IAA-PAMAM G4 nano-composite, preparation method and application of IAA-PAMAM G4 nano-composite

Abstract

The invention provides an IAA (Indoleacetic Acid)-PAMAM (Polyamidoamine) G4 nano-composite. The nano-composite is prepared by combining 3-indoleacetic acid and a fourth-generation polyamide-amine type dendrimer together in an ultrasonic manner. The invention further discloses a preparation method of the IAA-PAMAM G4 nano-composite and application of the IAA-PAMAM G4 nano-composite in prevention or treatment of chronic obstructive pulmonary diseases. According to the IAA-PAMAM G4 nano compound disclosed by the invention, the water solubility and the stability of 3-indoleacetic acid are effectively improved, and the anti-inflammatory effect of the 3-indoleacetic acid is enhanced. The IAA-PAMAM G4 nano-composite disclosed by the invention can directly act on the lung in an aerosol inhalation manner, so that the inflammatory response is relieved, and the lung function is improved. In a mouse chronic obstructive pulmonary disease model, a remarkable effect is shown, the pathological change of chronic obstructive pulmonary diseases is relieved, and the lung function is remarkably improved. In addition, the IAA-PAMAM G4 nano-composite also shows an inhibition effect on expression of inflammatory factors in an in-vitro cell experiment, and the cell inflammation level is reduced.

Description

Technical Field

[0001] The invention belongs to the field of medicine and relates to drugs related to chronic obstructive pulmonary disease, specifically a novel IAA-PAMAM G4 nanocomposite, a preparation method and application thereof in chronic obstructive pulmonary disease. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is one of the major public health problems worldwide, and its morbidity and mortality continue to rise. COPD is mainly characterized by airflow limitation, which is related to factors such as tobacco smoke and air pollution. The treatment of COPD mainly focuses on relieving symptoms, reducing acute exacerbations, improving quality of life and reducing the risk of death, including drug therapy and non-drug therapy. However, due to the large heterogeneity among patients, the demand for individualized treatment has not yet been met.

[0003] In the pathological process of COPD, key factors such as airway inflammation and oxidative stress play a significant role, but the specific molecular mechanisms and pathophysiological processes are not yet fully understood. The lack of clear biomarkers and diagnostic methods in the early stage led to the fact that many patients had impaired lung function when they were diagnosed.

[0004] The development of nanotechnology has provided a new approach to the treatment of lung diseases. Nano drug delivery systems can increase drug concentration in the lungs and enhance efficacy. The current challenge is to design nano systems that can improve drug solubility and stability and achieve targeted drug delivery to the lungs. Summary of the invention

[0005] The purpose of the present invention is to increase the solubility of 3-indoleacetic acid in water by using fourth-generation polyamide-amine dendrimers based on the deficiencies in the prior art, and to prepare a composite in an atomized dosage form and evaluate its application effect by atomization, and to achieve the beneficial effects of reducing inflammation and restoring lung function.

[0006] The invention provides an IAA-PAMAM G4 nanocomposite, which is composed of 3-indoleacetic acid and a fourth-generation polyamide-amine dendrimer.

[0007] 3-Indoleacetic acid (IAA) is a natural product that plays a key role in plant growth and development. In recent years, studies have found that 3-indoleacetic acid may also affect physiological functions in the human body by affecting cell proliferation, differentiation, apoptosis and other pathways. In particular, 3-indoleacetic acid shows potential therapeutic effects in regulating immune responses and reducing inflammation. However, as an organic compound with important activity, IAA has poor solubility in water. Although IAA is easily soluble in ethanol, acetone and ether, and slightly soluble in chloroform, clinical applications still require the selection of appropriate solvents and carriers. In addition, the specific odor of 3-indoleacetic acid affects the acceptance and use of IAA in clinical applications.

[0008] In order to overcome this limitation, the present invention adopts the fourth-generation polyamide-amine dendrimer (PAMAM G4) as a carrier. The fourth-generation polyamide-amine dendrimer is a synthetic dendrimer with a highly branched structure and multifunctional surface groups, which can effectively improve the solubility and bioavailability of drugs.

[0009] Preferably, the nanocomposite is prepared by combining 3-indoleacetic acid with a fourth-generation polyamidoamine dendrimer by ultrasound.

[0010] Preferably, the dosage form of the nanocomposite is one or more of capsules, tablets, granules, solutions, suspensions, gels, sprays or aerosols. If an aerosol dosage form is used and aerosol inhalation is used, the complex can act directly on the lungs to quickly relieve local inflammation, reduce airway obstruction, and thus improve respiratory function.

