Nano preparation for delivering antibiotics based on plant extracts and application thereof

Abstract

The invention relates to a nano preparation for delivering antibiotics based on plant extracts and application of the nano preparation, and belongs to the technical field of nano preparations. In order to solve the problems of low bioavailability and low action efficiency of oral administration or intravenous injection of antibiotics in the lung disease treatment process, the invention provides a nano preparation for delivering antibiotics based on plant extracts. The mass volume ratio of the antibiotics to the plant extract in the nano preparation is (1-10) mg: (20-100) mu L. The nano preparation disclosed by the invention can obviously inhibit the migration of fibroblasts, has an obvious bacteriostatic effect and can reduce the density of microorganisms in lungs; positive charges carried by the material are combined with negative charges on the surface of a bacterial cell membrane, transmembrane substance transfer on the bacterial cell membrane is affected, bacterial metabolism and reproduction are affected, adjustment of pulmonary symbiotic bacteria is better achieved, and the pulmonary fibrosis progress can be relieved. The nano preparation has the advantages of small toxic and side effects, good patient compliance, high encapsulation efficiency and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano preparations, and in particular relates to a nano preparation for delivering antibiotics based on plant extracts and an application thereof. Background Art

[0002] Changes in lung flora are associated with the occurrence and development of a variety of respiratory diseases. Researchers believe that in healthy lungs, there is a steady-state balance of microbial "in and out migration". When people suffer from respiratory diseases, the balance of airway flora is also broken, and the cilia in the lungs become dysfunctional, mucus secretion increases, and bacterial migration is enhanced.

[0003] There are usually two situations. The first is that the number of microorganisms in the lungs remains unchanged or decreases, but the abundance increases, and the overall microbial load increases. The second is that due to the influence of multiple factors, other pathogens appear in the lungs, resulting in an increase in the number of microorganisms in the lungs. Foreign pathogens directly cause disease or symbiotically regulate the abnormal growth of opportunistic pathogens in the lungs. A study showed that the bacterial load in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis (IPF) was about twice that of the control group, among which Streptococcus, Neisseria, Haemophilus, and Veillonella were all higher than those in the control group, and were independently associated with IPF. It has also been observed in animal models that the lung microbial flora is associated with IPF.

[0004] In addition to their antibacterial activity, some commonly used antibiotics in clinical practice also have immunomodulatory effects, which can reduce the mortality and hospitalization rate of acute exacerbations in IPF patients, inhibit the differentiation of myofibroblasts and the development of pulmonary fibrosis, and play an anti-fibrotic role. However, the bioavailability of oral antibiotics is low, and after intravenous infusion, antibiotics are rapidly distributed throughout the body, and their efficiency in the lesion site is low. At the same time, the antibacterial spectrum of antibiotics is limited, and their regulatory effect on the lung microbial flora has certain limitations. Combination therapy can make up for this defect, but multiple administrations often increase the burden on patients.

[0005] Another difficulty in clinical treatment of lung diseases is that pathogenic bacteria form biofilms to block the penetration of antibiotics, making the bacteria extremely resistant. Therefore, providing a drug that can meet the needs of clinical applications, effectively regulate lung symbiotic bacteria, and penetrate and destroy biofilms has become a technical problem that urgently needs to be solved in the medical field. Summary of the invention

[0006] In order to solve the problems of low bioavailability and low action efficiency of oral or intravenous antibiotics, the present invention provides a nanoformulation for delivering antibiotics based on plant extracts and its application.

[0007] The technical solution of the present invention:

[0008] A nanoformulation for delivering antibiotics based on plant extracts, wherein the mass volume ratio of the antibiotics to the plant extracts in the nanoformulation is 1-10 mg:20-100 μL.

[0009] Furthermore, the antibiotic is at least one of penicillin antibiotics, cephalosporin antibiotics, macrolide antibiotics, aminoglycoside antibiotics, quinolone antibiotics, tetracycline antibiotics, sulfonamide antibiotics, rifamycin antibiotics or glycopeptide antibiotics.

[0010] Furthermore, the antibiotic is at least one of azithromycin, moxifloxacin, doxycycline, neomycin or rapamycin.

[0011] Furthermore, the plant extract is at least one of tea tree oil, luteolin, vitamin E or bitter almond oil.

[0012] Furthermore, the particle size of the nano preparation is in the range of 50 to 300 nm, and the PDI is in the range of 0.1 to 0.4.

[0013] Furthermore, the preparation method of the nanoformulation comprises the following steps:

[0014] Step 1: dissolving antibiotics, phospholipids and cholesterol in an organic solvent, removing the organic solvent in the obtained mixed system by rotary evaporation under reduced pressure at 40-45°C, and drying the obtained film in a constant temperature vacuum oven at 37-45°C overnight;

[0015] Step 2: adding a hydration liquid to the film dried overnight in step 1 to hydrate it, adding a stabilizer to the hydration system, performing water bath ultrasound to cause the film to fall off, and performing ultrasound treatment at a power of 20 to 1000 kHz for 5 to 20 minutes under ice bath conditions to obtain a lipid suspension;

[0016] Step 3: Add a surfactant to the plant extract, mix thoroughly and then add to the lipid suspension obtained in step 2, perform water bath sonication until transparent, further perform sonication for 5 to 20 minutes, and perform ultrafiltration to obtain a nano preparation.

[0017] Furthermore, in step 1, the molar ratio of antibiotics, phospholipids and cholesterol is 1-30:50-95:3-30; the phospholipid is at least one of dipalmitoylphosphatidylcholine DPPC, distearoylphosphatidylcholine DSPC, dioleoylphosphatidylcholine DOPC, dimyristoylphosphatidylethanolamine DMPE, dipalmitoylphosphatidylethanolamine DPPE, distearoylphosphatidylethanolamine DSPE or soybean lecithin.

[0018] Furthermore, in step 2, the hydration liquid is at least one of pure water, physiological buffer, sucrose solution, trehalose solution, glucose solution, mannitol solution or Ringer's solution; and in step 3, the surfactant is one of anionic surfactant or nonionic surfactant.

[0019] Furthermore, the method further comprises step 4, adding an equal volume of chitosan solution with a mass concentration of 0.01 to 100 mg / mL to the ultrasonic system obtained in step 3, magnetically stirring at 400 rpm for 1 to 2 hours, and ultrafiltration to obtain the nano preparation.

