Florfenicol-polyethylene glycol conjugate as well as preparation method and application thereof

Abstract

The invention discloses a florfenicol-polyethylene glycol conjugate and a preparation method and application thereof, and belongs to the technical field of high polymer chemistry, the florfenicol-polyethylene glycol conjugate has the following structural formula: # imgabs0 # n is 17-113, florfenicol is combined with a high-molecular polymer methoxypolyethylene glycol to form the florfenicol-polyethylene glycol conjugate, and the florfenicol-polyethylene glycol conjugate is used for preparing the florfenicol-polyethylene glycol conjugate. The problems that florfenicol is low in absorptivity, prone to being affected by metabolic enzymes and short in half-life period are solved in a nano-drug delivery mode. The florfenicol-polyethylene glycol conjugate disclosed by the invention can be used for effectively treating poultry bacterial infection, not only can be used for improving the curative effect of a medicine, but also can be used for reducing the dosage and side effects of the medicine, and a safer and more effective treatment means is provided for the poultry breeding industry.

Description

Technical Field

[0001] The invention belongs to the technical field of polymer chemistry, and in particular relates to a florfenicol-polyethylene glycol conjugate and a preparation method and application thereof. Background Art

[0002] According to the World Organization for Animal Health (OIE), approximately 1% to 5% of poultry worldwide are affected by bacterial infections each year. This means that millions of poultry die or become sick every year due to bacterial infections. Poultry bacterial diseases refer to poultry diseases caused by various pathogens. In the process of poultry farming, bacteria are transmitted in various ways, which may come from feed, water, air, equipment, breeders and other links. These bacteria can cause various diseases in poultry, such as Escherichia coli disease, Salmonellosis, Pasteurellosis, etc., which bring huge economic losses to the farming industry.

[0003] Florfenicol is a broad-spectrum antibacterial drug that is often used in the veterinary field, especially for the treatment of bacterial infections in poultry. However, there are some problems with the traditional Florfenicol treatment method, such as low absorption rate, susceptibility to metabolic enzymes, short half-life, etc. Therefore, it is necessary to provide a solution to the above problems. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a florfenicol-polyethylene glycol conjugate and a preparation method and application thereof.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] One of the technical solutions of the present invention:

[0007] The present invention provides a florfenicol-polyethylene glycol conjugate (mPEG-SS-FF), the structural formula of which is as follows:

[0008]

[0009] Among them, n is 17-113, and n is preferably 17, 45 or 113.

[0010] The present invention combines florfenicol with a high molecular polymer polyethylene glycol monomethyl ether to form a florfenicol-polyethylene glycol conjugate, and solves the problems of low absorption rate of florfenicol, susceptibility to the influence of metabolic enzymes and short half-life in the form of nano drug delivery. The florfenicol-polyethylene glycol conjugate of the present invention can effectively treat poultry bacterial infection, not only can improve the efficacy of the drug, but also can reduce the dosage and side effects of the drug, and provide a safer and more effective treatment method for the poultry farming industry.

[0011] The second technical solution of the present invention:

[0012] The present invention also provides a method for preparing the above-mentioned florfenicol-polyethylene glycol conjugate, comprising the following steps:

[0013] S1. Dissolve 3,3′-dithiodipropionic acid (DTDP) in acetyl chloride, stir evenly, and reflux at 70-80° C. After the reaction is completed, remove the solvent by rotary evaporation, precipitate the product with ether, and dry to obtain dithiodipropionic anhydride (DTDPA);

[0014] S2. The DTDPA and polyethylene glycol monomethyl ether (mPEG) are dissolved in anhydrous dimethyl sulfoxide (DMSO), 4-dimethylaminopyridine (DMAP) and triethylamine (TEA) are added, and the mixture is reacted at 30-40° C. for 24-48 hours under nitrogen (N2) protection. After the reaction, glacial ether is added, and mPEG-SS-COOH is obtained after vacuum drying;

[0015] S3. The mPEG-SS-COOH and florfenicol (FF) are dissolved in DMSO, DMAP and N,N'-dicyclohexylcarbodiimide (DCC) are added, and the mixture is reacted at 0-30°C for 24-96h under N2 protection. The reaction product is dialyzed in water and freeze-dried to obtain the mPEG-SS-FF.

[0016] In step S2, the molar ratio of the mPEG to the DTDPA is 1:(1-20); and / or

[0017] The sum of the amounts of DMAP and TEA is 2 to 20 times the amount of DTDPA.

[0018] In step S2, the molecular weight of the mPEG is 750, 2000 or 5000.

[0019] In step S3, the ratio of the amount of the FF substance to the amount of the mPEG-SS-COOH substance is 1:(1-10).

[0020] In step S3, the sum of the amounts of DMAP and DCC is 2 to 20 times the amount of FF.

[0021] The third technical solution of the present invention:

[0022] The present invention also provides an antibacterial drug, the active ingredient of which is the florfenicol-polyethylene glycol conjugate.

