Antibacterial peptide and application thereof

Abstract

The invention relates to an antibacterial peptide and application thereof. The antibacterial peptide comprises a hydrophobic self-assembly promoting sequence, an OppA binding peptide sequence and a glycosylation modification group from an N terminal to a C terminal. The antibacterial peptide disclosed by the invention can be self-assembled to form stable nano-particles, so that the antibacterial peptide can be deposited on the epithelium of a respiratory tract infected by bacteria in a direct aerosol inhalation manner, and can efficiently penetrate through the mucous membrane layer of the respiratory tract to realize bacterial targeting. After being combined with bacteria, the antibacterial peptide can be transferred into cells by OppA and inhibit biosynthesis of bacterial peptidoglycan and capsular polysaccharide, so that the bacteria are killed, the bacterial infection capability is reduced, and the generation of drug resistance is inhibited. Therefore, the antibacterial peptide disclosed by the invention can be used for resisting gram-positive bacteria, particularly staphylococcus aureus, and is further used for preventing or treating acute or chronic infection caused by gram-positive bacteria, such as mucous membrane infection, including lung injury, pneumonia, meningitis, osteoarthritis, conjunctivitis and the like.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals, and specifically relates to an antimicrobial peptide and application thereof. Background Art

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of hospital-acquired pneumonia [1] and ventilator-associated pneumonia [2] It is one of the most common pathogens of the disease, with a mortality rate of up to 55% depending on the population being investigated. [3] Although systemic administration of glycosylated peptide-based antimicrobial substances such as vancomycin is listed as the first-line drug for the treatment of pneumonia caused by Staphylococcus aureus in clinical guidelines [4] , this type of treatment is usually difficult to achieve appropriate therapeutic effects. After systemic administration (intravenous or oral), although the antibacterial drug has a high blood concentration in the blood, it is difficult for it to penetrate into the lung tissue, which ultimately leads to insufficient drug accumulation in the lung epithelium to kill the customized bacteria. At the same time, long-term high-dose use of glycopeptide antibiotics may cause nephrotoxicity, especially for patients with renal insufficiency. [5] In the current clinical cases of Staphylococcus aureus infection, strains resistant to glycopeptide antibiotics have been isolated, proving that bacteria are developing resistance to this type of treatment. [6] .

[0003] In order to solve the above difficulties in treating bacterial pneumonia, the pharmaceutical industry has focused on the development of inhalable preparations of various antibiotics in recent years. [7] Direct inhalation of antibiotics can maximize the accumulation of drugs on the respiratory surface. Unfortunately, the development of inhaled preparations that rely on antibiotics as active ingredients and add various excipients has been repeatedly frustrated. So far, the U.S. Food and Drug Administration (FDA) has approved two inhaled preparations based on tobramycin and aztreonam, respectively. [8,9] However, the active ingredients of these two preparations mainly exert antibacterial activity against Gram-negative bacteria, and have limited antibacterial activity against Gram-positive bacteria (such as MRSA). In terms of positive bacteria, the development of inhalable vancomycin dry powder preparation (AeroVancTM, NCT03181932) has attracted much attention. However, it failed in the recent Phase III clinical trial because it failed to significantly improve the lung function of patients with pneumonia caused by Gram-positive bacteria.

[0004] In summary, the aforementioned formulation process that relies on existing antibiotics has limited ability to combat the development of drug resistance, so it is urgent to develop inhalable antibacterial substances for Gram-positive bacterial lung infections.

[0005] References

[0006] 1 Kollef,M.H.et al.Epidemiology and outcomes of health-care-associated pneumonia:results from a large US database of culture-positivepneumonia.Chest 128,3854-3862,doi:10.1378 / chest.128.6.3854(2005).

[0007] 2 Lee,M.S.,Walker,V.,Chen,L.F.,Sexton,D.J.&Anderson,D.J.Theepidemiology of ventilator-associated pneumonia in a network of communityhospitals:a prospective multicenter study.Infection control and hospitalepidemiology 34,657-662,doi:10.1086 / 670991(2013).

[0008] 3 DeRyke,C.A.,Lodise,T.P.,Jr.,Rybak,M.J.&McKinnon,P.S.Epidemiology,treatment,and outcomes of nosocomial bacteremic Staphylococcus aureuspneumonia.Chest 128,1414-1422,doi:10.1378 / chest.128.3.1414(2005).

[0009] 4 Ferrara,A.M.Treatment of hospital-acquired pneumonia caused bymethicillin-resistant Staphylococcus aureus.International journal ofantimicrobial agents 30,19-24,doi:10.1016 / j.ijantimicag.2007.02.011(2007).

[0010] 5 Bamgbola,O.Review of vancomycin-induced renal toxicity:anupdate.Therapeutic advances in endocrinology and metabolism 7,136-147,doi:10.1177 / 2042018816638223(2016).

[0011] 6 Hiramatsu,K.Vancomycin-resistant Staphylococcus aureus:a new modelof antibiotic resistance.The Lancet.Infectious diseases 1,147-155,doi:10.1016 / s1473-3099(01)00091-3(2001).

[0012] 7 Quon,B.S.,Goss,C.H.&Ramsey,B.W.Inhaled antibiotics for lower airwayinfections.Annals of the American Thoracic Society 11,425-434,doi:10.1513 / AnnalsATS.201311-395FR(2014).

[0013] 8 Brogden,R.N.&Heel,R.C.Aztreonam.Areview of its antibacterialactivity,pharmacokinetic properties and therapeutic use.Drugs 31,96-130,doi:10.2165 / 00003495-198631020-00002(1986).

[0014] 9 Muhlebach, MS et al. Treatment intensity and characteristics of MRSAinfection in CF. Journal of cystic fibrosis: official journal of the European Cystic Fibrosis Society 10, 201-206, doi:10.1016 / j.jcf.2011.02.004(2011). Summary of the invention

[0015] The present invention aims to develop a new self-formulated antibacterial molecule based on single-molecule self-assembly, which can withstand the atomization process after forming a specific nanostructure and efficiently pass through the lung mucosal barrier to achieve efficient targeting of bacteria colonized in the lung epithelium. Since the molecular structure involved in the present invention does not cover the molecular structure of existing antibiotics, its new antibacterial mechanism is expected to combat various existing bacterial resistance.

[0016] The present invention constructs a glycosylated antimicrobial peptide targeting the periplasmic oligopeptide-binding protein (OppA) of Staphylococcus aureus, which can be transported into Staphylococcus aureus cells by OppA and regulates the metabolic process of aminoglycans, ultimately achieving excellent antibacterial effects.

[0017] Furthermore, the glycosylated antimicrobial peptides of the present invention can self-assemble to construct hydrophilic and electrically neutral nanoparticles, which can be directly administered to the respiratory tract in the form of a suspension by atomization, and can efficiently penetrate the biological barrier of the lungs to achieve targeting of Gram-positive bacteria colonized in the lung epithelium and alveolar immune cells, thereby significantly improving lung drug retention and improving the therapeutic effect after local administration, providing sufficient lung antibacterial ability, and inhibiting the progression of lung inflammation.