[0011] Preferably, the source of 3-indoleacetic acid is a tryptophan metabolite.

[0012] The present invention also provides a method for preparing the above-mentioned IAA-PAMAM G4 nanocomposite, comprising the following steps:

[0013] Step S1, dissolving the fourth-generation polyamide-amine type dendrimer in water to obtain an aqueous solution of the fourth-generation polyamide-amine type dendrimer;

[0014] Step S2, adding 3-indoleacetic acid to the aqueous solution of the fourth-generation polyamide-amine dendrimer obtained in step S1, dissolving it, and obtaining a mixed solution;

[0015] Step S3, placing the mixed solution obtained in step S2 in an ultrasonic generator to perform ultrasonic reaction to obtain a reaction solution;

[0016] Step S4, removing unreacted 3-indoleacetic acid and fourth-generation polyamidoamine dendrimers from the reaction solution obtained in step S3 by centrifugation and dialysis to obtain a pure IAA-PAMAM G4 nanocomposite.

[0017] Preferably, the ultrasonic reaction time in step S3 is 1 to 4 hours.

[0018] The present invention also discloses the use of the above-mentioned IAA-PAMAM G4 nanocomposite in preventing or treating chronic obstructive pulmonary disease.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] 1. The IAA-PAMAM G4 nanocomposite of the present invention effectively combines 3-indoleacetic acid with the fourth-generation polyamide-amine dendrimer through ultrasound technology to form a nanocomposite with high drug loading capacity and good stability. The design of the nanocomposite increases the solubility of 3-indoleacetic acid in water and gives full play to its anti-inflammatory properties. In addition, due to the nanometer size and surface characteristics of the fourth-generation polyamide-amine dendrimer, the nanocomposite can avoid systemic side effects and improve the safety of treatment.

[0021] 2. The IAA-PAMAM G4 nanocomplex of the present invention exhibits significant therapeutic effects in a mouse model of chronic obstructive pulmonary disease. HE staining and pathological analysis revealed that aerosol inhalation of the IAA-PAMAM G4 nanocomplex can effectively reduce lung inflammation in mice, reduce the formation of pulmonary bullae, and significantly improve lung function. In addition, the results of ELISA and qPCR tests consistently showed that the levels of inflammatory factors (such as TNF-α, IL-6, and CXCL-8) in the lung tissue of mice were significantly reduced after treatment, further verifying the therapeutic effect. Single-cell sequencing analysis of lung tissue further revealed the effects of treatment on immune cell populations, providing a strong scientific basis for the use of IAA-PAMAM G4 nanocomplexes in the treatment of lung diseases, especially chronic obstructive pulmonary disease.

[0022] 3. The IAA-PAMAM G4 nanocomplex of the present invention also showed an inhibitory effect on the expression of inflammatory factors in in vitro cell experiments, reducing the level of cellular inflammation. In A549 and BEAS-2B cells, the expression levels of proinflammatory factors such as IL-6, IL-8 and IL-1β decreased after treatment with the IAA-PAMAM G4 nanocomplex. Transcriptome sequencing results showed that genes related to macrophage polarization in the IAA-PAMAM G4 group were upregulated, indicating that IAA-PAMAM G4 may play a role in immune regulation by regulating the phenotype and function of macrophages and promoting their transformation to an anti-inflammatory phenotype.

[0023] 4. The drug dosage form formed by the present invention can be a liquid reagent, which can be inhaled by atomization. In terms of lung diseases, the atomized inhaler can deliver the drug directly to the respiratory mucus and alveoli, so that the drug concentration of the local tissue drug is high, thereby exerting a therapeutic effect. As a method of local administration of the respiratory tract, compared with the systemic administration method, it can avoid the drug being destroyed by the gastrointestinal tract and the first-pass effect of the liver, so systemic adverse reactions are less common. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the reaction process of the IAA-PAMAM G4 nanocomposite of the present invention.

[0025] Figure 2 Schematic diagram showing the solubility comparison between the IAA and IAA-PAMAM G4 nanocomplexes of the present invention.

[0026] Figure 3 Schematic diagram of the ultraviolet absorption spectrum of the IAA and IAA-PAMAM G4 nanocomplex of the present invention.

[0027] Figure 4 and Figure 5 Schematic diagram of the absorption spectrum of the IAA-PAMAM G4 nanocomposite prepared in different ratios of the present invention.

[0028] Figure 6 Schematic diagram of the effect of aqueous solutions of different concentrations of the fourth-generation polyamide-amine dendrimers of the present invention on the growth and reproduction of A549 cells.