[0023] The fourth technical solution of the present invention:

[0024] The present invention also provides the use of the florfenicol-polyethylene glycol conjugate in the preparation of antibacterial drugs.

[0025] Preferably, the antibacterial drug is an anti-Escherichia coli drug.

[0026] Compared with the prior art, the present invention has the following advantages and technical effects:

[0027] In the present invention, florfenicol and mPEG are linked by a disulfide bond, and the drug has redox-sensitive properties and can be interrupted by specific redox substances, thereby releasing the small molecule drug florfenicol from the polymer carrier.

[0028] The florfenicol-polyethylene glycol conjugate of the invention is a high molecular polymer, which is composed of a hydrophilic PEG segment and a hydrophobic florfenicol segment, has amphiphilicity, and self-assembles into nanoparticles in an aqueous solution.

[0029] The florfenicol-polyethylene glycol conjugate of the present invention has a low hemolysis rate and good biocompatibility. After continuous testing for one week, the change range of the potential and particle size is less than 5%, showing good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0031] Figure 1 This is the H NMR spectrum of the florfenicol-polyethylene glycol conjugate prepared in Example 1;

[0032] Figure 2 This is the infrared absorption spectrum of the florfenicol-polyethylene glycol conjugate prepared in Example 1;

[0033] Figure 3 The particle size (a) and potential (b) data of the florfenicol-polyethylene glycol conjugate prepared in Example 1 at different times;

[0034] Figure 4 The thermogravimetric diagrams of the florfenicol-polyethylene glycol conjugate prepared in Example 1, wherein (a) is a TGA thermogravimetric curve diagram, and (b) is a DTG differential thermogravimetric diagram;

[0035] Figure 5 The release rate curve of the florfenicol-polyethylene glycol conjugate prepared in Example 1 in different concentrations of dithiothreitol;

[0036] Figure 6 The hemolysis rate of the florfenicol-polyethylene glycol conjugate prepared in Example 1 at different concentrations, and the inset is an image of the hemolysis supernatant;

[0037] Figure 7The antibacterial effect of the florfenicol-polyethylene glycol conjugate prepared in Example 1 on Escherichia coli, with the concentration unit being μg / mL.

[0038] Figure 8 The average fluorescence intensity of the florfenicol-polyethylene glycol conjugate prepared in Example 1 inhibiting Escherichia coli for 12 h and 48 h, and the inset is the fluorescence site.

[0039] Fig. 9 The pharmacokinetics of the florfenicol-polyethylene glycol conjugate prepared in Example 1 in mice, wherein (a) is a calibration curve of florfenicol in plasma measured using HPLC, and (b) is the concentration of florfenicol in plasma of healthy mice after a single oral administration of commercially available florfenicol tablets and the conjugate. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Unless otherwise specified, the room temperature in the present invention is 25±2°C.

[0046] All raw materials and reagents used in the examples of the present invention were purchased from commercial sources, and all reagents used were of analytical grade.

[0047] In some embodiments of the present invention, a florfenicol-polyethylene glycol conjugate (mPEG-SS-FF) is provided, and the structural formula is as follows:

[0048]

[0049] Among them, n is 17-113, and n is preferably 17, 45 or 113.

[0050] The present invention combines florfenicol with a high molecular polymer polyethylene glycol monomethyl ether to form a florfenicol-polyethylene glycol conjugate, polyethylene glycol and florfenicol are connected by a disulfide bond, and the problems of low absorption rate of florfenicol, susceptibility to the influence of metabolic enzymes and short half-life are solved in the form of nano drug delivery. The florfenicol-polyethylene glycol conjugate of the present invention can effectively treat poultry bacterial infection, not only can improve the efficacy of the drug, but also can reduce the dosage and side effects of the drug, and provide a safer and more effective treatment method for the poultry farming industry.

[0051] In some embodiments of the present invention, a method for preparing the above-mentioned florfenicol-polyethylene glycol conjugate (mPEG-SS-FF) is provided, comprising the following steps:

[0052] S1. Dissolve 3,3′-dithiodipropionic acid (DTDP) in acetyl chloride, stir evenly, and reflux at 70-80°C. After the reaction is completed, remove the solvent by rotary evaporation, precipitate the product with ether, and dry it to obtain dithiodipropionic anhydride (DTDPA). The reaction route is as follows:

[0053]

[0054] When the product is precipitated with diethyl ether, the volume of the diethyl ether is 5-50 times, preferably 5-15 times, the volume of the product, and the preferred drying method is vacuum drying;

[0055] S2. The DTDPA and polyethylene glycol monomethyl ether (mPEG) are dissolved in anhydrous dimethyl sulfoxide (DMSO), 4-dimethylaminopyridine (DMAP) and triethylamine (TEA) are added, and the mixture is reacted at 30-40° C. for 24-48 hours under N2 protection. After the reaction, glacial ether is added, and mPEG-SS-COOH is obtained after vacuum drying. The reaction route is as follows:

