Nano preparation for regulating mechanical microenvironment of idiopathic pulmonary fibrosis as well as preparation method and application of nano preparation

Abstract

The invention discloses a nano preparation for regulating an idiopathic pulmonary fibrosis mechanical microenvironment. According to the nano preparation, a zeolite imidazole framework material-8 is used as a carrier, the outer layer of the nano preparation is coated with a polydopamine coating, RGD targeting peptide is combined, and two drugs are synergistically loaded, so that dual regulation and control on the mechanical microenvironment of the lung are realized. The functionalized metal organic framework is used as a drug delivery carrier, and zinc ions and drugs can be responsively released in an acid environment. On one hand, the mechanical tension of the lung is reduced by repairing the damaged type II alveolar epithelial cells and promoting the differentiation of the damaged type II alveolar epithelial cells; on the other hand, lung mechanical signal transmission is inhibited, the contractility of myofibroblasts is reduced, and excessive deposition of extracellular matrixes is reduced. Finally, the nano preparation plays a dual role, the steady state of a lung mechanical microenvironment is recovered, and a brand new strategy and method are provided for treatment of idiopathic pulmonary fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of nano-biomedicine, and in particular to a nano-preparation for regulating the mechanical microenvironment of idiopathic pulmonary fibrosis, and a preparation method and application thereof. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by substantial changes in the structure, composition, and stiffness of lung tissue, leading to permanent deterioration of lung function. As an inherently mechanosensitive organ, the lung is subjected to continuous mechanical forces throughout the development of IPF, gradually leading to deterioration of its structure and function. On the one hand, repetitive mechanical injury weakens the regenerative capacity of type II alveolar epithelial cells and hinders their differentiation into type I alveolar epithelial cells, thereby increasing the mechanical tension of the lung. At the same time, this injury also triggers cytoskeletal remodeling, leading to increased vascular permeability. On the other hand, through mechanical signaling, external force stimulation can trigger the aggregation of integrin receptors around fibroblasts, activate myofibroblasts by incorporating α-smooth muscle actin into stress fibers, enhance their contractility, and lead to excessive accumulation of extracellular matrix, causing a surge in tissue mechanical stress, destroying the mechanical microenvironment of the lung, impairing lung respiratory function, and continuously exacerbating the severity of idiopathic pulmonary fibrosis. The U.S. Food and Drug Administration has previously approved two oral drugs for the treatment of pulmonary fibrosis: pirfenidone and nintedanib. These drugs mainly work by inhibiting inflammatory responses and regulating biochemical signaling of fibroblasts. However, they do not effectively address the substantial destruction of the mechanical microenvironment of the lung caused by changes in mechanical forces. In addition, oral administration makes it difficult for drugs to be accurately targeted to sites with disturbed mechanical microenvironment and accumulate efficiently, thus limiting their clinical efficacy.

[0003] Therefore, it is necessary to develop other drugs with different mechanisms of action to treat pulmonary fibrosis.

[0004] Fasudil (FSD) is a new drug with a wide range of pharmacological effects. It was approved for marketing by Japan's PMDA in 1995 and approved by NMPA in China in 2010. Its molecular structure is a 5-isoquinoline sulfonamide derivative. It is an effective Rho kinase inhibitor and vasodilator. It dilates blood vessels by increasing the activity of myosin light chain phosphatase, reduces the tension of endothelial cells, improves brain tissue microcirculation, does not produce and aggravates brain steal, and can antagonize inflammatory factors, protect nerves from apoptosis, and promote nerve regeneration. The drug is mainly used to improve the symptoms of ischemic cerebrovascular diseases caused by cerebral vasospasm after subarachnoid hemorrhage. In pulmonary fibrosis, it can inhibit the production of TGF-β1, CTGF, α-SMA, etc., and inhibit the activation of the TGF-β1 / Smad 3 pathway and the RhoA / ROCK pathway, reducing the development of pulmonary fibrosis. However, its administration method is injection, which makes it difficult to accurately target the drug and achieve efficient accumulation in the lungs.

