A nanoparticle complex targeting small airway disease of the lung and its preparation method and application

Abstract

The application belongs to the technical field of biological medicine, and provides a nanometer particle complex for targeting small airway disease, and a preparation method and application thereof.The nanometer particle complex is a mannotriose complex nanometer particle made of mannotriose, hyaluronate and liposome.The nanometer delivery system is constructed by using self-assembly method, mannotriose, hyaluronate and liposome.The mannotriose has inhibitory effect on neutral elastase, can improve immunity, resist tumor, reduce inflammatory reaction, etc.The nanometer particle loaded with mannotriose has better stability and bioavailability, can be targeted to be enriched in the lung, and has more significant effects of reducing inflammatory reaction and relieving respiratory dysfunction, etc.Moreover, the preparation method and application steps of the mannotriose complex nanometer particle are relatively simple, and are also very suitable for the development and application of the nanometer drug for targeting small airway disease, etc.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a nanoparticle complex for targeting small airway disease and a preparation method and application thereof. BACKGROUND

[0002] The simple use of conventional western medicine treatment for chronic obstructive pulmonary disease (COPD) has poor compliance, many adverse reactions and unsatisfactory effects, and pulmonary rehabilitation treatment can improve dyspnea, health status and exercise tolerance of patients in the stable phase of COPD and improve the quality of life, among which respiratory function exercise enhances the strength of respiratory muscles, improves respiratory efficiency and enhances the function of lung respiration. The respiration of the lung is related to the function of the vital energy running along the respiratory tract and the function of the blood and qi running through the heart vessels, and the generation and deficiency of the vital energy are related to the function of the lung, spleen and kidney. Traditional Chinese medicine believes that the deficiency of the vital energy is the deficiency of the stable phase of COPD, and the representative prescription of the Bu Yuan Decoction is the Bu Yuan Decoction (drug composition: Bai Zhi, Chen Pi, Dang Gui, Sheng Ma, Chai Hu, Suan Zhe each 10g, Dang Shen, Shan Zhu Yu each 15g, Huang Qi 30g, Zhi Gan Cao 6g).

[0003] The mass spectrum of the classic prescription Bu Yuan Decoction contains 2554 compounds, and the non-target data contains 3623 compounds. Through the compound name and CAS number in the PubChem database and literature, the SMILES is obtained, and finally 4564 compounds with SMILES data are obtained. Among them, 1464 are amino acids and their derivatives, 216 are benzene and its derivatives, 65 are alcohols and amines, 278 are phenolic acids, 48 are glycerophospholipids, 7 are glycerol lipids, 136 are nucleotides and their derivatives, 539 are flavonoids, 32 are quinones, 175 are lignans and coumarins, 488 are other types, 5 are sphingolipids, 22 are tannins, 4 are tryptamines, choline and pigments, 327 are alkaloids, 200 are terpenes, 284 are organic acids, 96 are heterocyclic compounds, 19 are steroids, 31 are fatty acyls, and 128 are lipids.

[0004] Manninotriose is a sugar compound, white crystalline powder, derived from Radix Rehmanniae Preparata. Manninotriose is the highest oligosaccharide component in Radix Rehmanniae Preparata. It can be prepared by hydrolysis of stachyose. Manninotriose has the pharmacological activities of promoting the proliferation of hematopoietic cells, improving immunity, reducing blood sugar, and resisting tumors.

[0005] Hyaluronic acid is an acidic mucopolysaccharide, D-glucuronic acid and N-acetylglucosamine are connected by beta-1, 3-glycosidic bond, and the disaccharide units are connected by beta-1, 4-glycosidic bond. The disaccharide unit can be up to 25000. The molecular weight of hyaluronic acid in the body is from 1 kilo to 20 million daltons. Hyaluronic acid shows many important physiological functions in the body due to its unique molecular structure and physicochemical properties. Studies have shown that hyaluronic acid is also very important for muscles, can promote muscle stem cells to start muscle repair, such as lubricating joints, regulating the permeability of blood vessel wall, regulating protein, water and electrolyte diffusion and transport, promoting wound healing, etc.