[0056]

[0057] The molar ratio of mPEG to DTDPA is 1:(1-20), more preferably 1:(1-10), and most preferably 1:1; the sum of the molar amounts of DMAP and TEA is 2-20 times the molar amount of DTDPA, the molecular weight of mPEG is 750, 2000 or 5000, glacial ether is added to precipitate mPEG-SS-COOH, and the vacuum drying time is 24 hours;

[0058] S3. Dissolve mPEG-SS-COOH and florfenicol (FF) in DMSO, add DMAP and N,N'-dicyclohexylcarbodiimide (DCC), react at 0-30°C for 24-96h under N2 protection, dialyze the reaction product in water, and freeze-dry to obtain the mPEG-SS-FF, the ratio of the amount of FF to the amount of mPEG-SS-COOH is 1:(1-10), and the sum of the amounts of DMAP and DCC is 2-20 times the amount of FF; dialysis in water is to remove DMSO.

[0059] The florfenicol-polyethylene glycol conjugate provided in the embodiment of the present invention can be used to prepare antibacterial drugs, and is particularly suitable for preparing anti-Escherichia coli drugs.

[0060] It should be pointed out that the matters not described in detail in the present invention are all conventional operating means in the art and are not the focus of the present invention. For example, specific methods such as vacuum drying and freeze drying are all completed using conventional methods.

[0061] The technical solution of the present invention is further illustrated by the following embodiments.

[0062] Example 1

[0063] This embodiment provides a method for preparing a florfenicol-polyethylene glycol conjugate:

[0064] S1. Preparation of DTDPA (dithiodipropionic anhydride):

[0065] 5 g of DTDP (3,3′-dithiodipropionic acid) was dissolved in 50 mL of acetyl chloride, stirred at 600 r / min, and then refluxed at 75°C for 7 h. After the reaction, the solvent was removed by a vacuum rotary evaporator, and the product was precipitated with 5 times the volume of ether, and the product was dried under vacuum to obtain DTDPA.

[0066] S2、mPEG 750 Preparation of -SS-COOH:

[0067] 96 mg (0.5 mmol) of DTDPA and 375 mg (0.5 mmol) of mPEG with a molecular weight of 750 were dissolved in 1.5 mL of anhydrous DMSO (dimethyl sulfoxide); then, 61 mg (0.5 mmol) of DMAP (4-dimethylaminopyridine) and 70 μL (0.5 mmol) of TEA (triethylamine) were added evenly under stirring, and the mixed solution was maintained at 35 ° C. The reaction was ensured to be complete under N2 protection. After 24 hours, 5 volumes of ice ether were added to the mixed solution to precipitate mPEG. 750 -SS-COOH, filtered, and dried under vacuum for 24 h to obtain mPEG 750 -SS-COOH.

[0068] S3. Preparation of florfenicol-polyethylene glycol conjugate (mPEG-SS-FF):

[0069] Under the protection of nitrogen, the mPEG obtained by S2 was 750 438.4 mg (0.2 mmol) of -SS-COOH and 71.64 mg (0.2 mmol) of florfenicol were dissolved in 3 mL of DMSO, and 24.4 mg (0.2 mmol) of DMAP and 41.3 mg (0.2 mmol) of DCC (N, N'-dicyclohexylcarbodiimide) were added as catalysts. After reacting at 25°C for 48 h, the reaction product was dialyzed in water to remove DMSO, and the final product mPEG-SS-FF was obtained by freeze drying. Its structural formula is as follows:

[0070] n is 17.

[0071] Example 2

[0072] This embodiment provides a method for preparing a florfenicol-polyethylene glycol conjugate:

[0073] S1. Preparation of DTDPA:

[0074] 5 g of DTDP was dissolved in 50 mL of acetyl chloride, stirred at 600 r / min, and then refluxed at 75°C for 7 h. After the reaction, the solvent was removed by a vacuum rotary evaporator, and the product was precipitated with 20 times excess volume of ether, and the product was dried under vacuum to obtain DTDPA.

[0075] S2、mPEG 2000 Preparation of -SS-COOH:

[0076] 96 mg (0.5 mmol) of DTDPA and 1000 mg (0.5 mmol) of mPEG with a molecular weight of 2000 were dissolved in 1.5 mL of anhydrous DMSO; then, 61 mg (0.5 mmol) of DMAP and 70 μL (0.5 mmol) of TEA were added evenly under stirring, and the mixed solution was kept at 35 ° C. The reaction was ensured to be complete under N2 protection. After 24 hours, 20 times the volume of ice ether was added to the mixed solution to precipitate mPEG. 2000 -SS-COOH, filtered, and dried under vacuum for 24 h to obtain mPEG 2000 -SS-COOH.

