An antimicrobial peptide targeting Gram-negative bacteria and chitosan nanoparticles and applications thereof

Abstract

The present invention provides an antimicrobial peptide targeting Gram-negative bacteria and its chitosan nanoparticles and applications. The amino acid sequence of the antimicrobial peptide FR5 is shown in SEQ ID No.1. The present invention uses the amino acid fragment of myeloid differentiation protein MD-2 binding to LPS as the basic fragment and utilizes the amino acid substitution method to construct the antimicrobial peptide FR5 targeting Gram-negative bacteria. The antimicrobial peptide FR5 was coated with chitosan by the ion crosslinking method, and chitosan nanoparticles FR5-CS-NPs were successfully constructed to improve the protease resistance of FR5. The present invention prolongs the release time of the antimicrobial peptide, thereby achieving a long-lasting antibacterial effect, overcoming the limitations of the antimicrobial peptide in practical applications, and promoting the industrialization process of the antimicrobial peptide.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and specifically relates to an antimicrobial peptide targeting Gram-negative bacteria and chitosan nanoparticles and applications thereof. Background Art

[0002] Antimicrobial peptides are a class of small molecule polypeptides with broad-spectrum antimicrobial activity that are ubiquitous in organisms. They have the characteristics of high biological activity, green and no residue. This property makes antimicrobial peptides have antimicrobial ability against both pathogens and probiotics. Therefore, designing antimicrobial peptides with targeted antimicrobial ability can effectively act against bacteria. The amino acid fragment of myeloid differentiation protein MD-2 that binds to LPS has weak antimicrobial activity against Gram-negative bacteria. By modifying this fragment through amino acid substitution, it is expected to obtain a new antimicrobial peptide with targeted effect on Gram-negative bacteria.

[0003] However, antimicrobial peptides have a short half-life and cannot achieve long-term antibacterial effects. They are easily degraded by proteases in the digestive tract. With the continuous advancement of nanotechnology, encapsulating antimicrobial peptides into nanoparticles is expected to improve their bioavailability and enhance their stability in the body. Most traditional nanoparticles are quickly swallowed by the reticuloendothelial system in the body, while chitosan, a material for preparing nanoparticles, overcomes this shortcoming and can prolong the drug's circulation time in the body, achieving better antibacterial effects. Summary of the invention

[0004] Based on the above shortcomings, the present invention provides an antimicrobial peptide FR5 targeting Gram-negative bacteria, which has the function of targeting Gram-negative bacteria.

[0005] The technical solution adopted by the present invention is as follows: an antimicrobial peptide FR5 targeting Gram-negative bacteria, whose amino acid sequence is shown in SEQ ID No. 1, and whose C-terminus is amidated with -NH2.

[0006] Another object of the present invention is to provide a method for preparing an antimicrobial peptide FR5 targeting Gram-negative bacteria, the steps of which are as follows:

[0007] S1: using amino acid substitution method, arginine was introduced into the 4th and 7th positions of the amino acid fragment of myeloid differentiation protein MD-2 binding to LPS, the resulting polypeptide sequence is shown in SEQ ID No. 1, and its C-terminus was amidated with -NH2;

[0008] S2: using solid phase chemical synthesis method, using a peptide synthesizer to obtain a peptide resin, and then cutting the obtained peptide resin with TFA to obtain a polypeptide; and then after reverse phase high performance liquid chromatography purification and mass spectrometry identification, the preparation of the polypeptide is completed;

[0009] S3: The polypeptide is then subjected to minimum inhibitory concentration detection and hemolytic activity detection, and is finally named antimicrobial peptide FR5.

[0010] Another object of the present invention is to provide a chitosan nanoparticle FR5-CS-NPs targeting Gram-negative bacteria, comprising the antimicrobial peptide FR5 as described above, which solves the problem that the antimicrobial peptide is easily degraded by proteases in the digestive tract.