[0006] However, there is no report on the combination of mannotriose and hyaluronic acid, and the use of liposome nanocarrier system for lung small airway diseases. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application provides a kind of nano particle complex for targeting lung small airway disease and its preparation method and application, which aims to solve the problems mentioned in the background art. The present application constructs a nano delivery system by self-assembly method with mannotriose, hyaluronic acid and liposome. Mannotriose can improve immunity, anti-tumor, reduce inflammatory response, etc. The use of nano delivery system makes the nano particle loaded with mannotriose have better stability and bioavailability, can be targeted to enrich in the lung, and the effect of reducing inflammation and relieving respiratory dysfunction is more significant. Moreover, the preparation method and application of mannotriose complex nano particle in the present application are relatively simple, and it is also very suitable for the development, popularization and application of nano drug for lung tissue damage repair and inflammation reduction.

[0008] In the first aspect, the present application provides a kind of nano particle complex for targeting lung small airway disease, which is mannotriose complex nano particle made of mannotriose, hyaluronic acid and liposome;The nano particle complex inhibits neutrophil elastase.

[0009] Preferably, the mannotriose is screened from the active ingredients that have an effect on neutrophil elastase based on the mass spectrum data of Buyuan Decoction.

[0010] Preferably, the liposome is any one or more of soybean lecithin, mesenchymal stem cell exosome, exosome fusion liposome, polyamidoamine, mesoporous silica or cyclodextrin polymer.

[0011] Preferably, the active ingredients that have an effect on neutrophil elastase are screened from the mass spectrum data of Buyuan Decoction, and the specific steps are as follows:

[0012] Step S1, establishing a neutrophil elastase machine learning model, and using the neutrophil elastase machine learning model to predict the activity of a compound;

[0013] Step S2, using molecular docking screening to select compounds with good effects on neutrophil elastase, and calculating the free binding energy between the compounds and neutrophil elastase;

[0014] Step S3, according to the results of step S1 and step S2, selecting compounds predicted to be active by the neutrophil elastase machine learning model and having good interactions predicted by the molecular docking model;

[0015] Step S4, sequentially performing molecular docking and calculating free binding energy with neutrophil elastase as the receptor;

[0016] Step S5, according to the calculation result of the free binding energy, it is predicted that mannotriose has an effect on neutrophil elastase.

[0017] In a second aspect, the application provides a preparation method of a nanoparticle complex targeting small airway disease, comprising the following steps:

[0018] A mixed solution is prepared by mixing a hyaluronic acid-ethanol solution with a concentration of 1-20 mg / ml and a liposome-ethanol solution with a concentration of 10-200 mg / ml at a volume ratio of 1:(1-40), and mesenchymal stem cell exosomes are added for incubation for 15 minutes. A mixed solution 1 exosome-fused liposome is obtained.

[0019] A mixed solution II is prepared by adding a mannotriose-ethanol solution with a concentration of 1-10 mg / ml to the mixed solution I, and the volume ratio of the mannotriose-ethanol solution to the mixed solution I is 1:(1-20).

[0020] The mixed solution II is added dropwise to a pure water system by a microfluidic chip during stirring to prepare a mixed solution III.

[0021] The mixed solution III is continuously dialyzed in pure water, and the solution after dialysis is centrifuged to remove the precipitate, and then passed through a liposome propeller, and the obtained supernatant is a mannotriose complex nanoparticle.

[0022] Preferably, after the mixed solution III is prepared, magnetic stirring dispersion, continuous stirring, and ultrasonic treatment are performed; the magnetic stirring dispersion is performed at room temperature; the continuous stirring time is 0.2-0.5 hours; the ultrasonic treatment is 150-300 W probe type ultrasonic treatment under a 36-38℃ water bath, and the ultrasonic treatment time is 5-25 min.

[0023] Preferably, the pH value of the manninotriose complex nanoparticle solution is adjusted with hydrochloric acid until the pH value reaches 6.5-7.5, and then 1,2-pentanediol is added to a final concentration of 0.05-0.1 mg / ml.

[0024] In a third aspect, the present application provides a use of the nanoparticle complex targeting small airway disease of the lung in the preparation of a drug for treating inflammatory lung injury.

[0025] In a fourth aspect, the present application provides a use of the nanoparticle complex targeting small airway disease of the lung in the preparation of a neutrophil elastase inhibitor.

