Multi-effect microrhin chitosan nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a multi-effect microrhin chitosan nanoparticle as well as a preparation method and application thereof, the chitosan nanoparticle comprises more than one lasso peptide, has a multi-effect synergistic antibacterial effect, and realizes a broad-spectrum antibacterial effect. The chitosan nanoparticles prepared by the invention can also comprise arginine, so that the acid-base tolerance of the lasso peptide is improved, and the application bottleneck of adding the lasso peptide for livestock and poultry feeding is broken through. In addition, the chitosan nanoparticles prepared by the preparation method disclosed by the invention also have relatively high safety.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to pleiotropic microcin chitosan nanoparticles and a preparation method and application thereof. Background Art

[0002] The problem of drug resistance in Enterobacteriaceae caused by the abuse of antibiotics has attracted the attention of the international community. In order to reduce dependence on antibiotics and ensure the health of livestock and poultry, antibiotic-free breeding has gradually become the future development direction of my country's animal husbandry. Previously, there was no ideal alternative antibiotic product to deal with the common Enterobacteriaceae in livestock and poultry breeding, and microcin (Mcc) showed great potential in dealing with antibiotic resistance. Lasso peptides (LPs) are a class of small molecule microcins (<10kDa) with a "lasso" structure assembled by ribosomes and post-translationally modified, with stable and efficient bactericidal biological activity. Common lasso peptides mainly include MccJ25, MccY, and Klebsidin. After entering the bacterial cell, they inhibit the activity of RNA polymerase to produce a highly efficient bactericidal effect (MIC = 0.001-1.0μg / mL). Previous studies have found that the new natural lasso peptide Microcin Y (MccY) has obvious complementary bactericidal advantages against Salmonella and Shigella compared to MccJ25, while MccJ25 and MccY are insensitive to Klebsiella, but the lasso peptide Klebsidin has a certain inhibitory effect on Klebsiella. Compared with other lasso peptides such as Citrocin, MccY, MccJ25 and Klebsidin have strong antibacterial activity against Enterobacteriaceae and Gram-positive bacteria (MIC = 0.001-20.0μg / mL). Although lasso peptides have the advantage of efficient bactericidal, the bacteria targeted by a single lasso peptide have targeting specificity; secondly, lasso peptide microcins are generally acid-base instability, and sensitivity to digestive enzymes has become the biggest obstacle in the process of lasso peptide feeding. Summary of the invention

[0003] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention provides a multi-effect microbactin chitosan nanoparticle, which contains a plurality of different lasso peptides coupled simultaneously, synergizes the specific antibacterial effects between the various lasso peptides, and achieves a broad-spectrum antibacterial effect. In addition, this nanoparticle can improve the acid-base tolerance and safety of the lasso peptide, so as to break through the application bottleneck of adding lasso peptide to livestock and poultry feed.

[0004] In order to achieve the above object of the invention, the first aspect of the present invention provides a chitosan (CN) nanoparticle, wherein the chitosan nanoparticle comprises chitosan and one or more lasso peptides, wherein the lasso peptide has a carboxyl group, and the amino group in the chitosan structure is connected to the carboxyl group of the lasso peptide through an amide bond.

[0005] Lasso peptides (LPs) are natural polypeptide products found in bacteria. They are a class of biologically active peptides with a lasso configuration that are synthesized by ribosomes and produced through post-translational modification. The lasso peptide structure usually contains 14-33 amino acid residues. The first amino acid at the N-terminus forms a large amide ring with the 7th, 8th or 9th amino acid side chain, and the amino acid at the C-terminus passes through this large amide ring to form a signature "lasso" structure. Lasso peptides can be divided into four categories based on the number of disulfide bonds they contain and the positions of their formation (e.g. Figure 8 As shown). The lasso peptides whose C-terminal peptide chain is connected to the large peptide ring by two disulfide bonds are defined as Class I lasso peptides (Class I); if the peptide tail passes through the peptide ring, there is no disulfide bond between the C-terminal peptide chain and the large peptide ring, but the lasso topology is stabilized by spatial interaction, it is called Class II lasso peptide (Class II); if there is only one disulfide bond between the C-terminal sequence of the peptide tail and the peptide ring, the lasso peptide is divided into Class III or Class IV lasso peptides (Class III or Class IV) according to the different positions of the disulfide bond formation. Specifically, the disulfide bond of Class III lasso peptides connects the peptide ring to the peptide tail, while the disulfide bond of Class IV only exists at the peptide tail. The four types of lasso peptides shown all have carboxyl groups.

[0006] In some embodiments of the present invention, the molar ratio of chitosan to lasso peptide is 5:(3-4).

[0007] In some embodiments of the invention, the lasso peptides include MccJ25, MccY, Klebsidin, Citrocin, Lihuanodin, Synthetase B1, Astexin 1, Koveensin, MccS and MccC7.

