Ciprofloxacin nano prodrug as well as preparation method and application thereof

Abstract

The invention relates to the field of biomedicine, particularly provides a ciprofloxacin nano prodrug as well as a preparation method and application thereof, and aims to solve the problem that fusobacterium nucleatum infection in macrophages is difficult to effectively remove. For the purpose, the preparation method of the ciprofloxacin nano prodrug comprises the following steps: reacting 3-maleimidopropionic acid hydroxyl succinimide ester with amino-polyethylene glycol-carboxyl to obtain an intermediate 1; carrying out amidation reaction on the intermediate 1 and polypeptide FRRG to obtain an intermediate 2; carrying out amidation reaction on the intermediate 2 and ciprofloxacin to obtain an intermediate 3; the intermediate 3 and polypeptide IL4Rpep-1 are subjected to a thiol-ene click reaction, and the ciprofloxacin prodrug molecule is obtained; ciprofloxacin prodrug molecules are assembled into the ciprofloxacin nano prodrug through a hydrophilic-hydrophobic effect. The preparation method is simple and convenient, low in cost, high in product purity and suitable for large-scale production. The prepared ciprofloxacin nano prodrug has good biocompatibility, can effectively kill fusobacterium nucleatum in macrophages through a cascade approach of targeted uptake and response release, and is expected to provide an effective strategy for treatment of colorectal cancer related to fusobacterium nucleatum in cells.

Description

Technical Field

[0001] The present invention relates to the biomedical field, and specifically provides a ciprofloxacin nano prodrug and a preparation method and application thereof. Background Art

[0002] Various human tumor tissues harbor microbiota of varying abundance and diversity, primarily residing within cancer cells and immune cells. The carcinogenic Fusobacterium nucleatum has been shown to be a facultative intracellular bacterium, capable of surviving for days within macrophages. Studies have shown that intracellular forms of Fusobacterium nucleatum play a crucial role in the formation of an immunosuppressive microenvironment and in the development and metastasis of colorectal cancer. However, previous therapeutic strategies to enhance antitumor responses through microbiota manipulation have focused solely on extracellular bacteria, overlooking the potential for intracellular parasitic forms. Because antibiotics have difficulty freely penetrating cells, strategies to deliver them intracellularly via drug carriers offer a promising approach to enhance their efficacy. While these strategies effectively enhance macrophage penetration and retention, drug release is often nonselective, potentially associated with toxic side effects on normal cells. Furthermore, intracellular bacteria are often metabolically quiescent, whereas most antibiotics are only effective against metabolically active bacteria. Consequently, the application of antibiotics, a typical antimicrobial therapy, in manipulating the gut microbiota is limited.

[0003] Due to its intracellular parasitic characteristics, intracellular Fusobacterium nucleatum is difficult to be effectively killed by antimicrobial agents with poor membrane permeability. Even antimicrobial agents that can enter cells find it difficult to specifically identify infected cells and exert selective inhibitory effects on intracellular Fusobacterium nucleatum. Especially for intestinal application scenarios where probiotics and pathogenic bacteria coexist, the targeting of intracellular Fusobacterium nucleatum is an important factor affecting the therapeutic effect of intracellular Fusobacterium nucleatum infection associated with colorectal cancer. Therefore, how to accurately target infected host cells in the area of ​​colorectal cancer and achieve efficient internalization, and then specifically detect and eliminate colorectal cancer-related intracellular Fusobacterium nucleatum without causing toxicity to the host, may be a key issue that needs to be urgently addressed in the treatment of colorectal cancer infected with intracellular Fusobacterium nucleatum. Summary of the Invention

[0001] Given the important role of intracellular Fusobacterium nucleatum in the development and progression of colorectal cancer, this patent designs a ciprofloxacin prodrug molecule (IL4Rpep-1-FRRG-CIP) that can achieve infection-specific intracellular delivery. The prodrug molecule is composed of the M2 macrophage targeting peptide IL4Rpep-1, the cathepsin B-cleavable linker FRRG, and ciprofloxacin. IL4Rpep-1-FRRG-CIP can self-assemble into nanoparticles MTCP-NPs. MTCP-NPs use IL4Rpep-1 to target and enter IL4-overexpressing Fusobacterium nucleatum-infected macrophages, and release ciprofloxacin on demand under the specific cleavage of intracellular overexpressed cathepsin B, thereby efficiently killing Fusobacterium nucleatum in macrophages. This novel ciprofloxacin prodrug molecule is expected to provide a new and effective strategy for the treatment of colorectal cancer by specifically eliminating tumor-associated intracellular Fusobacterium nucleatum.

