Florfenicol nano-liposome modified by lactoferrin as well as preparation method and application of florfenicol nano-liposome

Abstract

The invention discloses a lactoferrin modified florfenicol nano-liposome (R-FL) as well as a preparation method and application thereof. The R-FL is prepared by adopting a technology of combining an improved reversed-phase solvent evaporation method and chemical coupling. The R-FL disclosed by the invention is strong in slow and controlled release capability and good in biocompatibility, has excellent in-vitro and in-vivo antibacterial effects, and can be used for inhibiting the formation of a biofilm by precisely damaging the structure of the biofilm of the drug-resistant salmonella typhimurium in a targeting manner. The R-FL can effectively regulate the intestinal flora disorder caused by the drug-resistant salmonella typhimurium, so that the intestinal flora is recovered to a normal level. The R-FL can be used as a novel nano preparation with great potential for resisting drug-resistant salmonella typhimurium disease, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a lactoferrin-modified florfenicol nanoliposome and a preparation method and application thereof, in particular to a florfenicol nanoliposome capable of inhibiting multi-drug resistant pathogenic bacteria (resistant to multiple antibiotics) and a preparation method and application thereof, and belongs to the technical field of liposome preparations. BACKGROUND

[0002] Salmonella belongs to gram-negative bacilli and belongs to Enterobacteriaceae. Because Salmonella widely exists in nature, it often causes gastrointestinal diseases in animals and humans. Salmonella infection can cause various diseases, including gastroenteritis, bacteremia, sepsis and local infection, and can cause animal death in severe cases. About one-fourth of the global incidence and mortality associated with diarrhea is related to Salmonella. Salmonella enhances its resistance to antibiotics through biofilm, enabling it to survive for a long time in the host or environment. Due to its widespread resistance to antibiotics such as ampicillin, streptomycin, tetracycline and florfenicol, it has become a major challenge in the field of public health.

[0003] Florfenicol is a broad-spectrum amphenicol 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 has significant inhibitory activity against multi-drug resistant gram-negative bacteria, including Escherichia coli, Acinetobacter baumannii and Klebsiella pneumoniae. In the treatment of digestive diseases, florfenicol has good efficacy against intestinal infections caused by pathogenic bacteria such as Salmonella and Escherichia coli, but its oral bioavailability is low due to its bitter taste and susceptibility to gastric acid degradation, limiting its clinical use. In addition, bacterial resistance is one of the important factors limiting its widespread use. Studies have shown that gram-negative bacteria can escape the inhibitory effect of florfenicol through various mechanisms, including upregulation of drug efflux pumps, target modification (such as ribosome binding site mutation) and biofilm formation.

[0004] Long-term and large-scale use of florfenicol can lead to drug resistance in some bacteria. Therefore, rational use of florfenicol, development of new formulations and exploration of combination therapy regimens have become a hot research topic. Florfenicol formulations are diverse, mainly including oral and topical preparations. Oral formulations need to overcome the first-pass effect and gastrointestinal degradation, and often use microencapsulation technology or combination with lipid carriers to improve bioavailability. Today, the development of new delivery systems for florfenicol has solved the limitations of traditional oral formulations in terms of targeting, stability and efficacy.

[0005] At present, the nano delivery system of florfenicol mainly includes core-shell composite nanogel and other nano carrier systems, and alginate hydrogel and other hydrogel sustained-release systems. Compared with other nano delivery systems, liposomes have excellent biocompatibility and can protect drugs and have other advantages. Liposomes are a kind of nanoscale vesicular structure formed by phospholipid bilayer membrane, which has a hydrophilic core and a hydrophobic membrane layer, and can load various types of drugs or nucleic acid molecules (such as mRNA, siRNA). The core structure of the liposome is a lipid bilayer, which is formed depending on the self-assembly characteristics of the amphiphilic lipid molecules. In order to improve the targeting and stability of the liposome, the surface of the liposome is often modified. Commonly used modification materials include polyethylene glycol (PEG), antibodies and peptides.

[0006] Although liposomes show strong potential in antibacterial therapy, their clinical translation still faces great challenges. Large-scale production of liposomes needs to solve the problems of stability, drug loading efficiency and cost. In addition, the rapid evolution of drug-resistant bacteria requires the design of liposomes to adapt to new antibacterial mechanisms according to the overexpression of efflux pumps or changes in membrane components. In addition, since the liposome does not have obvious targeting for drug release in the body, the use of lactoferrin with intestinal targeting can achieve targeted delivery.

[0007] Lactoferrin is a multifunctional glycoprotein belonging to the transferrin family, which is widely present in the external secretions of mammals (such as milk, saliva, tears) and neutrophils. Lactoferrin can limit the availability of iron to pathogenic microorganisms through its superior iron-binding capacity, thereby inhibiting the growth of bacteria, fungi and viruses. Lactoferrin is an important part of the innate immune system and can directly destroy bacterial cell membranes and enhance the bactericidal effect of antibiotics. Lactoferrin can bind to lactoferrin receptors on the surface of intestinal epithelial cells to achieve efficient targeted delivery. In addition, lactoferrin has the ability to protect small intestinal epithelial cells and enhance the integrity of the intestinal barrier. Therefore, lactoferrin can target the inflamed colon cells to significantly improve the uptake efficiency of the inflamed colon cells, and a low dose can achieve a therapeutic effect. SUMMARY

[0008] The purpose of the present application is to solve the problems existing in the prior art, the first purpose of the present application is to provide a lactoferrin modified florfenicol nano-liposome which can inhibit multiple drug-resistant pathogenic bacteria (resistant to multiple antibiotics), the second purpose of the present application is to provide a preparation method of the lactoferrin modified florfenicol nano-liposome, and the third purpose of the present application is to provide an application of the lactoferrin modified florfenicol nano-liposome in preparing antibiotic drugs.

[0009] Technical solution: The lactoferrin modified florfenicol nanoliposome provided by the application is characterized in that the florfenicol nanoliposome is prepared from the following components with the following mass ratio: DSPE-PEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol 2000): soybean lecithin: florfenicol: cholesterol is (1-3): (18-22): (28-36): (2-6), and the mass ratio of the florfenicol nanoliposome to lactoferrin is (0.3-2): 1.

[0010] Further, preferably, the mass ratio of DSPE-PEG2000: soybean lecithin: florfenicol: cholesterol is (1.5-2.5): (19-21): (31-34): (3-5), and most preferably, the mass ratio of DSPE-PEG2000: soybean lecithin: florfenicol: cholesterol is 2:20:32:4, and preferably, the mass ratio of the florfenicol nanoliposome to lactoferrin is 1:1.

[0011] Further, the average hydrodynamic size of the lactoferrin modified florfenicol nanoliposome is 170.5-195.0 nm, preferably 171.7±1.2 nm, the Zeta potential value is -37.1--50.0 mV, preferably -49.8±0.2 mV, the polydispersity coefficient is 0.24-0.38, preferably 0.26±0.02, the encapsulation rate of the lactoferrin modified florfenicol nanoliposome is 94.84%-97.39%, and the drug loading of florfenicol is 3.00%-4.10%.

[0012] The preparation method of the lactoferrin modified florfenicol nanoliposome provided by the application comprises the following steps:

[0013] (1) Dissolve DSPE-PEG2000, soybean lecithin and cholesterol in anhydrous ethanol, ultrasonically dissolve, rotary evaporate, and obtain blank liposomes.

[0014] (2) Swell florfenicol in a mixed solution of acetone and water, then add to the blank liposomes, ultrasonically dissolve, centrifuge, wash, resuspend in water, add lactoferrin, and ultrasonically dissolve under heating.

