Florfenicol nano-liposome as well as preparation method and application thereof

Abstract

The invention discloses florfenicol nano-liposome and a preparation method and application thereof, the florfenicol nano-liposome is prepared from soybean lecithin, florfenicol and cholesterol according to the mass ratio of (10-30): 4: 5, florfenicol, cholesterol and soybean lecithin are dissolved in an organic solvent, rotary evaporation is performed, water is added for dissolution, and then ultrasonication is performed to obtain the florfenicol nano-liposome. The average particle size of the florfenicol liposome prepared by the preparation method disclosed by the invention is 249.6 to 638.2 nm, the Zeta potential value of the florfenicol liposome is-13.7 to-46.3 mV, and the polydispersity coefficient of the florfenicol liposome is 0.22 to 0.86. The entrapment rate is 88.31%-92.94%, the drug loading capacity is 3.21%-6.29%, the minimum inhibitory concentration to a salmonella standard strain is 4 mu g / mL, and the minimum bactericidal concentration to the salmonella standard strain is 8 mu g / mL. The minimum inhibitory concentration of the salmonella drug-resistant strain is 8 [mu] g / mL, and the minimum bactericidal concentration of the salmonella drug-resistant strain is 34 [mu] g / mL.

Description

Technical Field

[0001] The present invention relates to a florfenicol nanoliposome and a preparation method and application thereof, in particular to a florfenicol nanoliposome capable of inhibiting multidrug-resistant pathogens (resistant to multiple antibiotics) and a preparation method and application thereof, belonging to the technical field of liposome preparations. Background Art

[0002] Salmonella is a Gram-negative bacillus and an important member of the Enterobacteriaceae family, widely distributed in nature and in the intestines of animals. Salmonella infection can cause asymptomatic infection, acute gastroenteritis, or invasive disease, and its specific manifestations depend on pathogen and host factors. Salmonella is mainly transmitted through contaminated food and water, especially animal-derived foods such as meat and eggs, and plant-derived foods such as fruits and vegetables. In addition, Salmonella can also be transmitted through environmental water sources and irrigation water. Salmonella is one of the common foodborne pathogens that can cause serious illness and even death in humans and animals. Due to its widespread drug resistance and ability to spread through the food chain, this pathogen has become one of the major challenges facing global public health security.

[0003] Florfenicol is a broad-spectrum amide antibiotic and a synthetic derivative of chloramphenicol. Due to its unique chemical structure and mechanism of action, it is widely used in the livestock industry to treat bacterial infections. Florfenicol exhibits broad-spectrum antimicrobial activity, with strong inhibitory effects against Gram-positive and Gram-negative bacteria, as well as some anaerobic bacteria. As a chloramphenicol alternative, florfenicol is approved worldwide for the treatment of animal diseases. Florfenicol's mechanism of action primarily involves inhibiting bacterial protein synthesis. Its target site is located in the peptidyl transferase center of the large ribosomal subunit, which partially overlaps with the chloramphenicol binding site. However, bacteria can develop resistance through modifications to ribosomal RNA, which reduces florfenicol's ability to bind to the ribosome. This modification also confer cross-resistance to eight other classes of ribosome-targeting antibiotics, such as lincosamides and oxazolidinones. Furthermore, florfenicol has drawbacks such as poor solubility and low oral bioavailability, which require further investigation.

[0004] There are many types of florfenicol preparations, mainly including injections, oral preparations and topical preparations. Injections are one of the most commonly used dosage forms of florfenicol, with the characteristics of rapid onset of action and high bioavailability. However, this dosage form has problems such as strong irritation at the injection site and a short drug half-life. In clinical practice, its solubility and sustained-release effect are often improved by adjusting oily solvents (such as soybean oil) or complex surfactants. Powders, premixes and solutions are suitable for group administration (such as poultry or aquaculture). Oral preparations need to overcome the first-pass effect and gastrointestinal degradation problems, and often use microencapsulation technology or combine with lipid carriers to improve bioavailability.

[0005] Liposomes are a type of nanoscale vesicle structure formed by a phospholipid bilayer membrane. They have a hydrophilic core and a hydrophobic membrane layer, capable of loading a variety of drugs or nucleic acid molecules (such as mRNA and siRNA). As nanocarriers, liposomes can significantly improve the targeting and bioavailability of antibiotics through biomimetic design. This can reduce the distribution of antibiotics in non-target tissues, thereby reducing the risk of drug resistance and systemic toxicity. In addition, liposomes can directly disrupt the bacterial outer membrane through surface modification, enhancing antibiotic permeability.

