A polypeptide monomer molecule MBP, polypeptide co-assembled nanoparticles and their application

Abstract

The present invention belongs to the field of polypeptide and biomedicine technology, and specifically relates to a polypeptide monomer molecule MBP, polypeptide co-assembled nanoparticles and their applications. The polypeptide co-assembled nanoparticles of the present invention target bacterial outer membrane lipopolysaccharide with the help of the lipopolysaccharide (LPS) targeting unit in the polypeptide monomer molecule MBP, overcome the protective effect of bacterial permeability barrier, and penetrate the membrane to reach the cell; with the help of the thiol oxidase (DsbA) targeting unit in the polypeptide monomer molecule EIP, target the DsbA enzyme in the bacterial cell, strongly bind to the bacterial DsbA enzyme, and transform the nanoparticle state of the polypeptide co-assembled nanoparticles into nanofibers, so that the nanocomposite has a stronger competitive binding ability of the DsbA enzyme, inhibits the activity of the DsbA enzyme, and causes the synthesis of the downstream key enzyme β-lactamase to be blocked, thereby exerting the antibiotic adjuvant effect and realizing the precise delivery of antibiotics.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptides and biomedicine, and specifically relates to a polypeptide monomer molecule MBP, polypeptide co-assembled nanoparticles and applications thereof. Background Art

[0002] Antimicrobial resistance (AMR) has become a major medical problem faced by countries around the world and is one of the top ten global public health threats to human health. The range of antibiotic targets is very limited. The vast majority of approved antibiotics mainly target DNA / RNA synthesis, protein synthesis, membrane integrity or cell wall synthesis, etc. All of these antibiotics have produced varying degrees of resistance, such as the emergence of "super bacteria". At present, the global development of new antibiotics is slow and the process of drug resistance is accelerating. Therefore, there is an urgent need to develop therapies with new mechanisms of action to evade existing resistance mechanisms and not cause drug resistance.

[0003] β-lactam antibiotics are the first line of defense for antibiotic treatment and one of the most commonly used antibiotics in clinical practice. Currently, they have developed serious drug resistance. The main reason for resistance to β-lactam antibiotics is the production of β-lactamase in Gram-negative bacteria, which can hydrolyze antibiotics and cause resistance. In response to the drug resistance caused by this enzyme, a variety of competitive inhibitors of β-lactamase have been launched on the market, which can competitively inhibit β-lactamase and thus restore the sensitivity of bacteria to antibiotics. Such inhibitors mainly include sulbactam sodium and zobactam sodium. However, reports show that there is also a problem of failure of adjuvants of this type of antibiotics in clinical practice, and its inactivation mechanism is still unclear.

[0004] In recent years, peptides have become "potential molecules" to combat bacterial resistance due to their good biocompatibility and low toxicity. With the rapid development of peptide self-assembly nanotechnology, a variety of new self-assembly nanopeptides have been designed and prepared to address the shortcomings of peptide drugs. They have significant in vitro and in vivo antibacterial activity enhancement and anti-protease degradation capabilities, demonstrating their unique advantages as new safe and efficient antibacterial drugs. The design and research of new self-assembly nanopeptide drugs is becoming an international frontier hotspot.

[0005] In recent years, the strategy of targeting bacterial virulence has become a new model of antibiotic adjuvant therapy. Targeting virulence without directly killing bacteria can protect the host's endogenous microbiota and reduce the possibility of bacterial resistance. The latest studies have shown that thiol oxidase (DsbA) is a key protease that assists the folding of β-lactamase in the bacterial extracytoplasmic environment. Therefore, inhibiting the activity of DsbA protease can impair the stability of downstream β-lactamase, which is a new method for developing the next generation of antibiotic adjuvants to treat drug-resistant infections. However, due to the protective effect of the permeability barrier of the outer membrane of Gram-negative bacteria, DsbA enzyme inhibitors are difficult to reach intracellular targets. Summary of the invention

[0006] The purpose of the present invention is to provide a polypeptide monomer molecule MBP, polypeptide co-assembled nanoparticles and applications thereof. The polypeptide monomer molecule MBP can overcome the protective effect of the bacterial permeability barrier, assist the polypeptide co-assembled nanoparticles to cross the bacterial outer membrane to achieve accurate delivery of antibiotics and / or antibiotic adjuvants into cells, assist in reversing antibiotic resistance, and restore antibiotic efficacy.

[0007] The invention provides a polypeptide monomer molecule MBP, comprising a hydrophobic unit, an enzyme cleavage unit, a connecting unit and a lipopolysaccharide targeting unit; the hydrophobic unit, the enzyme cleavage unit, the connecting unit and the lipopolysaccharide targeting unit are sequentially connected through an amide bond; the lipopolysaccharide targeting unit comprises a polypeptide, a small molecule compound, a protein or a nucleic acid aptamer with a function of targeting bacterial outer membrane lipopolysaccharide.

[0008] Preferably, the hydrophobic unit comprises palmitic acid, stearic acid or cholesterol;

[0009] The enzymatic cleavage unit is a tripeptide, the amino acid sequence of the tripeptide is Cys-XX, and X is any amino acid;

[0010] The connecting unit is a polypeptide, and the first amino acid at the N-terminus of the polypeptide is Cys.