[0005] 7,8-Dihydroxyflavone (7,8-DHF) is a plant-derived flavonoid compound that can mimic the function of brain-derived neurotrophic factor (BDNF). It can be used as a specific small molecule agonist of tropomyosin receptor kinase B (TrkB) to activate signaling pathways downstream of TrkB, such as MAPK / ERK, PI3K / Akt and PKC, which is essential for promoting neuronal survival, growth and synaptic plasticity. It is a functional compound for neurological diseases and weight control. Studies have shown that it can promote the regeneration of alveolar epithelial cells in a variety of mouse lung injury models, thereby accelerating lung tissue repair, and has good application prospects. The drug is currently reported to be used only to treat lung injury, and there is no report on the treatment of pulmonary fibrosis. In addition, there is currently no literature report on the combined use of Sudil and 7,8-dihydroxyflavone to treat pulmonary fibrosis.

[0006] Aerosol inhalation therapy refers to the process of converting liquid drugs into aerosol microparticles through an aerosol inhalation device, which are then inhaled into the airway through the mouth or mask to exert therapeutic effects. Compared with oral, intramuscular and intravenous administration, aerosol inhalation therapy has the advantages of rapid onset, good efficacy, few systemic adverse reactions, and no need for deliberate cooperation from patients, because the drugs act directly on the target organs.

[0007] Therefore, the present invention develops an aerosol inhalation preparation (two-drug combination) that can accurately locate the damaged area of ​​the lung and effectively regulate the mechanical microenvironment of the lung, in order to improve the effectiveness of the treatment of idiopathic pulmonary fibrosis. Summary of the invention

[0008] Purpose of the invention: In order to overcome the shortcomings of existing treatment technologies, the first purpose of the present invention is to provide a combination of fasudil and 7,8-dihydroxyflavone for the treatment of pulmonary fibrosis; the second purpose of the present invention is to provide an atomized inhalation agent (nano preparation) that effectively regulates the mechanical microenvironment for the treatment of idiopathic pulmonary fibrosis; the third purpose of the present invention is to provide the use of the above-mentioned nano preparation for the treatment of idiopathic pulmonary fibrosis by regulating the mechanical microenvironment.

[0009] Technical solution: The present invention provides the use of fasudil in combination with 7,8-dihydroxyflavone in the preparation of a drug for treating idiopathic pulmonary fibrosis.

[0010] More specifically:

[0011] A nano preparation for treating idiopathic pulmonary fibrosis comprising the above-mentioned mechanical microenvironment regulating agent, wherein the nano preparation comprises a nano carrier and a loaded drug, wherein the loaded drug is a drug for promoting alveolar epithelial repair and an anti-fibrosis drug.

[0012] The drug for promoting the repair of alveolar cells is 7,8-dihydroxyflavone.

[0013] The anti-pulmonary fibrosis drug is a drug that inhibits mechanical signal transduction in fibroblasts, and the drug is fasudil.

[0014] The nanocarrier is composed of zeolitic imidazole acid framework-8 (ZIF-8) synthesized from zinc nitrate and 2-methylimidazole, and synergistically loads two drugs. The polydopamine (PDA) coating grafted on the outer layer of the nanoformulation not only improves biocompatibility and prevents drug leakage, but can also be further grafted with RGD peptides to specifically target damaged type II alveolar epithelial cells and myofibroblasts, thereby accurately regulating the mechanical microenvironment of diseased cells and reducing toxic side effects on other normal organs. After administration, the zinc ions in the nanoformulation are responsively released in an acidic environment, activating the zinc transporter 8 / silent information regulator 1 pathway, and synergistically repairing damaged type II alveolar epithelial cells with 7,8-dihydroxyflavone, promoting their differentiation, and thereby reducing the mechanical tension of the lungs. In addition, fasudil destroys the actin cytoskeleton, reduces the contractility of myofibroblasts, and reduces the release of extracellular matrix, thereby synergistically improving the mechanical microenvironment of the lungs.

[0015] Furthermore, the mass ratio of zinc nitrate to 2-methylimidazole is preferably 1:6; the input amount of polydopamine is 0.05-0.4 mg / mL, more preferably 0.2 mg / mL; the input amount of RGD peptide is 0.2-1 mg / mL, more preferably 0.4 mg / mL.

[0016] Furthermore, the sequence of the peptide segment is Cyclo (RGDfC).

[0017] Preferably, the drug loading in the anti-fibrosis nanoformulation is between 5% and 15%, and the particle size of the nanoformulation is between 20 and 500 nm.

