Fatty acid modified nano antibacterial peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of polypeptide drugs in biochemistry, and discloses three antibacterial peptide analogues as well as a preparation method and application thereof, the sequence of an antibacterial peptide P-1 is CH3CO-GGGENIKKILSKIKLLK-NH2, the sequence of an antibacterial peptide P-2 is CH3CH2CH2CO-GGGENIKKILSKIKLLK-NH2, the sequence of an antibacterial peptide P-3 is CH3 (CH2) 6CO-GGGENIKKILSKIKLLK-NH2, the carboxyl ends of polypeptides are subjected to amidation, and the polypeptides are all positively charged when the pH is equal to 7. The artificially synthesized antibacterial peptide disclosed by the invention shows a remarkable sterilization effect on multidrug resistant staphylococcus epidermidis. P-1, P-2 and P-3 have small molecular weight, are convenient to synthesize, have broad-spectrum bactericidal activity, are self-assembled into nanoparticles, have low cytotoxicity, resist trypsin degradation and the like, and have wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide drugs in biochemistry, and relates to fatty acid-modified nano antimicrobial peptides and their preparation method and application, and particularly to the use of antimicrobial peptides in the preparation of drugs for resisting multidrug-resistant Staphylococcus infection. Background Art

[0002] Antibiotics are effective weapons for treating bacterial infections. Various types of antibiotics are widely used clinically to treat infectious diseases. However, due to the lack of an effective management and control system and the improper use of antibiotics, the level of bacterial resistance has risen rapidly, the types and number of resistant strains have increased rapidly, and the mortality rate from bacterial infections has also increased year by year. The World Health Organization (WHO) has listed antibiotic resistance as one of the three major public health threats of the 21st century. According to reports, approximately 700,000 people die each year due to drug-resistant bacteria. In addition, it is estimated that by 2050, direct and indirect deaths caused by drug-resistant bacteria will reach 10 million and 300 million, respectively, exceeding the current number of deaths related to cancer. Therefore, there is an urgent need to develop new antimicrobial agents to meet this challenge.

[0003] Staphylococcus epidermidis is a normal flora that often colonizes the surface of humans and animals and cavities connected to the outside world. It is a coagulase-negative Gram-positive bacterium. Current studies have shown that coagulase-negative Staphylococci (CNS) have become a common pathogen of hospital infections. Staphylococcus epidermidis is the most common CNS in clinical practice. It is an important opportunistic pathogen that can cause serious acquired infections in immunocompromised patients. In recent years, with the widespread use of antibiotics and the increase in the types of antimicrobial drugs, the resistance rate of methicillin-resistant Staphylococcus epidermidis (MRSE) has increased. The China Bacteria Detection Network's report for the first half of 2024 showed that the detection rate of clinical strains of methicillin-resistant Staphylococcus epidermidis was as high as 77.4%. The characteristic of Staphylococcus epidermidis that it easily adheres to the surface of medical devices to form biofilms makes treatment more difficult, and it has now become one of the important pathogens of hospital infections.

[0004] Antimicrobial peptides (AMPs) are naturally occurring polypeptides produced by organisms. They typically exhibit broad-spectrum antimicrobial activity against bacteria, viruses, fungi, and parasites, with strong specificity, low risk of drug resistance, and immunomodulatory effects. However, natural AMPs suffer from low antimicrobial activity, high cytotoxicity, rapid degradation in the body, and short half-lives. Therefore, researchers are increasing the clinical potential of natural AMPs by adjusting their amino acid sequence, charge, polarity, adding saturated fatty acid chains, or introducing chemical modifications. Clear guiding strategies and practical examples are still needed for the modification of AMPs. Summary of the Invention

[0005] Purpose of the invention: To solve the existing problem of treating multidrug-resistant epidermidis infections, the present invention provides novel antimicrobial peptides P-1, P-2 and P-3 having good antimicrobial activity against multidrug-resistant Staphylococcus epidermidis, low cytotoxicity, and resistance to trypsin degradation, and their use.

[0006] To achieve the above research objectives, the present invention proposes:

[0007] The fatty acid-modified nanopeptides, P-1, P-2, and P-3, are acylated at the amino terminus and amidated at the carboxyl terminus. P-1 is acetylated at the amino terminus of the antimicrobial peptide GGGENIKKILSKIKKLLK-NH2, P-2 is butyrylated at the amino terminus of the antimicrobial peptide GGGENIKKILSKIKKLLK-NH2, and P-3 is octanoylated at the amino terminus of the antimicrobial peptide GGGENIKKILSKIKKLLK-NH2. This alters the hydrophobicity of the peptide chain while enhancing its antimicrobial activity.

