Polypeptide monomer molecule MBP, polypeptide co-assembled nanoparticle and use thereof

Abstract

The present application belongs to the technical field of polypeptides and biomedicine, and specifically relates to a polypeptide monomer molecule MBP, a polypeptide co-assembled nanoparticle and the use thereof. In the polypeptide co-assembled nanoparticle of the present application, a lipopolysaccharide (LPS)-targeting unit in the polypeptide monomer molecule MBP is used to target a lipopolysaccharide on a bacterial outer membrane, the protective effect of the bacterial permeability barrier is overcome, and the nanoparticle translocates across the membrane and enters a cell. A thiol oxidase (DsbA)-targeting unit in a polypeptide monomer molecule EIP in the polypeptide co-assembled nanoparticle is used to target a bacterial intracellular DsbA enzyme, the bacterial DsbA enzyme is strongly bound, and the polypeptide co-assembled nanoparticle undergoes a transition from a nanoparticle into a nanofiber, so that the nano composition has a stronger capacity of competitively binding to the DsbA enzyme, and the activity of the DsbA enzyme is inhibited, which causes the synthesis of a downstream key resistance enzyme β-lactamase to be blocked, thereby exerting the effect of an antibiotic adjuvant and realizing accurate delivery of antibiotics.

Description

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

[0001] This application claims priority to the Chinese patent application filed with the Patent Office of China on January 25, 2024, with application number CN202410107411.4 and invention name “A polypeptide monomer molecule MBP, polypeptide co-assembled nanoparticles and their applications”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application 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. Background Art

[0003] Antimicrobial resistance (AMR) has become a major medical challenge facing 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 primarily target DNA / RNA synthesis, protein synthesis, membrane integrity, or cell wall synthesis, all of which have induced varying degrees of resistance, as exemplified by the emergence of "superbugs." Currently, global research and development of new antibiotics is slow, while the progression of drug resistance is accelerating. Therefore, there is an urgent need to develop therapies with novel mechanisms of action that can circumvent existing resistance mechanisms without leading to drug resistance.

[0004] β-lactam antibiotics, as the first line of defense in antibiotic therapy, are one of the most commonly used antibiotics in clinical practice, but they have now developed serious resistance. The main cause of resistance to β-lactam antibiotics is the production of β-lactamases within Gram-negative bacteria, which can hydrolyze antibiotics and lead to resistance. To address the resistance caused by this enzyme, a variety of β-lactamase competitive inhibitors have been marketed. They can competitively inhibit β-lactamase, thereby restoring bacterial sensitivity to antibiotics. These inhibitors mainly include sulbactam sodium and zobactam sodium. However, reports have shown that the adjuvants of these antibiotics also fail in clinical practice, and their inactivation mechanism is still unclear.

[0005] In recent years, peptides have become a promising candidate for combating bacterial drug resistance due to their excellent biocompatibility and low toxicity. With the rapid development of peptide self-assembly nanotechnology, a variety of novel self-assembling nanopeptides have been designed and prepared to address the shortcomings of peptide drugs. These novel self-assembling nanopeptides have demonstrated significant enhancements in both in vitro and in vivo antimicrobial activity and resistance to protease degradation, demonstrating their unique advantages as new, safe, and highly effective antimicrobial agents. The design and research of novel self-assembling nanopeptide drugs is becoming a hot topic internationally.

[0006] 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 microbiome and reduce the possibility of bacterial resistance. Recent studies have shown that thiol oxidase (DsbA) is a key protease in the bacterial extracytoplasmic environment that assists in the folding of β-lactamase. Therefore, inhibiting DsbA protease activity can impair the stability of downstream β-lactamases and is a new approach to 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 find it difficult to reach intracellular targets. Summary of the Invention

[0007] The purpose of the present application is to provide a polypeptide monomer molecule MBP, polypeptide co-assembled nanoparticles and their applications, wherein 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 the precise delivery of antibiotics and / or antibiotic adjuvants into the cells, assist in reversing antibiotic resistance, and restore antibiotic efficacy.