[0006] Key factors in liposome preparation include the selection of membrane materials, preparation methods, particle size control, and solvent residue treatment. Liposome membrane materials mainly include phospholipids and cholesterol. Phospholipids such as phosphatidylcholine (PC) are commonly used membrane materials, with the most common being dipalmitoylcholine (DPPC), distearoylcholine (DSPC), and dimyristoylphosphatidylcholine (DMPC). Cholesterol, as an amphiphilic neutral lipid, can penetrate into the phospholipid membrane at high concentrations, enhancing the stability of liposomes.

[0007] There are many methods for preparing liposomes, including solvent injection, reverse evaporation, and microfluidics. The liposomes prepared by these methods have a large particle size and need to be granulated through thin film extrusion produced by high-pressure homogenization or thin film dispersion methods, and purified by ultrafiltration to obtain liposomes with uniform particle size that meet safety requirements. During the preparation process, the dissolution of phospholipids may require the use of highly toxic organic solvents. Solvent residues need to comply with the relevant regulations of the International Conference on Harmonization (ICH). Extrusion and ultrafiltration are key processes that affect particle size and solvent residue. These processes can ensure the safety and effectiveness of liposome products. Summary of the Invention

[0008] Purpose of the invention: In order to solve the problems existing in the prior art, the first purpose of the present invention is to provide a florfenicol nanoliposome that can inhibit multidrug-resistant pathogens (resistant to multiple antibiotics). The second purpose of the present invention is to provide a method for preparing the florfenicol nanoliposome. The third purpose of the present invention is to provide the use of the florfenicol nanoliposome in the preparation of antibiotic drugs.

[0009] Technical solution: The florfenicol nanoliposomes described in the present invention can inhibit multidrug-resistant pathogens (resistant to multiple antibiotics), which are made of the following components in a mass ratio: soybean lecithin: florfenicol: cholesterol is (10-30):4:5, preferably soybean lecithin: florfenicol: cholesterol is (15-25):4:5, and the optimal mass ratio of soybean lecithin, florfenicol and cholesterol is 20:4:5.

[0010] The florfenicol nanoliposomes have an average particle size of 249.6 to 638.2 nm, a zeta potential of -13.7 to -46.3 mV, preferably -46.3 ± 0.7 mV, a polydispersity coefficient of 0.22 to 0.86, preferably 0.22 ± 0.03, an encapsulation efficiency of 88.31% to 94.19%, and a florfenicol drug loading of 3.21% to 6.69%.

[0011] The method for preparing the florfenicol nanoliposomes of the present invention comprises the following steps:

[0012] Florfenicol, cholesterol and soybean lecithin were dissolved in an organic solvent, evaporated by rotary evaporation, added with water, dissolved by ultrasonication, and crushed by ultrasonication.

[0013] The mass volume ratio of florfenicol to the organic solvent is (0.1-1):(20-30) g / mL, preferably 0.4:24 g / mL. The volume ratio of water to the organic solvent is (0.5-1.5):(0.5-1.5), and the organic solvent is a methanol-chloroform mixture with a volume ratio of (0.5-1.5):(0.5-1.5), preferably 1:1. The rotary evaporation temperature is 25-50°C, preferably 30-40°C, and most preferably 35°C; the rotary evaporation time is 25-40 minutes, preferably 30 minutes. The ultrasonic dissolution time is 2-6 minutes, preferably 3 minutes. The ultrasonic crushing voltage is 200-300 mV, preferably 280 mV, and the ultrasonic crushing time is 2-6 minutes, preferably 4 minutes.

[0014] The invention discloses an application of the florfenicol nanoliposomes in the preparation of antibiotic drugs.

[0015] The pathogens are one or more of Gram-positive bacteria, Gram-negative bacteria or mycoplasma, and Gram-negative bacteria include one or more of Salmonella, Escherichia coli or Shigella.

[0016] The florfenicol nanoliposomes prepared by the present invention can effectively solve the problems of florfenicol such as poor solubility and low oral bioavailability, which lead to its large clinical dosage and the problem of drug resistance. As nanocarriers, liposomes can significantly improve the targeting and bioavailability of antibiotics through biomimetic design. The distribution of florfenicol in non-target tissues is reduced, thereby reducing the risk of drug resistance and systemic toxicity. In addition, liposomes can directly destroy the bacterial outer membrane through surface modification, enhancing the permeability of florfenicol.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0018] (1) The preparation method of the present invention is simple and easy to operate, and belongs to a type of solubilization technology, which is also the most economical. The appearance of the prepared florfenicol nanoliposomes is a relatively uniform, thick, light yellow milky liquid, with an optimal Zeta potential of -46.3±0.7mV. The higher Zeta potential can better ensure that the florfenicol nanoliposomes can stably exert their antibacterial effect. Observation under an electron microscope shows that the particles are relatively uniform in size and have a smooth surface. The minimum inhibitory concentration for the standard strain of Salmonella is 4μg / mL, and the minimum bactericidal concentration is 8μg / mL. It can effectively inhibit the growth of Salmonella at a lower concentration, solving the problem of antibiotic resistance caused by excessive use of antibiotics, which reduces the effectiveness of clinical antibacterial drugs. In addition, the liposomes can directly destroy the bacterial outer membrane through surface modification, thereby enhancing antibiotic permeability.