[0011] Preferably, the structure of the polypeptide monomer molecule MBP is as shown in Formula I.

[0012] The present invention provides a polypeptide co-assembled nanoparticle, wherein the polypeptide co-assembled nanoparticle comprises a polypeptide monomer molecule EIP and a polypeptide monomer molecule MBP as described in the above technical solution;

[0013] The polypeptide monomer molecule EIP includes a hydrophobic unit, an assembly unit, a connection unit and a thiol oxidase targeting unit; the hydrophobic unit, the assembly unit, the connection unit and the thiol oxidase targeting unit are sequentially connected through an amide bond; the thiol oxidase targeting unit includes a polypeptide, a small molecule compound, a protein or a nucleic acid aptamer that targets and inhibits thiol oxidase.

[0014] Preferably, the molar ratio of the polypeptide monomer molecule MBP to the polypeptide monomer molecule EIP is 0:1 to 1:20, and the molar ratio is not 0.

[0015] Preferably, in the polypeptide monomer molecule EIP, the hydrophobic unit includes palmitic acid, stearic acid or cholesterol; the assembly unit is a pentapeptide, and the amino acid sequence of the pentapeptide is Phe-Phe-Val-Leu-Al a; the connecting unit is a tripeptide, and the amino acid sequence of the tripeptide is Gly-Gly-Gly; the thiol oxidase targeting unit is a nonapeptide, and the amino acid sequence of the nonapeptide is Pro-Ser-Pro-Phe-Ala-Thr-Cys-Asp-Phe.

[0016] The present invention also provides the use of the polypeptide monomer molecule MBP described in the above technical solution or the polypeptide co-assembled nanoparticles described in the above technical solution in one or more of antibiotic delivery, antibiotic adjuvant delivery and reversal of antibiotic resistance.

[0017] The present invention also provides an antibiotic-loaded polypeptide co-assembled nanoparticle, which comprises the polypeptide co-assembled nanoparticle described in the above technical solution and the antibiotic contained in the polypeptide co-assembled nanoparticle.

[0018] Preferably, in the antibiotic-loaded polypeptide co-assembled nanoparticles, the concentration of the polypeptide co-assembled nanoparticles is 0.005 mM to 1 mM; the concentration of the antibiotic is 0 to 1 mM, and the concentration of the antibiotic is not zero.

[0019] The present invention also provides a method for preparing the antibiotic-loaded polypeptide co-assembled nanoparticles described in the above technical solution, comprising the following steps: subjecting a solution of polypeptide monomer molecules MBP, a solution of polypeptide monomer molecules EIP and a solution of antibiotics to ultrasonic treatment in an aqueous phase to obtain an ultrasonic treatment solution;

[0020] The ultrasonic treatment solution is dialyzed in PBS buffer, and the obtained retentate contains the antibiotic polypeptide-loaded co-assembled nanoparticles.

[0021] Beneficial effects:

[0022] The present invention provides a polypeptide monomer molecule MBP, comprising a hydrophobic unit, an enzyme cleavage unit, a linking unit and a lipopolysaccharide targeting unit; the hydrophobic unit, the enzyme cleavage unit, the linking unit and the lipopolysaccharide targeting unit are sequentially connected through an amide bond; the lipopolysaccharide targeting unit comprises a polypeptide, a small molecule compound or a protein having a function of targeting bacterial outer membrane lipopolysaccharide. The present invention utilizes the lipopolysaccharide (LPS) targeting unit in the polypeptide monomer molecule MBP to target bacterial outer membrane lipopolysaccharide, overcome the protective effect of the bacterial permeability barrier, and achieve accurate delivery of antibiotic adjuvants including bacterial thiol oxidase (DsbA) inhibitors into cells.

[0023] Based on the advantages of the polypeptide monomer molecule MBP, the present invention also provides a polypeptide co-assembled nanoparticle, which comprises a polypeptide monomer molecule EIP and the polypeptide monomer molecule MBP described in the above technical scheme; the polypeptide monomer molecule EIP comprises a hydrophobic unit, an assembly unit, a connecting unit and a thiol oxidase targeting unit; the hydrophobic unit, the assembly unit, the connecting unit and the thiol oxidase targeting unit are sequentially connected through an amide bond; the thiol oxidase targeting unit comprises a polypeptide, a small molecule compound or a protein that targets and inhibits thiol oxidase. The polypeptide co-assembled nanoparticles of the present invention reach cells with the help of the membrane-penetrating effect of the polypeptide monomer molecule MBP; the DsbA enzyme in the bacterial cell is targeted by the thiol oxidase targeting unit in the polypeptide monomer molecule EIP, and the bacterial DsbA enzyme is strongly combined to transform the nanoparticle state of the polypeptide co-assembled nanoparticles into nanofibers, so that the nanocomposite has a stronger competitive binding ability for the Ds bA enzyme, inhibits the activity of the DsbA enzyme, and causes the synthesis of the downstream key enzyme for drug resistance (β-lactamase) to be blocked, thereby exerting the antibiotic adjuvant effect and realizing the precise delivery of antibiotics; at the same time, the polypeptide monomer molecule EIP can shield the excessive surface positive charge of some polypeptide monomer molecules MBP, thereby reducing cytotoxic side effects.