[0029] Figure 7 Schematic diagram of the effect of aqueous solutions of different concentrations of the fourth-generation polyamide-amine dendrimers of the present invention on the growth and reproduction of BEAS-2B cells.

[0030] Figure 8 This is a schematic diagram of the effect of the culture time of different substances of the present invention on the growth and reproduction of A549 cells.

[0031] Fig. 9 This is a schematic diagram of the effect of the culture time of different substances of the present invention on the growth and reproduction of BEAS-2B cells.

[0032] Fig.10 Schematic diagram of macrophage phagocytosis of the IAA-PAMAM G4 nanocomplex of the present invention.

[0033] Fig.11 and Fig.12 Schematic diagram of the distribution of the IAA PAMAM G4 nanocomplex of the present invention in mice.

[0034] Figures 13 to 15HE staining of mouse lung tissue sections in the examples of the present invention reflects the improvement of IAA PAMAM G4 on COPD lung pathology.

[0035] Fig.16 This is a schematic diagram of an in vivo mouse experiment in an embodiment of the present invention reflecting that IAA PAMAMG4 alleviates the lung function of chronic obstructive pulmonary disease.

[0036] Figures 17 to 20 This is a schematic diagram of the mouse lung tissue RT-qPCR experiment in the example of the present invention, which reflects that IAA PAMAMG4 reduces the expression of inflammatory factors in lung tissue.

[0037] Figure 21 to Figure 23 This is a schematic diagram of the mouse lung tissue ELISA experiment in the example of the present invention, which reflects that IAA PAMAMG4 reduces the expression of inflammatory factors in lung tissue.

[0038] Figures 24 to 27 This is a schematic diagram of the cell RT-qPCR experiment in the embodiment of the present invention reflecting that IAA PAMAMG4 reduces the expression of inflammatory factors in cells. DETAILED DESCRIPTION

[0039] The present invention proposes an IAA-PAMAM G4 nanocomposite to solve the key problem of chronic obstructive pulmonary disease treatment. 3-Indoleacetic acid (IAA) has anti-inflammatory properties, but poor water solubility. The fourth-generation polyamidoamine dendrimer (PAMAMG4) as a nanocarrier can improve the water solubility and bioavailability of 3-Indoleacetic acid, and achieve efficient loading and targeted release.

[0040] The IAA-PAMAM G4 complex showed good biocompatibility in mouse models, could effectively reduce lung inflammation, improve lung function, and had clear targeting, reducing the risk of damage to other organs.

[0041] This invention provides a new perspective for the treatment of chronic obstructive pulmonary disease and a new idea for the design of nano drug delivery systems. By regulating immune response and reducing inflammation to improve lung function, it is expected to bring fundamental therapeutic effects to patients with chronic obstructive pulmonary disease and improve their quality of life. Future research is expected to make this innovative drug delivery system based on nanotechnology widely used in clinical practice, bringing new hope to COPD patients.

[0042] The present invention will be further described below in conjunction with the accompanying drawings and examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to the described content can be applied to the method of the present invention.

[0043] Example 1: Construction of IAA-PAMAM G4 Nanocomposite and Study on Biocompatibility

[0044] 1.1. Preparation of IAA-PAMAM G4 nanocomposites

[0045] Reference Figure 1 The reaction principle includes the following preparation steps:

[0046] Step S1, dissolving fourth-generation polyamide-amine (PAMAM) dendritic macromolecules in deionized water to first prepare a high-concentration fourth-generation polyamide-amine dendritic macromolecule stock solution, the concentration of the stock solution is 100 mg / mL; then diluting the stock solution into an aqueous solution of the fourth-generation polyamide-amine dendritic macromolecule for standby use, the concentration of the diluted aqueous solution is 2 mg / mL;

[0047] Step S2, adding 4 mg of 3-indoleacetic acid to an aqueous solution of a fourth-generation polyamide-amine dendrimer having a volume of 1.4 mL and a concentration of 2 mg / mL (i.e., the aqueous solution prepared in step 1), and dissolving the 3-indoleacetic acid to obtain a mixed solution;

[0048] Step S3, placing the mixed solution prepared in step S2 in a FS30D water bath ultrasonic generator, and ultrasonically treating it at a frequency of 40 Hz for 2 hours to perform an ultrasonic reaction, and obtaining a reaction solution after the ultrasonic reaction is completed;

[0049] Step S4, removing the unreacted 3-indoleacetic acid and the fourth-generation polyamidoamine dendrimer from the reaction solution obtained in step S3 by centrifugation and dialysis, and the remainder is the IAA-PAMAM G4 nanocomposite.

