CN119326717B

CN119326717B - A nano-formulation for delivering antibiotics based on plant extracts and its application

Info

Publication number: CN119326717B
Application number: CN202411691193.XA
Authority: CN
Inventors: 请求不公布姓名
Original assignee: Harbin Medical University
Current assignee: Harbin Medical University
Priority date: 2024-11-25
Filing date: 2024-11-25
Publication date: 2025-11-18
Anticipated expiration: 2044-11-25
Also published as: CN119326717A

Abstract

The invention relates to a nano preparation for delivering antibiotics based on plant extracts and application thereof, belonging to the technical field of nano preparations. In order to solve the problems of low bioavailability and low acting efficiency of oral or intravenous 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 extracts in the nano preparation is 1-10 mg:20-100 mu L. The nano preparation can remarkably inhibit migration of fibroblasts, has an obvious antibacterial effect, can reduce microbial density in the lung, combines positive charges with negative charges on the surface of bacterial cell membranes, influences transmembrane substance transport on the bacterial cell membranes, influences bacterial metabolism and reproduction, better realizes regulation of symbiotic bacteria of the lung, and can relieve the progress of pulmonary fibrosis. The nano preparation has the advantages of small toxic and side effects, good patient compliance, higher encapsulation efficiency and the like.

Description

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

Technical Field

The invention belongs to the technical field of nano preparations, and particularly relates to a nano preparation for delivering antibiotics based on a plant extract and application thereof.

Background

Changes in the pulmonary flora are associated with the development and progression of a variety of respiratory diseases. Researchers believe that there is a steady state balance of microbial "migration in and out" in the healthy lung, and when people suffer from respiratory diseases, the balance of the airway flora is also broken, and the cilia of the lung become dysfunctional, mucus secretion is increased, and bacterial migration is enhanced.

Two general conditions occur, the first being that the microbial species in the lungs themselves are unchanged or reduced but the abundance is increased, and the overall microbial load is increased. The second is the occurrence of other pathogens in the lungs due to a variety of factors, resulting in increased microbial species in the lungs, direct pathogenic or symbiotic modulation of opportunistic pathogens in the otherwise lung. One study showed that the bacterial load in bronchoalveolar lavage fluid from patients with idiopathic pulmonary fibrosis (Idiopathic Pulmonary Fibrosis, IPF) was about 2 times that of the control group, with streptococcus, neisseria, haemophilus, veillonella being higher than the control group and associated with IPF independence. The association of the pulmonary microbiota with IPF was also observed in animal models.

Certain clinically commonly used antibiotics have antibacterial activity and also have an immunoregulatory effect, can reduce acute exacerbation mortality and hospitalization rate of IPF patients, inhibit myofibroblast differentiation and development of pulmonary fibrosis, and play an anti-fibrosis role. However, the bioavailability of the oral antibiotics is low, and the antibiotics are rapidly distributed throughout the body after intravenous drip administration, so that the effect efficiency at focus positions is low. Meanwhile, antibiotics have limited antibacterial spectrum and have certain limitation on the regulation effect of the microbial flora of the lung. Combination therapy can compensate for this deficiency, but multiple administrations often increase the burden on the patient.

Another difficulty in the clinical treatment of pulmonary diseases is that pathogenic bacteria block the penetration of antibiotics by forming a biofilm, so that the bacteria therein have extremely strong resistance. Therefore, the medicine which can meet the clinical application requirements, effectively regulate the symbiotic bacteria of the lung and penetrate and destroy the biological membrane is an urgent technical problem to be solved in the medicine field.

Disclosure of Invention

In order to solve the problems of low bioavailability and low acting efficiency of oral or intravenous antibiotics, the invention provides a nano preparation for delivering antibiotics based on plant extracts and application thereof.

The technical scheme of the invention is as follows:

The nano preparation for delivering the antibiotics based on the plant extracts has the mass-volume ratio of the antibiotics to the plant extracts of 1-10 mg to 20-100 mu L.

Further, the antibiotic is at least one of penicillin antibiotics, cephalosporin antibiotics, macrolide antibiotics, aminoglycoside antibiotics, quinolone antibiotics, tetracycline antibiotics, sulfanilamide antibiotics, rifamycin antibiotics or glycopeptide antibiotics.

Further, the antibiotic is at least one of azithromycin, moxifloxacin, doxycycline, neomycin or rapamycin.

Further, the plant extract is at least one of tea tree oil, luteolin, vitamin E or bitter almond oil.

Further, the particle size range of the nano preparation is 50-300 nm, and the PDI is 0.1-0.4.

Further, the preparation method of the nano preparation comprises the following steps:

dissolving antibiotics, phospholipids and cholesterol in an organic solvent, removing the organic solvent in the obtained mixed system by reduced pressure rotary evaporation at the temperature of 40-45 ℃, and drying the obtained film in a constant-temperature vacuum box at the temperature of 37-45 ℃ overnight;

Step two, adding a hydration liquid into the film which is dried overnight in the step one for hydration, adding a stabilizing agent into a hydration system, enabling the film to fall off by water bath ultrasonic, and carrying out ultrasonic treatment for 5-20 min under the ice bath condition at 20-1000 kHz power to obtain a lipid suspension;

And thirdly, adding a surfactant into the plant extract, fully and uniformly mixing, adding the mixture into the lipid suspension obtained in the second step, performing water bath ultrasonic treatment until the lipid suspension is transparent, performing ultrasonic treatment for 5-20 min, and performing ultrafiltration to obtain the nano preparation.

Further, in the first step, the molar ratio of the antibiotics to the phospholipids to the cholesterol is 1-30:50-95:3-30, wherein the phospholipids are at least one of dipalmitoyl phosphatidylcholine DPPC, distearoyl phosphatidylcholine DSPC, dioleoyl phosphatidylcholine DOPC, dimyristoyl phosphatidylethanolamine DMPE, dipalmitoyl phosphatidylethanolamine DPPE, distearoyl phosphatidylethanolamine DSPE or soybean phospholipids.

