A pirfenidone nanocomposite based on tetrahedral framework nucleic acid and its preparation method and use

Abstract

The present invention provides a pirfenidone nanocomposite based on tetrahedral framework nucleic acid and its preparation method and use, which belongs to the field of nucleic acid molecule drugs. The complex can effectively relieve lung inflammation, balance oxidative stress, inhibit fibroblast activation and EMT process, and reduce the expression of type I collagen, α-SMA, fibronectin, vimentin, NLRP3, NOX4 and p-Smad2 / 3 protein, inhibit ROS and significantly promote the expression of E-cadherin and HO-1 protein, and the treatment of pulmonary fibrosis is excellent. In addition, the nanocomposite has high biosafety and low toxicity, which helps to improve the survival rate of patients. The nanocomposite prepared by the present invention can significantly reduce the oral dose and frequency of pirfenidone by aerosol administration, effectively reducing drug toxicity. The present invention provides a new strategy for the treatment of idiopathic pulmonary fibrosis, with broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of nucleic acid molecule drugs, and in particular relates to a pirfenidone nanocomposite based on tetrahedral framework nucleic acid, a preparation method and application thereof. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible lung disease, and its treatment currently faces major challenges. Existing treatment options are relatively limited and are mainly administered orally. Pirfenidone (PFD), a therapeutic drug for IPF, can improve lung dysfunction to a certain extent and help improve the quality of life of patients. However, there are some problems with PFD, such as poor water solubility, currently only available in the form of capsules and tablets, and can only be administered orally. The dose required for oral administration is high and cumbersome (801 mg each time, 3 times a day), bioavailability is reduced, and it may cause serious side effects such as indigestion and systemic allergic reactions, which greatly limits its clinical application value.

[0003] tFNAs is a tetrahedral structure formed by four DNA single strands through complementary base pairing. It can be used as a nano drug delivery carrier. The nano drug delivery carrier has high synthesis efficiency, simple synthesis steps and good biocompatibility. China's authorized invention patent CN 112587544 B "The use of DNA tetrahedral framework nucleic acids in the preparation of drugs for the treatment of fibrotic diseases" discloses that DNA tetrahedral framework nucleic acids can inhibit the process of cell fibrosis by inhibiting the production of extracellular matrix and cell epithelial-mesenchymal transition.

[0004] However, due to the special structure of tFNA and its certain activity, whether it can produce a synergistic effect after carrying pirfenidone needs further study. Summary of the invention

[0005] The object of the present invention is to provide a pirfenidone nanocomposite based on tetrahedral framework nucleic acid and a preparation method and use thereof.

[0006] The invention provides a tetrahedral framework nucleic acid nanocomplex, which is a complex of DNA tetrahedral framework nucleic acid loaded with a drug for treating pulmonary fibrosis.

[0007] Furthermore, the DNA tetrahedral framework nucleic acid is formed by base complementary pairing of single-stranded DNA molecules with sequences as described in SEQ ID NO.1 to 4; the drug for treating pulmonary fibrosis is one or more of pirfenidone, nintedanib, prednisone, prednisone acetate tablets, cyclophosphamide, and azathioprine.

[0008] Furthermore, it is obtained by mixing DNA tetrahedral framework nucleic acid and pirfenidone and then incubating them.

[0009] Furthermore, during the incubation process, the molar concentration ratio of the DNA tetrahedral framework nucleic acid to pirfenidone is 1 μM:0.5-10 mM; preferably 1 μM:5 mM.

[0010] Furthermore, the method for synthesizing the DNA tetrahedral framework nucleic acid comprises the following steps: adding four DNA single strands into TM buffer, maintaining at 95°C for 10 min, cooling to 4°C and maintaining for more than 20 min, preferably, the molar ratio of the four DNA single strands is 1:1:1:1.

[0011] The present invention also provides a method for preparing the above-mentioned tetrahedral framework nucleic acid nanocomplex, which comprises the following steps:

[0012] The DNA tetrahedral framework nucleic acid and pirfenidone are added into a solvent, mixed and incubated to obtain the product.

[0013] Further, the solvent is TM buffer;

[0014] And / or, the reaction temperature is 4°C;

[0015] And / or, the reaction time is 10 to 14 hours.

[0016] The present invention also provides a use of the above tetrahedral framework nucleic acid nanocomplex in preparing a drug for preventing and / or treating pulmonary fibrosis.

[0017] Furthermore, the pulmonary fibrosis is idiopathic pulmonary fibrosis; and / or the drug is a drug that reduces lung coefficient, reduces hydroxyproline level, reduces inflammatory infiltration, reduces tissue damage and structural fibrosis, reduces EMT, inhibits the expression of type I collagen, a-SMA, fibronectin, vimentin, NLRP3, NOX4 protein, p-smad2 / 3 protein, inhibits ROS, and promotes the expression of E-cadherin and HO-1 protein.

[0018] The present invention also provides a pharmaceutical preparation for preventing and / or treating pulmonary fibrosis, which comprises the above-mentioned nanocomposite and pharmaceutically acceptable excipients; preferably, the preparation is an atomizer.

[0019] The present invention provides a tetrahedral framework nucleic acid-based pirfenidone nanocomposite and its preparation method and use. The synergistic effect between PFD and tFNA in the complex can effectively relieve lung inflammation, balance oxidative stress, inhibit fibroblast activation and EMT process, and reduce the expression of type I collagen, α-SMA, fibronectin, vimentin, NLRP3, NOX4 and p-Smad2 / 3 protein, inhibit ROS and significantly promote the expression of E-cadherin and HO-1 protein, and the treatment of pulmonary fibrosis is excellent. The nanocomposite prepared by the present invention can significantly reduce the oral dose and frequency of pirfenidone by aerosol administration, effectively reducing drug toxicity. In addition, the nanocomposite exhibits good biosafety and low toxicity, and improves the survival rate of patients. The present invention provides a new treatment strategy for idiopathic pulmonary fibrosis, which has good application prospects.

[0020] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0021] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The synthesis and characterization results of tFNA and Pt. a) Schematic diagram of the preparation of Pt. b) The successful synthesis of tFNA and Pt was verified by PAGE. c) The successful synthesis of tFNA and Pt was verified by HPEC. d) TEM image shows the triangular structure of Pt (scale bar: 50nm).

[0023] Figure 2 Characterization results of tFNA and Pt. a) AFM image shows the successful synthesis of Pt (scale bar: 330nm and 60nm). b) Spectra of tFNA, Pt and PFD between 200 and 400nm. c) Zeta potential of tFNA and Pt determined by DLS. d) Molecular size of tFNA and Pt determined by DLS.