[0020] Application of a nanoformulation for delivering antibiotics based on plant extracts in the preparation of drugs for treating lung diseases.

[0021] Beneficial effects of the present invention:

[0022] The present invention provides a nanoformulation based on plant extracts for delivering antibiotics, which can significantly inhibit the migration of fibroblasts, has obvious antibacterial effects, can reduce the density of microorganisms in the lungs, inhibit and destroy the biofilm of pathogenic bacteria, and significantly reduce the drug resistance of pathogenic bacteria, thereby more effectively exerting antibacterial effects. The nanoformulation of the present invention carries a positive charge combined with the negative charge on the surface of the bacterial cell membrane, affects the transmembrane substance transport on the bacterial cell membrane, affects bacterial metabolism and reproduction, better realizes the regulation of lung symbiotic bacteria, and alleviates the progress of pulmonary fibrosis. The nanoformulation of the present invention can effectively improve the survival rate of mice with pulmonary fibrosis, improve their living conditions, keep their weight steadily rising, and is effective in treatment by regulating the symbiotic flora.

[0023] The nano preparation method provided by the present invention enhances the stability of plant extracts, solves the problems that plant extracts are unstable, volatile, and sensitive to oxygen, light, and temperature. At the same time, chitosan is soluble in weak acid solutions, and can form positively charged cationic groups in acidic solutions, while the antibiotics encapsulated in the plant extracts have negative charges and are combined with them through electrostatic adsorption. The atomized inhalation nano preparation prepared by the present invention has a small and uniform particle size, is suitable for atomized inhalation administration, and at the same time, the nano preparation has the advantages of antibacterial and anti-fibrosis properties, small toxic and side effects, good patient compliance, and a high encapsulation rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 TEM image of the TTO@AZM-CS nanoformulation prepared in Example 6;

[0025] Figure 2 This is the particle size distribution diagram of the TTO@AZM-CS nanoformulation prepared in Example 6;

[0026] Figure 3 The antibiotic standard curve established for the present invention;

[0027] Figure 4 Comparison of cell scratch states in cell scratch experiments, with scale bars of 200 μm in all figures;

[0028] Figure 5 This is a comparison chart of cell migration rates in the cell scratch experiment;

[0029] Figure 6 This is a comparison chart of the culture results of broth culture medium in the minimum inhibitory concentration test;

[0030] Figure 7 This is a photo of the TSA blood plate in the inhibition zone test;

[0031] Figure 8 This is a comparison chart of the sizes of inhibition zones in the inhibition zone test;

[0032] Fig. 9 This is a comparison chart of the experimental results of different drugs inhibiting bacterial biofilm formation;

[0033] Fig.10 This is a comparison chart of experimental results of different drugs destroying bacterial biofilm;

[0034] Fig.11 This is a comparison chart of the experimental results of different drug concentrations of nano-preparation in Example 6 for destroying bacterial biofilm;

[0035] Fig.12 The experimental results of the nano-preparation in Example 6 for destroying bacterial biofilm at different action times are shown;

[0036] Fig.13 This is an experiment to test the effects of different nanoformulations on the viability of fibroblasts induced by TGF-β;

[0037] Fig.14 This is a comparison chart of the 21-day survival curves of mice in different treatment groups in the animal experiment;

[0038] Fig.15 This is a comparison chart of the weight changes of mice in different treatment groups in the animal experiment;

[0039] Fig.16 This is a comparison of the lung tissue morphology, H&E staining and Masson staining of mice in different treatment groups in the animal experiment. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0041] Example 1

[0042] This embodiment provides a nanoformulation for delivering antibiotics based on plant extracts and a preparation method thereof.

[0043] The components of the nanoformulation in this embodiment are azithromycin AZM, dipalmitoylphosphatidylcholine DPPC, hydrating liquid pure water, plant extract tea tree oil TTO and surfactant Tween-80. The tea tree oil in this embodiment is purchased from Maclean Company, and the product number is 68647-73-4.

[0044] The preparation method of the nanoparticle preparation of this embodiment comprises the following steps:

[0045] Step 1. Accurately weigh 2 mg of azithromycin, 20 mg of dipalmitoylphosphatidylcholine DPPC, and 2 mg of cholesterol, dissolve in 1 mL of a mixed organic solvent prepared by chloroform and methanol in a volume ratio of 3:1, remove the organic solvent by rotary evaporation at 40°C for 2 h, and dry in a constant temperature vacuum incubator at 37°C overnight.

[0046] Step 2: Add 250 μL of pure water to the film that has been dried overnight in step 1 and hydrate for 1 hour. Use water bath ultrasound to remove the film. Use probe ultrasound at a power of 700 KHz for 5 minutes in an ice bath to obtain a clear lipid suspension.

[0047] Step 3: Take 50 μL of tea tree oil and 160 μL of Tween-80, mix thoroughly and add to the lipid suspension obtained in step 2, add to a Millipore ultrafiltration centrifuge tube after ultrasonic treatment at 700 kHz for 5 minutes, centrifuge for 10 minutes at 4000 rpm, collect the supernatant from the cannula, record it as TTO@AZM nanoformulation, seal it with nitrogen, and store it in a refrigerator at 4°C away from light.

[0048] Example 2

[0049] This embodiment provides a nanoformulation for delivering antibiotics based on plant extracts and a preparation method thereof.

[0050] The components of the nanoformulation in this example are moxifloxacin, dipalmitoylphosphatidylcholine, hydration liquid pure water, plant extract vitamin E and surfactant Tween-80.

[0051] The preparation method of the nanoparticle preparation of this embodiment comprises the following steps:

[0052] Step 1. Accurately weigh 3 mg of moxifloxacin, 20 mg of dipalmitoylphosphatidylcholine, and 2 mg of cholesterol, dissolve in 1 mL of a mixed organic solvent prepared by chloroform and methanol in a volume ratio of 3:1, remove the organic solvent by rotary evaporation at 40°C for 2 h, and dry in a constant temperature vacuum incubator at 37°C overnight.

[0053] Step 2: Add 230 μL of pure water to the film dried overnight in step 1 and hydrate for 1 hour. Use water bath ultrasound to remove the film. Use probe ultrasound at 800 KHz for 5 minutes in an ice bath to obtain a clear lipid suspension.