[0050] 1.2 Comparison of the solubility of 3-indoleacetic acid and IAA-PAMAM G4 nanocomplex in water

[0051] See also Figure 2 , a camera was used to record the macroscopic appearance changes of the dissolution of 3-indoleacetic acid and IAA-PAMAM G4 nanocomplex in water. It can be seen that after 12 hours and 48 hours, the solubility of IAA-PAMAM G4 nanocomplex in water was significantly better than that of 3-indoleacetic acid in water.

[0052] 1.3. Determination of the absorption spectrum of IAA-PAMAM G4 nanocomposite

[0053] The absorption spectrum of the IAA-PAMAM G4 nanocomposite was measured by UV-visible spectrometer to determine the shift of the maximum absorption light of the ultraviolet absorption value of 3-indoleacetic acid due to the addition of the fourth-generation polyamidoamine dendrimer.

[0054] In order to evaluate the drug solubilizing ability of the fourth-generation polyamidoamine dendrimer, a certain amount of 3-indoleacetic acid (2 mg) and aqueous solutions of the fourth-generation polyamidoamine dendrimers of different concentrations (the concentrations are shown in Table 1) were ultrasonically prepared according to the preparation method described in 1.1. The resulting reaction solution was centrifuged at 10,000 rpm for 5 minutes to remove the insoluble precipitate, and the value of 3-indoleacetic acid at the maximum absorption light in the supernatant was quantified by UV-visible spectroscopy.

[0055] Table 1 Mixed solution ratio of IAA-PAMAM G4 nanocomposite

[0056]

[0057] like Figure 4 and Figure 5 The results showed that the addition of the fourth-generation polyamide-amine dendrimer caused the maximum absorption peak of 3-indoleacetic acid to shift from 224 nm to 229 nm, and at a PAMAM G4 concentration of 2 mg / mL, the solution was already saturated and no undissolved particles existed.

[0058] Example 2: Biocompatibility and safety of IAA-PAMAM G4 nanocomposites

[0059] In order to evaluate the biocompatibility of the fourth-generation polyamidoamine dendrimer and IAA PAMAM G4 nanocomplex, this example uses CCK-8 reagent to detect the cytotoxicity and proliferation of A549 cells and BEAS-2B cells.

[0060] In order to evaluate the biosafety of the fourth-generation polyamidoamine dendrimer and IAA PAMAM G4 nanocomplex, this example used Raw264.7 cells to perform a cellular uptake assay.

[0061] 2.1 Biocompatibility

[0062] 1) Effects of different concentrations of aqueous solutions of four-generation polyamidoamine dendrimers on cells

[0063] A549 cells and BEAS-2B cells in the logarithmic growth phase were collected and cultured at 9×10 4 / well of cells were inoculated in two 96-well plates, and 200 μL of DMEM complete medium was added. The two cells were cultured in an incubator at 37°C and 5% CO2 until the density reached 30%. The old medium was discarded, and 200 μL of different concentrations of fourth-generation polyamide-amine dendrimer aqueous solution (0 mg / mL (NC), 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL) was added. According to the volume of the culture medium, CCK-8 reagent was added at a ratio of 1:10, incubated at 37°C for 4 hours, and the absorbance at 450 nm was measured using a microplate reader.

[0064] like Figure 6 and Figure 7 The results showed that different concentrations of fourth-generation polyamide-amine dendrimer aqueous solutions had no effect on the growth and reproduction of A549 cells and BEAS-2B cells.

[0065] 2) Effects of different substances on cells during culture

[0066] A549 cells and BEAS-2B cells in the logarithmic growth phase were cultured at 9×10 4 / well of cells were inoculated in 96-well plates, 200 μL DMEM complete medium was added, and the culture medium was replaced when the density reached 30% in an incubator at 37°C and 5% CO2. 200 μL of 2mg / mL of 4th generation polyamide-amine dendrimer aqueous solution, 200 μL of 2mg / mL of IAA-PAMAM G4 nanocomplex aqueous solution and a control (NC) group without addition were added, and cultured for 1 day, 3 days and 5 days respectively. CCK-8 reagent was added at a ratio of 1:10 according to the volume of the culture medium, and incubated at 37°C for 4 hours. The absorbance value (absorbance) at 450nm was measured using an enzyme reader.

[0067] like Figure 8 and Fig. 9 The results showed that the growth and proliferation of A549 cells and BEAS-2B cells were not affected with the increase of co-culture time.