[0008] Furthermore, all three antimicrobial peptide analogs were positively charged at pH = 7.

[0009] The preparation method of fatty acid-modified nano antimicrobial peptides adopts solid-phase organic synthesis, utilizes Fmoc-protected amino acid strategy, and SPPS solid-phase synthesis technology to complete peptide synthesis, cleavage, oxidation, and purification to obtain the target product.

[0010] The specific method is: using the Fmoc protection strategy and Fmoc-Linker MBHA resin as a carrier, the synthesis process starts from the C-terminus of the peptide chain and constructs the peptide chain by stepwise coupling of amino acids until the synthesis of the entire peptide sequence is completed. After each coupling of amino acids, color detection is performed to ensure the adequacy of the reaction and the correctness of the peptide chain;

[0011] After the synthesis is completed, the peptide resin enters the cleavage stage. The cleavage uses a TFA series of reagents added to the peptide resin at low temperature and stirred for 2.5 hours to ensure the removal of the protecting group and the release of the polypeptide. After the cleavage reaction is completed, it is precipitated with ice ether and washed multiple times to remove the solvent and impurities to obtain a crude product. The next purification step uses C-18 column packing for reverse phase chromatography purification, and finally the ESI-MS method is used to identify its molecular weight and purity.

[0012] Furthermore, the Fmoc-Linker MBHA resin S=0.3 mmoL / g.

[0013] Furthermore, during the synthesis process, 20% Pip / DMF solution and DMF solution were used to wash and react the resin.

[0014] Furthermore, when coupling amino acids, the formula of AA / DIEA / TBTU is used to activate and connect the amino acids to ensure the smooth extension of the peptide chain.

[0015] Furthermore, the TFA series reagents are FA:H2O:EDT:TIS=95:1:2:2, and the low temperature is 0-10°C.

[0016] Furthermore, the reverse phase chromatography purification includes dissolving the crude product after cleavage, using 0.1% TFA / H2O and 0.1% TFA / CAN as mobile phases, and separating the target product by gradient elution.

[0017] Application of fatty acid-modified nanoantimicrobial peptides in the treatment of multidrug-resistant Staphylococcus epidermidis.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The synthetic antimicrobial peptides described in this study demonstrate significant bactericidal activity against multidrug-resistant Staphylococcus epidermidis. P-1, P-2, and P-3 exhibit broad application prospects due to their low molecular weight, ease of synthesis, broad-spectrum bactericidal activity, ability to self-assemble into nanoparticles, low cytotoxicity, and resistance to trypsin degradation.

[0020] 1. Improved Antimicrobial Activity: This invention effectively alters the hydrophobicity of the antimicrobial peptide GGGENIKKILSKIKKLLK by fatty acylation of the amino terminus and amidation of the carboxyl terminus. This modification strategy significantly enhances the antimicrobial activity and self-assembly properties of the antimicrobial peptides (P-1, P-2, and P-3), resulting in a strong inhibitory effect against multidrug-resistant Staphylococcus epidermidis, providing a new and effective approach to addressing the increasingly serious problem of drug-resistant bacterial infections.

[0021] 2. Optimized Charge Properties: All three antimicrobial peptide analogs are positively charged at pH 7. This positive charge facilitates the interaction of the antimicrobial peptides with the negatively charged components on the bacterial cell membrane surface, enhancing their affinity and penetration into the bacterial cell membrane. This further enhances the antimicrobial peptides' antimicrobial efficacy, enabling them to more effectively disrupt bacterial cell membrane structures and exert their antimicrobial effects.

[0022] 3. Improved Self-Assembly Performance: All three antimicrobial peptides self-assembled into nanoparticles at a concentration of 1.56 μM, demonstrating excellent self-assembly capabilities. P-1, modified with acetic acid, self-assembled into spherical nanoparticles approximately 20 nm in size, exhibiting minimal particle size and optimal uniformity, making it suitable for the development of nano-drug delivery systems or antimicrobial agents. P-2 and P-3, modified with butyric acid and octanoic acid, formed nanoparticles measuring 50–200 nm, exhibiting diverse morphologies, including square prisms (P-2) and spherical shapes (P-3), respectively, with poor uniformity.