[0024] Figure 3 Results of cell uptake experiments. Flow cytometric analysis results of Cy5-labeled tFNA and Pt in NIH / 3T3 cells a) and quantitative results b); Flow cytometric analysis of Cy5-labeled tFNA and Pt in A549 cells c) and semi-quantitative results d). * indicates that there is a statistical difference between the tFNA group and the Pt group. * P<0.05,** P<0.01, *** P<0.001.

[0025] Figure 4 Fluorescence images of cell uptake experiments. a) Immunofluorescence images of cellular uptake of Cy5-labeled tFNA and Pt in NIH / 3T3, b) Immunofluorescence images of cellular uptake of Cy5-labeled tFNA and Pt in A549. (Cy5: red; cytoskeleton: green; cell nucleus: blue) (scale bar: 20 μm).

[0026] Figure 5 The in vivo biodistribution of Pt on IPF and the experimental schematic diagram. a) IVIS images of the lungs after intratracheal administration (tFNA and Pt) a) and drug quantification results b). c) Schematic diagram of mouse modeling and drug treatment process. * indicates that there is a statistical difference between the tFNA group and the Pt group. * P<0.05, ** P<0.01.

[0027] Figure 6 The in vivo biological therapeutic effect of Pt on IPF. a) Comparison of lung coefficients in each group. b) Hydroxyproline content in lung tissue. c) Results of H&E staining and Masson trichrome staining of mice in each group after the experiment.

[0028] Figure 7 Figure 3 shows the in vivo biological therapeutic effect of Pt on IPF. a) Ashcroft score was used to quantify the degree of fibrosis and structural distortion. b) Quantitative results of collagen volume fraction (CVF) in Masson trichrome stained lung sections. c) Western blot of E-cadherin, collagen type I, vimentin, and α-SMA in lung tissue after different treatments. *** indicates statistical difference compared with the Sham group, ### indicates statistical difference compared with the model group, and $$$ indicates statistical difference compared with the Pt-high group. *** P<0.001, ### P<0.001, $$$ P<0.001

[0029] Figure 8 Fluorescence staining results. a) Double immunofluorescence staining of type I collagen (red) and α-SMA (green) in lung sections, and 3D thermal imaging to reconstruct the fluorescence intensity of type I collagen and α-SMA. b) Double immunofluorescence staining of E-cadherin (red) and vimentin (green) in lung sections, and 3D thermal imaging to reconstruct the fluorescence intensity of E-cadherin and vimentin.

[0030] Fig. 9 Schematic diagram of the experimental process.

[0031] Fig.10 Pt attenuated BLM-induced immune cell recruitment and oxidative stress abnormalities. a) Flow cytometry detection of CD11b in lung tissue + F4 / 80 + CD206 + Macrophages b), MHCⅡ + CD11c + Dendritic cells c) and CD11b + Gr-1 + d) Infiltration of MDSCs.

[0032] Fig.11 a), b) and c) are CD11b + F4 / 80 + CD206 + Macrophage MHCⅡ + CD11c + Dendritic cells and CD11b + Cr-1 + Data analysis of changes in the number of MDSCs. *** indicates a statistical difference compared with the Sham group, ### indicates a statistical difference compared with the Model group, and $$$ indicates a statistical difference compared with the Pt-high group. *** P < 0.001, ## P<0.01, ### P < 0.001, $ P<0.05, $$ P<0.01, $$$ P<0.001.

[0033] Fig.12 a), b), and c) are immunofluorescence images of NLRP3, NOX4, and HO-1 expression in lung sections, and 3D thermal imaging for reconstruction of NLRP3, NOX4, and HO-1 fluorescence intensity. *** indicates a statistical difference compared with the Sham group, ### indicates a statistical difference compared with the Model group, and $$$ indicates a statistical difference compared with the Pt-high group. *** P < 0.001, ## P<0.01, ### P < 0.001, $ P<0.05, $$ P<0.01, $$$ P<0.001.

[0034] Fig.13 a), b) and c) show the levels of MDA, SOD and H2O2 after intratracheal administration, respectively.

[0035] Fig.14The efficacy test of Pt in vivo and its safety assessment. a) Schematic diagram of the experimental process. On the 10th day after BLM modeling, the survival status of mice was checked every day and the body weight was recorded. The experiment ended on the 28th day. b) Graph of the survival percentage of mice receiving the designated treatment (n=12 per group). c) The body weight of mice was measured every day after intratracheal aerosolization in each group. d) Schematic diagram of the experimental design for evaluating the safety of platinum by intratracheal aerosol administration.

[0036] Fig.15 For the safety assessment results, a) H&E staining images of heart (scale bar: 500μm and 100μm), liver (scale bar: 1000μm and 100%), spleen liver (scale bar: 1000μm and 100%), and kidney liver (scale bar: 1000μm and 100μm) tissues obtained from mice after treatment in each drug group. bg) Blood biochemical analysis results of serum obtained from mice after different treatments to evaluate heart, liver, and kidney function (heart function parameters: HDLC4; liver function parameters: ALTL, ASTL, BILT3; kidney function parameters: UA2, TP2). ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen, serum biochemical parameters.

[0037] Fig.16 Pt protects NIH / 3T3 and A549 from TGF-β1-induced damage. a) Schematic diagram of the process of Pt inhibiting IPF in vitro. b) Western blot analysis of functional biomarkers of fibrosis (type I collagen, α-SMA) in NIH / 3T3 cells. c) Statistical analysis of the relative gray values ​​of the bands in the results of b) (n=3). d) Western blot analysis of markers related to fibrosis (fibronectin, α-SMA) and EMT (E-cadherin, vimentin) in A549. e) Statistical analysis of the relative gray values ​​of the bands in the results of d) (n=3).

[0038] Fig.17 Results of Pt protecting NIH / 3T3 and A549 from TGF-β1-induced injury. a)-d) Statistical results of Western blot analysis of markers associated with fibrosis (fibronectin, α-SMA) and EMT (E-cadherin, vimentin) in A549. e) Immunofluorescence images of type I collagen and α-SMA expression in NIH / 3T3, and 3D thermal imaging for reconstruction of type I collagen and α-SMA fluorescence intensity. f) Immunofluorescence images of E-cadherin and fibronectin expression in A549, and 3D thermal imaging results for reconstruction of E-cadherin and fibronectin fluorescence intensity.

[0039] Fig.18The statistical results of immunofluorescence images are as follows: The relative fluorescence intensity of immunofluorescence images of type I collagen (a) and α-SMA (b) in NIH / 3T3 cells was statistically analyzed. The relative fluorescence intensity of immunofluorescence images of E-cadherin (c) and fibronectin (d) in A549 cells was statistically analyzed.