[0068] 2.2 Biosafety

[0069] Construction of nanocarriers with fluorescence imaging function: First, the fourth-generation polyamide-amine dendrimer was dissolved in deionized water to obtain a 100 mg / mL fourth-generation polyamide-amine dendrimer aqueous solution. At the same time, indocyanine green (ICG) was dissolved in methanol to prepare an ICG solution with a concentration of 0.00083 mg / mL, and the ICG solution was added to the fourth-generation polyamide-amine dendrimer aqueous solution (the molar ratio of PAMAM G4 / ICG was 1:3) to ensure that each PAMAM G4 molecule had enough ICG molecules for labeling. The mixture was stirred for 24 hours at room temperature in the dark and dialyzed with distilled water using a 1 kDa dialysis bag to remove unreacted indocyanine green. The purified product was obtained by freeze drying. PAMAM G4-ICG was dissolved in deionized water to obtain a 2 mg / mL PAMAM G4-ICG aqueous solution for use (stored in the dark). 2 mg of 3-indoleacetic acid and 700 μL of 2 mg / mL PAMAM G4-ICG were mixed by ultrasound to obtain an IAA-PAMAM G4-ICG solution at an ultrasound frequency of 40 Hz for 2 hours.

[0070] Evaluation of cellular uptake of four-generation polyamidoamine dendrimers: Raw264.7 cells (mouse mononuclear macrophages) were used to evaluate the uptake of four-generation polyamidoamine dendrimers.

[0071] Raw264.7 cells in the logarithmic growth phase were taken and 1×10 6 / well of cells were inoculated in a confocal microscope-specific culture dish, 2.5 mL of DMEM complete medium was added, and after culturing to a density of 50% in an incubator at 37°C and 5% CO2, 2.5 mL of fresh DMEM complete medium was replaced, and 1.25 μL of the IAA-PAMAMG4-ICG mixture prepared above was added to the culture medium. Subsequently, Raw264.7 cells were cultured in an environment of 37°C and 5% CO2 for 1 hour, 2 hours, 4 hours, and 8 hours, and then cell culture was terminated to observe the effects of different times on cell uptake. 30 minutes before terminating the culture, Lyso-Tracker Red, a lysosomal red fluorescent probe, was diluted with DMEM complete medium at a ratio of 1:15000 and added to the culture medium. Then, 20 minutes before terminating the culture, DAPI (diluted with DMEM complete medium at a ratio of 1:99) was used to incubate the cells. After terminating the culture, the cells were washed three times with PBS to remove excess probes and culture medium residues that did not enter the cells. The cells were captured and imaged using a confocal laser scanning microscope.

[0072] like Fig.10The results showed that IAA-PAMAM G4 nanocomplexes could be cleared by macrophages through phagocytosis.

[0073] 2.3 Biodistribution of IAA PAMAM G4 nanocomplex

[0074] Mouse preparation: 2 mg / mL PAMAM G4-ICG solution was ultrasonically mixed with 3-indoleacetic acid in proportion to form a uniform mixture. Subsequently, the mixture was ultrasonically atomized and inhaled by mice. In addition, a batch of mice was prepared and the prepared mixture was injected into the mice by intraperitoneal injection. After the atomization and intraperitoneal injection were completed, the mice were returned to the original SPF environment for feeding and anesthetized at 12 hours, 24 hours, and 48 hours.

[0075] To monitor and evaluate the distribution and fluorescence intensity of the drug in vivo, an IVIS200 small animal imaging system (Xenogen, Alameda, CA) was used to take pictures of anesthetized mice. The fluorescence emission signal was then normalized to the total efficiency to allow comparison and analysis of data at different time points. Images were acquired using Living Image 2.5 software (Xenogen, Alameda), and imaging data were further processed and analyzed using this software.

[0076] like Fig.11 and Fig.12 The results showed that for mice that inhaled the mixture through aerosolization, the fluorescence was mainly distributed in the lungs of the mice, while for mice that were intraperitoneally injected with the mixture, the fluorescence was mainly distributed near the injection point, and corresponding results were obtained in various tissues and organs.

[0077] Example 3: Study on the anti-inflammatory effect of IAA-PAMAM G4 nanocomplex in vivo and in vitro

[0078] 3.1 Establishment of experimental animal model

[0079] C57BL / 6 mice were purchased from Weitong Lihua Experimental Animal Co., Ltd., and 5-week-old male mice were selected and raised in an SPF environment. The mice were regularly subjected to a 12-hour light / dark cycle, with a room temperature of 20-25°C and a relative humidity of 50-70%. This ensures that the circadian rhythm of the mice is maintained and that their physiology and behavior are normal. In this study, a series of experimental groups were designed in this embodiment to evaluate the effect of IAA-PAMAM G4 nanocomplexes in the treatment of chronic obstructive pulmonary disease and their anti-inflammatory mechanism.