[0019] (2) Antibiotics with low bioavailability, such as florfenicol, lack targeting because Gram-negative bacteria can escape the inhibitory effects of florfenicol through a variety of mechanisms, including upregulation of drug efflux pumps, target modification (such as ribosome binding site mutations), and biofilm formation. The florfenicol nanoliposomes prepared by the present invention are novel drug carriers. By encapsulating the drug in liposomes to form a liquid drug delivery system with a nanometer particle size, it is a convenient method for reducing antibiotic resistance. The liposomes can also effectively enhance the targeting of florfenicol and allow targeted treatment of specific bacteria such as Salmonella, which is a potential application method for treating bacterial infections using antibiotics.

[0020] (3) The liposomes of the present invention also have controlled release properties. By adjusting their composition and preparation process, the liposomes can achieve continuous or slow release of drugs. This property helps to simulate the physiological secretion process, reduce the frequency of drug use in animals, and improve animal compliance. The soybean lecithin in the liposome preparation material has good biocompatibility and high safety, so the problem of solvent residue can be effectively solved. The preparation method used in the present invention is a thin film dispersion method. The film produced in this method is extruded for granulation and purified by ultrafiltration to obtain liposomes with uniform particle size that meet safety requirements for use. In addition, cholesterol, as an amphiphilic neutral lipid, can penetrate into the phospholipid membrane at high concentrations and enhance the stability of the liposomes. The cholesterol used in the present invention is relatively high in concentration, so it can greatly enhance the stability of the liposomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a particle size distribution diagram of florfenicol nanoliposomes prepared in Example 3;

[0022] Figure 2 This is the Zeta potential distribution diagram of the florfenicol nanoliposomes prepared in Example 3;

[0023] Figure 3 This is the apparent morphology of the florfenicol nanoliposomes prepared in Example 3;

[0024] Figure 4 is a transmission electron micrograph of the florfenicol nanoliposomes prepared in Example 3;

[0025] Figure 5 is a graph showing the in vitro simulated gastric fluid release kinetics of florfenicol and florfenicol nanoliposomes in Example 6;

[0026] Figure 6 3 is a graph showing the in vitro simulated intestinal fluid release kinetics of florfenicol and florfenicol nanoliposomes in Example 6. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0028] Florfenicol API: Florfenicol powder, purchased from Shanghai MacLean Biochemical Technology Co., Ltd., approval number: Veterinary Drug C15102601;

[0029] Florfenicol commercially available drug: Florfenicol soluble powder, purchased from Hefei Qianfang Animal Health Technology Co., Ltd.; approval number: veterinary drug number 120152539.

[0030] The Salmonella standard strain ATCC13311 was purchased from China Veterinary Microbiology and Culture Collection (CVCC).

[0031] Example 1

[0032] (1) Weigh appropriate amounts of florfenicol, cholesterol, and soybean lecithin, add an appropriate amount of methanol-chloroform solvent (1:1, v / v) to completely dissolve them, and obtain a mixed solution;

[0033] (2) removing the organic solvent from the mixed solution obtained by dissolving in step (1) by rotary evaporation under reduced pressure, adding an appropriate amount of pure water, and completely dissolving under appropriate conditions to obtain a primary product;

[0034] (3) The primary product obtained in step (2) is subjected to ultrasonic treatment to obtain light yellow milk-like florfenicol liposomes.

[0035] Optimization process of preparation conditions:

[0036] (1) Determine the optimal ratio of florfenicol to methanol-chloroform

[0037] 0.4 mg of florfenicol was dissolved in 20 to 30 mL of methanol-chloroform respectively. It was finally found that 0.4 mg of florfenicol was dissolved in 24 mL of methanol-chloroform to achieve a better dissolution effect.

[0038] (2) Optimization of the ratio of florfenicol to cholesterol and soy lecithin

[0039] Soy lecithin, florfenicol, and cholesterol were dissolved in 24 mL of methanol-chloroform at a mass ratio of 20:4:5. The optimized ratios of florfenicol to cholesterol and soybean lecithin in the nanoliposomes were determined by zeta potential and PDI measurements, as shown in Table 1. The optimization criteria were primarily based on selecting a formulation with a high zeta potential and a PDI value <0.3.

