A nano-formulation based on plant extract for delivery of antibiotics and its use

Abstract

The present application relates to a kind of nanometer preparation based on plant extract delivery antibiotic and its application, belong to nanometer preparation technical field.To solve the problem of low bioavailability and low efficiency in the treatment of pulmonary diseases by oral or intravenous injection of antibiotic, the present application provides a kind of nanometer preparation based on plant extract delivery antibiotic.The mass-volume ratio of antibiotic and plant extract in nanometer preparation is 1-10 mg:20-100 μL.The nanometer preparation of the present application can significantly inhibit the migration of fibroblasts, has obvious bacteriostatic effect, and can reduce the microbial density in lung;Its positive charge is combined with the negative charge on the surface of bacterial cell membrane, affects the transport of transmembrane substances on bacterial cell membrane, affects bacterial metabolism and reproduction, better realizes the regulation of lung symbiotic bacteria, and can alleviate the progression of pulmonary fibrosis.The nanometer preparation of the present application has the advantages of small toxic and side effects, good patient compliance, high encapsulation rate and the like.

Description

Technical Field

[0001] This invention belongs to the field of nanoparticle technology, and particularly relates to a nanoparticle formulation for delivering antibiotics based on plant extracts and its application. Background Technology

[0002] Changes in the lung microbiota are linked to the development and progression of various respiratory diseases. Researchers believe that in a healthy lung, there is a homeostatic balance of microbial migration in and out. When people suffer from respiratory diseases, this balance is disrupted, leading to dysfunction of the cilia in the lungs, increased mucus secretion, and enhanced bacterial migration.

[0003] Two scenarios typically occur. The first is that the types of lung microorganisms remain unchanged or decrease, but their abundance increases, leading to an overall increased microbial load. The second is due to various factors, including the introduction of other pathogens into the lungs, resulting in an increase in lung microbial diversity. These foreign pathogens may directly cause disease or symbiotically regulate the abnormal growth of opportunistic pathogens already present in the lungs, leading to disease. One study showed that the bacterial load in bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis (IPF) was approximately twice that of the control group, with higher levels of Streptococcus spp., Neisseria spp., Haemophilus spp., and Veillonella spp., all independently associated with IPF. A similar association between lung microbiota and IPF has been observed in animal models.

[0004] Some commonly used antibiotics, in addition to their antibacterial activity, also have immunomodulatory effects, which can reduce the mortality and hospitalization rates of acute exacerbations in IPF patients, inhibit myofibroblast differentiation and the development of pulmonary fibrosis, and exert anti-fibrotic effects. However, oral antibiotics have low bioavailability, while intravenously administered antibiotics rapidly distribute throughout the body, resulting in low efficacy at the lesion site. At the same time, antibiotics have a limited antibacterial spectrum, limiting their ability to regulate the pulmonary microbiota. Combination therapy can compensate for this deficiency, but multiple administrations often increase the burden on patients.

[0005] Another challenge in the clinical treatment of lung diseases is that pathogenic bacteria form biofilms that block the penetration of antibiotics, resulting in highly resistant bacteria. Therefore, providing a drug that meets clinical application needs, effectively regulates lung symbiotic bacteria, and can penetrate and disrupt biofilms has become an urgent technical problem to be solved in the pharmaceutical field. Summary of the Invention

[0006] To address the issues of low bioavailability and low efficacy of oral or intravenous antibiotics, this invention provides a nano-formulation for delivering antibiotics based on plant extracts and its applications.

[0007] The technical solution of the present invention:

[0008] A nano-formulation for delivering antibiotics based on plant extracts, wherein the mass-to-volume ratio of antibiotics to plant extracts in the nano-formulation 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 range of the nano-formulation is 50–300 nm, and the PDI is 0.1–0.4.

[0013] Furthermore, the preparation method of the nano-formulation includes the following steps:

[0014] Step 1: Dissolve antibiotics, phospholipids, and cholesterol in an organic solvent, and remove the organic solvent from the resulting mixture by rotary evaporation under reduced pressure at 40-45°C. Place the resulting film in a vacuum oven at 37-45°C and dry overnight.

[0015] Step 2: Add hydration solution to the film that has been dried overnight in Step 1 for hydration, add stabilizer to the hydration system, use water bath sonication to detach the film, and use ice bath sonication at 20-1000kHz power for 5-20 minutes to obtain lipid suspension.

[0016] Step 3: Add surfactant to plant extract, mix thoroughly, add to lipid suspension obtained in step 2, sonicate in water bath until transparent, further sonicate for 5-20 minutes, and ultrafilter to obtain nano-formulation.

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

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

[0019] Furthermore, the process includes step four: adding an equal volume of chitosan solution with a mass concentration of 0.01–100 mg / mL to the ultrasonic system obtained in step three, stirring magnetically at 400 rpm for 1–2 hours, and then ultrafiltration to obtain the nano-formulation.