[0024] Based on the advantages of the polypeptide co-assembled nanoparticles, the present invention provides an antibiotic-loaded polypeptide co-assembled nanoparticle, which comprises the polypeptide co-assembled nanoparticles described in the above technical solution and the antibiotics contained in the inner cavity of the polypeptide co-assembled nanoparticles. The antibiotic-loaded polypeptide co-assembled nanoparticles of the present invention can accurately deliver antibiotics into bacterial cells, significantly improve the efficacy of drugs, reduce the dosage, and reduce the occurrence of toxic and side effects; the antibiotic-loaded polypeptide co-assembled nanoparticles can reverse the drug resistance of antibiotics including bacterial β-lactam antibiotics, extend the service life of antibiotics, and have great potential in treating drug-resistant bacterial infections. At the same time, the antibiotic-loaded polypeptide co-assembled nanoparticles can encapsulate different types of hydrophobic antibiotics and have the universality of antibiotic delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0026] Figure 1 This is a schematic diagram of the structure of the polypeptide monomer molecule EIP in Example 1;

[0027] Figure 2 This is a schematic diagram of the structure of the polypeptide monomer molecule MBP in Example 1;

[0028] Figure 3 This is a transmission electron microscopy image of the polypeptide co-assembled nanoparticles in Example 1;

[0029] Figure 4 This is a transmission electron microscopy image of the polypeptide co-assembled nanoparticles loaded with antibiotics in Example 2;

[0030] Figure 5 This is a diagram showing the results of the cytotoxicity experiment in Example 3;

[0031] Figure 6 This is the transmission electron microscopy experimental result of the deformation of DsbA enzyme-triggered polypeptide co-assembled nanoparticles in Example 6. DETAILED DESCRIPTION

[0032] The invention provides a polypeptide monomer molecule MBP, comprising a hydrophobic unit, an enzyme cleavage unit, a connecting unit and a lipopolysaccharide targeting unit; the hydrophobic unit, the enzyme cleavage unit, the connecting unit and the lipopolysaccharide targeting unit are sequentially connected through an amide bond; the lipopolysaccharide targeting unit comprises a polypeptide, a small molecule compound or a protein having a function of targeting bacterial outer membrane lipopolysaccharide.

[0033] In the present invention, the lipopolysaccharide targeting unit is preferably a polypeptide having the function of targeting bacterial outer membrane lipopolysaccharide, and the amino acid sequence of the polypeptide targeting bacterial outer membrane lipopolysaccharide is preferably Lys-Lys-Arg-Ala-Lys-Lys-Phe-Phe-Lys-Lys-Pro-Arg-Val-Ile-Gly-Val-Ser-Ile-Pro-Phe (SEQ ID NO.1); the lipopolysaccharide targeting unit can specifically target the lipopolysaccharide in the bacterial outer membrane, and then pass through the bacterial outer membrane to overcome the bacterial permeability barrier. The hydrophobic unit of the present invention preferably includes palmitic acid, stearic acid or cholesterol, more preferably palmitic acid; the hydrophobic unit has the effect of enhancing the hydrophobicity of the molecule and balancing the hydrophilic and hydrophobic balance of the molecule in the polypeptide monomer molecule MBP. The enzymatic cleavage unit of the present invention is preferably a tripeptide, and the amino acid sequence of the tripeptide is preferably Cys-XX, wherein X is any amino acid; the enzymatic cleavage unit has the effect of being specifically recognized and cut off by the DsbA enzyme in the polypeptide single molecule MBP. The connecting unit of the present invention is preferably a polypeptide, and the first amino acid of the polypeptide is preferably Cys; the amino acid sequence of the polypeptide is preferably Pro-Ser-Pro-Phe-Ala-Thr-Cys-Asp-Phe (SEQ ID NO.2); the connecting unit has the function of providing a disulfide bond synthesis site in the polypeptide single molecule MBP, and connecting the enzymatic cleavage module and the targeting module.

[0034] In the present invention, the structure of the polypeptide monomer molecule MBP is shown in Formula I: The molecular formula of the structural formula shown in Formula I is C 150 H 249 N 39 O 29 S2.

[0035] In the present invention, the hydrophobic unit, the enzyme cleavage unit, the linking unit and the lipopolysaccharide targeting unit are sequentially linked via amide bonds. The linking process and conditions are not particularly limited and conventional linking processes and conditions in the art may be used.

[0036] The present invention utilizes the lipopolysaccharide (LPS) targeting unit in the polypeptide monomer molecule MBP to target the bacterial outer membrane lipopolysaccharide, overcome the protective effect of the bacterial permeability barrier, and achieve precise delivery of antibiotic adjuvants including bacterial thiol oxidase (DsbA) inhibitors into cells.

[0037] The present invention also provides a polypeptide co-assembled nanoparticle, wherein the polypeptide co-assembled nanoparticle comprises a polypeptide monomer molecule EIP and a polypeptide monomer molecule MBP as described in the above technical solution;

[0038] The polypeptide monomer molecule EIP includes a hydrophobic unit, an assembly unit, a connection unit and a thiol oxidase targeting unit; the hydrophobic unit, the assembly unit, the connection unit and the thiol oxidase targeting unit are sequentially connected through an amide bond; the thiol oxidase targeting unit includes a polypeptide, a small molecule compound or a protein that targets and inhibits thiol oxidase.