[0026] The present application has the following technical effects: The present application constructs a nanodelivery system by self-assembly method using manninotriose, hyaluronan and liposomes. Manninotriose has inhibitory effect on neutrophil elastase, can improve immunity, anti-tumor, reduce inflammatory reaction, etc. The manninotriose-loaded nanoparticle has better stability and bioavailability, can be targeted to accumulate in the lung, and has more significant effects of reducing inflammatory reaction and relieving respiratory dysfunction. Moreover, the preparation method of the manninotriose complex nanoparticle and the operation steps in application are relatively simple, and it is also very suitable for the development and application of the nanoparticle drug targeting small airway disease of the lung, etc. BRIEF DESCRIPTION OF DRAWINGS

[0027] The exemplary embodiments of the present application can be more completely understood by reference to the following drawings:

[0028] Figure 1 The average particle size of the manninotriose complex nanoparticle prepared in Example 1 of the present application;

[0029] Figure 2 The zeta potential of the manninotriose complex nanoparticle prepared in Example 1 of the present application;

[0030] Figure 3 The TEM transmission electron microscope negative staining scanning graph of the manninotriose complex nanoparticle in Example 1 of the present application;

[0031] Figure 4 The interaction graph between the target neutrophil elastase (1h1b) and manninotriose in Example 1 of the present application;

[0032] Figure 5 The structure of manninotriose;

[0033] Figure 6 The in vivo imaging graph of the enrichment of the manninotriose complex nanoparticle in the lung of a mouse in Example 2 of the present application;

[0034] Figure 7 The Masson staining result graph in Example 5 of the present application;

[0035] Figure 8 Figure 1 is a graph showing the results of detecting the content of IL-1β in the bronchoalveolar lavage fluid by the ELISA kit in Example 5 of the present application; wherein:

[0036] Figure 8 Figure 1 is a graph showing the results of detecting the content of IL-1β in the bronchoalveolar lavage fluid by the ELISA kit in Example 5 of the present application; wherein:

[0037] Figure 8 Figure 1 is a graph showing the results of detecting the content of IL-1β in the bronchoalveolar lavage fluid by the ELISA kit in Example 5 of the present application; wherein:

[0038] Figure 9 Figure 1 is a graph showing the results of detecting the content of IL-1β in the bronchoalveolar lavage fluid by the ELISA kit in Example 5 of the present application; wherein: DETAILED DESCRIPTION

[0039] In order to make the technical problems to be solved by the present application, technical solutions and beneficial effects more clearly understood, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs; the terms used herein are only for the purpose of describing specific examples and not to limit the present application.

[0041] In some embodiments, the present application provides a nanocomposite targeting small airway disease, which is a mannotriose complex nanocomposite made of mannotriose, hyaluronic acid sodium and liposome; the nanocomposite inhibits neutrophil elastase.

[0042] Specifically, the mannotriose is an active ingredient that has an effect on neutrophil elastase, which is screened from the mass spectrum data of the Busheng Decoction.

[0043] Specifically, the liposome is any one or more of soybean lecithin, mesenchymal stem cell exosome, exosome fusion liposome, polyamidoamine (PAMAM), mesoporous silica or cyclodextrin polymer.

[0044] Specifically, the active ingredient that has an effect on neutrophil elastase is screened from the mass spectrum data of the Busheng Decoction, and the specific steps are as follows:

[0045] Step S1, establishing a neutrophil elastase machine learning model, and using the neutrophil elastase machine learning model to predict the activity of the compound;

[0046] Step S2, using molecular docking to screen compounds with good effects on neutrophil elastase, and calculating the free binding energy between the compound and neutrophil elastase;

[0047] Step S3, according to the results of step S1 and step S2, selecting compounds predicted to be active by the neutrophil elastase machine learning model and having good interaction by the molecular docking model;

[0048] Step S4, using neutrophil elastase as the receptor, sequentially performing molecular docking and calculating the free binding energy;

[0049] Step S5, according to the calculation results of the free binding energy, it is predicted that mannotriose has an effect on neutrophil elastase, and the calculation results are shown in Table 1.