[0015] Further, in step (1), the ultrasonic dissolution time is 16-24 min, and the liquid is completely evaporated by rotary evaporation under reduced pressure. In step (2), the volume ratio of acetone to water is 5:95, the solid-liquid ratio of florfenicol to the mixed solution is (28-36):(6-10) mg / mL, preferably 32:8 mg / mL; the mass-volume ratio of florfenicol to deionized water for resuspending the washed liposomes is (28-36):(6-10) mg / mL, preferably (28-36):(7-9) mg / mL, and the mass-volume ratio of florfenicol to deionized water for resuspending the washed liposomes is (28-36):8 mg / mL. The ultrasonic dissolution time is 6-15 min, preferably 9-12 min, and most preferably 10 min. The ultrasonic dissolution temperature is 35-50℃, the heating water bath ultrasonic dissolution time is 6-15 min, preferably 9-12 min, and most preferably 10 min.

[0016] The lactoferrin-modified florfenicol nano-liposome can be used for preparing an antibiotic drug.

[0017] Further, the pathogen is one or more of gram-positive bacteria, gram-negative bacteria or mycoplasma. The gram-negative bacteria include one or more of Salmonella, Escherichia coli or Shigella.

[0018] The lactoferrin-modified florfenicol nano-liposome prepared by the present application can effectively solve the problems of stability, drug loading efficiency and cost in large-scale production of liposomes. In addition, the rapid evolution of drug-resistant bacteria requires that the design of liposomes should adapt to new antibacterial mechanisms according to the overexpression of efflux pumps or the dynamic changes of membrane components. Lactoferrin is an important component of the innate immune system, which can directly destroy bacterial cell membranes and enhance the bactericidal effect of florfenicol. Lactoferrin can bind to lactoferrin receptors on the surface of intestinal epithelial cells to achieve efficient targeted delivery. This receptor-mediated targeting mechanism enables lactoferrin and its related drug carriers to precisely act on specific sites in the intestinal tract, thereby improving the therapeutic effect and reducing side effects.

[0019] Advantages: Compared with the prior art, the present application has the following significant advantages:

[0020] (1) The preparation method is simple and easy to operate, belongs to a kind of cosolvent technology, which can effectively reduce production cost. The liposome prepared by the method combining chemical coupling and thin film hydration method has the following advantages: ① Targeting modification accuracy and flexibility. On the one hand, through chemical coupling, lactoferrin and other targeting molecules can be accurately connected to the surface of the liposome. On the other hand, by adjusting the mass ratio or adding proportion of the reactants, the antibody / ligand density on the surface of the liposome can be accurately controlled, which directly affects the target cell binding strength and internalization rate. ② Enhanced structural stability. Compared with the physical embedding of the insertion method, the chemical coupling forms a covalent bond connection (such as amide bond, thioether bond), which effectively prevents the ligand from falling off in the body circulation, ensuring the persistence of the targeting function. ③ Preparation flexibility and controllability. The modular process design and quantitative optimization of reaction conditions are specific. ④ Industrialization adaptability. The chemical coupling process is compatible with conventional liposome production lines, which can integrate fluorescence labeling, targeting ligand and drug delivery functions in a single carrier, realizing the design of diagnosis and treatment integration.

[0021] (2) The prepared lactoferrin modified florfenicol nanoliposome is a semi-transparent light yellow liquid, the optimal hydrodynamic size is 171.7±1.2nm, and the optimal Zeta potential is-49.8±0.2mV. Higher Zeta potential can better ensure that the florfenicol nanoliposome can stably exert antibacterial effect. Under electron microscope, it presents a three-dimensional bowl-shaped structure, and the shape is relatively uniform. The minimum inhibitory concentration for Salmonella standard strain is 2μg / mL, and the minimum bactericidal concentration is 4μg / mL, which can effectively inhibit the growth of Salmonella at a lower concentration. The minimum inhibitory concentration for drug-resistant Salmonella is 4μg / mL, and the minimum bactericidal concentration is 8μg / mL, which solves the problem of antibiotic resistance due to overuse of antibiotics, and reduces the effectiveness of clinical antibacterial drugs. In addition, lactoferrin in the liposome is an important part of the innate immune system, which can directly destroy the bacterial cell membrane and enhance the bactericidal effect of florfenicol. Lactoferrin has the ability to protect small intestinal epithelial cells and enhance the integrity of the intestinal barrier. Lactoferrin can reduce intestinal barrier damage by regulating the expression of intercellular tight junction proteins, thereby playing an important role in diseases such as inflammatory bowel disease. The liposome can effectively inhibit the intestinal flora disorder caused by drug-resistant murine typhus Salmonella, thereby restoring the balance of intestinal flora.

[0022] (3) Low bioavailability antibiotics such as florfenicol lack targeting because gram-negative bacteria can evade florfenicol inhibition through multiple mechanisms, including upregulation of drug efflux pumps, modification of the target (e.g., ribosome binding site mutations), and biofilm formation. The lactoferrin-modified florfenicol nano-liposome prepared in the present application belongs to a new type of drug carrier. Lactoferrin has many advantages as a drug carrier, specifically, it has good biocompatibility and degradability, and can be metabolized into non-toxic products in vivo. Lactoferrin has multiple receptor binding sites on its surface, enabling specific targeting. Lactoferrin activates immune cell receptors such as neutrophils, promotes granule release and secretion of inflammatory factors such as IL-1β, and regulates the composition of skin and gut microbiota. In terms of inflammatory targeting drug delivery, lactoferrin can recognize lactoferrin receptors overexpressed at inflammatory sites and low-pH environments. This property makes it an ideal carrier for the treatment of inflammatory diseases. In terms of intestinal targeting drug delivery, lactoferrin can specifically recognize lactoferrin receptors on the surface of intestinal epithelial cells.

[0023] (4) The lactoferrin-modified florfenicol nano-liposome in the present application also has controlled release properties. Liposomes can achieve sustained or slow release of drugs by adjusting their composition and preparation process. In addition, although liposomes show great potential in antibacterial therapy, their clinical translation still faces great challenges. Large-scale production of liposomes needs to address issues of stability, drug loading efficiency, and cost. In addition, the rapid evolution of drug-resistant bacteria requires liposome design to adapt to new antibacterial mechanisms according to the dynamic changes in efflux pump overexpression or membrane composition. The present application uses lactoferrin to modify the liposome, thus greatly enhancing the stability of the liposome. In addition, lactoferrin can also protect the drug from the damaging environment of the gastrointestinal tract, improving the stability and bioavailability of the drug. Lactoferrin can inhibit the proliferation of drug-resistant bacteria by depriving them of iron ions needed for growth, thereby indirectly protecting human and animal health. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a preparation process diagram of the lactoferrin-modified florfenicol nano-liposome in Example 3;

[0025] Figure 2 is a particle size distribution diagram of the lactoferrin-modified florfenicol nano-liposome prepared in Example 3;

[0026] Figure 3 is a Zeta potential distribution diagram of the lactoferrin-modified florfenicol nano-liposome prepared in Example 3;

[0027] Figure 4 is an apparent morphology diagram of the lactoferrin-modified florfenicol nano-liposome prepared in Example 3;

[0028] Figure 5 is a transmission electron microscope photograph of the lactoferrin-modified florfenicol nanoliposome prepared in Example 3;

[0029] Figure 6 is a Fourier transform infrared spectrogram of phospholipid-polyethylene glycol (DSPE-PEG2000), cholesterol (CHO), soy lecithin (SL), florfenicol (FFC), florfenicol nanoliposome (FL), and lactoferrin-modified florfenicol nanoliposome (R-FL) in Example 3;

[0030] Figure 7 is a graph of in vitro simulated gastric fluid release kinetics of florfenicol (FFC), florfenicol nanoliposome (FL), and lactoferrin-modified florfenicol nanoliposome (R-FL) in Example 6;

[0031] Figure 8 is a graph of in vitro simulated intestinal fluid release kinetics of florfenicol (FFC), florfenicol nanoliposome (FL), and lactoferrin-modified florfenicol nanoliposome (R-FL) in Example 6;

[0032] Figure 9 is a graph of the minimum inhibitory concentration determination results of florfenicol raw material (FFC-API), florfenicol commercial drug (FFC-CAM), FL, and R-FL against Salmonella standard strain ATCC13311 in Example 7;

[0033] Figure 10 is a graph of the minimum inhibitory concentration determination results of florfenicol raw material (FFC-API), florfenicol commercial drug (FFC-CAM), FL, and R-FL against Salmonella clinical strain 18s-2 in Example 9.

