Bacterial recognition ligand

Abstract

Provided is a ligand molecule against bacterial membranes, the ligand molecule including a compound having a ketone, a tertiary amine, and a single bond or an alkylene group spacer between the ketone and the tertiary amine.

Description

Bacteria-recognizing ligands

[0001] The present invention relates to a ligand molecule that recognizes the surface of bacteria for the purpose of delivering drugs to bacteria.

[0002] Traditionally, bacterial infections have been treated with antibiotics. However, the emergence of drug-resistant bacteria due to the overuse of drugs has become a problem in recent years. According to the WHO, it is estimated that by 2050, 10,000,000 people will die annually from drug-resistant bacteria (Non-Patent Document 1). Therefore, even if new drugs are developed, they may not be an effective solution because they contribute to the acquisition of further resistance. Therefore, drug delivery systems (DDS) have been attracting attention as a method to improve the effectiveness of existing drugs. Although ligands that specifically target bacterial membranes have been reported (Non-Patent Document 2), they were unable to recognize Gram-negative bacteria. Because Gram-negative bacteria are the main cause of sepsis, a severe symptom induced by bacterial infection, targeting Gram-negative bacteria is extremely important.

[0003]

[0004] Yunjiang Jiang, et al. Advanced Drug Delivery Reviews 170 (2021) 261-280 Anupam Bandyopadhyay, et al. Nature Communications 6 (2015) 6561-6569

[0005] Given the above background, there was a need to develop a ligand molecule that recognizes bacterial cells but not mammalian cells.

[0006] Therefore, the present inventors conducted extensive research to solve the above problems, and as a result, they succeeded in finding that a compound having a ketone and a tertiary amine can solve the above problems, and have completed the present invention. That is, the present invention is as follows: [1] A ligand molecule for a bacterial membrane, comprising a compound having a ketone and a tertiary amine, and having a spacer of a single bond or an alkylene group between the ketone and the tertiary amine. [2] The compound is represented by the following formula I: [In the formula, R １ represents an amino group, a hydroxyl group, or a carboxyl group, and R ２represents a tertiary amine, m represents an integer, and n represents an integer from 0 to 10. [3] The ligand molecule according to [1], wherein the compound represented by formula I is represented by the following formula II: [4] A complex comprising the ligand molecule according to any one of [1] to [3] and a target substance to be delivered to the bacterial membrane. [5] The complex according to [4], wherein the target substance is a drug. [6] The complex according to [5], wherein the drug is supported on a delivery carrier. [7] A pharmaceutical composition comprising the complex according to any one of [4] to [6]. [8] A method for delivering a ligand molecule to bacteria, comprising the step of contacting the ligand molecule according to any one of [1] to [3] with bacteria to bind the ligand molecule to the bacterial membrane. [9] A method for contacting the complex according to [4] with bacteria to deliver the target substance in the complex to bacteria.

[10] A method for contacting the complex according to [5] or [6] with bacteria to deliver the drug in the complex to bacteria.

[11] A kit for delivering a target substance to the bacterial membrane, comprising the ligand molecule according to any one of [1] to [3].

[0007] The present invention provides a ligand molecule that recognizes bacterial cells. The ligand molecule of the present invention makes it possible to deliver a target substance to the bacterial cell membrane, rather than to the mammalian cell membrane. Furthermore, the enhanced delivery ability to bacteria can improve the specificity and efficacy of antibiotics and other drugs against bacteria.