[0080] Thirty C57BL / 6 mice (male) were randomly divided into 5 groups.

[0081] Control group: 40 μL of phosphate buffered saline (PBS) was instilled intranasally once a week for 4 weeks.

[0082] Disease group: A suspension containing 50 μg / kg of lipopolysaccharide (LPS) and 60 U / kg of elastase (ELT) was intranasally instilled once a week for 4 consecutive weeks, with a total volume of 40 μL.

[0083] The group treated with aerosolized IAA-PAMAM G4 nanocomplexes: First, mice received a mixture of 50 μg / kg of LPS and 60 U / kg of ELT drugs once a week by nasal drops, with each dose of 40 μL for 4 consecutive weeks; starting from the third week, mice were further treated with aerosolized IAA-PAMAM G4 nanocomplexes, once every two days, for 14 days. The concentration of 3-indoleacetic acid was set at 50 mg / kg, and the concentration of the fourth-generation polyamide-amine dendrimer aqueous solution was 2 mg / mL.

[0084] Intraperitoneal injection of 3-indoleacetic acid treatment group (ip): received a suspension containing 50μg / kg LPS and 60U / kg ELT once a week by nasal drops, each dose was 40μL, for 4 weeks; starting from the third week of the experiment, mice also received intraperitoneal injection of 3-indoleacetic acid treatment, once every two days, for 14 days. 3-indoleacetic acid was 50mg / kg.

[0085] The group treated with aerosol inhalation of the fourth-generation polyamide-amine dendrimers: First, they received a suspension containing 50 μg / kg of LPS and 60 U / kg of ELT once a week through nasal drops, with each dose of 40 μL for 4 weeks; from the third week, the experimental mice began to receive aerosol inhalation of the fourth-generation polyamide-amine dendrimers, once every two days, for 14 days. The concentration of the fourth-generation polyamide-amine dendrimers was selected to be 2 mg / mL.

[0086] 3.2. HE staining of mouse lung tissue sections.

[0087] Lung tissue samples were collected from each group of mice. First, the lung tissue was simply rinsed with PBS, and then the lung tissue was placed in a 4% paraformaldehyde solution and fixed at room temperature for 24 hours. Paraffin embedding was then completed through dehydration, transparency, and paraffin infiltration. The paraffin-embedded tissue was then cut into thin slices and stained with hematoxylin and eosin (HE staining). HE-stained lung tissue sections were observed and scored, with special attention paid to the pathological degree of alveolar inflammation and emphysema.

[0088] Alveolar inflammation score: The number and distribution of inflammatory cells in the alveoli and interstitium were counted and evaluated. No infiltration was scored as 0 points, mild infiltration was scored as 1 point, moderate infiltration was scored as 2 points, and severe infiltration was scored as 3 points.

[0089] Scoring of emphysema pathological degree: Two fields of view were collected under a 10×10 magnification microscope, avoiding large blood vessels and bronchi for evaluation. An “×” was drawn in the center of each image, and the number of alveolar intervals (Numbers, Ns) and the number of alveolar in each field of view (Number of alveolar, Na) passing through the cross line were counted, the total length of the “×” line (Length, L) was measured, and the mean linear intercept (MLI) of the lung was calculated: MLI = L / Ns (measures the expansion of the alveolar space, which can be used to measure emphysema).

[0090] like Figures 13 to 15 The results showed that both the nebulized inhalation treatment group (IAA-G4(ihn)) and the intraperitoneal injection treatment group (IAA(ip)) achieved reduced inflammatory cell infiltration and relief of pulmonary bullae.

[0091] like Fig.16 The results showed that IAA PAMAM G4 can alleviate the lung function of chronic obstructive pulmonary disease, where FEV1 / FVC is the percentage of forced expiratory volume in the first second to forced vital capacity.

[0092] 3.3 RT-qPCR assay to detect the content of inflammatory factors in lung tissue

[0093] 1) Extraction of tissue RNA

[0094] After euthanasia, the lung tissues of the first four groups of mice in 3.2 above were taken. After the tissue samples were weighed, they were ground in the lysis solution, left to stand at room temperature for 5 minutes, and an equal volume of anhydrous ethanol was added and stirred thoroughly. The sample mixture was added to a centrifuge tube and centrifuged at 4000xg for 1 minute at room temperature. 500μL of washing solution was added to the centrifuge tube and centrifuged at 12000xg for 1 minute at room temperature. The centrifuge tube was transferred to a new EP tube, opened and dried for 2 minutes, 20-30μL of washing solution was added to the centrifuge tube, and centrifuged at 12000xg for 1 minute at room temperature. The centrifuge tube was discarded, and the centrifuge product was RNA.