[0040] Fig.19 Pt inhibits inflammation and oxidative stress by regulating the smad2 / 3 signaling pathway. a) Schematic diagram showing the entire process of Pt from the trachea to the lesion site, thereby exerting anti-fibrotic effects. b) Western blot analysis of functional biomarkers of inflammatory factors (NLRP3), oxidative stress (NOX4), and antioxidants (HO-1) in NIH / 3T3, c)-e) Statistical analysis of the relative gray values ​​of inflammatory factors (NLRP3), oxidative stress (NOX4), and antioxidants (HO-1) in NIH / 3T3 (n=3).

[0041] Fig. 20 a) Fluorescence image of ROS production detected by DCFH-DA fluorescent probe in NIH / 3T3, and 3D thermal imaging for reconstruction of DCFH-DA fluorescence intensity. b) Flow cytometry analysis for comparison of ROS fluorescence intensity of each group of cell samples, and statistical analysis of relative fluorescence intensity. c) Western blot results of NLRP3, NOX4 and HO-1 protein expression in mouse lung tissue sections, and statistical analysis of relative gray values ​​of NLRP3, NOX4 and HO-1 protein and p-smad2 / 3 protein bands (n=3). * indicates statistical difference compared with the control group, # indicates statistical difference compared with the TGF-β1 cell model group, and $ indicates statistical difference compared with the Pt-high group. *** P < 0.001, ## P<0.01, ### P < 0.001, $ P<0.05, $$ P<0.01, $$$ P<0.001.

[0042] Fig.21 a) Western blot results and statistical analysis of p-smad2 / 3 protein expression in NIH / 3T3 (n=3). b) Western blot results of p-smad2 / 3 protein expression in mouse lung tissue. c) Immunofluorescence images of p-sd2 / 3 expression in lung sections, and 3D thermal imaging for reconstruction of p-sd2 / 3 fluorescence intensity. * indicates statistical difference with control or Sham group, # indicates statistical difference with TGF-β1 cell model group or animal model group, and $ indicates statistical difference with Pt-high group. ***P < 0.001, ## P<0.01, ### P < 0.001, $ P<0.05, $$ P<0.01, $$$ P<0.001. DETAILED DESCRIPTION

[0043] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0044] Example 1. Preparation of Pirfenidone Nanocomplex Based on Tetrahedral Framework Nucleic Acid

[0045] 1. Experimental Methods

[0046] 1. Preparation of tFNA

[0047] The synthesis of tetrahedral framework nucleic acid-based pirfenidone nanocomplexes consisted of two steps, e.g. Figure 1 As shown in a.

[0048] Dissolve four DNA single strands (S1, S2, S3, S4) in TM buffer (10mM Tris-HCl, 50mM MgCl2, pH=8.0), the final concentration of the four DNA single strands is 1000nM, mix thoroughly, quickly heat to 95°C for 10 minutes, then quickly cool to 4°C and maintain for more than 20 minutes. Under the above temperature control, the four single strands self-assemble in the system according to the principle of complementary base pairing, and a DNA tetrahedral framework nucleic acid can be obtained. The sequences of the four single strands (S1, S2, S3, S4) are shown in Table 1:

[0049] Table 1 Single-stranded DNA sequence for preparing tFNA

[0050]

[0051] 2. Preparation of tetrahedral framework nucleic acid-pirfenidone nanocomplex

[0052] The molar concentration ratio of tFNA to PFD was 1 μM:5 mM, and they were dissolved in TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0), where the concentration of tFNA was 1 μM. The mixture was incubated at 4°C for 8 h to obtain a tetrahedral framework nucleic acid-pirfenidone nanocomplex, named PFD-tFNA, also known as Pt.

[0053] Example 2 Preparation of Pirfenidone Nanocomplex Based on Tetrahedral Framework Nucleic Acid

[0054] 1. Experimental Methods

[0055] 1. Preparation of tFNA The preparation method of tFNA is the same as that in Example 1.

[0056] 2. Preparation of tetrahedral framework nucleic acid-pirfenidone nanocomplex

[0057] The molar concentration ratio of tFNA to PFD was 1 μM:3 mM, dissolved in TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0), wherein the concentration of tFNA was 1 μM, and incubated at 4°C for 8 h to obtain a tetrahedral framework nucleic acid-pirfenidone nanocomplex, named PFD-tFNA, also known as Pt-low.

[0058] The following experimental examples demonstrate the beneficial effects of Pt prepared in Example 1 of the present invention.

[0059] Experimental Example 1: Pt synthesis verification and characterization

[0060] 1. Experimental methods

[0061] The successful synthesis of tFNA was verified by polyacrylamide gel electrophoresis (PAGE), and the successful synthesis of Pt was verified by high performance capillary electrophoresis (HPCE). The appearance of Pt nanoparticles was observed by transmission electron microscopy (TEM) and atomic force microscopy (AFM).

[0062] The UV absorption peaks of Pt, tFNA and PFD were observed through UV-visible absorption spectroscopy experiments to verify whether Pt was successfully synthesized.

[0063] Dynamic light scattering (DLS) was used to detect the zeta potential and average particle size of tFNA and Pt.

[0064] ①PAGE: The successfully synthesized tFNA was first characterized by 8% polyacrylamide gel electrophoresis. The sample was loaded and run at a constant voltage of 100V for 80 minutes, and then stained and exposed using 1:50 Goldview.

[0065] ②DLS:tFNA was diluted to 250 nM with double distilled water and then observed on the ZETAPals analyzer.

[0066] ③AFM: tFNA was diluted to 20 nM with TM buffer solution, and the surface morphology of tFNA nanoparticles was observed and characterized by Shimadzu SPM-9700 atomic force microscope in tapping scanning mode.

[0067] ④TEM: Transmission electron microscopy was used to observe the microstructure of tFNA, showing that tFNA nanomaterials were small particles with a particle size of 10 nm and uniform distribution.

[0068] ⑤Zeta potential: Zetasizer Nano ZS90 (Malvern Instruments Ltd, UK) was used to measure the potential of single-stranded DNA and tFNA.

[0069] 2. Experimental results

[0070] The molecular weight of a single-stranded DNA is about 50 bp. During the synthesis of tFNA, the molecular weight gradually increases, and the molecular weight of the final tFNA is 200 bp. After loading PFD, the mobility remains basically unchanged. Therefore, the molecular weight of Pt is roughly equivalent to that of tFNA, which is also about 200 bp. The PAGE results are shown in Figure 2. Figure 1 As shown in b, it can be seen that tFNA was successfully synthesized.