[0023] 4. Efficient Synthesis: The solid-phase organic synthesis method, utilizing Fmoc-protected amino acids and SPPS solid-phase synthesis technology, offers significant advantages in preparing antimicrobial peptide analogs. This method, which begins at the carboxyl terminus of the peptide chain and progressively couples amino acids to build the peptide chain, provides an efficient and controllable synthesis process. Colorimetric detection after each amino acid coupling ensures the adequacy of the reaction and the correctness of the peptide chain, significantly improving synthesis efficiency and product accuracy while reducing the risk of synthesis failure.

[0024] 5. Good cleavage and purification effects: During the cleavage stage, a specific ratio of TFA series reagents (FA:H2O:EDT:TIS=95:1:2:2) is used to react at low temperature (0-10°C), which can not only effectively remove the protective group, but also ensure the smooth release of the polypeptide, reducing the degradation of the polypeptide and the occurrence of side reactions. Through ice ether precipitation and multiple washings, the solvent and impurities can be efficiently removed to obtain a crude product with higher purity. Subsequently, C-18 column packing is used for reverse phase chromatography purification, and 0.1% TFA / H2O and 0.1% TFA / CAN are used as mobile phases for gradient elution, which can accurately separate the target product, further improve the purity of the product, and ensure that the final antimicrobial peptide analogue has high quality and activity.

[0025] 6. Reasonable selection of resin and reagents: Fmoc-Linker MBHA resin was selected as the carrier, with an exchange capacity of S = 0.3 mmoL / g, providing a suitable carrier environment for peptide chain synthesis, ensuring the stability and efficiency of peptide chain synthesis. During the synthesis process, the resin was cleaned and reacted with 20% Pip / DMF solution and DMF solution, which can effectively remove unreacted reagents and impurities and ensure the smooth progress of the reaction. When coupling amino acids, the AA / DIEA / TBTU formula was used for amino acid activation and access. This formula can effectively promote the coupling reaction between amino acids, ensure the smooth extension of the peptide chain, and improve the synthesis efficiency and product yield.

[0026] 7. Broad Application Prospects: Fatty acid-modified nanopeptides P-1, P-2, and P-3 have significant potential for treating multidrug-resistant Staphylococcus epidermidis. As drug-resistant bacterial infections become increasingly serious, the effectiveness of traditional antibiotics has gradually declined. The antimicrobial peptide analogs of this invention offer a new option for treating multidrug-resistant Staphylococcus epidermidis infections and are expected to play an important role in clinical treatment, improving patient outcomes and prognosis. They possess broad market application prospects and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the HPLC detection chart of the modified peptide P-1;

[0028] Figure 2 This is the HPLC detection chart of the modified peptide P-2;

[0029] Figure 3 This is the HPLC detection chart of the modified peptide P-3;

[0030] Figure 4 is the ESI-MS detection image of the modified peptide P-1;

[0031] Figure 5 is the ESI-MS detection image of the modified peptide P-1;

[0032] Figure 6 is the ESI-MS detection image of the modified peptide P-1;

[0033] Figure 7 is the circular dichroism spectrum of the modified peptide P-1 in water and 50% TFE;

[0034] Figure 8 is the circular dichroism spectrum of the modified peptide P-2 in water and 50% TFE;

[0035] Figure 9 is the circular dichroism spectrum of the modified peptide P-3 in water and 50% TFE;

[0036] Figure 10 It is a graph of the hemolytic activity of antimicrobial peptides P-1, P-2, and P-3;

[0037] Figure 11 The results are as follows: the effects of P-1, P-2, and P-3 on RAW264.7 cell viability;

[0038] Figure 12 These are scanning electron micrographs of 1.56 μM antimicrobial peptides, A is P-1, B is P-2, and C is P-3;

[0039] Figure 13 This is the bactericidal kinetic curve of antimicrobial peptide P-1 against drug-resistant Staphylococcus epidermidis;

[0040] Figure 14 This is the bactericidal kinetic curve of antimicrobial peptide P-2 against drug-resistant Staphylococcus epidermidis;

[0041] Figure 15 This is the bactericidal kinetic curve of antimicrobial peptide P-3 against drug-resistant Staphylococcus epidermidis;

[0042] Figure 16 The results of scanning electron microscopy observation of the morphological effects of P-1, P-2, and P-3 on multidrug-resistant Staphylococcus epidermidis are shown. A is the control group, which is multidrug-resistant Staphylococcus epidermidis without antimicrobial peptide treatment; B, C, and D are morphological images of multidrug-resistant Staphylococcus epidermidis treated with 1×MIC antimicrobial peptides P-1, P-2, and P-3 for 1 hour.