[0002] In order to solve the above problems, in a first aspect, the present invention provides a method for preparing a ciprofloxacin nano-prodrug, comprising the following steps:

[0003] 3-Maleimidopropionic acid hydroxysuccinimide ester reacts with amino-polyethylene glycol-carboxyl under amidation reaction conditions to obtain intermediate 1;

[0004] The intermediate 1 is reacted with a cathepsin B-cleavable peptide linker FRRG under amidation reaction conditions to obtain an intermediate 2;

[0005] The intermediate 2 is reacted with ciprofloxacin under amidation reaction conditions to obtain intermediate 3;

[0006] The intermediate 3 is reacted with the M2 macrophage targeting peptide IL4Rpep-1 under thiol-ene click reaction conditions to obtain a ciprofloxacin prodrug molecule;

[0007] The ciprofloxacin prodrug molecules are assembled through hydrophilic-hydrophobic interactions to obtain ciprofloxacin nanoprodrugs.

[0008] In the technical solution of the preparation method of the above-mentioned ciprofloxacin nano-prodrug, the synthesis of intermediate 1 specifically includes the following steps:

[0009] The amino-tetraethylene glycol-carboxyl group is completely dissolved in dichloromethane to form a first solution; the maleimide active ester is dissolved in dichloromethane to form a second solution; the obtained first solution is slowly added dropwise to the second solution, and N,N-diisopropylethylamine is added, and the reaction is stirred at a constant temperature of 25°C for 16 hours; the reaction progress is monitored by thin layer chromatography, and a dichloromethane / methanol mixed solvent with a volume ratio of 10:1 is used as a developing solvent; after the reaction is completed, the dichloromethane is removed using a rotary evaporator at 35°C and 0.1 MPa; the resulting product is purified by silica gel column chromatography to collect the target components; the collected components are concentrated and dried to obtain intermediate 1, which is maleimide carboxyl functionalized polyethylene glycol;

[0010] The molar ratio of amino-tetraethylene glycol-carboxyl to maleimide active ester is 1:1.0-1.05; the amount of N,N-diisopropylethylamine added is 3.5-4 times the molar amount of amino-tetraethylene glycol-carboxyl.

[0011] In the technical solution of the preparation method of the above-mentioned ciprofloxacin nano-prodrug, the synthesis of intermediate 2 specifically includes the following steps:

[0012] The intermediate 1, carbodiimide hydrochloride, and N-hydroxysuccinimide were dissolved in anhydrous dimethylformamide and reacted at a constant temperature of 37°C for 12 hours to obtain activated product 1; the cathepsin B-cleavable peptide linker FRRG was dissolved in 500 μL dimethylformamide, and N,N-diisopropylethylamine was added to form a reaction solution; the resulting reaction solution was slowly added dropwise to the activated product 1, and the reaction was continued at 37°C for 12 hours; after the reaction, the dimethylformamide was removed using a rotary evaporator at 40°C and 0.1 MPa; the resulting product was dissolved in an appropriate amount of dichloromethane and purified by silica gel column chromatography to collect the target fraction; the collected fraction was concentrated and dried to obtain a white powdery intermediate 2;

[0013] The molar ratio of carbodiimide hydrochloride to N-hydroxysuccinimide is 1:1.0-1.05; and the added amount of N,N-diisopropylethylamine is 3.5-4 times the molar amount of the cathepsin B-cleavable peptide linker FRRG.

[0014] In the technical solution of the preparation method of the above-mentioned ciprofloxacin nano-prodrug, the synthesis of intermediate 3 specifically includes the following steps:

[0015] Intermediate 2, carbodiimide hydrochloride, and N-hydroxysuccinimide were dissolved in anhydrous dimethyl sulfoxide and reacted at 4°C for 1 h under nitrogen protection, then the temperature was raised to 37°C and the reaction was continued for 12 h to obtain activated product 2; ciprofloxacin hydrochloride was dissolved in dimethyl sulfoxide, and N,N-diisopropylethylamine was added to form a reaction solution; the resulting reaction solution was slowly added dropwise to activated product 2, and the reaction was continued at 37°C for 12 h; the resulting reaction solution was transferred to a dialysis bag and dialyzed using dimethyl sulfoxide as the dialyzate for 3 days; the dialyzed solution was freeze-dried to obtain yellow powdered intermediate 3;

[0016] The molar ratio of carbodiimide hydrochloride to N-hydroxysuccinimide is 1:1.0-1.05; the amount of N,N-diisopropylethylamine added is 3.5-4 times the molar amount of ciprofloxacin hydrochloride; the dialysis process is preferably carried out at 25° C., and the molecular weight cut-off of the dialysis bag is 500 Da.