[0018] In view of this, one of the objects of the present invention is to provide an antimicrobial peptide or a salt thereof which comprises a self-assembly-promoting sequence, an OppA binding peptide and a C-terminal glycosylation modification from the N-terminus to the C-terminus.

[0019] On the other hand, another object of the present invention is to provide an antimicrobial peptide or a salt thereof, which comprises a self-assembly promoting sequence from the N-terminus to the C-terminus, and an OppA binding peptide.

[0020] On the other hand, another object of the present invention is to provide a pharmaceutical composition comprising the above antimicrobial peptide or a salt thereof.

[0021] On the other hand, another object of the present invention is to provide the use of the above antimicrobial peptide or its salt in the treatment of Gram-positive bacterial infection.

[0022] Technical Solution

[0023] In one aspect, the present invention provides an antimicrobial peptide or a salt thereof, wherein the antimicrobial peptide comprises, from the N-terminus to the C-terminus, a hydrophobic self-assembly-promoting sequence, an OppA-binding peptide sequence and a glycosylation modification group.

[0024] In an embodiment, the N-terminal hydrophobic self-assembly-promoting sequence can be represented by the structure shown in the following formula I:

[0025]

[0026] wherein Xaa-Xaa- represents a dipeptide linker selected from FF and FW, Indicates where the hydrophobic membrane permeable sequence is attached to the N-terminus of the OppA binding peptide sequence.

[0027] FF represents a dipeptide linker consisting of two phenylalanine amino acids, and FW represents a dipeptide linker consisting of one phenylalanine and one tryptophan.

[0028] The OppA binding peptide refers to a positively charged 3-9 peptide that can bind to periplasmic oligopeptide binding protein (OppA), for example, 3, 4, 5, 6, 7, 8, 9 peptides or a combination thereof, which is a lysine or arginine-rich sequence.

[0029] In a specific embodiment, the OppA binding peptide is a positively charged 6-9 peptide comprising the amino acid sequence KKKHKR (SEQ ID No.: 1).

[0030] In a specific embodiment, the amino acid sequence of the OppA binding peptide is KKKHKR.

[0031] In a specific embodiment, the glycosylation modification group can be selected from a monosaccharide group, a disaccharide group, a polysaccharide group and a chitosan fragment group, and is preferably selected from D-mannosamine, D-glucosamine and D-galactosamine groups and the like.

[0032] In a specific embodiment, the salt may be any suitable pharmaceutically acceptable salt, such as acetate or hydrochloride, but is not limited thereto.

[0033] In a specific embodiment, the antimicrobial peptide is represented by the following formula, sometimes referred to as BF8Man below:

[0034]

[0035] In the present invention, amino acids represented by single letters have the meanings generally used in the art.

[0036] In another aspect, the present invention provides an antimicrobial peptide or a salt thereof, wherein the antimicrobial peptide comprises a hydrophobic self-assembly-promoting sequence and an OppA binding peptide sequence from the N-terminus to the C-terminus.

[0037] The descriptions of the hydrophobic self-assembly-promoting sequence and the OppA-binding peptide are as described above.

[0038] In a specific embodiment, the antimicrobial peptide is represented by the following formula, sometimes referred to as BF8 below:

[0039]

[0040] The preparation method of the antimicrobial peptide according to the present invention is not particularly limited. For example, a polypeptide including a dipeptide linker sequence and an OppA binding peptide sequence can be first prepared by using polypeptide solid phase synthesis (SPPS), then biphenyl acetylation is performed, and finally glycosylation is performed. Based on the knowledge of the structure of the antimicrobial peptide according to the present invention, those skilled in the art can synthesize the antimicrobial peptide according to the present invention using any suitable method, such as a method in the prior art or developing a new synthesis method.

[0041] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the antimicrobial peptide or a salt thereof, and a pharmaceutically acceptable excipient.

[0042] In a specific embodiment, the dosage form of the pharmaceutical composition is not particularly limited, and can be prepared into any suitable dosage form, such as sprays, solutions, and suspensions, but not limited thereto. Those skilled in the art can select a suitable excipient according to the dosage form to be prepared, such as selecting a suitable propellant when preparing a spray, and selecting a suitable stabilizer when preparing a suspension.

[0043] The glycosylated polypeptide of the present invention can form stable nanoparticles with a diameter of about 20 nm by single-component self-assembly without the participation of auxiliary materials, which helps to deposit on the respiratory epithelial layer infected with bacteria by direct atomization inhalation, and efficiently penetrate the respiratory mucosal layer to achieve bacterial targeting. After binding to bacteria, the glycosylated polypeptide can be transported into the cell by OppA and inhibit the biosynthesis of bacterial peptidoglycan and capsular polysaccharide, thereby killing bacteria, reducing bacterial infection ability, and inhibiting the generation of drug resistance. In addition to treating bacterial pneumonia, the glycosylated polypeptide of the present invention is expected to be applied to mucosal infections caused by various Gram-positive bacteria, such as pneumonia, bacterial conjunctivitis, osteoarthritis, meningitis, etc.

[0044] Therefore, in another aspect, the present invention provides use of the above antimicrobial peptide or its salt in the preparation of antimicrobial drugs.

[0045] In a specific embodiment, the antibacterial drug is used to fight against Gram-positive bacteria, particularly Staphylococcus aureus.

[0046] In a specific embodiment, the antibacterial drug is used to prevent or treat acute or chronic infections caused by Gram-positive bacteria, such as mucosal infections, including lung injury, pneumonia, meningitis, osteoarthritis and conjunctivitis, but not limited thereto.

[0047] Beneficial Effects

[0048] The present invention intercepts a positively charged hexapeptide from the amino-rich positively charged sequence of the bacterial EspP signal peptide, performs hydrophobic modification on the sequence at the N-terminus to ensure the self-assembly ability of the molecule; performs monosaccharide modification on the C-terminus to increase the hydrophilicity of the C-terminus, optimize the nanostructure morphology, and ensure the hydrophilicity and electrical neutrality of the assembly surface. The finally formed glycosylated polypeptide molecule can efficiently target Gram-positive bacterial cells after penetrating into the lung epithelial layer, and be transported to the cytoplasm to achieve antibacterial activity.

[0049] The present invention has the following beneficial effects:

[0050] (1) The polypeptide constructed by the present invention can achieve high-affinity targeting to the periplasmic oligopeptide-binding protein (OppA) by intercepting a specific positively charged hexapeptide sequence and performing simple chemical modification on it. The number of amino acids in the final antimicrobial peptide BF8Man obtained is 8, the preparation cost is low, and it is easy to mass produce.

[0051] (2) The glycosylated polypeptide molecules constructed in the present invention can form BF8Man nanoparticles through a self-preparation process of single-molecule assembly. No auxiliary materials are required in the related process, and the final suspension can be directly administered by atomization inhalation, avoiding a special preparation process.

[0052] (3) The BF8Man nanoparticles constructed in the present invention have good stability, can withstand various shear forces during the atomization process, maintain a stable morphology and do not change the original antibacterial activity.