[0040] (3) The organic solution was transferred to a distillation flask and subjected to rotary evaporation. The evaporation temperature was set at 25-50°C to evaporate the methanol. It was found that 30-40°C was sufficient. After 25-40 minutes, a lipid film appeared on the surface of the distillation flask. Ultrapure water was then added to the distillation flask to dissolve the transparent lipid film. The amount of ultrapure water added was 20-30 mL. The amount of ultrapure water added was determined by measuring the Zeta potential and PDI value. The Zeta potential and PDI values were similar when the amount of ultrapure water added was 20-30 mL. Therefore, the amount of ultrapure water added was determined to be 24 mL. The distillation flask needed to be ultrasonically dissolved in an ultrasonic cleaner for 2-6 minutes to obtain the primary product of nanoliposomes. Ultrasonic dissolution was performed in an ultrasonic cleaner for 1-8 minutes. It was found that the lipid film could be dissolved by ultrasonic dissolution in an ultrasonic cleaner for 2-6 minutes.

[0041] (4) The primary product of nanoliposomes needs to be ultrasonically disrupted using an ultrasonic cell disruptor (200-300 mV, 2-6 min) to obtain the final product of florfenicol nanoliposomes. The ultrasonic cell disruptor is screened by (150-400 mV, 1-10 min) and (200-300 mV, 2-6 min) conditions.

[0042] Table 1 Prescription screening and parameter measurement results of florfenicol nanoliposomes (n=3, mean±SD)

[0043]

[0044] According to the screening in Table 1 above, it can be seen that the mass ratio of soybean lecithin, florfenicol and cholesterol is 15-25:4:5, which has a better effect, and the most preferred ratio is 20:4:5.

[0045] Therefore, the final determination was that when the mass ratio of soybean lecithin, florfenicol, and cholesterol was 15-25:4:5, that is, 0.4g of florfenicol, 0.5g of cholesterol, and 15-25g of soybean lecithin were dissolved in 24mL of methanol-chloroform, rotary evaporated at 25-50°C for 25-40min, 20-30mL of ultrapure water was added, and ultrasonic dissolution was performed for 2-6min to obtain a primary product. The primary product was then ultrasonicated at 200-300mV for 2-6min to obtain florfenicol nanoliposomes with the best effect. In particular, the florfenicol nanoliposomes obtained by dissolving 0.4g of florfenicol, 0.5g of cholesterol, and 20g of soybean lecithin in 24mL of methanol-chloroform had the best effect.

[0046] Example 2

[0047] Materials: Florfenicol 0.4 g, cholesterol 0.5 g, soybean lecithin 15 g, methanol-chloroform 24 mL, ultrapure water 24 mL

[0048] Preparation method: Dissolve 0.4 mg of florfenicol, 0.5 g of cholesterol and 15 g of soy lecithin in 24 mL of methanol-chloroform to obtain a mixed solution. The mixed solution is then transferred to a distillation flask and rotary evaporated at 35°C. At the same time, the methanol will evaporate in a sealed state. After 30 minutes, a lipid film will appear on the surface of the distillation flask. Add 24 mL of ultrapure water to the distillation flask to dissolve the lipid film. At the same time, the distillation flask needs to be ultrasonically dissolved in an ultrasonic cleaning machine for 3 minutes to obtain the primary product of polymer micelles. Finally, the primary product of nanoliposomes needs to be ultrasonically crushed using an ultrasonic cell crusher (280 mV, 4 minutes) to obtain the final product of florfenicol nanoliposomes.

[0049] The particle size and Zeta potential of the florfenicol nanoliposomes prepared in this example were detected using a Malvern laser particle size analyzer. The results showed that the particle size of the florfenicol nanoliposomes was 301.6±19.1 nm, the Zeta potential value was -13.7 mV±1.1, and the polydispersity coefficient was 0.29±0.18.

[0050] The entrapment efficiency of the florfenicol nanoliposomes prepared in this example was determined by high performance liquid chromatography to be 88.67%±0.36, and the drug loading was 6.41%±0.28.

[0051] Example 3

[0052] Materials: Florfenicol 0.4 g, cholesterol 0.5 g, soybean lecithin 20 g, methanol-chloroform 24 mL, ultrapure water 24 mL

[0053] Preparation method: Dissolve 0.4 mg of florfenicol, 0.5 g of cholesterol and 20 g of soy lecithin in 24 mL of methanol-chloroform to obtain a mixed solution. The mixed solution is then transferred to a distillation flask and rotary evaporated at 35°C. At the same time, methanol will evaporate in a sealed state. After 30 minutes, a lipid film will appear on the surface of the distillation flask. Add 24 mL of ultrapure water to the distillation flask to dissolve the lipid film. At the same time, the distillation flask needs to be ultrasonically dissolved in an ultrasonic cleaning machine for 3 minutes to obtain the primary product of polymer micelles. Finally, the primary product of nanoliposomes needs to be ultrasonically crushed using an ultrasonic cell crusher (280 mV, 4 minutes) to obtain the final product of florfenicol nanoliposomes.