[0008] 2 shows the results of FT-IR measurement of a sample containing BBM and PE. １３ 1 shows the results of measurement by C NMR. (Continuation of FIG. 2) A combination of PE and BBM, or a combination of PE and BBM with a control ligand, was mixed, and the results of measurement by C NMR. FIG. 3 shows the results of measurement by C NMR. １1H NMR results (continuation of FIG. 3). A combination of PE and BBM, or a combination of PE and BBM with a control ligand, was mixed, １4 is a diagram showing the results of H NMR measurement. Figure 4 is a diagram showing the results of synthesis of Cy5-8armPEG40k-Ligands. (Continued from Figure 4) A diagram showing the results of synthesis of Cy5-8armPEG40k-Ligands. (Continued from Figure 4) A diagram showing the results of synthesis of Cy5-8armPEG40k-Ligands. (Continued from Figure 4) A diagram showing the results of synthesis of Cy5-8armPEG40k-Ligands. (Continued from Figure 4) A diagram showing the results of synthesis of Cy5-8armPEG40k-Ligands. (Continued from Figure 4) A diagram showing the results of synthesis of Cy5-8armPEG40k-Ligands. Figure 5 is a fluorescence microscope image when Cy5-8armPEG40k-Ligands was administered to Escherichia coli stained with Hoechst. (Continued from Figure 5) Fluorescence microscope image when Cy5-8armPEG40k-Ligands were administered to Escherichia coli stained with Hoechst. (Continued from Figure 5) Fluorescence microscope image when Cy5-8armPEG40k-Ligands were administered to Escherichia coli stained with Hoechst. (Continued from Figure 5) Fluorescence microscope image when Cy5-8armPEG40k-Ligands were administered to Escherichia coli stained with Hoechst. Figure 6 is a fluorescence microscope image when Cy5-8armPEG40k-Ligands were administered to Staphylococcus aureus stained with Hoechst. (Continued from Figure 6) Fluorescence microscope image when Cy5-8armPEG40k-Ligands were administered to Staphylococcus aureus stained with Hoechst. (Continued from Figure 6) Fluorescence microscope image when Cy5-8armPEG40k-Ligands were administered to Staphylococcus aureus stained with Hoechst. (Continued from Figure 6) Fluorescence microscope image when Cy5-8armPEG40k-Ligands were administered to Staphylococcus aureus stained with Hoechst. Figure 7 shows fluorescence microscope images when Cy5-8armPEG40k-BBM with different BBM introduction rates was administered to Escherichia coli stained with Hoechst. (Continued from Figure 7) Fluorescence microscope images when Cy5-8armPEG40k-BBM with different BBM introduction rates was administered to Escherichia coli stained with Hoechst. (Continuation of Figure 7) Fluorescence microscope images of E. coli stained with Hoechst when Cy5-8armPEG40k-BBM with different BBM incorporation rates was administered.(Continued from Figure 7) Fluorescence microscope images of Cy5-8armPEG40k-BBM having different BBM introduction rates administered to Escherichia coli stained with Hoechst. Figure 8 shows fluorescence microscope images of Cy5-8armPEG40k-BBM having different BBM introduction rates administered to Staphylococcus aureus stained with Hoechst. (Continued from Figure 8) Fluorescence microscope images of Cy5-8armPEG40k-BBM having different BBM introduction rates administered to Staphylococcus aureus stained with Hoechst. (Continued from Figure 8) Fluorescence microscope images of Cy5-8armPEG40k-BBM having different BBM introduction rates administered to Staphylococcus aureus stained with Hoechst. (Continued from Figure 8) Fluorescence microscope images of Cy5-8armPEG40k-BBM with different BBM introduction rates administered to Staphylococcus aureus stained with Hoechst. Figure 9 shows fluorescence microscope images of free BBM and Cy5-8armPEG40k-Ligands administered to Escherichia coli stained with Hoechst. (Continued from Figure 9) Fluorescence microscope images of free BBM and Cy5-8armPEG40k-Ligands administered to Escherichia coli stained with Hoechst. (Continued from Figure 9) Fluorescence microscope images of free BBM and Cy5-8armPEG40k-Ligands administered to Escherichia coli stained with Hoechst. Figure 10 shows fluorescence microscopy images of free BBM and Cy5-8armPEG40k-Ligands administered to Staphylococcus aureus stained with Hoechst. (Continued from Figure 10) Fluorescence microscopy images of free BBM and Cy5-8armPEG40k-Ligands administered to Staphylococcus aureus stained with Hoechst. (Continued from Figure 10) Fluorescence microscopy images of free BBM and Cy5-8armPEG40k-Ligands administered to Staphylococcus aureus stained with Hoechst. Figure 11 shows fluorescence microscopy images of Cy5-BBM, a small molecule in which BBM is directly bound to the fluorescent dye Cy5, administered to Staphylococcus aureus stained with Hoechst. (Continuation of Figure 11) Fluorescence microscope image of the small molecule Cy5-BBM, in which BBM is directly bound to the fluorescent dye Cy5, administered to Staphylococcus aureus stained with Hoechst.(Continued from Figure 11) Fluorescence microscope image of the small molecule Cy5-BBM, in which BBM is directly bound to the fluorescent dye Cy5, administered to Staphylococcus aureus stained with Hoechst. (Continued from Figure 11) Fluorescence microscope image of the small molecule Cy5-BBM, in which BBM is directly bound to the fluorescent dye Cy5, administered to Staphylococcus aureus stained with Hoechst. A diagram showing the synthesis results of BBM bound to the fluorescent dye FITC via ethylenediamine. A diagram showing the remaining fluorescence intensity after adding FITC-BBM or unmodified fluorescein as a control, and PBS to well plates whose bottoms are coated with lipids having different PE / PC mixture ratios, and washing. A diagram showing the remaining fluorescence intensity after adding Cy5-8armPEG20k-Ligands to well plates whose bottoms are coated with lipids having different PE / PC mixture ratios, and washing. Figure 15 is a fluorescence microscope image when Cy5-8armPEG20k-Ligands are administered to E. coli stained with Hoechst at pH 6. (Continued from Figure 15) Fluorescence microscope image when Cy5-8armPEG20k-Ligands are administered to E. coli stained with Hoechst at pH 6. (Continued from Figure 15) Fluorescence microscope image when Cy5-8armPEG20k-Ligands are administered to E. coli stained with Hoechst at pH 6. (Continued from Figure 15) Fluorescence microscope image when Cy5-8armPEG20k-Ligands are administered to E. coli stained with Hoechst at pH 6. (Continuation of Figure 15) Fluorescence microscope image when Cy5-8armPEG20k-Ligands were administered to E. coli stained with Hoechst at pH 6. Figure 16 is a fluorescence microscope image when Cy5-8armPEG20k-Ligands were administered to E. coli stained with Hoechst at pH 7.4. (Continuation of Figure 16) Fluorescence microscope image when Cy5-8armPEG20k-Ligands were administered to E. coli stained with Hoechst at pH 7.4. (Continuation of Figure 16) Fluorescence microscope image when Cy5-8armPEG20k-Ligands were administered to E. coli stained with Hoechst at pH 7.4.(Continued from Figure 16) Fluorescence microscope image of E. coli stained with Hoechst after administration of Cy5-8armPEG20k-Ligands at pH 7.4. (Continued from Figure 16) Fluorescence microscope image of E. coli stained with Hoechst after administration of Cy5-8armPEG20k-Ligands at pH 7.4. Figure 17 is a fluorescence microscope image of E. coli stained with Hoechst after administration of Cy5-8armPEG20k-Ligands at pH 8. (Continued from Figure 17) Fluorescence microscope image of E. coli stained with Hoechst after administration of Cy5-8armPEG20k-Ligands at pH 8. (Continued from Figure 17) Fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Escherichia coli at pH 8. (Continued from Figure 17) Fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Escherichia coli at pH 8. (Continued from Figure 17) Fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Escherichia coli at pH 8. Figure 18 is a fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Staphylococcus aureus at pH 6. (Continued from Figure 18) Fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Staphylococcus aureus at pH 6. (Continued from Figure 18) Fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Staphylococcus aureus at pH 6. (Continued from Figure 18) Fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Staphylococcus aureus at pH 6. (Continued from Figure 18) Fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Staphylococcus aureus at pH 6. FIG. 19 shows fluorescence microscopy images of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Staphylococcus aureus at pH 7.4.(Continued from Figure 19) Fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Staphylococcus aureus stained with Hoechst at pH 7.4. (Continued from Figure 19) Fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Staphylococcus aureus stained with Hoechst at pH 7.4. (Continued from Figure 19) Fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Staphylococcus aureus stained with Hoechst at pH 7.4. (Continued from Figure 19) Fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Staphylococcus aureus stained with Hoechst at pH 7.4. Figure 20 shows a fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Staphylococcus aureus at pH 8. (Continued from Figure 20) A fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Staphylococcus aureus at pH 8. (Continued from Figure 20) A fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Staphylococcus aureus at pH 8. (Continued from Figure 20) A fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Staphylococcus aureus at pH 8. (Continued from Figure 20) A fluorescence microscope image of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Staphylococcus aureus at pH 8. (Continuation of Figure 20) Fluorescence microscopy images of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Staphylococcus aureus at pH 8. Figure 21 shows fluorescence microscopy images of FITC-BBM, a small molecule in which BBM is bound to the fluorescent dye FITC via EDA, administered to Hoechst-stained Escherichia coli or Staphylococcus aureus at pH 6. (Continuation of Figure 21) Fluorescence microscopy images of FITC-BBM, a small molecule in which BBM is bound to the fluorescent dye FITC via EDA, administered to Hoechst-stained Escherichia coli or Staphylococcus aureus at pH 6. (Continuation of Figure 21) Fluorescence microscope images of the small molecule FITC-BBM, in which BBM is bound to the fluorescent dye FITC via EDA, administered to Escherichia coli or Staphylococcus aureus stained with Hoechst at pH 6.(Continued from Figure 21) Fluorescence microscopy images of the small molecule FITC-BBM, in which BBM is bound to the fluorescent dye FITC via EDA, administered to Escherichia coli or Staphylococcus aureus stained with Hoechst at pH 6. Figure 22 shows fluorescence microscopy images of the small molecule FITC-BBM, in which BBM is bound to the fluorescent dye FITC via EDA, administered to Escherichia coli or Staphylococcus aureus stained with Hoechst at pH 7.4. (Continued from Figure 22) Fluorescence microscopy images of the small molecule FITC-BBM, in which BBM is bound to the fluorescent dye FITC via EDA, administered to Escherichia coli or Staphylococcus aureus stained with Hoechst at pH 7.4. (Continued from Figure 22) Fluorescence microscopy images of the small molecule FITC-BBM, in which BBM is bound to the fluorescent dye FITC via EDA, administered to Escherichia coli or Staphylococcus aureus stained with Hoechst at pH 7.4. (Continued from Figure 22) Fluorescence microscopy images of the small molecule FITC-BBM, in which BBM is bound to the fluorescent dye FITC via EDA, administered to Escherichia coli or Staphylococcus aureus stained with Hoechst at pH 7.4. Figure 23 shows fluorescence microscopy images of the small molecule FITC-BBM, in which BBM is bound to the fluorescent dye FITC via EDA, administered to Escherichia coli or Staphylococcus aureus stained with Hoechst at pH 8. (Continued from Figure 23) Fluorescence microscope images of the small molecule FITC-BBM, in which BBM is bound to the fluorescent dye FITC via EDA, administered to Escherichia coli or Staphylococcus aureus stained with Hoechst at pH 8. (Continued from Figure 23) Fluorescence microscope images of the small molecule FITC-BBM, in which BBM is bound to the fluorescent dye FITC via EDA, administered to Escherichia coli or Staphylococcus aureus stained with Hoechst at pH 8. (Continued from Figure 23) Fluorescence microscope images of the small molecule FITC-BBM, in which BBM is bound to the fluorescent dye FITC via EDA, administered to Escherichia coli or Staphylococcus aureus stained with Hoechst at pH 8. 1 shows the residual Cy5 fluorescence intensity after administering Cy5-8armPEG20k-Ligands to Escherichia coli or Staphylococcus aureus and washing away the unadsorbed free polymer at pH 6, 7.4, or 8. FIG. 1 shows the results of conjugating BBM to the antibiotic gemifloxacin.3 shows bacterial growth when Gem-BBM, which is gemifloxacin bound to a BBM, was administered to Escherichia coli and the unadsorbed free drug was removed after 1 hour of co-culture. 3 shows bacterial growth when Gem-BBM, which is gemifloxacin bound to a BBM, was administered to Staphylococcus aureus and the unadsorbed free drug was removed after 1 hour of co-culture. 3 shows the results of measuring cell viability when Gem-BBM was administered to mammalian cells, including cancer cells and healthy cells, to verify side effects of Gem-BBM. 3 shows the membrane structures of bacteria and mammalian cells. 3 shows the interaction between a ligand and a bacterial membrane. 3 shows the results of confirming Schiff base formation by FT-IR. 3 shows a scheme illustrating a method for preparing a Vcm-DMAX complex. 33 shows the structure of Vcm-DMAX. １ 33 shows the results of H-NMR measurement. １ 34 is a diagram showing the growth curve (OD) and CFU of each bacterium. (Continuation of FIG. 34) A diagram showing the growth curve (OD) and CFU of each bacterium.