[0020] Application of a plant extract-based nano-formulation for delivering antibiotics in the preparation of drugs for treating lung diseases.

[0021] The beneficial effects of this invention are:

[0022] This invention provides a nano-formulation for delivering antibiotics based on plant extracts. It significantly inhibits fibroblast migration, exhibits marked antibacterial activity, reduces lung microbial density, inhibits and disrupts the biofilm of pathogenic bacteria, and significantly reduces antibiotic resistance, thus exerting a more effective antibacterial effect. The nano-formulation carries a positive charge that binds to the negative charge on the bacterial cell membrane surface, affecting transmembrane transport, bacterial metabolism, and reproduction, thereby better regulating lung symbiotic bacteria and alleviating the progression of pulmonary fibrosis. By regulating the symbiotic microbiota, this nano-formulation effectively improves the survival rate of mice with pulmonary fibrosis, enhances their overall health, and promotes stable weight gain, demonstrating therapeutic efficacy.

[0023] The nano-formulation preparation method provided by this invention enhances the stability of plant extracts, solving the problems of instability, volatility, and sensitivity to oxygen, light, and temperature. Simultaneously, chitosan dissolves in weakly acidic solutions, forming positively charged cationic groups in the acidic environment, while the antibiotics encapsulated in the plant extracts have a negative charge, binding to them through electrostatic adsorption. The nebulized inhalation nano-formulation prepared by this invention has a small and uniform particle size, suitable for nebulized inhalation administration. Furthermore, this nano-formulation possesses advantages such as antibacterial and antifibrotic properties, low toxicity and side effects, good patient compliance, and high encapsulation efficiency. Attached Figure Description

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

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

[0026] Figure 3 Antibiotic standard curves established for this invention;

[0027] Figure 4 The images show a comparison of cell scratch patterns in a cell scratch assay. The scale bars in the images are all 200 μm.

[0028] Figure 5 A comparison of cell migration rates in a cell scratch assay;

[0029] Figure 6 A comparative graph showing the culture results of broth culture medium in the minimum inhibitory concentration test;

[0030] Figure 7 Photographs of TSA blood plates during the inhibition zone test;

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

[0032] Figure 9 Comparison of experimental results on the inhibition of bacterial biofilm formation by different drugs;

[0033] Figure 10 A comparative diagram showing the experimental results of different drugs disrupting bacterial biofilms;

[0034] Figure 11 This is a comparison of the experimental results of different drug concentrations of the nano-formulation used in Example 6 disrupting bacterial biofilms.

[0035] Figure 12 Figure 6 shows the experimental results of the nano-formulation in Example 6 disrupting bacterial biofilms at different contact times.

[0036] Figure 13 Experiments were conducted to assess the activity of different nano-formulations against TGF-β-induced fibroblasts.

[0037] Figure 14 A comparison of 21-day survival curves of mice in different treatment groups in animal experiments;

[0038] Figure 15 A comparison chart of weight changes in mice under different treatment groups in animal experiments;

[0039] Figure 16 Comparison of lung tissue morphology, H&E staining, and Masson staining in mice under different treatment groups in animal experiments. Detailed Implementation

[0040] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise 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 nano-formulation for delivering antibiotics based on plant extracts and its preparation method.

[0043] In this embodiment, the nano-formulation consists of azithromycin (AZM), dipalmitoylphosphatidylcholine (DPPC), purified water as a hydration solution, tea tree oil (TTO) as a plant extract, and the surfactant Tween-80. The tea tree oil in this embodiment was purchased from McLean, product number 68647-73-4.

[0044] The preparation method of the nano-formulation in this embodiment includes the following steps:

[0045] Step 1: Accurately weigh 2 mg azithromycin, 20 mg dipalmitoylphosphatidylcholine (DPPC), and 2 mg cholesterol, dissolve them 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 under reduced pressure at 40 °C for 2 h, and dry overnight in a vacuum incubator at 37 °C.

[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 sonication to detach the film. Then, use probe sonication at 700 kHz power for 5 minutes under ice bath conditions to obtain a clear lipid suspension.

[0047] Step 3: Take 50 μL of tea tree oil and 160 μL of Tween-80, mix them thoroughly, and add them to the lipid suspension obtained in Step 2. Sonicate at 700 kHz for 5 min, then add to a Millipore ultrafiltration centrifuge tube and centrifuge for 10 min at 4000 rpm. Collect the supernatant from the tube and label it as TTO@AZM nano-formulation. Seal it with nitrogen and store it in a refrigerator at 4°C in the dark.

[0048] Example 2

[0049] This embodiment provides a nano-formulation for delivering antibiotics based on plant extracts and its preparation method.

[0050] In this embodiment, the nano-formulation consists of moxifloxacin, dipalmitoylphosphatidylcholine, hydrated water, plant extract vitamin E, and surfactant Tween-80.