[0018] The method for preparing the above-mentioned drug-loaded nanoformulation for regulating the mechanical microenvironment for treating idiopathic pulmonary fibrosis comprises the following steps:

[0019] (1) Preparation of drug-loaded ZIF-8: First, 2-methylimidazole and two drugs were dissolved in methanol and stirred thoroughly until uniformly mixed. Subsequently, zinc nitrate was added to the solution to form drug-loaded ZIF-8 nanoparticles loaded with the two drugs through the action of metal coordination bonds. The obtained nanoparticles were centrifuged, washed and set aside.

[0020] (2) Preparation of PDA-coated nanoparticles: The drug-loaded ZIF-8 nanoparticles synthesized above were resuspended in Tris-HCl buffer, and then dopamine hydrochloride was added and stirred to react to form nanoparticles with a PDA coating to prevent drug leakage.

[0021] (3) Preparation of RGD peptide-grafted nanoparticles: The PDA-coated nanoparticles were resuspended in Tris-HCl buffer, and RGD peptide was added and stirred to react. After the reaction was completed, centrifugation and washing were performed to finally obtain RGD peptide-grafted nanoparticles.

[0022] Furthermore, in the step (1), the mass ratio of zinc nitrate to 2-methylimidazole is preferably 1:6;

[0023] In step (2), the amount of polydopamine added is 0.05-0.4 mg / mL, more preferably 0.2 mg / mL;

[0024] In step (3), the amount of RGD peptide added is 0.2 to 1 mg / mL, more preferably 0.4 mg / mL.

[0025] Furthermore, in step (1), the loaded drugs are 7,8-dihydroxyflavone and fasudil; preferably, the loading amount of 7,8-dihydroxyflavone is 1 to 15%, and the loading amount of fasudil is 1 to 15%;

[0026] The nano preparation is used in preparing a drug for treating idiopathic pulmonary fibrosis.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0028] (1) Fasudil is an anti-pulmonary fibrosis drug that inhibits the conduction of mechanical signals in fibroblasts. It can reduce the contractility of myofibroblasts by destroying the actin cytoskeleton, thereby reducing the release of extracellular matrix. 7,8-Dihydroxyflavone can repair damaged type II alveolar epithelial cells, promote their differentiation, and thus reduce the mechanical tension of the lungs. The present invention creatively combines two drugs with different mechanisms of action to treat idiopathic pulmonary fibrosis. After calculation of the synergy index (CI), the CI value is less than 1, and it is believed that the two have a synergistic effect. Furthermore, the combined drug administration strategy adopted by the present invention has a good ability to regulate the mechanical microenvironment of the lungs, promote the repair and differentiation of type II alveolar epithelial cells, thereby reducing their mechanical tension; at the same time, it destroys the actin cytoskeleton of myofibroblasts, inhibits the conduction of intracellular mechanical sensitive signals, breaks the vicious interaction between myofibroblasts and the rigid extracellular matrix, and restores the disordered mechanical microenvironment of the lungs to a steady state. The present invention takes the disordered mechanical force level in idiopathic pulmonary fibrosis as a therapeutic target, improves the therapeutic effect of idiopathic pulmonary fibrosis, and provides a new approach for reversing the treatment of idiopathic pulmonary fibrosis.

[0029] (2) In order to obtain a better therapeutic effect, the present invention is prepared into an atomized inhalation agent (nanoformulation), that is, the present invention uses ZIF-8 with good biocompatibility as a drug carrier to load two drugs to regulate the mechanical microenvironment of the lungs, and at the same time, through the inspiration of "polydopamine PDA on the surface of natural mussels", creatively constructs a PDA coating to wrap nanoparticles to prevent drug leakage, and is modified by RGD peptide. The nanoformulation not only targets damaged type II alveolar epithelial cells and myofibroblasts rich in integrin αvβ6 through RGD peptide, but also releases zinc ions under acidic conditions to repair damaged type II alveolar epithelial cells. The dual drugs synergistically regulate the mechanical microenvironment of fibrosis, while reducing the mechanical tension of damaged type II alveolar epithelial cells and the contractile force of myofibroblasts to reduce the formation of stiff extracellular matrix, thereby achieving the purpose of reversing idiopathic pulmonary fibrosis. As an aerosol inhalation agent, when treating pulmonary fibrosis, the drug needs to reach the alveoli directly, so there are requirements for the particle size of the aerosol inhalation agent. The prior art shows that particles with a diameter less than 500nm can penetrate deep into the alveoli, and particles larger than 500nm generally stay in the airways. Therefore, the present invention controls the particle size to be less than 500nm, about 20-500nm, to achieve a good therapeutic effect.