[0008] The present application provides a polypeptide monomer molecule MBP, comprising a hydrophobic unit, an enzymatic cleavage unit, a linking unit and a lipopolysaccharide targeting unit; the hydrophobic unit, the enzymatic 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, a protein or a nucleic acid aptamer having the function of targeting bacterial outer membrane lipopolysaccharide.

[0009] Preferably, the hydrophobic unit comprises palmitic acid, stearic acid or cholesterol;

[0010] The enzymatic cleavage unit is a tripeptide, the amino acid sequence of the tripeptide is Cys-XX, and X is any amino acid;

[0011] The linking unit is a polypeptide, and the first amino acid at the N-terminus of the polypeptide is Cys.

[0012] Preferably, the amino acid sequence of the lipopolysaccharide targeting unit is shown as SEQ ID NO.1.

[0013] Preferably, the structure of the polypeptide monomer molecule MBP is as shown in Formula I.

[0014] The present application 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;

[0015] The polypeptide monomer molecule EIP includes 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 includes a polypeptide, a small molecule compound, a protein or a nucleic acid aptamer that targets and inhibits thiol oxidase.

[0016] Preferably, the molar ratio of the polypeptide monomer molecule EIP to the polypeptide monomer molecule MBP is 3:7 to 1:9.

[0017] Preferably, the molar ratio of the polypeptide monomer molecule EIP to the polypeptide monomer molecule MBP is 3:7, 2:8 or 1:9.

[0018] 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.

[0019] The present application 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.

[0020] Preferably, the antibiotics include β-lactam antibiotics, cephalosporin antibiotics, quinolone antibiotics or polymyxins.

[0021] The present application 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 encapsulated in the polypeptide co-assembled nanoparticle.

[0022] 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 0.

[0023] Preferably, the antibiotics include β-lactam antibiotics, cephalosporin antibiotics, quinolone antibiotics or polymyxins.

[0024] The present application also provides a method for preparing the antibiotic-loaded polypeptide co-assembled nanoparticles described in the above technical solution, comprising the following steps: ultrasonically treating a solution of polypeptide monomer molecules MBP, a solution of polypeptide monomer molecules EIP, and a solution of an antibiotic in an aqueous phase to obtain an ultrasonically treated solution;

[0025] The ultrasonic treatment solution is dialyzed in PBS buffer, and the obtained retentate contains the antibiotic polypeptide-loaded co-assembled nanoparticles.

[0026] Preferably, the ultrasonic treatment is performed at a power of 100 to 600 W and for a time of 0 to 45 minutes.

[0027] Preferably, the molecular weight cut-off of the dialysis bag is 1000 Da.

[0028] Preferably, the dialysis time is 0 to 4 hours. Beneficial effects:

[0029] The present application provides a polypeptide monomer molecule MBP, comprising a hydrophobic unit, an enzymatic cleavage unit, a linker unit, and a lipopolysaccharide targeting unit; the hydrophobic unit, enzymatic cleavage unit, linker unit, and lipopolysaccharide targeting unit are sequentially connected via amide bonds; the lipopolysaccharide targeting unit comprises a polypeptide, small molecule compound, or protein that has the function of targeting bacterial outer membrane lipopolysaccharide. The present application utilizes the lipopolysaccharide (LPS) targeting unit in the polypeptide monomer molecule MBP to target bacterial outer membrane lipopolysaccharide, overcoming the protective effect of the bacterial permeability barrier and achieving precise delivery of antibiotic adjuvants, including bacterial thiol oxidase (DsbA) inhibitors, into cells.