[0051] The preparation method of the nano-formulation in this embodiment includes the following steps:

[0052] Step 1: Accurately weigh 3 mg of moxifloxacin, 20 mg of dipalmitoylphosphatidylcholine, and 2 mg of cholesterol, dissolve them 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 under reduced pressure at 40 °C for 2 h, and dry overnight in a vacuum incubator at 37 °C.

[0053] Step 2: Add 230 μL of pure water to the film that has been dried overnight in Step 1 and hydrate for 1 hour. Use water bath sonication to detach the film. Then, use probe sonication at 800 kHz power for 5 minutes under ice bath conditions to obtain a clear lipid suspension.

[0054] Step 3: Take 80 μL of vitamin E and 20 μL of Tween-80 and add them to the lipid suspension obtained in Step 2. Sonicate in a water bath until transparent, and then further sonicate with a probe at 800 kHz for 5 min. Add the mixture to a Millipore ultrafiltration centrifuge tube and centrifuge for 10 min at 4000 rpm. Collect the supernatant from the tube to obtain the nano-formulation. Seal with nitrogen and store in a refrigerator at 4°C in the dark.

[0055] Example 3

[0056] This embodiment provides a nano-formulation for delivering antibiotics based on plant extracts and its preparation method.

[0057] In this embodiment, the nano-formulation consists of doxycycline, dipalmitoylphosphatidylcholine, hydrated water, bitter almond oil (plant extract), and glycerin (surfactant).

[0058] The preparation method of the nano-formulation in this embodiment includes the following steps:

[0059] Step 1: Accurately weigh 4.5 mg doxycycline, 20 mg dipalmitoylphosphatidylcholine, and 2 mg cholesterol, dissolve them 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 under reduced pressure at 40 °C for 2 h, and dry overnight in a vacuum incubator at 37 °C.

[0060] Step 2: Add 240 μL of pure water to the film that has been dried overnight in Step 1 and hydrate for 1 hour. Use a water bath to sonicate the film to detach it. Then, use a probe-type sonicator at 400 kHz power 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 them thoroughly, and add them to the lipid suspension obtained in Step 2. Sonicate in a water bath until transparent, and then further sonicate with a probe at 400 kHz for 5 min. Add the mixture to a Millipore ultrafiltration centrifuge tube, centrifuge for 10 min at 4000 rpm, and collect the supernatant from the tube to obtain the nano-formulation. Seal with nitrogen and store in a refrigerator at 4°C in the dark.

[0062] Example 4

[0063] This embodiment provides a nano-formulation for delivering antibiotics based on plant extracts and its preparation method.

[0064] In this embodiment, the nano-formulation consists of neomycin, dipalmitoylphosphatidylcholine, hydrated water, bitter almond oil (plant extract), and surfactant Tween-80.

[0065] The preparation method of the nano-formulation in this embodiment includes the following steps:

[0066] Step 1: Accurately weigh 6 mg of neomycin, 20 mg of dipalmitoylphosphatidylcholine, and 2 mg of cholesterol, dissolve them 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 under reduced pressure at 40 °C for 2 h, and dry overnight in a vacuum incubator at 37 °C.

[0067] 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 sonication to detach the film. Then, use probe sonication at 400 kHz power 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 them thoroughly, and add them to the lipid suspension obtained in Step 2. Sonicate in a water bath until transparent, and then further sonicate with a probe at 400 kHz for 5 min. Add the mixture to a Millipore ultrafiltration centrifuge tube and centrifuge for 10 min at 4000 rpm. Collect the supernatant from the tube to obtain the nano-formulation. Seal with nitrogen and store in a refrigerator at 4°C in the dark.

[0069] Example 5

[0070] This embodiment provides a nano-formulation for delivering antibiotics based on plant extracts and its preparation method.

[0071] In this embodiment, the nano-formulation consists of rapamycin, dipalmitoylphosphatidylcholine, hydrated water, luteolin plant extract, and surfactant Tween-80.

[0072] The preparation method of the nano-formulation in this embodiment includes the following steps:

[0073] Step 1: Accurately weigh 10 mg of rapamycin, 20 mg of dipalmitoylphosphatidylcholine, and 2 mg of cholesterol, dissolve them 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 under reduced pressure at 40 °C for 2 h, and dry overnight in a vacuum incubator at 37 °C.

[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 sonication to detach the film. Then, use probe sonication at 300 kHz power 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, and then 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, and collect the supernatant from the tube to obtain the nano-formulation. Seal with nitrogen and store in a refrigerator at 4°C in the dark.

[0076] Example 6

[0077] This embodiment provides a nano-formulation for delivering antibiotics based on plant extracts and its preparation method.