[0030] (3) The present invention utilizes the characteristics of ZIF-8 nanoformulation, such as easy preparation, high drug loading efficiency, and pH-responsive biodegradation, to achieve rapid release of drugs and zinc ions. The biocompatible PDA coating on the carrier can prevent drug leakage. Meanwhile, the grafting of RGD peptide can target target cells with mechanical microenvironment disorders and increase the enrichment of drugs in the target area. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the preparation process of the nanoformulation of the present invention;

[0032] Figure 2 It is a drug prescription screening diagram of the nanoformulation in Example 1. A is the XRD diagram of 7,8-DHF@ZIF-8 at different 7,8-DHF concentrations, B is the loading capacity of ZIF-8 for 7,8-DHF at different 7,8-DHF concentrations, C is the XRD diagram of FSD@ZIF-8 at different FSD concentrations, and D is the loading capacity of FSD@ZIF-8 at different FSD concentrations.

[0033] Figure 3 This is a prescription screening diagram of the polydopamine coating of the nanoformulation in Example 1;

[0034] Figure 4 The appearance, particle size and potential diagram of the nanoformulation in Example 2; A is a scanning electron microscope image of ZDFPR NPs, B is a transmission electron microscope image of ZDFPR NPs, C is a particle size distribution diagram of ZDFPR, and D is a Zeta potential diagram of ZDFPR NPs;

[0035] Figure 5 The STEM-HAADF image (A), EDS elemental image (B) and XRD image (C) of the nanoformulation in Example 3;

[0036] Figure 6 The zinc ion release diagram of the nanoformulation in Example 4 under different pH conditions;

[0037] Figure 7 Figures 5 are cell uptake diagrams of the nanoformulations in different cells, wherein A is a cell uptake diagram of different nanoformulations in damaged type II alveolar epithelial cells, and B is a cell uptake diagram of different nanoformulations in myofibroblasts;

[0038] Figure 8 This is an analysis of the mechanical microenvironment of alveolar epithelial cells synergistically regulated by the nanoformulation in Example 7, wherein A is an image of cell elasticity detected by atomic force microscopy, and B is the Young's modulus value of type II alveolar epithelial cells treated with different nanoformulations;

[0039] Fig. 9 This is the analysis of the mechanical microenvironment of fibroblasts synergistically regulated by the nanoformulation in Example 8, wherein A is the height map of fibroblasts treated with different nanoformulations under atomic force microscopy. B is the Young's modulus value of myofibroblasts at different nanoformulations;

[0040] Fig.10This is an analysis of the effect of the nanoformulation in improving fibrosis in vivo in Example 9. A is an H&E staining image of fibrotic mice treated with different nanoformulations, B is a Masson trichrome staining image of fibrotic mice treated with different nanoformulations, and C is the Ashcroft score of fibrotic mice treated with different nanoformulations. DETAILED DESCRIPTION

[0041] In order to more clearly illustrate the technical content of the present invention, it is described in detail with reference to the accompanying drawings and specific embodiments. Obviously, the enumerated embodiments are only preferred implementation schemes of the present technical solution, and other technical solutions that can be obviously derived by technicians in this field based on the disclosed technical content still belong to the protection scope of the present invention.

[0042] Example 1 Preparation of Nanoparticles

[0043] like Figure 1 The preparation process of the nanoformulation is shown in FIG. In the following nanoformulation name abbreviations: Z is a ZIF-8 nanocarrier, including zinc nitrate and 2-methylimidazole, D is 7,8-dihydroxyflavone, F is fasudil, P is a polydopamine coating, and R is an RGD targeting peptide.

[0044] 1. The preparation method of ZDF nanoparticles is as follows:

[0045] ZDF nanoparticles were synthesized using a simple one-pot method. 0.1487 g zinc nitrate and 2.22 g 2-methylimidazole were dissolved in 1 mL and 9 mL methanol, respectively. Subsequently, 6 to 14 mg of 7,8-dihydroxyflavone and fasudil were added to the 2-methylimidazole solution and stirred thoroughly until completely dissolved. After that, the zinc nitrate solution was added dropwise and continued to stir for 20 minutes, followed by centrifugation at 14,000 rpm for 10 minutes. The precipitate was collected and washed three times to obtain ZDF nanoparticles. In order to optimize the drug loading, the maximum absorption wavelengths of the two drugs were determined by UV spectroscopy, and a standard curve was established. Figure 2 As shown, 10 mg / mL is the optimal co-encapsulation concentration of the two drugs. Due to the partial peak overlap in the UV full wavelength scan after drug loading, the drug loading of 7,8-dihydroxyflavone was further measured by high performance liquid chromatography to be 14.69% ± 1.36%, and the drug loading of fasudil was 13.85% ± 1.28%.