[0030] Based on the advantages of the polypeptide monomer molecule MBP, the present application also provides a polypeptide co-assembled nanoparticle, which includes a polypeptide monomer molecule EIP and the polypeptide monomer molecule MBP described in the above technical solution; the polypeptide monomer molecule EIP includes a hydrophobic unit, an assembly unit, a connecting unit and a thiol oxidase targeting unit; the hydrophobic unit, assembly unit, connecting unit and 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. The polypeptide co-assembled nanoparticles described in the present application reach the cell with the help of the membrane-penetrating effect of the polypeptide monomer molecule MBP; with the help of the thiol oxidase targeting unit in the polypeptide monomer molecule EIP, it targets the DsbA enzyme in the bacterial cell, strongly binds to the bacterial DsbA enzyme, and transforms 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 leads to the obstruction of the synthesis of the downstream key enzyme for drug resistance (β-lactamase), thereby exerting an 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.

[0031] Based on the advantages of the polypeptide co-assembled nanoparticles, the present application provides an antibiotic-loaded polypeptide co-assembled nanoparticle, which includes the polypeptide co-assembled nanoparticles described in the above technical solution and the antibiotics encapsulated in the inner cavity of the polypeptide co-assembled nanoparticles. The antibiotic-loaded polypeptide co-assembled nanoparticles described in the present application 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-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 universal applicability for antibiotic delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments.

[0033] FIG1 is a schematic diagram of the structure of the polypeptide monomer molecule EIP in Example 1;

[0034] FIG2 is a schematic diagram of the structure of the polypeptide monomer molecule MBP in Example 1;

[0035] FIG3 is a transmission electron micrograph of polypeptide co-assembled nanoparticles in Example 1;

[0036] FIG4 is a transmission electron micrograph of the antibiotic-loaded polypeptide co-assembled nanoparticles in Example 2;

[0037] FIG5 is a diagram showing the results of a cytotoxicity experiment in Example 3;

[0038] Figure 6 is a transmission electron microscopy experimental result of the deformation of DsbA enzyme-triggered polypeptide co-assembled nanoparticles in Example 6. DETAILED DESCRIPTION

[0039] The present application provides a polypeptide monomer molecule MBP, comprising a hydrophobic unit, an enzymatic cleavage unit, a linking unit and a lipopolysaccharide targeting unit; the hydrophobic unit, the enzymatic 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 the function of targeting bacterial outer membrane lipopolysaccharide.

[0040] In the present application, 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, thereby passing through the bacterial outer membrane and overcoming the bacterial permeability barrier. The hydrophobic unit described in the present application 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 and hydrophobic balance of the molecule in the polypeptide monomer molecule MBP. The enzymatic cleavage unit described in the present application is preferably a tripeptide, and the amino acid sequence of the tripeptide is preferably Cys-XX, where X is any amino acid; the enzymatic cleavage unit has the function of being specifically recognized and cut off by the DsbA enzyme in the polypeptide single molecule MBP. The linking unit described in the present application 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 linking 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.

[0041] In the present application, 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.

[0042] In the present application, the hydrophobic unit, the enzymatic cleavage unit, the linking unit and the lipopolysaccharide targeting unit are sequentially connected via amide bonds. The connection process and conditions are not particularly limited and conventional connection processes and conditions in the art can be used.

[0043] This application 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 precise delivery of antibiotic adjuvants including bacterial thiol oxidase (DsbA) inhibitors into cells.

[0044] The present application 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;

[0045] The polypeptide monomer molecule EIP includes a hydrophobic unit, an assembly unit, a linking unit and a thiol oxidase targeting unit; the hydrophobic unit, the assembly unit, the linking 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.

[0046] In the polypeptide monomer molecule EIP described in the present application, the hydrophobic unit 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 (hydrophobic force) of the molecule. The assembly unit described in the present application 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 effect of providing a driving force (hydrogen bond, π-π interaction) for molecular assembly. The connecting unit described in the present application is preferably a tripeptide, and the amino acid sequence of the tripeptide is preferably Gly-Gly-Gly; the connecting unit has the effect of connecting and spacing the assembly module and the targeting module. The thiol oxidase targeting unit described in the present application 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 inhibition of the synthesis of the downstream key enzyme for drug resistance (β-lactamase), thereby exerting an antibiotic adjuvant effect and reversing antibiotic resistance.