[0043] Figure 17 is the stability of antimicrobial peptide P-1 under trypsin incubation;

[0044] Figure 18 is the stability of antimicrobial peptide P-2 under trypsin incubation;

[0045] Figure 19 The stability of antimicrobial peptide P-3 under trypsin incubation. DETAILED DESCRIPTION

[0046] The following examples may help those skilled in the art to better understand the present invention. The examples are only used to illustrate the present invention and do not impose any limitation on the present invention.

[0047] Example 1 Preparation of antimicrobial peptides P-1, P-2, and P-3:

[0048] The antimicrobial peptide is acetylated, butyrylated, and octanoylated at the amino terminus of the polypeptide chain of the antimicrobial peptide GGGENIKKILSKIKKLLK-NH2, thereby changing the hydrophobicity of the peptide chain and improving its antimicrobial activity.

[0049] Table 1 Sequence information of P-1, P-2, and P-3

[0050]

[0051] Chemical synthesis method of P-1, P-2 and P-3: According to the designed amino acid sequence (Table 1), the solid phase synthesis Fmoc method was used to synthesize the full sequence. This process uses solid phase synthesis (SPPS) to synthesize the target peptide, adopts Fmoc protection strategy, and uses Fmoc-Linker MBHA resin (S=0.3mmoL / g) as a carrier. The synthesis process starts from the C-terminus of the peptide chain and constructs the peptide chain by step-by-step coupling of amino acids until the synthesis of the entire peptide sequence is completed. During the synthesis process, the resin is washed and reacted with 20% Pip / DMF solution and DMF solution. When coupling amino acids, the AA / DIEA / TBTU formula is used for amino acid activation and access to ensure smooth extension of the peptide chain. Color development detection is performed after each coupling to ensure the adequacy of the reaction and the correctness of the peptide chain.

[0052] After the synthesis is completed, the peptide resin enters the cleavage stage. The cleavage uses a TFA series of reagents (TFA:H2O:EDT:TIS=95:1:2:2). These reagents are added to the peptide resin at low temperature (0-10°C) and stirred for 2.5 hours to ensure the removal of the protecting group and the release of the peptide. After the cleavage reaction is completed, ice ether precipitation is used and washed multiple times to remove the solvent and impurities to obtain a crude product. The next purification step uses C-18 column packing for reverse phase chromatography purification. After the cleavage crude product is dissolved, 0.1% TFA / H2O and 0.1% TFA / CAN are used as mobile phases to separate the target product by gradient elution. During the purification process, the peak of the target product is detected by reverse HPLC to ensure that the purity of the target peptide is higher than 95%. Unqualified products will be purified again, and finally the qualified main peak will be collected and freeze-dried under reduced pressure to obtain a peptide powder with a purity higher than 95%. Finally, the ESI-MS method is used to identify its molecular weight and purity to ensure that the target peptide is obtained with extremely high purity, providing reliable polypeptide materials for subsequent application research or experiments.

[0053] like Figure 1-3 As shown in Figure 2, the purity of the modified peptides P-1, P-2, and P-3 can reach 95% or above. Figure 4-6 As shown, the relative molecular masses of P-1, P-2 and P-3 identified by ESI-MS were 2208.5, 2036.5 and 2092.5, respectively.

[0054] Example 2 Determination of the secondary structure of antimicrobial peptides P-1, P-2, and P-3

[0055] Circular dichroism (CD) can quickly and accurately obtain information on the secondary structure of biological macromolecules such as proteins. Measurements in solution are closer to their physiological state. The secondary structure of P-1, P-2, and P-3 was detected using the CD method. The colorimetric cell was cleaned with 2 mol / L dilute hydrochloric acid and rinsed with a large amount of ultrapure water before use. 50% TFE dissolved in ultrapure water was transferred to the colorimetric cell, and the wavelength range of the scanning spectrum was set to 190-260 nm, the path length was 2 nm, and the measurement was performed at room temperature with a scanning speed of 50 nm / min, a response time of 2 s, and a spectral bandwidth of 1 nm. The spectrum was measured and the scanning baseline was recorded as the background value. Solutions of 0.125 mg / mL of P-1, P-2, and P-3 dissolved in ultrapure water and 50% TFE (v / v) were transferred to the colorimetric cell, the spectrum was measured under the above conditions, and the average value of four scans was collected.