[0054] The particle size distribution of the florfenicol nanoliposomes prepared in this example is shown in the figure below: Figure 1 As shown, the obtained micelles were subjected to nanoliposome measurement using a Malvern laser particle size analyzer, and the average particle size was measured to be 638.2±17.5nm. The Zeta potential distribution diagram is shown in Figure 2 As shown, the obtained micelles were measured by potential using a Malvern laser particle size analyzer, and the measured Zeta potential value was -46.3±0.7mV, indicating that the system had good stability; the polydispersity index (PDI) was 0.22±0.03, indicating that the particle size was uniform.

[0055] The morphology of the florfenicol nanoliposomes prepared in this example was observed and found to be Figure 3 As shown in Figure 2, the prepared FL is a relatively uniform, thick, light yellow milky liquid. Further transmission electron microscopy analysis of its microscopic morphology is performed, and the transmission electron microscopy photos are shown in Figure 2. Figure 4 As shown. Figure 4It can be seen that the FL observed under transmission electron microscopy showed a relatively uniform particle size and smooth surface spheres. The entrapment efficiency of the florfenicol nanoliposomes prepared in this example was determined by high performance liquid chromatography to be 92.94% ± 0.86 and the drug loading was 6.29% ± 0.22.

[0056] Example 4

[0057] Materials: Florfenicol 0.4 g, cholesterol 0.5 g, soybean lecithin 25 g, methanol-chloroform 24 mL, ultrapure water 24 mL

[0058] Preparation method: Dissolve 0.4 mg of florfenicol, 0.5 g of cholesterol and 25 g of soy lecithin in 24 mL of methanol-chloroform to obtain a mixed solution. The mixed solution is then transferred to a distillation flask and rotary evaporated at 35°C. At the same time, methanol will evaporate under a closed state. After 30 minutes, a lipid film will appear on the surface of the distillation flask. Add 24 mL of ultrapure water to the distillation flask to dissolve the lipid film. At the same time, the distillation flask needs to be ultrasonically dissolved in an ultrasonic cleaning machine for 3 minutes to obtain the primary product of polymer micelles. Finally, the primary product of nanoliposomes needs to be ultrasonically crushed using an ultrasonic cell crusher (280 mV, 4 minutes) to obtain the final product of florfenicol nanoliposomes.

[0059] The particle size of the florfenicol nanoliposomes prepared in this example was measured using a Malvern laser particle size analyzer: the particle size of the florfenicol nanoliposomes prepared in this example was 249.6±62.7 nm, the Zeta potential value was -22.4±0.3 mV, and the polydispersity coefficient was 0.86±0.12.

[0060] The entrapment efficiency of the florfenicol nanoliposomes prepared in this example was determined by high performance liquid chromatography to be 93.23%±0.96, and the drug loading was 3.40%±0.19.

[0061] Example 5

[0062] Materials: Florfenicol 0.4 g, cholesterol 0.25 g, soybean lecithin 20 g, methanol-chloroform 24 mL, ultrapure water 24 mL

[0063] Preparation method: Dissolve 0.4 mg of florfenicol, 0.25 g of cholesterol and 20 g of soy lecithin in 24 mL of methanol-chloroform to obtain a mixed solution. The mixed solution is then transferred to a distillation flask and rotary evaporated at 35°C. At the same time, the methanol will evaporate in a sealed state. After 30 minutes, a lipid film will appear on the surface of the distillation flask. Add 24 mL of ultrapure water to the distillation flask to dissolve the lipid film. At the same time, the distillation flask needs to be ultrasonically dissolved in an ultrasonic cleaning machine for 3 minutes to obtain the primary product of polymer micelles. Finally, the primary product of nanoliposomes needs to be ultrasonically crushed using an ultrasonic cell crusher (280 mV, 4 minutes) to obtain the final product of florfenicol nanoliposomes.

[0064] The particle size of the florfenicol nanoliposomes prepared in this example was measured using a Malvern laser particle size analyzer: the particle size of the florfenicol nanoliposomes prepared in this example was 649.6±32.7 nm, the Zeta potential value was -15.4±0.3 mV, and the polydispersity coefficient was 0.56±0.12.