[0054] Step 3: Take 80 μL of vitamin E and 20 μL of Tween-80, add them to the lipid suspension obtained in step 2, and sonicate in a water bath until transparent. Further, use probe ultrasound at 800 kHz for 5 minutes, then add them to a Millipore ultrafiltration centrifuge tube, centrifuge for 10 minutes at 4000 rpm, collect the supernatant from the cannula to obtain the nanopreparation, seal it with nitrogen, and store it in a refrigerator at 4°C away from light.

[0055] Example 3

[0056] This embodiment provides a nanoformulation for delivering antibiotics based on plant extracts and a preparation method thereof.

[0057] The components of the nanoformulation in this example are doxycycline, dipalmitoylphosphatidylcholine, hydrating liquid pure water, plant extract bitter almond oil and surfactant glycerol.

[0058] The preparation method of the nanoparticle preparation of this embodiment comprises the following steps:

[0059] Step 1. Accurately weigh 4.5 mg of doxycycline, 20 mg of dipalmitoylphosphatidylcholine, and 2 mg of cholesterol, dissolve in 1 mL of a mixed organic solvent prepared by chloroform and methanol in a volume ratio of 3:1, remove the organic solvent by rotary evaporation at 40°C for 2 h, and dry in a constant temperature vacuum incubator at 37°C overnight.

[0060] Step 2: Add 240 μL of pure water to the film that has been dried overnight in step 1, hydrate for 1 hour, and then perform water bath ultrasound to remove the film. Further, perform probe ultrasound at a power of 400 kHz for 5 minutes under ice bath conditions to obtain a clear lipid suspension.

[0061] Step 3: Take 70 μL of bitter almond oil and 150 μL of glycerol, mix well and add to the lipid suspension obtained in step 2, sonicate in a water bath until transparent, further sonicate with a probe at 400 kHz for 5 min, add to a Millipore ultrafiltration centrifuge tube, centrifuge for 10 min at 4000 rpm, collect the supernatant from the cannula to obtain the nanopreparation, seal with nitrogen, and store in a refrigerator at 4°C away from light.

[0062] Example 4

[0063] This embodiment provides a nanoformulation for delivering antibiotics based on plant extracts and a preparation method thereof.

[0064] The components of the nanoformulation in this embodiment are neomycin, dipalmitoylphosphatidylcholine, hydrating liquid pure water, plant extract bitter almond oil and surfactant Tween-80.

[0065] The preparation method of the nanoparticle preparation of this embodiment comprises the following steps:

[0066] Step 1. Accurately weigh 6 mg of neomycin, 20 mg of dipalmitoylphosphatidylcholine, and 2 mg of cholesterol, dissolve in 1 mL of a mixed organic solvent prepared by chloroform and methanol in a volume ratio of 3:1, remove the organic solvent by rotary evaporation at 40°C for 2 h, and dry in a constant temperature vacuum incubator at 37°C overnight.

[0067] Step 2: Add 250 μL of pure water to the film dried overnight in step 1 for hydration for 1 hour, remove the film by water bath ultrasound, and further perform probe ultrasound treatment at a power of 400 kHz for 5 minutes under ice bath conditions to obtain a clear lipid suspension.

[0068] Step 3: Take 50 μL of bitter almond oil and 160 μL of Tween-80, mix well and add to the lipid suspension obtained in step 2, sonicate in a water bath until transparent, further sonicate with a probe at 400 kHz for 5 min, add to a Millipore ultrafiltration centrifuge tube, centrifuge for 10 min at 4000 rpm, collect the supernatant from the cannula to obtain the nanopreparation, seal with nitrogen, and store in a refrigerator at 4°C away from light.

[0069] Example 5

[0070] This embodiment provides a nanoformulation for delivering antibiotics based on plant extracts and a preparation method thereof.

[0071] The components of the nanoformulation in this example are rapamycin, dipalmitoylphosphatidylcholine, hydration solution pure water, plant extract luteolin and surfactant Tween-80.

[0072] The preparation method of the nanoparticle preparation of this embodiment comprises the following steps:

[0073] Step 1: Accurately weigh 10 mg of rapamycin, 20 mg of dipalmitoylphosphatidylcholine, and 2 mg of cholesterol, dissolve in 1 mL of a mixed organic solvent prepared by chloroform and methanol in a volume ratio of 3:1, remove the organic solvent by rotary evaporation at 40°C for 2 h, and dry in a constant temperature vacuum incubator at 37°C overnight.

[0074] Step 2: Add 260 μL of pure water to the film that has been dried overnight in step 1 and hydrate for 1 hour. Use water bath ultrasound to remove the film. Use probe ultrasound at a power of 300 KHz for 5 minutes under ice bath conditions to obtain a clear lipid suspension.

[0075] Step 3: Take 100 μL of luteolin and 150 μL of Tween-80, mix thoroughly and add to the lipid suspension obtained in step 2, sonicate in a water bath until transparent, further sonicate with a probe at 300 kHz for 5 min, add to a Millipore ultrafiltration centrifuge tube, centrifuge for 10 min at 4000 rpm, collect the supernatant from the cannula to obtain the nanopreparation, seal with nitrogen, and store in a refrigerator at 4°C away from light.

[0076] Example 6

[0077] This embodiment provides a nanoformulation for delivering antibiotics based on plant extracts and a preparation method thereof.

[0078] The components of the nanoformulation in this embodiment are azithromycin, dipalmitoylphosphatidylcholine DPPC, hydrating liquid pure water, plant extract tea tree oil TTO, surfactant Tween-80 and chitosan CS. The tea tree oil in this embodiment is purchased from Maclean Company, and the product number is 68647-73-4.

[0079] The preparation method of the nanoparticle preparation of this embodiment comprises the following steps:

[0080] Step 1. Accurately weigh 2 mg of azithromycin, 20 mg of dipalmitoylphosphatidylcholine DPPC, and 2 mg of cholesterol, dissolve in 1 mL of a mixed organic solvent prepared by chloroform and methanol in a volume ratio of 3:1, remove the organic solvent by rotary evaporation at 40°C for 2 h, and dry in a constant temperature vacuum incubator at 37°C overnight.