[0047] The structure of the polypeptide monomer molecule EIP described in the present application is preferably as shown in Formula II:

[0048] Formula II; the molecular formula of the structural formula shown in Formula II is C 99 H 143 N 17 O23 S.

[0049] In the polypeptide monomer molecule EIP described in the present application, the hydrophobic unit, assembly unit, linker 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.

[0050] In the present application, the molar ratio of the polypeptide monomer molecule EIP to the polypeptide monomer molecule MBP 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.

[0051] The preparation method of the polypeptide co-assembled nanoparticles described in the present application 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;

[0052] 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;

[0053] 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.

[0054] The present application 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 described in the present application preferably includes dimethyl sulfoxide or an aqueous solution of dimethyl sulfoxide.

[0055] After obtaining the polypeptide monomer molecule MBP solution and the polypeptide monomer molecule EIP solution, the present application preferably subjects the polypeptide monomer molecule MBP solution and the polypeptide monomer molecule EIP solution to water bath sonication to obtain a water bath sonication solution. The power of the water bath sonication in the present application 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 minutes, more preferably 15 minutes.

[0056] After obtaining the water-bath ultrasonic treatment solution, the present application preferably subjects the water-bath ultrasonic treatment solution to ice-water ultrasonic treatment in water or PBS phosphate buffer to obtain the ice-water ultrasonic treatment solution. The pH value of the PBS phosphate buffer described in the present application is preferably 7.4. The power of the ice-water ultrasonic treatment described in the present application is preferably 100-300W, more preferably 200-250W; the time is preferably 0-40 minutes, more preferably 30 minutes. The ice-water bath has the effect of cooling the solution and preventing the nanoparticles from being damaged by excessive ultrasonic temperature.

[0057] After obtaining the ice-water bath ultrasonic treatment solution, the present application preferably allows the ice-water bath ultrasonic treatment solution to stand to obtain a standing ultrasonic treatment solution. The standing time in the present application is preferably 0 to 1.5 hours, more preferably 1 hour.

[0058] After obtaining the ultrasonic treatment liquid, the present application 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 used for dialysis in the present application is preferably 1000 Da. The dialysis time in the present application is preferably 0 to 4 hours, more preferably 1 hour. The pH value of the PBS buffer in the present application is preferably 7.4. The dialysis described in the present application can remove free polypeptide single molecules.

[0059] The polypeptide co-assembled nanoparticles described in the present application reach the cell with the help of the membrane-penetrating effect of the polypeptide monomer molecule MBP; with the help of the thiol oxidase targeting unit in the polypeptide monomer molecule EIP, it targets the DsbA enzyme in the bacterial cell, strongly binds to the bacterial DsbA enzyme, and transforms 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 leads to the obstruction of the synthesis of the downstream key enzyme for drug resistance (β-lactamase), 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.

[0060] Based on the above advantages, the present application 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.

[0061] The present application 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.

[0062] The polypeptide co-assembled nanoparticles and antibiotics described in the present application are preferably encapsulated through hydrophobic interactions. The concentration of the polypeptide co-assembled nanoparticles described in the present application 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 described in the present application 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.

[0063] The present application also provides a method for preparing the antibiotic-loaded polypeptide co-assembled nanoparticles described in the above technical solution, comprising the following steps: ultrasonically treating a solution of polypeptide monomer molecules MBP, a solution of polypeptide monomer molecules EIP, and a solution of an antibiotic in an aqueous phase to obtain an ultrasonically treated solution;

[0064] The ultrasonic treatment solution is dialyzed in PBS buffer, and the obtained retentate contains the antibiotic polypeptide-loaded co-assembled nanoparticles.

[0065] The present application 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 described in the present application preferably includes dimethyl sulfoxide or an aqueous solution of dimethyl sulfoxide.

[0066] After obtaining the polypeptide monomer molecule MBP solution, polypeptide monomer molecule EIP solution, and antibiotic solution, the present application 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 ultrasonic treatment power of the present application is preferably 100 to 600 W, more preferably 100 to 300 W, and more preferably 200 to 250 W; the ultrasonic treatment time is preferably 0 to 45 minutes, more preferably 30 minutes.