[0095] 2) Total RNA reverse transcription

[0096] Table 2 Reverse transcription system

[0097]

[0098] Reverse transcription reaction conditions: 42°C for 15 minutes, 95°C for 30 seconds, and cDNA is obtained after the reverse transcription stage is completed.

[0099] 3) RT-qPCR test

[0100] Dilute the cDNA obtained in 3.2 above 5 times with sterilized ddH2O and prepare the qPCR reaction system:

[0101] Table 3 Configuration of qPCR reaction system

[0102]

[0103]

[0104] Table 4 qPCR reaction procedure

[0105]

[0106] Centrifuge the qPCR plate for 2 minutes before testing.

[0107] 4) Experimental results analysis

[0108] Calculation formula:

[0109] △Ct=Ct(target gene)-Ct(reference gene)

[0110] △△Ct=△Ct(experimental group)-△Ct(control group)

[0111] RQ=2- △△Ct

[0112] like Figures 17 to 20 The results showed that the mRNA expression levels of TNF-α, IL-6, CXCL-15, and IL-1β showed that both treatment groups had reduced inflammation.

[0113] 5) ELISA

[0114] To quantitatively detect the expression levels of cytokine proteins in mouse lung tissue homogenates, enzyme-linked immunosorbent assay (ELISA), a standardized biochemical analysis method, was used.

[0115] Sample preparation: The collected mouse lung tissue samples were appropriately processed and homogenized using a tissue homogenizer to obtain homogenized samples.

[0116] The data were analyzed after preparation of cytokine standard curve, antibody coating, blocking, sample and standard addition, detection antibody addition, substrate addition, stop reaction, and optical density measurement. The concentration of cytokines in lung tissue homogenate was calculated based on the standard curve and the OD value of the sample, and statistical analysis was performed.

[0117] like Figure 21 to Figure 23 The results showed that after treatment with IAA-PAMAM G4, the protein expression levels of IL1β, IL-6, and CXCL-15 decreased. The levels of IL1β, IL-6, and CXCL-15 in the lung tissue of mice treated with nebulized inhalation were lower than those in the intraperitoneal treatment group.

[0118] Example 4: Establishing a cell model and detecting the content of inflammatory factors in cells

[0119] 4.1 Establishment of cell model

[0120] A549 cells and BEAS-2B cells need to be stored in DMEM culture medium, supplemented with 10% fetal bovine serum and penicillin (100 μg / mL)-streptomycin (100 μg / mL), and cultured at 37°C in a humidified environment with 5% carbon dioxide.

[0121] Preparation of modeling liquid: Commercially available Double Happiness cigarettes were used to prepare CSE (smoke extract) using a CSE preparation device (including a sterile operating table, a smoking device, a sterile collection device, an absorption liquid, and cigarettes). The preparation process is as follows: Light cigarettes (a total of 5 cigarettes), and guide the smoke through a sterile catheter into a volume containing 50 ml of DMEM, and continuously draw it through a syringe drive device so that each cigarette burns for 5 minutes. The dissolution of smoke in the absorption liquid matches the speed of drawing so that the chemical components in the smoke can be dissolved in the DMEM culture medium, and 1 cigarette is extracted for every 10 mL of culture medium. In addition, the pH of the extract was adjusted to 7.4 ± 0.5, and undissolved particulate matter was removed by sterile filtration (0.22 μm filter membrane) to obtain a 100% CSE stock solution, which was then diluted to 10% CSE, and 10% fetal bovine serum and penicillin (100 μg / mL)-streptomycin (100 μg / mL) were added, followed by the addition of LPS (600 ng / mL) according to the volume of 10% CSE.

[0122] Model establishment and use: A549 and BEAS-2B cells in the logarithmic growth phase were inoculated in two 6-well plates at a cell volume of 1×106 / well. After the cells grew adherently in the well plate and reached a density of 50%, the medium was changed.

[0123] In this application, we designed a series of cell-based experimental groups with the aim of deeply evaluating the efficacy of IAA-PAMAM G4 nanocomplexes in the treatment of chronic obstructive pulmonary disease (COPD).

[0124] Control group: discard the old DMEM complete medium and replace with new 2.5 mL DMEM complete medium.

[0125] Disease group: discard the old DMEM complete medium and replace with new 2.5 mL of 10% CSE culture medium (containing 600 ng / mL LPS) containing penicillin (100 μg / mL)-streptomycin (100 μg / mL) and 10% fetal bovine serum.