[0078] In this embodiment, the nano-formulation consists of azithromycin, dipalmitoylphosphatidylcholine (DPPC), purified water as a hydration solution, tea tree oil (TTO) as a plant extract, Tween-80 as a surfactant, and chitosan (CS). The tea tree oil in this embodiment was purchased from McLean, product number 68647-73-4.

[0079] The preparation method of the nano-formulation in this embodiment includes the following steps:

[0080] Step 1: Accurately weigh 2 mg azithromycin, 20 mg dipalmitoylphosphatidylcholine (DPPC), and 2 mg cholesterol, dissolve them 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 under reduced pressure at 40 °C for 2 h, and dry overnight in a vacuum incubator at 37 °C.

[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 sonication to detach the film. Then, use probe sonication at 800 kHz power for 5 minutes under ice bath conditions to obtain a clear lipid suspension.

[0082] Step 3: Take 50 μL of tea tree oil and 160 μL of Tween-80, mix them thoroughly, and add them to the lipid suspension obtained in Step 2. Sonicate in a water bath until transparent, and then further treat with probe-type sonication at 800 kHz power for 5 min; 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. After stirring magnetically at 400 rpm for 2 hours, add the mixture to a Millipore ultrafiltration centrifuge tube and centrifuge for 10 minutes at 4000 rpm. Collect the supernatant from the tube and label it as TTO@AZM-CS nano-formulation. Seal it with nitrogen and store it in a refrigerator at 4°C in the dark.

[0084] Example 7

[0085] This embodiment provides a nano-formulation for delivering antibiotics based on plant extracts and its preparation method.

[0086] In this embodiment, the components of the nano-formulation are moxifloxacin, dipalmitoylphosphatidylcholine, hydrated water, plant extract vitamin E, surfactant Tween-80, and chitosan CS.

[0087] The preparation method of the nano-formulation in this embodiment includes the following steps:

[0088] Step 1: Accurately weigh 2 mg of moxifloxacin, 20 mg of dipalmitoylphosphatidylcholine, and 2 mg of cholesterol, dissolve them 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 under reduced pressure at 40 °C for 2 h, and dry overnight in a vacuum incubator at 37 °C.

[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. Use water bath sonication to detach the film. Then, use probe sonication at 800 kHz power 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 Ween-80, mix them thoroughly, and add them to the lipid suspension obtained in Step 2. Sonicate in a water bath until transparent, and then further sonicate with probe-type ultrasound at 800 kHz power for 5 min; to obtain an ultrasound 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 hours, then add to a Millipore ultrafiltration centrifuge tube, centrifuge for 10 minutes at 4000 rpm, collect the supernatant from the tube to obtain the nano-formulation, seal with nitrogen, and store in a refrigerator at 4°C in the dark.

[0092] Example 8

[0093] This embodiment provides a nano-formulation for delivering antibiotics based on plant extracts and its preparation method.

[0094] In this embodiment, the nano-formulation consists of doxycycline, dipalmitoylphosphatidylcholine, hydrated water, bitter almond oil (plant extract), Tween-80 surfactant, and chitosan.

[0095] The preparation method of the nano-formulation in this embodiment includes the following steps:

[0096] Step 1: Accurately weigh 9 mg doxycycline, 20 mg dipalmitoylphosphatidylcholine, and 2 mg cholesterol, dissolve them 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 under reduced pressure at 40 °C for 2 h, and dry overnight in a vacuum incubator at 37 °C.

[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. Use water bath sonication to detach the film. Then, use probe sonication at 600 kHz power 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 them thoroughly, and add them to the lipid suspension obtained in Step 2. Sonicate in a water bath until transparent, and then further treat with probe-type sonication at 600 kHz power for 5 min; 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 hours, then add to a Millipore ultrafiltration centrifuge tube. Centrifuge for 10 minutes at 4000 rpm. Collect the supernatant from the tube to obtain the nano-formulation. Seal with nitrogen and store in a refrigerator at 4°C in the dark.

[0100] Example 9

[0101] This embodiment provides a nano-formulation for delivering antibiotics based on plant extracts and its preparation method.

[0102] In this embodiment, the nano-formulation consists of neomycin, dipalmitoylphosphatidylcholine, hydrated water, tea tree oil (a plant extract), and the surfactant Tween-80.

[0103] The preparation method of the nano-formulation in this embodiment includes the following steps:

[0104] Step 1: Accurately weigh 2 mg of neomycin, 20 mg of dipalmitoylphosphatidylcholine, and 2 mg of cholesterol, dissolve them 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 under reduced pressure at 40 °C for 2 h, and dry overnight in a vacuum incubator at 37 °C.

[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. Use water bath sonication to detach the film. Then, use probe sonication at 200 kHz power 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 them thoroughly, and add them to the lipid suspension obtained in Step 2. Sonicate in a water bath until transparent, and then further treat with probe-type sonication at 200 kHz power for 5 min; 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 hours, then add to a Millipore ultrafiltration centrifuge tube, centrifuge for 10 minutes at 4000 rpm, collect the supernatant from the tube to obtain the nano-formulation, seal with nitrogen, and store in a refrigerator at 4°C in the dark.