[0009] 1. Overview In the present invention, we focus on phosphoethanolamine and its analogous structure (PE), which are commonly present in both Gram-negative and Gram-positive bacteria. Because this structure is not present on the surface of mammalian cells (Zhaoyu Li, et al. Cell Metabolism 3 (2006) 321-331), bacteria can be selectively targeted using molecules that specifically recognize PE. Therefore, the present invention provides ligand molecules that can recognize the bacterial surface to enhance the targeting of drugs, polymeric compounds, and nanoparticles. The ligand molecules of the present invention can recognize the phosphoethanolamine structure uniquely present on the outer surface of bacteria, thereby allowing various molecules to selectively bind to the bacterial surface rather than to mammalian cells. The ligand molecules of the present invention are molecules that bind to bacterial cell membranes and are referred to as "Bacteria Binding Moieties" (BBMs).

[0010] The surface (outer membrane) of bacterial cells contains lipoteichoic acid (LTA) in Gram-positive bacteria and lipopolysaccharide (LPS) in Gram-negative bacteria, and these LTA and LPS contain phosphatidylethanolamine (PE) (Figure 29). In contrast, the outer membrane surface of mammalian cells does not contain PE (Figure 29). Therefore, in the present invention, BBM is designed to target PE on the outer membrane of bacterial cells.

[0011] In this invention, we used a molecule containing a tertiary amine and a ketone as the PE-recognizing molecule (BBM) (details will be described later). This BBM allows the positive charge of the tertiary amine to approach the negative charge of the phosphate of PE through electrostatic interaction, and simultaneously, the ketone of the BBM and the primary amine of PE form a Schiff base, allowing interaction via a covalent bond (Figure 30).

[0012] 2. Ligand Molecule (BBM) The ligand molecule of the present invention includes a compound having a ketone and a tertiary amine, and having a single bond or an alkylene group spacer between the ketone and the tertiary amine. The number of carbon atoms in the alkylene group spacer is not particularly limited, but is, for example, an integer of 1 to 10, preferably an integer of 1 to 4, and more preferably 2.

[0013] In one embodiment of the present invention, such compounds have, for example, the following formula I: It is shown by R １ represents an amino group, a hydroxyl group, or a carboxyl group, and R ２ represents a tertiary amine. Preferably, R １ is a carboxyl group, and R ２ is dimethylamine or diethylamine. m represents any integer, and n represents an integer of 0 to 10. m is preferably an integer of 0 to 10, more preferably an integer of 0 to 4, and even more preferably 2. Furthermore, n is preferably an integer of 0 to 4, and even more preferably 2.

[0014] In the present invention, the compound represented by the above formula (I) is preferably represented by the following formula II: The compound represented by formula II is 6-dimethylamino-4-ketohexanoic acid. The compound represented by formula II is also called "DMAX."

[0015] The ligand molecules of the present invention can be produced or obtained by any method known to those skilled in the art. The compound represented by formula I may be a synthetic product or a commercially available product.

[0016] In the present invention, to allow the ligand molecule to interact with the bacterial cell membrane, the bacteria may be suspended in an aqueous solution such as a phosphate buffer solution, and the ligand molecule may be added thereto, followed by stirring and mixing for 30 seconds or more.

[0017] 3. BBM-target substance complex The present invention provides a complex comprising the ligand molecule and a target substance to be delivered to the bacterial membrane.

[0018] The target substance to be delivered to the bacterial membrane is not particularly limited, and may be any type of drug delivery carrier, such as a drug, protein, antibody, low-molecular-weight compound, or polymeric micelle. Here, the target substance may be supported on a delivery carrier. "Supported" refers to a state in which the target substance is contained in a delivery carrier and / or a state in which the target substance is attached or bound to a delivery carrier by chemical means (e.g., chemical bonding) and / or physical means. Specific examples of chemical bonding include covalent bonding, metallic bonding, coordinate bonding, ionic bonding, hydrogen bonding, and intermolecular forces. Physical means may include any suitable immobilization method other than chemical means. Specific examples include adsorption, embedding, impregnation, etc. When a target substance is supported on a delivery carrier, the delivery carrier may be bound to a ligand molecule, rather than the target substance itself being bound to a ligand molecule. The type of delivery carrier is not limited, as long as it can bind to the ligand molecule without impairing its pharmacological activity.

[0019] Examples of delivery carriers include polymeric micelles, liposomes, and polyion complexes. Polymeric micelles refer to polymer aggregates formed by the self-association of block copolymers (diblock or multiblock copolymers each having one or more blocks with different physicochemical properties) composed of polymer chains with different physicochemical properties, such as hydrophilicity, hydrophobicity, and charge. Typically, they are particles with a core-shell structure formed by the autonomous multimolecular association of block copolymers in which hydrophilic and hydrophobic polymer chains are linked.

[0020] Liposomes are vesicles formed by two molecular membrane layers. The molecular membrane is usually a bilayer membrane made of phospholipids. Polyion complexes are particulate polymer aggregates formed by the autonomous multi-molecular association of a block copolymer in which a hydrophilic polymer chain is linked to an anionic polymer chain, and a block copolymer in which a hydrophilic polymer chain is linked to a cationic polymer chain (e.g., polyglutamic acid, polyaspartic acid, etc.), driven by electrostatic interactions.

[0021] In the present invention, the target substance to be carried by the delivery carrier is preferably a drug. In the present invention, the target substance or the delivery carrier carrying the target substance (hereinafter also referred to as "target substance, etc.") is preferably capable of binding to the ligand molecule of the present invention without impairing the pharmacological action. Furthermore, since the binding between the target substance, etc. and the ligand molecule preferably does not impair the pharmacological action, it is preferable that the functional group of the target substance, etc. is not involved in the pharmacological mechanism. The binding between the target substance and the ligand molecule is expected to improve the bacterial targeting ability. Furthermore, by binding the delivery carrier carrying the target substance to the BBM, further improvement of the bacterial targeting ability is expected.