[0034] Figure 11 is a graph of the effect of R-FL on biofilm observed by scanning electron microscope in Example 11;

[0035] Figure 12 is a graph of the effect of R-FL on biofilm observed by laser scanning confocal microscope in Example 12;

[0036] Figure 13 is a schematic diagram of the establishment process of a Salmonella typhimurium infected mouse model and the therapeutic effect of R-FL on Salmonella typhimurium infected mice in Example 13;

[0037] Figure 14 is a graph of the effect of R-FL on the inflammatory response caused by drug-resistant Salmonella typhimurium analyzed by HE staining technology (main organs) in Example 13;

[0038] Figure 15Figure is the graph of the effect of R-FL on the inflammatory response caused by drug-resistant Salmonella typhimurium in Example 13 using HE staining technology (intestinal tissue);

[0039] Figure 16 Figure is the graph of the effect of R-FL on the inflammatory response caused by drug-resistant Salmonella typhimurium in Example 13 using immunohistochemical technology (liver and spleen);

[0040] Figure 17 Figure is the graph of the effect of R-FL on the biofilm of drug-resistant Salmonella typhimurium in the intestines of mice in Example 13 using scanning electron microscopy;

[0041] Figure 18 Figure is the graph of the effect of R-FL on the biofilm of drug-resistant Salmonella typhimurium in the intestines of mice in Example 13 using metabolomics analysis;

[0042] Figure 19 Figure is the graph of the results of the intestinal microbial targeting effect of R-FL on drug-resistant Salmonella typhimurium infected mice in Example 13 using IVIS live imaging analysis;

[0043] Figure 20 Figure is the graph of the effect of R-FL on the intestinal flora disorder caused by drug-resistant Salmonella typhimurium in Example 13 using 16sRNA sequencing analysis. DETAILED DESCRIPTION

[0044] The technical solutions of the present application will be further described below in combination with the drawings.

[0045] Florfenicol raw material: Florfenicol powder, purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., approval number: Veterinary Drug C15102601;

[0046] Florfenicol on the market: Florfenicol soluble powder, purchased from Hefei Qiangfang Animal Health Technology Co., Ltd.; approval number: Veterinary Drug 120152539.

[0047] Salmonella standard strain ATCC13311 was purchased from the National Veterinary Microbial Bacteria (Toxin) Culture Collection Center (CVCC).

[0048] Example 1

[0049] (1) 1-3 mg of DSPE-PEG2000, 18-22 mg of soybean lecithin, and 2-6 mg of cholesterol were precisely weighed with an analytical balance, dissolved in 2-6 mL of anhydrous ethanol, and ultrasonicated for 16-24 min until the solids were completely dissolved. The liquid was evaporated to complete volatilization under reduced pressure using a rotary evaporator at 35-50°C, and the blank liposomes were attached to the walls of a round-bottom flask.

[0050] (2) Precisely take 28-36 mg FFC and dissolve in 6-10 mL acetone-water (5:95, V / V) solution, after swelling, ultrasonic 6-15 min until the liposome completely from the wall of the bottle, can be prepared to get light yellow transparent liposome, recorded as FL.

[0051] (3) The liposome is transferred to the ultrafiltration tube (100KD), centrifuged at 5000 rpm 10℃ for 1h, the obtained product is added to deionized water and washed 3 times, the washed liposome is resuspended in 8 mL deionized water; the liposome is mixed with lactoferrin according to the mass ratio of (0.3-2):1, and then ultrasonic 6-15 min in water bath, to obtain lactoferrin modified florfenicol nanoliposome (R-FL), which is stored at 4℃ for standby.

[0052] Optimization process of preparation conditions:

[0053] (1) Optimization of the proportion of liposome components

[0054] Precisely take 1-3 mg DSPE-PEG2000, 18-22 mg soybean lecithin and 2-6 mg cholesterol with an analytical balance, dissolve in 2-6 mL anhydrous ethanol, ultrasonic 16-24 min until the solid is completely dissolved, evaporate to liquid completely with a rotary evaporator, and the blank liposome is pasted on the wall of the round bottom flask. Finally, it is found that 2 mg DSPE-PEG2000, 20 mg soybean lecithin and 4 mg cholesterol are precisely weighed with an analytical balance, dissolved in 4 mL anhydrous ethanol, ultrasonic 20 min until the solid is completely dissolved, evaporated to liquid completely with a rotary evaporator, and the blank liposome is pasted on the wall of the round bottom flask. The obtaining of the blank liposome film is attributed to the best proportion of DSPE-PEG2000, soybean lecithin, cholesterol and anhydrous ethanol.

[0055] (2) Optimization of the proportion of florfenicol liposome preparation

[0056] Precisely take 28-36 mg FFC and dissolve in 6-10 mL acetone-water (5:95, V / V) solution, after swelling, ultrasonic 6-15 min until the liposome completely from the wall of the bottle, can be prepared to get light yellow transparent liposome. Finally, it is found that 32 mg FFC is precisely weighed and dissolved in 8 mL acetone-water (5:95, V / V) solution, after swelling, ultrasonic 10 min until the liposome completely from the wall of the bottle, can be prepared to get light yellow transparent liposome. The obtaining of the light yellow transparent liposome is attributed to the best proportion of FFC and acetone-water (5:95, V / V) solution.

[0057] (3) Optimization of the proportion of florfenicol and lactoferrin

[0058] The liposome was transferred to an ultrafiltration tube (100KD), centrifuged at 5000 rpm at 10°C for 1h, and the resulting product was washed with deionized water 3 times. The washed liposome was resuspended in 8mL deionized water. The liposome was mixed with lactoferrin at a mass ratio of (0.3-2):1, and then ultrasonicated in a water bath for 6-15min to obtain lactoferrin-modified florfenicol nanoliposomes (R-FL), which were stored at 4°C for later use. Finally, the liposome was transferred to an ultrafiltration tube (100KD), centrifuged at 5000 rpm at 10°C for 1h, and the resulting product was washed with deionized water 3 times. The washed liposome was resuspended in 8mL deionized water. The liposome was mixed with lactoferrin at a mass ratio of 1:1, and then ultrasonicated in a water bath for 10min to obtain lactoferrin-modified florfenicol nanoliposomes (R-FL), which were stored at 4°C for later use.

[0059] The lactoferrin-modified florfenicol nanoliposomes were prepared by a combination of chemical coupling and the thin film hydration method (as shown in Figure 1 The optimization of the ratio of florfenicol to lactoferrin was determined by measuring the hydrodynamic size, Zeta potential and PDI, as shown in Table 1. The optimization criteria mainly selected the smallest hydrodynamic size, the highest absolute value of Zeta potential, and a PDI value <0.3.

[0060] Table 1 Prescription screening and parameter measurement results of lactoferrin-modified florfenicol nanoliposomes (n=3, mean±SD)

[0061]

[0062] Different mass ratios of liposomes and lactoferrin were mixed to modify the FL to prepare R-FL, and then the HD, ZP and PDI were determined. As shown in Table 1, the lactoferrin-modified liposome R-FL has a smaller hydrodynamic size, but the insufficient modification of lactoferrin leads to aggregation of the liposome, weak charge coverage and slightly poor uniformity. Excessive modification of ZP may cause charge shielding or loose structure. DSPE-PEG2000, soy lecithin and cholesterol are low in price and low in required amount in this process, which can save costs well.