[0071] Capillary electrophoresis (HPEC) Figure 1 As shown in c, the characteristics of tFNA and Pt were confirmed, indicating that Pt was successfully synthesized.

[0072] The appearance and morphology of Pt nanoparticles were observed by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Figure 1 d and AFM results are shown in Figure 2 As shown in a, the Pt morphology in the image is a triangular structure.

[0073] UV-Visible absorption spectra Figure 2 As shown in b, it can be seen that the absorption wavelength of tFNA is 260 nm, and the absorption wavelength of Pt and PFD is 310 nm, indicating that PFD is successfully loaded on tFNA to form a composite material Pt.

[0074] Dynamic light scattering (DLS) results are shown in Figure 2. Figure 2 c- Figure 2 As shown in d, Figure 2 c shows that both tFNA and Pt are negatively charged. Figure 2 d shows the average particle size of tFNA and Pt, both of which are around 7 nm. PFD can be embedded in the tetrahedral framework structure of tFNA through an embedding binding mode, and tFNA successfully loads PFD.

[0075] The experimental results show that the present invention successfully constructs a pirfenidone nanocomplex based on tetrahedral framework nucleic acid.

[0076] Experimental Example 2: Cellular Uptake Experiment

[0077] 1. Experimental Methods

[0078] Detect the entry of Pt into mouse embryonic fibroblasts (NIH / 3T3) and human alveolar epithelial cells (A549). Specifically: Cy5-tFNA and Cy5-Pt (prepared according to the method described in Example 1, S1 replaced with S1-Cy5) with Cy5 (red fluorescent group) were incubated with mouse embryonic fibroblasts (NIH / 3T3) and human alveolar epithelial cells (A549) for 6 hours, where the concentrations of Cy5-tFNA and Cy5-Pt were both 250nM, and then the cells were fixed with 4% paraformaldehyde, and the cytoskeleton and cell nucleus were stained with phalloidin (green) and DAPI (blue), respectively. Finally, confocal microscopy was used for observation and image collection.

[0079] Immunofluorescence and flow cytometry (FCM) were used to detect the entry of Pt into fibroblasts (NIH / 3T3) and human alveolar epithelial cells (A549).

[0080] 2. Experimental Results

[0081] The FCM results of the cell uptake experiment are as follows Figure 3 a- Figure 3 d As shown in the results, the uptake level of tFNA and Pt by cells reached more than 85% at 6 h and more than 90% at 12 h.

[0082] The uptake level of tFNA and Pt by A549 cells reached 99% at 12 h. In addition, the uptake level of Pt by A549 cells and NIH / 3T3 cells was significantly higher than that of the tFNA group, a finding that was consistent with the results of immunofluorescence images and statistical analysis ( Figure 4 a, Figure 4 b).

[0083] The experimental results show that the pirfenidone nanocomplex based on tetrahedral framework nucleic acid can be taken up by fibroblasts and alveolar epithelial cells, and its ability to be taken up is better than that of simple tFNA, making it an excellent biological preparation. In addition, PFD has poor water solubility and is not easy to enter the lungs and be taken up by cells. It can be seen that in the Pt provided by the present invention, tFNA and PFD synergistically enhance the cell's uptake of the composite drug.

[0084] Experimental Example 3: In vivo experiment

[0085] 1. Experimental Methods

[0086] (I) Construction of mouse model

[0087] IPF model mice were established by administering BLM to 8-month-old C57BL / 6J mice via aerosolization.

[0088] Specifically, each mouse was given a single dose of 2.5 mg / kg bleomycin (BLM) by aerosol inhalation. The sham control group (sham) inhaled an equal amount of normal saline by tracheal aerosol inhalation. The mice were randomly divided and administered on the 10th day after BLM treatment.

[0089] (II) Experimental Grouping

[0090] Sham control group (sham), 8 mice in each group: 50 μL normal saline was given each time.

[0091] IPF model mice were randomly divided into 5 groups (8 mice in each group), and the specific operation was as follows:

[0092] Vehicle group: 50 μL of normal saline (0.9% sodium chloride) was administered each time;

[0093] Pt-low group: 50 μL of Pt-low (1 μM) prepared in Example 2 was administered each time;

[0094] Pt-high group: 50 μL of Pt (1 μM) prepared in Example 1 was administered each time;

[0095] tFNA group: 50 μL of tFNA (1 μM) was administered each time;

[0096] PFD group: 50 μL of PFD (5 mM) was administered each time.

[0097] All of the above were administered via bronchial atomization, with the first administration starting on the 10th day of modeling and then every 3 days until the end of the experiment on the 21st day. After the experiment, related experiments were conducted.

[0098] (III) Detection methods

[0099] 1. Pt accumulation in the lungs

[0100] The mice in the tFNA group and the Pt-high group were killed 0.5h, 1h, 3h, 6h and 12h after the first administration, respectively, and the in vivo imaging system (IVIS) was used to detect the distribution of the drug in the lungs, heart, liver, spleen and kidneys of the mice in the tFNA group and the Pt-high group.

[0101] 2. Pathological collagen detection

[0102] In IPF patients, the amount of pathological collagen is two to three times higher than normal levels. Excessive collagen accumulates in the interstitial tissue of the lungs, which not only makes the lungs heavier, but also seriously affects the normal exchange of gases. Therefore, blocking the continued production of pathological collagen is crucial to controlling IPF. The lung coefficient and hydroxyproline level are key indicators of collagen deposition. In this experiment, the lung coefficient was obtained by dividing the lung weight by the body weight, and the hydroxyproline level was measured by the hydroxyproline detection kit.

[0103] At the end of treatment on day 21, Masson staining of lung sections was performed to observe the distribution of collagen in lung tissue, and the collagen volume fraction was calculated, and the fibrosis score was calculated using the Ashcroft method.

[0104] 3. Detection of epithelial-mesenchymal transition (EMT) and fibrosis markers

[0105] At the end of treatment on day 21, the expression levels of epithelial-mesenchymal transition (EMT) and fibrosis markers were detected by immunoblotting and immunofluorescence.

[0106] 4. Lung immune microenvironment examination

[0107] At the end of treatment on day 21, lung tissues were lysed into living single cells, and then various types of living cells were labeled with corresponding antibodies, and CD11b was detected in the lungs of each group of mice by flow cytometry (FCM). + F4 / 80+CD206+ cells, MHCII+CD11c+ cells, Gr-1 + CD11b + cells, CD4 + CD69 + Cells and CD8 + CD66 + Changes in cell number, detection diagram as shown Fig. 9 shown.