[0081] Step 2: Add 250 μL of pure water to the film that has been dried overnight in step 1 and hydrate for 1 hour. Use water bath ultrasound to remove the film. Use probe ultrasound at a power of 800 KHz for 5 minutes in an ice bath to obtain a clear lipid suspension.

[0082] Step 3: Take 50 μL of tea tree oil and 160 μL of Tween-80, mix thoroughly and add to the lipid suspension obtained in step 2, sonicate in a water bath until transparent, and further sonicate with a probe at 800 KHz for 5 minutes to obtain an ultrasonic system with a total volume of 500 μL.

[0083] Step 4: Add 500 μL of 10 mg / mL chitosan solution to the ultrasonic system obtained in step 3, stir magnetically at 400 rpm for 2 h, then add to a Millipore ultrafiltration centrifuge tube, centrifuge for 10 min at 4000 rpm, collect the supernatant from the cannula, record it as TTO@AZM-CS nanoformulation, seal it with nitrogen, and store it in a refrigerator at 4°C away from light.

[0084] Example 7

[0085] This embodiment provides a nanoformulation for delivering antibiotics based on plant extracts and a preparation method thereof.

[0086] The components of the nanoformulation in this example are moxifloxacin, dipalmitoylphosphatidylcholine, hydration liquid pure water, plant extract vitamin E, surfactant Tween-80 and chitosan CS.

[0087] The preparation method of the nanoparticle preparation of this embodiment comprises the following steps:

[0088] Step 1. Accurately weigh 2 mg of moxifloxacin, 20 mg of dipalmitoylphosphatidylcholine, and 2 mg of cholesterol, dissolve in 1 mL of a mixed organic solvent prepared by chloroform and methanol in a volume ratio of 3:1, remove the organic solvent by rotary evaporation at 40°C for 2 h, and dry in a constant temperature vacuum incubator at 37°C overnight.

[0089] Step 2: Add 230 μL of pure water to the film that has been dried overnight in step 1 and hydrate for 1 hour. Add 40 μL of glycerol to the hydration system, and use water bath ultrasound to remove the film. Further, use probe ultrasound at a power of 800 KHz for 5 minutes under ice bath conditions to obtain a clear lipid suspension.

[0090] Step 3: Take 80 μL of vitamin E and 150 μL of Tween-80, mix thoroughly and add to the lipid suspension obtained in step 2, sonicate in a water bath until transparent, and further sonicate with a probe at 800 KHz for 5 minutes to obtain an ultrasonic system with a total volume of 500 μL.

[0091] Step 4: Add 500 μL of 15 mg / mL chitosan solution to the ultrasonic system obtained in step 3, stir magnetically at 400 rpm for 2 h, then add to a Millipore ultrafiltration centrifuge tube, centrifuge for 10 min at 4000 rpm, collect the supernatant from the cannula to obtain the nanoformulation, seal with nitrogen, and store in a refrigerator at 4°C away from light.

[0092] Example 8

[0093] This embodiment provides a nanoformulation for delivering antibiotics based on plant extracts and a preparation method thereof.

[0094] The components of the nanoformulation in this example are doxycycline, dipalmitoylphosphatidylcholine, hydration solution pure water, plant extract bitter almond oil, surfactant Tween-80 and chitosan.

[0095] The preparation method of the nanoparticle preparation of this embodiment comprises the following steps:

[0096] Step 1. Accurately weigh 9 mg of doxycycline, 20 mg of dipalmitoylphosphatidylcholine, and 2 mg of cholesterol, dissolve in 1 mL of a mixed organic solvent prepared by chloroform and methanol in a volume ratio of 3:1, remove the organic solvent by rotary evaporation at 40°C for 2 h, and dry in a constant temperature vacuum incubator at 37°C overnight.

[0097] Step 2: Add 240 μL of pure water to the film that has been dried overnight in step 1 and hydrate for 1 hour. Add 40 μL of glycerol to the hydration system, and use water bath ultrasound to remove the film. Further, use probe ultrasound at a power of 600 KHz for 5 minutes under ice bath conditions to obtain a clear lipid suspension.

[0098] Step 3: Take 70 μL of bitter almond oil and 150 μL of Tween-80, mix well and add to the lipid suspension obtained in step 2, sonicate in a water bath until transparent, and further sonicate with a probe at 600 KHz for 5 minutes to obtain an ultrasonic system with a total volume of 500 μL.

[0099] Step 4: Add 500 μL of 20 mg / mL chitosan solution to the ultrasonic system obtained in step 3, stir magnetically at 400 rpm for 2 h, then add to a Millipore ultrafiltration centrifuge tube, centrifuge for 10 min at 4000 rpm, collect the supernatant from the cannula to obtain the nanoformulation, seal with nitrogen, and store in a refrigerator at 4°C away from light.

[0100] Example 9

[0101] This embodiment provides a nanoformulation for delivering antibiotics based on plant extracts and a preparation method thereof.

[0102] The components of the nanoformulation in this example are neomycin, dipalmitoylphosphatidylcholine, hydrating liquid pure water, plant extract tea tree oil and surfactant Tween-80.

[0103] The preparation method of the nanoparticle preparation of this embodiment comprises the following steps:

[0104] Step 1. Accurately weigh 2 mg of neomycin, 20 mg of dipalmitoylphosphatidylcholine, and 2 mg of cholesterol, dissolve in 1 mL of a mixed organic solvent prepared by chloroform and methanol in a volume ratio of 3:1, remove the organic solvent by rotary evaporation at 40°C for 2 h, and dry in a constant temperature vacuum incubator at 37°C overnight.

[0105] Step 2: Add 250 μL of pure water to the film that has been dried overnight in step 1 and hydrate for 1 hour. Add 40 μL of glycerol to the hydration system, and use water bath ultrasound to remove the film. Further, use probe ultrasound at a power of 200 kHz for 5 minutes under ice bath conditions to obtain a clear lipid suspension.

[0106] Step 3: Take 50 μL of tea tree oil and 160 μL of Tween-80, mix thoroughly and add to the lipid suspension obtained in step 2, sonicate in a water bath until transparent, and further sonicate with a probe at 200 KHz for 5 minutes to obtain an ultrasonic system with a total volume of 500 μL.