[0039] In the polypeptide monomer molecule EIP of the present invention, the hydrophobic unit preferably includes palmitic acid, stearic acid or cholesterol, more preferably palmitic acid; the hydrophobic unit has the function of enhancing the hydrophobicity of the molecule and balancing the hydrophilic-hydrophobic balance (hydrophobic force) of the molecule. The assembly unit of the present invention is preferably a pentapeptide, and the amino acid sequence of the pentapeptide is preferably Phe-Phe-Val-Leu-Ala (SEQ ID NO.3); the assembly unit has the function of providing a driving force (hydrogen bond, π-π interaction) for molecular assembly. The connecting unit of the present invention is preferably a tripeptide, and the amino acid sequence of the tripeptide is preferably Gly-Gly-Gly; the connecting unit has the function of connecting and spacing the assembly module and the targeting module. The thiol oxidase targeting unit of the present invention is preferably a nonapeptide, and the amino acid sequence of the nonapeptide is preferably Pro-Ser-Pro-Phe-Ala-Thr-Cys-Asp-Phe (SEQ ID NO. 2); the thiol oxidase targeting unit can selectively target and inhibit thiol oxidase, resulting in the obstruction of the synthesis of the downstream key enzyme for drug resistance (β-lactamase), thereby exerting an antibiotic adjuvant effect and reversing antibiotic resistance.

[0040] The structure of the polypeptide monomer molecule EIP of the present invention is preferably as shown in Formula II,

[0041]

[0042] Formula II; the molecular formula of the structural formula shown in Formula II is C 99 H 143 N 17 O 23 S.

[0043] In the polypeptide monomer molecule EIP of the present invention, the hydrophobic unit, assembly unit, connection unit and thiol oxidase targeting unit are sequentially connected through amide bonds. The connection process and conditions are not particularly limited and conventional connection processes and conditions in the art can be used.

[0044] In the present invention, the molar ratio of the polypeptide monomer molecule MBP to the polypeptide monomer molecule EIP is preferably 0:1 to 1:20, more preferably 1:1 to 1:10, and more preferably 3:7 to 1:9; the molar ratio is not 0.

[0045] The method for preparing the polypeptide co-assembled nanoparticles of the present invention preferably comprises: dissolving the polypeptide monomer molecule MBP and the polypeptide monomer molecule EIP in a good solvent to obtain a polypeptide monomer molecule MBP solution and a polypeptide monomer molecule EIP solution respectively;

[0046] The polypeptide monomer molecule MBP solution and the polypeptide monomer molecule EIP solution are subjected to water bath ultrasonic treatment to obtain a water bath ultrasonic treatment solution;

[0047] The water bath ultrasonic treatment solution is subjected to ice water bath ultrasonic treatment in water or PBS phosphate buffer to obtain the polypeptide co-assembled nanoparticles.

[0048] The present invention preferably dissolves the polypeptide monomer molecule MBP and the polypeptide monomer molecule EIP in a good solvent to obtain a polypeptide monomer molecule MBP solution and a polypeptide monomer molecule EIP solution, respectively. The good solvent of the present invention preferably includes dimethyl sulfoxide or an aqueous solution of dimethyl sulfoxide.

[0049] After obtaining the polypeptide monomer molecule MBP solution and the polypeptide monomer molecule EIP solution, the present invention preferably performs water bath ultrasonic treatment on the polypeptide monomer molecule MBP solution and the polypeptide monomer molecule EIP solution to obtain a water bath ultrasonic treatment solution. The power of the water bath ultrasonic treatment of the present invention is preferably 100 to 600 W, more preferably 100 to 300 W, and more preferably 200 to 250 W; the time is preferably 0 to 30 min, and more preferably 15 min.

[0050] After obtaining the water bath ultrasonic treatment liquid, the present invention preferably performs ice water bath ultrasonic treatment on the water bath ultrasonic treatment liquid in water or PBS phosphate buffer to obtain ice water bath ultrasonic treatment liquid. The pH value of the PBS phosphate buffer of the present invention is preferably 7.4. The power of the ice water bath ultrasonic treatment of the present invention is preferably 100 to 300 W, more preferably 200 to 250 W; the time is preferably 0 to 40 min, more preferably 30 min. The ice water bath has the effect of cooling and preventing the damage of the nanoparticles caused by excessive ultrasonic temperature.

[0051] After obtaining the ice-water bath ultrasonic treatment solution, the present invention preferably allows the ice-water bath ultrasonic treatment solution to stand to obtain a standing ultrasonic treatment solution. The standing time of the present invention is preferably 0 to 1.5 hours, more preferably 1 hour.

[0052] After obtaining the ultrasonic treatment liquid, the present invention preferably dialyzes the ultrasonic treatment liquid after standing in PBS buffer to obtain a retentate, which is the polypeptide co-assembled nanoparticles. The molecular weight cutoff of the dialysis bag for dialysis of the present invention is preferably 1000Da. The dialysis time of the present invention is preferably 0 to 4h, more preferably 1h. The pH value of the PBS buffer of the present invention is preferably 7.4. The dialysis of the present invention can remove free polypeptide single molecules.