[0056] The results are as follows Figure 7-9As shown, the secondary structures of P-1, P-2, and P-3 were analyzed in water and 50% TFE solutions. Based on the experimentally measured relationship between wavelength and ellipticity, the secondary structure content of the antimicrobial peptide analogs was analyzed using the circular dichroism spectroscopy analysis website (http: / / cbdm-01.zdv.uni-mainz.de / ~andrade / k2d3 / ). The results are shown in Table 2 below.

[0057] In the 50% TFE phase, P-1, P-2, and P-3 exhibited a positive peak near 190 nm and negative peaks at approximately 208 nm and 220 nm, respectively. All three exhibited α-helical structures, while the α-helical content was lower in the aqueous phase. This suggests that TFE significantly promotes α-helical formation in these peptides in a simulated membrane environment. In the aqueous phase, P-1, P-2, and P-3 exhibited a mixed α-helical and β-sheet structure, but in the 50% TFE phase, they completely transitioned to α-helical structures. This transition generally indicates that the peptide possesses strong membrane binding ability and potential antimicrobial activity in a simulated membrane environment.

[0058] Table 2 Secondary structure content of P-1, P-2, and P-3 in water and 50% TFE

[0059]

[0060] Example 3 Determination of hemolytic activity of antimicrobial peptides P-1, P-2, and P-3

[0061] Freshly collected human blood was diluted with 0.9% saline to a 2% human red blood cell suspension. Antimicrobial peptides P-1, P-2, and P-3 were dissolved in 0.9% saline to final concentrations ranging from 200 μM to 12.5 μM. 750 μL of the peptide solution was mixed with 750 μL of human red blood cells in equal volumes. 0.9% saline was used as a negative control, and 0.1% Triton X-100 was used as a positive control. All groups were incubated in a 37°C water bath for 1 hour. After removal, the cells were centrifuged at 1500 rpm for 10 minutes. 200 μL of the supernatant was added to a 96-well cell culture plate, and the absorbance of each well was measured at 414 nm using a microplate reader. The hemolytic activity was calculated according to the following formula: hemolytic activity = {(Fn-F0) / (Ft-F0)}×100% (Fn is the absorbance of the experimental group, Ft is the absorbance of the positive control group, and F0 is the absorbance of the negative control group).

[0062] Depend on Figure 10 It can be seen that the P-1 and P-2 antimicrobial peptides did not cause hemolysis of human red blood cells even at a high concentration of 200 μM, and the P-3 antimicrobial peptide only caused 15.93% hemolysis at 200 μM.

[0063] Example 4 Cytotoxicity assay of antimicrobial peptides P-1, P-2, and P-3

[0064] The cytotoxicity of P-1, P-2, and P-3 was detected using the CCK-8 assay. RAW264.7 cells were plated at 5×10 4 cells / mL were plated and cultured in 96-well plates with a volume of 100 μL per well. Three replicates were set up and cultured at 37°C and 5% CO2 for 24 hours. The experiments were divided into the following groups: the drug-added group consisted of the wells containing cell suspension, CCK-8 solution and antimicrobial peptide solution; the control group consisted of the wells containing cell suspension and CCK-8 solution; the blank group consisted of the wells containing culture medium and CCK-8 solution. P-1, P-2 and P-3 were added to the corresponding systems of the experimental groups to give final concentrations of 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM and 100 μM, respectively, and cultured at 37°C and 5% CO2 for 24 hours. 10 μL of CCK-8 reagent was added to each well, mixed and cultured at 37°C in the dark for 2 hours. The OD values ​​were determined using an enzyme-labeled instrument. 450 Calculate cell viability.

[0065] The experimental results are as follows Figure 11 As shown, P-1 and P-2 had little effect on RAW 264.7 cell viability at concentrations up to 100 μM, with cell survival rates exceeding 80%. At a P-3 concentration of 6.25 μM, RAW 264.7 cell survival rates reached 80% and above. This suggests that the engineered peptides P-1 and P-2 exhibit minimal cytotoxicity to RAW 264.7 cells.