[0053] (4) The glycosylation strategy used in the present invention enables the self-assembled BF8Man nanoparticles to obtain surface hydrophilicity and electrical neutrality, significantly reducing the interaction between the polypeptide molecules and mucins in the respiratory mucosa, allowing the polypeptide nanoparticles to penetrate the respiratory mucosa layer, and ensuring the antibacterial activity of the glycosylated polypeptide in the microenvironment of the damaged airways and alveoli.

[0054] (5) The glycosylated antimicrobial peptides constructed in the present invention can be used to treat bacterial infections other than the respiratory tract, especially infections in areas with abundant mucosa or extracellular matrix, such as the treatment of bacterial conjunctivitis, meningitis and osteoarthritis.

[0055] (6) The glycosylated antimicrobial peptide constructed in the present invention is a new antimicrobial molecule that can be self-formulated based on single-molecule self-assembly. After forming a specific nanostructure, the molecule can withstand the atomization process and efficiently pass through the lung mucosal barrier to achieve efficient targeting of bacteria colonized in the lung epithelium.

[0056] (7) The molecular structure of the glycosylated antimicrobial peptide constructed by the present invention does not include the molecular structure of existing antibiotics, and its new antibacterial mechanism is expected to combat the drug resistance of various existing bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments, but the present invention is not limited thereto:

[0058] Figure 1 : Antibacterial efficiency of glycosylated antimicrobial peptide BF8Man and control peptide BF8 in killing free MRSA. A is a statistical graph of the antibacterial efficiency of BF8Man and BF8 in killing MRSA resuspended in PBS at a final concentration of 256 μM, ND means not detected. B is a representative optical photo of the plate coating in experiment A. C is the antibacterial ability of the peptide in the simulated airway and alveolar microenvironment in vitro.

[0059] Figure 2 : The broad spectrum of antimicrobial activity and the ability to inhibit drug resistance of the glycosylated antimicrobial peptide BF8Man. A is the minimum inhibitory concentration (MIC) of BF8Man and BF8 for various Gram-positive / negative bacteria as shown in the figure. B is the ability of BF8Man to inhibit drug resistance after 30 generations of continuous culture of MRSA compared with ciprofloxacin.

[0060] Figure 3 :Toxicity test of glycosylated antimicrobial peptide BF8Man on mammalian cells. A is the cytotoxicity of BF8Man and BF8 on normal human lung bronchial epithelial cells (BEAS-2B) at the concentrations shown in the figure. B is the hemolytic ability test of BF8Man and BF8 (320 μM) on New Zealand white rabbit blood.

[0061] Figure 4 :EDA energy spectrum determination of fluorine (F 18 )-substituted glycosylated antimicrobial peptide BF8Man in the cytoplasm of MRSA. Control is untreated MRSA.

[0062] Figure 5 :BF8Man and periplasmic oligopeptide binding protein (OppA) affinity determination. A is the endothermic and exothermic energy change diagram of the affinity between BF8Man and OppA determined by isothermal thermal titration experiment. B is the various thermodynamic values ​​calculated from A.

[0063] Figure 6 : Volcano plot of BF8Man conjugated with biotin binding to various proteins in the bacterial cytoplasm.

[0064] Figure 7 :Proteomic analysis proves that BF8Man affects the key proteins required for peptidoglycan synthesis by regulating the bacterial aminoglycan metabolism process. A is the KEGG pathway analysis of untreated bacteria, BF8-treated bacteria and BF8Man bacteria. B is the protein interaction map of bacteria treated with BF8Man.

[0065] Figure 8 :Material characterization of BF8Man nanoparticles and BF8 nanofibers. A is a transmission electron microscopy image showing the self-assembly morphology of BF8Man and BF8 at 256μM. B is the surface Zeta potential measurement of BF8Man and BF8 nanostructures. C is the critical micelle concentration (CMC) measurement of BF8Man and BF8 molecules.

[0066] Fig. 9 :Stability and activity test of BF8Man nanoparticles after atomization. A is a schematic diagram of measuring the particle size of BF8Man nanoparticles before and after atomization. B is the particle size distribution of BF8Man before atomization, sprayed after atomization, and remaining in the atomization cup. C is a schematic diagram of measuring the antibacterial ability of BF8Man after atomization. D is the residual number of MRSA after BF8Man (256μM) treats MRSA after atomization. E is the antibacterial efficiency of BF8Man in killing MRSA after atomization.

[0067] Fig.10 :In vitro evaluation of the ability of BF8Man nanoparticles and BF8 nanofibers to penetrate the lung mucosa. A is a schematic diagram of the in vitro construction of an artificial mucus model to evaluate the efficiency of peptide assemblies in penetrating the mucosa. B is a diagram of the relative fluorescence intensity of Cy5.5-labeled BF8Man and BF8 that successfully penetrated the mucosa in the lower gelatin layer.

[0068] Fig.11 : Immunofluorescence staining demonstrated the distribution of BF8Man on the surface of healthy airways and MRSA-infected airways.

[0069] Fig.12 : Immunofluorescence staining demonstrated the distribution of BF8Man in immune cells of healthy lung tissue and MRSA-infected lung tissue.

[0070] Fig.13 :BF8Man aerosol treatment alleviates MRSA infection in mouse lungs. A is a schematic diagram of the experiment of MRSA infection in mice, peptide aerosol inhalation treatment and evaluation of in vivo antibacterial effect. B is the content of MRSA in mouse bronchoalveolar lavage fluid one day after peptide aerosol administration (two days after infection).

[0071] Fig.14 : HE staining sections of the lungs of MRSA-infected mice three days after peptide aerosol administration.

[0072] Fig.15 :Peptide aerosol treatment relieves lung inflammation in piglets caused by MRSA infection. A is a schematic diagram of MRSA local infection, peptide aerosol treatment, hematological evaluation and lung inflammatory cell evaluation in piglets. B is a statistical chart of body temperature changes in piglets after MRSA infection and peptide aerosolization. C is a statistical chart of the total amount of white blood cells in piglet bronchoalveolar lavage fluid. D is a statistical chart of the change in the proportion of neutrophils in piglet bronchoalveolar lavage fluid.

[0073] Fig.16 :In vivo safety evaluation of piglets with continuous nebulization of BF8Man for 14 days. A is a schematic diagram of the use of a piglet model to evaluate the safety of continuous nebulization of BF8Man. B is a statistical chart of the number of nebulizations per minute in piglets during continuous nebulization. C is a statistical chart of the number of red blood cells in piglet blood during continuous nebulization. D is a statistical chart of the number of white blood cells in piglet blood during continuous nebulization. E is a statistical chart of the proportion of neutrophils in piglet blood during continuous nebulization.

[0074] Fig.17 : Continuous BF8Man aerosolization did not affect the thickening of the piglet's airway and the mucus secretion capacity of the lung epithelium. A is the Masson-Trichrome and Periodic Acid-Schiff stain (PAS) staining of the piglet's airway after 14 days of continuous aerosolization (day 15). B is the statistics of the average collagen layer thickness around the piglet's airway. C is the average number of goblet cells in the piglet's airway epithelium. DETAILED DESCRIPTION

[0075] The present invention will be described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments are further descriptions of the technical solutions of the present invention and do not indicate that the present invention is limited to these embodiments.