Further, the hydration liquid in the second step is at least one of pure water, physiological buffer solution, sucrose solution, trehalose solution, glucose solution, mannitol solution or ringer's solution, and the surfactant in the third step is one of anionic surfactant or nonionic surfactant.

And further, adding chitosan solution with the equal volume and mass concentration of 0.01-100 mg/mL into the ultrasonic system obtained in the step III, magnetically stirring at 400rpm for 1-2 hours, and performing ultrafiltration to obtain the nano preparation.

Use of a nano-formulation for delivering antibiotics based on plant extracts for the preparation of a medicament for the treatment of pulmonary diseases.

The invention has the beneficial effects that:

The invention provides a nano preparation for delivering antibiotics based on plant extracts, which can remarkably inhibit migration of fibroblasts, has an obvious antibacterial effect, can reduce the microbial density in the lung, inhibit and destroy the biofilm of pathogenic bacteria, remarkably reduce the drug resistance of pathogenic bacteria, and further effectively exert the antibacterial effect. The nano preparation provided by the invention carries positive charges and combines with negative charges on the surface of a bacterial cell membrane, so that the trans-membrane substance transport on the bacterial cell membrane is influenced, the metabolism and propagation of bacteria are influenced, the regulation of symbiotic bacteria of the lung is better realized, and the progress of pulmonary fibrosis is relieved. The nano preparation can effectively improve the survival rate of the pulmonary fibrosis mice and improve the survival condition of the mice by adjusting symbiotic flora, so that the weight of the mice can be stably increased, and the treatment is effective.

The preparation method of the nano preparation provided by the invention enhances the stability of the plant extract and solves the problems that the plant extract is unstable and volatile and is sensitive to oxygen, light and temperature. Meanwhile, chitosan is dissolved in a weak acid solution, positive cationic groups can be formed in the acid solution, and antibiotics carried by the plant extracts have negative charges and are combined with the antibiotics through electrostatic adsorption. The aerosol inhalation nano preparation prepared by the invention has smaller and even particle size, is suitable for aerosol inhalation administration, and simultaneously has the advantages of antibacterial and anti-fibrosis performance, small toxic and side effects, good patient compliance, higher encapsulation efficiency and the like.

Drawings

FIG. 1 is a TEM image of the TTO@AZM-CS nano-preparation prepared in example 6;

FIG. 2 is a graph showing the particle size distribution of TTO@AZM-CS nano-preparation prepared in example 6;

FIG. 3 is a standard curve of an antibiotic according to the present invention;

FIG. 4 is a graph showing the comparison of cell scratch states in a cell scratch test, with the scale of 200 μm;

FIG. 5 is a graph showing cell mobility contrast in a cell scratch experiment;

FIG. 6 is a graph showing comparison of culture results of broth in a minimum inhibitory concentration test;

FIG. 7 is a photograph of TSA platelets in a zone of inhibition test;

FIG. 8 is a graph showing comparison of the size of the inhibition zone in the inhibition zone test;

FIG. 9 is a graph comparing experimental results of inhibition of bacterial biofilm formation by different drugs;

FIG. 10 is a graph comparing experimental results of bacterial biofilm disruption by different drugs;

FIG. 11 is a graph showing the comparison of the experimental results of the bacterial biofilm disruption at different drug concentrations for the nano-formulation of example 6;

FIG. 12 is a graph showing the results of experiments on bacterial biofilm disruption by the nano-formulation of example 6 at various time of action;

FIG. 13 is an experiment of activity of different nano-formulations on TGF-beta induced fibroblasts;

FIG. 14 is a graph showing comparison of 21 natural survival curves of mice in different treatment groups in animal experiments;

FIG. 15 is a graph showing comparison of weight changes of mice in different treatment groups in animal experiments;

FIG. 16 is a comparison of lung tissue morphology, H & E staining and Masson staining of mice from different treatment groups in animal experiments.

Detailed Description

The following embodiments are used for further illustrating the technical scheme of the present invention, but not limited thereto, and all modifications and equivalents of the technical scheme of the present invention are included in the scope of the present invention without departing from the spirit and scope of the technical scheme of the present invention. The process equipment or apparatus not specifically mentioned in the following examples are all conventional equipment or apparatus in the art, and the raw materials and the like used in the examples of the present invention are commercially available unless otherwise specified, and the technical means used in the examples of the present invention are all conventional means well known to those skilled in the art.

Example 1

The present example provides a nano-formulation for delivering antibiotics based on plant extracts and a method of preparing the same.

The components of the nano preparation in the embodiment are azithromycin AZM, dipalmitoyl phosphatidylcholine DPPC, water solution pure water, plant extract tea tree oil TTO and a surfactant Tween-80. Tea tree oil in this example was purchased from mikrin corporation under the product number 68647-73-4.

The preparation method of the nano preparation of the embodiment comprises the following steps:

Step one, precisely weighing 2mg of azithromycin, 20mg of dipalmitoyl phosphatidylcholine DPPC and 2mg of cholesterol, dissolving in 1mL of mixed organic solvent prepared by chloroform and methanol according to a volume ratio of 3:1, removing the organic solvent by rotary evaporation under reduced pressure at 40 ℃ for 2 hours, and drying overnight in a vacuum incubator at a constant temperature of 37 ℃.

And step two, adding 250 mu L of pure water into the film which is dried overnight in the step one, hydrating for 1h, enabling the film to fall off by water bath ultrasonic, and further performing ultrasonic treatment for 5min by probe ultrasonic under the ice bath condition at 700KHz power so as to obtain clear lipid suspension.

And step three, absorbing 50 mu L of tea tree oil and 160 mu L of Tween-80, adding the tea tree oil and 160 mu L of Tween-80 into the lipid suspension obtained in the step two after fully mixing, adding the lipid suspension into a Millipore ultrafiltration centrifuge tube after carrying out ultrasonic treatment for 5min at 700KHz power, centrifuging for 10min at the rotating speed of 4000rpm, collecting the upper liquid from the sleeve, marking the upper liquid as a TTO@AZM nano preparation, sealing with nitrogen, and preserving in a refrigerator at a dark place at 4 ℃.