[0066] Example 5 Scanning electron microscopy observation of 1.56 μM antimicrobial peptides P-1, P-2, and P-3

[0067] 1.56 μM of P-1, P-2, and P-3 were prepared in ultrapure water, sonicated in an ultrasonic water bath for 2 min, centrifuged at 1000 rpm for 1 min, smeared and spread on a glass slide, quickly dried with nitrogen, ion sputtered with gold, and images observed using a scanning electron microscope.

[0068] like Figure 12As shown, antimicrobial peptide P-1 at a concentration of 1.56 μM self-assembles into spherical nanoparticles approximately 20 nm in size. The images reveal uniform particle morphology, densely and regularly distributed, demonstrating good self-assembly ability and high nucleation efficiency. Antimicrobial peptide P-2, at the same concentration, forms nanoparticles ranging in size from 50–200 nm, exhibiting square or quasi-square-prism structures. The particles are regular in shape and present in a moderate number, indicating a certain degree of self-assembly orientation, though their uniformity is slightly inferior to that of P-1. Antimicrobial peptide P-3 also forms particles with a size range of 100–200 nm, but primarily spherical or elliptical shapes. The particles vary in size and are relatively dispersed, indicating weaker self-assembly consistency. It is speculated that the hydrophobic chain length interferes with the self-assembly process.

[0069] Example 6 Antimicrobial Activity Detection of Antimicrobial Peptides P-1, P-2, and P-3

[0070] The multidrug-resistant Staphylococcus epidermidis MRSE 1208 strain was inoculated into 10 mL of liquid culture medium and placed in a shaker at 37°C and 150 rpm overnight. The bacteria that were cultured to the logarithmic growth phase were diluted to 2 × 10 5 CFU / mL for later use, and use the half-dilution method to prepare an aqueous solution of antimicrobial peptides with a final concentration of 100 μM-1.56 μM.

[0071] The experiment was divided into three groups: the experimental group included antimicrobial peptides and bacterial suspension, antimicrobial peptides and LB medium; the negative control group included bacterial suspension and ultrapure water, and the blank control group included ultrapure water and LB medium. Equal amounts of bacterial suspension were mixed with different concentrations of P-1, P-2, P-3 and Gentamicin, placed in a sterile 96-well plate, incubated at 37°C for 18 hours, and each sample (OD) was measured using a microplate reader (Varioskan Flash Microplate Reader, Thermo Scientific Co, Beijing, China). 600 ) was measured using the absorbance of the assay. The MIC was defined as the lowest drug concentration that inhibited bacterial growth by 95% or more. Each condition was repeated three times.

[0072] As shown in Table 3, P-1, P-2, and P-3 exhibited broad and rapid antibacterial activity against a wide range of strains, including sensitive and multidrug-resistant Gram-positive and Gram-negative bacteria, such as drug-resistant Staphylococcus epidermidis, drug-resistant Acinetobacter baumannii, drug-resistant Enterococcus faecalis, and drug-resistant Staphylococcus aureus. The MICs of P-1, P-2, and P-3 against these pathogens ranged from 1.56 to 50 μmol / L. In particular, the MIC values ​​for both sensitive and multidrug-resistant Staphylococcus epidermidis were only 1.56 μmol / L.

[0073] Table 3 Antibacterial activity of antimicrobial peptides P-1, P-2, and P-3

[0074]

[0075] Example 7 Bactericidal kinetics of P-1, P-2, and P-3 against multidrug-resistant Staphylococcus epidermidis

[0076] Drug-resistant Staphylococcus epidermidis MRSE 1208 was identified and its antibiotic susceptibility tested by the clinical laboratory of the First Affiliated Hospital of Dalian Medical University. The strain was resistant to ampicillin, cefazolin, ciprofloxacin, erythromycin, imipenem, levofloxacin, penicillin, ceftazidime, and rifampicin; moderately sensitive to tetracycline and vancomycin; and sensitive to chloramphenicol and sulfamethoxazole.