[0077] Preparation of S3, mPEG-SS-FF:

[0078] Under the protection of nitrogen, the mPEG obtained by S2 was 2000 1169 mg (0.2 mmol) of -SS-COOH and 716.4 mg (2 mmol) of florfenicol were dissolved in 3 mL of DMSO, and 24.4 mg (0.2 mmol) of DMAP and 41.3 mg (0.2 mmol) of DCC were added as catalysts. After reacting at 25°C for 24 h, the reaction product was dialyzed in water to remove DMSO, and the final product mPEG-SS-FF was obtained by freeze drying. Its structural formula is as follows:

[0079] n is 45.

[0080] Example 3

[0081] This embodiment provides a method for preparing a florfenicol-polyethylene glycol conjugate:

[0082] S1. Preparation of DTDPA:

[0083] 5 g of DTDP was dissolved in 50 mL of acetyl chloride, stirred at 600 r / min, and then refluxed at 75° C. for 7 h. After the reaction, the solvent was removed by a vacuum rotary evaporator, and the product was precipitated with 50 times excess volume of ether, and the product was dried under vacuum to obtain DTDPA.

[0084] S2、mPEG 5000 Preparation of -SS-COOH:

[0085] 96 mg (0.5 mmol) of DTDPA and 2500 mg (0.5 mmol) of mPEG with a molecular weight of 5000 were dissolved in 1.5 mL of anhydrous DMSO; then, 61 mg (0.5 mmol) of DMAP and 70 μL (0.5 mmol) of TEA were added evenly under stirring, and the mixed solution was kept at 35 ° C. The reaction was ensured to be complete under N2 protection. After 24 hours, 50 times the volume of ice ether was added to the mixed solution to precipitate mPEG. 5000 -SS-COOH, filtered, and dried under vacuum for 24 h to obtain mPEG 5000 -SS-COOH.

[0086] Preparation of S3, mPEG-SS-FF:

[0087] Under the protection of nitrogen, the mPEG obtained by S2 was 5000 2922 mg (0.2 mmol) of -SS-COOH and 71.64 mg (0.2 mmol) of florfenicol were dissolved in 3 mL of DMSO, and 244 mg (2 mmol) of DMAP and 413 mg (2 mmol) of DCC were added as catalysts. After reacting at 25°C for 24 h, the reaction product was dialyzed in water to remove DMSO, and the final product mPEG-SS-FF was obtained by freeze drying. Its structural formula is as follows:

[0088] n is 113.

[0089] Example 4

[0090] This embodiment provides a method for preparing a florfenicol-polyethylene glycol conjugate:

[0091] S1. Preparation of DTDPA:

[0092] 5 g of DTDP was dissolved in 50 mL of acetyl chloride, stirred at 600 r / min, and then refluxed at 75° C. for 7 h. After the reaction, the solvent was removed by a vacuum rotary evaporator, and the product was precipitated with 50 times excess volume of ether, and the product was dried under vacuum to obtain DTDPA.

[0093] S2、mPEG 2000 Preparation of -SS-COOH:

[0094] 96 mg (0.5 mmol) of DTDPA and 1000 mg (0.5 mmol) of mPEG with a molecular weight of 2000 were dissolved in 1.5 mL of anhydrous DMSO; then, 61 mg (0.5 mmol) of DMAP and 70 μL (0.5 mmol) of TEA were added evenly under stirring, and the mixed solution was kept at 35 ° C. The reaction was ensured to be complete under N2 protection. After 24 hours, 20 times the volume of ice ether was added to the mixed solution to precipitate mPEG. 2000 -SS-COOH, filtered, and dried under vacuum for 24 h to obtain mPEG 2000 -SS-COOH.

[0095] Preparation of S3, mPEG-SS-FF:

[0096] Under the protection of nitrogen, the mPEG obtained by S2 was 2000 -SS-COOH 1169 mg (0.2 mmol) and florfenicol 71.64 mg (0.2 mmol) were dissolved in 3 mL DMSO, and catalyst 24.4 mg (0.2 mmol) DMAP and 41.3 mg (0.2 mmol) DCC were added. After reacting at 25°C for 48 h, the reaction product was dialyzed in water to remove DMSO, and the final product mPEG-SS-FF was obtained by freeze drying.

[0097] Example 5

[0098] This embodiment provides a method for preparing a florfenicol-polyethylene glycol conjugate:

[0099] S1. Preparation of DTDPA:

[0100] 5 g of DTDP was dissolved in 50 mL of acetyl chloride, stirred at 600 r / min, and then refluxed at 75° C. for 7 h. After the reaction, the solvent was removed by a vacuum rotary evaporator, and the product was precipitated with 50 times excess volume of ether, and the product was dried under vacuum to obtain DTDPA.