Example 2

The components of the nano preparation in the embodiment are moxifloxacin, dipalmitoyl phosphatidylcholine, water solution pure water, plant extract vitamin E and a surfactant Tween-80.

step one, precisely weighing 3mg of moxifloxacin, 20mg of dipalmitoyl phosphatidylcholine and 2mg of cholesterol, dissolving in 1mL of a mixed organic solvent prepared by chloroform and methanol according to a volume ratio of 3:1, performing rotary evaporation under reduced pressure at 40 ℃ for 2 hours to remove the organic solvent, and drying overnight in a vacuum incubator at a constant temperature of 37 ℃.

And step two, adding 230 mu L of pure water into the film which is dried overnight in the step one, hydrating for 1h, enabling the film to fall off by water bath ultrasonic, and further performing ultrasonic treatment for 5min by probe ultrasonic under the ice bath condition at 800KHz power so as to obtain clear lipid suspension.

And thirdly, sucking 80 mu L of vitamin E and 20 mu LTween-80, adding the mixture into the lipid suspension obtained in the second step, performing ultrasonic treatment on the mixture in a water bath until the mixture is transparent, performing ultrasonic treatment on the mixture by probe type ultrasonic waves at 800KHz power for 5min, adding the mixture into a Millipore ultrafiltration centrifuge tube, centrifuging the mixture for 10min at the rotating speed of 4000rpm, collecting the upper liquid from the sleeve, thus obtaining the nano preparation, sealing the nitrogen, and preserving the nano preparation in a refrigerator at a dark place at the temperature of 4 ℃.

Example 3

The components of the nano preparation in the embodiment are doxycycline, dipalmitoyl phosphatidylcholine, hydration liquid pure water, plant extract bitter almond oil and surfactant glycerol.

Step one, precisely weighing 4.5mg of doxycycline, 20mg of dipalmitoyl phosphatidylcholine and 2mg of cholesterol, dissolving in 1mL of mixed organic solvent prepared by chloroform and methanol according to a volume ratio of 3:1, removing the organic solvent by rotary evaporation under reduced pressure at 40 ℃ for 2 hours, and drying overnight in a vacuum incubator at a constant temperature of 37 ℃.

And step two, adding 240 mu L of pure water into the film which is dried overnight in the step one, hydrating for 1h by using water bath ultrasonic waves to enable the film to fall off, and further performing ultrasonic treatment for 5min by using probe type ultrasonic waves under the ice bath condition at 400KHz power so as to obtain clear lipid suspension.

And thirdly, absorbing 70 mu L of bitter almond oil and 150 mu L of glycerol, fully and uniformly mixing, adding the mixture into the lipid suspension obtained in the second step, carrying out ultrasonic treatment on the mixture in a water bath until the mixture is transparent, further carrying out ultrasonic treatment on the mixture by probe type ultrasonic waves at 400KHz power for 5min, adding the treated mixture into a Millipore ultrafiltration centrifuge tube, centrifuging the mixture for 10min at the rotating speed of 4000rpm, collecting the upper liquid from the sleeve, thus obtaining the nano preparation, sealing the nitrogen, and preserving the nano preparation in a refrigerator at the dark temperature of 4 ℃.

Example 4

The components of the nano preparation in the embodiment are neomycin, dipalmitoyl phosphatidylcholine, hydration liquid pure water, plant extract bitter almond oil and a surfactant Tween-80.

Step one, precisely weighing 6mg of neomycin, 20mg of dipalmitoyl phosphatidylcholine and 2mg of cholesterol, dissolving in 1mL of mixed organic solvent prepared by chloroform and methanol according to the volume ratio of 3:1, removing the organic solvent by rotary evaporation under reduced pressure at 40 ℃ for 2h, and drying overnight in a vacuum incubator at a constant temperature of 37 ℃.

And step two, adding 250 mu L of pure water into the film which is dried overnight in the step one, hydrating for 1h, enabling the film to fall off by water bath ultrasonic, and further performing ultrasonic treatment for 5min by probe ultrasonic under the ice bath condition at 400KHz power so as to obtain clear lipid suspension.

And thirdly, absorbing 50 mu L of bitter almond oil and 160 mu L of Tween-80, fully and uniformly mixing, adding the mixture into the lipid suspension obtained in the second step, carrying out water bath ultrasonic treatment until the lipid suspension is transparent, further carrying out probe type ultrasonic treatment at 400KHz power for 5min, adding the obtained product into a Millipore ultrafiltration centrifuge tube, centrifuging for 10min, collecting the upper liquid from a sleeve at the rotating speed of 4000rpm, thus obtaining the nano preparation, sealing the nitrogen, and preserving the nano preparation in a refrigerator at a temperature of 4 ℃ in a dark state.

Example 5

The components of the nano preparation in the embodiment are rapamycin, dipalmitoyl phosphatidylcholine, hydration liquid pure water, plant extract luteolin and a surfactant Tween-80.

step one, precisely weighing 10mg of rapamycin, 20mg of dipalmitoyl phosphatidylcholine and 2mg of cholesterol, dissolving in 1mL of mixed organic solvent prepared by chloroform and methanol according to the volume ratio of 3:1, removing the organic solvent by rotary evaporation under reduced pressure at 40 ℃ for 2h, and drying overnight in a vacuum incubator at a constant temperature of 37 ℃.

And step two, adding 260 mu L of pure water into the film which is dried overnight in the step one for hydration for 1h, carrying out water bath ultrasonic treatment to enable the film to fall off, and carrying out probe ultrasonic treatment for 5min under the ice bath condition under the power of 300KHz so as to obtain clear lipid suspension.