[0108] Example 10

[0109] This embodiment provides a nano-formulation for delivering antibiotics based on plant extracts and its preparation method.

[0110] In this embodiment, the nano-formulation consists of rapamycin, dipalmitoylphosphatidylcholine, hydrated water, stabilizer glycerol, plant extract bitter almond oil, and surfactant Tween-80.

[0111] The preparation method of the nano-formulation in this embodiment includes the following steps:

[0112] Step 1: Accurately weigh 2 mg of rapamycin, 20 mg of dipalmitoylphosphatidylcholine, and 2 mg of cholesterol, dissolve them 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 under reduced pressure at 40 °C for 2 h, and dry overnight in a vacuum incubator at 37 °C.

[0113] 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 sonication to detach the film. Then, use probe sonication at 200 kHz power for 5 minutes under ice bath conditions to obtain a clear lipid suspension.

[0114] Step 3: Take 50 μL of bitter almond oil and 150 μL of Tween-80, mix them thoroughly, and add them to the lipid suspension obtained in Step 2. Sonicate in a water bath until transparent, and then further treat with probe-type sonication at 200 kHz power for 5 min; 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 hours, then add to a Millipore ultrafiltration centrifuge tube. Centrifuge for 10 minutes at 4000 rpm. Collect the supernatant from the tube to obtain the nano-formulation. Seal with nitrogen and store in a refrigerator at 4°C in the dark.

[0116] I. Transmission electron microscopy characterization of the nano-formulation prepared in this invention.

[0117] The TTO@AZM-CS nanoparticles prepared in Example 6 were examined using transmission electron microscopy (TEM) with negative staining. Specifically, the TTO@AZM-CS nanoparticle solution prepared in Example 6 was dropped onto a copper grid, dried, and then 1 drop of 2% phosphotungstic acid staining solution was added to the grid for staining for 30 seconds. The staining solution was then removed, and the grid was dried before TEM imaging. The TEM images of the nanoparticles prepared in Example 6 are shown below. Figure 1 As shown, the TTO@AZM-CS nanoformulation has a regular spherical shape.

[0118] II. Determination of Particle Size and Zeta Potential of the Nanoparticles Prepared in this Invention

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

[0120] Table 1

[0121]

[0122]

[0123] Smaller nanoscale sizes allow nanomaterials to achieve greater overall drug volume accumulation and deeper penetration depth. Plant extracts, acting as essential oils, can be inserted into the lipid membrane surface, increasing membrane fluidity and weakening membrane strength. After probe-type ultrasound, lipid vesicles are more likely to rupture, thereby reducing the particle size of the nanomolecular probe.

[0124] III. Examination of the encapsulation efficiency of the nano-formulation prepared in this invention

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

[0126] Azithromycin solutions with concentrations of 3000, 1500, 750, 375, 187.5, and 93.75 μg / mL were prepared sequentially using methanol solution. HPLC analysis was performed, and a linear regression equation was plotted with azithromycin concentration (X) on the x-axis and peak area (Y) on the y-axis. A standard curve was then constructed. Figure 3 .

[0127] The regression equation is y = 1322.8x + 10.393, and the correlation coefficient R0 is... 2 =0.9995, indicating that azithromycin has a good linear relationship in the range of 3.75-3000 μg / mL.

[0128] Encapsulation efficiency was determined using ultrafiltration. 50 μL of the nano-formulations prepared in Examples 1 and 6 were added to 950 μL of methanol solution to demulsify, and the mixture was thoroughly shaken to ensure complete demulsification. The azithromycin content was determined by HPLC, which represents the mass of the nano-formulation loaded, denoted as W. The drug loading and encapsulation efficiency were calculated using the following formulas:

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

[0130] In the formula: W 总 W represents the total amount of azithromycin. L This refers to the total mass of the nano-formulation.

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

[0132] IV. Investigating the cell migration ability of the nano-formulation prepared by this invention.

