

[0099] The antibacterial properties of the antimicrobial peptide preparation assembled with cucurbituril

[10] were determined by a quantitative bacterial killing method. The results showed that the antimicrobial properties were significantly improved compared with those of a concentration of 7.1 × 10⁻⁶. 7 ±0.04*10 7 After 20 min of treatment with E. coli at CFU / mL, the antimicrobial peptide preparation assembled from cucurbituril

[10] prepared in Example 5 decreased to 1.25*10. 2 ±0.04*10 2 CFU / mL, while the control group of unassembled antimicrobial short peptides had a concentration of 6.9*10. 5 ±0.04*10 5 CFU / mL; with a concentration of 7.5*10 7 ±0.04*10 7 After 20 min of treatment with Staphylococcus aureus at CFU / mL, the antimicrobial peptide preparation assembled with cucurbituril

[10] decreased to 1.81*10.2 ±0.03*10 2 The concentration of CFU / mL was 6.49*10⁻⁶ CFU / mL, while the control group of unassembled antimicrobial short peptides had a concentration of 6.49*10⁻⁶ CFU / mL. 5 ±0.03*10 5 CFU / mL; with a concentration of 4.29*10 6 ±0.04*10 6 After co-culturing Candida albicans with CFU / mL for 20 min, the antimicrobial peptide preparation assembled with cucurbituril

[10] decreased to 1.49*10. 2 ±0.03*10 2 The concentration of CFU / mL was higher than that of the unassembled antimicrobial short peptide control group, which was 5.3*10. 4 ±0.02*10 4 CFU / mL. Cucurbita

[10] forms a host-guest ternary complex with its large hydrophobic cavity and short peptide, which shows strong bactericidal effect against Gram-positive, Gram-negative bacteria and fungi.

[0100] The antimicrobial activity of the cucurbitacin

[10] -assembled antimicrobial peptide preparation was detected by determining the MIC value using the micro-broth dilution method. *Escherichia coli*, *Pseudomonas aeruginosa*, and *Acinetobacter baumannii* were cultured in Luria-Bertani broth, while *Staphylococcus aureus* was cultured in tryptone soybean broth. The results showed that the minimum inhibitory concentration (MIC) of the cucurbitacin

[10] -assembled antimicrobial peptide preparation prepared in Example 5 was 2 μg / mL against *Escherichia coli*, 4 μg / mL against *Pseudomonas aeruginosa*, 4 μg / mL against *Acinetobacter baumannii*, and 2 μg / mL against *Staphylococcus aureus*. In contrast, the MIC of the unassembled antimicrobial short peptide control group against all four bacteria was 256 μg / mL. This indicates that the cucurbitacin

[10] -assembled antimicrobial peptide preparation prepared in Example 5 has significant antimicrobial effects against *Escherichia coli*, *Pseudomonas aeruginosa*, *Acinetobacter baumannii*, and *Staphylococcus aureus*.

[0101] The antimicrobial activity of the cucurbitacin

[10] -assembled antimicrobial peptide preparation prepared in Example 5 against drug-resistant bacteria was tested by double dilution. Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) were cultured in tryptone soybean broth. The results showed that the minimum inhibitory concentration (MIC) of the cucurbitacin

[10] -assembled antimicrobial peptide preparation against MRSA was 4 μg / mL, and against MRSE it was 8 μg / mL. In contrast, the MIC of the unassembled antimicrobial peptide control group against both drug-resistant bacteria was 256 μg / mL. These results demonstrate that the antimicrobial effect of the cucurbitacin

[10] -assembled antimicrobial peptide preparation against drug-resistant bacteria was 32–64 times that of the unassembled antimicrobial peptide, indicating that the antimicrobial activity of the antimicrobial peptide was significantly enhanced.

Claims

1. A supramolecularly assembled antimicrobial peptide formulation, characterized in that: The antimicrobial peptide preparation is assembled from cucurbituril and antimicrobial short peptides through host-guest interaction; wherein, both ends of the antimicrobial short peptide sequence are phenylalanine residues, and cucurbituril is included with the phenylpropyl groups at the ends of the antimicrobial short peptide to form a supramolecular structure; the antimicrobial short peptide is one of FLVELLEVLF, FVVDIWIDVVF, FLALEIIELALF, FGGDWDGGF or FELLELLEF; the cucurbituril is one or a combination of several of cucurbituril[8], cucurbituril[10] or cucurbituril[14].

2. The supramolecular assembled antimicrobial peptide formulation according to claim 1, characterized in that: The amino acid residues in the antibacterial short peptide sequence are arranged symmetrically.

3. The method for preparing the supramolecular assembled antimicrobial peptide formulation according to claim 1, characterized in that, The steps include the following: (1) Prepare antimicrobial short peptides with specific sequences using the Fmoc solid-phase synthesis method; (2) The prepared antibacterial short peptides are added to a solvent to obtain a short peptide solution; (3) Mix cucurbituril aqueous solution with short peptide solution and incubate to obtain antimicrobial peptide preparation.

4. The method for preparing the supramolecular assembled antimicrobial peptide formulation according to claim 3, characterized in that: In step (2), the solvent is one of water, methanol, ethanol, isopropanol or DMSO.

5. The method for preparing supramolecularly assembled antimicrobial peptide formulations according to claim 3, characterized in that: In step (2), the short peptide solution contains short peptides with a mass concentration of 0.01 mg / mL to 2 mg / mL.

6. The method for preparing the supramolecular assembled antimicrobial peptide formulation according to claim 3, characterized in that: In step (3), the mass concentration of cucurbituril in the aqueous solution is 0.001 mg / mL; the mass ratio of the short peptide to cucurbituril is 3:1 to 1:

5.

7. The method for preparing the supramolecular assembled antimicrobial peptide formulation according to claim 3, characterized in that: In step (3), the incubation time is no less than 30 minutes.

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