[0107] Step 4: Add 500 μL of 30 mg / mL chitosan solution to the ultrasonic system obtained in step 3, stir magnetically at 400 rpm for 2 h, then add to a Millipore ultrafiltration centrifuge tube, centrifuge for 10 min at 4000 rpm, collect the supernatant from the cannula to obtain the nanoformulation, seal with nitrogen, and store in a refrigerator at 4°C away from light.

[0108] Example 10

[0109] This embodiment provides a nanoformulation for delivering antibiotics based on plant extracts and a preparation method thereof.

[0110] The components of the nanoformulation in this embodiment are rapamycin, dipalmitoylphosphatidylcholine, hydrating liquid pure water, stabilizer glycerol, plant extract bitter almond oil and surfactant Tween-80.

[0111] The preparation method of the nanoparticle preparation of this embodiment comprises the following steps:

[0112] Step 1. Accurately weigh 2 mg of rapamycin, 20 mg of dipalmitoylphosphatidylcholine, and 2 mg of cholesterol, dissolve in 1 mL of a mixed organic solvent prepared by chloroform and methanol in a volume ratio of 3:1, remove the organic solvent by rotary evaporation at 40°C for 2 h, and dry in a constant temperature vacuum incubator at 37°C overnight.

[0113] Step 2: Add 260 μL of pure water to the film dried overnight in step 1 and hydrate for 1 hour. Use water bath ultrasound to remove the film. Use probe ultrasound at a power of 200 KHz for 5 minutes in an ice bath to obtain a clear lipid suspension.

[0114] Step 3: Take 50 μL of bitter almond oil and 150 μL of Tween-80, mix well and add to the lipid suspension obtained in step 2, sonicate in a water bath until transparent, and further sonicate with a probe at 200 KHz for 5 minutes to obtain an ultrasonic system with a total volume of 500 μL.

[0115] Step 4: Add 500 μL of 20 mg / mL chitosan solution to the ultrasonic system obtained in step 3, stir magnetically at 400 rpm for 2 h, then add to a Millipore ultrafiltration centrifuge tube, centrifuge for 10 min at 4000 rpm, collect the supernatant from the cannula to obtain the nanoformulation, seal with nitrogen, and store in a refrigerator at 4°C away from light.

[0116] 1. Investigate the transmission electron microscopy characterization of the nanoformulation prepared by the present invention.

[0117] The TTO@AZM-CS nanoformulation prepared in Example 6 was tested by transmission electron microscopy using negative staining technology. The specific method is as follows: the TTO@AZM-CS nanoformulation solution prepared in Example 6 was dripped onto a copper mesh, and after drying, 1 drop of 2% phosphotungstic acid staining solution was dripped onto the copper mesh for staining for 30 seconds, the staining solution was removed, and after drying, TEM photography was performed. The TEM of the nanoformulation prepared in Example 6 is shown in FIG. Figure 1 As shown, the TTO@AZM-CS nanoformulation has a regular spherical appearance.

[0118] 2. Determination of particle size and zeta potential of the nanoparticles prepared in the present invention

[0119] The particle size, PDI and Zeta potential of the nanoformulations prepared in Examples 1-10 were measured using a Malvern Nano ZS Zetasizer. 3 μL of the nanoformulations prepared in each example were added to 3000 μL of ultrapure water to ensure uniform dispersion. An appropriate amount of the nanoformulation aqueous solution was placed in a cuvette, and the hydrated particle size, polydispersity index (PDI) and Zeta potential of the nanoformulation were measured by dynamic light scattering analysis. Table 1 shows the DLS characterization of the nanoformulations obtained in Examples 1-10. Figure 2 This is the particle size distribution diagram of the TTO@AZM-CS nanoformulation prepared in Example 6.

[0120] Table 1

[0121]

[0122]

[0123] Smaller nanometer size enables nanomaterials to achieve greater total drug volume accumulation and deeper penetration depth. Plant extracts, as essential oils, can be inserted into the surface of lipid membranes, increasing membrane fluidity and weakening membrane strength. After probe ultrasound, lipid vesicles are more likely to split, thereby reducing the particle size of nanomolecular probes.

[0124] 3. Investigation of the encapsulation efficiency of the nanoformulation prepared by the present invention

[0125] A standard curve of azithromycin was established and the encapsulation efficiency of azithromycin in the nanoformulation was determined.

[0126] Azithromycin solutions with drug concentrations of 3000, 1500, 750, 375, 187.5, and 93.75 μg / mL were prepared with methanol solution in sequence. After HPLC detection, a linear regression equation was made with the concentration of azithromycin (X) as the horizontal axis and the peak area (Y) as the vertical axis to draw a standard curve, as shown in the figure. Figure 3 .

[0127] The regression equation is y=1322.8x+10.393, and the correlation coefficient is R 2 =0.9995, indicating that azithromycin has a good linear relationship in the range of 3.75-3000 μg / mL.

[0128] The encapsulation efficiency was determined by ultrafiltration. 50 μL of the nanoformulations prepared in Example 1 and Example 6 were taken and added to 950 μL of methanol solution to break the emulsions. The mixture was fully shaken and mixed to completely break the emulsions. The azithromycin content was determined by HPLC, which was the mass of the nanoformulation loaded, denoted as W. The drug loading and encapsulation efficiency were calculated according to the following formula:

[0129] Encapsulation efficiency EE=W / W 总 X100%; drug loading DL=W / W L X100%

[0130] Where: W 总 is the total amount of azithromycin, W L is the total mass of the nanoparticles.

[0131] The encapsulation efficiency of the nanoformulation of Example 1 was calculated to be 60.1±0.03%, and the drug loading was 4.7±0.006%; the encapsulation efficiency of the nanoformulation of Example 2 was 59.1±0.001%, and the drug loading was 4.5±0.003%; the encapsulation efficiency of the nanoformulation of Example 3 was 57.5±0.01%, and the drug loading was 4.0±0.006%; the encapsulation efficiency of the nanoformulation of Example 4 was 61.3±0.03%, and the drug loading was 3.8±0.009%; the encapsulation efficiency of the nanoformulation of Example 5 was 56.9±0.07%, and the drug loading was 4.1±0.002 %; the encapsulation efficiency of the nano-preparation in Example 6 is 61.3±0.05%, and the drug loading is 4.4±0.003%; the encapsulation efficiency of the nano-preparation in Example 7 is 55.2±0.07%, and the drug loading is 4.4±0.006%; the encapsulation efficiency of the nano-preparation in Example 8 is 59.2±0.05%, and the drug loading is 4.1±0.003%; the encapsulation efficiency of the nano-preparation in Example 9 is 59.1±0.03%, and the drug loading is 4.6±0.001%; ​​the encapsulation efficiency of the nano-preparation in Example 10 is 56.6±0.03%, and the drug loading is 4.4±0.005%.