[0101] S2、mPEG 2000 Preparation of -SS-COOH:

[0102] 192 mg (1 mmol) of DTDPA and 1000 mg (0.5 mmol) of mPEG with a molecular weight of 2000 were dissolved in 1.5 mL of anhydrous DMSO; then, 61 mg (0.5 mmol) of DMAP and 70 μL (0.5 mmol) of TEA were added evenly under stirring. The mixed solution was maintained at 35 ° C and the reaction was completed under N2 protection. After 24 hours, 20 times the volume of ice ether was added to the mixed solution to precipitate mPEG. 2000-SS-COOH. Then filter and dry under vacuum for 24h to obtain mPEG 2000 -SS-COOH.

[0103] Preparation of S3, mPEG-SS-FF:

[0104] Under the protection of nitrogen, the mPEG obtained by S2 was 2000 -SS-COOH 1169 mg (0.2 mmol) and florfenicol 71.64 mg (0.2 mmol) were dissolved in 3 mL DMSO, and catalyst 24.4 mg (0.2 mmol) DMAP and 41.3 mg (0.2 mmol) DCC were added. After reacting at 25°C for 48 h, the reaction product was dialyzed in water to remove DMSO, and the final product mPEG-SS-FF was obtained by freeze drying.

[0105] Example 6

[0106] This embodiment provides a method for preparing a florfenicol-polyethylene glycol conjugate:

[0107] S1. Preparation of DTDPA:

[0108] 5 g of DTDP was dissolved in 50 mL of acetyl chloride, stirred at 600 r / min, and then refluxed at 75°C for 7 h. After the reaction, the solvent was removed by a vacuum rotary evaporator, and the product was precipitated with 5 times the volume of ether, and the product was dried under vacuum to obtain DTDPA.

[0109] S2、mPEG 750 Preparation of -SS-COOH:

[0110] 96 mg (0.5 mmol) of DTDPA and 375 mg (0.5 mmol) of mPEG with a molecular weight of 750 were dissolved in 1.5 mL of anhydrous DMSO; then, 610 mg (5 mmol) of DMAP and 700 μL (5 mmol) of TEA were added evenly under stirring, and the mixed solution was maintained at 35 ° C. The reaction was ensured to be complete under N2 protection. After 24 hours, 5 volumes of ice ether were added to the mixed solution to precipitate mPEG. 750 -SS-COOH, filtered, and dried under vacuum for 24 h to obtain mPEG 750 -SS-COOH.

[0111] Preparation of S3, mPEG-SS-FF:

[0112] Under the protection of nitrogen, the mPEG obtained by S2 was 750-SS-COOH 438.4 mg (0.2 mmol) and florfenicol 71.64 mg (0.2 mmol) were dissolved in 3 mL DMSO, and catalysts 244 mg (2 mmol) DMAP and 413 mg (2 mmol) DCC were added. After reacting at 25°C for 24 h, the reaction product was dialyzed in water to remove DMSO, and the final product mPEG-SS-FF was obtained by freeze drying.

[0113] Example 7

[0114] This embodiment provides a method for preparing a florfenicol-polyethylene glycol conjugate:

[0115] S1. Preparation of DTDPA:

[0116] 5 g of DTDP was dissolved in 50 mL of acetyl chloride, stirred at 600 r / min, and then refluxed at 75°C for 7 h. After the reaction, the solvent was removed by a vacuum rotary evaporator, and the product was precipitated with 5 times the volume of ether, and the product was dried under vacuum to obtain DTDPA.

[0117] S2、mPEG 750 Preparation of -SS-COOH:

[0118] 96 mg (0.5 mmol) of DTDPA and 375 mg (0.5 mmol) of mPEG with a molecular weight of 750 were dissolved in 1.5 mL of anhydrous DMSO; then, 610 mg (5 mmol) of DMAP and 700 μL (5 mmol) of TEA were added evenly under stirring, and the mixed solution was maintained at 35 ° C. The reaction was ensured to be complete under N2 protection. After 48 hours, 5 volumes of ice ether were added to the mixed solution to precipitate mPEG. 750 -SS-COOH, filtered, and dried under vacuum for 24 h to obtain mPEG 750 -SS-COOH.

[0119] Preparation of S3, mPEG-SS-FF:

[0120] Under the protection of nitrogen, the mPEG obtained by S2 was 750 -SS-COOH 438.4 mg (0.2 mmol) and florfenicol 71.64 mg (0.2 mmol) were dissolved in 3 mL DMSO, and catalysts 244 mg (2 mmol) DMAP and 413 mg (2 mmol) DCC were added. After reacting at 25°C for 96 h, the reaction product was dialyzed in water to remove DMSO, and the final product mPEG-SS-FF was obtained by freeze drying.

[0121] Figure 1This is the hydrogen nuclear magnetic resonance spectrum of mPEG-SS-FF in Example 1. Analysis shows that the characteristic peak of -CH2-CH2- in mPEG appears at a chemical shift of 3.65ppm; the characteristic peak of -CH2- of 3,3'-dithiodipropionic acid appears at a chemical shift of 3.38ppm; and the characteristic peaks of two groups of symmetrical -CH- on the benzene ring of florfenicol appear at a chemical shift of 7.25-7.30ppm; indicating that the target product was successfully obtained using the above method.