[0063] Therefore, the R-FL with the mass ratio of FL to lactoferrin of 1:1 was selected for subsequent experiments. The hydrodynamic size of R-FL was 171.7±1.2 nm, the Zeta potential was -49.8±0.2 mV (in general, the absolute value of Zeta potential greater than 30 mV indicates that the material has good stability), and the PDI value was 0.26±0.02. Compared with FL, R-FL was still negatively charged and had a larger absolute value, indicating that it had more stable properties. The above results show that the R-FL has smaller particle size, more stable properties, and better characterization.

[0064] Example 2

[0065] Materials: DSPE-PEG2000 2 mg, soy lecithin 20 mg, cholesterol 4 mg, anhydrous ethanol 4 mL, FFC 32 mg, acetone-water (5:95, V / V) 8 mL, and the mass ratio of liposomes to lactoferrin was 0.5:1

[0066] Preparation method: accurately weigh 2 mg of DSPE-PEG2000, 20 mg of soy lecithin, and 4 mg of cholesterol with an analytical balance, dissolve them in 4 mL of anhydrous ethanol, and ultrasonicate for 20 min until the solids are completely dissolved. Evaporate the solution under reduced pressure using a rotary evaporator until the liquid is completely evaporated. Coat the blank liposomes on the wall of a round-bottom flask. Accurately weigh 32 mg of FFC and dissolve it in 8 mL of acetone-water (5:95, V / V) solution. After swelling, ultrasonicate for 10 min until the liposomes completely detach from the flask wall. A light yellow transparent liposome is obtained. Transfer the liposome to an ultrafiltration tube (100KD), centrifuge at 5000 rpm and 10°C for 1 h. The obtained product is washed with deionized water for 3 times. The washed liposome is resuspended in 8 mL of deionized water. Mix the liposome with lactoferrin according to the mass ratio of 0.5:1, and ultrasonicate for 10 min in a water bath. The lactoferrin-modified florfenicol liposome (R-FL) is obtained and stored at 4°C for standby.

[0067] The particle size and Zeta potential of the lactoferrin-modified florfenicol liposome (R-FL) prepared in this example were detected by Malvern laser particle size analyzer. The results showed that the hydrodynamic size of R-FL was 185.5±5.3 nm, the Zeta potential value was -38.6±1.5 mV±1.1, and the polydispersity coefficient was 0.34±0.04. The absolute value of the Zeta potential of the liposome is less than 30 mV, indicating that the material has lower stability.

[0068] The encapsulation efficiency of the lactoferrin-modified florfenicol nanoliposome prepared in this example was determined by high performance liquid chromatography to be 97.17%±0.22, and the drug loading was 3.11%±0.11.

[0069] Example 3

[0070] Materials: DSPE-PEG2000 2mg, soy lecithin 20mg, cholesterol 4mg, anhydrous ethanol 4mL, FFC 32mg, acetone-water (5:95, v / v) 8mL, liposomes to lactoferrin protein mass ratio 1:1

[0071] Preparation method: Accurately weigh 2 mg of DSPE-PEG2000, 20 mg of soybean lecithin, and 4 mg of cholesterol using an analytical balance. Dissolve them in 4 mL of anhydrous ethanol and sonicate for 20 min until the solid is completely dissolved. Evaporate under reduced pressure using a rotary evaporator until the liquid is completely evaporated. Place blank liposomes against the wall of a round-bottom flask. Accurately weigh 32 mg of FFC and dissolve it in 8 mL of acetone-water (5:95, V / V) solution. After sufficient swelling, sonicate for 10 min until the liposomes completely detach from the flask wall to obtain pale yellow transparent liposomes. Transfer the liposomes to an ultrafiltration tube (100 KD) and centrifuge at 5000 rpm at 10℃ for 1 h. Wash the product three times with deionized water and resuspend the washed liposomes in 8 mL of deionized water. Mix the liposomes with lactoferrin at a 1:1 mass ratio and sonicate in a water bath for 10 min to obtain lactoferrin-modified florfenicol liposomes (R-FL), which are stored at 4℃ for later use.

[0072] The particle size distribution of the lactoferrin-modified florfenicol nanoliposomes prepared in this embodiment is shown in the figure below. Figure 2 As shown, the obtained micelles were analyzed using a Malvern laser particle size analyzer to determine the nanoliposome size, and the average hydrodynamic size was found to be 171.7 ± 1.2 nm. The Zeta potential distribution is shown in the figure. Figure 3 As shown, the potential of the obtained liposomes was measured using a Malvern laser particle size analyzer. The measured Zeta potential value was -49.8±0.2mV, indicating that the system has good stability. The polydispersity index (PDI) was 0.26±0.02, indicating that the particle size was uniform.

[0073] The morphology of the lactoferrin-modified florfenicol nanoliposomes prepared in this embodiment was observed, and it was found that, for example... Figure 4 As shown, the prepared R-FL appears as a translucent, light yellow liquid to the naked eye. Further analysis of its microstructure using transmission electron microscopy (TEM) yielded the following TEM images: Figure 5 As shown. By Figure 5 As observed under transmission electron microscopy, the R-FLs modified with lactoferrin exhibit a three-dimensional, bowl-shaped structure with relatively uniform shape. The encapsulation efficiency of the lactoferrin-modified florfenicol nanoliposomes prepared in this example was determined by high-performance liquid chromatography to be 96.75% ± 0.39, and the drug loading was 4.02% ± 0.08.

[0074] Example 4

[0075] Materials: DSPE-PEG2000 2 mg, soybean lecithin 20 mg, cholesterol 4 mg, anhydrous ethanol 4 mL, FFC 32 mg, acetone-water (5:95, V / V) 8 mL, the mass ratio of liposome to lactoferrin is 1.5:1

[0076] Preparation method: precisely weigh 2 mg of DSPE-PEG2000, 20 mg of soybean lecithin and 4 mg of cholesterol with an analytical balance, dissolve them in 4 mL of anhydrous ethanol, and ultrasonicate for 20 min until the solids are completely dissolved. Evaporate the liquid completely under reduced pressure using a rotary evaporator, and then coat the blank liposome on the wall of a round-bottom flask. Precisely weigh 32 mg of FFC and dissolve it in 8 mL of acetone-water (5:95, V / V) solution, and then ultrasonicate for 10 min until the liposome completely falls off the wall of the flask. A light yellow transparent liposome is thus prepared. Transfer the liposome to an ultrafiltration tube (100KD), and centrifuge at 5000 rpm at 10°C for 1 h. The obtained product is washed with deionized water for 3 times, and then resuspended in 8 mL of deionized water. Mix the liposome with lactoferrin according to a mass ratio of 1.5:1, and then ultrasonicate in a water bath for 10 min to obtain lactoferrin-modified florfenicol liposomes (R-FL). Store the product at 4°C for later use.

[0077] The lactoferrin-modified florfenicol nanoliposomes prepared in this example were detected for particle size using a Malvern laser particle size analyzer. The hydrodynamic size of the lactoferrin-modified florfenicol nanoliposomes prepared in this example was 190.2±4.8 nm, the Zeta potential value was -47.5±0.8 mV, and the polydispersity coefficient was 0.29±0.03.

[0078] The encapsulation efficiency of the florfenicol nanoliposomes prepared in this example was determined to be 95.15%±0.31 using high-performance liquid chromatography, and the drug loading was 3.82%±0.05. The encapsulation efficiency and drug loading of the liposomes in this example were lower than those in Example 3.