[0050] Table 1 Free binding energy calculation results

[0051]

[0052] In some embodiments, the present application provides a preparation method of a nanoparticle complex targeting small airway diseases, comprising the following steps:

[0053] A mixed solution is prepared by mixing a hyaluronic acid-ethanol solution with a concentration of 1-20 mg / ml and a liposome-ethanol solution with a concentration of 10-200 mg / ml at a volume ratio of 1:(1-40), and mesenchymal stem cell exosomes are added for incubation for 15 minutes. A mixed solution 1 exosome-fused liposome is obtained.

[0054] A mixed solution II is prepared by adding a mannotriose-ethanol solution with a concentration of 1-10 mg / ml to the mixed solution I, and the volume ratio of the mannotriose-ethanol solution to the mixed solution I is 1:(1-20);

[0055] The mixed solution II is added dropwise to a pure water system by microfluidic chip during stirring to prepare a mixed solution III;

[0056] The mixed solution III is continuously dialyzed in pure water, and the solution after dialysis is centrifuged to remove the precipitate, and then passed through a liposome propeller. The supernatant obtained is a mannotriose complex nanoparticle.

[0057] Specifically, after the mixed solution III is prepared, magnetic stirring dispersion, continuous stirring, and ultrasonic treatment are performed; the magnetic stirring dispersion is performed at room temperature; the continuous stirring time is 0.2-0.5 hours; the ultrasonic treatment is 150-300 W probe type ultrasonic treatment under a 36-38 °C water bath, and the ultrasonic treatment time is 10-30 min.

[0058] Specifically, the manninotriose complex nanoparticle solution is adjusted to a pH value of 6.5-7.5 using hydrochloric acid, and then 1,2-pentanediol is added to a final concentration of 0.05-0.1 mg / ml.

[0059] In some embodiments, the present application provides use of the nanoparticle complex targeting small airway disease of the lung in the preparation of a drug for treating inflammatory lung injury.

[0060] In some embodiments, the present application provides use of the nanoparticle complex targeting small airway disease of the lung in the preparation of a neutrophil elastase inhibitor.

[0061] Example 1:

[0062] (1) Take manninotriose 1 mg, HA hyaluronic acid 1 mg, and soybean lecithin 20 mg, and dissolve them in 1 ml of ethanol (100%) respectively; adjust the pH value to about 7.0 using 0.5% hydrochloric acid by mass fraction to prepare a manninotriose-ethanol solution with a concentration of 1 mg / ml, a hyaluronic acid-ethanol solution with a concentration of 1 mg / ml, and a soybean lecithin-ethanol solution with a concentration of 20 mg / ml. Add mesenchymal stem cell exosomes 2 mg / ml. Incubate for 15 minutes. Obtain a soybean lecithin exosome fusion liposome mixture. The manninotriose is purchased from Shanghai Taiwei Pharmaceutical Co., Ltd., CAS: 13382-86-0; the exosomes are purchased from ThermoFisher. The hyaluronic acid is purchased from MERCK Company, item number 924474; and the soybean lecithin is purchased from Shanghai Taiwei Pharmaceutical Co., Ltd., CAS; 8002-43-5.

[0063] (2) Mix the hyaluronic acid-ethanol solution with a concentration of 1 mg / ml and the soybean lecithin-ethanol solution with a concentration of 20 mg / ml at a volume ratio of 1:1, add mesenchymal stem cell exosomes 2 mg / ml, and incubate for 15 minutes to prepare mixture I. The mixing method is magnetic rotation in a water bath for 15 minutes.

[0064] (3) Mix mixture I and the manninotriose-ethanol solution with a concentration of 1 mg / ml at a volume ratio of 1:1 to prepare mixture II. The mixing method is also magnetic rotation in a water bath for 15 minutes.

[0065] (4) Take 100 ml of pure water system, use a microfluidic chip, and slowly flow mixture II drop by drop into the stirring 100 ml pure water solution at a flow rate of 10 s / drop, then magnetically stir and disperse at room temperature, then continue stirring for 0.2 hours, and finally set the 300w probe type ultrasonic for 15 minutes under 37°C water bath to prepare mixture III.