[0122] Figure 2 The infrared absorption spectrum of mPEG-SS-FF in Example 1 shows that the wavelengths of the mPEG-SS-FF in the range of 1450 to 1600 cm -1 The vibration absorption peak of the benzene ring appeared and florfenicol existed in the polymer, indicating that the target product was successfully obtained by the above method.

[0123] The particle size and potential measurement data of mPEG-SS-FF in Test Example 1 for seven consecutive days were as follows: the sample was dispersed in water, and its particle size and Zeta potential were measured at room temperature using a Zeta particle size analyzer (SALD, 2300, SHIMADZU). The test results are shown in Figure 3 From the results, it can be seen that the particle size of the florfenicol derivatives with disulfide bonds is smaller than that of the florfenicol derivatives without disulfide bonds, and tends to be stable, with little fluctuation for seven consecutive days; from the potential results, the modified florfenicol nanoparticles are all negatively charged, indicating that hydrophilic materials can increase the repulsive force between nanoparticles, prevent aggregation, and thus improve the stability of the drug.

[0124] Figure 4 The thermogravimetric curve of mPEG-SS-FF in Example 1 shows that the TGA curve of florfenicol shows two stages of weight loss; it is obvious that the drug molecules are decomposed at a temperature of 150-290°C and destroyed at a temperature of 290-690°C. The florfenicol derivative containing a disulfide bond also has two stages of weight loss. At a temperature of 150-330°C, the weight is reduced, which may be due to the residual water physically adsorbed on the sample surface and the breaking and reorganization of the chemical bonds inside the structure; at a temperature of 330-440°C, the compound further decomposes, and the oxygen-containing functional group may be decomposed, and the weight is reduced by 80%.

[0125] The release rate curve of mPEG-SS-FF in different concentrations of dithiothreitol in Test Example 1, the specific test process is: establish a calibration curve of fluorofenicol, and measure the UV-visible absorption spectra of fluorofenicol solutions of different concentrations (0.02, 0.018, 0.016, 0.012 and 0.01 mg / mL). Select dithiothreitol with concentrations of 1 mm and 10 mm as redox stimulants. A dialysis bag (MWCO 500) containing 10 mL of mPEG-SS-FF (5 mg / mL) solution is placed in a DTT solution of a specific concentration. Take 3 mL of the release medium at a specified time (2h, 4h, 8h, 12h, 24h, 48h) to measure the absorbance, and add an equal volume of DTT solution. According to the measured absorbance and the standard curve, the drug release curve is obtained. The release rate results are shown in Figure 5 It can be seen that the release rate is faster at 10mmol, and it tends to be stable after 25h, and no burst release phenomenon is observed.

[0126] The hemolysis rate of mPEG-SS-FF at different concentrations in Test Example 1 was tested as follows: whole blood of healthy chickens was collected in anticoagulation tubes using the venous blood method. The blood was centrifuged at 3000 rpm for 15 minutes and washed 3 times with saline. The separated red blood cells were resuspended in a sterile saline solution to form a red blood cell suspension (2%). The sample was diluted with saline of different concentrations (100-1000 μg / mL). The coupling liquid (1 mL), saline (1 mL) and 20 μL RBC suspension were mixed. Distilled water and saline were used as positive and negative controls. The mixed solution was incubated in a 37°C water bath for 1 hour, centrifuged at 4000 rpm for 10 minutes, and the absorbance of the supernatant was measured at 576 nm. The hemolysis rate was calculated as follows.

[0127] Hemolysis rate (%) = (Ap-An) / (As-An) × 100%;

[0128] Where Ap, An, and As are the absorbance values ​​of the sample, negative control, and positive control, respectively. Figure 6 It can be seen that there is no obvious hemolysis phenomenon when the concentration is 1000μg / mL, and the hemolysis rate is less than 5%, showing good biocompatibility.

[0129] The nuclear magnetic resonance hydrogen spectrum, infrared absorption spectrum, particle size and potential measurement data for seven consecutive days, thermogravimetric curve, release rate curve in different concentrations of dithiothreitol, and hemolysis rate of the florfenicol-polyethylene glycol conjugate prepared in Examples 2 to 7 were not significantly different from those of the florfenicol-polyethylene glycol conjugate in Example 1.