[0011] Another object of the present invention is to provide a method for preparing the chitosan nanoparticles FR5-CS-NPs as described above, the method steps are as follows:

[0012] S1: Preparation of chitosan solution: Prepare 1% acetic acid solution by volume with sterile water, dissolve 30 mg chitosan powder in the acetic acid solution, and finally dissolve chitosan at a concentration of 1 mg / mL. Stir magnetically at 25°C and 210 rpm for 30 min, and adjust the pH of the chitosan solution to 5.5 with 10 M NaOH solution for later use;

[0013] S2: Preparation of nanoparticles: Prepare 1 mg / mL sodium tripolyphosphate solution, add 60 μg / mL antimicrobial peptide FR5 to the prepared chitosan solution, and add the sodium tripolyphosphate solution dropwise to the chitosan solution under magnetic stirring, wherein the volume ratio of the chitosan solution to the sodium tripolyphosphate solution is 3:1, continue magnetic stirring at 25°C and 210 rpm for 2 h, and spontaneously form a chitosan nanoparticle suspension;

[0014] S3: Purification and freeze drying: The prepared nanoparticle suspension is centrifuged at 4°C, 14000×g for 45 min, the precipitate is washed with sterile water, and the supernatant is discarded to obtain a gel-like precipitate, which is freeze-dried using a freeze dryer to complete the preparation of chitosan nanoparticles.

[0015] Furthermore, in the chitosan nanoparticles FR5-CS-NPs described above, the molecular weight of chitosan is in the range of 50-190 kDa, and the degree of deacetylation is 75-85%.

[0016] Another object of the present invention is to provide the use of the antimicrobial peptide FR5 targeting Gram-negative bacteria as described above in the preparation of drugs for treating and / or preventing infectious diseases caused by Gram-negative bacteria.

[0017] Furthermore, the Gram-negative bacteria mentioned above are Escherichia coli or Salmonella typhimurium.

[0018] Another object of the present invention is to provide the use of the chitosan nanoparticles FR5-CS-NPs targeting Gram-negative bacteria as described above in the preparation of drugs for treating and / or preventing infectious diseases caused by Gram-negative bacteria.

[0019] Furthermore, the Gram-negative bacteria mentioned above are Escherichia coli or Citrobacter rodentium.

[0020] Another object of the present invention is to provide a drug suitable for treating and / or preventing Gram-negative bacterial infection, wherein the drug contains the chitosan nanoparticles FR5-CS-NPs as described above.

[0021] The present invention has the following beneficial effects and advantages: the antimicrobial peptide FR5 of the present invention has targeted antimicrobial activity against Gram-negative bacteria. The present invention adopts chitosan as a nano-wall material to construct nanoparticles coated with antimicrobial peptides, which can improve the stability of antimicrobial peptides in proteases and artificial gastrointestinal fluids, prolong the release time of antimicrobial peptides, and thus achieve a long-term antibacterial effect, overcome the limitations of antimicrobial peptides in practical applications, and promote the industrialization process of antimicrobial peptides. At the same time, the antimicrobial time is extended through the nano-sustained release effect, and the addition cost is reduced, and it can be widely used in the fields of medicine, food, agriculture, industry, animal husbandry, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the chromatogram of antimicrobial peptide FR5;

[0023] Figure 2 is the mass spectrum of antimicrobial peptide FR5;

[0024] Figure 3 This is the hemolytic activity diagram of the antimicrobial peptide FR5;

[0025] Figure 4 Figure 2 is a diagram of chitosan solution; A: the diagram of chitosan solution before construction; B: the diagram of FR5-CS-NPs solution after construction;

[0026] Figure 5 is the in vitro release kinetics of FR5 from FR5-CS-NPs;

[0027] Figure 6 This is a graph showing the effect of FR5-CS-NPs on Caco-2 cell viability. Specific implementation methods

[0028] The present invention is further described by taking the following examples:

[0029] Example 1

[0030] Arginine was introduced into the 4th and 7th positions of the amino acid fragment of myeloid differentiation protein MD-2 that binds to LPS, and the resulting polypeptide sequence was shown in SEQ ID No. 1 or Table 1, and its C-terminus was amidated with -NH2. A peptide resin was obtained by solid phase chemical synthesis using a peptide synthesizer, and the obtained peptide resin was cut with TFA to obtain polypeptide FR5; then, the peptide was purified by reverse phase high performance liquid chromatography (such as Figure 1as shown) and after mass spectrometry identification (as Figure 2 The preparation of polypeptide FR5 is completed. The sequence, molecular weight and charge number of polypeptide FR5 are shown in Table 1.

