Enrofloxacin composite nanogel capable of promoting intestinal absorption and preparation method of enrofloxacin composite nanogel

Abstract

The invention discloses enrofloxacin composite nanogel for promoting intestinal absorption and a preparation method of the enrofloxacin composite nanogel. The enrofloxacin composite nanogel comprises 0.1-3% of sodium alginate; 0.1 to 1% of carboxymethyl chitosan; 0.05 to 0.5 percent of mint exosome; and 0.05 to 1 percent of enrofloxacin. According to the composite nanogel disclosed by the invention, the mint exosome and the enrofloxacin are prevented from being damaged by gastric juice through a shell structure of sodium alginate, so that the stability of the mint exosome and the enrofloxacin in the gastric juice is improved; after the enrofloxacin composite nanogel reaches the intestinal tract, the sodium alginate is used as a shell and is subjected to pH responsive swelling hydrolysis, and the wrapped carboxymethyl chitosan-enrofloxacin nanoparticles and the mint exosome are released; the released mint exosome opens close connection of intestinal cells, enrofloxacin is slowly released from carboxymethyl chitosan-enrofloxacin nanoparticles to achieve a slow release effect, and the mint exosome and the enrofloxacin are synergistically targeted to promote absorption of enrofloxacin in intestinal tracts, so that the drug concentration of enrofloxacin in plasma is enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary drug preparation, and specifically relates to an enrofloxacin composite nanogel that promotes intestinal absorption by opening cell tight junctions through mint exosomes and a preparation method thereof. Background Art

[0002] As a quinolone antibiotic for animals, enrofloxacin has excellent antibacterial activity, but its permeability in animals is poor and its bioavailability is low. Mint exosomes inhibit the expression of tight junction proteins (ZO-1, Claudin-5, Occludin and E-cadherin) and open the tight junctions between cells, which helps the absorption of enrofloxacin and improves its low bioavailability. However, mint exosomes and enrofloxacin have poor stability in gastric juice, so a delivery system is urgently needed to prevent mint exosomes and enrofloxacin from being destroyed by gastric juice, thereby improving the stability of mint exosomes and enrofloxacin in gastric juice.

[0003] As a commonly used and efficient oral delivery strategy, the nanogel delivery system can effectively prevent drugs from being destroyed by gastric juice, thereby improving the stability of drugs in gastric juice. Among them, sodium alginate, as a hydrogel material, has high stability in gastric juice and can protect the loaded drugs from being destroyed by gastric juice, thereby improving the stability of the loaded drugs in gastric juice. However, sodium alginate will swell and hydrolyze rapidly in the intestinal tract, and cannot achieve the effect of slow release.

[0004] Since carboxymethyl chitosan is often used as a gel material to encapsulate drugs to achieve a sustained release effect, the present invention encapsulates enrofloxacin with carboxymethyl chitosan to form carboxymethyl chitosan-enrofloxacin nanoparticles; then, the carboxymethyl chitosan-enrofloxacin nanoparticles and the extracted mint exosomes are added to sodium alginate to form a sodium alginate / mint exosome / carboxymethyl chitosan-enrofloxacin composite nanogel. The shell structure of sodium alginate can prevent mint exosomes and enrofloxacin from being destroyed by gastric juice, thereby improving the stability of mint exosomes and enrofloxacin in gastric juice; after reaching the intestine, the enrofloxacin composite nanogel undergoes pH-responsive swelling and hydrolysis, and the encapsulated carboxymethyl chitosan-enrofloxacin nanoparticles and mint exosomes are released; the released mint exosomes open the tight junctions of intestinal cells, and enrofloxacin is slowly released from the carboxymethyl chitosan-enrofloxacin nanoparticles to achieve a sustained release effect, and the two synergistically target and promote the absorption of enrofloxacin in the intestine, thereby enhancing the drug concentration of enrofloxacin in plasma. Summary of the invention

[0005] The present invention encapsulates enrofloxacin with carboxymethyl chitosan to form carboxymethyl chitosan-enrofloxacin nanoparticles; then, the carboxymethyl chitosan-enrofloxacin nanoparticles and the extracted mint exosomes are added to sodium alginate at the same time to form a sodium alginate / mint exosome / carboxymethyl chitosan-enrofloxacin composite nanogel, which can prevent the exosomes and enrofloxacin from being destroyed by gastric juice and improve their stability; after the enrofloxacin composite nanogel reaches the intestine, the sodium alginate, as an outer shell, undergoes pH-responsive swelling and hydrolysis, and the encapsulated carboxymethyl chitosan-enrofloxacin nanoparticles and mint exosomes are released; the released mint exosomes open the tight junctions of intestinal cells, and enrofloxacin is slowly released from the carboxymethyl chitosan-enrofloxacin nanoparticles to achieve a sustained release effect, and the two synergistically promote the absorption of enrofloxacin in the intestine in a targeted manner, thereby enhancing the drug concentration of enrofloxacin in plasma.