[0046] 2. The preparation method of ZDFP nanoparticles is as follows:

[0047] The synthesized ZDF nanoparticles were resuspended in Tris-HCl buffer at pH 8.5. Then, dopamine hydrochloride was added in a concentration gradient of 0.05 to 0.4 mg / mL and stirred for 1 h to obtain ZDFP nanoparticles. The mixture was then centrifuged at 14,000 rpm for 10 min, the precipitate was collected, and washed three times to obtain ZDFP nanoparticles. Figure 3 As shown in the figure, with the increase of dopamine concentration, the absorbance of ZDFP gradually increased and the color of the solution deepened, which indicated that dopamine was successfully modified on the surface of ZDF. Through the analysis of absorbance, 0.2 mg / mL was finally determined to be the optimal concentration for dopamine encapsulation.

[0048] 3. The preparation method of ZDFPR is as follows:

[0049] The synthesized ZDFP nanoparticles were resuspended in Tris-HCL buffer at pH = 8.5. Subsequently, RGD peptide was added to the ZDFP nanoparticle solution at a concentration range of 0.2-1.0 mg / mL and stirred for 2 hours to obtain ZDFPR nanoparticles. Next, centrifuge at 14000 rpm for 10 minutes, collect the precipitate, and wash three times to finally obtain ZDFPR nanoparticles. According to the experimental results, the drug loading of ZDFPR nanoparticles was between 1% and 10%, and the particle size was between 50 and 500 nm. As shown in Table 1, the particle size distribution and drug loading were comprehensively analyzed to determine that 0.4 mg / mL was the optimal addition concentration of RGD peptide. This step ensures the stable preparation of ZDFPR nanoparticles and optimizes their drug loading efficiency.

[0050] Table 1 is the results of the screening of the RGD peptide formulations in Example 1

[0051]

[0052] Example 2 Investigation of the appearance and particle size of nanoformulations

[0053] The appearance of the nanoparticles was observed by scanning electron microscopy (SEM, SU8010, Hitachi, Japan) and transmission electron microscopy (TEM, JEM-200CX, Jeol, Japan). The average particle size, particle size distribution and Zeta potential of the samples were measured using a Malvern Zetasizer Nano ZS. Figure 4 The analysis results showed that ZDFPR nanoparticles had a regular dodecahedral structure. The average diameter of ZIF-8 nanoparticles was 112.90±3.34nm, which increased to 179.76±2.66nm after drug loading. The change in zeta potential further confirmed the successful modification of PDA coating and RGD peptide.

[0054] Example 3 Characterization of the structure and surface modification of nanoformulations

[0055] X-ray diffraction (XRD) patterns of ZIF-8 nanoparticles were characterized using an X-ray powder diffractometer (Rigaku, SmartLab SE, Japan), and X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, Thermo, USA) was used to analyze the elemental distribution. Figure 5 The XRD results showed that the diffraction peaks of ZIF-8 nanoparticles were consistent with their corresponding crystal structure simulation patterns. The slight deviation caused by drug encapsulation indicated that the drug molecules might be embedded in the micropores of ZIF-8. After PDA and RGD coating, part of the diffraction pattern of ZDFPR nanoparticles was obscured, indicating the success of chemical modification. XPS analysis revealed characteristic peaks such as C1s, O 1s, N 1s, S2p and Zn 2p, which together with the energy spectrum analysis of scanning electron microscopy confirmed the successful loading of drugs and the effective coating of PDA and RGD.

[0056] Example 4 In vitro release behavior of nanoformulations

[0057] The ZDFPR nanoparticles were dispersed in 50 mL of PBS solution with different pH values ​​(7.4 and 5.5) and gently shaken at 100 rpm at 37 °C. 1 mL of supernatant was taken out at set time points (1, 2, 4, 6, 12 and 24 h) for centrifugation, and an equal volume of fresh PBS solution was added. The quantitative analysis of zinc ion release was performed using inductively coupled plasma mass spectrometry (ICP-MS, ICAP-QC, Thermo, USA). Figure 6 The results showed that ZDFPR nanoparticles showed significant pH-responsive controlled release behavior under acidic conditions, with a zinc ion release rate of up to 82.2% within 24 hours, indicating that it has excellent release properties in an acidic environment.