[0031] Table 1 Amino acid sequence, molecular weight and charge number

[0032]

[0033] Example 2

[0034] Minimum inhibitory concentration test:

[0035] The minimum inhibitory concentration (MIC) of the peptides was determined by the standard microbroth dilution method. The bacteria were incubated at 37°C overnight with constant shaking at 220 rpm and then transferred to new MHB until the logarithmic phase of growth. The logarithmic phase bacteria were diluted to 10 5 CFU / ml. 50 μL of peptides of different concentrations (final concentration of peptides was 1-64 μM) and 50 μL of bacterial suspension were added to each well of a 96-well plate. The negative control was MHB medium without bacteria, and the positive control was MHB medium with bacteria. The 96-well plate was placed in a 37°C constant temperature incubator for 18-20 hours. The minimum inhibitory concentration was the concentration when no turbidity was observed at the bottom of the well with the naked eye. The results are shown in Table 2.

[0036] Table 2 Antimicrobial activity of antimicrobial peptides

[0037]

[0038] The results showed that the minimum inhibitory concentration of antimicrobial peptide FR5 against Gram-negative bacteria was 2-8 μM, and it had no antibacterial ability against Gram-positive bacteria.

[0039] Example 3

[0040] To evaluate the safety of antimicrobial peptide FR5, the hemolytic behavior of the peptide against human red blood cells (hRBC) was studied in the concentration range of 1-64 μg / mL.

[0041] 2 mL of fresh blood from a healthy person was collected and centrifuged at 4°C, 3000r for 10 min, then the supernatant was discarded and the red blood cells were collected. The collected red blood cells were then washed 3 times with PBS buffer under the same centrifugal conditions, and finally resuspended with 10 mL PBS for use. Take a sterile 96-well culture plate, use PBS buffer as the diluent, add the antimicrobial peptide stock solution with an initial concentration of 5.12 mg / mL to the diluent of the 96-well culture plate, perform a 2-fold gradient dilution, and then add 50 μL of red blood cell suspension to each well, and incubate in a 37°C incubator for 1 hour. Then the 96-well culture plate was centrifuged at 4°C, 1000×g for 10 min; 50 μL of the supernatant after centrifugation was aspirated and transferred to a new sterile 96-well culture plate, and the absorbance at 570 nm was measured using an ELISA reader. 50 μL of red blood cells plus 50 μL of 0.1% Triton X-100 was used as a positive control, and 50 μL of red blood cells plus 50 μL of PBS buffer was used as a negative control. Figure 3 The biocompatibility of antimicrobial peptide FR5 was evaluated by the minimum concentration (MHC) of antimicrobial peptide when it caused 5% hemolysis of human red blood cells, and its cell selectivity was further evaluated by calculating the therapeutic index (TI) of FR5, as shown in Table 3.

[0042] Table 3 Hemolytic activity and therapeutic index of antimicrobial peptides

[0043]

[0044] Example 4

[0045] The preparation method of antimicrobial peptide-chitosan nanoparticles is as follows:

[0046] S1: Preparation of chitosan solution: Prepare 1% acetic acid solution by volume with sterile water, dissolve 30 mg chitosan powder in the acetic acid solution, and finally dissolve chitosan at a concentration of 1 mg / mL. Stir magnetically at 25°C and 210 rpm for 30 min, and adjust the pH of the chitosan solution to 5.5 with 10 M NaOH solution for later use;

[0047] S2: Preparation of nanoparticles: Prepare 1 mg / mL sodium tripolyphosphate solution, add 60 μg / mL antimicrobial peptide FR5 to the prepared chitosan solution, and add the sodium tripolyphosphate solution dropwise to the chitosan solution under magnetic stirring, wherein the volume ratio of the chitosan solution to the sodium tripolyphosphate solution is 3:1, continue magnetic stirring at 25°C and 210 rpm for 2 h, and spontaneously form a chitosan nanoparticle suspension;

[0048] S3: Purification and freeze drying: The prepared nanoparticle suspension was centrifuged at 4°C, 14000×g for 45 min, and the precipitate was washed with sterile water. The supernatant was discarded to obtain a gel-like precipitate, which was freeze-dried using a freeze dryer to complete the preparation of chitosan nanoparticles. Figure 4 As shown, the successfully constructed nanoparticle solution transforms from clear and transparent to a milky white suspension with no visible precipitation.

[0049] Example 5

[0050] Parameter characterization of CS-NPs and FR5-CS-NPs:

[0051] The successfully prepared CS-NPs and FR5-CS-NPs were dissolved in 4 mL of sterile water to a final concentration of 40 μg / mL, and the parameter characterization of the nanoparticles was measured. The results are shown in Table 4.