[0006] To achieve the above object, the present invention provides the following technical solutions: An enrofloxacin composite nanogel for promoting intestinal absorption comprises the following raw materials in percentage by mass: 0.1-3% sodium alginate; 0.1-1% carboxymethyl chitosan; 0.05-0.5% mint exosomes; and 0.05-1% enrofloxacin.

[0007] Preferably, the raw materials are included in the following mass percentages: sodium alginate 0.2-2%; carboxymethyl chitosan 0.2-0.8%; mint exosomes 0.1-0.4%; enrofloxacin 0.1-0.8%.

[0008] More preferably, the raw materials are included in the following mass percentages: sodium alginate 0.5-1.5%; carboxymethyl chitosan 0.3-0.6%; mint exosomes 0.2-0.3%; enrofloxacin 0.2-0.5%.

[0009] The most preferred method comprises the following raw materials in percentage by weight: 1.0% sodium alginate; 0.5% carboxymethyl chitosan; 0.25% mint exosomes; 0.3% enrofloxacin; and the balance is water.

[0010] The preparation method of the enrofloxacin composite nanogel comprises the following steps: (1) adding the enrofloxacin to ethanol and stirring to dissolve, thereby obtaining a solution 1; (2) adding the carboxymethyl chitosan into water and stirring to dissolve to obtain solution 2; (3) adding the sodium alginate into water and stirring to dissolve to obtain solution 3; (3) Slowly adding the solution 1 into the solution 2 and stirring evenly to obtain a solution 4; (4) Solution 4 and mint exosomes were slowly added to solution 3 at the same time and stirred (0.5 h-2 h) to form a nanogel, thereby obtaining enrofloxacin composite nanogel.

[0011] The beneficial effects of the present invention are: (1) The present invention encapsulates enrofloxacin with carboxymethyl chitosan to form carboxymethyl chitosan-enrofloxacin nanoparticles; then, the carboxymethyl chitosan-enrofloxacin nanoparticles and the extracted mint exosomes are added to sodium alginate to form a sodium alginate / mint exosome / carboxymethyl chitosan-enrofloxacin composite nanogel.

[0012] (2) The enrofloxacin composite nanogel of the present invention prevents the mint exosomes and enrofloxacin from being destroyed by gastric juice through the shell structure of sodium alginate, thereby improving the stability of the mint exosomes and enrofloxacin in gastric juice; after the enrofloxacin composite nanogel reaches the intestine, the sodium alginate as the shell undergoes pH-responsive swelling and hydrolysis, and the encapsulated carboxymethyl chitosan-enrofloxacin nanoparticles and mint exosomes are released; the released mint exosomes open the tight junctions of intestinal cells, and enrofloxacin is slowly released from the carboxymethyl chitosan-enrofloxacin nanoparticles to achieve a sustained release effect, and the two synergistically promote the absorption of enrofloxacin in the intestine, thereby enhancing the drug concentration of enrofloxacin in plasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for use are briefly introduced below.

[0014] Figure 1 This is a schematic diagram of the principle of the enrofloxacin composite nanogel in Example 5 opening the cell tight junctions through the mint exosomes to promote intestinal absorption; Figure 2 is a transmission electron microscopic diagram of the mint exosomes used in Examples 1-5; Figure 3 is a transmission electron microscopic diagram of the carboxymethyl chitosan-enrofloxacin nanoparticles obtained in step (4) of Example 5; Figure 4 is a transmission electron microscopic diagram of the enrofloxacin composite nanogel in Example 5; Figure 5 is a schematic diagram of the particle size distribution of peppermint exosomes, carboxymethyl chitosan-enrofloxacin nanoparticles and enrofloxacin composite nanogel in Example 5; Figure 6 is a schematic diagram of the in vitro release of enrofloxacin composite nanogel in Example 5; Figure 7This is a schematic diagram of the qualitative detection of the expression of ZO-1, Claudin-5, Occludin and E-cadherin by enrofloxacin composite nanogel and mint exosomes in Example 5 by laser confocal microscopy; Figure 8 This is a schematic diagram of the quantitative detection of the expression of ZO-1, Claudin-5, Occludin and E-cadherin by RT-qPCR of the enrofloxacin composite nanogel and mint exosomes in Example 5; Fig. 9 This is a schematic diagram of the drug concentration of enrofloxacin soluble powder and enrofloxacin composite nanogel in pig plasma in the pharmacokinetic experiment. DETAILED DESCRIPTION

[0015] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0016] Preparation of mint exosomes used in the following examples: (1) Wash the mint leaves and stems with ultrapure water, crush the mint leaves and stems with a juicer or grinder, collect the juice and remove the residue; Recommendation: Use ultrapure water to wash the equipment and wear gloves.