[0053] The polypeptide co-assembled nanoparticles of the present invention reach cells with the help of the membrane-penetrating effect of the polypeptide monomer molecule MBP; the DsbA enzyme in the bacterial cell is targeted by the thiol oxidase targeting unit in the polypeptide monomer molecule EIP, and the bacterial DsbA enzyme is strongly combined to transform the nanoparticle state of the polypeptide co-assembled nanoparticles into nanofibers, so that the nanocomposite has a stronger competitive binding ability for the DsbA enzyme, inhibits the activity of the DsbA enzyme, and causes the synthesis of the downstream key enzyme for drug resistance (β-lactamase) to be blocked, thereby exerting the antibiotic adjuvant effect and realizing the precise delivery of antibiotics; at the same time, the polypeptide monomer molecule EIP can shield the excessive surface positive charge of some polypeptide monomer molecules MBP, thereby reducing cytotoxic side effects.

[0054] Based on the above advantages, the present invention also provides the use of the polypeptide monomer molecule MBP described in the above technical solution or the polypeptide co-assembled nanoparticles described in the above technical solution in one or more of antibiotic delivery, antibiotic adjuvant delivery and reversal of antibiotic resistance, preferably including the use of the polypeptide co-assembled nanoparticles in antibiotic delivery, antibiotic adjuvant delivery and reversal of antibiotic resistance.

[0055] The present invention also provides an antibiotic-loaded polypeptide co-assembled nanoparticle, which comprises the polypeptide co-assembled nanoparticle described in the above technical solution and the antibiotic contained in the inner cavity of the polypeptide co-assembled nanoparticle.

[0056] The polypeptide co-assembled nanoparticles and antibiotics of the present invention are preferably encapsulated by hydrophobic interactions. The concentration of the polypeptide co-assembled nanoparticles of the present invention is preferably 0.005mM to 1mM, more preferably 0.005mM to 0.5mM, and more preferably 0.0125mM to 0.2mM; the concentration of the antibiotic is preferably 0 to 1mM, more preferably 0.01mM to 0.1mM, and more preferably 0.05mM to 0.1mM; the concentration of the antibiotic is not 0. The antibiotics of the present invention preferably include hydrophobic antibiotics or hydrophilic antibiotics, more preferably hydrophobic antibiotics; the hydrophobic antibiotics preferably include β-lactam antibiotics, cephalosporin antibiotics, quinolone antibiotics or polymyxins, and more preferably β-lactam antibiotics.

[0057] The present invention also provides a method for preparing the antibiotic-loaded polypeptide co-assembled nanoparticles described in the above technical solution, comprising the following steps: subjecting a solution of polypeptide monomer molecules MBP, a solution of polypeptide monomer molecules EIP and a solution of antibiotics to ultrasonic treatment in an aqueous phase to obtain an ultrasonic treatment solution;

[0058] The ultrasonic treatment solution is dialyzed in PBS buffer, and the obtained retentate contains the antibiotic polypeptide-loaded co-assembled nanoparticles.

[0059] The present invention preferably dissolves the polypeptide monomer molecule MBP, the polypeptide monomer molecule EIP and the antibiotic in a good solvent to obtain a polypeptide monomer molecule MBP solution, a polypeptide monomer molecule EIP solution and an antibiotic solution, respectively. The good solvent of the present invention preferably includes dimethyl sulfoxide or an aqueous solution of dimethyl sulfoxide.

[0060] After obtaining the polypeptide monomer molecule MBP solution, polypeptide monomer molecule EIP solution and antibiotic solution, the present invention ultrasonically treats the polypeptide monomer molecule MBP solution, polypeptide monomer molecule EIP solution and antibiotic solution in an aqueous phase to obtain an ultrasonically treated solution. The power of the ultrasonic treatment of the present invention is preferably 100 to 600 W, more preferably 100 to 300 W, and more preferably 200 to 250 W; the time of the ultrasonic treatment is preferably 0 to 45 min, and more preferably 30 min.

[0061] After obtaining the ultrasonic treatment liquid, the present invention preferably dialyzes the ultrasonic treatment liquid in a PBS buffer to obtain a retentate. The molecular weight cutoff of the dialysis bag for dialysis of the present invention is preferably 1000Da. The dialysis of the present invention is preferably light-proof dialysis, because it is to avoid the instability of antibiotics in light and degradation. The dialysis time of the present invention is preferably 0 to 4 hours, more preferably 1 hour. The pH value of the PBS buffer of the present invention is preferably 7.4. The dialysis of the present invention can remove free antibiotics and polypeptide single molecules.

[0062] After obtaining the retentate, the present invention preferably centrifuges the retentate to obtain the supernatant to obtain the antibiotic polypeptide-loaded co-assembled nanoparticles. The centrifugation speed of the present invention is preferably 6000 g and the time is preferably 5 min.