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

[0134] First, use a marker to draw horizontal lines on the back of the 6-well plate, 1 cm apart, with the lines even and parallel, passing through the wells; then, insert NIH-3T3 cells at a density of 2x10⁻¹². 5 Cells were seeded at a density of [number] cells / well in 6-well plates and incubated overnight. Cell scratches were artificially created by streaking the cells perpendicularly to the back of the plate using a 200 μL pipette tip. Cells were washed with PBS to remove cell debris. Different groups of solutions (Control group, TGF-β1 group, Example 6-TTO@AZM-CS group, AZM group) were added to each well. The cell scratches were observed and photographed under a microscope at 0 hours. After induction with TGF-β1 for 1 hour, the cells were replaced with serum-free DMEM, and photographed again after 24 hours. Figure 4 and Figure 5 As shown. Figure 5 The formula for calculating cell migration rate is 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 5This invention demonstrates that the synthesized nano-formulation can significantly inhibit fibroblast migration. Different drug components exhibit varying degrees of inhibition. Using plant extracts (plant essential oils) as carriers demonstrates significantly better inhibition of fibroblast migration than antibiotics alone. This is because, in addition to azithromycin, chitosan and tea tree oil also possess antibacterial properties. Chitosan is a natural antibacterial agent. Under acidic conditions, through electrostatic adsorption, the amino cations on the chitosan chains combine with the negative charges on the bacterial cell membrane surface, affecting transmembrane transport and thus bacterial metabolism and reproduction, exhibiting good antibacterial effects against Gram-negative bacteria. Tea tree oil is also a broad-spectrum natural antibacterial agent, thus contributing to a combined antibacterial effect.

[0137] V. Investigating the minimum inhibitory concentration (MIC) of the nano-formulation prepared in this invention.

[0138] Based on a literature review, DNA was isolated from the lungs of mice that underwent intratracheal administration of saline or BLM for 16S rDNA gene sequencing. Gram-negative Bacteroides was the most upregulated bacterial phylum in the pulmonary fibrosis model, with Bacteroidaceae and Prevotellaceae being the two families with the most significant upregulation within Bacteroidetes. Therefore, we selected *Bacteroides ovatus* ATCC 8483 for antibacterial experiments.

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

[0140] The TTO@AZM-CS nanoparticles and azithromycin AZM from Example 6 were serially diluted using modified minced meat medium. The mass concentrations of AZM in wells 1 through 7 were 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 μg / mL, respectively. Well 8 served as a positive control, containing only bacterial culture without antibiotics. Well 9 served as a negative control, containing only broth medium. 10 μL of bacterial culture was inoculated into 90 μL of a 96-well plate containing different concentration gradients of TTO@AZM-CS nanoparticles or AZM. The 96-well plates were incubated at 37°C for 24 hours. After 24 hours, bacterial growth was observed visually. The concentration at which the wells showed almost no turbidity and no bacterial growth was defined as the MIC. The results are as follows: Figure 6 As shown.

[0141] Nanoparticles synthesized using plant extracts (plant essential oils) as carriers exhibit more significant antibacterial effects than antibiotics alone. When plant extracts (plant essential oils) are applied to bacteria cultured in small dishes, antibiotics at a concentration of 0.625 μg / mL already show significant antibacterial effects. However, commonly available commercial antibiotics only begin to show slight antibacterial effects at a concentration of 1.25 μg / mL. This indicates that TTO@AZM-CS nanoparticles possess superior antibacterial activity.

[0142] VI. Investigating the antibacterial zone of the nano-formulation prepared in this invention.

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

[0144] A single colony was picked from a TSA blood agar plate using an inoculation loop and diluted with broth medium until the OD value was approximately 0.5 (turbidity). Drug-containing filter paper discs were prepared, with three groups: the TTO@AZM-CS novel nano-formulation group, the AZM group, and the Control group. 20 μL of each component solution was added to the discs, and they were allowed to dry. Using a sterile cotton swab, 100 μL of bacterial suspension was applied to the top of the TSA blood agar plate in a zigzag pattern to spread the suspension evenly. The filter paper discs were then placed on the corresponding marked positions on the plate, and gently pressed down with tweezers. The plates were incubated anaerobically for 16-20 hours. Results were as follows: Figure 7 As shown, observe the inhibition zone around the filter paper and measure its diameter with calipers. Figure 8 As shown.

[0145] Figure 7 and Figure 8 The results showed that, at the same concentration of azithromycin, the TTO@AZM-CS nano-formulation exhibited a higher antibacterial effect. The inhibition zone diameter of the TTO@AZM-CS group was 1.46 times that of the AZM group. This indicates that the novel nano-scale drug synthesized using plant extracts as a carrier in this invention has a significant antibacterial effect, with an inhibition zone significantly larger than that of conventional commercial antibiotics.

[0146] VII. Experiments to investigate the inhibition of bacterial biofilm formation by the nano-formulation prepared in this invention.

[0147] Pseudomonas aeruginosa was selected as the main pathogen of lung infection to study the ability of the nano-formulation prepared in this invention to inhibit bacterial biofilm formation.