[0132] 4. Investigation of the Cell Migration Ability of the Nanoformulation Prepared by the Present Invention

[0133] The cell scratch assay is a simple and common method to study cell migration ability.

[0134] First, use a marker pen to draw horizontal lines on the back of the 6-well plate, with 1 cm between each line. The lines should be even and parallel, and cross the wells. NIH-3T3 cells were plated at 2x10 5 Cells were inoculated at a density of 1000 cells / well in a 6-well plate and incubated overnight. A 200 μL pipette tip was used to draw lines perpendicular to the back of the well plate to artificially create cell scratches. The cells were rinsed with PBS to remove cell debris, and different group solutions (Control group, TGF-β1 group, Example 6-TTO@AZM-CS group, AZM group) were added to each well. The cell scratch state at 0 hour was observed under a microscope and photographed. After 1 hour of induction with TGF-β1, serum-free DMEM was replaced, and photographs were taken 24 hours later. Figure 4 and Figure 5 shown. Figure 5 The cell migration rate was calculated as follows:

[0135] Cell migration rate = (0 hour scratch area - 24 hour scratch area) / 0 hour scratch area × 100%

[0136] One of the reasons for the continuous progression of fibrosis is the migration of fibroblasts. Figure 4 and Figure 5It is shown that the nano-preparation synthesized by the present invention can significantly inhibit the migration of fibroblasts. Different drug components have different inhibition conditions. The inhibition of fibroblast migration by using plant extracts (plant essential oils) as carriers is much better than the simple use of antibiotics. This is because, in addition to azithromycin, chitosan and tea tree oil also have antibacterial effects. Chitosan is a natural antibacterial agent. Under acidic conditions, through electrostatic adsorption, the amino cations on the chitosan chain are combined with the negative charge on the surface of the bacterial cell membrane, affecting the transmembrane substance transport on the bacterial cell membrane, affecting bacterial metabolism and reproduction, and having a better antibacterial effect on Gram-negative bacteria. Tea tree oil is also a broad-spectrum natural antibacterial agent, so it can play a combined antibacterial effect.

[0137] V. Investigation of the Minimum Inhibitory Concentration (MIC) of the Nanoformulation Prepared by the Present Invention

[0138] Through literature research, DNA was isolated from the lungs of experimental mice that received intratracheal delivery of saline or BLM for 16S rDNA gene sequencing. Gram-negative Bacteroidetes was the most upregulated bacterial phylum in the pulmonary fibrosis model, and Bacteroidaceae and Prevotellaceae were the two most significantly upregulated families in the Bacteroidetes phylum. Therefore, we selected Bacteroides ovatus ATCC 8483 for antibacterial experiments.

[0139] The minimum inhibitory concentration (MIC) of AZM and the TTO@AZM-CS nanoformulation of Example 6 against Bacteroides ovatus was determined by the broth microdilution method. MIC is an indicator for detecting the antibacterial activity of antibacterial drugs. The smaller the MIC value, the stronger the antibacterial activity.

[0140] The TTO@AZM-CS nanoformulation and azithromycin AZM of Example 6 were diluted in multiple proportions with modified minced meat culture medium. The mass concentrations of AZM in the 1st to 7th wells were 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 μg / mL, respectively; the 8th well was a positive control, with no antibacterial agent and only bacterial solution; the 9th well was a negative control, containing only broth culture medium. 10 μL of bacterial solution was inoculated into 90 μL of a 96-well plate containing TTO@AZM-CS nanoformulation or AZM with different concentration gradients. The 96-well plate was placed in an incubator at 37°C and incubated for 24 hours. After 24 hours, the bacterial growth was observed with the naked eye, and the concentration at which there was almost no turbidity in the small well and no bacterial growth was defined as the MIC. The results are as follows Figure 6 shown.

[0141] The nanoformulation synthesized by using plant extracts (plant essential oils) as carriers has a more obvious antibacterial effect than the simple use of antibiotics. When plant extracts (plant essential oils) act on bacteria cultured in small dishes, the antibiotic concentration of 0.625μg / mL has a significant antibacterial effect, but ordinary commercial antibiotics only begin to show a slight antibacterial effect when the concentration reaches 1.25μg / mL, which shows that TTO@AZM-CS nanoformulation has better antibacterial activity.

[0142] VI. Inhibition zone experiment of the nanoformulation prepared by the present invention

[0143] The inhibition zone experiment was used to detect the growth inhibition of Bacteroides ovatus by azithromycin AZM and the TTO@AZM-CS nanoformulation of Example 6.

[0144] Use an inoculation loop to pick up a single colony on the TSA blood plate and dilute it with broth culture medium to an OD value of about 0.5 turbidity; make drug-containing filter paper, and the experiment is set up in three groups, TTO@AZM-CS new nanoformulation group, AZM group, and Control group, add 20μL of different component solutions respectively, and wait for the paper to dry; use a sterile cotton swab to dip 100μL of bacterial solution, and evenly draw zigzag lines along the starting position above the TSA blood plate to evenly spread the bacterial solution; place the filter paper pieces at the corresponding positions marked on the plate, press the filter paper pieces gently with tweezers, and culture them under anaerobic conditions for 16-20 hours. The results are as follows Figure 7 As shown, observe the antibacterial zone around the filter paper and measure the diameter of the antibacterial zone with a caliper. Figure 8 shown.

[0145] Figure 7 and Figure 8 It shows that under the same mass concentration of azithromycin, the TTO@AZM-CS nanoformulation exhibits a higher antibacterial effect. From the inhibition zone, it can be seen that the diameter of the inhibition zone produced by the TTO@AZM-CS group is 1.46 times that of the AZM group. This shows that the new nanoscale drug synthesized by using plant extracts as carriers in the present invention has a very obvious antibacterial effect, and the inhibition zone is significantly larger than that of ordinary commercial antibiotics.