[0065] The florfenicol nanoliposome prepared in the present embodiment was measured to have an encapsulation efficiency of 86.23% ± 0.96 by high performance liquid chromatography, and a drug loading of 3.10% ± 0.06. Compared to the cholesterol content in Example 3, the cholesterol content in the present embodiment was relatively low, resulting in uneven distribution and poor stability of the liposome. Cholesterol, as an amphipathic neutral lipid, can penetrate into the phospholipid membrane at high concentrations, thereby enhancing the stability of the liposome. Therefore, the cholesterol content in the formula is relatively higher, and the cholesterol content of 0.5g in the selection embodiment 3 is more suitable.

[0066] Example 6 In vitro release kinetics test

[0067] The florfenicol nanoliposomes (FL) and pure florfenicol (FFC) prepared in Example 2-4 were subjected to in vitro release kinetics tests, and the in vitro release kinetics curves were obtained using high performance liquid chromatography (see Cheng Peipei, Yang Yajun, Liu Xiwang, et al. Establishment of an HPLC method for determination of florfenicol in a new compound florfenicol injection [J]. Advances in Veterinary Medicine, 2014, 35(10): 52-56).

[0068] 3.0 mL of a 2 mg / mL FL solution and 3 mL of a FFC solution in an acetone-water mixture (5:95, v / v) were accurately measured and placed in dialysis bags with molecular weight cutoffs ranging from 8000 to 14000. The dialysis bags were then placed in a reagent bottle containing 300 mL of release medium (simulated gastric fluid at pH 12 and simulated intestinal fluid at pH 6.8, respectively, prepared according to the standards of the Chinese Pharmacopoeia). The solutions were shaken in a thermostatic water bath at (37 ± 0.5)°C and 100 rpm. 3 mL of release medium was sampled at 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 10 h, and 20 h, and an equal volume of fresh release medium was added to maintain a constant volume. The sampled release medium was filtered through a 0.22 μm aqueous filter, and the FFC concentration was determined by HPLC.

[0069] The concentration of florfenicol in the release medium was determined by high performance liquid chromatography, and the release percentage was calculated and the release curve was drawn to study the release characteristics of the drug-loaded liposomes. The test process of FFC is the same as that of FL. The results are shown in Figure 2. Figure 5 and Figure 6 As shown, compared with florfenicol, the same in simulated gastric fluid ( Figure 5 ) and simulated intestinal fluid ( Figure 6 ) (under normal physiological conditions), the florfenicol nanoliposomes prepared by the present invention have obvious sustained-release and controlled-release effects.

[0070] Example 7 In vitro minimum inhibitory concentration test of florfenicol nanoliposomes (FL) against Salmonella standard strains

[0071] After culturing the Salmonella standard strain ATCC13311 for 4-6 hours, the turbidity of the bacterial solution was adjusted to 0.5 McFarland turbidimetric concentration (equivalent to 1×10 8 CFU / mL), and diluted it 1:100 with MH broth to obtain a bacterial suspension for inoculation in the in vitro minimum inhibitory concentration test.

[0072] The florfenicol nanoliposomes prepared in Example 2-4 were subjected to an in vitro minimum inhibitory concentration test. The florfenicol raw material (FFC-API), the commercial florfenicol drug (FFC-CAM), the florfenicol liposomes in the reference document (FL-Reference, referred to as FL-R, see Zhengzhou Houyi Pharmaceutical Co., Ltd. A method for preparing florfenicol liposomes: 201210325291.2 [P]. 2014-06-11.) and the florfenicol nanoliposomes (FL) prepared in Example 2-4 were mixed with MH broth to make the final florfenicol concentrations of 2, 4, 8, 16, 32, and 64 μg / mL. The growth of the Salmonella standard strain ATCC13311 added to the above drug-containing solution was observed by naked eye to determine the minimum inhibitory concentration.

[0073] The broth microdilution method was used, mainly using MH liquid medium to dilute the FFC-API, FFC-CAM, and FL solutions in series to prepare a series of drug solutions with a concentration gradient of 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL. Salmonella was inoculated into a 96-well plate containing the above drug solution so that the final bacterial solution concentration in each well was approximately 5×10 8 CFU / mL. A positive control well containing no drug and a negative control well containing sterile solution were also set up. The 96-well plate was incubated in a 37°C incubator for 16-20 hours, and bacterial growth in each well was observed. The lowest drug concentration at which no bacterial growth was observed by naked eye was defined as the MIC value for the antimicrobial drug against Salmonella. The results are shown in Table 2.