[0022] The method for binding the target substance or the like to the ligand molecule (BBM) is not particularly limited. １) or a functional group added to the BBM (for example, a functional group of a linker compound described below) can be used to bond with a functional group of a target substance or the like. When a functional group is added to the BBM, for example, a BBM to which the functional group is added via a linker can be used. A BBM to which a functional group is added via a linker can be produced, for example, by reacting a linker compound having functional groups at both ends with the BBM and bonding one of the functional groups of the linker compound to the functional group of the BBM.

[0023] The method for binding a BBM to a delivery carrier carrying a target substance is not particularly limited, but includes adding a functional group to a portion of the delivery carrier (e.g., the end of a polymer constituting the delivery carrier) as needed, and binding the functional group to a functional group of the BBM. For example, the BBM can be bound to the end of a polymer constituting a polymer micelle, liposome, or polyion complex (e.g., the end of a diblock copolymer (e.g., a hydrophilic block) in the case of a polymer micelle), thereby binding the BBM to the delivery carrier carrying a target substance.

[0024] For example, if the target substance has an amino group (NH ２ ) or hydroxyl group (OH), it can be bonded to the carboxyl group (COOH) of the BBM. For example, when the BBM is 6-dimethylamino-4-ketohexanoic acid shown in Formula II, the amino group (NH ２ ) or the COOH of BBM can be directly bonded to the hydroxyl group (OH).

[0025] For example, if the target substance has a carboxyl group (COOH), the hydroxyl group (OH) or amino group (NH ２ For example, when the BBM is 6-dimethylamino-4-ketohexanoic acid shown in Formula II, the carboxyl group (COOH) of the target substance can be bound to an amino group (NH ２ By adding NH ) or hydroxyl group (OH), it is possible to bind target substances to BBM. ２A divalent amine (e.g., ethylenediamine) having the formula: １ ) to form a structure in which the target substance or the like is bound to the BBM via a linker.

[0026] In addition, in the present invention, a hydrophilic polymer can be attached to the ligand molecule to evaluate its bacterial recognition function, which makes it possible to control the pharmacokinetics of the target substance in the body, and is expected to achieve improved drug circulation in the blood, controlled drug release, and organ-selective drug delivery.

[0027] Examples of hydrophilic polymer compounds include polyethylene glycol, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, polyvinylacetamide, polyamine, poly(4-styrenesulfonic acid), poly(allylamine hydrochloride), poly(vinylsulfonic acid, sodium salt), poly(diallyldimethylammonium chloride), poly(2-methacryloyloxyethylphosphorylcholine), etc., which may be used alone or in combination of two or more. It is preferable to add functional groups to both ends or one end of the hydrophilic polymer compound.

[0028] In a preferred embodiment of the present invention, the hydrophilic polymer compound is polyethylene glycol (PEG). PEG may be linear or multi-branched. The number of branches in the multi-branched form is not particularly limited, and examples include bi-branched, tri-branched, tetra-branched, penta-branched, hexa-branched, hepta-branched, and octa-branched forms. PEG can also be a star polymer or a dendrimer. It is preferable to add functional groups to both ends or one end of PEG.

[0029] Examples of functional groups that can be added to hydrophilic polymer compounds (e.g., PEG) include, but are not limited to, thiol, acrylate, amine (amino group, secondary amine, tertiary amine), biotin, aldehyde, maleimide, succinimidyl carboxymethyl ester, succinimidyl glutarate ester, and the like.

[0030] The method for binding a hydrophilic polymer compound (e.g., PEG) to a ligand molecule (BBM) is not particularly limited. Similar to the method for binding the target substance or the like to the ligand molecule (BBM), the functional group (e.g., R １ ) or the functional group added to the BBM can be used to bind to a functional group of a target substance or the like (for example, the functional group of the linker compound described above).

[0031] It is also possible to bind both a target substance, etc. and a hydrophilic polymer compound to the ligand molecule (BBM). For example, by a method similar to that described above, a hydrophilic polymer compound can be bound to the ligand molecule (BBM) directly or via a linker, and a target substance, etc. can be further bound to the hydrophilic polymer compound directly or via a linker. In addition, any other compound can be bound to the ligand molecule (BBM). In addition to the target substance, etc. and / or the hydrophilic polymer compound, any other compound can be bound to the ligand molecule (BBM).

[0032] In some embodiments, the target substance is a drug. The type of drug is not particularly limited, and examples include antibacterial agents and antibiotics.

[0033] 4. Pharmaceutical Composition When a drug is used as the target substance in the conjugate of the present invention, it can be used in treatments (e.g., antibacterial therapy with antibiotics, diagnosis by identifying the focus of infection, etc.) by introducing a drug or antibody into bacteria that cause various bacterial diseases (e.g., infectious diseases, sepsis, etc.). Therefore, the present invention can also provide a pharmaceutical composition containing the conjugate, and a method for treating various diseases (e.g., infectious diseases) using the conjugate.

[0034] The pharmaceutical composition can be prepared by a conventional method using an appropriate selection of excipients, fillers, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizing agents, antiseptics, flavoring agents, soothing agents, stabilizers, isotonic agents, and the like that are commonly used in pharmaceutical manufacturing.

[0035] The pharmaceutical composition of the present invention can be administered by injection, including systemic administration via conventional routes such as intravenous or intraarterial routes, as well as local administration via intramuscular, intraarticular, subcutaneous, or intradermal routes. In this case, the composition is typically provided in a unit-dose ampule or multi-dose container, and may be in the form of a powder that can be reconstituted with a suitable carrier, such as sterile water, upon use. These dosage forms may also contain additives commonly used in pharmaceutical formulations. The dosage varies widely depending on the therapeutic purpose, age of the recipient, route of administration, and frequency of administration. The amount of active ingredient contained in the pharmaceutical composition of the present invention can be determined appropriately by those skilled in the art. For example, the effective dose of the pharmaceutical composition of the present invention, when administered in combination with an appropriate diluent and a pharmacologically acceptable carrier, is 160 mg to 460 mg per kg of body weight per administration, administered at intervals of one day to six weeks.

[0036] 5. Delivery Method The present invention provides a method for delivering a ligand molecule to bacteria, which comprises the steps of contacting a ligand molecule with bacteria and causing the ligand molecule to bind to the bacterial membrane. The present invention also provides a method for delivering a target substance to bacteria, which comprises the steps of contacting a complex of a ligand molecule and a target substance with bacteria and causing the target substance in the complex to bind to the bacterial membrane.

[0037] "Contact" refers to reacting the bacteria and the ligand molecule so that they electrostatically interact with each other and form a Schiff base, and examples of such contact include mixing a solution containing the bacteria with a solution containing the ligand molecule, or adding a solid ligand molecule or a complex containing a ligand molecule to a solution containing the bacteria. The reaction can be carried out by stirring at room temperature for 30 seconds or more, or by shaking at 37°C for 30 seconds or more.

[0038] 6. Kit for delivering a target substance to a bacterial membrane The kit of the present invention is characterized by including the ligand molecule. The kit can be preferably used for treating, for example, infectious diseases and sepsis.

[0039] In the kit of the present invention, the storage state of the complex is not limited, and a state such as a solution or powder can be selected taking into consideration its stability (storage) and ease of use. The kit of the present invention may contain other components in addition to the ligand molecule. Examples of other components include various buffers, a target substance to be introduced into cells via the bacterial cell surface, a lysis buffer, and instructions for use (instruction manual). The kit of the present invention is used to prepare a complex with a target substance to be introduced into target cells, and the prepared complex can be used as a target substance delivery device to the outer surface of target bacterial cells.