And thirdly, absorbing 100 mu L of luteolin and 150 mu LTween-80, adding the mixture into the lipid suspension obtained in the second step after fully and uniformly mixing, carrying out water bath ultrasonic treatment until the lipid suspension is transparent, further carrying out probe type ultrasonic treatment at 300KHz power for 5min, adding the obtained product into a Millipore ultrafiltration centrifuge tube, centrifuging for 10min at the rotating speed of 4000rpm, collecting the upper liquid from the sleeve, thus obtaining the nano preparation, sealing the nano preparation with nitrogen, and storing the nano preparation in a refrigerator at the temperature of 4 ℃ in a dark place.

Example 6

The components of the nano preparation in the embodiment are azithromycin, dipalmitoyl phosphatidylcholine DPPC, water solution pure water, plant extract tea tree oil TTO, a surfactant Tween-80 and chitosan CS. Tea tree oil in this example was purchased from mikrin corporation under the product number 68647-73-4.

And step two, adding 250 mu L of pure water into the film which is dried overnight in the step one, hydrating for 1h, enabling the film to fall off by water bath ultrasonic, and further performing ultrasonic treatment for 5min by probe ultrasonic under the ice bath condition at 800KHz power so as to obtain clear lipid suspension.

And thirdly, sucking 50 mu L of tea tree oil and 160 mu L of Tween-80, fully and uniformly mixing, adding the mixture into the lipid suspension obtained in the second step, performing water bath ultrasonic treatment until the mixture is transparent, and performing probe ultrasonic treatment for 5min at 800KHz power to obtain an ultrasonic system with the total volume of 500 mu L.

And step four, adding 500 mu L of 10mg/mL chitosan solution into the ultrasonic system obtained in the step three, magnetically stirring at 400rpm for 2 hours, adding into a Millipore ultrafiltration centrifuge tube, centrifuging for 10 minutes, collecting the upper liquid from the sleeve at 4000rpm, recording the upper liquid as TTO@AZM-CS nano preparation, sealing with nitrogen, and storing in a refrigerator at a temperature of 4 ℃ in a dark place.

Example 7

The components of the nano preparation in the embodiment are moxifloxacin, dipalmitoyl phosphatidylcholine, pure water of hydration liquid, vitamin E of plant extract, a surfactant Tween-80 and chitosan CS.

step one, precisely weighing 2mg of moxifloxacin, 20mg of dipalmitoyl phosphatidylcholine and 2mg of cholesterol, dissolving in 1mL of a mixed organic solvent prepared by chloroform and methanol according to a volume ratio of 3:1, performing rotary evaporation under reduced pressure at 40 ℃ for 2 hours to remove the organic solvent, and drying overnight in a vacuum incubator at a constant temperature of 37 ℃.

And step two, adding 230 mu L of pure water into the film which is dried overnight in the step one for hydration for 1h, adding 40 mu L of glycerol into a hydration system, enabling the film to fall off by water bath ultrasonic, and further carrying out probe ultrasonic treatment for 5min under the ice bath condition at 800KHz power so as to obtain clear lipid suspension.

And thirdly, sucking 80 mu L of vitamin E and 150 mu LTween-80, adding the mixture into the lipid suspension obtained in the second step after fully mixing, performing water bath ultrasonic treatment until the lipid suspension is transparent, and performing probe ultrasonic treatment for 5min at 800KHz power to obtain an ultrasonic system with the total volume of 500 mu L.

And step four, adding 500 mu L of a chitosan solution with the concentration of 15mg/mL into the ultrasonic system obtained in the step three, magnetically stirring at 400rpm for 2 hours, adding into a Millipore ultrafiltration centrifuge tube, centrifuging for 10 minutes at the rotation speed of 4000rpm, collecting the upper liquid from the sleeve, and obtaining the nano preparation, sealing with nitrogen, and storing in a refrigerator at the temperature of 4 ℃ in a dark place.

Example 8

The components of the nano preparation in the embodiment are doxycycline, dipalmitoyl phosphatidylcholine, hydration liquid pure water, plant extract bitter almond oil, a surfactant Tween-80 and chitosan.

step one, 9mg of doxycycline, 20mg of dipalmitoyl phosphatidylcholine and 2mg of cholesterol are precisely weighed and dissolved in 1mL of mixed organic solvent prepared by chloroform and methanol according to the volume ratio of 3:1, the organic solvent is removed by rotary evaporation under reduced pressure at 40 ℃ for 2 hours, and the mixture is dried in a vacuum incubator at the constant temperature of 37 ℃ for overnight.

And step two, adding 240 mu L of pure water into the film which is dried overnight in the step one for hydration for 1h, adding 40 mu L of glycerol into a hydration system, carrying out water bath ultrasonic treatment to enable the film to fall off, and carrying out probe ultrasonic treatment for 5min under the ice bath condition at 600KHz power so as to obtain clear lipid suspension.

And thirdly, absorbing 70 mu L of bitter almond oil and 150 mu L of Tween-80, fully and uniformly mixing, adding the mixture into the lipid suspension obtained in the second step, performing water bath ultrasonic treatment until the lipid suspension is transparent, and performing probe ultrasonic treatment for 5min at 600KHz power to obtain an ultrasonic system with the total volume of 500 mu L.

And step four, adding 500 mu L of a 20mg/mL chitosan solution into the ultrasonic system obtained in the step three, magnetically stirring at 400rpm for 2 hours, adding into a Millipore ultrafiltration centrifuge tube, centrifuging for 10 minutes at 4000rpm, collecting the upper liquid from the sleeve, and obtaining the nano preparation, sealing with nitrogen, and storing in a refrigerator at a temperature of 4 ℃ in a dark place.