[0052] Table 4 Characterization parameters of CS-NPs and FR5-CS-NPs

[0053]

[0054] As can be seen from Table 4, the particle size of CS-NPs is 183.0±1.6nm, and the particle size of FR5-CS-NPs is 292.6±4.2nm. FR5-CS-NPs exhibit a larger particle size than CS-NPs. In this method, the PDI of CS-NPs and FR5-CS-NPs are 0.180 and 0.241, respectively, and the Zeta potential is 28.0±0.7mV and 26.5±0.8mV, respectively. The drug loading and encapsulation efficiency of FR5-CS-NPs are 8.9±0.3% and 75.2±2.5%, respectively.

[0055] Example 6

[0056] In vitro release kinetics of FR5-CS-NPs:

[0057] FR5-CS-NPs were dispersed in 10 mL of 10 mM PBS solution to a final concentration of 0.25 mg / mL and incubated at 37°C with shaking. 1 mL of solution samples were taken at 0.25, 0.5, 1, 1.5, 2, 4, 6, 12, 18 and 24 h, and an equal amount of 10 mM PBS solution was immediately added. The samples were centrifuged at 4°C, 14000 × g for 5 min to collect the supernatant. The amount of FR5 released from FR5-CS-NPs was determined by the method recommended by the BCA protein concentration assay kit. The in vitro release kinetics of FR5 from FR5-CS-NPs is shown in the figure. Figure 5As shown in the figure, it can be seen that the release rate of FR5 in FR5-CS-NPs can be divided into two stages. The early release is slow, and the cumulative release rate in the first 1.5 hours is only 5.04%. After 2 hours, the release rate increases rapidly, and at the 12th hour, the cumulative release rate reaches 63.06%, and at the 24th hour, the cumulative release rate reaches 93.43%.

[0058] Example 7

[0059] Antibacterial activity of FR5-CS-NPs:

[0060] Inoculate the bacterial solution into fresh MHB broth for overnight culture, and transfer the overnight cultured bacterial solution into fresh MHB until the bacteria grow to the logarithmic phase. Use MHB to adjust the concentration of the bacterial solution, and finally adjust the colony count of the obtained bacterial solution to about 10 6 CFU / mL for standby use. Filter the sterile MHB liquid culture medium with a 0.22μM water filter membrane, add the filtered MHB to a 1.5mL EP tube, add 335μL to the first tube, and add 500μL to tubes 2-7. Add 670μL of antimicrobial peptide FR5 and FR5-CS-NPs (the initial concentration of FR5 is 30μg / mL; the initial concentration of FR5-CS-NPs is 1.5mg / mL) to the first tube, blow continuously with a pipette, mix it thoroughly, then aspirate 500μL and add it to the second tube, blow continuously, mix it thoroughly, then add it to the third tube, dilute it to the fifth tube in a continuous multiple ratio, aspirate 500μL and discard it after mixing thoroughly, then add the prepared bacterial solution, and set up 3 parallels for each sample. The group without drug addition was used as the positive control, and the group without bacteria addition was used as the negative control. The 1.5 mL EP tube was incubated in a 37°C incubator for 18-20 h. The lowest drug concentration at which no visible bacterial growth was observed was the minimum antibacterial concentration (MAC) of the drug against the test bacteria. The minimum antibacterial concentrations of FR5 and FR5-CS-NPs are shown in Table 5.

[0061] Table 5 Antibacterial activity of FR5 and FR5-CS-NPs

[0062]

[0063]

[0064] As can be seen from Table 5, the antimicrobial peptide FR5 has strong antibacterial activity (5-10 μg / mL) against E. coli ATCC 25922, E. coli ATCC K88, and C. rodentium. The MAC value of FR5-CS-NPs against all tested bacteria was 250 μg / mL.

[0065] Example 8

[0066] Protease stability of antimicrobial peptides FR5 and FR5-CS-NP:

[0067] Prepare 2 mg / mL protease solutions respectively, and the proteases to be tested include: trypsin and chymotrypsin. Incubate the samples to be tested with 2 mg / mL protease at different concentrations at 37°C for 1, 6 and 12 hours and then boil for 15 minutes. After boiling, cool to room temperature and determine the MAC value of the sample according to the steps in Example 7. The effect of enzyme treatment on the antibacterial activity of the drug is determined based on the change in MAC value. The protease stability of antimicrobial peptide FR5 and FR5-CS-NPs is shown in Table 6.