[0008] In some embodiments of the invention, the lasso peptide comprises MccY and MccJ25.

[0009] In some embodiments of the invention, the lasso peptide comprises MccY and Klebsidin.

[0010] In some embodiments of the invention, the lasso peptide comprises MccJ25 and Klebsidin.

[0011] In some embodiments of the invention, the lasso peptide comprises MccY, MccJ25 and Klebsidin.

[0012] In the present invention, CN nanoparticles comprising a single MccY lasso peptide (CN-MccY) have no antibacterial effect on Serratia marcescens and Klebsiella pneumoniae; CN nanoparticles comprising a single MccJ25 lasso peptide (CN-MccJ25) have no antibacterial effect on Salmonella typhimurium; CN nanoparticles comprising a single Klebsidin lasso peptide (CN-Klebsidin) have no antibacterial effect on Salmonella typhimurium.

[0013] Further, in the present invention, the combined use of MccY and MccJ25 exhibits an additive inhibitory effect on Salmonella enteritidis, Salmonella typhimurium, Serratia marcescens and Klebsiella pneumoniae, and a synergistic inhibitory effect on Staphylococcus aureus; the combined use of MccY and Klebsidin exhibits an additive inhibitory effect on Salmonella enteritidis, Salmonella typhimurium, Serratia marcescens, and a synergistic inhibitory effect on Klebsiella pneumoniae and Staphylococcus aureus; the combined use of MccJ25 and Klebsidin exhibits an additive inhibitory effect on Salmonella enteritidis, Klebsiella pneumoniae pneumoniae and Escherichia coli showed additive inhibition, and showed synergistic inhibition against Serratia marcescens. Therefore, CN nanoparticles (CN-LPs) including MccY, MccJ25 and Klebsidin lasso peptide showed synergistic effects at least against Klebsiella pneumoniae, Staphylococcus aureus and Serratia marcescens, and showed additive inhibition against Salmonella enteritidis and Salmonella typhimurium.

[0014] In the present invention, CN nanoparticles (CN-LPs) including MccY, MccJ25 and Klebsidin lasso peptide extended the antibacterial activity of microcin to major foodborne pathogens Salmonella, Serratia and Klebsiella.

[0015] In some embodiments of the present invention, the molar ratio of CN, MccY, MccJ25 and Klebsidin is 5:(1-2):1:1.

[0016] In some embodiments of the present invention, the chitosan nanoparticles further include arginine (Arg), and the amino group in the chitosan structure is connected to the carboxyl group of arginine via an amide bond.

[0017] In some embodiments of the present invention, the molar ratio of CN to Arg is (1-10):1.

[0018] In the present invention, the arginine is coupled to chitosan via an amide bond, thereby improving the acid-base stability of the chitosan nanoparticles.

[0019] In some embodiments of the present invention, the particle size of the chitosan nanoparticles is 100-200 nm.

[0020] In a second aspect of the present invention, a method for preparing chitosan nanoparticles is provided, wherein the chitosan nanoparticles according to the first aspect of the present invention are prepared by the method.

[0021] In some embodiments of the present invention, the preparation method comprises the following steps:

[0022] 1) mixing a 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution and an N-hydroxysuccinimide solution (NHS) to obtain an EDCN mixed solution;

[0023] 2) dissolving chitosan and lasso peptide in the acetylation solution respectively, and mixing them evenly to obtain chitosan acetylation solution and lasso peptide acetylation solution;

[0024] 3) adding the EDCN mixed solution in step 1) to the chitosan acetylated solution and the lasso peptide acetylated solution in step 2) respectively, and mixing them evenly to obtain a chitosan-EDCN mixed solution and a lasso peptide-EDCN mixed solution;

[0025] 4) The chitosan-EDCN mixed solution in step 3) and the lasso peptide-EDCN mixed solution are sequentially mixed, and stirred sufficiently to complete the esterification reaction, thereby preparing the chitosan nanoparticles.

[0026] In some embodiments of the present invention, the step 2) further comprises the preparation of an arginine (Arg) acetylation solution, and the preparation step comprises: dissolving arginine in the acetylation solution and mixing uniformly to obtain the arginine acetylation solution.

[0027] In some embodiments of the present invention, the step 3) further comprises the preparation of an arginine-EDCN mixed solution, the preparation step comprising: adding the EDCN mixed solution in step 1) to the arginine acetylation solution in step 2), mixing uniformly, and obtaining an arginine-EDCN mixed solution.

[0028] In some embodiments of the present invention, the step 4) further comprises mixing with the arginine-EDCN mixed solution in step 3).

[0029] In some embodiments of the present invention, the acetylation solution includes sodium acetate solution, acetyl chloride solution, acetic anhydride solution and glacial acetic acid solution.

[0030] In some embodiments of the present invention, the acetylation solution comprises sodium acetate buffer (SAB).