[0017] In the technical solution of the above-mentioned method for preparing ciprofloxacin nano-prodrug, the synthesis of ciprofloxacin prodrug molecules specifically includes the following steps:

[0018] The intermediate 3 and the polypeptide IL4Rpep-1 were dissolved in water, and a reducing agent, tri(2-formylethyl)phosphine hydrochloride, was added, and the reaction was carried out at a constant temperature of 25°C for 12 hours to obtain a reaction solution. The obtained reaction solution was transferred to a dialysis bag and dialyzed against a pH 7.4 phosphate buffer for 3 days. The dialyzed solution was freeze-dried to obtain a white powdery ciprofloxacin prodrug molecule.

[0019] The amount of the reducing agent added is 1.2-1.5 times the molar amount of the intermediate; the dialysis process is preferably carried out at 4° C., and the molecular weight cut-off of the dialysis bag is 500 Da.

[0020] In the technical solution of the above-mentioned method for preparing the ciprofloxacin nano-prodrug, the preparation of the ciprofloxacin nano-prodrug specifically comprises the following steps:

[0021] Ciprofloxacin prodrug molecules are dissolved in 0.5 mL of dimethyl sulfoxide to form a first solution; the resulting first solution is added dropwise to 2.5 mL of deionized water under ultrasonic conditions; after the addition is complete, the mixed solution is subjected to ultrasonic treatment for 30 minutes to allow the ciprofloxacin prodrug molecules to self-assemble into nanostructures through hydrophilic-hydrophobic interactions; the resulting solution is placed in a dialysis bag and dialyzed with deionized water to remove the dimethyl sulfoxide, thereby obtaining ciprofloxacin nanoprodrugs with uniform particle size distribution;

[0022] The ultrasonic treatment is preferably carried out at a frequency of 40 kHz and a power of 100 W.

[0023] In a second aspect, the present invention provides a ciprofloxacin nano-prodrug, which is prepared by any one of the methods for preparing the ciprofloxacin nano-prodrug.

[0024] In a third aspect, the present invention also provides a use of the above-mentioned ciprofloxacin nanoprodrug for specifically recognizing and killing Fusobacterium nucleatum in macrophages.

[0025] The beneficial effects of the present invention are as follows:

[0026] The ciprofloxacin nano-prodrug provided by the present invention has a simple preparation process, mild preparation conditions, low cost, high product purity, and is suitable for large-scale production.

[0027] The ciprofloxacin nanoprodrug has good biocompatibility and can specifically recognize and target macrophages through the surface-modified IL-4 receptor ligand polypeptide, significantly increasing the drug's accumulation concentration in the target cells. It specifically releases active ciprofloxacin only under the action of macrophage lysosomal cathepsin B, effectively reducing toxic side effects caused by nonspecific release. It has excellent clearance efficiency for Fusobacterium nucleatum in macrophages and is not prone to inducing bacterial resistance.

[0028] The ciprofloxacin nanoprodrug can effectively eliminate Fusobacterium nucleatum in macrophages and is expected to become a new therapeutic strategy for treating colorectal cancer caused by Fusobacterium nucleatum infection in macrophages. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The preferred embodiments of the present invention are described below with reference to the accompanying drawings, in which:

[0030] Figure 1 is the H NMR spectrum of intermediate 1 in Example 1;

[0031] Figure 2 is the H NMR spectrum of intermediate 2 in Example 2;

[0032] Figure 3 is the H NMR spectrum of intermediate 3 in Example 3;

[0033] Figure 4 is the H NMR spectrum of IL4Rpep-1-FRRG-CIP in Example 4;

[0034] Figure 5 is a transmission electron microscopy image of MTCP-NPs in Example 5;

[0035] Figure 6 is the CIP release curve in Example 6;

[0036] Figure 7 is a quantitative data graph of killing free Fusobacterium nucleatum in Example 6;

[0037] Figure 8 This is a flow cytometry quantitative data diagram of MTCP-NPs entering macrophages in Example 6;

[0038] Figure 9 is a quantitative data graph of killing Fusobacterium nucleatum in macrophages in Example 6;

[0039] Figure 10 The present invention is a flow chart of a method for preparing a ciprofloxacin nano-prodrug according to an embodiment. DETAILED DESCRIPTION

[0040] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0041] The present invention provides a method for preparing a ciprofloxacin (CIP) nanoprodrug, comprising the following steps:

[0042] 3-Maleimidopropionic acid hydroxysuccinimide ester was reacted with amino-polyethylene glycol-carboxyl group under amidation reaction conditions to obtain intermediate 1 (Ma1-PEG4-COOH).