[0068] Table 6 Effect of FR5 and FR5-CS-NPs on the antibacterial activity of E.coli ATCC 25922 after protease treatment

[0069]

[0070] As can be seen from Table 6, after 1, 6 and 12 hours of treatment with chymotrypsin and trypsin, FR5 lost its antibacterial activity against E.coli ATCC25922 (MAC>80μg / mL), and the MAC value of FR5-CS-NPs remained unchanged at 250μg / mL; after 12 hours of treatment, the antibacterial activity decreased by 2 times, and the MAC value was 500μg / mL. This is because FR5 was not released from the nanoparticles in a short time, and the chitosan coating prevented FR5 from being hydrolyzed by proteases. However, as the treatment time increased, the release rate of FR5 increased, and the released FR5 was hydrolyzed by proteases, resulting in a decrease in the antibacterial activity of FR5-CS-NPs, which is consistent with the in vitro release kinetics results.

[0071] Example 9

[0072] Stability of antimicrobial peptide FR5 and FR5-CS-NPs in artificial gastrointestinal fluid:

[0073] Artificial gastric juice and intestinal juice were mixed with antimicrobial peptide FR5 and FR5-CS-NPs (v / v=1:1), and the final FR5 concentration was 30 μg / mL (in the intestinal juice stability test, the FR5 concentration was 300 μg / mL after mixing with the digestive juice), and the FR5-CS-NPs concentration was 1.5 mg / mL. In a 37°C incubator, the artificial gastric juice mixture was incubated for 0.5 h, 1 h and 3 h, and the artificial intestinal juice mixture was incubated for 0.5 h, 1 h, 3 h, 6 h and 12 h. The minimum antibacterial concentration of the sample was determined according to the steps in Example 7. The effect of enzyme treatment on the antibacterial activity of the drug was determined based on the change in the minimum antibacterial concentration. The artificial gastrointestinal juice stability of FR5 and FR5-CS-NPs is shown in Tables 7 and 8.

[0074] Table 7 Effect of FR5 and FR5-CS-NPs on the antibacterial activity of E.coli ATCC 25922 after artificial gastric juice treatment

[0075]

[0076] Table 8 Effect of FR5 and FR5-CS-NPs on the antibacterial activity of E.coli ATCC 25922 after artificial intestinal fluid treatment

[0077]

[0078] As can be seen from Tables 7 and 8, the antibacterial activity of FR5 decreased after treatment with artificial gastric juice. After 0.5 h of treatment, its MAC value increased from 5 μg / mL to 10 μg / mL, and after 1 h and 3 h of treatment, the MAC value increased to 20 μg / mL. After 3 h of treatment with artificial gastric juice, the MAC value of FR5-CS-NPs increased from 250 μg / mL to 500 μg / mL. After 0.5, 1, 3, 6 and 12 h of treatment with artificial intestinal juice, FR5 lost its antibacterial activity. After 0.5 h of treatment with artificial intestinal juice, the MAC value of FR5-CS-NPs was still 250 μg / mL. After 1, 3, 6 and 12 h of treatment, its antibacterial activity decreased, and the MAC value increased from 250 μg / mL to 500 μg / mL. This may be because FR5 was not released from the nanoparticles in a short time, and as time went on, the released FR5 was hydrolyzed by the protease in the intestinal juice, resulting in a decrease in its antibacterial activity.

[0079] Example 10

[0080] Cell viability assay of FR5-CS-NPs:

[0081] Caco-2 cells in the logarithmic growth phase were obtained and digested with trypsin. The cells were counted and the number of cells was 2.6×10 4 / mL cell density was inoculated into a 96-well plate, 100μL per well, and control and FR5-CS-NPs stimulation groups of different concentrations (100, 200, 400, 800μg / mL, respectively) were set up, and 6 replicate wells were set up for each group. After 24 hours of cell culture, the waste liquid was aspirated with a pipette, and 100μL of different concentrations of FR5-CS-NPs were added to each well. 100μL of DMEM high-glucose medium containing 2% fetal bovine serum was added to the control group. After 24 hours of culture, 25μL of 5mg / mL MTT was added to each well, incubated for 4 hours, the supernatant was aspirated, 150μL DMSO was added to each well, and the plate was placed in an incubator for 10 minutes. The OD value was measured at 570nm with an enzyme marker. The determination of cell viability by FR5-CS-NPs is shown in the figure below. Figure 6 As shown, FR5-CS-NPs at concentrations of 100-800 μg / mL had no effect on cell viability.