[0074] Table 2 Minimum inhibitory concentration of FL against Salmonella standard strains

[0075]

[0076] As shown in Table 2, the MIC values of FFC-CAM, FFC-API, and FL-R against the Salmonella standard strain were all 8 μg / mL, while the MIC value of FL prepared in Examples 2-4 against ATCC13311 was 4-6 μg / mL, especially the MIC value of FL prepared in Example 3 against ATCC13311 was 4 μg / mL. The MIC value of FFC-API against ATCC13311 was within the quality control range, indicating that the test results were reliable.

[0077] Example 8 In vitro minimum bactericidal concentration test of FL against Salmonella standard strain

[0078] The Salmonella standard strain ATCC13311 was cultured for 4-6 hours and the turbidity of the bacterial solution was adjusted to 0.5 McFarland turbidimetric concentration (1×108 CFU / mL), which was diluted 1:100 with MH broth to obtain a bacterial suspension for inoculation in the in vitro minimum bactericidal concentration test.

[0079] The florfenicol nanoliposomes prepared in Example 2-4 were subjected to an in vitro minimum bactericidal concentration test. Using the micro-two-fold broth dilution method, the florfenicol API, the commercially available florfenicol drug, FL-R, and the florfenicol nanoliposomes prepared in Example 2-4 were mixed with MH broth, and the Salmonella standard strain ATCC13311 was added. After incubation, the mixed solution was aspirated and added to a tryptic soy agar medium for growth. The number of colonies in each tryptic soy agar medium was recorded to determine the minimum inhibitory concentration.

[0080] After determining the MIC, 100 μL of culture medium without bacterial growth was pipetted onto the plates, with three replicates per well. After complete absorption of the liquid, the plates were incubated at 37°C for 18–24 hours. Colonies were then counted. The minimum bactericidal concentration (MBC) was determined if the colony count was less than or equal to the minimum concentration of 0.1% of the inoculum per well (≤2 colonies per plate). The results are shown in Table 3.

[0081] Table 3 Minimum bactericidal concentration of FL against Salmonella standard strains

[0082]

[0083] As shown in Table 3, post-culture MBC values determined by colony counts showed that FFC-API, FFC-CAM, and FL-R had MBC values of 16 μg / mL, 64 μg / mL, and 16 μg / mL, respectively. The MBC values for FL prepared in Examples 2-4 were 12 μg / mL, 8 μg / mL, and 12 μg / mL, respectively. These results demonstrate that compared to FFC-API, FFC-CAM, and FL-R, FL prepared in Examples 2-4, particularly Example 3, exhibited stronger inhibitory activity against Salmonella growth. In summary, FL exhibits excellent antibacterial activity.

[0084] Example 9 Minimum inhibitory concentration test of FL against drug-resistant strains of Salmonella

[0085] After culturing the drug-resistant Salmonella strains (as shown in Table 4) for 4 to 6 h, the turbidity of the bacterial solution was adjusted to 0.5 McFarland turbidimetric concentration (equivalent to 1×10 8 CFU / mL), which were diluted 1:100 with MH broth to obtain five groups of bacterial suspensions for inoculation in the in vitro minimum inhibitory concentration test.

[0086] Table 4 Information on drug-resistant strains of Salmonella

[0087] Strain name Serotype Strain source 18s～2 Salmonella Typhimurium CMCC(B)50094 Shu1 Salmonella Typhimurium CMCC(B)50093 Shu2 Salmonella Typhimurium CMCC(B)50093 S8 Salmonella Typhi VIP(S)10032 S15 Salmonella Typhi VIP(S)10032

[0088] Note: The strains in Table 4 were purchased from China Veterinary Microbiology and Virus Culture Collection Center (CVCC).

[0089] The florfenicol API (FFC-API), the commercial florfenicol drug (FFC-CAM), the florfenicol liposomes in the reference document (FL-Reference, abbreviated as FL-R, see Zhengzhou Houyi Pharmaceutical Co., Ltd. A method for preparing florfenicol liposomes: 201210325291.2[P].2014-06-11.), and the florfenicol nanoliposomes (FL) prepared in Example 2-4 were mixed with MH broth to obtain final florfenicol concentrations of 8, 16, and 32 μg / mL. The growth of drug-resistant Salmonella strains added to the above drug-containing solutions was observed by visual observation to determine the minimum inhibitory concentration.

[0090] The minimum inhibitory concentration (MIC) of florfenicol nanoliposomes was determined using a two-fold broth microdilution method: 100 μL of the aforementioned solutions containing varying concentrations of florfenicol API, commercially available florfenicol, FL-R, and FL were added to the first well of a 96-well plate. A gradient dilution was performed with MH broth, followed by addition of 90 μL of MH broth. Each well was inoculated with 10 μL of the bacterial suspension previously used for inoculation. A negative control (broth alone) and a positive control (no drug) were also set up. After capping and incubation at 37°C for 18–24 hours, the culture medium was visually observed. The minimum inhibitory concentration (MIC) for resistant Salmonella strains was determined. The results are shown in Table 5.