[0146] VII. Experimental study on the inhibition of bacterial biofilm formation by the nanoparticles prepared by the present invention

[0147] Pseudomonas aeruginosa was selected as the main pathogen of lung infection to study the ability of the nanoformulation prepared by the present invention to inhibit the formation of bacterial biofilm.

[0148] Pseudomonas aeruginosa was mixed with TTO@AZM-CS NPs prepared in Example 6, TTO@AZMNPs prepared in Example 1, AZM NPs, TTO NPs, CS NPs, AZM solution and PBS, respectively, and added to a 96-well plate and incubated at 37°C for 24 hours. Crystal violet staining was then performed to determine the cell membrane. Free bacteria that did not form a film were discarded, and 100 μL of methanol was added to each well to fix at room temperature for 15 minutes, the methanol was aspirated and discarded, and the wells were naturally air-dried. Then, stain with 0.05% crystal violet stain for 5 minutes, wash off excess dye with deionized water, and place the 96-well plate in an oven to dry. Subsequently, 33% acetic acid solution was added to each well, and the plate was vibrated for 15 minutes to completely dissolve the dye. The OD value of the crystal violet stain at 570 nm was measured with an enzyme reader, and the total biofilm ratio was calculated by comparing with the negative control group. The results are shown as follows Fig. 9 As shown, compared with the unmedicated PA group, the TTO@AZM-CS NPs prepared in Example 6, the TTO@AZM NPs prepared in Example 1, AZM NPs, TTO NPs, CS NPs and AZM solution all have the ability to inhibit the formation of bacterial biofilm, but TTO@AZM-CS NPs has the best inhibitory effect.

[0149] 8. Experimental study on the destruction of bacterial biofilm by the nanoparticles prepared by the present invention

[0150] Pseudomonas aeruginosa was inoculated into a 96-well plate and cultured at 37°C for 48 hours to allow it to form a mature bacterial biofilm. 100 μL of supernatant was discarded from each well, and then TTO@AZM-CS NPs prepared in Example 6, TTO@AZM NPs prepared in Example 1, AZM NPs, TTO NPs, CS NPs, AZM solution and PBS with the same probe concentration were added to the 96-well plate and cultured at 37°C for 24 hours. Crystal violet staining was then performed according to the biofilm formation inhibition experiment, and the results are shown as follows: Fig.10 As shown in the figure, TTO@AZM-CS NPs had the best biofilm destruction effect.

[0151] IX. Experimental study on the destruction of bacterial biofilm by nanoparticles prepared by the present invention at different concentrations

[0152] Pseudomonas aeruginosa was inoculated into a 96-well plate and cultured at 37°C for 48 hours to allow it to form a mature bacterial biofilm. 100 μL of supernatant was discarded from each well, and then 0.75, 1.5, 3, 6, 12, 24, 48 and 96 μg / mL of TTO@AZM-CS NPs prepared in Example 6 and PBS were added to the 96-well plate and cultured at 37°C for 24 hours. Crystal violet staining was then performed according to the biofilm formation inhibition experiment, and the results are shown in FIG. Fig.11As shown, the higher the concentration of TTO@AZM-CS NPs, the better the effect of destroying bacterial biofilm.

[0153] 10. Experimental study on the destruction of bacterial biofilm by nanoparticles prepared by the present invention at different action times

[0154] Pseudomonas aeruginosa was inoculated into a 96-well plate and cultured at 37°C for 48 hours to allow it to form a mature bacterial biofilm. 100 μL of supernatant was discarded from each well, and the TTO@AZM-CS NPs prepared in Example 6 were added to the 96-well plate and cultured at 37°C for 72 hours. Transmission electron microscopy images were taken at 0 hours, 12 hours, 24 hours, and 72 hours, respectively. The results are shown in FIG. Fig.12 As shown in the figure, the untreated Pseudomonas aeruginosa presents a typical short rod shape, with a clear and smooth cell wall, a complete cell structure, and evenly distributed cytoplasm in the bacteria. Obvious flagella can be seen and the bacterial capsule can be vaguely seen. After 24 hours, the cell wall is not visible, the cytoplasm in the bacteria is uneven, and cavities begin to appear. The nuclear membrane structure is blurred, and the core begins to dissolve. After 48 hours, the cell structure is further damaged. Almost all the cells used have cavities and are almost empty shells. The cell wall structure of some cells is blurred and discontinuous, and the contents are leaking or malnourished. After 72 hours, the bacteria even completely shattered and disintegrated, and the bacterial morphology is almost invisible.

[0155] XI. Experimental study on the effect of the nanoformulation prepared by the present invention on the activity of fibroblasts induced by TGF-β

[0156] Cell viability was assessed by MTT assay. NIH-3T3 cells were cultured in 96-well plates at 5 × 10 3 The cells were seeded at a density of 10 cells per well overnight, and the cells were induced with serum-free DMEM containing or not containing 10 ng / ml TGF-β1 for 24 h. Then, the TGF-β1-induced and non-induced cells were incubated with TTO NPs, AZMNPs, CS NPs, TTO@AZM NPs prepared in Example 1, and TTO@AZM-CS NPs prepared in Example 6 at concentrations of 0.375, 0.75, 1.5, 3, 6, 12, 24, 48, and 96 μg / mL for 24 h, and the untreated cells were used as controls. Then the culture medium was discarded, 120 μL of MTT solution was added to each well, and the cells were incubated at 37°C in the dark for 4 hours. The MTT solution was discarded and dried, and 150 μL of DMSO was added to each well to dissolve the formazan, and the OD value was measured at 490 nm using a microplate reader. All experiments were performed independently at least 3 times, and the results are shown in the following table. Fig.13 As shown, the nanoformulation prepared by the present invention has an inhibitory effect on the cell viability of TGF-β-induced fibroblasts, and the TTO@AZM-CS NPs prepared in Example 6 has a better effect.

[0157] 12. The in vivo efficacy and safety of the nanoformulation prepared by the present invention were investigated through in vivo animal experiments.

[0158] In order to study the anti-fibrosis ability of aerosol inhaled nanoformulations at the in vivo level and the advantages of aerosol inhalation, a bleomycin-induced pulmonary fibrosis mouse animal model was established.