[0079] Example 5 Fourier infrared spectrum determination of R-FL

[0080] The R-FL prepared in Example 3 was pre-frozen in a refrigerator at-80℃ for 24 h, and then the completely frozen R-FL was placed in a vacuum freeze dryer, and after the pressure was reduced to below 20 Pa by starting the vacuum pump, the R-FL was dried for 48 h until it became a freeze-dried powder. Then, the DSPE-PEG2000, soy lecithin, cholesterol, FFC, FL, lactoferrin, and R-FL freeze-dried powder were each mixed with potassium bromide to form transparent thin sheets, which were then scanned by infrared spectroscopy to obtain the infrared absorption spectrum. By analyzing the wave number and intensity of the characteristic absorption peaks and comparing with the standard spectrum library, the chemical structure and functional groups of the sample were qualitatively or quantitatively analyzed. The results are shown in Figure 6 .

[0081] Figure 6 are the Fourier infrared spectra of R-FL, FL, lactoferrin, DSPE-PEG2000, CHO, FFC, and SL in Example 3, wherein a is R-FL, b is FL, c is lactoferrin, d is DSPE-PEG2000, e is CHO, f is FFC, and g is SL. As can be seen from Figure 6 , the successful synthesis of FL can be verified by the retention of the C-F characteristic peak at 1280 cm -1 and the benzene ring C=C vibration peak at 1600-1620 cm -1 , indicating that the drug FFC is effectively loaded in the liposome. After further modification, R-FL has a new amide I band (α-helix structure) at 1650 cm -1 and a PEG ether bond peak at 1100 cm -1 , confirming that DSPE-PEG2000 and lactoferrin have been successfully coupled to the surface of the liposome. In addition, the superposition of the characteristic peaks of soy lecithin at 2920 / 2850 cm -1 alkyl chain peak, cholesterol at 3400 cm -1 hydroxyl peak, and other components indicates that the structure is intact during the synthesis process and no significant degradation of the components has occurred. The above results show that the retention of the C-F peak indicates that SL and CHO successfully loaded FFC to prepare FL, and the introduction of new functional groups amide I band and PEG ether bond indicates that lactoferrin modified FL to obtain R-FL.

[0082] Example 6: In vitro release kinetics test

[0083] The nanoliposomes of florfenicol (FL), the lactoferrin-modified florfenicol liposomes (R-FL), and pure florfenicol (FFC) prepared in Example 3 were subjected to an in vitro release kinetics test, and the high-performance liquid chromatography method (see Cheng, P. P., Yang, Y. J., Liu, H. X., et al. Establishment of HPLC method for determination of florfenicol in new compound florfenicol injection [J]. Animal Husbandry Progress, 2014, 35(10): 52-56) was used to obtain the in vitro release kinetics curve.

[0084] Precise amount of R-FL and FL solution with concentration of 2 mg / mL 3.0 mL and FFC solution dissolved in acetone-water solution (5:95, V / V) 3 mL were placed in dialysis bags with molecular weight cut-off of 8000-14000. Then, the dialysis bags were placed in reagent bottles containing 300 mL of release medium (pH 12 simulated gastric juice and pH 6.8 simulated intestinal juice, respectively, which were prepared according to the standard of Chinese Pharmacopoeia). The solution was oscillated in a constant temperature water bath (37±0.5)℃, 100 r / min. 3 mL of release medium was taken 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 keep the volume constant. The sampled release medium was filtered through a 0.22 μm water phase filter membrane, and the concentration of FFC was determined by HPLC. The drug release percentage was calculated, and the release curve was drawn to explore the release characteristics of the drug-loaded liposome.

[0085] The concentration of florfenicol in the release medium was determined by high performance liquid chromatography, and finally the release percentage was calculated and the release curve was drawn to study the release characteristics of the drug-loaded liposome. The test process of FFC and FL was the same as that of R-FL, and the results were shown in Figure 7 and Figure 8 Compared with florfenicol, the sustained-release and controlled-release effect of the lactoferrin modified florfenicol nano-liposome prepared in the present application was obvious in simulated gastric juice ( Figure 7 ) and simulated intestinal juice ( Figure 8 ) (under normal physiological environment).

[0086] Example 7 In vitro minimum inhibitory concentration test of lactoferrin modified florfenicol nano-liposome (R-FL) on Salmonella standard strain

[0087] After 4-6 h of culture, the turbidimeter was used to adjust the turbidity of the bacterial solution to 0.5 McFarland turbidity concentration (equivalent to 1×10 8 CFU / mL) with MH broth, and the bacterial suspension was obtained by 1:100 dilution of the bacterial solution with MH broth for inoculation of in vitro minimum inhibitory concentration test.

[0088] The minimum inhibitory concentration test in vitro was carried out on the lactoferrin-modified florfenicol nanoliposomes prepared in Example 2-4. Florfenicol raw material (FFC-API), florfenicol commercial drug (FFC-CAM), florfenicol nanoliposomes (FL) and the lactoferrin-modified florfenicol nanoliposomes (R-FL) prepared in Example 2-4 were mixed with MH broth to make the final concentration of florfenicol 2, 4, 8, 16, 32, 64 μg / mL, respectively. The growth of Salmonella standard strain ATCC13311 added to the above drug-containing solution was observed by naked eye to determine the minimum inhibitory concentration.

[0089] The micro-broth dilution method was used to prepare a series of drug solutions with 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, 0.5 μg / mL by diluting FFC-API, FFC-CAM, FL and R-FL in MH liquid medium by different times. 8 The Salmonella was inoculated into the 96-well plate containing the above drug solution to make the final concentration of bacterial solution about 5x10 Figure 9

[0090] Table 2 Minimum inhibitory concentration of R-FL on Salmonella standard strain

[0091]

[0092]

[0093] As shown in Table 2, the MIC value of FFC-CAM and FFC-API on Salmonella standard strain was 8 μg / mL, the MIC value of FL on ATCC13311 was 4 μg / mL, and the MIC value of R-FL prepared in Example 2-4 on ATCC13311 was 2-4 μg / mL, especially the MIC value of R-FL prepared in Example 3 on ATCC13311 was 2 μg / mL. The MIC value of FFC-API on ATCC13311 was within the quality control range, indicating that the test results were reliable.

[0094] Example 8 Minimum bactericidal concentration test in vitro of R-FL on Salmonella standard strain

[0095] ​The standard strain of Salmonella ATCC13311 was cultured for 4-6 h, and the turbidity of the bacterial solution was adjusted to 0.5 McFarland turbidity (1 x 10 8 CFU / mL) with MH broth. The bacterial suspension was obtained by diluting the solution 1:100 with MH broth for use in the in vitro minimum bactericidal concentration test.

[0096] The lactoferrin-modified prepared in Example 2-4 was subjected to the in vitro minimum bactericidal concentration test. The raw material drug of florfenicol, the commercial drug of florfenicol, FL, and the lactoferrin-modified florfenicol nanoliposomes prepared in Example 2-4 were mixed with MH broth, and the standard strain of Salmonella ATCC13311 was added. After incubation, the mixed solution was taken and inoculated on trypticase soy agar medium, and the number of colonies on the trypticase soy agar medium was recorded to determine the minimum bactericidal concentration.

[0097] After the MIC was measured, 100 μL of the culture solution without bacterial growth was taken and inoculated on a plate, and 3 replicates were set for each well. After the liquid was completely absorbed, the plate was incubated at 37°C for 18-24 h, and colony counting was performed. If the number of colonies was less than or equal to 0.1% of the minimum concentration of the amount of bacteria inoculated in each well (the number of colonies on the plate ≤ 2), it was the minimum bactericidal concentration (MBC). The results are shown in Table 3.

[0098] Table 3 Minimum bactericidal concentration of R-FL on the standard strain of Salmonella

[0099]

[0100]

[0101] As shown in Table 3, the MBC values of FFC-API, FFC-CAM, FL, and R-FL prepared in Example 2-4 were 16 μg / mL, 64 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, and 4 μg / mL, respectively (standard: if the number of colonies is less than or equal to 0.1% of the minimum concentration of the amount of bacteria inoculated in each well (the number of colonies on the plate ≤ 2), the number of colonies is shown in the table). The above results show that, compared with FFC-API, FFC-CAM, and FL, R-FL prepared in Example 2-4 has stronger inhibitory ability on the growth of Salmonella, especially Example 3. In summary, R-FL has good bacteriostatic effect.