[0077] To evaluate the tolerance and persistence of P-1, P-2, and P-3 against multidrug-resistant Staphylococcus epidermidis, we conducted a bactericidal kinetics experiment and extracted the minimum duration forkilling (MDK) from the Time-Kill curve. Two strains of Acinetobacter baumannii were cultured overnight at 37°C to the logarithmic growth phase and diluted to OD 600 A value of 10 -3 For later use. For the experimental group, 10 μL of P-1, P-2, or P-3 at final concentrations of 1×MIC, 2×MIC, 4×MIC, and 8×MIC, respectively, was added to 990 μL of the diluted bacterial solution and incubated in a 37°C constant temperature incubator. After incubation at 37°C at different time points, the cells were removed and centrifuged at 4000 rpm for 10 minutes. The supernatant was discarded, and the cells were washed three times with PBS to remove the antimicrobial peptides. The cells were diluted and plated. For the control group, 1 mL of the diluted bacterial solution was incubated at the same time points as the experimental group. After serial dilution, the cells were plated and incubated at 37°C constant temperature incubator for 18-24 hours before colonies were counted.

[0078] The experimental results are as follows Figure 13-15 As shown in the figure. The control group consisted of multidrug-resistant Staphylococcus epidermidis that were not treated with antimicrobial peptides. The bacterial count increased with incubation time. In the experimental group, antimicrobial peptide P-2 completely killed the drug-resistant S. epidermidis at a concentration of 8× the MIC within 30 minutes, indicating a MDK value of 30 minutes for P-2 against sensitive Acinetobacter baumannii. P-3 completely killed the drug-resistant S. epidermidis at a concentration of 4× the MIC within 30 minutes. Within 2 hours, P-1 had no significant inhibitory effect on drug-resistant S. epidermidis. The bactericidal effects of P-1, P-2, and P-3 against drug-resistant S. epidermidis showed concentration- and time-dependent trends.

[0079] Example 8 Scanning electron microscopy observation of the effects of P-1, P-2, and P-3 on the morphology of drug-resistant Staphylococcus epidermidis

[0080] The cell morphology of drug-resistant Staphylococcus epidermidis was observed using scanning electron microscopy (SEM). 5 CFU / mL) and treated with 1×MIC of peptides P-1, P-2, and P-3 at 37°C for 1 h. The bacterial samples were smeared and spread on a glass slide, quickly blown dry with nitrogen gas, and sputtered with gold ions. The images were observed using a scanning electron microscope.

[0081] like Figure 16 As shown, the control group without peptide ( Figure 16 A) The bacteria are complete in morphology, compact and full in structure, and have intact cell membranes. When 1×MIC antimicrobial peptide P-1 ( Figure 16 B) After 1 hour of treatment, a lot of P-1 nanoparticles were obviously enriched on the surface of bacterial cell membranes. The morphological changes of drug-resistant Staphylococcus epidermidis were not obvious, which may not be achieved by breaking the membrane. When 1×MIC antimicrobial peptides P-2 and P-3 were added and treated for 1 hour ( Figure 16 C and Figure 16 D) The bacterial cell membrane surface becomes concave, wrinkled, and convex, leading to leakage of intracellular substances. These results indicate that P-1, P-2, and P-3 have strong antibacterial activity against multidrug-resistant Acinetobacter baumannii.

[0082] Example 9: HPLC Detection of the Enzymatic Stability of P-1, P-2, and P-3 in Trypsin

[0083] Prepare 0.2 mg / mL pancreatic enzyme solution for use. To better simulate the in vivo environment, the peptides and trypsin solutions used were prepared with PBS buffer. Antimicrobial peptides P-1, P-2, and P-3 were prepared into 25 mm stock solutions for use. The antimicrobial peptides were mixed with trypsin at a volume ratio of 1:15, i.e., 5 μL of antimicrobial peptide and 75 μL of trypsin were mixed and placed in a 37°C incubator for 0 h, 2 h, and 4 h, respectively. After incubation, 160 μL each of pre-cooled 15% TFA aqueous solution and acetonitrile were added to the sample, mixed evenly, and allowed to stand at 4°C for 20 min. The sample was centrifuged at 4°C and 13,000 rpm for 10 min to precipitate the protein. The supernatant was collected and filtered using a filter membrane. The HPLC instrument parameters were as follows: flow rate 1 mL / min, phase A was 0.1% TFA in water, phase B was 0.1% TFA in acetonitrile, and data were collected for analysis using a C18 loading column (5 mm, 0.46×25 cm).

[0084] according to Figure 17-19Analysis of the data in Tables 4, 5, and 6 shows that P-1, P-2, and P-3 all exhibited varying degrees of degradation during trypsin incubation, with the degree of degradation increasing with prolonged incubation time. Specifically, after 4 hours of incubation, P-1 had a residual rate of 53.21% and a degradation rate of 46.79%. P-2 had a higher residual rate of 79.75% and a degradation rate of 20.25%. P-3 had a residual rate of 72.33% and a degradation rate of 27.67%. Compared to P-1, P-2 and P-3 exhibited stronger stability against enzymatic degradation, particularly P-2. This suggests that P-2 and P-3 are suitable candidates for antimicrobial peptides, better able to resist degradation by trypsin after prolonged exposure.