[0031] In the third aspect of the present invention, there is provided use of the chitosan nanoparticles according to the first aspect of the present invention in preparing a product having an antibacterial effect.

[0032] In some embodiments of the present invention, the bacteria include Salmonella, Serratia and Klebsiella.

[0033] In some embodiments of the present invention, the Salmonella spp. includes Salmonella enteritidis and Salmonella typhimurium.

[0034] In some embodiments of the invention, the Serratia genus includes Serratia marcescens.

[0035] In some embodiments of the invention, the Klebsiella genus includes Klebsiella pneumoniae.

[0036] In some embodiments of the present invention, the product includes feed, preservatives, disinfectants and medicines for external wound infections.

[0037] The beneficial effects of the present invention are:

[0038] 1) The chitosan nanoparticles prepared by the present invention, including chitosan and more than one lasso peptide, have multi-effect synergistic antibacterial effects on a variety of bacteria, and synergize the specific antibacterial effects between a variety of different lasso peptides, thereby achieving a broad-spectrum antibacterial effect.

[0039] 2) The chitosan nanoparticles prepared by the present invention are also connected with arginine (Arg) via an amide bond, thereby improving the acid-base tolerance of lasso peptide and breaking through the application bottleneck of adding lasso peptide to livestock and poultry feed.

[0040] 3) The chitosan nanoparticles prepared by the present invention have high safety and will not cause hemolysis even at a high concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the design diagram of the CN-LPs-Arg synthesis route in Example 1.

[0042] Figure 2is the characterization analysis result of the nanomaterial CN-LPs-Arg in Example 2; wherein Figure 2 A is the FTIR analysis result of CN, Figure 2 B is the FTIR analysis result of CN-LPs-Arg, Figure 2 C is the particle size analysis result of CN-LPs-Arg, Figure 2 D is the Zeta potential characterization analysis diagram of CN-LPs-Arg.

[0043] Figure 3 is CN( Figure 3 A)、CN-Arg( Figure 3 B) and CN-LPs-Arg( Figure 3 C) Transmission electron microscopy images of three types of CN nanoparticles.

[0044] Figure 4 is the result of the physical and chemical stability test of CN nanoparticles in Example 4; wherein Figure 4 A-4C are the UPLC-Q-TOF mass spectrometry results of MccY, Klebsidin and MccJ25, Figure 4 D-4E is the stability analysis result of MccY at 95℃, pH=2.0 and pH=12.0. Figure 4 F is the stability analysis result of CN-LPs-Arg at 95℃, pH=2.0 and pH=12.0. Figure 4 G is the antibacterial activity of CN-LPs-Arg after treatment at 95°C, pH = 2.0 and pH = 12.0, determined by spot method.

[0045] Figure 5 These are the spot plate test results of CN, Arg, and various CN nanoparticles (CN-Arg, CN-MccY, CN-MccJ25, CN-Klebsidin, CN-LPs, and CN-LPs-Arg), showing the antibacterial effects determined by the spot assay; **p<0.01, ***p<0.001, ****p<0.0001.

[0046] Figure 6 This is the experimental process of FICI combined antibacterial analysis of lasso peptides MccY, MccJ25, and Klebsidin.

[0047] Figure 7 is the biocompatibility analysis result of CN nanoparticles; Figure 7 A shows the morphology of red blood cells after CN nanoparticle treatment; Figure 7 B: The cytotoxicity of CN nanoparticles on HEK293T cells was evaluated by MTT assay; Figure 7 C is the hemolytic effect of CN nanoparticles on red blood cells.

[0048] Figure 8 Schematic diagram of the structures of four types of lasso peptides. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0050] The instrument information used in this example is shown in Table 1 below:

[0051] Table 1 Instrument information used

[0052]

[0053]

[0054] Statistical analysis GraphPad Prism 8.0 software was used for statistical analysis, and the statistical results within the group were expressed as Mean ± SD. One-way analysis of variance was used for comparisons between two groups and three or more groups. Student's t-test was used to compare significant differences between groups, and each treatment was repeated 3 times independently.

[0055] Example 1: Chemical synthesis of CN-LPs-Arg

[0056] In this example, secretory proteins were obtained by the E. coli prokaryotic expression system, and three lasso peptides (MccY, MccJ25 and Klebsidin) were prepared by HPLC. The preparation method is shown in Reference 1. The multifunctional nanomaterials were obtained by coupling with chitosan (CN) through chemical reaction. Figure 1 The synthesis process is described in detail. The amino group of CN is connected to the carboxyl group of the lasso peptide through an amide bond to synthesize a variety of CN nanoparticles; the carboxyl groups of MccY, MccJ25 and Klebsidin are activated by EDC and then coupled with CN in sequence to form CN-LPs. In addition, in order to improve the acid and alkali resistance, Arg is introduced to combine with CN-LPs to form CN-LPs-Arg. The amino group of CN-LPs and the carboxyl group of Arg react in the presence of water-soluble EDC to form a new amide bond. The optimized formula of these nanomaterials achieves an encapsulation efficiency of more than 60% w / w in CN and encapsulates approximately 90% of the lasso peptide. The synthesis process of CN-LPs-Arg is as follows:

[0057] The expression and preparation of the three lasso peptides were as follows: the vector pET-28a(+) was introduced into BL21(DE3) competent cells to obtain BL21(DE3) bacteria containing pET-28a(+), the BL21(DE3) bacteria containing pET-28a(+) were revived in an LB plate with a kanamycin concentration of 30 μg / mL, and a loopful of bacteria was inoculated in a 100 mL plate containing In a 250 ml Erlenmeyer flask containing LB medium, kanamycin and isopropyl-β-D-thiogalactoside (IPTG) (the final concentration of kanamycin is 30 μg / mL, and the final concentration of IPTG is 0.1 M) are added, and the flask is placed in a shaker for culture (culture conditions are: 200 r / min, 37°C, 14 h); the bacterial solution after expression is collected, and after centrifugation at 5000 r / min for 20 min, the supernatant after centrifugation is collected, and filtered using a 0.22 μm filter membrane to obtain a supernatant filtrate, and then reverse solid phase extraction technology is used to enrich the supernatant with a C18 column of SPE, and then Q-TOF is used to identify the expressed target protein, and then preparative HPLC is used to collect and purify the target protein, and vacuum distillation is used to concentrate it, and finally the target protein is dried, weighed and quantified using a freezing method.

[0058] The synthesis of CN-LPs-Arg is mainly divided into two steps: In the first step, MccY, MccJ25 and Klebsidin are coupled with CN step by step. Prepare the reaction reagents: 0.5mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) solution and 0.25mM N-hydroxysuccinimide solution (NHS), mixed in a volume ratio of 1:1 to form EDCN solution. Prepare 0.1M sodium acetate buffer (SAB, pH=6.4). Weigh CN and dissolve it in SAB to prepare a 1mg / mL CN acetylation solution. Add 100μL EDCN to activate the amino group of CN, and the volume ratio of EDCN solution to CN acetylation solution is 1:10 to generate CN-EDCN solution. Dissolve 1mg of the three lasso peptides in SAB to obtain 1mg / mL lasso peptide SAB solution. 100 μL of EDCN solution was added to the lasso peptide SAB solution to activate the lasso peptide carboxyl group. The volume ratio of EDCN solution to lasso peptide SAB solution was 1:10, and MccY-EDCN solution, MccJ25-EDCN solution and Klebsidin-EDCN solution were generated respectively. 1 mL of MccY-EDCN solution was added to 1 mL of CN-EDCN solution, and stirred well to complete the esterification reaction to generate a CN-MccY EDCN solution. 1 mL of MccJ25-EDCN solution was added to 2 mL of CN-MccY EDCN solution, and stirred to cause an esterification reaction to generate a CN-MccY-MccJ25 EDCN solution. Then 1 mL of Klebsidin-EDCN solution was added to 3 mL of CN-MccY-MccJ25 EDCN solution, and stirred well to complete the esterification reaction, and a CN-LPs EDCN solution was generated.

[0059] In the second step, Arg is coupled with the amino group of CN under certain conditions to form a new amide bond, so that CN-LPs completes amino acid modification to synthesize CN-LPs-Arg, wherein the introduction of Arg can further improve the acid and alkali stability of CN-LPs. Specifically, 1 mg of Arg is dissolved in 1 mL of SAB solution to prepare a 1 mg / mL Arg SAB solution, and the EDCN solution (100 μL) prepared in the first step is added dropwise to activate the carboxyl group of Arg, the volume ratio of EDCN solution to Arg SAB solution is 1:10, and an Arg-EDCN solution is generated. 1 mL of Arg-EDCN solution is added to 1 mL of EDCN solution of CN-LPs, and distilled water is filled into a MWCO 2000 benzoylation dialysis tube, and dialyzed continuously for 3 times for 24 hours to remove excess reactants and by-products. Finally, CN-LPs-Arg is obtained and the resulting solution is freeze-dried for standby use.

[0060] The preparation method of CN-MccY, CN-Arg, CN-MccJ25 and CN-Klebsidin as control samples is similar to the preparation method of CN-LPs-Arg mentioned above. The EDCN solutions of CN-MccY, CN-Arg, CN-MccJ25 and CN-Klebsidin are prepared by activation of EDCN and esterification reaction with CN-EDCN solution in the first step, and then dialysis and freeze-drying are used in the second step to obtain CN-MccY, CN-Arg, CN-MccJ25 and CN-Klebsidin.