Example 9

The components of the nano preparation in the embodiment are neomycin, dipalmitoyl phosphatidylcholine, hydration liquid pure water, plant extract tea tree oil and a surfactant Tween-80.

step one, precisely weighing 2mg of neomycin, 20mg of dipalmitoyl phosphatidylcholine and 2mg of cholesterol, dissolving in 1mL of mixed organic solvent prepared by chloroform and methanol according to the volume ratio of 3:1, removing the organic solvent by rotary evaporation under reduced pressure at 40 ℃ for 2h, and drying overnight in a vacuum incubator at a constant temperature of 37 ℃.

And step two, adding 250 mu L of pure water into the film which is dried overnight in the step one for hydration for 1h, adding 40 mu L of glycerol into a hydration system, enabling the film to fall off by water bath ultrasonic, and further carrying out probe ultrasonic treatment for 5min under the ice bath condition at 200KHz power so as to obtain clear lipid suspension.

And thirdly, sucking 50 mu L of tea tree oil and 160 mu L of Tween-80, fully and uniformly mixing, adding the mixture into the lipid suspension obtained in the second step, performing water bath ultrasonic treatment until the mixture is transparent, and performing probe ultrasonic treatment for 5min at 200KHz power to obtain an ultrasonic system with the total volume of 500 mu L.

And step four, adding 500 mu L of 30mg/mL chitosan solution into the ultrasonic system obtained in the step three, magnetically stirring at 400rpm for 2 hours, adding into a Millipore ultrafiltration centrifuge tube, centrifuging for 10 minutes at 4000rpm, collecting the upper liquid from the sleeve, and obtaining the nano preparation, sealing with nitrogen, and storing in a refrigerator at a temperature of 4 ℃ in a dark place.

Example 10

The components of the nano preparation in the embodiment are rapamycin, dipalmitoyl phosphatidylcholine, hydration liquid pure water, stabilizer glycerol, plant extract bitter almond oil and a surfactant Tween-80.

Step one, precisely weighing 2mg of rapamycin, 20mg of dipalmitoyl phosphatidylcholine and 2mg of cholesterol, dissolving in 1mL of mixed organic solvent prepared by chloroform and methanol according to the volume ratio of 3:1, removing the organic solvent by rotary evaporation under reduced pressure at 40 ℃ for 2h, and drying overnight in a vacuum incubator at a constant temperature of 37 ℃.

And step two, adding 260 mu L of pure water into the film which is dried overnight in the step one for hydration for 1h, carrying out water bath ultrasonic treatment to enable the film to fall off, and carrying out probe ultrasonic treatment for 5min under the ice bath condition at 200KHz power so as to obtain clear lipid suspension.

And thirdly, sucking 50 mu L of bitter almond oil and 150 mu L of Tween-80, fully and uniformly mixing, adding the mixture into the lipid suspension obtained in the second step, performing water bath ultrasonic treatment until the lipid suspension is transparent, and performing probe ultrasonic treatment for 5min at 200KHz power to obtain an ultrasonic system with the total volume of 500 mu L.

1. The transmission electron microscope characterization of the nano-preparation prepared by the invention is examined.

The TTO@AZM-CS nano preparation prepared in the embodiment 6 is subjected to transmission electron microscopy detection by utilizing a transmission electron microscopy negative dyeing technology, and the specific method comprises the steps of dropwise adding the TTO@AZM-CS nano preparation solution prepared in the embodiment 6 onto a copper wire, drying, dropwise adding 1 drop of 2% phosphotungstic acid dyeing liquid onto the copper wire for 30s, sucking the dyeing liquid, drying, and performing TEM shooting. TEM of the nano-preparation prepared in example 6 is shown in FIG. 1, and the TTO@AZM-CS nano-preparation has a regular spherical appearance.

2. The particle size and Zeta potential of the nano-preparation prepared by the invention are measured

The particle size, PDI and Zeta potential of the nanoformulations prepared in examples 1-10 were measured using a Markov nanoparticle size analyzer (MalvernNano ZS Zetasizer). Taking 3 mu L of the nano preparation prepared in each example, respectively adding into 3000 mu L of ultrapure water for dilution, ensuring uniform dispersion, taking a proper amount of nano preparation aqueous solution, placing into a cuvette, and measuring the hydration particle size, the polydisperse index (PDI) and the Zeta potential of the nano preparation by dynamic light scattering analysis. Table 1 shows DLS characterization of the nano-formulations obtained in examples 1-10, and FIG. 2 shows the particle size distribution diagram of the TTO@AZM-CS nano-formulations prepared in example 6.

TABLE 1

Smaller nanoscales can allow nanomaterials to achieve greater total volume accumulation of drug and deeper penetration depth. The plant extract can be used as an essential oil to be inserted into the surface of a lipid membrane, so that the fluidity of the membrane is increased, the strength of the membrane is weakened, and after probe type ultrasonic treatment, lipid vesicles are easier to split, so that the particle size of a nano molecular probe is reduced.

3. Investigation of the encapsulation efficiency of the nano-preparation prepared by the invention

Establishing an azithromycin standard curve, and measuring the encapsulation efficiency of the azithromycin in the nano preparation.

Azithromycin solutions with drug concentrations of 3000, 1500, 750, 375, 187.5 and 93.75 mug/mL are sequentially prepared by using methanol solution, and a standard curve is drawn by taking the concentration (X) of the azithromycin as an abscissa and the peak area (Y) as an ordinate as a linear regression equation through HPLC detection, wherein the standard curve is shown in figure 3.

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

The encapsulation efficiency was measured by ultrafiltration, 50 μl of the nano-formulations prepared in example 1 and example 6 were respectively absorbed, and added into 950 μl of methanol solution for demulsification, followed by thoroughly shaking and mixing to completely rupture. The content of azithromycin is measured by HPLC, namely the mass of the loaded nano preparation is recorded as W, and the drug loading and encapsulation efficiency are calculated according to the following formula:

Encapsulation efficiency ee=w/W _{Total (S)} x100%, drug loading dl=w/W _L x100%

Wherein W _{Total (S)} is the total amount of azithromycin medicine, and W _L is the total mass of the nano preparation.