[0040] EXAMPLES The present invention will be explained in more detail below with reference to examples, although the scope of the present invention is not limited to these examples.

[0041] Synthesis of Cy5-8armPEG40k-Ligands: 6-dimethylamino-4-ketohexanoic acid (represented by Formula II) was used as the BBM. This BBM was commercially available (https: / / www.sigmaaldrich.com / JP / ja / product / aldrich / s936413). Using a model molecule with multiple BBMs exposed on the outer layer, an 8-branched polyethylene glycol (8armPEG) was prepared, with its termini modified with a fluorescent dye (Cy5) and BBM. The 8-branched polyethylene glycol allows multiple ligands and fluorescent molecules to be simultaneously introduced into a single molecule, and was used to evaluate the ability of BBM to deliver target substances.

[0042] 8-arm PEG-NH with a molecular weight of 40,000 (40 kDa) ２ (200 mg) was dissolved in DMF (10 ml), and then sulfo-Cy5-NHS (3.9 mg, 1 eq to 8 arm PEG40k-NH ２ After the reaction, the mixture was dialyzed in pure water and freeze-dried to obtain blue powder of Cy5-8armPEG40k-NH ２ obtained.

[0043] Cy5-8armPEG40k-NH ２(50 mg) was dissolved in DMF (2.5 ml), and BBM (9.2 mg, 5 eq to amines), EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, 34 mg, 5 eq to BBM), and NHS (N-Hydroxysuccinimide, 5 mg, 1 eq to BBM) were added. After stirring at 4°C for 17 hours, the mixture was dialyzed in pure water and lyophilized to obtain blue powdery Cy5-8armPEG40k-BBM. Note that temperatures in this specification are in degrees Celsius (°C).

[0044] I. Materials and Methods 1. Confirmation of Schiff Base Formation by FTIR The formation of a Schiff base between the bacterial recognition ligand BBM (6-dimethylamino-4-ketohexanoic acid hydrochloride) and the bacterial membrane model molecule PE (phosphoethanolamine) was verified using Fourier transform infrared spectroscopy (FT-IR). BBM (30 mg / ml) and PE (20 mg / ml, 1 eq. to BBM) were dissolved in pure water, shaken at 37°C for 1 hour, and then freeze-dried to obtain a powdered sample. The resulting sample was mixed with 100 mg of potassium bromide at a ratio of 1 mg to 1 mg to prepare tablets, which were then measured by FT-IR.

[0045] 2. Evaluation of Schiff base formation by NMR The formation of a Schiff base by BBM and PE was confirmed by NMR. １ H-NMR and １３ PE (20 mg / ml) and BBM (30 mg / ml) were dissolved in deuterated PBS (pH 7.4), and the mixture was shaken and stirred at 37°C for 1 hour. １ H-NMR and １３ As a negative control group, ketone (19 mg / ml), dimethylamine (22 mg / ml), or acetic acid (8.6 mg / ml) at the same molar concentration was mixed with PE (20 mg / ml), and the mixture was shaken and stirred at 37°C for 1 hour. １ H-NMR and １３Measurements were carried out by C-NMR. Here, "Ketone" refers to 5-oxohexanoic acid, and "Dimethylamine" refers to 3-(Dimethylamino)propionic acid hydrochloride. Each is composed of a partial structure of DMAX, with 5-oxohexanoic acid having a keto group of DMAX and 3-(Dimethylamino)propionic acid having a dimethylamine group, and therefore, in this specification, they are referred to as "Ketone" and "Dimethylmine," respectively.

[0046] 3. Synthesis of Cy5-8armPEG40k-Ligands Using a model molecule with multiple BBMs exposed on the outer layer, we prepared eight-branched polyethylene glycol (PEG) with terminals modified with a fluorescent dye and BBM. ２ (200 mg) was dissolved in DMF (10 ml), and then sulfo-Cy5-NHS (3.9 mg, 1 eq to 8 arm PEG40k-NH ２ After the reaction, the mixture was dialyzed in pure water and freeze-dried to obtain blue powder of Cy5-8armPEG40k-NH ２ obtained.

[0047] Cy5-8armPEG40k-NH ２ Cy5-8armPEG40k-BBM (50 mg) was dissolved in DMF (2.5 ml), and BBM (9.2 mg, 5 eq to amines), EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, 34 mg, 5 eq to BBM), and NHS (N-Hydroxysuccinimide, 5 mg, 1 eq to BBM) were added. After stirring at 4°C for 17 hours, the mixture was dialyzed in pure water and lyophilized to obtain blue powder Cy5-8armPEG40k-BBM.

[0048] Similarly, Cy5-8armPEG40k-NH ２(50 mg) was dissolved in DMF (2.5 ml), and ketone (5-oxohexanoic acid, 5.7 mg, 5 eq to amines), EDC (34 mg, 5 eq to ketone), and NHS (5 mg, 1 eq to ketone) were added. After stirring at 4°C for 17 hours, the mixture was dialyzed in pure water and lyophilized to obtain blue powdery Cy5-8armPEG-Ketone.

[0049] Similarly, Cy5-8armPEG40k-NH ２ Cy5-8armPEG-Dimethylamine (50 mg) was dissolved in DMF (2.5 ml), and dimethylamine (3-(dimethylamino)propionic acid, 6.7 mg, 5 eq. to amines), EDC (34 mg, 5 eq. to dimethylamine), and NHS (5 mg, 1 eq. to dimethylamine) were added. After stirring at 4°C for 17 hours, the mixture was dialyzed in pure water and lyophilized to obtain blue powder of Cy5-8armPEG-Dimethylamine.

[0050] Cy5-8armPEG40k-NH ２ (50 mg) was dissolved in DMF (2.5 ml), and triethylamine (13 μl, 11 eq to amines) and acetic anhydride (8.3 μl, 10 eq to amines) were added. After stirring at 4°C for 17 hours, the mixture was dialyzed in pure water and lyophilized to obtain blue powder Cy5-8armPEG-Ac. The obtained Cy5-8armPEG40k-Ligands were analyzed by nuclear magnetic resonance (NMR) spectroscopy. １ The introduction of Cy5 and various ligands was confirmed by H-NMR. Furthermore, the progress of the reaction was confirmed by quantifying the residual amines of the 8-arm PEG using fluorescamine. Additionally, the zeta potential of the resulting polymer was measured to evaluate the change in zeta potential due to the introduction of the ligands.

[0051] 4. Evaluation of Bacterial Recognition Ability by Cy5-8armPEG40k-BBM Escherichia coli or Staphylococcus aureus was cultured in LB medium. The diluted bacterial suspension (OD = 0.3, 50 μl) was centrifuged (5000 rpm, 5 min) to remove the medium, and a PBS solution of Cy5-8armPEG40k-Ligands (2 mg / ml, 50 μl) was added. After incubation at 37°C for 1 hour, the supernatant was removed by centrifugation (5000 rpm, 5 min). This washing procedure was repeated twice, after which the cells were resuspended in PBS, and the bacteria were fluorescently stained with Hoechst staining. The prepared samples were imaged using a confocal laser microscope to evaluate the bacterial recognition ability of BBM.