[0061] Example 2: Characterization and analysis of CN-LPs-Arg

[0062] The conjugation efficiency of CN-LPs-Arg, CN-Arg and CN was studied using a Vertex 70 Fourier Transform Infrared Spectrometer (FTIR). The spectral range was 4000-15000cm -1 The resolution is 4cm -1 , scanned 64 times. The structures of CN and CN-LPs-Arg were tested by FT-IR spectrum. The results are as follows Figure 2 A and 2B. The characteristic peaks of NH in CN are located at 2850 and 2930 cm -1 Nearby, CN-LPs are at 2921.99 and 2858.79 cm -1 At, CN-LPs-Arg is at 2923.30-2871.22cm -1 The stretching bands of -COOH in CN-LPs and CN-LPs-Arg were observed to be approximately 3359.96 cm -1 and 3351.45cm -1 . At 1735cm -1 A new peak related to the CN structure and carboxyl reaction appeared at 1700-1750 cm-1, which was represented by the characteristic peak of -C=O, confirming the successful reaction between the amino group of CN and the carboxyl group of lasso peptide. The infrared absorption peak of -C=O generally appears at 1700-1750 cm-1. -1 The infrared absorption peak is weak due to the high degree of CN deacetylation. The vibration peak of the amide group in the CN-LPs-Arg is obvious. The particle size and zeta potential of the synthesized compound were measured using a Malvern ZS-90 Zetasizer at 25°C. The static average particle size of the modified CN-LPs-Arg increased slightly to between 200-400nm. Figure 2C. The lower molecular weight of CN-LPs-Arg leads to more uniform pore morphology and lower pore polydispersity. The zeta potential values ​​of the prepared nanomaterials are between -22.0 and 22.0 mV, as shown in Figure 2 As shown in D.

[0063] Example 3: Transmission electron microscopy analysis of CN-LPs-Arg

[0064] The CN-LPs-Arg nanoparticles were resuspended in 0.1M sodium acetate solution to obtain a 1mg / mL CN-LPs-Arg nanoparticle suspension. 20μL of the suspension was dropped into a 200-mesh grid and incubated at room temperature for 10min. Then, 2% phosphotungstic acid was used for negative staining for 3min, and the remaining liquid was filtered out with filter paper. The samples were observed using HT7700 and Hitachi Su-8010 transmission electron microscopes. The structures and particle sizes of the three nanoparticles of CN, CN-Arg and CN-LPs-Arg are shown in Figure 2. Figure 3 As shown. Figure 3 In A, the particle size of CN is between 10-50nm; Figure 4 B and Figure 4 In C, the particle sizes of CN-LPs and CN-LPs-Arg are between 100-200 nm, respectively.

[0065] Example 4: Physical and chemical stability test of CN-LPs-Arg

[0066] In order to investigate the stability of chitosan nanoparticles, acid-base polarity test and high temperature stability test were carried out.

[0067] First, the molecular weights of the three lasso peptides were analyzed by UPLC-Q-TOF mass spectrometry. The three lasso peptides were expressed and prepared according to Example 1. The results of UPLC-Q-TOF mass spectrometry analysis showed that the precise molecular weight of MccY was 2224.0165 m / z, corresponding to (M+3H) +3 and (M+2H) +2 The ionization molecular weights are 742.6794m / z and 1113.5188m / z ( Figure 4 A). Klebsidin consists of only 19 amino acids, with a molecular weight of 2032.0015 m / z, corresponding to (M+2H) +2 The ionization molecular weight is 1017.5097m / z( Figure 4 B) By Figure 4 C shows that the precise molecular weight of MccJ25 is 2106.0223m / z, corresponding to (M+3H) +3 The ionization molecular weight is 703.3494 m / z.

[0068] The experimental steps of the high temperature stability test are as follows: MccY was dissolved in 0.1 M SAB solution to prepare a 1 mg / mL MccY SAB solution, and then MccY was divided into two groups, the experimental group was treated at 95°C for 2 h, and the control group was placed at room temperature for 2 h.

[0069] The experimental steps of the acid-base polarity test are as follows: 1 mg / mL MccY SAB solution was divided into three groups, the first group was placed in a hydrochloric acid solution with a pH of 2.0 for 2 h, the second group was placed in a sodium hydroxide solution with a pH of 12.0 for 2 h, and the third control group was not treated in any way.

[0070] Then, the lasso peptide MccY was detected using 280nm UV scanning spectrum. Figure 4 As shown in Figures D and 4E, the degradation of the lasso peptide MccY was mainly caused by acid-base treatment at pH = 2.0 and pH = 12.0, while high temperature treatment had no effect on the MccY protein; the acid treatment weakened the characteristic absorption peak of MccY, that is, the acid treatment led to partial peptide degradation; in addition, additional degradation peaks were observed in the spectrum of 12.7-13.3 min after alkali treatment.