[0067] After obtaining the ultrasonic treatment liquid, the present application preferably dialyzes the ultrasonic treatment liquid in PBS buffer to obtain a retentate. The molecular weight cutoff of the dialysis bag for dialysis described in the present application is preferably 1000Da. The dialysis described in the present application is preferably light-proof dialysis, because it avoids the instability of antibiotics exposed to light and degradation. The dialysis time described in the present application is preferably 0 to 4 hours, more preferably 1 hour. The pH value of the PBS buffer described in the present application is preferably 7.4. The dialysis described in the present application can remove free antibiotics and polypeptide single molecules.

[0068] After obtaining the retentate, the present application preferably centrifuges the retentate and collects the supernatant to obtain the antibiotic polypeptide-loaded co-assembled nanoparticles. The centrifugation speed is preferably 6000 g and the time is preferably 5 min.

[0069] The antibiotic-loaded polypeptide co-assembled nanoparticles described herein can precisely deliver antibiotics into bacterial cells, significantly improving drug efficacy, reducing dosage, and minimizing toxic and side effects. These antibiotic-loaded polypeptide co-assembled nanoparticles can reverse antibiotic resistance, including in bacterial β-lactam antibiotics, and extend the lifespan of antibiotics, demonstrating significant potential in treating drug-resistant bacterial infections. Furthermore, these antibiotic-loaded polypeptide co-assembled nanoparticles can encapsulate different types of hydrophobic antibiotics, demonstrating universal applicability for antibiotic delivery.

[0070] In order to further illustrate the present application, the technical solutions provided in the present application are described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present application.

[0071] Example 1

[0072] A method for preparing polypeptide co-assembled nanoparticles, comprising the following steps:

[0073] 1) The structural formula of the polypeptide monomer molecule EIP is shown in FIG1 , and the structural formula of the polypeptide monomer molecule MBP is shown in FIG2 . The polypeptide monomer molecule EIP and the polypeptide monomer molecule MBP were respectively commissioned to Jier Biochemical (Shanghai) Co., Ltd. and prepared by the polypeptide solid phase synthesis method.

[0074] 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 and shake 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 of PBS phosphate buffer (pH 7.4), vortexed and mixed, and ultrasonicated in an ice water bath at 240W for 30 minutes, and then allowed to stand for 1 hour.

[0075] 2) Preparation of transmission electron microscopy samples using the negative staining method: 10 μL of the co-assembled nanoparticles at different ratios from step 1) was dropped onto a carbon-supported copper grid (100 mesh). After allowing to settle for 5 minutes, the droplet was removed using disposable filter paper. 10 μL of 2% uranyl acetate was then dropped onto the copper grid after the droplet was removed. After allowing to settle for 1 minute, the stain was again removed using disposable filter paper. The samples were observed and photographed using a biological transmission electron microscope (HT 7800). The results are shown in Figure 3.

[0076] 3) Determination of particle size and potential by dynamic light scattering (DLS): 200 μL of the co-assembled nanoparticles prepared in step 1) at different ratios were placed in a potential cup and a particle size cup, respectively, and then placed in a dynamic light scattering particle size analyzer (Malvern, ZS90) to measure the particle size and potential. The results are shown in Table 1.

[0077] Table 1 Statistical results of appearance, particle size and potential of co-assembled nanoparticles in Example 1

[0078] Figure 3 and Table 1 show that the particle size of nanoparticles self-assembled from single-molecule MBP peptides is 24.9 ± 4.5 nm. As the ratio of single-molecule EIP peptides increases (1:9 to 3:7), the particle size gradually increases, indicating co-assembly of MBP and EIP. Further increases in the EIP ratio (5:5 to 8:2) lead to the formation of partial or even complete nanofibers, which are not conducive to crossing the bacterial outer membrane barrier. Zeta potential significantly influences nanoparticle stability. Generally, higher potentials reduce sedimentation, coagulation, and aggregation, resulting in a more stable system. Stability requirements are achieved when the potential reaches 30 mV.