[0108] 5. Detection of NADPH oxidase-4 and heme oxygenase-1 expression

[0109] At the end of treatment on day 21, the expression levels of NOX4 and HO-1 in lung tissue sections were detected by immunofluorescence.

[0110] 6. Detect MDA, SOD and H2O2 levels

[0111] At the end of treatment on day 21, the levels of MDA, SOD and H2O2 were measured.

[0112] 2. Experimental Results

[0113] 1. Pt accumulation in the lungs

[0114] The distribution of tFNA and Pt in the lungs of mice and the quantitative results of the accumulation of tFNA and Pt in the lungs were evaluated at 0.5 h, 1 h, 3 h, 6 h and 12 h after administration. Figure 5 a and Figure 5 As shown in Figure b, it can be seen that at 0.5h, 1h, 3h, 6h and 12h of administration, the accumulation of Pt in the lungs was significantly higher than that of tFNA. In addition, PFD has poor water solubility and poor dispersibility, and is not easy to enter the lungs and be taken up by cells, while the accumulation of Pt in the lungs is significantly higher than that of tFNA. The experimental results show that PFD and tFNA have a synergistic effect and enhance the ability of lung absorption.

[0115] 2. Pathological collagen test results

[0116] The lung coefficient results of each group are as follows Figure 6 The results of hydroxyproline levels are shown in a. Figure 6 As shown in b, it can be seen that the lung coefficient and hydroxyproline level of the vehicle group were significantly higher than those of the sham control group, indicating that the IPF model mice were successfully constructed. The Pt-high group was significantly lower than the vehicle group, tFNA group, and PFD group, and was lower than the Pt-low group, and was closest to the sham control group. PFD and tFNA had a synergistic effect, which greatly reduced the lung coefficient and hydroxyproline level.

[0117] Masson staining of lung sections, the distribution of collagen in lung tissue is shown in the following figure. Figure 6 c, the collagen volume fraction is Figure 7 Ashcroft results quantifying the degree of fibrosis and structural distortion are shown in b. Figure 7 As shown in a. Figure 6 c It can be seen that compared with the sham control group, the lungs of the vehicle group had obvious inflammatory infiltration, tissue damage and structural fibrosis, which once again proved that the IPF model mouse was successfully established. After treatment, the pathological symptoms of each drug group were alleviated to varying degrees, among which Pt-high had the most significant therapeutic effect. Figure 7 b It can be seen that the collagen volume fraction of each drug group was significantly lower than that of the vehicle group. The Pt-high group was closest to the sham control group and was significantly lower than that of the vehicle group, Pt-low group, tFNA group and PFD group.

[0118] 3. Detection of epithelial-mesenchymal transition (EMT) and fibrosis markers

[0119] The results of immunoblotting and immunofluorescence were as follows Figure 7As shown in Figure c, compared with the sham control group, the contents of EMT markers and fibrosis markers in tFNA lung tissue changed significantly, the fibrosis markers type I collagen and a-SMA increased significantly, the EMT marker E-cadherin decreased, and another EMT marker vimentin increased. At the same time, the Pt-high group had the best effect in improving fibrosis and inhibiting EMT in fibrotic lungs. It is worth mentioning that the treatment effect of the Pt-low and PFD groups was limited, and the efficacy of the Pt-high group was significantly different compared with the Pt-low and PFD groups.

[0120] Figure 8 Type I collagen (red), α-SMA (green) ( Figure 8 a), Double immunofluorescence staining results of E-cadherin (red) and vimentin (green) ( Figure 8 b).

[0121] 4. Lung immune microenvironment test results

[0122] CD11b was detected by flow cytometry + F4 / 80+CD206+ cells, MHCII+CD11c+ cells, Gr-1 + CD11b + cells, CD4 + CD69 + Cells and CD8 + CD66 + The changes in cell number are shown in Fig.10 a- Fig.10 c, quantitative data such as Fig.11 a- Fig.11 As shown in c. The results of FCM show that BLM disrupts the balance of the lung immune microenvironment, such as CD11b + F4 / 80 + CD206 + Cells, MHCII + CD11c+ cells, Gr-1 + CD11b + cells, CD4 + CD69 + Cells and CD8 + CD69 + The number of cells increased, and these cells increased by 1.80 times, 3.16 times, 3.17 times, 1.93 times, and 2.65 times, respectively. At the same time, the number of the above immune cells decreased in each group after drug treatment, among which the Pt-high group had the most significant ability to inhibit the increase in the number of immune cells, which was closest to the level of the sham control group. The results showed that Pt has a regulatory effect on various immune cells in the lungs of pulmonary fibrosis.

[0123] Fig.12 The immunofluorescence images and statistical analysis of a showed that all drug groups could significantly reduce the expression of NLRP3, a marker of inflammatory response. Similarly, the Pt-high group had the best anti-inflammatory inhibitory ability.

[0124] The experimental results showed that the tetrahedral framework nucleic acid-based pirfenidone nanocomplex Pt can better regulate the immune microenvironment homeostasis and anti-inflammatory ability.

[0125] 5. Expression results of NADPH oxidase-4 and heme oxygenase-1

[0126] Oxidative stress is one of the key pathogenesis of IPF, and its biomarkers play an important role in disease diagnosis and monitoring. NADPH oxidase-4 (NOX4) is one of the most important sources of ROS in the body, especially expressed significantly in the lesion area of ​​IPF. NOX4 not only promotes the fibrosis process, but is also widely present in a variety of lung cells. It aggravates the pathological changes of IPF by enhancing the expression of fibrosis-related markers such as type I collagen, α-SMA and fibronectin. Heme oxygenase-1 (HO-1) is a key regulator of antioxidant therapy for pulmonary fibrosis and plays a core role in defending against oxidative and inflammatory damage to the lungs.

[0127] Immunofluorescence was used to detect the expression levels of NOX4 and HO-1 in lung tissue sections. Fig.12 b and Fig.12 As shown in c, in the vehicle group, NOX4 expression increased and HO-1 expression decreased, indicating that BLM disrupted the oxidative defense homeostasis in the lung and the IPF model mice were successfully established. Compared with the drug administration, the Pt-high group showed an excellent ability to regulate the imbalance of oxidative stress.

[0128] 6. MDA, SOD and H2O2 level results

[0129] Malondialdehyde (MDA), the final product of lipid peroxidation, and MDA content indirectly indicate the extent of oxidative damage. Hydrogen peroxide (H2O2) is a strong oxidant that can be metabolized to produce ROS. The release of large amounts of ROS can stimulate fibroblast activation and lead to lung damage. Superoxide dismutase (SOD) plays a vital role in scavenging oxygen free radicals and inhibiting lipid peroxidation, and its antioxidant properties play a key role in combating oxidative stress.