[0071] The CN-LPs-Arg nanoparticles were further tested for stability using high temperature stability tests and acid-base polarity tests. The CN-LPs-Arg nanoparticles were dissolved in 0.1 M SAB solution to prepare a 1 mg / mL CN-LPs-Arg SAB solution. The remaining experimental steps were as described above. The CN-LPs-Arg nanoparticles were then tested using a 280 nm UV scanning spectrum. The results are as follows: Figure 4 As shown in Figure F, the CN-LPs-Arg nanoparticles treated with acid and alkali, high temperature and the control group all showed characteristic peaks at 12.2 min, 12.7 min and 13.6 min, respectively. The above results indicate that CN-LPs-Arg nanoparticles can improve the stability of lasso peptide in acid, alkali and high temperature environments. CN-LPs-Arg nanoparticles are stable in acid, alkali and high temperature environments and have acid, alkali and high temperature resistance.

[0072] After 0.1 mg / mL CN-LPs-Arg was treated for 2 h at 95 °C, pH = 2.0 and pH = 12.0, the bactericidal activity was determined by spot assay. 600=0.8 Test bacteria (Salmonella typhimurium, ST53, information as shown in Table 2) were taken and cultured in LB solid medium. Then the treated lasso peptide was diluted into 6 concentrations and spotted on 6 sector areas. The drug concentrations of wells 1 to 6 were: area 1: 10.0 μM, area 2: 5.0 μM, area 3: 1.0 μM, area 4: 0.5 μM, area 5: 0.2 μM, area 6: 0.04 μM. The cells were cultured at 37°C for 12 h. The results were as follows. Figure 4 As shown in G, high temperature treatment resulted in a 5-fold decrease in activity (0.2 μM), while the activities of the two groups treated with acid and alkali were comparable to those of the control group (0.04 μM). It can be seen that chitosan arginine nanoparticles CN-LPs-Arg have nanoparticle stability and acid-base buffering effects, and the acid-base resistance of the three lasso peptides in CN-LPs-Arg is improved.

[0073] Example 5: Antibacterial activity of CN-LPs-Arg

[0074] The synthetic samples (CN, Arg, CN-Arg, CN-MccY, CN-MccJ25, CN-Klebsidin, CN-LPs and CN-LPs-Arg) were tested by spot plate test to demonstrate the antibacterial effect determined by spot method. The strains to be tested included Salmonella Enteritidis (SE63), Salmonella Typhimurium (ST53), Serratia marcescens (SMATCC14756) and Klebsiella pneumoniae (KP07), and the specific strain information is shown in Table 2. The specific steps include: culturing the strains on LB agar plates overnight, then transferring the strains to LB broth and culturing at 37°C until OD 600 The value reached 0.8. After that, the bacterial solution was diluted at a ratio of 1:1000 and added to LB solid culture medium containing 0.5% agar, and prepared into a plate at 40°C. Then each LB plate was divided into 8 sector-shaped areas. From the 1st to the 8th area, 10-20 μL of drug was used for spot plating, and 0.05 μM of the following drugs were inoculated respectively: 1. CN, 2. Arg, 3. CN-Arg, 4. CN-MccY, 5. CN-MccJ25, 6. CN-Klebsidin, 7. CN-LPs, 8. CN-LPs-Arg. After culturing for 24 hours, the antibacterial diameter was observed and recorded. In the spot plate test, the results of the inhibition zone determination of CN nanoparticles on each strain are shown in Table 3.

[0075] Table 2 Strains used in Example 5

[0076]

[0077] Table 3 Determination of inhibition zone of CN nanoparticles in spot plate test

[0078]

[0079]

[0080] Furthermore, the statistical results of the spot test are as follows Figure 5 As shown in the figure, CN and Arg alone have no inhibitory effect on these microorganisms, CN nanoparticles with single lasso peptides (CN-MccY, CN-MccJ25, CN-Klebsidin) have specific inhibitory effects on specific strain types, and CN nanoparticles with three lasso peptides (CN-LPs) have inhibitory effects on Salmonella enteritidis (SE63), Salmonella typhimurium (ST53), Serratia marcescens (SMATCC14756) and Klebsiella pneumoniae (KP07), that is, CN-LPs achieve multi-effect synergistic inhibitory effects on different fungi. Compared with CN nanoparticles with single lasso peptides (CN-MccY, CN-MccJ25, CN-Klebsidin), the corresponding single lasso peptide concentration in CN-LPs at the same concentration is reduced to about 1 / 3 of that in CN nanoparticles with single lasso peptide, so the diameter of the inhibition zone for some strains is smaller. Compared with CN-LPs, CN nanoparticles further coupled with Arg (CN-LPs-Arg) can further improve the antibacterial effect against different fungi.