[0052] To evaluate the change in binding ability due to the multivalent effect of BBM, we prepared 8-armPEG40k-nBBMs (n = 7, 4, 2, 0) with different numbers of BBMs at the termini and the remaining termini modified with acetic acid (Ac). Similar experiments were performed using the resulting 8-armPEG40k-nBBMs. Subsequently, we confirmed the competitive inhibitory effect of the presence of free BBM, demonstrating that bacterial recognition was due to the BBM. Similar experiments were performed using 8-armPEG40k-BBM BBMs with 0 equivalent, 1 equivalent (0.074 mg / ml), and 2 equivalents (0.15 mg / ml) of free BBM added.

[0053] 5. Evaluation of bacterial recognition ability by Cy5-BBM To confirm that modifying a drug molecule with BBM improves the drug's bacterial targeting ability, Cy5-BBM was prepared as a drug-BBM binding molecule model by modifying Cy5 with BBM. BBM (1.4 mg, 5 eq to Cy5), EDC (5.2 mg, 5 eq to BBM), and NHS (0.76 mg, 1 eq to BBM) were added to a DMF solution (1 mg / ml) of sulfo-Cy5-amine (1 mg). After stirring at 4°C for 17 hours, the mixture was purified using a PD10 column to obtain Cy5-BBM. To prepare Cy5-Ac as a control molecule, triethylamine (2 μl, 11 eq. to amines) and acetic anhydride (1.3 μl, 10 eq. to amines) were added to a DMF solution (1 mg / ml) of sulfo-Cy5-amine (1 mg). After stirring at 4°C for 17 hours, the mixture was purified using a PD column to obtain Cy5-Ac. The resulting Cy5-BBM and Cy5-Ac were used in the same experiment as in 3.

[0054] 6. Synthesis of FITC-BBM To confirm that modifying a drug molecule with BBM improves the drug's bacterial targeting ability, FITC was modified with BBM via ethylenediamine (EDA) to prepare FITC-BBM as a drug-BBM binding molecular model. FITC (50 mg) was dissolved in THF (500 μl) and added dropwise to an excess of EDA (5 ml). The mixture was stirred at room temperature for 17 hours, and after the reaction, the mixture was added dropwise to diethyl ether. The precipitate was then collected by filtration to obtain FITC-EDA.

[0055] BBM (50 mg) was added to dichloromethane (5 ml), and oxalyl chloride (1 ml, 50 eq to BBM) was added dropwise while stirring. The mixture was stirred at room temperature for 17 hours, and after the reaction, dichloromethane and oxalyl chloride were removed under reduced pressure using a vacuum pump to obtain BBM-Cl. The obtained BBM-Cl was again dissolved in dichloromethane (5 ml, 10 mg / ml), and a BBM-Cl solution (1.9 ml, 2 eq to amines) was added to a THF solution (20 mg / ml) of FITC-EDA (20 mg). After stirring at room temperature for 17 hours, the mixture was purified using an LH20 column to obtain FITC-BBM. The reaction and purification results were as follows:１ This was confirmed by H-NMR.

[0056] 7. Evaluation of PE Recognition Ability of BBM Using Lipid-Coated Plates The PE targeting ability of BBM was quantified using glass well plates immobilized with bacterial membrane lipids. A chloroform solution of DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), the target lipid of BBM, and a chloroform solution of DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine), a component of the outer membrane of mammalian cells (5 mg / ml each), were mixed at DOPE:DOPC ratios of 1:0, 0.8:0.2, 0.6:0.4, 0.2:0.8, and 0:1, and 20 μl of the mixture was dropped into each well. After drying the well plate under reduced pressure, 20 μL of a PBS solution of Cy5-8armPEG40k-Ligands, FITC-BBM, and Fluorescein (2 mg / mL), and PBS were added. After standing at 37°C for 1 hour, each well was washed with PBS. 100 μL of PBS was added, and the fluorescence intensity of Cy5 or Fluorescein remaining in the well was measured using a plate reader.

[0057] 8. Evaluation of bacterial recognition ability by Cy5-8armPEG20k-BBM and FITC-BBM 8armPEG20k-NH ２ Cy5-8armPEG20k-Ligands were prepared using the same reaction described in "2. Evaluation of Schiff Base Formation by NMR." Escherichia coli or Staphylococcus aureus were cultured in LB medium, and the diluted bacterial suspension (OD = 0.3, 50 μl) was centrifuged (5000 rpm, 5 min) to remove the medium. A PBS solution of Cy5-8armPEG20k-Ligands and FITC-BBM (pH = 6, 7.4, 8, 2 mg / ml, 50 μl) was added. After incubation at 37°C for 1 hour, the supernatant was removed by centrifugation (5000 rpm, 5 min). This washing procedure was repeated twice, followed by resuspension in PBS, and the bacteria were fluorescently stained with Hoechst dye. The prepared samples were imaged using a confocal laser microscope to evaluate the bacterial recognition ability of the BBM.

[0058] 9. Quantification of Bacterial-Adsorbed Polymers To quantify drug delivery to bacteria via BBM, the amount of fluorescently labeled polymer adsorbed to bacteria was measured. Escherichia coli or Staphylococcus aureus was cultured in LB medium. The diluted bacterial suspension (OD = 0.3, 50 μl) was centrifuged (5000 rpm, 5 min) to remove the medium, and a PBS solution of Cy5-8armPEG20k-Ligands (pH = 6, 7.4, 8, 2 mg / ml, 50 μl) was added. After incubation at 37°C for 1 hour, the supernatant was removed by centrifugation (5000 rpm, 5 min). This washing procedure was repeated twice, followed by resuspension in PBS, and the bacteria were fluorescently stained with Hoechst staining. The prepared samples were treated with a PBS solution of PFA to fix the bacteria, and the fluorescence intensity from the remaining polymer was measured using a plate reader. In addition, a similar experiment was performed under conditions in which 1 equivalent of free BBM or PE was added to the BBM of 8armPEG20k-BBM, confirming that bacterial adsorption by the polymer was a reaction mediated by BBM and PE.

[0059] 10. Preparation of Gem-BBM To verify the bacterial targeting ability of drug-BBM when BBM is conjugated to a drug molecule, the antibiotic gemifloxacin mesylate (Gem) was modified with BBM. BBM (50 mg) was added to dichloromethane (5 ml), and oxalyl chloride (1 ml, 50 eq to BBM) was added dropwise while stirring. The mixture was stirred at room temperature for 17 hours. After the reaction, the dichloromethane and oxalyl chloride were removed under reduced pressure using a vacuum pump to obtain BBM-Cl. The resulting BBM-Cl was dissolved again in dichloromethane (5 ml, 10 mg / ml), and a dichloromethane solution of BBM-Cl (1.9 ml, 2 eq to amines) was added to a THF solution (20 mg / ml) of Gem (20 mg). After the reaction, the precipitate was collected by filtration and washed with dichloromethane to obtain Gem-BBM. １ The results of the reaction and purification were confirmed by H-NMR and FT-IR.

[0060] 11. Evaluation of the antibacterial activity of Gem-BBM To verify the bacterial targeting ability and antibacterial activity of Gem-BBM, antibacterial tests were conducted under washed and unwashed conditions. Escherichia coli or Staphylococcus aureus were cultured in LB medium, and Gem or Gem-BBM (0.5 mg / ml) was added to the diluted bacterial suspension (OD = 0.01, 600 μl). After co-culture for 1 hour, non-adsorbed free drug was removed by centrifugation (5000 rpm, 5 min), and the sample was resuspended in 600 μl of LB medium. A sample without washing was also prepared as a positive control. Bacterial growth after drug addition was monitored by OD measurement, and the bacterial suspension 6 hours after drug addition was analyzed by 10 ８ After diluting twice, 100 μl of the solution was applied to an LB agar medium, and the number of colonies after 17 hours of culture was measured to obtain the CFU value, from which the number of viable bacteria was calculated.