[0130] Application Example 1 In vitro inhibition of Escherichia coli experiment

[0131] The filter paper diffusion method was used to determine the inhibition zone: first, the bacteria were revived, 100 μL of bacterial suspension was drawn into a centrifuge tube, and then 10 mL of liquid LB culture medium was added, placed in a 37°C shaker at 180 r / min for 12 hours, and then placed in a 4°C refrigerator for standby use. Next, the drug solution was prepared, and florfenicol (FF) and its derivatives (i.e., florfenicol-polyethylene glycol conjugate) were dissolved in ethanol to prepare a mother solution with a concentration of 1 mg / mL, which was then diluted into 4 concentration gradients, namely 32 μg / mL, 64 μg / mL, 128 μg / mL, and 256 μg / mL of drug solution concentrations. Take 200 μL of different concentrations of drug solution and add it to a small filter paper (punched into small circular paper pieces of the same diameter by a hole puncher, and then sterilized by high pressure). After volatilization and drying to remove excess solvent, all the drug solution is attached to the filter paper. Then, an in vitro antibacterial experiment is carried out to prepare solid LB culture medium, pour plates, and inoculate bacteria; 100 μL of bacterial solution is dripped onto each plate, spread evenly and placed in a clean bench to air dry for 2 hours. After the bacterial solution is dry, place the filter paper with the drug solution added in the center of the culture medium, divide it into portions, and place it in a 37°C incubator for 24 hours to measure its antibacterial diameter. The antibacterial effect of the florfenicol-polyethylene glycol conjugate prepared in Example 1 on Escherichia coli is shown in FIG. Figure 7 , wherein CK is a blank control group, and only PBS is added. It can be seen that the disulfide-bonded florfenicol-polyethylene glycol conjugate prepared by the present invention has good antibacterial activity, and florfenicol belongs to the third-generation chlorinase broad-spectrum antibacterial drugs, and its mechanism of action is to inhibit bacterial protein synthesis, because the florfenicol-polyethylene glycol conjugate of the present invention introduces a disulfide bond into the florfenicol molecule, and cleaves under redox conditions, so that the florfenicol carried by it is easier to enter the cell membrane, and bind to the 50S ribosomal subunit, thereby inhibiting the formation and extension of the peptide chain, preventing the synthesis of bacterial protein, and then preventing bacterial growth and reproduction.

[0132] Application Example 2 Bacterial uptake

[0133] First, the florfenicol-polyethylene glycol conjugate was fluorescently labeled by physical embedding. The conjugate (10 mg) and Nile red (1 mg) were dissolved in 0.5 mL DMSO and stirred for 12 hours. The mixture was then slowly added to 9.5 ml of water under ultrasound. The solution was sealed in a dialysis bag (MWCO 3500). The dialysis bag was dialyzed in deionized water for 24 hours to remove DMSO and free Nile red. The fluorescent marker (200 μL) was added to the E. coli solution (2.2 mL, 1×10 6 CFU mL -1), 37°C, 250 rpm, shaker culture for 12 h and 48 h, centrifuged at 2000 rpm for 10 min, washed 3 times with PBS, and then dispersed in 2 mL of solution. Fluorescence distribution in bacteria was collected using a fluorescence microscope, and the bacterial uptake of drugs was quantitatively evaluated using ImageJ software. Each experiment was repeated three times. The bacterial uptake of the florfenicol-polyethylene glycol conjugate prepared in Example 1 is shown in Figure 8 , fluorescence imaging technology was used to more intuitively detect the uptake of the conjugate by E. coli. After 12 hours of incubation, bacteria treated with mPEG-SS-FF showed obvious fluorescence signals on the surface and in the body. When the incubation time was extended to 48 hours, the fluorescence signals of bacteria treated with mPEG-SS-FF on the surface and in the body were significantly enhanced. Quantitative analysis of fluorescence intensity showed that the average fluorescence intensity of mPEG-SS-FF treatment increased by 1.6 times from 12h to 48h of incubation. This further shows that mPEG-SS-FF is easier to enter E. coli and increases the absorption rate. This may be related to the introduction of disulfide bonds that increase the flexibility of the conjugate and the compatibility of bacterial cell membranes.

[0134] Application Example 3 In vivo pharmacokinetic experiment

[0135] The concentration of florfenicol in plasma samples was determined by high performance liquid chromatography (HPLC). HPLC (Agilent 1260) was used for quantitative analysis of florfenicol, with a mobile phase of acetonitrile and water in a ratio of 27:73, a flow rate of 1 mL / min, a column temperature of 35 °C, and an injection volume of 10 μL. The concentration of florfenicol was detected by measuring the ultraviolet absorption at 223 nm. To establish a standard curve of florfenicol in blank plasma, florfenicol solutions of different concentrations (0.5, 2.5, 5, 12.5 and 25 μg / mL) were prepared, blank plasma (0.2 mL) was added and vortexed, and then extracted with acetonitrile three times, purged with nitrogen, dissolved in chromatographically pure acetonitrile (400 μL), and analyzed by HPLC. Each step was repeated 3 times. A standard curve of peak area and concentration was established.