[0043] The Mal-PEG4-COOH was reacted with a cathepsin B (CTSB) cleavable peptide linker FRRG under amidation reaction conditions to obtain intermediate 2 (Mal-FRRG-COOH).

[0044] The Mal-FRRG-COOH is reacted with CIP under amidation reaction conditions to obtain intermediate 3 (Mal-FRRG-CIP).

[0045] The Mal-FRRG-CIP is reacted with the M2 macrophage targeting peptide IL4Rpep-1 under thiol-ene click reaction conditions to obtain a CIP prodrug molecule (IL4Rpep-FRRG-CIP).

[0046] The IL4Rpep-FRRG-CIP was assembled through hydrophilic-hydrophobic interactions to obtain CIP nanoprodrugs (MTCP-NPs).

[0047] In Example 1, 3-maleimidopropionic acid hydroxysuccinimide ester is reacted with amino-polyethylene glycol-carboxyl under amidation reaction conditions to obtain Mal-PEG4-COOH, which specifically includes the following steps:

[0048] To a dry round-bottom flask, add maleimide active ester (25 mg, 94 μmol), anhydrous DCM (2 mL), and N,N-diisopropylethylamine (65 μL, 376 μmol). Dissolve amino-tetraethylene glycol-carboxyl (30 μL, 94 μmol) in DCM and add dropwise to the maleimide active ester solution after stirring. Stir at 25°C for 16 h. Analyze by thin-layer chromatography (DCM:MeOH = 9:1). After rotary evaporation of the DCM, purify the product by silica gel column chromatography (DCM:MeOH = 9:1) to obtain Mal-PEG4-COOH as a colorless oil.

[0049] The molar ratio of the amino-tetraethylene glycol-carboxyl group to the maleimide active ester is 1:1.0-1.05; and the added amount of the N,N-diisopropylethylamine is 3.5-4 times the molar amount of the amino-tetraethylene glycol-carboxyl group.

[0050] like Figure 1 As shown, H NMR spectrum data indicated the successful synthesis of Mal-PEG4-COOH.

[0051] Example 2:

[0052] This example takes the reaction of Mal-PEG4-COOH with the CTSB cleavable peptide linker FRRG under amidation reaction conditions to obtain Mal-FRRG-COOH as an example, and includes the following steps:

[0053] To a dry round-bottom three-necked flask, Mal-PEG4-COOH (30 mg, 72 μmol), carbodiimide hydrochloride (13.8 mg, 108 μmol), and N-hydroxysuccinimide (12.44 mg, 108 μmol) were added sequentially. The flask was pumped with an oil pump for 10 min to remove water vapor. 1 mL of anhydrous N,N-dimethylformamide (DMF) was added and the mixture was reacted at 4°C for 1 h under nitrogen, then at 37°C for 12 h to obtain activated product 1. FRRG (50.07 mg, 93.6 μmol) was quickly weighed and dissolved in 500 μL of DMF. 15 μL of N,N-diisopropylethylamine was then added and slowly added dropwise to the three-necked flask using a syringe. The reaction was continued for 12 h. After the reaction, DMF was removed by rotary evaporation, and the residue was dissolved with a small amount of DCM and purified by silica gel column chromatography (DCM:MeOH=8:2) to obtain white powder Mal-FRRG-COOH.

[0054] The molar ratio of the carbodiimide hydrochloride to N-hydroxysuccinimide is 1:1.0-1.05; and the added amount of the N,N-diisopropylethylamine is 3.5-4 times the molar amount of FRRG.

[0055] like Figure 2As shown, H NMR spectrum data indicated the successful synthesis of Mal-FRRG-COOH.