[0061] 12. Evaluation of Gem-BBM Cytotoxicity Using the CCK-8 Test In this section, we evaluated the toxicity of Gem-BBM to mammalian cells to verify potential side effects during treatment. Different concentrations of Gem-BBM or Gem were added to multiple mammalian cell lines, including cancer cells (HEK293, RAW264.3, DC2.4, HT29, U87, 4T1, B16F10), and after 1 hour of incubation, the number of viable cells was measured using the CCK-8 test.

[0062] II. Results The results are shown in Figures 1 to 28 and 31. Figure 1 shows the results of FT-IR measurement of a sample of BBM and PE mixed together. By mixing BBM and PE, a peak was observed at 1684 ( / cm) that was not observed when either BBM or PE was mixed together. This peak was due to a carbon-nitrogen double bond, indicating that a new covalent bond was generated when BBM and PE formed a base.

[0063] Figure 2 shows the results of mixing a combination of PE and BBM, or a combination of PE and BBM with a control ligand. １３This figure shows the results of C NMR measurements. By mixing BBM and PE, it was confirmed that the ketone-derived peak observed with BBM alone disappeared. On the other hand, this reaction was not observed with the control ligand having a partial structure of BBM. This shows that BBM binds to PE via a Schiff base due to the action of both the ketone and dimethylamine.

[0064] FIG. 3 shows the results of mixing a combination of PE and BBM, or a combination of PE and BBM with a control ligand. １ This figure shows the results of H NMR measurements. It was confirmed that by mixing BBM and PE, a new peak appeared that was not seen with BBM alone. On the other hand, this reaction was not observed with the control ligand having a partial structure of BBM. This indicates that BBM binds to PE via a Schiff base through the action of both ketone and dimethylamine.

[0065] FIG. 4 is a diagram showing the synthesis results of Cy5-8armPEG40k-Ligands; １ The results of measurements of 1 H NMR, fluorescamine, and zeta potential showed that the fluorescent dye Cy5 and various ligand molecules were introduced at the termini of the 8-branched PEG.

[0066] Figure 5 shows fluorescence microscopy images of Cy5-8armPEG40k-Ligands administered to Hoechst-stained E. coli. The molecules containing BBM as a ligand colocalized with the bacteria, whereas the other control ligands either did not detect Cy5-derived fluorescence or had lower fluorescence intensity than BBM. This indicates that BBM exerts its bacterial recognition ability through the action of both ketone and dimethylamine.

[0067] Figure 6 shows fluorescence microscopy images of Cy5-8armPEG40k-Ligands administered to Hoechst-stained Staphylococcus aureus. While molecules incorporating BBM as a ligand colocalized with the bacteria, other control ligands either did not detect Cy5-derived fluorescence or showed lower fluorescence intensity than BBM. This indicates that BBM exerts its bacterial recognition ability through the actions of both ketone and dimethylamine.

[0068] Figure 7 shows fluorescence microscopy images of Cy5-8armPEG40k-BBM with different BBM incorporation ratios administered to Hoechst-stained E. coli. The results confirmed that the bacterial recognition ability varied depending on the number of BBMs introduced at the polymer termini, demonstrating the effectiveness of the multivalent effect of BBM in bacterial recognition.

[0069] Figure 8 shows fluorescence microscopy images of Cy5-8armPEG40k-BBM with different BBM incorporation ratios administered to Hoechst-stained Staphylococcus aureus. The results confirmed that the bacterial recognition ability varied depending on the number of BBMs introduced at the polymer termini, demonstrating the effectiveness of the multivalent effect of BBM in bacterial recognition.

[0070] Figure 9 shows fluorescence microscopy images of free BBM and Cy5-8armPEG40k-Ligands administered to E. coli stained with Hoechst staining. It was confirmed that the colocalization rate with bacteria was lower in the presence of free BBM than in the absence of free BBM. This indicates that bacterial recognition by the polymer is due to the BBM mechanism.

[0071] Figure 10 shows fluorescence microscopy images of free BBM and Cy5-8armPEG40k-Ligands administered to Hoechst-stained Staphylococcus aureus. The presence of free BBM confirmed a lower colocalization rate with the bacteria than the absence of free BBM. This indicates that the recognition of bacteria by the polymer is due to the BBM.

[0072] Figure 11 shows a fluorescence microscopy image of the small molecule Cy5-BBM, in which BBM is directly conjugated to the fluorescent dye Cy5, administered to Staphylococcus aureus stained with Hoechst staining. The molecule with BBM as a ligand colocalized with the bacteria. On the other hand, no fluorescence from Cy5 was detected in the control Ac. This indicates that BBM has the ability to recognize bacteria even in the absence of a multivalent effect.

[0073] FIG. 12 shows the synthesis results of the fluorescent dye FITC bound to BBM via ethylenediamine. １ 1 H NMR showed that

[0074] Figure 13 shows the residual fluorescence intensity after washing with FITC-BBM, unmodified fluorescein (control), and PBS added to well plates whose bottoms were coated with lipids containing different PE / PC mixtures. When the results were normalized to the condition without PE in the lipid coating, FITC-BBM exhibited a PE concentration-dependent transition in fluorescence intensity, while the other controls were independent of PE concentration. This indicates that BBM actually recognizes PE.

[0075] Figure 14 shows the residual fluorescence intensity after adding Cy5-8armPEG20k-Ligands to well plates whose bottoms were coated with lipids at different PE / PC mixture ratios and washing. Under the same conditions, BBM showed the highest fluorescence intensity compared to the control group consisting of only the BBM partial structure, demonstrating the bacterial recognition ability due to the synergistic action of both ketone and dimethylamine.

[0076] Figure 15 shows fluorescence microscopy images of Cy5-8armPEG20k-Ligands administered to Hoechst-stained E. coli at pH 6. The molecules containing BBM as a ligand colocalized with the bacteria. In contrast, the other control ligands either did not detect Cy5-derived fluorescence or had lower fluorescence intensity than BBM. This indicates that BBM exerts its bacterial recognition ability through the action of both ketone and dimethylamine.

[0077] Figure 16 shows fluorescence microscopy images of Cy5-8armPEG20k-Ligands administered to Hoechst-stained E. coli at pH 7.4. The molecules containing BBM as a ligand colocalized with the bacteria. In contrast, the other control ligands either did not detect Cy5-derived fluorescence or had lower fluorescence intensity than BBM. This indicates that BBM exerts its bacterial recognition ability through the action of both ketone and dimethylamine.

[0078] Figure 17 shows fluorescence microscopy images of Cy5-8armPEG20k-Ligands administered to Hoechst-stained E. coli at pH 8. The molecules containing BBM as a ligand colocalized with the bacteria. In contrast, the other control ligands either did not detect Cy5-derived fluorescence or had lower fluorescence intensity than BBM. This indicates that BBM exerts its bacterial recognition ability through the action of both ketone and dimethylamine.

[0079] Figure 18 shows fluorescence microscopy images of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Staphylococcus aureus at pH 6. The molecules containing BBM as a ligand colocalized with the bacteria. In contrast, the other control ligands either did not detect Cy5-derived fluorescence or had lower fluorescence intensity than BBM. This indicates that BBM exerts its bacterial recognition ability through the action of both ketone and dimethylamine.