[0136] Blood sample collection and HPLC analysis. Healthy mice were randomly divided into a drug group and a control group, with three mice in each group, weighed and numbered. They were raised in a warm and ventilated environment for one week, fasted for one day, and then gavage was performed. The dose of florfenicol aqueous solution and florfenicol conjugate aqueous solution was 15 mg / kg, and mouse blood (0.5 mL) was collected at 1h, 2h, 4h, 8h, 12h, 24h and 48h after administration. The blood was immediately transferred to a centrifuge tube containing EDTA K2 anticoagulant and centrifuged (3000rpm) for 10min. The plasma sample (0.2mL) was transferred to a 2mL centrifuge tube, acetonitrile (400μL) was added, vortexed for 5min, and centrifuged (12000rpm) for 10min. After repeated vortexing and centrifugation, the supernatant was transferred to another test tube, blown with nitrogen, and chromatographic pure acetonitrile (400μL) was added to dissolve. The sample was vortexed (5 min) and centrifuged (12000 rpm, 10 min), filtered through a 0.22 μm microporous membrane and analyzed by HPLC. The pharmacokinetic curve of the florfenicol-polyethylene glycol conjugate prepared in Example 1 is shown in Fig. 9 The pharmacokinetic parameters are shown in Table 1 ((Mean±SD). 1 / 2ab : absorption half-life; T 1 / 2el : elimination half-life; Tmax: time required for the drug to reach maximum concentration; Cmax: maximum concentration; AUC 0～48 : area under the curve; CL: elimination rate; MRT: 0～48 : average residence time), the standard curve based on peak area and concentration showed a good linear relationship, and the correlation coefficient exceeded 0.999 ( Fig. 9 The temporal trend of mean plasma concentration of florfenicol is shown in (a). Fig. 9 As shown in (b). After a single oral dose of florfenicol tablets and conjugates, the time (T max ) were 5.33±2.309 and 3.33±1.155h, respectively. The maximum plasma concentration (C max ) were 10.77±0.836 and 6.48±0.324 μg / ml, respectively (Table 1). The substantial increase in Cmax highlights the enhanced systemic delivery achieved by the responsive conjugate formulation. The elimination half-life (T 1 / 2el ) were 31.61±0.122 and 28.42±0.157h, respectively, indicating that the responsive conjugate mPEG-SS-FF significantly prolonged the half-life of florfenicol. This indicates that the florfenicol-polyethylene glycol conjugate prepared by the present invention lasts longer in vivo and can lead to a sustained therapeutic effect.

[0137] Table 1

[0138]

[0139] The above are only preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A florfenicol-polyethylene glycol conjugate, characterized in that: The structural formula is as follows: Among them, n is 17 to 113.

2. The florfenicol-polyethylene glycol conjugate according to claim 1, characterized in that n is 17, 45 or 113.

3. A method for preparing the florfenicol-polyethylene glycol conjugate according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1. dissolving 3,3′-dithiodipropionic acid in acetyl chloride, stirring evenly, and reacting under reflux at 70-80° C., after the reaction is completed, removing the solvent by rotary evaporation, precipitating the product with ether, and drying to obtain dithiodipropionic anhydride; S2. dissolving the dithiodipropionic anhydride and polyethylene glycol monomethyl ether in anhydrous dimethyl sulfoxide, adding 4-dimethylaminopyridine and triethylamine, reacting at 30-40° C. for 24-48 h under nitrogen protection, adding glacial ether after the reaction, and vacuum drying to obtain mPEG-SS-COOH; S3. The mPEG-SS-COOH and florfenicol are dissolved in dimethyl sulfoxide, 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide are added, and the mixture is reacted at 0-30°C for 24-96 hours under nitrogen protection. The reaction product is dialyzed in water and freeze-dried to obtain the florfenicol-polyethylene glycol conjugate.

4. The method for preparing the florfenicol-polyethylene glycol conjugate according to claim 3, characterized in that: In step S2, the molar ratio of the polyethylene glycol monomethyl ether to the dithiodipropionic anhydride is 1:(1-20); and / or The sum of the amounts of the 4-dimethylaminopyridine and triethylamine is 2 to 20 times the amount of the dithiodipropionic anhydride.

5. The method for preparing the florfenicol-polyethylene glycol conjugate according to claim 4, characterized in that: In step S2, the molecular weight of the polyethylene glycol monomethyl ether is 750, 2000 or 5000.

6. The method for preparing the florfenicol-polyethylene glycol conjugate according to claim 3, characterized in that: In step S3, the ratio of the amount of the florfenicol to the amount of the mPEG-SS-COOH is 1:(1-10).

7. The method for preparing the florfenicol-polyethylene glycol conjugate according to claim 3, characterized in that: In step S3, the sum of the amounts of the 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide is 2 to 20 times the amount of the florfenicol.

8. An antibacterial drug, characterized in that: The active ingredient is the florfenicol-polyethylene glycol conjugate according to any one of claims 1 to 2.

9. Use of the florfenicol-polyethylene glycol conjugate according to any one of claims 1 to 2 in the preparation of antibacterial drugs.

10. The use according to claim 9, characterized in that: The antibacterial drug is an anti-Escherichia coli drug.

Images