[0076] Reagents and instruments

[0077] D-Mannosamine was purchased from Aladdin. Type II mucin and dipalmitoylphosphatidylcholine (DPPC) were purchased from Leyan Biotechnology. BCA protein assay kit (#P0010S) was purchased from Beyotime. Vancomycin hydrochloride (Van) was purchased from Sigma-Aldrich. TRIzol (#15596026) was purchased from Thermo Fisher. Methicillin-resistant Staphylococcus aureus ATCC43300, USA 300, CCTCC 16465; Staphylococcus aureus ATCC 25923; Escherichia coli ATCC 25922, ATCC700928; multidrug-resistant Escherichia coli BNCC 186732 were purchased from Hangzhou Baosai Biotechnology Co., Ltd. Human lung epithelial cells (BEAS-2B) were purchased from American Type Culture Collection (ATCC).

[0078] Example 1 Design and preparation of inhalable glycosylated antimicrobial peptide BF8Man:

[0079] In order to design and prepare an inhalable glycosylated antimicrobial peptide, the present invention prepared a polypeptide molecule constructed by modular design, a 6-amino acid positively charged fragment (KKKHKR) was extracted from the EspP signal peptide, and its N-terminus was linked to a hydrophobic self-assembly promoting sequence (expressed as: Bip-FF, where Bip represents F represents phenylalanine) for covalent coupling. Further, in order to achieve efficient pulmonary delivery of the polypeptide and enhance the antibacterial activity of the molecule, the C-terminus was modified with D-mannosamine (represented as Man).

[0080] All polypeptides involved in the present invention are prepared, synthesized and purified by Shanghai WuXi AppTec Pharmaceutical Development Co., Ltd.

[0081] First, a polypeptide with a sequence of FFKKKHKR (SEQ ID No.: 2) was prepared by peptide solid phase synthesis (SPPS), and then the biphenyl group was covalently bound to the N-terminus of the polypeptide using 4-biphenylacetic acid (#196487, Sigma-Aldrich) to prepare BF8 with a purity greater than 98% (analyzed by HPLC by Shanghai WuXi AppTec New Drug Development Co., Ltd.).

[0082] Next, the glycosylation process was completed using a liquid phase reaction. The cutting solution (trifluoroethanol: dichloromethane = 1:4, volume ratio) was mixed with the resin containing BF8 and cut for 2 hours to obtain BF8 with a protective group. Subsequently, vacuum concentration was performed, the solvent was removed and lyophilized. The BF8 with a protective group: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU): D-mannosamine was added to a nitrogen-protected round-bottom flask in a molar ratio of 1:2:2, followed by ultra-dry dimethylformamide (DMF) and stirring overnight. After the reaction was completed, the solvent was dried, and a cutting solution consisting of 95% trifluoroacetic acid, 2.5% triisopropylsilane (TIS) and 2.5% water was added, and the protective group was cut off after reaction for 2 hours. Finally, vacuum concentration was performed to remove the solvent to obtain a crude product, which was then separated and purified by HPLC. BF8Man was prepared with a purity greater than 98% (analyzed by HPLC by Shanghai WuXi AppTec New Drug Development Co., Ltd.).

[0083] To prepare Cy5.5-labeled BF8 peptide (Pep-3), Bip-FFK(Dde)KKKHKR (SEQ ID No.: 3) was synthesized using Fmoc-K(Dde)-OH (purchased from Gill Biochemical) according to the above solid phase synthesis method. The resin was then treated with 5% hydrazine hydrate (dissolved in DMF) for 10 minutes to remove the Dde protection, and the resin was washed three times with DMF and the solvent was removed. Cy5.5-N-hydroxysuccinimide ester (2 equiv.) and N,N-diisopropylethylamine (2 equiv.) were dissolved in 20 mL DMF, mixed with the resin, and reacted for 2 hours. Subsequently, DMF was used to wash three times and the solvent was removed. A cutting solution consisting of 95% trifluoroacetic acid, 2.5% triisopropylsilane (TIS) and 2.5% water was added and reacted for 2 hours to remove the protecting group. Finally, the solvent was removed by vacuum concentration to obtain a crude product, which was then separated and purified by HPLC. Cy5.5-BF8 was prepared with a purity greater than 98% (analyzed by HPLC by Shanghai WuXi AppTec Pharmaceuticals Co., Ltd.).

[0084] To prepare Cy5.5-labeled BF8Man peptide (Pep-4), the glycosylation process was completed using a liquid phase reaction. The cleavage solution (trifluoroethanol: dichloromethane = 1:4, volume ratio) was mixed with the resin containing Bip-FFK(Cy5.5)KKKHKR, cleaved for 2 hours, and then spin-dried. The Bip-FFK(Cy5.5)KKKHKR containing the protective group was obtained by precipitating with ether and filtering. The Bip-FFK(Cy5.5)KKKHKR with the protective group: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU): D-mannosamine were added to a nitrogen-protected round-bottom flask in a molar ratio of 1:2:2, and then ultra-dry dimethylformamide (DMF) was added and stirred overnight. After the reaction is completed, the solvent is dried by spin drying, and a cutting solution consisting of 95% trifluoroacetic acid, 2.5% triisopropylsilane (TIS) and 2.5% water is added, and the reaction is continued for 2 hours to cut off the protecting group. Finally, the solvent is removed by vacuum concentration to obtain a crude product, which is then separated and purified by HPLC. Cy5.5-BF8Man is prepared with a purity greater than 98% (analyzed by HPLC by Shanghai WuXi AppTec New Drug Development Co., Ltd.).

[0085] The prepared polypeptide sequences are shown in Table 1.

[0086] Table 1 Polypeptide sequences involved in the present invention

[0087] Serial number Peptide sequence abbreviation Pep-1 Bip-FFKKKHKR BF8 Pep-2 Bip-FFKKKHKR-Man BF8Man Pep-3 Bip-FFK(Cy5.5)KKKHKR Cy5.5-BF8 Pep-4 Bip-FFK(Cy5.5)KKKHKR-Man Cy5.5-BF8Man

[0088] Among them, Bip in the peptide sequence represents (4-biphenylacetyl), the other letters represent amino acids commonly understood in the art, and Man represents D-mannosamine.

[0089] Example 2 Determination of in vitro antibacterial ability of glycosylated polypeptides

[0090] To evaluate the ability of BF8Man to kill MRSA in PBS (pH = 7.4), 1 mL of 1 × 10 8 CFU mL -1 PBS suspension of MRSA was incubated with BF8 or BF8Man (final concentration 256 μM) for 4 hours, and then the amount of bacteria involved was determined by plate spreading method. Untreated bacteria were used as control. Figure 1 A and B.