[0102] Example 9 Minimum bactericidal concentration test of R-FL on drug-resistant strains of Salmonella

[0103] The drug-resistant strains of Salmonella (as shown in Table 4) were cultured for 4-6 h, and the turbidity of the bacterial solution was adjusted to 0.5 McFarland turbidity (equivalent to 1 x 108 CFU / mL), and five groups of bacterial suspensions were obtained by diluting the bacteria with MH broth at a ratio of 1:100 for inoculation of the in vitro minimum inhibitory concentration test.

[0104] Table 4 Salmonella drug-resistant strain information

[0105]

[0106]

[0107] Note: The strains in Table 4 were purchased from the National Veterinary Microbial Culture Collection (CVCC).

[0108] The florfenicol raw material (FFC-API), florfenicol commercial drug (FFC-CAM), florfenicol liposomes (FL), and lactoferrin-modified florfenicol nanoliposomes (R-FL) prepared in Examples 2-4 were mixed with MH broth to obtain a final florfenicol concentration of 8, 16, and 32 μg / mL, respectively. The growth of the Salmonella drug-resistant strains added to the above drug-containing solutions was observed by the naked eye to determine the minimum inhibitory concentration.

[0109] The minimum inhibitory concentration of the lactoferrin-modified florfenicol nanoliposomes was determined by the micro-doubling broth dilution method. In the first well of a 96-well plate, 100 μL of the above-prepared solution containing different concentrations of florfenicol raw material, florfenicol commercial drug, FL, and R-FL was added, and gradient dilution was performed by adding 90 μL of MH broth. Then, 10 μL of the bacterial suspension for inoculation was inoculated into each well. At the same time, negative controls (only broth medium) and positive controls (without drug) were set. After covering, the plate was incubated at 37°C for 18-24 h, and the lowest concentration of clear broth was observed by the naked eye, which was the minimum inhibitory concentration of the Salmonella drug-resistant strain. The test results are shown in Table 5 and Figure 10

[0110] Table 5 Minimum inhibitory concentration of FL against Salmonella drug-resistant strains

[0111]

[0112]

[0113] As shown in Table 5, the five clinical strains tested were resistant to FFC, and only 18s-2 was sensitive to FFC. When determining the MIC values of FFC-CAM, FFC-API, FL, and R-FL against Salmonella drug-resistant strain 18s-2, it was found that the MIC value of R-FL was relatively the smallest, which was 4 μg / mL. The results showed that R-FL had good antibacterial ability against Salmonella drug-resistant strain 18s-2.

[0114] ​Example 10 Minimum bactericidal concentration test of R-FL on Salmonella resistant strain

[0115] After the MIC value of R-FL on Salmonella resistant strain 18s-2 was determined, 4MIC, 2MIC, MIC of the bacterial-free culture solution were respectively taken and plated on the plate, and the MBC value was determined by the number of colonies. The results are shown in Table 6. Compared with FFC-CAM, FFC-API and FL, R-FL prepared in Example 3 has a relatively minimum MBC value of 8 μg / mL, and the results show that R-FL can effectively inhibit the growth of Salmonella resistant strain 18s-2 (the standard is: if the number of colonies is less than or equal to 0.1% of the amount of bacteria inoculated per well (the number of colonies on the plate ≤2), the number of colonies in the table is the number of colonies).

[0116] Table 6 Minimum bactericidal concentration of FL on Salmonella resistant strain

[0117]

[0118] In the subsequent experiments, FL and R-FL were prepared according to Example 3.

[0119] Example 11 Scanning electron microscope observation of the effect of R-FL on the biofilm formation ability of Salmonella

[0120] Salmonella clinical strain 18s-2 was inoculated into MH medium and placed in a constant temperature shaker, and cultured at 37°C and 120 r / min for 8h to the logarithmic growth phase of Salmonella. The turbidity of the bacterial solution was adjusted to 0.5 McFarland turbidity (equivalent to 1×10 8 CFU / mL) using sterile broth, and PBS solution, FFC-API, FFC-CAM, FL and R-FL solutions with FFC concentration of 16 μg / mL were added respectively, and incubated at 37°C for 1h, centrifuged at 6688 rpm for 10 min, the supernatant was discarded, and the bacteria were washed with PBS for 3 times, and then fixed with 2.5% glutaraldehyde solution for 4h; then gradient dehydration treatment was carried out with different concentration gradients of ethanol (such as 30%, 50%, 70%, 90%, 100%), each step for 10-15 min. After dehydration, the sample was dried by critical point drying instrument, and gold film was sprayed to enhance the conductivity. Finally, the sample was placed under scanning electron microscope, and the morphological characteristics of the biofilm were observed and photographed under appropriate accelerating voltage.

[0121] After the Salmonella clinical strain was treated with FFC-CAM, FFC-API, FL and R-FL, critical point drying was carried out, and the effect of the bacterial biofilm on the colony morphology was observed by scanning electron microscope. As Figure 11As shown, the drug-treated Salmonella all showed biofilm shrinkage and length shortening. Compared with FFC-CAM, FFC-API, FL, the strain after R-FL incubation showed more obvious shrinkage, especially the Salmonella co-incubated with R-FL became short, the biofilm showed shrinkage, and part of the biofilm showed deformation and damage. The above results showed that FL and R-FL could more effectively destroy the structure of bacterial biofilm than FFC-CAM and FFC-API.

[0122] Example 12 Laser scanning confocal microscope observation of the effect of R-FL on biofilm

[0123] Live and dead bacteria staining was used to compare the changes of biofilm before and after co-incubation of FFC-API, FFC-CAM, FL and R-FL with Salmonella clinical strains. The concentration of Salmonella clinical strains was 1x10 8 CFU / mL of Salmonella clinical strains were co-incubated with FFC-API, FFC-CAM, FL, R-FL and PBS solution at 37°C for 1h, centrifuged at 5000r / min for 10min, and the supernatant was discarded. Washed with sterile normal saline for 3 times and centrifuged. Mixed with a certain proportion of DMAO and PI dye mixture at room temperature in the dark for 30min. After staining, the dye was aspirated, and washed with sterile normal saline solution for 3 times. The stained biofilm was imaged by FV3000 laser scanning confocal microscope, and the effect of R-FL on Salmonella clinical strain biofilm was evaluated.

[0124] The destruction of FL, R-FL, FFC-CAM and FFC-API on the activity of Salmonella clinical strain biofilm was evaluated by laser confocal microscopic imaging technology. As shown Figure 12 (red), the mortality of bacteria after incubation with FL and R-FL was significantly increased, which indicated that both of them could accelerate the death of Salmonella by destroying the biofilm structure, and R-FL was particularly prominent in the destruction of biofilm. Further study found that the inhibition of R-FL on drug-resistant Salmonella biofilm formation was significantly better than that of FFC-API, FFC-CAM and FL groups, suggesting that it had better anti-biofilm activity.

[0125] Example 13

[0126] 1. Establishment process of mouse model of Salmonella typhimurium infection and therapeutic effect of R-FL on mice infected with Salmonella typhimurium

[0127] Forty-two 6-week-old SPF BALB / c healthy mice were randomly divided into 7 groups (half male and half female), 6 mice in each group, and the grouping and drug administration are shown in Table 7 and Table 8 below. Figure 13Salmonella clinical strain 18s-2 was cultured for 4-6 h, and the concentration of the bacterial solution was determined. The turbidity of the bacterial solution was adjusted to 0.5 McFarland turbidity (equivalent to 1 x 10 8 CFU / mL) using sterile MH broth. The bacterial solution was diluted 1:10 using MH broth and stored for later use. The mice in each group were administered 0.2 mL of the bacterial solution by gavage according to the administration schedule in Table 7, and the healthy control group was administered 0.2 mL of PBS solution by gavage. Each administration group was administered according to the respective administration schedule for 14 d. During the experiment, the number of sick, recovered, and dead mice in each group was observed and recorded in detail every day, and the morbidity, mortality, and cure rate of the animals in each group were calculated, respectively.