[0080] Figure 19 shows fluorescence microscopy images of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Staphylococcus aureus at pH 7.4. The molecules containing BBM as a ligand colocalized with the bacteria. In contrast, the other control ligands either did not detect Cy5-derived fluorescence or had lower fluorescence intensity than BBM. This indicates that BBM exerts its bacterial recognition ability through the action of both ketone and dimethylamine.

[0081] Figure 20 shows fluorescence microscopy images of Cy5-8armPEG20k-Ligands administered to Hoechst-stained Staphylococcus aureus at pH 8. The molecules containing BBM as a ligand colocalized with the bacteria. In contrast, the other control ligands either did not detect Cy5-derived fluorescence or exhibited lower fluorescence intensity than BBM. This indicates that BBM exerts its bacterial recognition ability through the action of both ketone and dimethylamine.

[0082] Figure 21 shows fluorescence microscopy images of the small molecule FITC-BBM, in which BBM is conjugated to the fluorescent dye FITC via EDA, administered to Escherichia coli or Staphylococcus aureus stained with Hoechst dye at pH 6. The molecules containing BBM as a ligand colocalized with the bacteria. On the other hand, the control fluorescein showed either no or low fluorescence intensity from Cy5. This indicates that BBM has the ability to recognize bacteria even in the absence of a multivalent effect.

[0083] Figure 22 shows fluorescence microscopy images of the small molecule FITC-BBM, in which BBM is conjugated to the fluorescent dye FITC via EDA, administered to Escherichia coli or Staphylococcus aureus stained with Hoechst dye at pH 7.4. The molecules containing BBM as a ligand colocalized with the bacteria. On the other hand, the control fluorescein showed either no or low fluorescence intensity from Cy5. This indicates that BBM has the ability to recognize bacteria even in the absence of a multivalent effect.

[0084] Figure 23 shows fluorescence microscopy images of the small molecule FITC-BBM, in which BBM is conjugated to the fluorescent dye FITC via EDA, administered to Escherichia coli or Staphylococcus aureus stained with Hoechst dye at pH 8. The molecules containing BBM as a ligand colocalized with the bacteria. On the other hand, the control fluorescein showed either no or low fluorescence intensity from Cy5. This indicates that BBM has the ability to recognize bacteria even in the absence of a multivalent effect.

[0085] Figure 24 shows the residual Cy5 fluorescence intensity after administering Cy5-8armPEG20k-Ligands to Escherichia coli or Staphylococcus aureus at pH 6, 7.4, or 8 and washing away the unadsorbed free polymer. The BBM-introduced molecules exhibited higher residual fluorescence intensity than the other control ligand-introduced polymers, demonstrating their high bacterial adsorption ability. Furthermore, the BBM polymer exhibited lower fluorescence intensity in the presence of free BBM or PE, indicating that bacterial recognition by the polymer is due to the interaction between BBM and PE.

[0086] FIG. 25 shows the results of binding BBM to the antibiotic gemifloxacin. １ The results of H NMR and FT-IR measurements showed that BBM was introduced into gemifloxacin.

[0087] Figure 26 shows bacterial growth when Gem-BBM, which is gemifloxacin bound to BBM, was administered to E. coli and the unadsorbed free drug was removed after 1 hour of co-culture. As a control, no difference in efficacy was observed between Gem-BBM and unmodified Gem when the free drug was not removed. However, when the free drug was removed, E. coli cells in the unmodified Gem administration condition grew. Meanwhile, OD and CFU measurements indicated that Gem-BBM inhibited growth.

[0088] 27 shows bacterial growth when Gem-BBM, which is gemifloxacin bound to BBM, was administered to Staphylococcus aureus, and the unadsorbed free drug was removed after 1 hour of co-culture. As a control, no difference in efficacy was observed between Gem-BBM and unmodified Gem when the free drug was not removed. However, when the free drug was removed, OD and CFU measurements showed that S. aureus in the unmodified Gem-administered condition showed more active bacterial growth than Gem-BBM.

[0089] Figure 28 shows the results of measuring cell viability when Gem-BBM was administered to mammalian cells, including cancer cells and healthy cells, to verify side effects of Gem-BBM. The results show that at the concentration used in the bactericidal test, approximately 80% of healthy cells survived, demonstrating safety of the treatment. Meanwhile, the survival rate of cancer cells was approximately 60% or higher.

[0090] 31 shows the results of confirming Schiff base formation by FT-IR. FT-IR showed that BBM forms a Schiff base with PE (left panel). In contrast, when a similar experiment was performed using the control ligand, Ketone, no peak indicating Schiff base formation was observed (right panel).

[0091] In Example 2, the efficacy of gemifloxacin (Example 1) and DMAX (BBM) bound to vancomycin (Vcm) was evaluated.

[0092] (1) Preparation of Vcm-DMAX Conjugate A scheme illustrating the preparation of the Vcm-DMAX conjugate is shown in Figure 32. To modify Vcm with DMAX, DMAX was activated with NHS ester, and unreacted DIPC was inactivated with mercaptoethanol. Vcm-DMAX was obtained by mixing this reaction solution with Vcm and then purified by dialysis.

[0093] (2) Vcm-DMAX １ H-NMR measurement Vcm-DMAX １ The results of H-NMR measurement are shown in FIG. １ The H-NMR measurement results showed that 70% of Vcm was modified with DMAX.

[0094] (3) Evaluation of bacterial growth. Similar to Gem-DMAX, bacterial growth was evaluated under post-dose washing conditions. Vcm-DMAX was administered at various concentrations to a medium containing Staphylococcus aureus (OD = 0.1, LB medium). After co-cultivation for 1 hour, the samples were washed twice by centrifugation (5000 rpm, 5 min), and the OD values ​​were measured after resuspension in LB medium. Furthermore, the bacterial suspension was plated on an agar medium after 6 hours of incubation to measure CFU.

[0095] Figure 34 shows the growth curves (OD) and CFU of each bacterium. The OD and CFU results confirmed the improved efficacy of DMAX modification, especially at concentrations of 50 μg / ml or higher. These results demonstrate that, similar to Gem-DMAX, the bacterial binding ability of DMAX allowed the drug to remain in the culture system even after washing, thereby exerting its efficacy. On the other hand, unmodified Vcm was removed by washing, resulting in its loss of efficacy.

Claims

1. A ligand molecule for bacterial membranes, comprising a compound having a ketone and a tertiary amine with a spacer of a single bond or an alkylene group between the ketone and the tertiary amine.

2. The compound has the following formula I: [In the formula, R １ represents an amino group, a hydroxyl group, or a carboxyl group, and R ２ represents a tertiary amine, m represents an integer, and n represents an integer of 0 to 10. The ligand molecule according to claim 1, 3. The compound of formula I is a compound of formula II:

3. The ligand molecule of claim 2, which is 6-dimethylamino-4-ketohexanoic acid represented by the formula:

4. A complex comprising the ligand molecule according to any one of claims 1 to 3 and a target substance to be delivered to the bacterial membrane.

5. The complex according to claim 4, wherein the target substance is a drug.

6. The complex of claim 5, wherein the drug is carried on a delivery carrier.

7. A pharmaceutical composition comprising the complex of claim 5.

8. A method for delivering a ligand molecule to bacteria, comprising the step of contacting the ligand molecule according to any one of claims 1 to 3 with bacteria and binding the ligand molecule to the bacterial membrane.

9. A method for delivering a target substance in the complex to bacteria by contacting the complex according to claim 4 with the bacteria.

10. A method for delivering a drug in the complex to bacteria, comprising contacting the complex according to claim 5 with bacteria.

11. A kit for delivering a target substance to a bacterial membrane, comprising the ligand molecule according to any one of claims 1 to 3.

Images