[0081] Example 6: FICI combined antibacterial analysis of MccY, MccJ25, and Klebsidin

[0082] In order to confirm the drug-induced antibacterial relationship between the three separate lasso peptides, the relationships between MccY and MccJ25, MccY and Klebsidin, and MccJ25 and Klebsidin were further explored. Figure 6 The experimental results are shown in Tables 4-6. FICI experiment: In each well of a 96-well plate, 50 μL of a specific concentration of lasso peptide A and 50 μL of a specific concentration of lasso peptide B were added in equal volumes. The concentration of lasso peptide was Figure 6 As shown. The well plate was incubated at 37°C for 24 hours to observe and record the MIC of each lasso peptide. Each experimental concentration was repeated three times to ensure the accuracy of the results. The interaction between lasso peptide A and lasso peptide B was evaluated according to the calculation formula of FICI (combined inhibition fraction). The calculation formula of FICI combined inhibition is as follows:

[0083]

[0084] When FICI > 2, the antagonistic effect of lasso peptides A and B is shown; when 1 < FICI ≤ 2, an irrelevant effect is shown; when 0.5 < FICI ≤ 1, the additive effect of lasso peptides A and B is shown; when FICI ≤ 0.5, the synergistic effect of lasso peptides A and B is shown.

[0085] Table 4 FICI numerical results of MccY and MccJ25

[0086]

[0087] Table 5 FICI numerical results of MccY and Klebsidin

[0088]

[0089] Table 6 FICI numerical results of MccJ25 and Klebsidin

[0090]

[0091] The results in Table 4, Table 5 and Table 6 show that: (1) In the drug detection of MccY and MccJ25, Salmonella enteritidis (SE63), Salmonella typhimurium (ST53), Serratia marcescens (SMATCC14756) and Klebsiella pneumoniae (KP07) showed an additive effect. (2) In the drug detection of MccY and Klebsidin, Salmonella enteritidis (SE63), Salmonella typhimurium (ST53), Serratia marcescens (SMATCC14756) showed an additive effect, and Klebsiella pneumoniae (KP07) showed a synergistic effect. (3) In the drug detection of MccJ25 and Klebsidin, Salmonella enteritidis (SE63), Klebsiella pneumoniae (KP07) showed an additive effect, Salmonella typhimurium (ST53) showed an irrelevant effect, and Serratia marcescens (SMATCC14756) showed a synergistic effect.

[0092] Example 7: Biocompatibility analysis of CN1.25-LPs-Arg

[0093] Evaluate the effect of CN-LPs-Arg on the activity of mammalian cells to evaluate its biocompatibility. The specific steps are as follows: Collect human red blood cells in a heparin-containing collection tube. The steps are as follows: Centrifuge to separate the serum at 750g for 15 min, wash the red blood cells 3 times with sterile PBS, and then dilute the red blood cells with PBS to make a 1% red blood cell suspension; then treat the cells with samples (CN-Arg, CN-LPs, CN-LPs-Arg) at a concentration of 10 μg / mL, and observe their morphological changes. Triton X-100 diluted with PBS is used as a positive control, and cells treated with PBS are used as a negative control.

[0094] The experimental results are as follows Figure 7 As shown in A, the negative control cells had normal morphology, while the positive control cells treated with Triton X-100 showed cell membrane lysis and cell death, and the cells treated with CN, CN-Arg, CN-LPs and CN-LPs-Arg did not cause obvious cell membrane lysis and hemolysis.

[0095] Next, the cytotoxicity of CN nanoparticles to HEK293T cells was evaluated by MTT assay. 293T cells were cultured at 37°C and 5% CO2 in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. 200 μL of the above 1% red blood cell suspension (adjusted to a concentration of 1.0×10 5 Cells / well were seeded in 96-well plates) and cultured for 24h. CN nanoparticles (CN-LPs-Arg and CN) were then resuspended and diluted in DMEM. The concentration gradient of CN nanoparticles was diluted to 0.5%, 1.0%, 5.0%, 10.0%, 20.0% according to the concentration of 20.0% (20mg / mL); the concentration gradient of CN-LPs-Arg drug was diluted to: 10μM, 20μM, 40μM, 80μM, 160μM, 320μM, and then 200μL of diluted CN nanoparticle solution was added to each well and incubated at 37°C, 5% CO2 for 24h. Triton X-100 diluted in PBS was used as a positive control, and cells treated with PBS were used as negative controls.

[0096] The qualitative results of CN nanoparticles toxicity to HEK293T cells are as follows Figure 7 As shown in B. Figure 7 B reflects the color change of red blood cells at different concentrations. The smaller the red color change, the lower the degree of hemolysis. The color change of red blood cells in the concentration ranges of 0.5%-20.0% and 10μM-80μM is not obvious, and the color change at the concentrations of 160μM and 320μM is large, indicating red blood cell lysis.