[0056] Example 3:

[0057] This embodiment takes the reaction of Mal-FRRG-COOH with CIP under amidation reaction conditions to obtain Mal-FRRG-CIP as an example, and includes the following steps:

[0058] To a dry, round-bottomed, three-necked flask, Mal-FRRG-COOH (25 mg, 53 μmol), carbodiimide hydrochloride (7.7 mg, 79 μmol), and N-hydroxysuccinimide (3.08 mg, 53 μmol) were added sequentially. The flask was pumped with an oil pump for 10 min to remove water vapor. Then, 1 mL of anhydrous dimethyl sulfoxide (DMSO) was added. Under nitrogen, the mixture was reacted at 4°C for 1 h, then at 37°C for 12 h to obtain activated product 2. CIP·HCl (29 mg, 159 μmol) was weighed and dissolved in 1.5 mL of DMSO. 10 μL of N,N-diisopropylethylamine was then added dropwise to the three-necked flask using a syringe. The reaction was continued for 12 h. After the reaction, the liquid in the flask was transferred to a 500 Da dialysis bag and dialyzed against 500 mL of DMSO for 3 days, with the DMSO replaced twice daily. After dialysis, the liquid in the dialysis bag was lyophilized to obtain yellow powder Mal-FRRG-CIP.

[0059] The molar ratio of carbodiimide hydrochloride to N-hydroxysuccinimide is 1:1.0-1.05; the amount of N,N-diisopropylethylamine added is 3.5-4 times the molar amount of CIP; the dialysis process is preferably carried out at 25° C., and the molecular weight cutoff of the dialysis bag is 500 Da.

[0060] like Figure 3 As shown, H NMR spectrum data indicated the successful synthesis of Mal-FRRG-CIP.

[0061] Example 4:

[0062] This example takes the reaction of Mal-FRRG-CIP with IL4Rpep-1 under thiol-ene click reaction conditions to obtain IL4Rpep-1-FRRG-CIP as an example, and includes the following steps:

[0063] Mal-FRRG-CIP (12.48 mg, 10 μmol), IL4Rpep -1 (2.324 mg, 2 μmol) was dissolved in 2 mL of H2O, and the reducing agent tri(2-formylethyl)phosphine hydrochloride (0.5734 mg, 2 μmol) was added. After reacting at 25°C for 12 h, the resulting solution was transferred to a 500 Da dialysis bag and dialyzed against 500 mL of phosphate buffered saline (PBS) for 3 days, with PBS changed twice daily. After dialysis, the liquid in the dialysis bag was lyophilized to obtain IL4Rpep-1-FRRG-CIP as a white powder.

[0064] The amount of the reducing agent added is 0.2-0.3 times the molar amount of Mal-FRRG-CIP; the dialysis process is preferably carried out at 4° C., and the molecular weight cut-off of the dialysis bag is 500 Da.

[0065] like Figure 4 As shown, H NMR spectrum data demonstrated the successful synthesis of IL4Rpep-1-FRRG-CIP.

[0066] Example 5:

[0067] This example takes IL4Rpep-1-FRRG-CIP as an example to assemble nanoparticles MTCP-NPs through hydrophilic-hydrophobic interactions, including the following steps:

[0068] IL4Rpep-1-FRRG-CIP was dissolved in 0.5 mL of DMSO to form a first solution; the obtained first solution was added dropwise into 2.5 mL of deionized water under ultrasonic conditions; after the addition was completed, the mixed solution was further ultrasonicated for 30 minutes to allow IL4Rpep-1-FRRG-CIP to self-assemble into nanostructures through hydrophilic-hydrophobic interactions; the obtained solution was placed in a dialysis bag and dialyzed with deionized water to remove DMSO to obtain CIP nanoprodrug MTCP-NPs with uniform particle size distribution.

[0069] The ultrasonic treatment is preferably carried out at a frequency of 40 kHz and a power of 100 W.

[0070] like Figure 5 As shown in the figure, transmission electron microscopy images show that MTCP-NPs have a spherical morphology, a particle size of about 200 nm, and good dispersion.

[0071] Comparative Example 1:

[0072] This comparative example takes the assembly of Mal-FRRG-CIP into nanoparticles CP-NPs through hydrophilic-hydrophobic interactions as an example, and includes the following steps:

[0073] Mal-FRRG-CIP was dissolved in 0.5 mL of DMSO to form a first solution; the resulting first solution was added dropwise to 2.5 mL of deionized water under ultrasonic conditions; after the addition was completed, the mixed solution was further ultrasonicated for 30 minutes to allow Mal-FRRG-CIP to self-assemble into nanostructures through hydrophilic-hydrophobic interactions; the resulting solution was placed in a dialysis bag and dialyzed with deionized water to remove DMSO, thereby obtaining CIP nanoprodrug CP-NPs with uniform particle size distribution.