[0079] Example 2

[0080] Cefoperazone (Cefoperazone, Cefo)-loaded peptide co-assembled nanoparticles (EMPs) 3-7Preparation of @Cefo): Accurately weigh 15.77 mg of the polypeptide monomer molecule EIP (prepared in Example 1) and dissolve it in 800 μL of DMSO. Vortex to dissolve it and prepare a 10 mM stock solution for later use. Accurately weigh 12.50 mg of the polypeptide monomer molecule MBP (prepared in Example 1) and dissolve it in 400 μL of DMSO. Vortex to dissolve it and prepare a 10 mM stock solution for later use. Accurately weigh 25.82 mg of Cefo antibiotic and dissolve it in 400 μL of DMSO. Vortex to dissolve it and prepare a 100 mM stock solution for later use. 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 ultrasonicate them in an ice water bath at 240W for 30 minutes. Then transfer them to a dialysis bag (MWCO: 1000Da), dialyze them in PBS buffer solution (pH = 7.4) in the dark for 1 hour to remove free antibiotics, and centrifuge them at 6000g for 5 minutes to obtain the supernatant for later use.

[0081] The electron microscopy sample preparation method and the particle size potential measurement method are the same as in Example 1. The electron microscopy test results are shown in FIG4 , and the test statistical results of appearance, particle size and potential are shown in Table 2.

[0082] Table 2 Statistical results of appearance, particle size and potential of the antibiotic-loaded polypeptide co-assembled nanoparticles in Example 2

[0083] It can be concluded from FIG4 and Table 2 that the morphology of Cefo-loaded polypeptide co-assembled nanoparticles is relatively uniform, and there is no significant difference in morphology from the blank polypeptide co-assembled nanoparticles (Example 1).

[0084] Example 3

[0085] Cytotoxicity assay, steps are as follows:

[0086] 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. 2 mL of 0.25% trypsin was then added. After digestion for 4 min, 2 mL of cell culture medium was immediately added to terminate the digestion. The cell suspension was repeatedly pipetted several times to make the cell suspension density uniform. The cell density of the cell suspension was calculated using a hemocytometer. The corresponding volume of complete culture medium was added according to the counting results. The final cell density was adjusted to 6 × 10 4 pieces / mL.

[0087] Take a 96-well plate and add 100 μL of mixed cell suspension (about 6×10 3The blank control group was not supplemented with cell suspension but only with 100 μL of culture medium or PBS buffer. The cells were cultured at 37°C and 5% CO2 until the cells were fully attached, which took about 24 hours.

[0088] The polypeptide self-assembled nanoparticles prepared in Example 1 were suspended in complete culture medium (EIP, BMP, EMP 3-7 ) were 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 was added to each well (each 100 μL contained 10 μL of CCK-8 solution), gently shaken to mix, and placed in the incubator for 4 hours. The absorbance value OD at a wavelength of 450 nm was detected by a microplate reader. 450 , the results are shown in Figure 5.

[0089] It can be concluded from Figure 5 that the polypeptide single molecule EIP, BMP and co-assembled nanoparticle cell EMP in this embodiment 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.

[0090] Example 4

[0091] The pharmacodynamics experiment (minimum inhibitory concentration experiment) has the following steps:

[0092] The minimum inhibitory concentration (MIC) value was determined by broth dilution method. MIC determination was performed on representative antibiotics currently commonly used in clinical practice: cefazolin sodium (Cefazolin, Cefa, first generation cephalosporin), cefuroxime sodium (Cefuroxime, Cefu, second generation cephalosporin), cefoperazone (Cefop, Cefo, third generation cephalosporin) and cefepime (Cefe, fourth generation cephalosporin). A single clone of clinically isolated multidrug-resistant Escherichia coli (E. coli MDR ESBL-1 (Clinical), E. coli MDR ESBL-1 (Clinical) was published in ACS Nano. 2022, 16, (12): 20545-20558.) was incubated 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.