[0058] Because the coordination bonds of nanoparticles can only be cleaved under acidic conditions, they are cleaved in the acidic lysosomal environment of the cells after being targeted to the cells. Therefore, the above results ensure that the drug is released after reaching the cells.

[0059] Example 5 Investigation of the targeting properties of nanoformulations

[0060] To evaluate the cellular uptake effect of different nanoparticles, experiments were conducted using damaged alveolar epithelial cells and myofibroblasts (cells under pathological conditions), in which fibroblasts were stimulated with 10 ng / mL TGF-β for 48 hours to promote their transformation into myofibroblasts. After the cells were seeded in a 35 mm glass-bottomed culture dish overnight, they were incubated with FITC-labeled ZDF, ZPR, ZDFP and ZDFPR nanoparticles for 6 hours. After washing with PBS, the cell nuclei were stained with DAPI and analyzed by laser confocal microscopy and flow cytometry. Figure 7 The results showed that the uptake of ZDFPR nanoparticles modified with RGD peptide was significantly increased, indicating that the interaction between cellular integrins and RGD cyclic peptide effectively promoted the targeting and internalization of nanoparticles to the damaged site, and ZDFPR nanoparticles were enriched in both alveolar epithelial cells and myofibroblasts.

[0061] Example 6 Determination of the synergistic effect of nanoformulations

[0062] To evaluate the synergistic effect of the two drugs in the nanoformulation, fibroblasts were cultured at a rate of 1×10 4 The cells were evenly inoculated in a 96-well plate at a density of 100 cells / well. When the cell density grew to more than 80% of the bottom of the well plate, different concentrations of ZDPR, ZFPR, and ZDFPR were added to the well plate at concentrations of 1.56 μg / mL, 3.13 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL, respectively. At the same time, a blank control group without cells and a negative control group without medication were set up, and 6 replicates were set up in each group. After 24 hours, the culture plate was removed, the drug-containing culture medium was removed, and 120 μL / well of MTT solution (1 mg / mL) was added using a spray gun, and the plate was placed in a cell culture incubator for further incubation for 4 hours. After incubation, remove the MTT solution, add 150 μL / well of DMSO solution using a spray gun, shake to completely dissolve the formazan, measure the absorbance (OD value) of each well at 490 nm using a multifunctional microplate reader, and calculate the synergistic index according to the inhibition rate of each group of drugs on cells according to the following formula:

[0063]

[0064] Among them, (Dx) 1 and (Dx) 2 represents the dose required for drug 1 and drug 2 to produce x% effect, respectively. 1 and D 2 They represent the doses of drug 1 and drug 2 required to produce the same effect when used in combination.

[0065] The results showed that the CI indexes of the two drugs in IC25, IC50, and IC75 were 0.60, 0.33, and 0.19, respectively, and the calculated values ​​were all less than 1, indicating that there was a synergistic effect between ZDPR and ZFPR, that is, when the two drugs acted together, the effect of ZDFPR was greater than the sum of the effects of each individual drug.

[0066] Example 7 Determination of the synergistic effect of nanoformulation on improving the mechanical microenvironment of alveolar epithelial cells

[0067] After 72 hours of stretch culture of type II alveolar epithelial cells that were not damaged or damaged by BLM (80 μM), ZPR, ZDPR, ZFPR and ZDFPR nanoparticles at a concentration of 6.25 μg / mL were added to study the effects of these nanoparticles on the mechanical tension of alveolar epithelial cells. The mechanical tension of these damaged cells was quantified by atomic force microscopy. Figure 8 The results showed that the Young's modulus of stretched type II alveolar epithelial cells was significantly higher than that of unstretched cells, indicating that stretching enhanced the mechanical tension of cells. After nanoparticle treatment, the mechanical tension of each sample decreased, among which the ZDFPR nanoparticles had the largest decrease, and there was no significant difference between the ZDFPR group and the normal group.

[0068] This result highlights the great potential of ZDFPR nanoparticles in alleviating mechanical tension in damaged alveolar epithelial cells.