[0074] Example 6:

[0075] Various performance tests were conducted on the above-mentioned MTCP-NPs. The specific experiments are as follows:

[0076] 1. CTSB responsive CIP release performance test

[0077] Two 2 mL MTCP-NPs solutions were prepared. CTSB was added to one of the solutions to a final concentration of 1 μg / mL, while the other solution served as a control without CTSB. Both samples were transferred to 500 Da molecular weight cutoff dialysis tubing and then immersed in a centrifuge tube containing 15 mL PBS. The tubes were shaken at a constant speed in a thermostatic oscillator (37 ± 0.5°C) to simulate drug release under physiological pH conditions. At predetermined time intervals (0, 0.17, 0.33, 0.5, 1, 2, 4, 6, 9, 12, 24, and 48 h), 300 μL of buffer was removed from the centrifuge tube and replaced with an equal volume of fresh PBS buffer. The absorbance of the sample solution at 278 nm was measured by UV-visible spectrophotometry, and the concentration of the released drug was calculated based on the CIP standard curve.

[0078] like Figure 6 As shown in the figure, in the presence of CTSB, the drug release of MTCP-NPs was significantly accelerated, and the cumulative release of CIP after 48 h reached 60%, proving that MTCP-NPs have CTSB-responsive drug release behavior.

[0079] 2. Extracellular antibacterial performance test

[0080] 100 μL of frozen Fusobacterium nucleatum solution was inoculated into 1 mL of liquid culture medium and cultured on a shaker at 37°C and 150 rpm for 24 h to revive the bacteria. The bacterial suspension was then adjusted to an OD of 600 A value of 0.1 is equivalent to about 10 8CFU / mL. 100 μL of the initial bacterial solution was added to 900 μL of liquid culture medium as a blank control. 100 μL of the initial bacterial solution and 100 μL of different treatments (CIP, MTCP-NPs, and MTCP-NPs+CTSB) were added to 800 μL of liquid culture medium and cultured at 37°C for 12 h. The OD of the treated bacteria was then measured by UV spectrophotometer. 600 value, to assess bacterial growth.

[0081] like Figure 7 As shown in the results, compared with the PBS group, the MTCP-NPs + CTSB group achieved a 90% inhibition rate against Fusobacterium nucleatum, close to the 95% inhibition rate of the CIP group. However, when MTCP-NPs were used alone, the inhibition effect on Fusobacterium nucleatum was not significant, with an inhibition rate of only 20%. This result indicates that in the presence of CTSB, MTCP-NPs can effectively release the antibiotic CIP, thereby exhibiting significant antibacterial activity.

[0082] 3. M2 macrophage uptake experiment

[0083] Fluorescent dye PI was loaded into MTCP-NPs to prepare PI@MTCP-NPs. 5 RAW264.7 cells were seeded into 6-well plates and treated with PI@MTCP-NPs. The cells were incubated at 37°C in a 5% CO2 / 95% air atmosphere for 3 hours. After incubation, the cells in the 6-well plates were rinsed with cold PBS and resuspended in 0.2 mL of PBS to form a single-cell suspension. The intracellular fluorescence intensity of PI was quantified by flow cytometry.

[0084] Comparative Example 2:

[0085] Fluorescent dye PI was loaded into CP-NPs to prepare PI@CP-NPs. 5 RAW264.7 cells were seeded into 6-well plates and treated with PI@CP-NPs. The cells were incubated at 37°C in a 5% CO2 / 95% air atmosphere for 3 hours. After incubation, the cells in the 6-well plates were rinsed with cold PBS and resuspended in 0.2 mL of PBS to form a single-cell suspension. The intracellular fluorescence intensity of PI was quantified by flow cytometry.

[0086] like Figure 8As shown in the results, compared with normal macrophages and the groups treated with free PI and PI@CP-NPs (PI positive rate was approximately 40%), the PI positive rate of F. nucleatum-infected macrophages treated with PI@MTCP-NPs was significantly increased, reaching 80%. This result indicates that MTCP-NPs can efficiently enter F. nucleatum-infected M2-related macrophages through receptor-ligand interaction-mediated endocytosis.

[0087] In another aspect, the present invention further provides a CIP prodrug nanoparticle, which is prepared by the method for preparing the prodrug nanoparticle according to any one of the above technical solutions.

[0088] The CIP nanoprodrug provided by the present invention has a simple preparation process, mild preparation conditions, low cost, high product purity, is suitable for large-scale production, and can be widely used in the fields of materials science, biology, medicine, etc.