[0093] Take a 96-well plate and add 100 μL of the above bacterial suspension and 100 μL of different drugs to each well. 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; set a series of sample concentrations, set 6 replicates for each concentration, set blank culture medium as blank group, and set no drug group as control group. After incubation at 37°C for 18 hours, measure OD 600nm The absorbance was measured, and the minimum concentration that inhibited bacterial growth was taken as the MIC value.

[0094] Table 3 MIC values ​​in Example 4

[0095] As can be seen from Table 3, clinically isolated strains all showed varying degrees of resistance to commonly used antibiotics (MIC>300 μM); and the polypeptide single molecule MBP and polypeptide co-assembled nanoparticles EMP in this example had certain antibacterial activity.

[0096] Example 5

[0097] The steps of antibiotic synergy experiment are as follows:

[0098] The broth dilution method was used to determine the synergistic effect of the peptides on the antibiotics cefazolin, cefuroxime, and cefoperazone. A monoclonal clinically isolated multidrug-resistant Escherichia coli (E. coli MDR ESBL-1 (Clinical)) was incubated in M63 broth medium at 37°C with a shaker at 180 rpm overnight. The next day, 1% of the bacterial suspension was added to fresh M63 broth medium and incubated until the absorbance OD 600nm =0.1(1.0×10 8 CFU / mL) and diluted to 1.5×10 6 CFU / mL.

[0099] Take a 96-well plate, add 100 μL of the above bacterial suspension to each well, 50 μL of the polypeptide co-assembled nanoparticles EMP prepared in Example 1 3-7 (final concentrations were 12.5, 6.25, 3.13, 1.56, and 0.78 μM, respectively), 50 μL of antibiotics (final concentrations were 25, 12.5, 6.25, 3.13, and 1.56 μM, respectively), 4 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 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.

[0100] Table 4 Statistical results of the synergistic effect of antibiotics in Example 5

[0101] It can be concluded from Table 4 that when antibiotics were used alone (Example 4, Table 3), bacteria showed obvious antibiotic resistance (MIC>300 μM); when peptide co-assembled nanoparticles (EMP 3-7 ), it has weak antibacterial activity; and when the two 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, with a sensitization factor of >100 times, indicating that the polypeptide co-assembled nanoparticles in this example have the effect of an antibiotic adjuvant, which can significantly reverse bacterial antibiotic resistance and restore bacterial sensitivity to antibiotics.

[0102] Example 6

[0103] 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 for 0.5, 1, and 4 hours, 10 μL of the sample was dripped onto a copper grid. After 10 minutes of incubation, the sample was discarded with filter paper. After staining with 2% uranyl acetate for 5 minutes, transmission electron microscopy was performed. The results are shown in Figure 6.

[0104] From Figure 6, we can conclude that EMP 3-7 In solution, it is a nanoparticle. When it interacts with DabA enzyme, the hydrophilic-hydrophobic balance changes. 3-7 The morphology was transformed from nanoparticles to nanofibers, and the number of nanofibers increased gradually over time.

[0105] It can be concluded from the above examples that the polypeptide co-assembled nanoparticles of the present application 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 antibiotic efficacy.

[0106] Although the above embodiment provides a detailed description of the present application, it is only a part of the embodiments of the present application, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present application.

Claims

1. A polypeptide monomer molecule MBP, comprising a hydrophobic unit, a cleavage unit, a linking unit, and a lipopolysaccharide targeting unit; the hydrophobic unit, the cleavage unit, the linking unit, and the lipopolysaccharide targeting unit are sequentially linked by amide bonds; the lipopolysaccharide targeting unit comprises a polypeptide, a small molecule compound, a protein, or a nucleic acid aptamer having the function of targeting the lipopolysaccharide on the outer membrane of bacteria.