Calculated for the nano-formulation of example 1, the encapsulation efficiency was 60.1.+ -. 0.03%, the drug loading was 4.7.+ -. 0.006%, the encapsulation efficiency was 59.1.+ -. 0.001%, the drug loading was 4.5.+ -. 0.003%, the encapsulation efficiency was 57.5.+ -. 0.01%, the drug loading was 4.0.+ -. 0.006%, the encapsulation efficiency was 61.3.+ -. 0.03%, the drug loading was 3.8.+ -. 0.009%, the encapsulation efficiency was 56.9.+ -. 0.07%, the drug loading was 4.1.+ -. 0.002%, the encapsulation efficiency was 61.3.+ -. 0.05%, the drug loading was 4.4.+ -. 0.003%, the encapsulation efficiency was 55.2.+ -. 0.07%, the drug loading was 4.4.+ -. 0.006%, the encapsulation efficiency was 59.2.+ -. 0.05%, the drug loading was 4.1.+ -. 0.003%, the encapsulation efficiency was 56.9.+ -. 0.03%, the drug loading was 4.03.+ -. 0.03%, and the drug loading was 4.6.03%, the encapsulation efficiency was 4.6.+ -. 0.001% and the drug loading was 4.3.+ -. 0.03%.

4. Examination of cell migration ability of the preparation of the nano-preparation of the invention

Cell scratch experiments are a common method for researching cell migration capacity, and are simple and easy to implement.

Firstly, a marker pen is used for drawing transverse lines on the back of a 6-hole plate, each line is 1cm apart, lines are uniform and parallel and cross through holes, NIH-3T3 cells are inoculated into the 6-hole plate at the cell density of 2x10 ⁵ cells/hole, the cells are incubated overnight, 200 mu L gun heads are used for drawing lines perpendicular to the back of the hole plate, cell scratches are artificially manufactured, PBS is used for moistening the cells to remove cell fragments, different groups of solutions (Control group, TGF-beta 1 group, example 6-TTO@AZM-CS group and AZM group) are added into each hole, the state of the cell scratches is observed under a microscope and photographed, after 1 hour induction is carried out by adding TGF-beta 1, serum-free DMEM is replaced for 24, and photographing is carried out as shown in fig. 4 and 5. The cell mobility calculation formula in fig. 5 is as follows:

Cell mobility= (0 hr scratch area-24 hr scratch area)/0 hr scratch area×100%

One of the reasons why fibrosis progresses is fibroblast migration, and fig. 4 and 5 illustrate that the synthesized nano-preparation of the present invention can significantly inhibit fibroblast migration, and that the inhibition of different pharmaceutical components is different, and the inhibition of fibroblast migration by using plant extracts (plant essential oils) as carriers is much better than that of the simple use of antibiotics, because chitosan and tea tree oil have antibacterial effects in addition to azithromycin. Chitosan is a natural antibacterial agent, and under the acidic condition, amino cations on a chitosan chain are combined with negative charges on the surface of a bacterial cell membrane through electrostatic adsorption, so that the trans-membrane substance transport on the bacterial cell membrane is influenced, the metabolism and the propagation of bacteria are influenced, and the antibacterial effect on gram-negative bacteria is good. Tea tree oil is also a broad-spectrum natural antibacterial agent, so that the tea tree oil can play a role in combined bacteriostasis.

5. Investigation of the Minimum Inhibitory Concentration (MIC) of the nano-formulations prepared according to the present invention

Through literature studies, DNA was isolated for 16S rDNA gene sequencing from the lungs of experimental mice that had either intratracheal delivery of normal saline or BLM. Gram-negative bacteroides are the most up-regulated bacteria in the pulmonary fibrosis model, bacteroidae (Bacteroidaceae) and Prevotella (Prevotellaceae) are the two most up-regulated families in bacteroidae (Bacteroidetes). So we choose the Bacteroides oval Bacteroides ovatusATCC 8483 to carry out the antibacterial experiment.

The Minimum Inhibitory Concentration (MIC) of the TTO@AZM-CS nano preparation of AZM and example 6 on Bacteroides ovalis Bacteroides ovatus was determined by a trace broth dilution method, wherein the MIC is an index for detecting the antibacterial activity of the antibacterial drug, and the smaller the MIC value, the stronger the antibacterial activity.

The TTO@AZM-CS nano preparation of example 6 and Azithromycin AZM were diluted by a double ratio with a modified minced medium, the mass concentrations of AZM from the 1 st well to the 7 th well were 10, 5, 2.5, 1.25, 0.625, 0.3125 and 0.15625 mug/mL respectively, the 8 th well was a positive control, no antibacterial agent was added, only the bacterial solution was added, the 9 th well was a negative control, and only the broth medium was contained. mu.L of the bacterial liquid was inoculated into 90. Mu.L of 96-well plates containing TTO@AZM-CS nanoformulations or AZM at different concentration gradients. The 96-well plates were placed in a 37 ℃ incubator for 24 hours. After 24 hours, bacterial growth was observed visually, the concentration in the wells at which little turbidity and no bacterial growth were observed was defined as MIC, and the results are shown in fig. 6.

The nano preparation synthesized by taking the plant extract (plant essential oil) as a carrier has more obvious antibacterial effect than the simple use of antibiotics. The plant extract (plant essential oil) is used for acting on bacteria cultivated in a small dish, the concentration of the antibiotics is 0.625 mug/mL, but the common commercial antibiotics only start to have a slight antibacterial effect when the concentration reaches 1.25 mug/mL, which indicates that the TTO@AZM-CS nano preparation has more excellent antibacterial activity.

6. Experiments for examining bacteriostasis circle of nano preparation prepared by the invention

The growth inhibition of Azithromycin AZM and the TTO@AZM-CS nano preparation of example 6 on Bacteroides ovalis Bacteroides ovatus is detected through a bacteriostasis zone experiment.