[0066] (5) Transfer the mixed solution III to a pre-treated dialysis bag (MWCO: 10-14 kDa), dialyze continuously in pure water for 12 h, and change the solution multiple times during the dialysis. After the dialysis is completed, transfer the solution in the dialysis bag to a centrifuge tube, centrifuge at 2700 rpm for 12 min to remove the precipitate, and pass through a liposome pusher (100 KDA). The obtained supernatant is the solution of the mannose tri-saccharide complex nanoparticles. Adjust the pH value of the solution of the mannose tri-saccharide complex nanoparticles using dilute hydrochloric acid (0.5% wt) until the pH value reaches about 7, and then add 1,2-pentanediol at a final concentration of 0.1 mg / ml to prevent the growth of microorganisms.

[0067] The particle size and potential of the prepared mannose tri-saccharide complex nanoparticles were observed using a Brookhaven laser particle size analyzer 90Plus, and the results are shown in Figures 1-2 Figure 3 The TEM transmission electron microscope negative staining scanning diagram of the mannose tri-saccharide complex nanoparticles is shown in Figure 3. Figure 4 The interaction diagram between the target neutrophil elastase (1h1b) and the mannose tri-saccharide in Example 1 of the present application is shown in Figure 4. Figure 5 The structure diagram of the mannose tri-saccharide is shown in Figure 5. The results show that the average particle size of the mannose tri-saccharide complex nanoparticles is 128 nm, and the zeta potential is -23.91 mv, indicating that the average particle size of the mannose tri-saccharide complex nanoparticles prepared by the present application is small, and the stability is good.

[0068] Example 2

[0069] (1) Take 10 mg of mannose, 10 mg of HA hyaluronic acid, and 100 mg of cyclodextrin polymer, and dissolve them in 1 ml of ethanol (100%); adjust the pH value to about 7.0 using 0.5% hydrochloric acid to prepare a mannose-ethanol solution with a concentration of 10 mg / ml, a hyaluronic acid-ethanol solution with a concentration of 10 mg / ml, and a cyclodextrin polymer-ethanol solution with a concentration of 100 mg / ml. Add mesenchymal stem cell exosomes at a concentration of 2 mg / ml. Incubate for 15 minutes. Obtain an exosome-fused cyclodextrin polymer liposome mixture. The mannose is purchased from Shanghai Taiwei Pharmaceutical Co., Ltd., CAS: 13382-86-0; the cyclodextrin polymer is purchased from Thermo Fisher; and the hyaluronic acid is purchased from MERCK Company, and the item number is 924474.

[0070] (2) Mix the hyaluronic acid-ethanol solution with a concentration of 10 mg / ml with the exosome-fused cyclodextrin polymer liposome I with a concentration of 100 mg / ml at a volume ratio of 1:40 in a water bath by magnetic rotation for 15 min.

[0071] ​(3) Take 500 ml of pure water system, use microfluidic chip, and flow the mixed solution II into the stirring 500 ml pure water solution at a flow rate of 10 s / drop, then disperse under magnetic stirring at room temperature, then continue stirring for 0.2 hours, and finally set the 300w probe type ultrasonic for 15 minutes under 37℃ water bath to prepare mixed solution III.

[0072] (4) Take 500 ml pure water system, use microfluidic chip, and flow the mixed solution II into the stirring 500 ml pure water solution at a flow rate of 10 s / drop, then disperse under magnetic stirring at room temperature, then continue stirring for 0.2 hours, and finally set the 300w probe type ultrasonic for 15 minutes under 37℃ water bath to prepare mixed solution III.

[0073] (5) Transfer the mixed solution III to the pre-processed dialysis bag (MWCO: 10-14 kDa), dialyze in pure water for 12 hours, and change the solution several times during the dialysis. After dialysis, transfer the solution in the dialysis bag to a centrifuge tube, centrifuge at 2700 rpm for 12 min to remove the precipitate, and pass through the liposome propeller (100KDA). The obtained supernatant is the mannose complex nanoparticle solution. Adjust the pH value of the mannose complex nanoparticle solution using dilute hydrochloric acid (0.5% wt) until the pH value reaches about 7, and then add 1,2-pentanediol with a final concentration of 0.1 mg / ml to prevent microbial growth.