[0063] The antibiotic polypeptide-loaded co-assembled nanoparticles of the present invention can accurately deliver antibiotics into bacterial cells, significantly improve drug efficacy, reduce the dosage, and reduce the occurrence of toxic and side effects; the antibiotic polypeptide-loaded co-assembled nanoparticles can reverse the drug resistance of antibiotics including bacterial β-lactam antibiotics, extend the service life of antibiotics, and have great potential in treating drug-resistant bacterial infections. At the same time, the antibiotic polypeptide-loaded co-assembled nanoparticles can encapsulate different types of hydrophobic antibiotics and have universal applicability in antibiotic delivery.

[0064] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0065] Example 1

[0066] A method for preparing polypeptide co-assembled nanoparticles, comprising the following steps:

[0067] 1) The structural formula of the polypeptide monomer molecule EIP is as follows Figure 1 As shown, the structural formula of the polypeptide monomer molecule MBP is as follows Figure 2 As shown, the polypeptide monomer molecule EIP and the polypeptide monomer molecule MBP were respectively commissioned to Gill Biochemical (Shanghai) Co., Ltd. and prepared by the polypeptide solid phase synthesis method.

[0068] Polypeptide co-assembled nanoparticles (EMPs) X：Y ) Preparation: Accurately weigh the prescribed amount of polypeptide monomer molecules EIP and polypeptide monomer molecules MBP, dissolve in DMSO, vortex to dissolve, and prepare a 10mM mother solution for use. Take the molar ratio X (EIP) : Y (MBP) The polypeptide monomer molecules EIP and MBP (respectively 10:0, 8:2, 7:3, 5:5, 3:7, 2:8, 1:9 and 0:10) were vortexed and thoroughly mixed, and then ultrasonicated in a water bath for 15 minutes, transferred to 1 mL PBS phosphate buffer (pH 7.4), vortexed and mixed, ultrasonicated at 240W for 30 minutes in an ice water bath, and then allowed to stand for 1 hour for use.

[0069] 2) Prepare transmission electron microscopy samples using the negative staining method: Take 10 μL of the co-assembled nanoparticles of different proportions in step 1) and drop them on a carbon-supported copper mesh (100 mesh). After standing and settling for 5 minutes, use a disposable filter paper to absorb the droplets. Take 10 μL of uranyl acetate (2%) and drop them on the copper mesh after absorbing the droplets. After standing for 1 minute, use a disposable filter paper to absorb the dye again. Use a biological transmission electron microscope (HT 7800) to observe and photograph. The results are as follows: Figure 3 shown.

[0070] 3) Determination of particle size and potential by dynamic light scattering (DLS) method: 200 μL of the co-assembled nanoparticles of different proportions prepared in step 1) were placed in a potential cup and a particle size cup respectively, and placed in a dynamic light scattering particle size analyzer (Malvern, ZS90) to determine the particle size and potential. The results are shown in Table 1.

[0071] Table 1 Statistical results of appearance, particle size and potential of co-assembled nanoparticles in Example 1

[0072]

[0073] Depend on Figure 3 From Table 1, it can be concluded that the particle size of the nanoparticles formed by the self-assembly of the polypeptide single molecule MBP is 24.9±4.5nm, and as the ratio of polypeptide single molecule EIP increases (1:9 to 3:7), the particle size gradually increases, indicating that MBP and EIP are co-assembled. As the EIP ratio further increases (5:5 to 8:2), some or even all of the nanofibers appear, and the nanofibers are not conducive to the barrier effect of crossing the bacterial outer membrane. Zeta potential has a great influence on the stability of nanoparticles. Generally, the higher the potential, the less likely it is to precipitate, condense and aggregate, and the system is stable; when the potential reaches 30mV, the stability requirements can be met.

[0074] Example 2

[0075] Cefoperazone (Cefoperazone, Cefo)-loaded peptide co-assembled nanoparticles (EMPs) 3-7 @Cefo) Preparation: Accurately weigh 15.77 mg of polypeptide monomer molecule EIP (prepared in Example 1), dissolve in 800 μL of DMSO, vortex to dissolve, prepare a 10 mM mother solution, and set aside. Accurately weigh 12.50 mg of polypeptide monomer molecule MBP (prepared in Example 1), dissolve in 400 μL of DMSO, vortex to dissolve, prepare a 10 mM mother solution, and set aside. Accurately weigh 25.82 mg of Cefo antibiotic, dissolve in 400 μL of DMSO, vortex to dissolve, prepare a 100 mM mother solution, and set aside. Take 3 μL of polypeptide monomer molecule EIP, 7 μL of polypeptide monomer molecule MBP and 0.9 μL of Cefo and mix them thoroughly, then transfer them to 1 mL of PBS phosphate buffer (pH = 7.4), vortex and mix them, and after ultrasonication at 240W in an ice water bath for 30 min, transfer them to a dialysis bag (MWCO: 1000Da), dialyze them in PBS buffer solution (pH = 7.4) in the dark for 1 h to remove free antibiotics, and centrifuge them at 6000g for 5 min to take the supernatant for use.

[0076] The electron microscope sample preparation method and particle size potential measurement method are the same as in Example 1. The electron microscope test results are as follows Figure 4 The statistical results of the appearance, particle size and potential are shown in Table 2.