[0128] Table 7: Grouping of test animals and administration schedule

[0129]

[0130] The mice were administered 0.2 mL of Salmonella solution at a concentration of 1 x 10 8 CFU / mL by gavage to establish a Salmonella-infected mouse model. The morbidity, mortality, and cure rate of the mice were calculated. On the 2nd day after gavage, the gavaged mice showed rough and disheveled fur, weight loss, and diarrhea, and individual mice showed intestinal bleeding. The morbidity of the mice was 100%, indicating that the Salmonella-infected mouse model had been successfully established. As shown in Table 8, the mortality of the mice in the negative group reached 66.67% on the 7th day; the mortality of the mice in the FFC-API and FFC-CAM groups reached 33.33% and 50%, respectively, on the 7th day; the mortality of the mice in the FL group was 33.33%, and the mortality of the mice in the R-FL group was 16.67%.

[0131] Table 8: Mortality of mice in each group

[0132]

[0133] As shown in Table 9, the cure rate of the mice in the negative group was the lowest, only 16.67%; the cure rate of the mice in the FFC-API and FFC-CAM groups was 50%; compared with the FFC-API and FFC-CAM groups, the cure rate of the mice in the FL and R-FL groups was higher, 66.67% and 83.33%, respectively. The above results show that R-FL has a better therapeutic effect than FFC-API, FFC-CAM, and FL.

[0134] Table 9: Cure rate of mice in each group

[0135]

[0136]

[0137] 2. HE staining and immunohistochemical technique were used to analyze the effect of R-FL on the inflammatory response induced by drug-resistant S. typhimurium

[0138] The mice were sacrificed by cervical dislocation after blood sampling from the eye frame, and the internal organs were observed for pathological changes. The heart, liver, spleen, lung, and kidney of the mice were removed and preserved in 4% paraformaldehyde. The preserved internal organs were used to make sections to observe their pathological changes. After fixation in 10% formalin, the tissues were sequentially subjected to routine sampling, dehydration, transparency, paraffin embedding, sectioning, and HE staining. The histopathological changes were observed under a light microscope, and photographs were taken. After immunohistochemical staining with interleukin-6 (IL-6) and interleukin-1 beta (IL-1β), the sections were observed under a light microscope, and the integrated optical density (IOD) of the target area was measured using Image-ProPlus software. The experimental data were expressed as mean ± standard deviation (Mean ± SD), and one-way ANOVA was performed using SPSS software. P < 0.05 was considered statistically significant.

[0139] As shown in Figure 14 , after gavage with the clinical strain of Salmonella 18s-2, the mice in the drug-un-treated group showed varying degrees of inflammatory cell infiltration in the heart, lung, liver, and spleen (black arrows indicate inflammatory cell infiltration). After treatment with FFC-API and FFC-CAM, the mice still showed more inflammatory cell infiltration in the lung, liver, and spleen. After treatment with FL, the mice showed only a small amount of inflammatory cell infiltration in the liver. However, after treatment with R-FL, no obvious pathological changes were observed in the heart, liver, spleen, lung, and kidney sections of the mice. This result indicates that R-FL has good biocompatibility.

[0140] As shown in Figure 15 , the cecum and colon of the mice in the challenge-only group without drug treatment also showed a large amount of inflammatory cell infiltration. After treatment with FFC-API and FFC-CAM, no obvious pathological changes were observed in the intestinal tract of the mice. After treatment with FL, some inflammatory cell infiltration was observed in the cecum and colon sections of the mice. After treatment with R-FL, no obvious pathological changes were observed in the intestinal tract sections. These results indicate that R-FL has good inhibitory effects on Salmonella and good therapeutic effects on Salmonella-infected mice, and it can protect the organs and tissues.

[0141] The expression levels of IL-6 and IL-1β in the liver and spleen of Salmonella-infected mice were analyzed using immunohistochemical techniques to evaluate the anti-inflammatory effects of R-FL. As shown in Figure 16As shown in FIG. 9, the darker the color of the slice, the higher the degree of positivity. The results showed that, compared with the drug untreated group, the FFC-API group and the FFC-CAM group, the FL group and the R-FL group could effectively inhibit the expression of inflammatory factors IL-1β and IL-6 in the liver; further studies showed that, compared with FL, R-FL could more effectively inhibit the expression of inflammatory factors. The above results were further verified in the spleen. The above results showed that R-FL could inhibit the expression of inflammatory factors IL-1β and IL-6, thereby reducing the inflammatory response of the body.

[0142] 2. Effect of R-FL on drug-resistant Salmonella typhimurium biofilm

[0143] Three mice were randomly selected from each group for autopsy. The duodenum of the mouse was taken, and the intestinal contents were cultured under sterile conditions. After centrifugation, the supernatant was discarded, 2.5% glutaraldehyde electron microscope fixing solution was added, the remaining bacteria in the centrifuge tube were dried by critical point drying, and the growth of intestinal bacterial biofilm was observed by scanning electron microscopy.

[0144] The colonies with salmonella characteristics were purified and cultured, and the bacterial biofilm in the contents was observed by scanning electron microscopy after fixation. As shown in FIG. 10, Figure 17 The bacillus surface in the negative group was smooth, the biofilm was complete, and it grew normally in the mouse intestine; the bacillus in the FFC-API and FFC-CAM groups appeared slight deformation; the salmonella in the FL group changed a little, the bacterial biofilm surface was not smooth, and the periphery was contracted; the bacillus in the R-FL group changed significantly, the bacterial biofilm structure was destroyed, and there were many depressions and wrinkles. The above results showed that R-FL had a significant destructive effect on salmonella biofilm in the mouse body.

[0145] 3. Metabolomics analysis of the effect of R-FL on drug-resistant Salmonella typhimurium biofilm in the mouse intestine

[0146] To further investigate the effect of R-FL on Salmonella typhimurium biofilm and understand the regularity of metabolite changes in different drug treatments, LC-MS full spectrum metabolic technology was used to analyze and identify the metabolites in the mouse intestine. As shown in FIG. 11, Figure 18 Figure 18 ​Figure 13 is a graph showing the effect of R-FL on drug-resistant Salmonella typhimurium biofilm in the intestines of mice using metabolomics analysis in Example 13, wherein A is a PCA plot, B is a PLS-DA plot, C and D are contrast plots of differential metabolites between groups, E is a Venn diagram (group contrast), F is a volcano plot (FL vs. negative), G is a volcano plot (R-FL vs. negative), H is a lollipop plot (FL vs. negative), I is a lollipop plot (R-FL vs. negative), J is a KEGG metabolic pathway (FL vs. negative), and K is a KEGG metabolic pathway (R-FL vs. negative). The different drug treatments were clearly separated in the PCA results, indicating that there were significant changes in metabolites in the samples after treatment, which was consistent with the physiological and biochemical indicators. There were significant differences in metabolites in different groups, and there were up-regulated and down-regulated metabolites between different groups (e.g. Figure 18 The intestinal metabolites of mice in the FL and R-FL groups were most different from those of mice in the negative group, mainly in the colon contents. Figure 18 Figs. F and G are volcano plot contrasts between the FTPM group and the negative group, and the FTPPM group and the negative group, which can more clearly reflect the types and significant differences of differential metabolites between the two groups. Figure 18 Figs. H and I are lollipop plots of differential metabolites between the FL group and the negative group, and the R-FL group and the negative group, which can reflect the types and significant differences of up-regulated and down-regulated metabolites. Figure 18 As shown in Figs. J and K, based on the KEGG database, metabolic pathway enrichment analysis was performed on the differential metabolites, and the differential metabolite pathways of the FL, R-FL, and negative groups were mainly enriched in the categories of metabolism, human diseases, biological systems, genetic information processing, cellular processes, and environmental information processing.