[0074] Example 3

[0075] (1) Take 1 mg of mannose, 1 mg of hyaluronic acid, and 30 mg of mesoporous silica, and dissolve them in 1 ml of ethanol (100%); adjust the pH value to about 7.0 using 0.5% by mass hydrochloric acid to prepare a mannose-ethanol solution with a concentration of 1 mg / ml, a hyaluronic acid-ethanol solution with a concentration of 1 mg / ml, and a mesoporous silica-ethanol solution with a concentration of 30 mg / ml. Add 2 mg / ml of mesenchymal stem cell exosomes. Incubate for 15 minutes to obtain an exosome-fused mesoporous silica liposome mixed solution. The mannose is purchased from Shanghai Taiwei Pharmaceutical Co., Ltd., with CAS: 13382-86-0; the hyaluronic acid is purchased from MERCK Company, with catalog number 924474;

[0076] (2) Mix the hyaluronic acid-ethanol solution with a concentration of 1 mg / ml and the mesoporous silica-ethanol solution with a concentration of 20 mg / ml to prepare mixed solution I, with a volume ratio of 0.5:3. Mix in a water bath by magnetic rotation for 15 min.

[0077] (3) Mix mixed solution I and the mannose-ethanol solution with a concentration of 1 mg / ml at a volume ratio of 5:1 to prepare mixed solution II. Mix in a water bath by magnetic rotation for 15 min.

[0078] (4) Take 100 ml pure water system, using microfluidic chip, with flow rate 10 s / drop, slowly drop the mixed solution II into the stirring 100 ml pure water solution, then disperse at room temperature under magnetic stirring, then continue stirring for 0.2 hours, finally set 300 w probe type ultrasonic for 15 min under 37℃ water bath, to prepare mixed solution III.

[0079] (5) Transfer the mixed solution III to the pre-processed dialysis bag (MWCO: 10-14 kDa), dialyze in pure water for 12 hours, and change the solution several times during the dialysis. After dialysis, transfer the solution in the dialysis bag to a centrifuge tube, centrifuge at 2700 rpm for 12 min to remove the precipitate, and pass through the liposome propeller (100KDA). The obtained supernatant is the mannose tri-saccharide complex nanoparticle solution. Adjust the pH value of the mannose tri-saccharide complex nanoparticle solution using dilute hydrochloric acid (0.5% wt) until the pH value reaches about 7, then add 1,2-pentanediol with a final concentration of 0.1 mg / ml to prevent microbial growth.

[0080] Example 4: Verify the effect of rat lung enrichment by using the mannose tri-saccharide complex nanoparticles prepared in Example 1

[0081] (1) Select healthy SPF level C57BL / 6 mice (male, 6 weeks old, body weight range 20±2g) 3 experimental mice are divided into 3 groups, 1 mouse in each group.

[0082] Group 1, blank control group (treated with the same volume of blank solvent, single aerosol needle tracheal administration of 20ul, observed for 2 days after administration) (N=1) 24 hours, 48 hours 2 points do live imaging photo detection.

[0083] Group 2, FITC+mannose tri-saccharide complex nanoparticle group, single aerosol needle tracheal administration of 20ul, observed for 2 days after administration) (N=1) 24 hours, 48 hours 2 points do live imaging photo detection.

[0084] Group 3, FITC fluorescent label group, single aerosol needle tracheal administration of 20ul, observed for 2 days after administration) (N=1) 24 hours, 48 hours 2 points do live imaging photo detection.

[0085] (2) Fluorescent label molecule FITC, weigh 5mg FITC dissolved in 0.1mL DMSO, take 20ul FITC solution for standby. Anesthesia: sevoflurane breathing anesthesia machine (manufacturer: Yugen Technology; model: ABM) Live imaging: instrument: live imaging instrument (manufacturer: Tanon; model: ABLX5 PRO); parameters: select NIR700 channel, the absorbance value at 497nm is quantified.

[0086] The live imaging results of the lung enrichment of the mannose tri-saccharide complex nanoparticles in mice are as follows:Figure 6 As shown, it can be seen that the individual fluorescent label groups are dispersed in each tissue organ, and the FITC-labeled mannose triose complex nanoparticles have better enrichment effect in the lung, and better metabolic efficiency after 48 hours.