[0130] MDA, H2O2 and SOD test results, such as Fig.13 a- Fig.13As shown in Figure c, it can be seen that BLM stimulation increased the levels of MDA and H2O2, indicating that strong lipid peroxidation occurred in the lungs of mice. Compared with the vehicle group, the Pt-high group significantly reduced the levels of MDA and H2O2, while the therapeutic ability of other drug groups was limited. At the same time, BLM reduced SOD activity to increase oxidative stress, while the Pt-high group was able to significantly enhance the SOD activity level of model mice and increase the ability of model mice to scavenge free radicals in the lungs.

[0131] It can be seen that the Pt-high group has the best ability to enhance immune regulation and resist oxidative damage, and can restore lung inflammation and oxidative stress biomarkers to almost normal levels.

[0132] There were no significant differences in lung coefficient, NOX4, E-cadherin, HO-1, MDA, H2O2 and SOD levels between the Pt-low group and the tFNA group and the PFD group, but there were significant differences in lung coefficient, NOX4, E-cadherin, HO-1, MDA, H2O2 and SOD levels between the Pt-high group and the tFNA group and the PFD group. The enhanced therapeutic effect could only be achieved when the molar concentration ratio of tFNA to PFD in Pt was 1μM:5mM.

[0133] The above experimental results show that Pt is more likely to accumulate in the lungs than tFNA and PFD. The lung index, hydroxyproline level, inflammatory infiltration, tissue damage and structural fibrosis, fibrosis markers type I collagen and a-SMA in the Pt-high group were significantly reduced compared with the vehicle group, tFNA group and PFD group, promoting the balance of oxidative stress, enhancing the immunoregulatory ability and anti-oxidative damage ability of model mice, and Pt can be used to treat idiopathic pulmonary fibrosis (IPF) with excellent results.

[0134] Experimental Example 4: Safety Testing of Pt

[0135] 1. Experimental Methods

[0136] (I) Construction of mouse model

[0137] Same as Example 3.

[0138] (II) Experimental Grouping

[0139] Each group had 12 mice. The first administration started on the 10th day after modeling, and then was administered every 3 days until the 28th day. The rest was the same as in Example 3. The schematic diagram of the experimental process is shown in Fig.14 As shown in a, during the modeling period, BLM was observed to cause mice to have reduced feeding activity, sunken fur, and dull and unkempt hair.

[0140] 2. Experimental Results

[0141] (I) Survival rate of mice

[0142] The survival rate of mice was counted at the end point, and it was found that the survival rate of the vehicle group was 16%, while the survival rates of the Pt-high group and the sham control group were 100%, and the survival rates of the Pt-low group, tFNA group, and PFD group were 91%, 58%, and 67%, respectively ( Fig.14 b). After the drug was administered to all groups, it was observed that the mice's spirits improved, their appetite increased, their hair gloss increased, and their quality of life was significantly improved.

[0143] On the 10th day of modeling, the body weight of mice after BLM administration was significantly reduced compared with the sham control group, indicating that the IPF mouse model was successfully established. From the first administration, the average body weight of mice in each group increased to varying degrees. Among them, the recovery of the Pt-high group was the most significant ( Fig.14 c).

[0144] (II) Safety results of Pt

[0145] Safety of major organs was assessed by histopathological examination and serum levels of related factors ( Fig.14 d) The morphology of the heart, liver, spleen, and kidney was analyzed by H&E staining. The overall and local views of each tissue are shown in Figure 4. Fig.15 As shown in a, it can be seen that no obvious damage was observed in the main organs of each group.

[0146] Blood biochemical analysis was performed on serum samples of mice in each group. Fig.15 b Heart function (HDLC4), Fig.15 c- Fig.15 eLiver function (ALTL, ASTL, BILT3) and renal function Fig.15 f- Fig.15 There was no significant difference in g(UA2, TP2) index between each drug-treated group and the sham control group, indicating that Pt has good safety.

[0147] The experimental results show that the pirfenidone nanocomplex based on tetrahedral framework nucleic acid can significantly improve the survival rate of IPF mice and improve the quality of life of IPF mice. At the same time, the pirfenidone nanocomplex based on tetrahedral framework nucleic acid can be directly delivered to the lungs through tracheal atomization, which is highly safe and can be used to treat IPF with excellent efficacy.

[0148] Experimental Example 4: Verification of Pt inhibiting the sustained activation of fibroblasts and inhibiting the EMT of epithelial cells

[0149] 1. Experimental Methods

[0150] (I) Pt inhibits the sustained activation of fibroblasts

[0151] The pathological process of idiopathic pulmonary fibrosis (IPF) is mainly characterized by the continuous activation of fibroblasts and epithelial-mesenchymal transition (EMT) induced by epithelial cells, which together promote the accumulation of myofibroblasts in the lesion area. In the over-repair stage of the disease, the expanded myofibroblasts produce a large amount of abnormal collagen (such as type I collagen), leading to excessive accumulation of extracellular matrix (ECM), which ultimately causes impaired lung function. TGF-β1 is a pro-fibrotic factor that induces a large number of fibroblasts to proliferate and differentiate into myofibroblasts in pulmonary fibrosis, and enhances fibroblast-mediated ECM contraction by stimulating contractile stress fibers (such as α-SMA).

[0152] The NIH / 3T3 cell line was derived from mouse embryonic fibroblasts and was obtained from Zhongqiao Xinzhou Biotechnology Co., Ltd. NIH / 3T3 cells (mouse embryonic fibroblasts) were stimulated with TGF-β1 (at a concentration of 10 ng / mL) to generate myofibroblasts that can be used as an in vitro model of pulmonary fibrosis.

[0153] Specifically: NIH / 3T3 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (cell-box) and 1% penicillin / streptomycin. All cells were incubated at 37°C and 5% carbon dioxide. The cells were divided into 6 groups, specifically:

[0154] The control group (control) did not undergo additional manipulation.

[0155] TGF-β1 group: TGF-β1 was added to the culture medium of the control group at a final concentration of 10 ng / mL and incubated.

[0156] T+Pt-low group: TGF-β1 was added to the culture medium of the control group at a final concentration of 10 ng / mL; at the same time, 250 nM of Pt-low prepared in Example 2, which contained 3 mM PFD and 250 nM tFNA, was added for incubation.