[0091] To evaluate the antibacterial ability of BF8Man in the airway microenvironment, MRSA was resuspended in PBS containing 0.3% or 0.5% (mass ratio) mucin to a final concentration of 5×10 7 CFU mL -1To evaluate the antibacterial ability of BF8Man in the alveolar microenvironment, MRSA was resuspended in 20 μg mL -1 and 60 μg mL -1 Dipalmitoylphosphatidylcholine (DPPC) was added to PBS to a final concentration of 5 × 10 7 CFU mL -1 Subsequently, the relevant bacterial suspensions were incubated with BF8Man or BF8 (final concentration of 256 μM) for four hours, and the remaining bacterial count was determined using the plate spreading method. Figure 1 C.

[0092] Figure 1 A and B show that at a concentration of 256 μM, BF8Man can kill 99.99% MRSA, while BF8 can kill 80.05% MRSA. This proves that BF8Man has good anti-MRSA activity and glycosylation modification can enhance the antibacterial ability of the self-assembling polypeptide skeleton.

[0093] Figure 1 The C showed that in the environment containing 0.3% and 0.5% mucin, the remaining survival number (CFU) of MRSA in the BF8Man treatment group was 19.16 and 8.71 times less than that in the BF8 treatment group. Similarly, in the environment of different concentrations of DPPC, BF8Man showed a higher bactericidal efficiency than BF8. This shows that glycosylation modification can significantly improve the antibacterial activity of this type of antimicrobial peptide molecules in the damaged lung physiological microenvironment.

[0094] Example 3 Determination of the broad-spectrum antimicrobial activity and ability to inhibit the generation of drug resistance of the glycosylated antimicrobial peptide BF8Man

[0095] To evaluate the broad-spectrum antibacterial activity of the peptides, various bacteria (methicillin-resistant Staphylococcus aureus ATCC 43300, USA 300, CCTCC 16465; Staphylococcus aureus ATCC 25923; Escherichia coli ATCC 25922, ATCC 700928; multidrug-resistant Escherichia coli BNCC 186732) in mid-logarithmic growth phase were diluted in LB medium to a final concentration of 1×10 5 CFUmL -1 Then, they were incubated with 8, 16, 32, 64, 128, 256 or 512 μM BF8 or BF8Man at 37°C for 24 hours. After incubation, the minimum inhibitory concentration (MIC) of the peptide for various bacteria was determined by measuring the turbidity at OD600. Figure 2 A.

[0096] To evaluate the ability of peptides to inhibit the generation of drug resistance, MRSA ATCC43300 was treated with BF8Man and ciprofloxacin at half the minimum inhibitory concentration, and the bacteria were passaged (one generation every 20 hours). Each new generation of bacteria is generated by the previous generation of bacteria. The experiment was passed for 30 generations, and the minimum inhibitory concentration of BF8Man and ciprofloxacin for each generation of bacteria was determined. The results are shown in Figure 2 B.

[0097] Figure 2 A shows that BF8Man has good antibacterial activity against various types of Staphylococcus aureus, with a MIC range of 4 to 32μM. Among them, the MIC of BF8Man against MRSA ATCC43300 is 8μM, which is 8 times higher than that of BF8. BF8Man has weaker antibacterial activity against three types of Gram-negative bacteria, with MICs of 32μM. The antibacterial ability of BF8 against Gram-positive or Gram-negative bacteria is significantly weaker than that of BF8Man.

[0098] Figure 2 Figure B shows that after 30 generations of resistance culture, the MIC value of ciprofloxacin for MRSA increased by 4 times, while the MIC value of BF8Man for MRSA did not increase significantly. This proves that compared with commercially available antibiotics, BF8Man can significantly inhibit the development of resistance in Gram-positive bacteria.

[0099] Example 3 Determination of Cell Tolerance and Hemolytic Ability of Glycosylated Antimicrobial Peptide BF8Man

[0100] To evaluate cell compatibility, human normal lung bronchial epithelial cells Beas-2B (CRL-9609) were cultured in 100 mm pre-coated culture dishes (430167, Corning) in RPMI-1640 (#11875093, Gibco) containing 10% fetal bovine serum and 1% penicillin-streptomycin (#15140122, Gibco). TM ). 4 Cells were seeded in 96-well plates and incubated for an additional 12 h. The medium was then replaced with fresh medium containing different concentrations of peptides (BF8Man, BF8) at 8, 16, 32, 64, 128, 256, or 512 μM, respectively. D-mannose was used as a control. After 24 h of co-incubation, cell viability was measured using the MTT assay. The results are shown in Figure 3 A.

[0101] To evaluate the hemolytic ability of the peptides, 2 mL of freshly drawn New Zealand rabbit blood was diluted 10-fold with PBS. Then, the red blood cell pellet was collected by centrifugation and washed 8 times with PBS, and then incubated with BF8Man, BF8 and Man (final concentration of 320 μM) at 37°C for 3 h. A positive control was set up with 1% Triton X-100. A negative control was set up with PBS. After the incubation was completed, the mixture was centrifuged at 10,000 rpm for 10 minutes, and the absorbance of the supernatant at 570 nm was measured to further evaluate the hemolysis rate. The results are shown in Figure 3 B.

[0102] Calculation method of hemolysis rate:

[0103] Hemolysis rate = (sample absorbance - average absorbance of PBS group) / (average absorbance of Triton X-100 group - average absorbance of PBS group) * 100%.

[0104] Figure 3 A shows that BF8Man can achieve a survival rate of more than 75% for normal human lung bronchial epithelial cells at a concentration of 256 μM. BF8 still did not show obvious toxicity to the cells when the final concentration reached 256 μM, indicating that in subsequent practical applications, the concentration of BF8Man should not exceed 256 μM.

[0105] Figure 3 B shows the hemolytic activity test results of the polypeptides, where BF8Man showed a final hemolysis rate of less than 5% at a concentration of 320 μM (40 times the minimum inhibitory concentration). Similarly, no obvious hemolysis was found in red blood cells treated with BF8. This result shows that BF8Man did not cause obvious hemolysis within the concentration range used (8-256 μM).

[0106] Example 4 Determination of the content of glycosylated antimicrobial peptides in MRSA cytoplasm

[0107] To detect the distribution of glycosylated peptides in free MRSA cells, 400 μL MRSA suspension (5×10 8 CFU mL -1 ) were incubated with fluorine-substituted BF8Man (256 μM) for 30 minutes at 37°C. The cells were then centrifuged at 13,000 rpm and washed three times with PBS. MRSA cells were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde and then sliced ​​for biological electron microscopy. Electron diffraction spectroscopy (EDS-mapping) was used to analyze the fluorine content in the cytoplasm of MRSA to reflect the uptake of BF8Man molecules by cells.

[0108] like Figure 4The results showed that compared with MRSA cells not treated with polypeptide (Control), there was a significant enhancement of fluorine signal in MRSA cells treated with fluorine-substituted BF8Man, indicating that BF8Man was transported into MRSA cells to exert subsequent antibacterial effects.