[0087] Example 5: Using the mannose triose complex nanoparticles prepared in Example 1, it is verified that the mannose triose complex nanoparticles can alleviate the inflammatory response of lipopolysaccharide (LPS)-induced lung injury in rats.

[0088] (1) 20 SPF Wistar rats, male, body weight 180-220 g. Randomly divided into 5 control group and model group, 5 mannose triose group + model group, 5 mannose triose complex nanoparticle group + model group;

[0089] (2) Lipopolysaccharide (LPS)-induced lung injury model in rats: lipopolysaccharide-induced acute lung injury in rats, 20 7-week-old clean male Sprague-Dawley (SD) rats, body weight 180-220 g, the rats were randomly divided into control group, model group, mannose triose group + model group, mannose triose complex nanoparticle group + model group. Each group of rats used lipopolysaccharide three times to establish a lung injury model, and lipopolysaccharide was administered on days 7, 14 and 21.

[0090] (3) Gross observation after lung injury: After lung injury, there is visible lung bleeding and exudation, which can directly reflect the degree of lung injury. The wet / dry weight ratio of the lung is a direct indicator of pulmonary edema and a sensitive indicator of the severity of lung injury. After lung injury occurs, a large amount of fluid exudes into the alveoli and interstitial lung, causing the weight of the lung to increase, while the dry weight of the lung is not affected. After the rats were sacrificed, the trachea was cut off, the whole lung was taken, the wet weight was measured, then the lung was placed in a 65°C constant temperature box for drying, and after 24 hours, the dry weight was taken, the wet / dry weight ratio of the rat lung was calculated, and the results are shown in Table 2.

[0091] (4) Histopathological observation after lung injury: The left lung was fixed with 4% paraformaldehyde, then the lung tissue was routinely dehydrated, paraffin-embedded, sectioned (thickness 5 μm), and the lung tissue was taken. Masson staining was used to observe the degree of inflammation and fibrosis after lung injury. After staining, the inflammatory cell infiltration, edema and damage of the whole lung tissue can be observed under a low-power microscope, and the degree of fibrosis.

[0092] (5) The expression level of the marker inflammation factor was detected: the lung lobe was dissected, and the bronchial intubation was performed. The bronchial alveolar lavage was performed 3 times with 1 ml of pre-cooled (4 degrees) sterile saline. The bronchial alveolar lavage fluid (BALF) was recovered into a sterile centrifuge tube, centrifuged at 1500 r / min for 10 min, and the supernatant was collected and stored at -80 degrees. The content of IL-6 and IL-1β in the bronchial alveolar lavage fluid was detected by an ELISA kit.

[0093] Table 2 Wet / dry mass ratio of lung tissue of rats

[0094]

[0095] Wherein, compared with the control group, *P<0.05.

[0096] The Masson staining results are shown in Figure 7 It can be seen that the manninotriose can improve the lung injury of rats induced by lipopolysaccharide, reduce the inflammatory response, and the treatment effect of the manninotriose complex nanoparticle wrapped by liposomes is better.

[0097] The content of IL-1β and IL-6 in the bronchial alveolar lavage fluid was detected by an ELISA kit. The detection results are shown in Figure 8 It can be seen that compared with the control group, the expression of IL-1β and IL-6 in the model group presents a certain upward trend. After the manninotriose drug intervention, the expression of IL-1β and IL-6 in the manninotriose group+model group presents a certain decrease. After the manninotriose complex nanoparticle wrapped by liposomes is intervened, the expression of IL-1β and IL-6 in the manninotriose complex nanoparticle group+model group decreases more obviously.

[0098] Example 6: Effect of the manninotriose complex nanoparticle prepared in Example 1 on the activity of neutrophil elastase

[0099] (1) Set blank holes (without adding samples and enzyme markers), standard holes and sample holes. Add 50 μL of standard samples with different concentrations in the standard holes. In the sample holes, add 40 μL of sample diluent, and then add 10 μL of the sample to be detected (the final dilution is 5 times), and mix gently.

[0100] (2) Seal the plate with a sealing film, incubate at 37 degrees for 30-60 minutes. Carefully remove the sealing film, discard the liquid, and add enough washing liquid to each hole. After standing for 30 seconds, discard it, and repeat 5 times, and pat dry.