[0159] Grouping and drug delivery:

[0160] The mice were randomly divided into: Control group, BLM group, azithromycin AZM group and TTO@AZM-CS group prepared in Example 6. The mice were randomly divided into groups according to the above groups, and the mice were treated with drugs every other day starting from the 7th day after bleomycin induction, for a total of 21 days.

[0161] Drug administration for each group:

[0162] Control group: mice were allowed to breathe freely in the nebulizer inhalation device and inhale normal saline in a conscious state;

[0163] BLM group: Bleomycin-induced mice (same as above) were allowed to breathe freely with aerosolized normal saline;

[0164] AZM group: AZM was orally administered to bleomycin-induced mice; the dosage was 100 μg.

[0165] TTO@AZM-CS group: The mice induced by bleomycin were allowed to breathe freely and aerosolized with an equal volume of normal saline of the TTO@AZM-CS nanoformulation prepared in Example 6, with a dosage of 100 μL and a concentration of 1 μg / μL.

[0166] After 21 days, the mice were euthanized and the blood and lung tissues of each group of mice were collected. The lung tissues were fixed in formalin and then subjected to H&E staining and Masson staining.

[0167] Fig.14 This is a comparison chart of the 21-day survival curves of mice in different treatment groups in the animal experiment; it can be seen from the survival curve that compared with the Control group, the mortality rate of mice in the BLM group was 75%, while the mortality rate of mice in the nebulized TTO@AZM-CS group and the oral AZM group was reduced. From the establishment of the IPF model to the end of 21 days of treatment, the survival rate of mice in the nebulized TTO@AZM-CS nanoformulation group remained at 70%.

[0168] Fig.15This is a comparison chart of the weight changes of mice in different treatment groups in the animal experiment; according to the weight change curve, it can be seen that the weight of mice in the Control group (healthy control group) has been steadily and continuously increasing, and the weight of mice in the BLM group (bleomycin modeling group) has gradually decreased, which indirectly proves that the pulmonary fibrosis mouse model has been successfully established. Compared with the BLM group, the weight of mice in the nebulized TTO@AZM-CS group and the oral AZM group began to gradually recover on the 7th to 9th day, and the average weight of mice in the nebulized TTO@AZM-CS group and the oral AZM group increased on the 21st day.

[0169] Fig.16 The figures are comparisons of lung tissue morphology, H&E staining and Masson staining of mice in different treatment groups in the animal experiment. By observing the alveolar structure, collagen deposition and tissue morphology, it can be seen that compared with the BLM group, the aerosol inhalation TTO@AZM-CS nanoformulation group and the oral AZM group weakened the increase in alveolar septum thickening caused by bleomycin, the damaged alveolar cavity structure gradually recovered, and the collagen deposition and parenchymal damage were reduced. The improvement of the aerosol inhalation TTO@AZM-CS nanoformulation group was more obvious.

Claims

1. A nanoformulation for delivering antibiotics based on plant extracts, characterized in that: The mass volume ratio of the antibiotic to the plant extract in the nano preparation is 1-10 mg:20-100 μL.

2. A nanoformulation for delivering antibiotics based on plant extracts according to claim 1, characterized in that: The antibiotic is at least one of penicillin antibiotics, cephalosporin antibiotics, macrolide antibiotics, aminoglycoside antibiotics, quinolone antibiotics, tetracycline antibiotics, sulfonamide antibiotics, rifamycin antibiotics or glycopeptide antibiotics.

3. A nanoformulation for delivering antibiotics based on plant extracts according to claim 1 or 2, characterized in that: The antibiotic is at least one of azithromycin, moxifloxacin, doxycycline, neomycin or rapamycin.

4. A nanoformulation for delivering antibiotics based on plant extracts according to claim 3, characterized in that: The plant extract is at least one of tea tree oil, luteolin, vitamin E or bitter almond oil.

5. A nanoformulation for delivering antibiotics based on plant extracts according to claim 4, characterized in that: The particle size of the nano preparation is in the range of 50 to 300 nm, and the PDI is in the range of 0.1 to 0.

4.

6. A nanoformulation for delivering antibiotics based on plant extracts according to claim 4, characterized in that: The preparation method of the nano preparation comprises the following steps: Step 1: dissolving antibiotics, phospholipids and cholesterol in an organic solvent, removing the organic solvent in the obtained mixed system by rotary evaporation under reduced pressure at 40-45°C, and drying the obtained film in a constant temperature vacuum oven at 37-45°C overnight; Step 2: adding a hydration liquid to the film dried overnight in step 1 to hydrate it, adding a stabilizer to the hydration system, performing water bath ultrasound to cause the film to fall off, and performing ultrasound treatment at a power of 20 to 1000 kHz for 5 to 20 minutes under ice bath conditions to obtain a lipid suspension; Step 3: Add a surfactant to the plant extract, mix thoroughly and then add to the lipid suspension obtained in step 2, perform water bath sonication until transparent, further perform sonication for 5 to 20 minutes, and perform ultrafiltration to obtain a nano preparation.

7. A nanoformulation for delivering antibiotics based on plant extracts according to claim 6, characterized in that: In step 1, the molar ratio of antibiotics, phospholipids and cholesterol is 1-30:50-95:3-30; the phospholipid is at least one of dipalmitoylphosphatidylcholine DPPC, distearoylphosphatidylcholine DSPC, dioleoylphosphatidylcholine DOPC, dimyristoylphosphatidylethanolamine DMPE, dipalmitoylphosphatidylethanolamine DPPE, distearoylphosphatidylethanolamine DSPE or soybean lecithin.

8. A nanoformulation for delivering antibiotics based on plant extracts according to claim 7, characterized in that: In step 2, the hydration liquid is at least one of pure water, physiological buffer, sucrose solution, trehalose solution, glucose solution, mannitol solution or Ringer's solution; in step 3, the surfactant is one of anionic surfactant or non-ionic surfactant.

9. A nanoformulation for delivering antibiotics based on plant extracts according to claim 8, characterized in that: The method further comprises the step of adding an equal volume of chitosan solution with a mass concentration of 0.01 to 100 mg / mL to the ultrasonic system obtained in the step of 3, magnetically stirring at 400 rpm for 1 to 2 hours, and ultrafiltration to obtain a nano preparation.

10. Use of a nanoformulation for delivering antibiotics based on plant extracts as claimed in any one of claims 1 to 9 in the preparation of a drug for treating lung diseases.

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