[0017] (2) Use gauze to filter out large particles of impurities in the juice and collect the filtrate.

[0018] (3) Filter twice: first filter once with a 0.45 μm filter membrane to remove larger particles, then filter once with a 0.22 μm filter membrane and collect the filtrate.

[0019] (4) Initial centrifugation: centrifuge at 10,000 g for 2 h to remove large particles in the filtrate; centrifuge again at 10,000 g for 2 h and collect the filtrate.

[0020] (5) Low-temperature ultracentrifugation: use an ultracentrifuge at 100,000 g for 2 h, remove the filter residue, and collect the filtrate.

[0021] (6) Density centrifugation and purification step: Use sucrose or exosome purification kits to remove non-vesicle impurities.

[0022] (7) Resuspend the exosomes with the intermediate layered structure in PBS.

[0023] Example 1: An enrofloxacin composite nanogel is composed of the following raw materials in percentage by mass: 3.0% sodium alginate; 1% carboxymethyl chitosan; 0.05% mint exosomes; 0.5% enrofloxacin; and the balance is sterile water. The preparation method specifically comprises the following steps: (1) adding the enrofloxacin to anhydrous ethanol and stirring to dissolve to obtain solution 1; (2) adding the carboxymethyl chitosan into sterile water and stirring to dissolve to obtain solution 2; (3) adding the sodium alginate into sterile water and stirring to dissolve, thereby obtaining solution 3; (3) Slowly adding the solution 1 into the solution 2 and stirring for 1 h to obtain a solution 4; (4) Solution 4 and mint exosomes were slowly added dropwise to solution 3 at the same time and stirred for 1 h to form a nanogel, thereby obtaining enrofloxacin composite nanogel.

[0024] Example 2: An enrofloxacin composite nanogel is composed of the following raw materials in percentage by mass: 0.5% sodium alginate; 0.5% carboxymethyl chitosan; 0.1% mint exosomes; 0.8% enrofloxacin; and the balance is sterile water. The preparation method is similar to that of Example 1.

[0025] Example 3: An enrofloxacin composite nanogel is composed of the following raw materials in percentage by mass: 0.5% sodium alginate; 0.5% carboxymethyl chitosan; 0.5% mint exosomes; 0.1% enrofloxacin; and the balance is sterile water. The preparation method is similar to that of Example 1.

[0026] Example 4: An enrofloxacin composite nanogel is composed of the following raw materials in percentage by mass: 1.0% sodium alginate; 0.5% carboxymethyl chitosan; 0.25% mint exosomes; 0.5% enrofloxacin; and the balance is sterile water. The preparation method is similar to that of Example 1.

[0027] Example 5: An enrofloxacin composite nanogel is composed of the following raw materials in percentage by mass: 1.0% sodium alginate; 0.5% carboxymethyl chitosan; 0.25% mint exosomes; 0.3% enrofloxacin; and the balance is sterile water. The preparation method is similar to that of Example 1.

[0028] The products obtained in the above examples were subjected to relevant characterization and testing: 1. Take an appropriate amount of the nanogels obtained in Examples 1-5 and observe whether the prepared enrofloxacin composite nanogels have uniform color and whether they form a gel. Among them, Example 1 has uneven color and does not form a gel; Examples 2-3 have uniform color but do not form a gel; the enrofloxacin composite nanogels prepared in Examples 4-5 have good uniformity and can form a stable gel, which can be used as a subsequent pharmacokinetic test agent.

[0029] 2. Morphological observation: After the mint exosomes, carboxymethyl chitosan-enrofloxacin nanoparticles and enrofloxacin composite nanogels in Example 5 were diluted 100 times, their microscopic morphology was observed by transmission electron microscopy. The results showed that the mint exosomes under transmission electron microscopy were double-layer three-dimensional saucer-like structures with a diameter of about 100 nm, such as Figure 2 As shown; Carboxymethyl chitosan-enrofloxacin nanoparticles under transmission electron microscopy are spherical structures with a diameter of about 50 nm, as shown Figure 3 As shown; Enrofloxacin composite nanogel under transmission electron microscopy is a network structure, such as Figure 4 shown.

[0030] 3. Determination of particle size: After the mint exosomes, carboxymethyl chitosan-enrofloxacin nanoparticles and enrofloxacin composite nanogels in Example 5 were diluted 100 times, their particle sizes were measured by Malvern nano laser particle size analyzer, and the average value was obtained after three times of dilution. The results showed that the average particle sizes of mint exosomes, carboxymethyl chitosan-enrofloxacin nanoparticles and enrofloxacin composite nanogels were 119.2±14.3 nm, 54.6±4.8 nm and 209.0±20.1 nm, respectively. Figure 5 shown.