[0109] Example 5 Glycosylated antimicrobial peptides are transported across bacterial membranes by binding to periplasmic oligopeptide transporter (OppA)

[0110] The dissociation constant between BF8Man and purified OppA protein was determined by isothermal titration (ITC). BF8Man and OppA were dissolved in HEPES buffer (10 mM) to a final concentration of 500 μg mL -1 and 50 μg mL -1 The BF8Man suspension was loaded into the detection cell of MigroCal PEAQ-ITC (Malvern), and OppA was loaded into the syringe. The temperature of the detection cell was set to 25°C. OppA was then dripped into BF8Man in 19 drops (3 μL per drop). Various thermodynamic constants were then calculated based on the heat changes during titration.

[0111] Results Figure 5 The dissociation constant (KD) between BF8Man and OppA is 1.76 μM, which is common in the binding of ligands and membrane receptors. The binding heat (△H is 4.59 KJ mol -1 ). These data prove that the glycosylated antimicrobial peptide BF8Man constructed by the present invention can efficiently bind to OppA and achieve the expected transbacterial cell membrane transport process.

[0112] Example 8 BF8Man binds to specific intracellular proteins in bacteria to affect transcription and translation processes

[0113] To verify the potential targets of BF8Man after being transported into the cytoplasm of MRSA, the Pull-Down assay was used to detect proteins that may bind to BF8Man.

[0114] The results are as follows Figure 6 As shown, BF8Man has the potential to bind to specific proteins on DNA polymerase, RNA polymerase and 70s ribosome complex, which indicates that BF8Man may affect the metabolic stability of bacteria by regulating processes such as DNA replication, transcription and translation, thereby killing bacteria.

[0115] Example 9 BF8Man regulates bacterial aminoglycan metabolism and ultimately inhibits peptidoglycan synthesis

[0116] Proteomic analysis was used to explore the regulatory mechanism of BF8Man on MRSA metabolism. PBS suspension containing MRSA (5×10 9 CFU mL -1 ) were incubated with BF8Man or BF8 (512 μM) for 4 hours, and the treated bacteria were centrifuged at 7000 rpm for 10 minutes (4°C) and washed three times with PBS. Label-free proteomics analysis was then completed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. The relevant data were subjected to KEGG pathway enrichment analysis (www.genome.jp / kegg / ) and protein interaction analysis (String v11.5).

[0117] The results are as follows Figure 7 As shown. KEGG pathway enrichment analysis showed that compared with BF8, BF8Man can significantly regulate the intracellular aminoglycan metabolic pathway. Subsequent studies have shown that BF8Man can significantly regulate proteins such as MurA and MurB, thereby affecting the biosynthesis of peptidoglycan using amino sugar UDP-GlcNAc as raw material.

[0118] Example 10 The self-assembly behavior of BF8Man molecules ensures the self-formulation process for inhalation therapy

[0119] 1) Preparation of BF8Man self-prepared nanoparticles: To prepare BF8Man self-prepared nanoparticles with a final concentration of 256μM, first weigh 3.77mg of BF8Man lyophilized powder and dissolve it in 1mL of distilled water to form a BF8Man stock solution with a concentration of 2560μM. Then, 100μL of the stock solution was taken and mixed evenly with 800μL of distilled water, and 100μL of 10×PBS was added to the system. After the prepared BF8Man suspension was mixed evenly using a vortex instrument, the suspension was ultrasonicated for 30min (ice bath) using a probe-type ultrasonic cell disruptor to obtain a suspension of BF8Man nanoparticles. This step can be referred to for the preparation of BF8Man suspensions of other concentrations. The only difference is that a suitable stock solution with a concentration of 10× the required concentration should be prepared first, then diluted to the required final concentration, and then ultrasonicated.

[0120] 2) Preparation of BF8 nanofibers: To prepare BF8 nanofibers with a final concentration of 256μM, first weigh 3.36mg of BF8 lyophilized powder and dissolve it in 1mL of distilled water to form a BF8 stock solution with a concentration of 2560μM. Then, take 100μL of the stock solution and mix it evenly with 800μL of distilled water, and then add 100μL of 10×PBS to the system. After mixing the prepared BF8 suspension evenly with a vortex instrument, oscillate at 25°C for 4 hours to obtain a suspension of BF8 nanofibers. This step can be used to prepare BF8 suspensions of other concentrations. The only difference is that a suitable stock solution with a concentration of 10× the required concentration should be prepared first, then diluted to the required final concentration, and then oscillated for 4 hours.

[0121] 3) Transmission electron microscopy morphology: The morphology of the peptide assembly was imaged using a Talos L120C G2 transmission electron microscope (ThermoFisher). 10 μL of the prepared suspension containing the peptide assembly was dropped onto a 200-mesh copper grid, then stained with uranyl acetate for 2 minutes and imaged.

[0122] Results Figure 8 As shown in A, the nanostructure formed by self-assembly of BF8Man at 256μM presents the morphology of spherical nanoparticles with a diameter of about 20nm. This is conducive to the intrapulmonary delivery of BF8Man and passing through the reticular pores in the lung mucosa. BF8 self-assembles to form a nanofiber morphology at 256μM, which indicates that the assembly of BF8 may be captured by the lung mucosa due to body resistance. The above results prove that for this polypeptide backbone, glycosylation modification helps to form a nanoparticle morphology that is conducive to direct inhalation.

[0123] 4) Determine the critical micelle concentration (CMC) of the polypeptide molecule: Use a fluorescence spectrophotometer (Edinburgh, F55) to determine the CMC value of the polypeptide. Dissolve BF8Man and BF8 in PBS, respectively, and then dilute 8 times by 2 times as shown in the figure. Adjust the pH of the suspension to 7. Mix 100 μL of the polypeptide suspension with 2 μL of rhodamine 6G (final concentration of 5 μM) and incubate for 24 hours. Use a spectrophotometer to read the wavelength of the maximum OD value (515nm-560nm) for CMC calculation.

[0124] Results Figure 8 The critical micelle concentration (CMC) values ​​of BF8Man and BF8 were 47.58 μM and 71.86 μM, respectively, indicating that C-terminal glycosylation modification can enhance the self-assembly ability of the polypeptide backbone.

[0125] 5) Determination of the surface Zeta potential of BF8Man and BF8 assemblies ( Figure 8B) shows that at each specified concentration, the surface potential of the nanostructure formed by BF8Man assembly tends to be electrically neutral, while the surface potential of BF8 is close to +15mV. This indicates that the hydrophilic D-mannosamine modification can shield the positive charge of the internal positively charged polypeptide during the process of self-assembly to form nanoparticles, which can reduce the hydrophobic interaction and electrostatic interaction between BF8Man nanoparticles and mucin, and increase the stability of the assembly.

[0126] Example 11 Stability and antibacterial activity of BF8Man nanoparticles after atomization

[0127] 1) To verify the stability of BF8Man nanoparticles during the nebulization process, 8 mL of the prepared BF8Man nanoparticle suspension (256 μM) was placed in a nebulizer cup, and then a compressed nebulizer (403M, Yuyue) was used for nebulization for 20 minutes. During this period, the liquid sprayed by the nebulization and the liquid remaining in the nebulizer cup were collected. The diameters of various types of nanoparticles were measured using the number distribution mode of the BI-200SM NanoBrook ZetaPALS analyzer. Unnebulized BF8Man nanoparticles were used as a control.