[0089] Furthermore, the present invention also provides a use of the CIP nanoprodrug as described above for clearing Fusobacterium nucleatum in macrophages.

[0090] Example 7:

[0091] Intracellular antibacterial performance test

[0092] RAW264.7 cells were infected with Fusobacterium nucleatum at an MOI of 20 for 2 hours and then cultured in 24-well plates at 37°C for 24 hours. Afterwards, they were treated with PBS, CIP, CP-NPs, or MTCP-NPs for 48 hours. Following treatment, cells were washed with sterile PBS and lysed with 0.1% Triton X-100 aqueous solution for 2 hours. The lysate was diluted with PBS, and 50 μL of the sample was evenly plated on blood agar plates to determine the number of residual bacterial colonies.

[0093] Comparative Example 3:

[0094] RAW264.7 cells were infected with Fusobacterium nucleatum at an MOI of 20 for 2 hours and then treated with PBS, CIP, or CP-NPs for 48 hours. After treatment, cells were washed with sterile PBS and lysed with 0.1% Triton X-100 in water for 2 hours. The lysate was diluted with PBS, and 50 μL of the sample was evenly plated on blood agar plates to determine the number of residual bacterial colonies.

[0095] In the case of adopting the above technical solution, the present invention can target M2 macrophages through the IL4Rpep - 1. Specific targeting of macrophages infected with Fusobacterium nucleatum; after entering the macrophages, under the action of CTSB overexpressed in the cells, the nanoparticles dissociate and release the antibiotic CIP, killing Fusobacterium nucleatum inside the macrophages. Figure 9 As shown in the results, MTCP-NPs showed the most significant killing effect on intracellular Fusobacterium nucleatum, with the clearance rate of Fusobacterium nucleatum in macrophages reaching 90% within 48 hours.

[0096] The CIP nanoprodrug can efficiently and specifically eliminate Fusobacterium nucleatum in macrophages, providing a new strategy for controlling intracellular Fusobacterium nucleatum infection.

Claims

1. A method for preparing a ciprofloxacin nano prodrug, characterized in that: The following steps are involved: 3-maleimidopropionic acid hydroxysuccinimide ester is reacted with amino-polyethylene glycol-carboxyl under amidation reaction conditions to obtain intermediate 1; The intermediate 1 is reacted with a cathepsin B-cleavable peptide linker FRRG under amidation reaction conditions to obtain an intermediate 2; The intermediate 2 is reacted with ciprofloxacin under amidation reaction conditions to obtain intermediate 3; The intermediate 3 is reacted with the M2 macrophage targeting peptide IL4Rpep-1 under thiol-ene click reaction conditions to obtain a ciprofloxacin prodrug molecule; The ciprofloxacin prodrug molecules are assembled through hydrophilic-hydrophobic interactions to obtain ciprofloxacin nano-prodrugs.

2. The method for preparing a ciprofloxacin nano prodrug according to claim 1, characterized in that: The synthetic intermediate 1 specifically comprises the following steps: The amino-tetraethylene glycol-carboxyl group is completely dissolved in dichloromethane to form a first solution; the maleimide active ester is dissolved in dichloromethane to form a second solution; the obtained first solution is slowly added dropwise to the second solution, and N, N-diisopropylethylamine is added, and the reaction is stirred at a constant temperature of 25°C for 16 hours; the reaction progress is monitored by thin layer chromatography, and a dichloromethane / methanol mixed solvent with a volume ratio of 10:1 is used as a developing agent; after the reaction is completed, the dichloromethane is removed by a rotary evaporator at 35°C and 0.1MPa; the obtained product is purified by silica gel column chromatography to collect the target components; the collected components are concentrated and dried to obtain an intermediate 1, wherein the intermediate 1 is maleimide carboxyl functionalized polyethylene glycol; The molar ratio of the amino-tetraethylene glycol-carboxyl group to the maleimide active ester is 1:1.0-1.05; and the added amount of the N,N-diisopropylethylamine is 3.5-4 times the molar amount of the amino-tetraethylene glycol-carboxyl group.