[0157] T+Pt-high group: TGF-β1 was added to the culture medium of the control group at a final concentration of 10 ng / mL; at the same time, 250 nM of Pt prepared in Implementation 1 was added, which contained PFD: 5 mM, tFNA: 250 nM, and incubated.

[0158] T+tFNA group: TGF-β1 was added to the culture medium of the control group at a final concentration of 10 ng / mL; 250 nM tFNA was also added for incubation.

[0159] T+PFD group: TGF-β1 was added to the culture medium of the control group at a final concentration of 10 ng / mL; 5 mM PFD was also added for incubation.

[0160] NIH / 3T3 cells were treated with TGF-β1 and each group of drugs for 24 hours, and the effects of each group of drugs on the expression of p-smad2 / 3 protein in the TGF-β1 / smad2 / 3 pathway were detected.

[0161] (II) Pt inhibits EMT of epithelial cells

[0162] E-cadherin is an epithelial cell marker that forms tight junctions between cells and helps prevent malignant cell proliferation. When the expression of E-cadherin is reduced, the tight junctions of epithelial cells are weakened, making these cells prone to becoming invasive and undergoing malignant proliferation. At the same time, the increase of mesenchymal markers such as fibronectin and vimentin further disrupts the junctions between epithelial cells and enhances the migration ability of cells with mesenchymal properties. This process plays a key role in the remodeling of lung tissue, especially in pathological conditions such as the development of pulmonary fibrosis. In short, the reduction of E-cadherin and the increase of mesenchymal markers jointly promote the pathological remodeling of lung tissue.

[0163] Therefore, inhibiting epithelial-mesenchymal transition (EMT) is critical for the treatment of idiopathic pulmonary fibrosis (IPF). It can effectively reduce the number of myofibroblasts transformed from epithelial cells and reduce their excessive production of extracellular matrix (ECM). TGF-β1 can trigger the EMT process in normal alveolar epithelial cells.

[0164] The A549 cell line is derived from human alveolar basal epithelial cells and is obtained from the American Type Culture Collection (ATCC). The present invention uses TGF-β1 (at a concentration of 10 ng / mL) to stimulate alveolar epithelial cells A549 to trigger the EMT process, and the cells are incubated and grouped with NIH / 3T3 cells. After 24 hours of incubation, the expression levels of E-cadherin, fibronectin, vimentin, and a-SMA in each group were analyzed by immunoblotting.

[0165] 2. Experimental Results

[0166] (I) Pt inhibits the sustained activation of fibroblasts

[0167] The results of immunoblot analysis of the expression of fibrosis markers type I collagen and a-SMA in each group are shown in Fig.16 b. Fig.16 As shown in c, immunofluorescence staining studies Fig.17e. Fig.17 f, the results showed that Pt inhibited TGF-β1-induced fibroblast activation in a dose-dependent manner, and the Pt-high group had the strongest anti-fibroblast differentiation ability (the lowest expression of type I collagen and a-SMA), which was significantly different from the tFNA group and the PFD group.

[0168] (II) Pt inhibits EMT of epithelial cells

[0169] The expression levels of E-cadherin, fibronectin, vimentin and a-SMA in each group were shown in Figure 2. Fig.16 As shown in Figure d, Pt has a significant blocking effect on the reprogramming of epithelial cells A549 to mesenchymal stromal cells induced by exogenous TGF-β1. This blocking effect is reflected in the upregulation of E-cadherin expression and the downregulation of fibronectin and vimentin expression, which are significantly different from the tFNA group and PFD group, proving that Pt has the best effect in inhibiting the EMT of epithelial cells.

[0170] At the same time, the Pt-high group can effectively degrade the stress fiber a-SMA in A549 and weaken the contraction of some ECMs, which is one of the keys to enhance the treatment of pulmonary fibrosis.

[0171] In addition, the experimental results of double immunofluorescence of fibronectin and E-cadherin are shown in the figure Fig.17 f, Fig.18 c and Fig.18 d Consistent with the above immunoblotting results, compared with the Pt-high group, the tFNA group and the PFD group had limited ability to inhibit the EMT of epithelial cells.

[0172] The experimental results showed that tFNA and PFD in the tetrahedral framework nucleic acid-based pirfenidone nanocomplex produced a synergistic effect. Compared with the tFNA group and the PFD group, the anti-fibroblast activation and EMT inhibition effects of the tetrahedral framework nucleic acid-based pirfenidone nanocomplex had significant differences. The tetrahedral framework nucleic acid-based pirfenidone nanocomplex can be used to treat IPF with excellent results.

[0173] Experimental Example 5: Pt regulates the smad2 / 3 signaling pathway and has anti-inflammatory and antioxidant effects

[0174] 1. Experimental methods (I) Anti-inflammatory and antioxidant effects of Pt

[0175] The regulatory effect of Pt on inflammation and oxidative stress was studied using the TGF-β1-induced NIH / 3T3 cell model. The specific method was the same as step (I) in the experimental method of Experimental Example 4.

[0176] (II) Pt inhibits the expression of phosphorylated Smad2 / 3 (p-Smad2 / 3)

[0177] Same anti-inflammatory and antioxidant effects as Pt.

[0178] 2. Experimental results (I) Anti-inflammatory and antioxidant effects of Pt

[0179] NLRP3, NOX4 and HO-1 immunoblotting experiments were performed as Fig.19 As shown in (b), Pt can significantly change the expression of TGF-β1-induced inflammatory and oxidative stress markers in NIH / 3T3 cells. Pt showed excellent ability to regulate the balance of immune response and oxidative stress. Compared with the tFNA group and PFD group, the expression of NLRP3 and NOX4 proteins in the Pt-high group was significantly reduced, and the expression of HO-1 protein was significantly increased.

[0180] In the course of idiopathic pulmonary fibrosis (IPF), inflammation and oxidative stress are two factors that are constantly increasing. In the inflammatory microenvironment, reactive oxygen species (ROS) levels increase, and excessive ROS can cause further cell damage and oxidative stress. At the same time, oxidative stress can also aggravate the inflammatory response and disrupt the balance of the immune environment. The vicious cycle of oxidative stress and inflammation is a key factor in the pathogenesis of IPF.

[0181] The study also found that TGF-β1 can stimulate the production of high levels of ROS, and these ROS-producing cells will increase the oxidative burden of each cell, further promoting the expression of fibrosis markers in lung fibroblasts and accelerating the development of IPF.