[0148] Pseudomonas aeruginosa was mixed with TTO@AZM-CS NPs prepared in Example 6, TTO@AZM NPs, AZM NPs, TTO NPs, CS NPs, AZM solution, and PBS prepared in Example 1, and added to 96-well plates. The plates were incubated at 37°C for 24 hours. Crystal violet staining was then performed to measure cell membrane activity. Unfilm-forming free bacteria were discarded, and 100 μL of methanol was added to each well for fixation at room temperature for 15 minutes. The methanol was then removed, and the plates were air-dried. The plates were then stained with 0.05% crystal violet solution for 5 minutes, washed with deionized water to remove excess dye, and the 96-well plates were dried in an oven. 33% acetic acid solution was then added to each well, and the plates were shaken for 15 minutes to ensure complete dye dissolution. The OD value of the crystal violet solution at 570 nm was measured using a microplate reader, and the total biofilm ratio was calculated by comparing it with the negative control group. The results are shown below. Figure 9 As shown, compared with the untreated PA group, the TTO@AZM-CS NPs prepared in Example 6, the TTO@AZM NPs, AZM NPs, TTO NPs, CS NPs and AZM solution prepared in Example 1 all have the ability to inhibit bacterial biofilm formation, but TTO@AZM-CS NPs have the best inhibitory effect.

[0149] 8. Experiments to investigate the disruption of bacterial biofilms by the nano-formulations prepared in this invention.

[0150] Pseudomonas aeruginosa was inoculated into 96-well plates and incubated at 37°C for 48 hours to allow for the formation of a mature bacterial biofilm. 100 μL of supernatant was discarded from each well. 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 were added to the 96-well plates at the same probe concentration, and the plates were incubated at 37°C for 24 hours. Crystal violet staining was then performed according to the biofilm inhibition assay. The results are shown below. Figure 10 As shown, TTO@AZM-CS NPs exhibit the best biofilm disruption effect.

[0151] IX. Experiments to investigate the disruption of bacterial biofilms by nano-formulations prepared in this invention at different concentrations.

[0152] Pseudomonas aeruginosa was inoculated into 96-well plates and incubated at 37°C for 48 hours to allow for the formation of 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, along with PBS, were added to the 96-well plates, and the plates were incubated at 37°C for 24 hours. Crystal violet staining was then performed according to the biofilm inhibition assay, and the results are shown below. Figure 11As shown, the higher the concentration of TTO@AZM-CS NPs, the better the effect on disrupting bacterial biofilms.

[0153] 10. Experiments to investigate the disruption of bacterial biofilms by the nano-formulations prepared in this invention at different treatment times.

[0154] Pseudomonas aeruginosa was inoculated into 96-well plates and incubated statically at 37°C for 48 hours to allow for the formation of 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 plates. The plates were incubated statically at 37°C for 72 hours. Transmission electron microscopy (TEM) was performed at 0 h, 12 h, 24 h, and 72 h. The results are as follows: Figure 12 As shown, untreated *Pseudomonas aeruginosa* exhibits typical short rod-like shapes with clear, smooth cell walls, intact cell structure, and uniform cytoplasm distribution. Visible flagella and a faintly visible bacterial capsule are also present. After 24 hours, the cell walls become invisible, the cytoplasm becomes heterogeneous, and cavitation begins to appear. The nuclear membrane structure becomes blurred, and the core begins to dissolve. After 48 hours, the cell structure is further damaged; almost all cells exhibit cavitation, resembling empty shells. The cell walls of some cells become blurred and discontinuous, with leakage of contents or malnutrition. After 72 hours, the bacteria even completely disintegrate, with almost no bacterial morphology remaining.

[0155] XI. An experiment to investigate the activity of the nano-formulation prepared in this invention against TGF-β-induced fibroblasts.

[0156] Cell viability was assessed using the MTT assay. NIH-3T3 cells were cultured in 96-well plates at a rate of 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 10 μg / mL overnight and induced for 24 h with serum-free DMEM containing or without 10 ng / mL TGF-β1. Then, TGF-β1-induced and uninduced cells were co-incubated for 24 h with TTO NPs, AZM NPs, 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. Untreated cells served as controls. The culture medium was then discarded, and 120 μL of MTT solution was added to each well. The cells were incubated at 37°C in the dark for 4 h. The MTT solution was then discarded and dried. 150 μL of LDMSO was added to each well to dissolve formazan, and the OD value was measured at 490 nm using a microplate reader. All experiments were performed independently at least three times, and the results are as follows: Figure 13 As shown, the nano-formulation prepared in this invention has an inhibitory effect on the cell viability of TGF-β-induced fibroblasts, and the TTO@AZM-CS NPs prepared in Example 6 have a better effect.

[0157] 12. The in vivo efficacy and safety of the nano-formulation prepared in this invention will be investigated through in vivo animal experiments.

[0158] To investigate the in vivo anti-fibrotic ability of nebulized nano-formulations and the advantages of nebulized inhalation, a mouse model of pulmonary fibrosis induced by bleomycin was established.

[0159] Grouping and drug delivery:

[0160] Mice were randomly assigned to four groups: Control group, BLM group, azithromycin AZM group, and TTO@AZM-CS group prepared in Example 6. Following these groupings, mice were treated every other day starting on day 7 after bleomycin induction, for a total of 21 days.