[0085] Table 4 Stability and degradation of antimicrobial peptide P-1 at different incubation times

[0086]

[0087] Table 5 Stability and degradation of antimicrobial peptide P-2 at different incubation times

[0088]

[0089] Table 6 Stability and degradation of antimicrobial peptide P-3 at different incubation times

[0090]

[0091] The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be all feasible embodiments of the present invention. Any obvious modifications made by a person skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. Fatty acid-modified nano antimicrobial peptide, characterized in that: They are P-1, P-2, and P-3, respectively. The amino terminus of the polypeptide is fatty acylated and the carboxyl terminus is amidated. P-1 is acetylated at the amino terminus of the polypeptide chain of the antimicrobial peptide GGGENIKKILSKIKKLLK-NH2, P-2 is butyrylated at the amino terminus of the polypeptide chain of the antimicrobial peptide GGGENIKKILSKIKKLLK-NH2, and P-3 is octanoylated at the amino terminus of the polypeptide chain of the antimicrobial peptide ENIKKILSKIKKLLK-NH2, which improves the antibacterial activity while changing the hydrophobicity of the peptide chain.

2. The fatty acid-modified nano antimicrobial peptide according to claim 1, characterized in that: All three antimicrobial peptide analogs were positively charged at pH 7. The three antimicrobial peptides could self-assemble into nanoparticles at 1.56 μM. P-1 formed uniform spherical particles of about 20 nm, while P-2 and P-3 formed diverse particles of 50-200 nm, and their uniformity was poorer than that of P-1.

3. A method for preparing a fatty acid-modified nano antimicrobial peptide, characterized in that: Solid-phase organic synthesis, Fmoc-protected amino acid strategy, and SPPS solid-phase synthesis technology were used to complete peptide synthesis, cleavage, oxidation, and purification to obtain the target product.

4. The method for preparing the fatty acid-modified nano antimicrobial peptide according to claim 3, wherein: The Fmoc protection strategy was adopted with Fmoc-Linker MBHA resin as the carrier. The synthesis process started from the C-terminus of the peptide chain and constructed the peptide chain by step-by-step coupling of amino acids until the synthesis of the entire peptide sequence was completed. Color development detection was performed after each coupling of amino acids to ensure the adequacy of the reaction and the correctness of the peptide chain. After the synthesis was completed, the peptide resin entered the cleavage stage. The cleavage used TFA series reagents added to the peptide resin at low temperature and stirred for 2.5 hours to ensure the removal of the protecting group and the release of the polypeptide. After the cleavage reaction was completed, ice ether precipitation was used and multiple washings were performed to remove the solvent and impurities to obtain a crude product. The next purification step used C-18 column packing for reverse phase chromatography purification, and finally the ESI-MS method was used to identify its molecular weight and purity.

5. The method for preparing the fatty acid-modified nano antimicrobial peptide according to claim 4, characterized in that: Fmo c-Linker MBHA resin S = 0.3 mmoL / g.

6. The method for preparing the fatty acid-modified nano antimicrobial peptide according to claim 4, wherein: During the synthesis process, 20% Pip / DMF solution and DMF solution were used to wash and react the resin.

7. The method for preparing the fatty acid-modified nano antimicrobial peptide according to claim 4, characterized in that: When coupling amino acids, the formula of AA / DIEA / TBTU is used to activate and connect amino acids to ensure the smooth extension of the peptide chain.

8. The method for preparing the fatty acid-modified nano antimicrobial peptide according to claim 4, characterized in that: The TFA series reagents are FA:H2O:EDT:TIS=95:1:2:2, low temperature 0-10℃.

9. The method for preparing the fatty acid-modified nano antimicrobial peptide according to claim 4, characterized in that: Reverse phase chromatography purification includes dissolving the crude product after cleavage, using 0.1% TFA / H2O and 0.1% TFA / CAN as mobile phases, and separating the target product by gradient elution.

10. Application of fatty acid modified nano antimicrobial peptides, characterized in that: Application in multidrug-resistant Staphylococcus epidermidis.

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