[0147] 4. IVIS live imaging analysis of R-FL targeting intestinal microorganisms in drug-resistant Salmonella typhimurium infected mice

[0148] Figure 19 Figure 14 is a graph showing the results of IVIS live imaging analysis of R-FL targeting intestinal microorganisms in drug-resistant Salmonella typhimurium infected mice in Example 13, wherein A is a live imaging graph of the main organs and intestinal tissues in drug-resistant Salmonella typhimurium infected mice, B is an ex vivo fluorescence image of the main organs of drug-resistant Salmonella typhimurium infected mice after oral administration of different drug formulations such as FFC-CAM, FFC-API, FL, and R-FL for 24 h, C is a fluorescence image of intestinal tissues of drug-resistant Salmonella typhimurium infected mice after oral administration of different drug formulations such as FFC-CAM, FFC-API, FL, and R-FL for 1 h and 2 h. As shown in Figure 19As shown in A-C, compared with the negative group (Drug untreated), the FFC-CAM group and the FFC-API group, the FL and R-FL groups have intestinal targeting and time dependence. Compared with the FL group, the R-FL group has more intestinal targeting, and the intestinal targeting is more obvious in the colon. In addition, compared with other groups, the R-FL group has more in the liver and spleen of the drug-resistant S. typhimurium infected mice, which indicates that the R-FL can effectively inhibit the inflammatory response caused by drug-resistant S. typhimurium, which is consistent with the results of immunohistochemical detection.

[0149] 5. Effect of R-FL on intestinal flora balance of mice infected with S. typhimurium

[0150] Figure 20 is a graph of the effect of R-FL on intestinal flora disorder caused by drug-resistant S. typhimurium analyzed by 16sRNA sequencing in Example 13, wherein A is a graph of the relative abundance of intestinal flora at each level, B and C are Venn diagrams of common and unique amplicon sequence variants (ASVs) in intestinal flora between groups, D is a graph of dominant flora in the intestine of drug-resistant S. typhimurium infected mice, E and F are PCA graphs, G is a graph of the relative abundance of dominant flora in the intestine of drug-resistant S. typhimurium infected mice, and H is a heatmap of drug-resistant S. typhimurium related signaling pathways. As shown in Figure 20 As shown in A-C, compared with the negative group (Drug untreated), the FFC-CAM group and the FFC-API group, the content of drug-resistant S. typhimurium in the FL and R-FL groups is significantly reduced. Figure 20 This result is further verified in D. As shown in Figure 20 As shown in D, there are three dominant flora in the intestine of drug-resistant S. typhimurium infected mice, including Shigella, Bacteroides and drug-resistant S. typhimurium, and the content of drug-resistant S. typhimurium in the intestine of the R-FL group mice is the lowest compared with other groups. In addition, as shown in E-H of 20, the PCA graph, the relative abundance graph and the drug-resistant S. typhimurium related signaling pathway heatmap results all show that the content of drug-resistant S. typhimurium in the intestine of the R-FL group mice is the lowest. The above results show that the R-FL can effectively inhibit the intestinal flora disorder caused by drug-resistant S. typhimurium and restore the intestinal flora balance.

[0151] From the above analysis results, the water dynamic size of the lactoferrin modified florfenicol nanoliposomes (R-FL) prepared by the present application is 170.5-195.0 nm, the zeta potential value is -37.1--50.0 mV, and the polydispersity coefficient is 0.24-0.38. This reflects that the mutual repulsion between the particles is good, the system is stable, and under electron microscopy, the liposomes modified by lactoferrin show a three-dimensional bowl-shaped structure, and the shape is relatively uniform. The encapsulation efficiency is 94.84%-97.79% and the drug loading capacity is 3.00%-4.10% as determined by high performance liquid chromatography. The lactoferrin modified florfenicol nanoliposomes prepared by the present application have excellent sustained release effect in simulated gastrointestinal fluid. The MIC and MBC of the product for Salmonella standard strain are 2 μg / mL and 4 μg / mL, respectively; and the product also has good antibacterial effect on clinical strains, with MIC of 4 μg / mL and MBC of 8 μg / mL. Laser scanning confocal microscopy and scanning electron microscopy observation show that R-FL can destroy the structure of Salmonella biofilm and effectively inhibit the formation of biofilm. R-FL has good therapeutic effect on murine typhoid salmonellosis, with high cure rate and low mortality rate of the diseased mice, and the physiological and biochemical indexes are within the normal range, indicating that it has good biocompatibility; immunohistochemical analysis shows that R-FL can effectively inhibit the inflammatory response caused by drug-resistant murine typhoid Salmonella, mainly by inhibiting the expression of key inflammatory factors such as IL-6 and IL-1β; scanning electron microscopy observation shows that R-FL can destroy the structure of biofilm in the mouse body, thereby inhibiting the production of drug-resistant murine typhoid Salmonella biofilm. In addition, the product can effectively regulate the intestinal flora disorder caused by drug-resistant murine typhoid Salmonella, so as to restore the intestinal flora to normal level.

Claims

1. A lactoferrin-modified florfenicol nanoliposome, characterized in that, Florfenicol nanoliposomes are made from the following components in the following mass ratio: DSPE-PEG2000: soybean lecithin: florfenicol: cholesterol in the ratio of (1-3): (18-22): (28-36): (2-6), and florfenicol liposomes: lactoferrin in the mass ratio of (0.3-2):

1.

2. The lactoferrin-modified florfenicol nanoliposomes according to claim 1, characterized in that, The average hydrodynamic size of lactoferrin-modified florfenicol liposomes was 170.5–195.0 nm, the zeta potential was -37.1–-50.0 mV, and the polydispersity index was 0.24–0.

38.

3. The lactoferrin-modified florfenicol nanoliposomes according to claim 2, characterized in that, The encapsulation efficiency of lactoferrin-modified florfenicol nanoliposomes ranged from 94.84% to 97.39%, and the drug loading was 3.00% to 4.10%.

4. The method for preparing lactoferrin-modified florfenicol nanoliposomes according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Dissolve DSPE-PEG2000, soybean lecithin and cholesterol in anhydrous ethanol, sonicate to dissolve, and rotary evaporate to obtain blank liposomes. (2) Florfenicol was swollen in a mixture of acetone and water, then added to blank liposomes, sonicated to dissolve, centrifuged, washed, resuspended in water, lactoferrin was added, and the mixture was heated and sonicated to dissolve.

5. The method for preparing lactoferrin-modified florfenicol nanoliposomes according to claim 4, characterized in that, In step (1), the ultrasonic dissolution time is 16-24 min, and the liquid is evaporated under reduced pressure until it is completely evaporated.

6. The method for preparing lactoferrin-modified florfenicol nanoliposomes according to claim 4, characterized in that, In step (2), the volume ratio of acetone to water is 5:95, the solid-liquid ratio of florfenicol to the mixed solution is (28-36):(6-10) mg / mL, and the mass-volume ratio of florfenicol to the deionized water used for suspending the washed lipids is (28-36):(6-10) mg / mL.

7. The method for preparing lactoferrin-modified florfenicol nanoliposomes according to claim 4, characterized in that, In step (2), the ultrasonic dissolution time is 6-15 min, the ultrasonic dissolution temperature is 35-50℃, and the ultrasonic dissolution time in the heated water bath is 6-15 min.

8. The use of lactoferrin-modified florfenicol nanoliposomes according to any one of claims 1-3 in the preparation of antibiotic drugs.

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

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

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