[0097] Use the 1% red blood cell suspension prepared above. The experimental group was composed of 100 μL of red blood cell suspension and 100 μL of CN-LPs-Arg sample (concentrations: 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, 320 μM) added to a microcentrifuge tube. The positive control was 100 μL of red blood cell suspension treated with Triton X-100 diluted in sterile PBS (final concentration = 0.5 mg / mL), and the negative control was PBS treated with red blood cell suspension. The control group and the experimental group were incubated at 37°C for 2 hours, and 50 μL of the supernatant was transferred to a 96-well plate after centrifugation. The absorbance of the supernatant of the control group and the experimental group at 540 nm was determined by spectrophotometry to determine the hemolysis rate. The percentage calculation formula of the hemolysis rate is as follows:

[0098] Hemolysis rate % = (Abs Compound -Abs Positive ) / (Abs Negative -Abs Blank )×100%,

[0099] Among them Abs Compound Indicates the absorbance of the CN nanoparticle sample to be tested (experimental group), Abs Positive Represents the absorbance of the positive control, Abs Negative Indicates the absorbance of the negative control, Abs Blank Indicates the absorbance of the blank control.

[0100] The results of hemolysis rate are as follows Figure 7 As shown in C, CN-LPs-Arg had no effect on erythrocyte activity at concentrations below 80 μM, and compared with the complete lysis of erythrocytes induced by Triton X-100, CN nanoparticles had no hemolytic toxicity. This indicates that CN-LPs-Arg nanoparticles are not sufficient to cause significant host side effects.

[0101] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0102] References

[0103] 1.Li,Y.,Han,Y.,Zeng,Z.,Li,W.,Feng,S.,&Cao,W.(2021).Discovery andBioactivity of the Novel Lasso Peptide Microcin Y.Journal of agricultural andfood chemistry,69(31),8758–8767.https: / / doi.org / 10.1021 / acs.jafc.1c02659.

Claims

1. A chitosan nanoparticle, characterized in that: The invention comprises chitosan and more than one lasso peptide, wherein the lasso peptide has a carboxyl group, and the amino group in the chitosan structure is connected with the carboxyl group of the lasso peptide through an amide bond.

2. The chitosan nanoparticles according to claim 1, characterized in that The lasso peptides include MccJ25, MccY, Klebsidin, Citrocin, Lihuanodin, Synthetase B1, Astexin 1, Koveensin, MccS and MccC7.

3. The chitosan nanoparticles according to claim 1, characterized in that The molar ratio of chitosan to lasso peptide is 5:(3-4).

4. The chitosan nanoparticles according to claim 1, characterized in that The chitosan nanoparticles also include arginine, and the amino group in the chitosan structure is connected to the carboxyl group of arginine through an amide bond.

5. The method for preparing chitosan nanoparticles according to any one of claims 1 to 3, comprising the steps of: 1) mixing a 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and an N-hydroxysuccinimide solution to obtain an EDCN mixed solution; 2) dissolving chitosan and lasso peptide in the acetylation solution respectively, and mixing them evenly to obtain chitosan acetylation solution and lasso peptide acetylation solution; 3) adding the EDCN mixed solution in step 1) to the chitosan acetylated solution and the lasso peptide acetylated solution in step 2) respectively, and mixing them evenly to obtain a chitosan-EDCN mixed solution and a lasso peptide-EDCN mixed solution; 4) The chitosan-EDCN mixed solution in step 3) and the lasso peptide-EDCN mixed solution are sequentially mixed, and stirred sufficiently to complete the esterification reaction, thereby preparing the chitosan nanoparticles.

6. The method for preparing chitosan nanoparticles according to claim 4, comprising the following steps: 1) mixing a 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and an N-hydroxysuccinimide solution to obtain an EDCN mixed solution; 2) dissolving chitosan, lasso peptide and arginine in the acetylation solution respectively, and mixing them evenly to obtain chitosan acetylation solution, lasso peptide acetylation solution and arginine acetylation solution; 3) adding the EDCN mixed solution in step 1) to the chitosan acetylated solution, the lasso peptide acetylated solution and the arginine acetylated solution in step 2) respectively, and mixing them evenly to obtain a chitosan-EDCN mixed solution, a lasso peptide-EDCN mixed solution and an arginine-EDCN mixed solution; 4) The chitosan-EDCN mixed solution in step 3) is sequentially mixed with the lasso peptide-EDCN mixed solution and the arginine-EDCN mixed solution, and the mixture is fully stirred to complete the esterification reaction, thereby preparing the chitosan nanoparticles.

7. The preparation method according to claim 5 or 6, characterized in that The acetylation solution includes sodium acetate solution, acetyl chloride solution, acetic anhydride solution and glacial acetic acid solution.

8. Use of the chitosan nanoparticles according to any one of claims 1 to 4 in the preparation of products with antibacterial effects.

9. The use according to claim 8, characterized in that The bacteria include Salmonella, Serratia and Klebsiella.

10. The method according to claim 8, characterized in that The products include feed, preservatives, disinfectants and medicines for external wound infections.

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