[0128] Results Fig. 9 B, the BF8Man nanoparticles sprayed out by atomization and remaining in the atomization cup showed similar particle size distribution compared with the un-atomized BF8Man nanoparticles, with a diameter of about 20nm and a similar aggregation index. This shows that the BF8Man nanoparticles can withstand the shear force during the atomization process and have good morphological stability.

[0129] 2) To verify the antibacterial activity of BF8Man after aerosolization, the sprayed BF8Man nanoparticles were collected during the aforementioned aerosolization process and then mixed with 1 mL of PBS suspension rich in MRSA (the final concentration of BF8Man was 256 μM; the final concentration of MRSA was 1×10 8 CFUmL -1 ) were incubated for 4 hours, and then the amount of bacteria involved was determined using the plate spread method. Untreated bacteria were used as a control.

[0130] Results Fig. 9 In D and E, the atomized BF8Man nanoparticles showed similar bactericidal activity as before atomization, both of which killed 99.99% MRSA. This shows that the atomization process does not affect the antibacterial activity of BF8Ma, ensuring the stability of the subsequent treatment process.

[0131] Example 12 Glycosylation modification enhances mucosal permeability of antimicrobial peptide backbone

[0132] To construct artificial lung mucus containing 0.5% mucin, 50 mg DNA (transiently transfected with pGPU6 / GFP / Neo Plasmid, purchased from Qingke Biotechnology), 25 mg mucin (type II), 25 mg sterile egg yolk emulsion (diluted to 50% with saline) and 100 μL RPMI 1640 medium were added to 5 mL of diethylenetriaminepentaacetic acid (5.9 μg mL -1 )、NaCl(5mg mL -1 ) and KCl (2.2 mg mL -1 ) in a suspension and shaken for 8 hours to obtain artificial lung mucus.

[0133] To prepare Cy5.5-labeled BF8Man nanoparticles with a final concentration of 256 μM, weigh Cy5.5-BF8Man lyophilized powder and BF8Man lyophilized powder, dissolve in distilled water, and prepare a Cy5.5-labeled BF8Man stock solution with a final concentration of 2560 μM (the molar ratio of Cy5.5-BF8Man to BF8Man is 1:7). Then, 100 μL of the stock solution was taken and mixed with 800 μL of distilled water, and 100 μL of 10×PBS was added to the system. After the prepared BF8Man suspension was mixed evenly using a vortex instrument, the suspension was ultrasonicated for 30 minutes (ice bath) using a probe-type ultrasonic cell disruptor to obtain a suspension of Cy5.5-labeled BF8Man nanoparticles.

[0134] To prepare Cy5.5-labeled BF8 nanofibers with a final concentration of 256 μM, weigh Cy5.5-BF8 lyophilized powder and BF8 lyophilized powder, dissolve in distilled water, and prepare a Cy5.5-labeled BF8 stock solution with a final concentration of 2560 μM (the molar ratio of Cy5.5-BF8 to BF8 is 1:7). Then, 100 μL of the stock solution was mixed with 800 μL of distilled water, and 100 μL of 10×PBS was added to the system. After the prepared BF8 suspension was mixed evenly using a vortex instrument, it was shaken at 25°C for 4 hours to obtain a suspension of Cy5.5-labeled BF8 nanofibers.

[0135] For in vitro mucosal penetration experiments, 400 μL of 10% gelatin solution was added to a 24-well plate. After cooling, 200 μL of freshly prepared artificial lung mucus was added to the surface of the gelatin. Subsequently, 100 μL of pre-assembled Cy5.5-labeled BF8Man nanoparticles or BF8 nanofibers were added and incubated for 24 hours. Fig.10The gelatin layer was then washed three times with PBS, mixed with 600 μL of water and ultrasonicated for 15 minutes. The resulting solution was used to measure its emission wavelength at 700-800 nm (excitation 680 nm). Gelatin without any fluorescent polypeptide added was used as a control.

[0136] The results are as follows Fig.10 As shown in B. The relative fluorescence intensity of BF8 nanofibers in the gelatin layer at 2 and 4 h was basically the same, indicating that the penetration process of BF8 nanofibers stopped within 2 hours. BF8Man nanoparticles can continue to penetrate the artificial mucus layer within 4 hours of the test. Compared with BF8, the relative fluorescence intensity of BF8Man in the gelatin layer increased by 2.27, 1.76, and 2.58 times at 0.5, 2, and 4 hours, respectively, which indicates that the hydrophilic and electrically neutral nanoparticle morphology formed after glycosylation modification helps to achieve rapid mucosal penetration.

[0137] Example 13 Distribution of BF8Man nanoparticles in vivo after inhalation

[0138] 1) Modeling of MRSA-induced pneumonia: 8-week-old Balb / c mice were anesthetized with 2.5% Avertin and then intrapulmonary instilled with MRSA suspension (50 μL, 5×10 7 CFU), and 24 hours later, a MRSA-infected pneumonia mouse model was obtained.

[0139] 2) Exploration of the in vivo distribution of BF8Man: MRSA-infected pneumonia mice or healthy mice were euthanized with carbon dioxide after nebulizing Cy5.5-labeled BF8Man nanoparticles (256 μM) for 15 minutes. The mouse lung tissue was removed and lavaged. The lung tissue was then immunofluorescently stained to observe the distribution of BF8Man molecules in the lung epithelium.

[0140] Fig.11 The results showed that in the airways of healthy mice, the fluorescence signal of BF8Man was located in the lower lung epithelial cells compared to Ac-α-Tubulin, which marked the lung mucosa. This indicates that BF8Man nanoparticles can penetrate the lung mucosa in mice and deliver BF8Man molecules into airway epithelial cells. In the MRSA-infected airways, the fluorescence signal of BF8Man overlapped well with the MRSA signal in the airway epithelial cells, indicating that BF8Man can efficiently target intracellular bacteria after being delivered across the mucosa to the epithelial cells, which ensures the ability of BF8Man to clear bacteria on the airway surface.

[0141] Fig.12 It was shown that in the lungs of healthy mice, BF8Man can be distributed in F4 / 80 +In alveolar macrophages. In healthy lungs, except for resident alveolar macrophages, the remaining immune cells are less abundant. In MRSA-infected lung tissue, a large number of CD45 + Inflammatory immune cells infiltrate the alveoli and contain high MRSA signals. It is worth mentioning that BF8Man can be taken up by these immune cells, ensuring the antibacterial activity of the peptide in alveolar immune cells.

[0142] Example 14 BF8Man efficiently clears MRSA from the lungs of pneumonia mice by aerosolization

[0143] To explore the in vivo antibacterial activity of aerosolized BF8Man, 8-week-old Balb / c mice were anesthetized with 2.5% Avertin and intrapulmonary instilled with MRSA suspension (50 μL, 5×10 7 CFU) (day 0), the model was successfully established when the mouse survival rate was less than 40% within 24-48 hours. 24 hours after MRSA infection in the lungs (day 1), the mice were nebulized with BF8Man nanoparticles (256μM) twice, each time for 20 minutes, with an interval of 8 hours between each nebulization. On the second day, the mice were euthanized and the alveolar lavage fluid was extracted from the mice, and then the residual bacteria were determined by plate coating. Vancomycin (Van) (10mg Kg -1 ) and nebulized inhalation of 0.9% saline were selected as controls.