[0077] Table 2 Statistical results of appearance, particle size and potential of polypeptide co-assembled nanoparticles loaded with antibiotics in Example 2

[0078] Inspection indicators result Appearance Spherical Particle size (nm) 48.7±9.2 Potential (mV) 29.0±5.0

[0079] Depend on Figure 4 It can be concluded from Table 2 that the Cefo-loaded polypeptide co-assembled nanoparticles have a relatively uniform morphology and have no significant difference from the morphology of the blank polypeptide co-assembled nanoparticles (Example 1).

[0080] Example 3

[0081] Cytotoxicity assay, steps are as follows:

[0082] Human umbilical vein endothelial cells (HUVECs) in the logarithmic growth phase were washed once with 2 mL of PBS buffer in a sterilized clean bench, and then 2 mL of 0.25% trypsin was added. After digestion for 4 min, 2 mL of cell culture medium was immediately added to terminate the digestion. The cell suspension was repeatedly blown several times to make the cell suspension density uniform. The cell density of the cell suspension was calculated using a hemocytometer. According to the counting results, the corresponding volume of complete culture medium was added, and the final cell density was adjusted to 6×10 4 Pieces / mL.

[0083] Take a 96-well plate and add 100 μL of the mixed cell suspension (about 6×10 3 The blank control group was not added with cell suspension, but only with 100 μL culture medium or PBS buffer. The cells were cultured at 37°C and 5% CO2 until the cells were completely attached to the wall, which took about 24 hours.

[0084] The polypeptide self-assembled nanoparticles prepared in Example 1 were suspended in complete culture medium (EIP, BMP, EMP 3-7 ) was added to a 96-well plate, 100 μL of the drug solution per well, so that the final concentrations of each well were 50, 25, 12.5, 6.25, 3.13, and 1.56 μM, respectively. The blank control group without cells was replaced with 100 μL of complete medium, and each group was set up with 6 replicate wells. After incubation for 24 hours, 20 μL of CCK-8 (10 μL of CCK-8 solution per 100 μL) was added to each well, and after gentle shaking and mixing, it was placed in an incubator for 4 hours, and the absorbance value OD at a wavelength of 450 nm was detected by an enzyme reader. 450 , the results are as follows Figure 5 shown.

[0085] Depend on Figure 5 It can be concluded that the polypeptide single molecule EIP, BMP and co-assembled nanoparticle cell EMP in this example 3-7 In the toxicity experiment, the cell survival rate was above 80%, indicating that both the polypeptide single molecule and the co-assembled nanoparticles had no obvious cytotoxicity.

[0086] Example 4

[0087] Pharmacodynamic experiment (minimum inhibitory concentration experiment), the steps are as follows:

[0088] The minimum inhibitory concentration (MIC) value was determined by broth dilution method. MIC determination was performed on the representative antibiotics currently commonly used in clinical practice, namely, Cefazolin sodium (Cefazolin, Cefa, first generation cephalosporin), Cefuroxime sodium (Cefuroxime, Cefu, second generation cephalosporin), Cefoperazone (Cefoperazone, Cefo, third generation cephalosporin) and Cefepime (Cefepime, Cefe, fourth generation cephalosporin). A monoclonal clinically isolated multidrug-resistant Escherichia coli (E. coli MDRESBL-1 (Clinical), E. coli MDR ESBL-1 (Clinical) was published in ACS Nano. 2022, 16, (12): 20545-20558.) was taken. Incubate in LB broth medium at 37°C with a shaker at 180 rpm overnight. The next day, 1% of the above bacterial suspension was added to fresh LB broth and cultured to a standard absorbance of OD 600nm =0.1(1.0×10 8 CFU / mL) and diluted to 1.5×10 6 CFU / mL.

[0089] Take a 96-well plate, add 100 μL of the above bacterial suspension to each well, and 100 μL of different drugs. The different drug conditions are shown in Table 3, among which EMP 3-7 、EMP 2-8 、EMP 1-9 and MBP were the samples obtained in Example 1; a series of sample concentrations were set, 6 replicates were set for each concentration, blank culture medium was set as the blank group, and the group without drug addition was set as the control group. After incubation at 37°C for 18 hours, OD was measured. 600nm The absorbance was measured and the minimum concentration that inhibited bacterial growth was taken as the MIC value.

[0090] Table 3 MIC values ​​in Example 4

[0091]

[0092] It can be seen from Table 3 that the clinically isolated strains have different degrees of resistance to the commonly used antibiotics in clinical practice (MIC>300 μM); and the polypeptide single molecule MBP and polypeptide co-assembled nanoparticles EMP in this example have certain antibacterial activity.

[0093] Example 5

[0094] Antibiotic synergy experiment, the steps are as follows:

[0095] Broth dilution method was used to determine the synergistic effect of peptides on antibiotics cefazolin, cefuroxime and cefoperazone. Monoclonal clinically isolated multidrug-resistant Escherichia coli (E. coli MDR ESBL-1 (Clinical)) was incubated in M63 broth medium at 37°C and 180 rpm overnight. The next day, 1% of the above bacterial suspension was taken in fresh M63 broth medium and cultured to a standardized absorbance OD 600nm =0.1(1.0×10 8 CFU / mL) and diluted to 1.5×10 6 CFU / mL.