[0069] Example 8 Determination of the synergistic effect of nanoformulation on improving the mechanical microenvironment of myofibroblasts

[0070] After culturing fibroblasts without TGF-β stimulation (10 ng / mL) or with TGF-β stimulation for 48 h, ZPR, ZDPR, ZFPR, and ZDFPR nanoparticles at a concentration of 6.25 μg / mL were added to study the effects of these nanoparticles on the mechanical microenvironment of myofibroblasts. AFM was used to evaluate the mechanical stiffness of myofibroblasts to different nanoparticle formulations. Fig. 9 As shown in the figure, after TGF-β stimulation, myofibroblasts showed contraction and flattening of the cell surface due to the activity of stress fibers, and the increase in Young's modulus indicated that the mechanical force on the cells was enhanced. However, after the addition of nanoparticles, the Young's modulus of each group decreased, among which the decrease in the ZDFPR NPs group was the largest, and there was no significant difference between the ZDFPR and normal groups. This result shows that ZDFPRNPs can effectively reduce the contractile force of myofibroblasts induced by TGF-β, thereby improving their pathological mechanical microenvironment.

[0071] Example 9 Analysis of the effect of nanoformulation in treating fibrotic mouse lungs

[0072] After anesthesia, mice were injected with a single intratracheal injection of 2.0U / kg of BLM, and an experimental pulmonary fibrosis model was established 14 days later. In the in vivo treatment study, normal mice were used as the blank control group, and the model group was given normal saline as a positive control. The drug-treated groups were ZPRNPs, ZDPRNPs, ZFPRNPs, and ZDFPR NPs (FSD: 25mg / kg; 7,8-DHF: 10mg / kg), and aerosol inhalation treatment was performed on days 15, 18, 21, 24, and 27 after modeling. After two weeks of treatment, the effect of each group of preparations in treating pulmonary fibrosis was evaluated by H&E staining, Masson staining analysis, and Ashcroft score.

[0073] like Fig.10 The results showed that compared with the BLM group, the ZDFPR group showed the most significant anti-pulmonary fibrosis effect, and there was almost no difference with the normal group.

Claims

1. Application of fasudil combined with 7,8-dihydroxyflavone in the preparation of drugs for the treatment of idiopathic pulmonary fibrosis.

2. A nanoformulation for regulating the mechanical microenvironment to treat idiopathic pulmonary fibrosis, the nanoformulation comprising a nanocarrier and a loaded drug, the loaded drug being a drug that promotes alveolar epithelial repair and an anti-fibrotic drug.

3. The nanoformulation according to claim 2, characterized in that: The drug for promoting alveolar cell repair is 7,8-dihydroxyflavone; and the anti-pulmonary fibrosis drug is fasudil.

4. The nanoformulation according to claim 2, characterized in that: The nano-carrier is composed of zeolite imidazole acid skeleton-8 synthesized from zinc nitrate and 2-methylimidazole; wherein the outer layer of the nano-carrier is grafted with polydopamine PDA and RGD peptide.

5. The nanoformulation according to claim 4, characterized in that: The mass ratio of zinc nitrate to 2-methylimidazole is preferably 1:6; the input amount of polydopamine is 0.05-0.4 mg / mL; and the input amount of RGD peptide is 0.2-1 mg / mL.

6. The nanoformulation according to claim 4, characterized in that: The sequence of the peptide segment is Cyclo (RGDfC).

7. The nanoformulation according to claim 2, characterized in that: The particle size of the nano preparation is 20-500nm.

8. The nanoformulation according to claim 4, characterized in that: The nano preparation is prepared according to the following steps: (1) Preparation of drug-loaded ZIF-8: First, 2-methylimidazole, fasudil and 7,8-dihydroxyflavone were dissolved in methanol and stirred until uniformly mixed; Subsequently, zinc nitrate is added to the solution to form drug-loaded ZIF-8 nanoparticles loaded with two drugs through the action of metal coordination bonds; the obtained nanoparticles are separated by centrifugation and washed for later use; (2) Preparation of PDA-coated nanoparticles: The drug-loaded ZIF-8 nanoparticles synthesized above were resuspended in Tris-HCl buffer, and then dopamine hydrochloride was added and stirred to react to form nanoparticles with a PDA coating to prevent drug leakage; (3) Preparation of RGD peptide-grafted nanoparticles: The PDA-coated nanoparticles were resuspended in Tris-HCl buffer, RGD peptide was added and the reaction was continued under stirring; after the reaction was completed, centrifugation and washing were performed to finally obtain RGD peptide-grafted nanoparticles.

9. Use of the nanoformulation according to claims 2-8 in the preparation of a drug for treating idiopathic pulmonary fibrosis.

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