The method comprises the steps of selecting single bacterial colonies on a TSA blood plate by an inoculating loop, diluting an OD value by a broth culture medium to be about 0.5 specific turbidity, preparing a medicated filter paper sheet, setting three groups of TTO@AZM-CS novel nano preparation groups, AZM groups and Control groups according to experiments, respectively adding 20 mu L of solutions with different components, waiting for drying of the paper sheet, dipping 100 mu L of bacterial liquid by a sterile cotton swab, uniformly marking the bacterial liquid in a zigzag manner along the initial position above the TSA blood plate, uniformly smearing the bacterial liquid, respectively placing the filter paper sheets at the corresponding positions of the plate marks, slightly pressing the filter paper sheets by forceps, culturing for 16-20 hours under anaerobic conditions, observing a bacteriostasis ring around the filter paper sheets, and measuring the diameter of the bacteriostasis ring by using a caliper, as shown in figure 8.

Fig. 7 and 8 show that the tto@azm-CS nano preparation shows a higher antibacterial effect under the condition that the mass concentration of azithromycin is the same, and the diameter of the antibacterial ring generated by the tto@azm-CS group is 1.46 times that of the AZM group as can be seen from the antibacterial ring. The novel nano-scale medicament synthesized by taking the plant extract as a carrier has obvious antibacterial effect, and the antibacterial circle is obviously larger than that of common commercial antibiotics.

7. Investigation of experiments for inhibiting bacterial biofilm formation by the nano preparation prepared by the invention

Pseudomonas aeruginosa (Pseudomonas aeruginosa) was selected as the primary pathogen for pulmonary infection to investigate the ability of the prepared nano-formulations to inhibit bacterial biofilm formation.

Pseudomonas aeruginosa was added to 96-well plates with TTO@AZM-CS NPs prepared in example 6, TTO@AZM NPs prepared in example 1, AZM NPs, TTO NPs, CS NPs, AZM solutions and PBS, respectively, and incubated at 37℃for 24 hours. Then, crystal violet staining was performed to measure cell membranes. The free bacteria without film formation were discarded, 100. Mu.L of methanol was added to each well and the mixture was fixed at room temperature for 15 minutes, the methanol was removed by suction, and the mixture was naturally air-dried. Then, the plate was dyed with 0.05% crystal violet dye for 5 minutes, and the excess dye was washed off with deionized water, and the plate was dried in an oven. Then 33% acetic acid solution was added to each well and the plate was shaken for 15 minutes to dissolve the dye completely. The OD value of the crystal violet dye solution at 570nm is measured by an enzyme-labeled instrument, the comparison with a negative control group is carried out, and the total biofilm proportion is calculated, and the result is shown in figure 9. The TTO@AZM-CS NPs prepared in example 6, the TTO@AZM NPs prepared in example 1, the AZM NPs, the TTO NPs, the CS NPs and the AZM solution all have the capacity of inhibiting bacterial biofilm formation, but the TTO@AZM-CS NPs inhibition effect is optimal compared with an unaddressed PA group.

8. Investigation of experiments of destroying bacterial biofilm by the nano preparation prepared by the invention

Pseudomonas aeruginosa was inoculated into 96-well plates and allowed to stand at 37℃for 48 hours to allow formation of mature bacterial biofilms. 100. Mu.L of the supernatant was pipetted off per well, and TTO@AZM-CS NPs prepared in example 6, TTO@AZM NPs prepared in example 1, AZM NPs, TTO NPs, CS NPs, AZM solutions and PBS were then added to 96-well plates at the same probe concentration, respectively, and incubated at 37℃for 24 hours. Then crystal violet staining detection was performed according to the experiment of inhibiting biofilm formation, and the result is shown in FIG. 10, in which the biofilm disruption effect of TTO@AZM-CS NPs was optimal.

9. Experiment for examining bacterial biofilm destruction of nano preparation prepared by the invention with different concentrations

Pseudomonas aeruginosa was inoculated into 96-well plates and allowed to stand at 37℃for 48 hours to allow formation of mature bacterial biofilms. 100. Mu.L of supernatant was pipetted per well, then TTO@AZM-CS NPs prepared in example 6 and PBS at 0.75, 1.5, 3, 6, 12, 24, 48 and 96. Mu.g/mL were added to 96 well plates, respectively, and incubated at 37℃for 24 hours. Then, crystal violet staining was performed according to the experiment of inhibiting biofilm formation, and the result is shown in FIG. 11, wherein the higher the TTO@AZM-CS NPs concentration, the better the effect of destroying the bacterial biofilm.

10. Experiments for examining the bacterial biofilm destruction of the nano preparation prepared by the invention with different action time

Pseudomonas aeruginosa was inoculated into 96-well plates and allowed to stand at 37℃for 48 hours to allow formation of mature bacterial biofilms. 100 mu L of supernatant was pipetted from each well, TTO@AZM-CS NPs prepared in example 6 were added to a 96-well plate, and were subjected to static culture at 37℃for 72h, and transmission electron microscopy was performed at 0h, 12h, 24h and 72h, respectively, as shown in FIG. 12, the untreated Pseudomonas aeruginosa exhibited typical short rod shape, clear and smooth cell walls, complete cell structures, uniform cytoplasmic distribution in the cells, and visible thin and visible bacterial capsules in the apparent flagellum. After 24 hours, the cell wall is invisible, the cytoplasm in the bacteria is not uniform, the cavity shape begins to appear, the nuclear membrane structure is fuzzy, and the core begins to dissolve. After 48 hours, the cell structure is further destroyed, almost all cells are in a hollow thallus approximate empty shell, and the cell wall structure of part of cells is fuzzy and discontinuous, and the content is leaked or malnourished. After 72 hours the bacteria even disintegrated completely, almost no bacterial morphology was seen.