[0101] (3) Add 50-100 μL of enzyme marker to each hole (except blank holes). Incubate again: seal the plate with a sealing film, incubate at 37 degrees for 30-60 minutes. Wash again, repeat 5 times.

[0102] (4) Add 50 μL chromogenic agent A and 50 μL chromogenic agent B to each well, mix gently, and develop color at 37°C for 10-15 minutes in the dark. Terminate the reaction by adding 50 μL of termination solution to each well, and the color of the reaction solution changes from blue to yellow.

[0103] (5) Zero with the blank hole, and measure the absorbance (OD value) of each hole at 450 nm wavelength with an enzyme marker. The measurement should be completed within 15 minutes after adding the termination solution.

[0104] The fluorescence content of neutrophil elastase in the medium was detected by a neutrophil elastase (NE) ELISA kit, and the detection results are shown in Table 1. Figure 9 As can be seen, compared with the control group, the expression of neutrophil elastase in the medium of the model group showed a certain upward trend. After the intervention of the mannose drug, the expression of neutrophil elastase in the medium of the mannose group + model group showed a certain decrease, and after the intervention of the liposome-encapsulated mannose complex nanoparticle, the expression of neutrophil elastase in the medium of the mannose complex nanoparticle group + model group decreased more obviously.

[0105] From the results of Examples 1-6, the present application draws the following conclusions: a nano delivery system is constructed by self-assembly method with mannose, hyaluronic acid and liposome. Mannose has an inhibitory effect on neutrophil elastase, can improve immunity, anti-tumor, reduce inflammatory response, etc. The mannose-loaded nanoparticle has better stability and bioavailability, can be targeted to accumulate in the lung, and has more significant effects of reducing inflammatory response and relieving respiratory dysfunction. Moreover, the preparation method and application operation steps of the mannose complex nanoparticle are relatively simple, and it is also very suitable for the development and application of targeted lung small airway disease nano drugs, etc.

[0106] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement and improvement within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. Use of a nanoparticle complex targeting small airway disease of the lung in the manufacture of a medicament for the treatment of inflammatory lung injury, characterized in that, The nanocomposite is a mannotriose complexed nanoparticle made of mannotriose, hyaluronic acid and liposome; The ratio of mannotriose, hyaluronic acid and liposome is as follows: a mixed solution I is prepared by mixing a 1-20 mg / ml hyaluronic acid-ethanol solution and a 10-200 mg / ml liposome-ethanol solution at a volume ratio of 1: (1-40); a mixed solution II is prepared by adding a 1-10 mg / ml mannotriose-ethanol solution into the mixed solution I, and the volume ratio of the mannotriose-ethanol solution to the mixed solution I is 1: (1-20).

2. Use according to claim 1, characterized in that, The preparation steps of the nanocomposite are as follows: a mixed solution I is prepared by mixing a 1-20 mg / ml hyaluronic acid-ethanol solution and a 10-200 mg / ml liposome-ethanol solution at a volume ratio of 1: (1-40); a mixed solution II is prepared by adding a 1-10 mg / ml mannotriose-ethanol solution into the mixed solution I, and the volume ratio of the mannotriose-ethanol solution to the mixed solution I is 1: (1-20); the mixed solution II is added dropwise into a pure water system by using a microfluidic chip during stirring to prepare a mixed solution III; the mixed solution III is continuously dialyzed in pure water; the solution after dialysis is centrifuged to remove the precipitate; then a liposome propeller is used, and the obtained supernatant is the mannotriose complexed nanoparticle.

3. Use according to claim 2, characterized in that, After the preparation of the mixed solution III, the processes of magnetic stirring dispersion, continuous stirring and ultrasonic treatment are carried out; the magnetic stirring dispersion is carried out at room temperature; the time of the continuous stirring is 0.2-0.5 hours; the ultrasonic treatment is 150-300 W probe type ultrasonic treatment under a 36-38 ℃ water bath, and the time of the ultrasonic treatment is 5-25 min.

4. Use according to claim 3, characterized in that, The pH value of the mannotriose complexed nanoparticle solution is adjusted by using hydrochloric acid until the pH value reaches 6.5-7.5, and then 1, 2-pentanediol with a final concentration of 0.05-0.1 mg / ml is added.

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