3. The method for preparing prodrug nanoparticles according to claim 1, characterized in that: The synthesis of intermediate 2 specifically includes the following steps: dissolving intermediate 1, carbodiimide hydrochloride and N-hydroxysuccinimide in anhydrous dimethylformamide, reacting at a constant temperature of 37°C for 12 hours to obtain activated product 1; dissolving cathepsin B cleavable peptide linker FRRG in 500 μL dimethylformamide, adding N,N-diisopropylethylamine to form a reaction solution; slowly adding the obtained reaction solution dropwise to activated product 1, and continuing to react at 37°C for 12 hours; after the reaction is completed, using a rotary evaporator to remove dimethylformamide at 40°C and 0.1MPa; dissolving the obtained product with an appropriate amount of dichloromethane, purifying it by silica gel column chromatography, and collecting the target component; concentrating and drying the collected component to obtain white powdery intermediate 2; The molar ratio of carbodiimide hydrochloride to N-hydroxysuccinimide is 1:1.0-1.05; the added amount of N,N-diisopropylethylamine is 3.5-4 times the molar amount of the cathepsin B cleavable peptide linker FRRG.

4. The method for preparing prodrug nanoparticles according to claim 1, characterized in that: The synthesis of intermediate 3 specifically includes the following steps: dissolving intermediate 2, carbodiimide hydrochloride and N-hydroxysuccinimide in anhydrous dimethyl sulfoxide, reacting at 4°C for 1 hour under nitrogen protection, then heating to 37°C and continuing to react for 12 hours to obtain activated product 2; dissolving ciprofloxacin hydrochloride in dimethyl sulfoxide, adding N,N-diisopropylethylamine to form a reaction solution; slowly adding the obtained reaction solution dropwise to activated product 2, and continuing to react at 37°C for 12 hours; transferring the obtained reaction solution to a dialysis bag, using dimethyl sulfoxide as a dialysis solution, and dialyzing for 3 days; freeze-drying the dialyzed solution to obtain yellow powder intermediate 3; The molar ratio of the carbodiimide hydrochloride to N-hydroxysuccinimide is 1:1.0-1.05; the amount of N,N-diisopropylethylamine added is 3.5-4 times the molar amount of ciprofloxacin hydrochloride; the dialysis process is preferably carried out at 25°C, and the molecular weight cutoff of the dialysis bag is 500Da.

5. The method for preparing prodrug nanoparticles according to claim 1, characterized in that: The synthesis of ciprofloxacin prodrug molecules specifically comprises the following steps: The intermediate 3 and the polypeptide IL4Rpep-1 were dissolved in water, and a reducing agent, tri(2-formylethyl)phosphine hydrochloride, was added, and the mixture was reacted at a constant temperature of 25° C. for 12 hours to obtain a reaction solution. The obtained reaction solution was transferred to a dialysis bag, and dialyzed with a pH 7.4 phosphate buffer for 3 days. The dialyzed solution was freeze-dried to obtain a white powdery ciprofloxacin prodrug molecule. The amount of the reducing agent added is 0.2-0.25 times the molar amount of the intermediate 3; the dialysis process is preferably carried out at 4°C, and the molecular weight cutoff of the dialysis bag is 500Da.

6. The method for preparing the ciprofloxacin nano prodrug according to claim 1, characterized in that: The preparation of ciprofloxacin nano prodrug specifically comprises the following steps: The ciprofloxacin prodrug molecules are dissolved in 0.5 mL of dimethyl sulfoxide to form a first solution; the obtained first solution is dripped into 2.5 mL of deionized water under ultrasonic conditions; after the dripping is completed, the mixed solution is subjected to ultrasonic treatment for 30 minutes to allow the ciprofloxacin prodrug molecules to self-assemble into nanostructures through hydrophilic-hydrophobic interactions; the obtained solution is placed in a dialysis bag, and the dimethyl sulfoxide is removed by dialysis with deionized water to obtain a ciprofloxacin nanoprodrug with uniform particle size distribution; The ultrasonic treatment is preferably carried out at a frequency of 40 kHz and a power of 100 W.

7. A ciprofloxacin nano prodrug, characterized in that: The ciprofloxacin nano prodrug is prepared by the preparation method described in any one of claims 1 to 6.

8. Use of the ciprofloxacin nano prodrug as claimed in claim 6 in the preparation of a drug for specifically recognizing and killing Fusobacterium nucleatum in macrophages.

9. The use according to claim 8, characterized in that: include: The ciprofloxacin nanoprodrug specifically binds to the IL-4 receptor on the surface of macrophages through its surface-modified polypeptide ligand to achieve targeted delivery; After the ciprofloxacin nano prodrug is internalized by macrophages, the active ciprofloxacin molecules are released under the action of lysosomal cathepsin B.

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