11. Investigation of the activity of the prepared nano-preparation on TGF-beta induced fibroblast

Cell viability was assessed by MTT assay. NIH-3T3 cells were seeded overnight in 96-well plates at a density of 5X 10 ³ cells per well and cells were induced for 24h with serum-free DMEM with or without 10ng/ml TGF-beta 1. TGF-. Beta.1-induced and uninduced cells were then incubated with TTO NPs, AZM NPs, CS 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. Mu.g/mL for 24h, and untreated cells were used as controls. The medium was then discarded, 120 μl of MTT solution was added to each well, incubated at 37 ℃ in the dark for 4 hours, blotted MTT solution was discarded, 150 μl of LDMSO dissolved formazan was added to each well, and OD was measured at 490nm using an enzyme-labeled instrument. All experiments were independently performed at least 3 times, and as shown in fig. 13, the nano preparation prepared by the invention has an inhibitory effect on the cell viability of TGF-beta induced fibroblasts, and the tto@azm-CS NPs prepared in example 6 has a better effect.

12. The in-vivo curative effect and the safety of the nano preparation prepared by the invention are inspected by in-vivo animal experiments.

In order to study the anti-fibrosis capability of the aerosol inhalation nano preparation at the living body level and the aerosol inhalation advantage, a bleomycin-induced pulmonary fibrosis mouse animal model is established.

Grouping and drug delivery:

Mice were randomly grouped: control group, BLM group, azithromycin AZM group and TTO@AZM-CS group prepared in example 6. The mice were randomized according to the above-described groupings and were given a total of 21 days of treatment starting on day-to-day intervals after bleomycin induction.

Each group was dosed:

a Control group for freely breathing and atomizing the mice in the atomized inhalation device to inhale physiological saline under the awake state;

BLM group, free breath nebulization of bleomycin-induced mice (supra) into physiological saline;

AZM group bleomycin-induced mice were orally administered AZM at a dose of 100 μg.

TTO@AZM-CS group TTO@AZM-CS nanoformulation prepared in example 6 by atomizing bleomycin-induced free breath of mice at an equivalent volume to normal saline was administered at a concentration of 1 μg/μl delivering 100 μl.

Mice were euthanized after 21 days, blood and lung tissue from each group was collected, and lung tissue was fixed in formalin, followed by H & E staining and Masson staining.

Fig. 14 is a graph showing comparison of 21 natural survival curves of mice in different treatment groups in animal experiments, and it can be seen from survival curves that compared with a Control group, the death rate of the mice in the BLM group is 75%, the death rate of the mice in the aerosol inhalation TTO@AZM-CS group and the mice in the oral AZM group are reduced, and the survival rate of the fraction of the aerosol inhalation TTO@AZM-CS nano preparation group is kept at 70% from the beginning of the establishment of an IPF model to the end of 21 days of treatment.

Fig. 15 is a comparison graph of weight changes of mice in different treatment groups in animal experiments, and according to the weight change curve, it can be seen that the weight of the mice in a Control group (healthy Control group) steadily and continuously rises, the weight of the mice in a BLM group (bleomycin modeling group) gradually decreases, and the success of establishing a pulmonary fibrosis mouse model is indirectly proved. The weights of the mice in the nebulized inhaled TTO@AZM-CS group and the oral AZM group began to recover gradually on days 7-9, and the average weights of the mice in the nebulized inhaled TTO@AZM-CS group and the oral AZM group increased on day 21, as compared to the BLM group.

Fig. 16 is a graph showing comparison of lung tissue morphology, H & E staining and Masson staining of mice in different treatment groups in animal experiments, and it is known that, by observing alveolar structure, collagen deposition and tissue morphology, the aerosol inhalation tto@azm-CS nano preparation group and oral AZM group weaken the increase of alveolar space thickening caused by bleomycin, the damaged alveolar space structure is gradually recovered, collagen deposition and substantial destruction are reduced, and the improvement of aerosol inhalation tto@azm-CS nano preparation group is more obvious than that of BLM group.

Claims

1. The nano preparation for delivering the antibiotics based on the plant extracts is characterized in that the mass volume ratio of the antibiotics to the plant extracts in the nano preparation is 1-10 mg:20-100 mu L, the plant extracts are tea tree oil, and the antibiotics are at least one of azithromycin, moxifloxacin, doxycycline, neomycin or rapamycin;

The preparation method of the nano preparation comprises the following steps:

And thirdly, adding a surfactant into the plant extract, fully and uniformly mixing, adding the mixture into the lipid suspension obtained in the second step, performing water bath ultrasonic treatment until the mixture is transparent, performing ultrasonic treatment for 5-20 min, and performing ultrafiltration to obtain the nano preparation.

2. The botanical extract based antibiotic delivery nano-formulation of claim 1, wherein the nano-formulation has a particle size in the range of 50-300 nm and a pdi in the range of 0.1-0.4.

3. The nano preparation for delivering antibiotics based on plant extracts according to claim 2, wherein in the first step, the molar ratio of the antibiotics to the phospholipids to the cholesterol is 1-30:50-95:3-30, and the phospholipids are at least one of dipalmitoyl phosphatidylcholine DPPC, distearoyl phosphatidylcholine DSPC, dioleoyl phosphatidylcholine DOPC, dimyristoyl phosphatidylethanolamine DMPE, dipalmitoyl phosphatidylethanolamine DPPE, distearoyl phosphatidylethanolamine DSPE or soybean phospholipids.

4. The plant extract-based antibiotic delivery nano-formulation according to claim 3, wherein 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 the second step, and the surfactant in the third step is one of an anionic surfactant or a nonionic surfactant.

5. The nano preparation for delivering antibiotics based on plant extracts according to claim 4, further comprising the step four of adding chitosan solution with the equal volume mass concentration of 0.01-100 mg/mL into the ultrasonic system obtained in the step three, and performing magnetic stirring for 1-2 h by 400 rpm, and performing ultrafiltration to obtain the nano preparation.

6. Use of a nano-formulation for the delivery of antibiotics based on plant extracts according to any of claims 1 to 5 for the preparation of a medicament for the treatment of pulmonary diseases, characterized in that said pulmonary diseases are pulmonary fibrosis.