[0182] This experiment used the DCFH-DA fluorescent probe to detect whether Pt intervention can inhibit the generation of ROS. The intensity of DCFH-DA fluorescence was detected using fluorescence microscopy and FCM, which is proportional to the concentration of ROS. Fluorescence image ( Fig. 20 a) and FCM results ( Fig. 20 b) It showed that TGF-β1 significantly increased the concentration of ROS, and the Pt-low group, tFNA group, and PFD group all effectively reduced the production of ROS, but the inhibitory effect was limited. The Pt-high group had a higher inhibitory effect than the Pt-low group, tFNA group, and PFD group.

[0183] The experimental results showed that the tetrahedral framework nucleic acid-based pirfenidone nanocomplex had significant anti-inflammatory and antioxidant capabilities, which were significantly different from the tFNA group and the PFD group. It could significantly inhibit the expression of NLRP3 and NOX4 proteins, promote the expression of HO-1 protein, and significantly inhibit ROS.

[0184] (II) Pt inhibits the expression of phosphorylated Smad2 / 3 (p-Smad2 / 3)

[0185] TGF-β1 is one of the main factors that promote Smad2 / 3 phosphorylation, and plays a key role in the activation of fibroblasts, epithelial-mesenchymal transition (EMT), and the production of extracellular matrix (ECM). In the process of idiopathic pulmonary fibrosis (IPF), the activation of the TGF-β1 / Smad signaling pathway triggers the overexpression of fibrosis-related genes, and its abnormal activation is the main mechanism leading to tissue fibrosis. Studies have shown that PFD can improve pulmonary fibrosis by inhibiting the activation of the TGF-β1 / Smad2 / 3 signaling pathway. In addition, tFNA can also regulate the development of fibrosis by inhibiting the abnormal upregulation of Smad2 / 3 protein phosphorylation.

[0186] In vitro immunoblotting results of p-Smad2 / 3 protein expression levels, cell experiments Fig.21 a and the p-smad2 / 3 protein expression levels of mice in each group on the 21st day of treatment in Experimental Example 3 are shown in Fig.21 As shown in b.

[0187] In the cell experiment, the expression level of p-smad2 / 3 protein in the TGF-β1 group was significantly increased compared with that in the control group, and the expression level of p-smad2 / 3 protein in each drug-treated group was significantly decreased compared with that in the TGF-β1 group. Among them, the Pt-high group had the best effect in inhibiting the expression of p-smad2 / 3 protein, which was significantly better than the tFNA group and the PFD group.

[0188] In the animal experiment, the p-smad2 / 3 protein expression level in the vehicle group was significantly higher than that in the sham control group, and the p-smad2 / 3 protein expression levels in each drug-treated group were significantly lower than those in the TGF-β1 group. Among them, the Pt-high group had the best effect in inhibiting the expression of p-smad2 / 3 protein, which was significantly better than the tFNA group and the PFD group.

[0189] In summary, Pt has an excellent inhibitory effect on smad2 / 3 phosphorylation, which is significantly better than tFNA and PFD.

[0190] The experimental results showed that the tetrahedral framework nucleic acid-based pirfenidone nanocomplex can inhibit smad2 / 3 phosphorylation and effectively enhance the immunomodulatory ability, anti-inflammatory and anti-oxidative damage effects of PFD and tFNA by regulating the TGF-β1 / smad pathway.

[0191] It can be seen from the above embodiments and experimental examples that the present invention relates to a nanocomposite based on tetrahedral framework nucleic acid (tFNA) and pirfenidone (PFD), its preparation method and application. The synergistic effect of PFD and tFNA in the nanocomposite effectively relieves lung inflammation, balances oxidative stress, inhibits fibroblast activation and EMT process, reduces specific protein expression, and promotes E-cadherin and HO-1 protein expression, thereby significantly improving pulmonary fibrosis. In addition, through aerosol administration, the bio-energy of the complex can significantly reduce the oral dose and frequency of PFD, reduce drug toxicity, and has the characteristics of good safety and low toxicity, which helps to improve patient survival rate. The present invention provides a new strategy for the treatment of idiopathic pulmonary fibrosis and has broad application prospects.

Claims

1. A tetrahedral framework nucleic acid nanocomplex for preventing and / or treating pulmonary fibrosis, characterized in that: It is a complex of DNA tetrahedral framework nucleic acid loaded with pirfenidone. The DNA tetrahedral framework nucleic acid is formed by base complementary pairing of single-stranded DNA molecules with sequences such as SEQ ID NO.1 to 4. The molar concentration ratio of the DNA tetrahedral framework nucleic acid and pirfenidone is 1 μM: 0.5-10 mM.

2. The tetrahedral framework nucleic acid nanocomplex according to claim 1, characterized in that: The invention is obtained by mixing DNA tetrahedral framework nucleic acid and pirfenidone and then incubating the mixture.

3. The tetrahedral framework nucleic acid nanocomplex according to claim 2, characterized in that: During the incubation process, the molar concentration ratio of the DNA tetrahedral framework nucleic acid to pirfenidone is 1 μM:5 mM.

4. The tetrahedral framework nucleic acid nanocomplex according to any one of claims 1 to 3, characterized in that: The method for synthesizing the DNA tetrahedral framework nucleic acid comprises the following steps: adding four DNA single strands into TM buffer, maintaining at 95° C. for 10 minutes, cooling to 4° C. and maintaining for more than 20 minutes, wherein the molar ratio of the four DNA single strands is 1:1:1:

1.

5. A method for preparing the tetrahedral framework nucleic acid nanocomplex according to any one of claims 1 to 4, characterized in that: It includes the following steps: The DNA tetrahedral framework nucleic acid and pirfenidone are added into a solvent, mixed and incubated to obtain the product.

6. The method according to claim 5, characterized in that: The solvent is TM buffer; And / or, the incubation temperature is 4°C; And / or, the incubation time is 10 to 14 hours.

7. Use of the tetrahedral framework nucleic acid nanocomplex according to any one of claims 1 to 4 in the preparation of a drug for preventing and / or treating pulmonary fibrosis.

8. The use according to claim 7, characterized in that: The pulmonary fibrosis is idiopathic pulmonary fibrosis; and / or the drug is a drug that reduces lung coefficient, reduces hydroxyproline level, reduces inflammatory infiltration, reduces tissue damage and structural fibrosis, reduces EMT, inhibits the expression of type I collagen, a-SMA, fibronectin, vimentin, NLRP3, NOX4 protein, p-smad2 / 3 protein, inhibits ROS, and promotes the expression of E-cadherin and HO-1 protein.

9. A pharmaceutical preparation for preventing and / or treating pulmonary fibrosis, characterized in that: It comprises the nanocomposite according to any one of claims 1 to 4 and pharmaceutically acceptable excipients.

10. The pharmaceutical preparation according to claim 9, characterized in that: The preparation is an aerosol.

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