[0082] In summary, antibacterial FR5 showed strong antibacterial ability against Gram-negative bacteria and weak antibacterial ability against Gram-positive bacteria. Chitosan coating improved the stability of antimicrobial peptide FR5 in proteases and artificial gastrointestinal fluid. Compared with monomeric antimicrobial peptide FR5, FR5-CS-NPs can still maintain good antibacterial activity in different proteases and artificial gastrointestinal fluid environments. The results of cell viability assay showed that FR5-CS-NPs were safe and non-toxic. The results of physicochemical parameters and morphological characterization showed that both FR5-CS-NPs and empty chitosan nanoparticles CS-NPs showed positive Zeta potential, while FR5-CS-NPs showed a larger particle size. The encapsulation rate of antimicrobial peptides in FR5-CS-NPs can reach 75.2±2.5%, and the drug loading is 8.9±0.3%. The results of in vitro release kinetics showed that chitosan-coated antimicrobial peptides have a sustained release effect. The results of antibacterial activity assay showed that FR5-CS-NPs have good antibacterial activity. The present invention provides a theoretical basis and technical support for realizing the application of antimicrobial peptides in medicine, food, agriculture, industry and animal husbandry.

Claims

1. An antimicrobial peptide FR5 targeting Gram-negative bacteria, characterized in that: Its amino acid sequence is shown in SEQ ID No. 1, and its C-terminus is amidated with -NH2.

2. The method for preparing a Gram-negative bacteria targeting antimicrobial peptide FR5 according to claim 1, characterized in that: The steps are as follows: S1: using amino acid substitution method, arginine was introduced into the 4th and 7th positions of the amino acid fragment of myeloid differentiation protein MD-2 binding to LPS, the resulting polypeptide sequence is shown in SEQ ID No. 1, and its C-terminus was amidated with -NH2; S2: solid phase chemical synthesis method is used to obtain peptide resin using a peptide synthesizer, and the obtained peptide resin is cut by TFA to obtain a polypeptide; after reverse phase high performance liquid chromatography purification and mass spectrometry identification, the preparation of the polypeptide is completed; S3: The polypeptide is then subjected to minimum inhibitory concentration detection and hemolytic activity detection, and is finally named antimicrobial peptide FR5.

3. A chitosan nanoparticle FR5-CS-NPs targeting Gram-negative bacteria, characterized in that: Comprising the antimicrobial peptide FR5 as claimed in claim 1.

4. The method for preparing chitosan nanoparticles FR5-CS-NPs according to claim 3, characterized in that: The steps are as follows: S1: Preparation of chitosan solution: Prepare 1% acetic acid solution by volume with sterile water, dissolve 30 mg chitosan powder in the acetic acid solution, and finally dissolve chitosan at a concentration of 1 mg / mL. Stir magnetically at 25°C and 210 rpm for 30 min, and adjust the pH of the chitosan solution to 5.5 with 10 M NaOH solution for later use; S2: Preparation of nanoparticles: Prepare 1 mg / mL sodium tripolyphosphate solution, add 60 μg / mL antimicrobial peptide FR5 to the prepared chitosan solution, and add the sodium tripolyphosphate solution dropwise to the chitosan solution under magnetic stirring, wherein the volume ratio of the chitosan solution to the sodium tripolyphosphate solution is 3:1, continue magnetic stirring at 25°C and 210 rpm for 2 h, and spontaneously form a chitosan nanoparticle suspension; S3: Purification and freeze drying: The prepared nanoparticle suspension is centrifuged at 4°C, 14000×g for 45 min, the precipitate is washed with sterile water, and the supernatant is discarded to obtain a gel-like precipitate, which is freeze-dried using a freeze dryer to complete the preparation of chitosan nanoparticles.

5. The chitosan nanoparticles FR5-CS-NPs according to claim 3, characterized in that: The molecular weight of chitosan ranges from 50 to 190 kDa, and the degree of deacetylation is 75% to 85%.

6. Use of an antimicrobial peptide FR5 targeting Gram-negative bacteria according to claim 1 in the preparation of a drug for treating and / or preventing infectious diseases caused by Gram-negative bacteria, wherein the Gram-negative bacteria is Escherichia coli or Salmonella typhimurium.

7. Use of the chitosan nanoparticles FR5-CS-NPs targeting Gram-negative bacteria according to claim 3 in the preparation of a drug for treating and / or preventing infectious diseases caused by Gram-negative bacteria, wherein the Gram-negative bacteria are Escherichia coli or Citrobacter rodentium.

8. A drug suitable for treating and / or preventing Gram-negative bacterial infection, characterized in that: The drug contains the chitosan nanoparticles FR5-CS-NPs as claimed in claim 3.

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