[0091] Table 5 Minimum inhibitory concentration of FL against drug-resistant strains of Salmonella

[0092]

[0093]

[0094]

[0095] As shown in the table, all five clinical isolates tested were resistant to FFC, with only 18s-2 being sensitive. Furthermore, when measuring the MICs of FFC-CAM, FFC-API, FL-R, and FL against the resistant Salmonella strain 18s-2, FL had the lowest MIC value, at 8 μg / mL. These results indicate that FL has excellent antibacterial activity against the resistant Salmonella strain 18s-2.

[0096] Example 10: Minimum bactericidal concentration test of FL against drug-resistant strains of Salmonella

[0097] After determining the MIC value of R-FL against the drug-resistant Salmonella strain 18s-2, culture fluid containing no bacterial growth at concentrations of 4 MIC, 2 MIC, and MIC was applied to plates, and the MBC values were determined by colony count. The results are shown in the table. Compared to FFC-CAM, FFC-API, and FL-R, FL prepared in Example 3 had the lowest MBC value of 34 μg / mL. This indicates that FL effectively inhibits the growth of the drug-resistant Salmonella strain 18s-2.

[0098] Table 6 Minimum bactericidal concentration of FL against drug-resistant strains of Salmonella

[0099]

[0100]

[0101] The above analysis results show that the florfenicol nanoliposomes prepared by the present invention have an average particle size of 249.6 to 638.2 nm and a zeta potential of -13.7 to -46.3 mV, reflecting the good mutual repulsion between the particles. The system is stable and has a polydispersity coefficient of 0.22 to 0.86. Under an electron microscope, the micelles exhibit a relatively uniform, smooth-surfaced sphere with no aggregation between the particles. The encapsulation efficiency measured by high performance liquid chromatography is 88.31% to 92.94% and the drug loading is 3.21% to 6.29%. The florfenicol nanoliposomes prepared by the present invention can play a very excellent sustained-release and controlled-release effect on florfenicol in simulated gastric and intestinal fluids. The minimum inhibitory concentration of this product for a standard strain of Salmonella is 4 μg / mL, and the minimum bactericidal concentration is 8 μg / mL. The minimum inhibitory concentration of this product against drug-resistant strains of Salmonella is 8μg / mL, and the minimum bactericidal concentration is 34μg / mL.

Claims

1. A florfenicol nanoliposome, characterized in that The florfenicol nanoliposomes are prepared from the following components in a mass ratio: soybean lecithin: florfenicol: cholesterol (10-30): 4:

5.

2. The florfenicol nanoliposome according to claim 1, wherein The average particle size of florfenicol liposomes is 249.6-638.2 nm, the Zeta potential value is -13.7-46.3 mV, and the polydispersity coefficient is 0.22-0.

86.

3. The florfenicol nanoliposome according to claim 2, wherein The encapsulation efficiency of florfenicol-TPGS liposomes was 88.31% to 92.94%, and the drug loading amount of florfenicol was 3.21% to 6.29%.

4. The method for preparing the florfenicol nanoliposome according to any one of claims 1 to 3, characterized in that: The following steps are involved: Florfenicol, cholesterol and soybean lecithin were dissolved in an organic solvent, evaporated by rotary evaporation, dissolved by ultrasonication in water, and crushed by ultrasonication.

5. The method for preparing florfenicol nanoliposomes according to claim 4, wherein The mass volume ratio of florfenicol to the organic solvent is (0.1-1):(20-30) g / mL, the volume ratio of water to the organic solvent is (0.5-1.5):(0.5-1.5), and the organic solvent is a methanol-chloroform mixture with a volume ratio of (0.5-1.5):(0.5-1.5).

6. The method for preparing florfenicol nanoliposomes according to claim 4, wherein The temperature of the rotary evaporation is 25-50° C., the time of the rotary evaporation is 25-40 minutes, and the time of the ultrasonic dissolution is 2-6 minutes.

7. The method for preparing florfenicol nanoliposomes according to claim 4, wherein The voltage of ultrasonic fragmentation is 200-300 mV, and the time of ultrasonic fragmentation is 2-6 minutes.

8. Use of the florfenicol nanoliposome according to any one of claims 1 to 3 in the preparation of antibiotic drugs.

9. The use according to claim 8, characterized in that The pathogen is one or more of Gram-positive bacteria, Gram-negative bacteria or mycoplasma.

10. The use according to claim 9, characterized in that Gram-negative bacteria include one or more of Salmonella, Escherichia coli, or Shigella.

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