2. The polypeptide monomer molecule MBP according to claim 1, wherein The hydrophobic unit comprises palmitic acid, stearic acid, or cholesterol; The cleavage unit is a tripeptide, and the amino acid sequence of the tripeptide is Cys-X-X, where X is any amino acid; The linking unit is a polypeptide, and the first amino acid at the N-terminus of the polypeptide is Cys.

3. The polypeptide monomer molecule MBP according to claim 1, wherein, The amino acid sequence of the lipopolysaccharide targeting unit is as shown in SEQ ID NO.

1.

4. The polypeptide monomer molecule MBP according to any one of claims 1 to 3, characterized in that, The structure of the polypeptide monomer molecule MBP is shown in Formula I:

5. A polypeptide co-assembled nanoparticle, characterized in that, The polypeptide co-assembled nanoparticles comprise a polypeptide monomer molecule EIP and the polypeptide monomer molecule MBP according to any one of claims 1 to 4; The polypeptide monomer molecule EIP comprises a hydrophobic unit, an assembly unit, a linking unit, and a thiol oxidase targeting unit; the hydrophobic unit, the assembly unit, the linking unit, and the thiol oxidase targeting unit are sequentially linked by amide bonds; the thiol oxidase targeting unit comprises a polypeptide, a small molecule compound, a protein, or a nucleic acid aptamer having the function of targeting and inhibiting thiol oxidase.

6. The polypeptide co-assembled nanoparticles according to claim 5, characterized in that, The molar ratio of the polypeptide monomer molecule EIP to the polypeptide monomer molecule MBP is 3:7 to 1:

9.

7. The polypeptide co-assembled nanoparticles according to claim 6, characterized in that, The molar ratio of the polypeptide monomer molecule EIP to the polypeptide monomer molecule MBP is 3:7, 2:8, or 1:

9.

8. The polypeptide co-assembled nanoparticles according to any one of claims 5 to 7, characterized in that, In the polypeptide monomer molecule EIP, the hydrophobic unit comprises 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-Ala; the linking 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.

9. Use of the polypeptide monomer molecule MBP according to any one of claims 1 to 4 or the polypeptide co-assembled nanoparticles according to any one of claims 5 to 8 in one or more of antibiotic delivery, antibiotic adjuvant delivery, and reversal of antibiotic resistance.

10. The application according to claim 9, wherein The antibiotics include β-lactam antibiotics, cephalosporin antibiotics, quinolone antibiotics, or polymyxins.

11. A polypeptide co-assembled nanoparticle loaded with antibiotics, characterized in that, The antibiotic-loaded polypeptide co-assembled nanoparticles comprise the polypeptide co-assembled nanoparticles according to any one of claims 5 to 8 and an antibiotic encapsulated in the polypeptide co-assembled nanoparticles.

12. The antibiotic-loaded polypeptide co-assembled nanoparticles according to claim 11, wherein 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 0.

13. The antibiotic-loaded polypeptide co-assembled nanoparticles according to claim 11 or 12, characterized in that, The antibiotics include β-lactam antibiotics, cephalosporin antibiotics, quinolone antibiotics, or polymyxins.

14. The preparation method of the antibiotic-loaded polypeptide co-assembled nanoparticles according to any one of claims 11 to 13, characterized in that, Comprising the following steps: subjecting a solution of the polypeptide monomer molecule MBP, a solution of the polypeptide monomer molecule EIP, and a solution of an antibiotic to ultrasonic treatment in an aqueous phase to obtain an ultrasonic treatment solution; Dialyze the ultrasonic treatment solution in PBS buffer, and the retentate contains the antibiotic-loaded polypeptide co-assembled nanoparticles.

15. The preparation method according to claim 14, characterized in that, The power of the ultrasonic treatment is 100 - 600 W, and the time is 0 - 45 min.

16. The preparation method according to claim 14, characterized in that, The molecular weight cut-off of the dialysis bag used for dialysis is 1000 Da.

17. The preparation method according to claim 14 or 16, characterized in that, The dialysis time is 0 - 4 h.

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