[0126] IAA treatment group: discard the old DMEM complete medium and replace it with new 2.5mL of 10% CSE culture medium (containing 600ng / mL LPS) containing penicillin (100μg / mL)-streptomycin (100μg / mL) and 10% fetal bovine serum, and add 1.25μL of IAA soaking solution (add 2mg IAA to 700μL H2O, place on a shaker for 24 hours, centrifuge and take the supernatant for later use).

[0127] IAA-PAMAM G4 treatment group: discard the old DMEM complete medium, replace with new 2.5 mL of 10% CSE culture medium (containing 600 ng / mL LPS) containing penicillin (100 μg / mL)-streptomycin (100 μg / mL) and 10% fetal bovine serum, and add 1.25 μL IAA-PAMAM G4 solution.

[0128] PAMAM G4 treatment group: discard the old DMEM complete medium and replace it with new 2.5 mL of 10% CSE culture medium (containing 600 ng / mL LPS) containing penicillin (100 μg / mL)-streptomycin (100 μg / mL) and 10% fetal bovine serum, and add 1.25 μL PAMAM G4 solution.

[0129] After culturing the cells in each group at 37°C and 5% carbon dioxide in a humidified environment for 48 hours, the culture was terminated.

[0130] 4.2. RT-qPCR assay to detect the content of inflammatory factors in cells

[0131] 1) Extraction of tissue RNA

[0132] A549 cells and BEAS-2B cells were inoculated in 6-well plates, and 12 hours later, modeling solution, IAA, IAA-PAMAM G4, and PAMAM G4 were added to the 6-well plates according to the above grouping. After 72 hours and when the cell confluence reached 90%, the culture medium was discarded, the cells were washed with PBS, 500 μL of lysis solution was added, and the mixture was shaken at room temperature for 1 to 2 minutes. An equal volume of anhydrous ethanol was added, and the mixture was added to the centrifuge column after being fully mixed. The mixture was centrifuged at 4000g for 2 minutes, the liquid was discarded, 500 μL of washing solution was added, and the mixture was centrifuged at 12000g for 2 minutes. The centrifuge column was removed and placed in a new RNase-free EP tube. 25 μL of elution solution was added to the centrifuge column, the column was left open for 3 minutes, and centrifuged at 12000g for 2 minutes. The resulting liquid was the extracted total RNA.

[0133] 2) RT-qPCR

[0134] The procedures of RNA reverse transcription and RT-qPCR are the same as those in 3.3 of Example 3.

[0135] Table 5 Primers for detecting inflammatory factors

[0136]

[0137] like Figure 24 to Figure 27 The results showed that the expression of IL-8 and IL-6 in the A549 cell 10% CSE+600ng / mL LPS+IAA-PAMAM G4 group was lower than that in the other two treatment groups. We also obtained similar results of inflammation reduction in BESA-2B cells.

[0138] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0139] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. An IAA-PAMAM G4 nanocomposite, characterized in that: The nanocomposite is composed of 3-indoleacetic acid and fourth-generation polyamide-amine type dendrimer.

2. The IAA-PAMAM G4 nanocomposite according to claim 1, characterized in that: The nanocomposite combines 3-indoleacetic acid and fourth-generation polyamide-amine dendrimers together by ultrasound.

3. The IAA-PAMAM G4 nanocomposite according to claim 1 or 2, characterized in that: The dosage form of the nanocomposite is one or more of capsules, tablets, particles, solutions, suspensions, gels, sprays or atomizers.

4. The IAA-PAMAM G4 nanocomposite according to claim 1 or 2, characterized in that: The source of the 3-indoleacetic acid is a tryptophan metabolite.

5. Use of the IAA-PAMAM G4 nanocomplex according to claim 1 in preventing or treating chronic obstructive pulmonary disease.

6. A method for preparing an IAA-PAMAM G4 nanocomposite, characterized in that: The following steps are included: Step S1, dissolving the fourth-generation polyamide-amine type dendrimer in water to obtain an aqueous solution of the fourth-generation polyamide-amine type dendrimer; Step S2, adding 3-indoleacetic acid to the aqueous solution of the fourth-generation polyamide-amine dendrimer obtained in step S1, dissolving it, and obtaining a mixed solution; Step S3, placing the mixed solution obtained in step S2 in an ultrasonic generator to perform ultrasonic reaction to obtain a reaction solution; Step S4, removing the unreacted 3-indoleacetic acid and the fourth-generation polyamidoamine dendrimer from the reaction solution obtained in step S3 by centrifugation and dialysis, and the remainder is the IAA-PAMAM G4 nanocomposite.

7. The method for preparing the IAA-PAMAM G4 nanocomposite according to claim 6, characterized in that: The ultrasonic reaction time in step S3 is 1 to 4 hours.

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