[0144] Fig.13 B shows that compared with inhaled saline, BF8Man successfully cleared 99.80% of MRSA in bronchoalveolar lavage fluid after two nebulized administrations, while BF8 could clear 93.61% of MRSA. The commonly used intravenous administration of vancomycin in clinical practice can clear 85.71% of MRSA. This shows that local inhalation of BF8Man has better antibacterial activity.

[0145] Example 15 BF8Man nanoparticle aerosolization prevents the development of lung damage and inflammation caused by infection

[0146] like Fig.14 The results showed that after histological analysis (HE staining) of mouse lung tissue on the fourth day, it was found that mice that inhaled saline by aerosol had obvious signs of airway and alveolar epithelial damage, including airway thickening, alveolar fusion, and a large number of immune cell infiltrations. It is worth mentioning that BF8Man can greatly slow down the progression of such damage and inflammatory processes because it can quickly clear MRSA from the lungs after aerosolization on the first day. However, compared with healthy mice, pneumonia mice that inhaled BF8Man still showed a certain degree of immune cell infiltration on the fourth day.

[0147] Example 16 BF8Man aerosolization alleviates the inflammatory progression of MRSA-induced pneumonia in piglets

[0148] like Fig.15 As shown in A, the present invention uses the MRSA infection model of Bama pigs to demonstrate the ability of BF8Man nanoparticle aerosol to treat bacterial pneumonia. To construct a piglet model of MRSA pneumonia infection, 7-month-old Bama piglets (about 20 kg) were anesthetized by intramuscular injection of 2.5 mL of Zota, placed in a supine position, and an incision was made about 6 cm below the Adam's apple, followed by blunt separation to expose the trachea. A puncture needle was used to inject 20 mL of MRSA suspension (10 8 CFU mL -1 The wound was then sutured. Hematological analysis and temperature measurement proved that the piglet pneumonia model was successfully established 3 days after MRSA injection.

[0149] On days 4-6, infected piglets were treated with BF8Man aerosol (256 μM) twice a day, 8 hours apart. Blood and bronchoalveolar lavage fluid were collected at designated times to evaluate the therapeutic effect. Aerosol inhalation of 0.9% saline was selected as a control.

[0150] The results are as follows Fig.15 As shown in the BD, on the third day after modeling, the body temperature of the piglets rose to nearly 41°C. After 3 consecutive days of BF8Man aerosolization, the body temperature of the piglets in the BF8Man group dropped to 39.7°C. Compared with before modeling (healthy), the total number of white blood cells in the alveolar lavage fluid increased significantly on the third day after infection, and the proportion of neutrophils in the alveolar lavage fluid increased to 71%. After treatment, the total number of white blood cells and the proportion of neutrophils in the alveolar lavage fluid of the piglets in the BF8Man group showed a faster decline, proving that BF8Man aerosolization can significantly shorten the course of the disease in the large animal lung infection model.

[0151] Example 17 Evaluation of the biosafety of continuous BF8Man aerosolization on the respiratory tract and systemic function of piglets

[0152] To evaluate the biosafety of BF8Man aerosolized treatment, 7-month-old Bama piglets (about 20 kg) continuously inhaled BF8Man nanoparticles (256 μM) for 14 days (twice a day, 8 hours apart each time). During this period, blood was collected at designated times and respiratory rates were counted. Lung tissue was collected on the 15th day and subjected to Masson-Trichrome staining and PAS staining. Aerosolized inhalation of 0.9% saline was selected as a control.

[0153] like Fig.16During the continuous nebulization process, the number of red blood cells in the blood of the piglets in the BF8Man group was not significantly lower than the lowest range of normal values, indicating that BF8Man nebulization did not cause significant hemolytic reactions in the body. The total number of white blood cells and the proportion of neutrophils in the blood fluctuated within the normal range, indicating that BF8Man nebulization did not cause obvious systemic inflammation. The respiratory rate per minute was not different from the normal value, indicating that no symptoms such as respiratory distress occurred.

[0154] like Fig.17 The results showed that after 14 days of continuous aerosolization, there was no significant difference in the collagen deposition around the terminal bronchi and the number of goblet cells on the airway surface in the piglets in the BF8Man group compared with the normal saline group. This shows that continuous BF8Man aerosolization did not lead to pulmonary fibrosis or increase the secretion of airway mucus, proving that continuous aerosol inhalation of BF8Man nanoparticles is safe for the respiratory tract.

Claims

1. An antimicrobial peptide or a salt thereof, wherein: The antimicrobial peptide comprises a hydrophobic self-assembly-promoting sequence, an OppA binding peptide sequence and a glycosylation modification group from the N-terminus to the C-terminus, or the antimicrobial peptide comprises a hydrophobic self-assembly-promoting sequence, an OppA binding peptide sequence from the N-terminus to the C-terminus.

2. The antimicrobial peptide or a salt thereof according to claim 1, wherein The N-terminal hydrophobic self-assembly promoting sequence is represented by the structure shown in the following formula I: wherein Xaa-Xaa- represents a dipeptide linker selected from FF and FW, Indicates where the hydrophobic membrane permeable sequence is attached to the N-terminus of the OppA binding peptide sequence.

3. The antimicrobial peptide or a salt thereof according to claim 1, wherein The OppA binding peptide is a positively charged 3-9 peptide that can bind to the periplasmic oligopeptide binding protein (OppA), particularly a positively charged 6-9 peptide comprising the amino acid sequence KKKHKR, more particularly the amino acid sequence is KKKHKR.

4. The antimicrobial peptide or a salt thereof according to claim 1, wherein The glycosylation modification group is selected from a monosaccharide group, a disaccharide group, a polysaccharide group and a chitosan fragment group, preferably selected from D-mannosamine, D-glucosamine and D-galactosamine groups.

5. The antimicrobial peptide or a salt thereof according to claim 1, wherein The antimicrobial peptide is:

6. A pharmaceutical composition comprising a therapeutically effective amount of the antimicrobial peptide or a salt thereof according to any one of claims 1 to 5, and a pharmaceutically acceptable excipient.

7. The pharmaceutical composition according to claim 6, wherein The dosage form of the pharmaceutical composition is selected from spray, solution and suspension.

8. Use of the antimicrobial peptide or a salt thereof according to any one of claims 1 to 5 in the preparation of antimicrobial drugs.

9. The use according to claim 8, wherein The antibacterial drug is used to fight against Gram-positive bacteria, especially Staphylococcus aureus.

10. The use according to claim 8, wherein The antibacterial drug is used to prevent or treat acute or chronic infections caused by Gram-positive bacteria, such as mucosal infections, including lung damage, pneumonia, meningitis, osteoarthritis and conjunctivitis.

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