[0031] 4. In vitro release: The enrofloxacin composite nanogel prepared in Example 5 was placed in simulated gastric juice and intestinal juice, and the release rate of enrofloxacin was calculated at different time points. The results showed that in the simulated gastric juice, 98%±1% of enrofloxacin was released at 4h; in the simulated intestinal juice, only 52%±5% of enrofloxacin was released at 4h, and only 64%±3% of enrofloxacin was released at 24h. Figure 6 shown.

[0032] 5. The cell density in the logarithmic growth phase is 5×10 5 Caco2 cells / mL were added to a 6-well plate. After the cells were stably attached to the wall, the culture medium containing mint exosomes and enrofloxacin composite nanogels at protein concentrations of 100, 50, and 20 μg / mL was replaced for incubation for 24 h. The PBS group was designed as a control. The expression of ZO-1, Claudin-5, Occludin, and E-cadherin was qualitatively and quantitatively detected by laser confocal microscopy and RT-qPCR, respectively. The results of laser confocal microscopy and RT-qPCR showed that compared with mint exosomes, enrofloxacin composite nanogels could significantly reduce the expression of ZO-1, Claudin-5, Occludin, and E-cadherin proteins, thus confirming that enrofloxacin composite nanogels can open cell tight junctions through the encapsulated mint exosomes, such as Figure 7-8 shown.

[0033] 6. Pharmacokinetic studies: (1) Materials and methods Drugs: Enrofloxacin soluble powder, enrofloxacin composite nanogel.

[0034] Experimental animals: 12 healthy three-way crossbred piglets.

[0035] Instruments: laser nanoparticle size analyzer, heat-collecting constant temperature magnetic stirrer, heating magnetic stirrer, freeze dryer, variable vacuum ultra-high resolution field emission scanning electron microscope, UV-visible spectrophotometer.

[0036] (2) Test methods Twelve pigs were fasted, weighed, and numbered 12 h before the experiment. Enrofloxacin soluble powder and enrofloxacin composite nanogel were accurately weighed or measured according to body weight and administered by oral gavage at a dose of 10 mg / kg. Blood was collected once before administration, and blood was collected at 0.5, 1, 2, 3, 4, 6, 8, 12, 24, and 48 h after oral gavage. The collected blood was added to a centrifuge tube containing sodium heparin, and plasma was prepared by centrifugation at 3500 r / min for 10 min, and the drug concentration in plasma was determined.

[0037] (3) Results After pigs were gavaged with enrofloxacin composite nanogel, the enrofloxacin concentration in plasma quickly reached a peak of 0.54±0.03 µg / mL at 2 h, and then slowly decreased. As a control, enrofloxacin soluble powder reached a peak of 0.73±0.09 µg / mL more quickly at 4 h, and then slowly decreased to 0.21±0.02 µg / mL 48 h after administration. Fig. 9 As shown. This shows that the prepared enrofloxacin composite nanogel can promote the absorption of enrofloxacin in the intestine, thereby enhancing the drug concentration of enrofloxacin in plasma.

[0038] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. An enrofloxacin composite nanogel for promoting intestinal absorption, characterized in that: The invention is prepared from the following raw materials in percentage by mass: 0.1-3% sodium alginate; 0.1-1% carboxymethyl chitosan; 0.05-0.5% mint exosomes; and 0.05-1% enrofloxacin.

2. The enrofloxacin composite nanogel according to claim 1, characterized in that: The invention comprises the following raw materials in percentage by weight: 0.2-2% sodium alginate; 0.2-0.8% carboxymethyl chitosan; 0.1-0.4% mint exosomes; and 0.1-0.8% enrofloxacin.

3. The enrofloxacin composite nanogel according to claim 1, characterized in that: The invention comprises the following raw materials in percentage by weight: 0.5-1.5% sodium alginate; 0.3-0.6% carboxymethyl chitosan; 0.2-0.3% mint exosomes; and 0.2-0.5% enrofloxacin.

4. The enrofloxacin composite nanogel according to claim 1, characterized in that: The invention comprises the following raw materials in percentage by weight: 1.0% sodium alginate; 0.5% carboxymethyl chitosan; 0.25% mint exosomes; 0.3% enrofloxacin; and the balance is water.

5. The method for preparing the enrofloxacin composite nanogel according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) adding the enrofloxacin to ethanol and stirring to dissolve, thereby obtaining a solution 1; (2) adding the carboxymethyl chitosan into water and stirring to dissolve to obtain solution 2; (3) adding the sodium alginate into water and stirring to dissolve to obtain solution 3; (3) Slowly adding the solution 1 into the solution 2 and stirring evenly to obtain a solution 4; (4) Solution 4 and mint exosomes are slowly added dropwise to solution 3 at the same time, and stirred to form a nanogel, thereby obtaining enrofloxacin composite nanogel.

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