[0096] Take a 96-well plate, add 100 μL of the above bacterial suspension to each well, and 50 μL of the polypeptide co-assembled nanoparticles EMP prepared in Example 1 3-7 (the final concentrations were 12.5, 6.25, 3.13, 1.56, and 0.78 μM, respectively), 50 μL of antibiotics (the final concentrations were 25, 12.5, 6.25, 3.13, and 1.56 μM, respectively), 4 replicate wells were set for each concentration, blank culture medium was set as the blank group, and the group without drug addition was set as the control group. After incubation at 37°C in the incubator for 18 h, the OD was measured 600nm The inhibition rate was calculated by measuring the absorbance. 加药组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )]×100%, the results are shown in Table 4.

[0097] Table 4 Statistical results of the synergistic effect of antibiotics in Example 5

[0098] antibiotic Sensitization multiple Cefa ＞100 Cefu ＞100 Cefo ＞100

[0099] It can be concluded from Table 4 that when antibiotics are used alone (Example 4, Table 3), bacteria have obvious antibiotic resistance (MIC>300μM); when peptide co-assembled nanoparticles (EMP 3-7 ), it has weak antibacterial activity; when the two drugs are used in combination, 3-7 At lower dose levels (3.12-6.25 μM), the MIC of the antibiotic can be reduced to 1.56 μM, and the sensitization factor is >100 times, indicating that the polypeptide co-assembled nanoparticles in this example have the effect of antibiotic adjuvant, which can significantly reverse the bacterial antibiotic resistance and restore the bacterial sensitivity to antibiotics.

[0100] Example 6

[0101] DsbA enzyme-triggered deformation experiment: 50 μM of the polypeptide prepared in Example 1 was co-assembled into nanoparticles EMP 3-7 After incubation with 50 μg / mL DsbA enzyme for 0.5 h, 1 h, and 4 h, 10 μL was aspirated and dropped onto the copper grid. After standing for 10 min, the liquid was discarded by filter paper. After staining with 2% uranyl acetate for 5 min, transmission electron microscopy was used to observe the results. Figure 6 shown.

[0102] Depend on Figure 6 It can be concluded that: EMP 3-7 In solution, it is a nanoparticle. When it interacts with DabA enzyme, the hydrophilic-hydrophobic balance changes, and EMP 3-7 The morphology was transformed from nanoparticles to nanofibers, and the number of nanofibers increased gradually over time.

[0103] It can be concluded from the above examples that the polypeptide co-assembled nanoparticles of the present invention can cross the bacterial outer membrane to achieve precise delivery of antibiotics and / or antibiotic adjuvants into cells, significantly improve the binding ability with the DsbA enzyme, inhibit the stability of downstream β-lactamase, thereby reversing drug resistance and restoring the efficacy of antibiotics.

[0104] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A polypeptide monomer molecule MBP, characterized in that: The structure of the polypeptide monomer molecule MBP is shown in Formula I: Formula Ⅰ.

2. A polypeptide co-assembled nanoparticle, characterized in that: The polypeptide co-assembled nanoparticles include polypeptide monomer molecules EIP and polypeptide monomer molecules MBP according to claim 1; The structural formula of the polypeptide monomer molecule EIP is shown in Formula II: Formula II; The molar ratio of the polypeptide monomer molecule EIP to the polypeptide monomer molecule MBP is 3:7, 2:8 or 1:

9.

3. Use of the polypeptide monomer molecule MBP according to claim 1 or the polypeptide co-assembled nanoparticles according to claim 2 in the preparation of one or more of drugs for antibiotic delivery, antibiotic adjuvant delivery and reversal of antibiotic resistance; the antibiotics include cefazolin, cefuroxime and cefoperazone.

4. An antibiotic polypeptide-loaded co-assembled nanoparticle, characterized in that: The antibiotic-loaded polypeptide co-assembled nanoparticles include the polypeptide co-assembled nanoparticles according to claim 3 and the antibiotics encapsulated in the polypeptide co-assembled nanoparticles; The molar ratio of the polypeptide monomer molecule EIP to the polypeptide monomer molecule MBP in the polypeptide co-assembled nanoparticles is 3:7; The antibiotics include cefazolin, cefuroxime and cefoperazone.

5. The antibiotic polypeptide-loaded co-assembled nanoparticles according to claim 4, characterized in that: In the antibiotic-loaded polypeptide co-assembled nanoparticles, the concentration of the polypeptide co-assembled nanoparticles is 0.005 mM~1 mM; the concentration of the antibiotic is 0~1 mM, and the concentration of the antibiotic is not 0.

6. The method for preparing the antibiotic polypeptide-loaded co-assembled nanoparticles according to claim 4 or 5, characterized in that: The method comprises the following steps: subjecting a solution of polypeptide monomer molecule MBP, a solution of polypeptide monomer molecule EIP and a solution of antibiotic to ultrasonic treatment in an aqueous phase to obtain an ultrasonic treatment solution; The ultrasonic treatment solution is dialyzed in PBS buffer, and the obtained retentate contains the antibiotic polypeptide-loaded co-assembled nanoparticles.

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