[0161] Dosage for each group:

[0162] Control group: Mice were allowed to breathe freely and inhale saline solution via a nebulizer while awake.

[0163] BLM group: Bleomycin-induced mice (as above) were given free breathing via nebulized saline.

[0164] AZM group: Mice induced by bleomycin were orally administered AZM at a dose of 100 μg.

[0165] TTO@AZM-CS group: TTO@AZM-CS nanoformulation prepared in Example 6 was prepared by nebulizing bleomycin-induced mice with free respiration and an equal volume of physiological saline. The dosage was 100 μL, and the concentration was 1 μg / μL.

[0166] Mice were euthanized 21 days later, and blood and lung tissue were collected from each group of mice. The lung tissue was fixed in formalin and then stained with H&E and Masson staining.

[0167] Figure 14 The survival curves of mice in different treatment groups at 21 days are shown in the animal experiment. The survival curves show that the mortality rate of mice in the BLM group was 75% compared with the Control group, while the mortality rates of mice in the nebulized TTO@AZM-CS group and the oral AZM group were reduced. From the establishment of the IPF model to the end of the 21-day treatment, the survival rate of mice in the nebulized TTO@AZM-CS nano-formulation group remained at 70%.

[0168] Figure 15This is a comparison of body weight changes in mice under different treatment groups during animal experiments. The body weight change curves show that the mice in the Control group (healthy control group) experienced a stable and continuous increase in body weight, while the mice in the BLM group (bleomycin modeling group) gradually lost body weight, indirectly demonstrating the successful establishment of the pulmonary fibrosis mouse model. Compared to the BLM group, the body weight of mice in the nebulized TTO@AZM-CS group and the oral AZM group gradually recovered from day 7 to 9, and the average body weight of mice in both groups increased on day 21.

[0169] Figure 16 The images show comparisons of lung tissue morphology, H&E staining, and Masson staining in mice from different treatment groups during animal experiments. Observation of alveolar structure, collagen deposition, and tissue morphology revealed that, compared with the BLM group, the nebulized TTO@AZM-CS nanoparticle formulation group and the oral AZM group reduced the increase in alveolar septal thickening induced by bleomycin, the damaged alveolar cavity structure gradually recovered, and collagen deposition and parenchymal damage were reduced. Moreover, the improvement was more significant in the nebulized TTO@AZM-CS nanoparticle formulation group.

Claims

1. A nano-formulation for delivering antibiotics based on plant extracts, characterized in that, The mass-to-volume ratio of antibiotic to plant extract in the nano-formulation is 1-10 mg: 20-100 µL, wherein the plant extract is tea tree oil; and the antibiotic is at least one of azithromycin, moxifloxacin, doxycycline, neomycin, or rapamycin. The preparation method of the nano-formulation includes the following steps: Step 1: Dissolve antibiotics, phospholipids, and cholesterol in an organic solvent, and remove the organic solvent from the resulting mixture by rotary evaporation under reduced pressure at 40-45 °C. Place the resulting film in a vacuum oven at 37-45 °C and dry overnight. Step 2: Add hydration solution to the film that has been dried overnight in Step 1 for hydration, add stabilizer to the hydration system, use water bath sonication to detach the film, and use ice bath sonication at 20~1000 kHz power for 5~20 min to obtain lipid suspension. Step 3: Add surfactant to plant extract, mix thoroughly, add to lipid suspension obtained in step 2, sonicate in water bath until transparent, further sonicate for 5-20 min, and ultrafilter to obtain nano-formulation.

2. The nano-formulation for delivering antibiotics based on plant extracts according to claim 1, characterized in that, The nanoparticles have a particle size range of 50-300 nm and a PDI of 0.1-0.

4.

3. The nano-formulation for delivering antibiotics based on plant extracts according to claim 2, characterized in that, In step one, the molar ratio of antibiotics, phospholipids, and cholesterol is 1~30:50~95:3~30; the phospholipid is at least one of dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearylphosphatidylethanolamine (DSPE), or soybean phospholipids.

4. The nano-formulation for delivering antibiotics based on plant extracts according to claim 3, characterized in that, In step two, the hydration solution is at least one of pure water, physiological buffer solution, sucrose solution, trehalose solution, glucose solution, mannitol solution, or Ringer's solution; in step three, the surfactant is one of anionic surfactant or nonionic surfactant.

5. A nano-formulation for delivering antibiotics based on plant extracts according to claim 4, characterized in that, The process also includes step four, adding an equal volume of chitosan solution with a mass concentration of 0.01~100 mg / mL to the ultrasonic system obtained in step three, stirring magnetically at 400 rpm for 1~2 h, and then ultrafiltration to obtain the nano-formulation.

6. The use of a nano-formulation based on plant extracts for delivering antibiotics as described in any one of claims 1-5 in the preparation of a medicament for treating lung diseases, characterized in that, The lung disease mentioned is pulmonary fibrosis.

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