Nano preparation for blocking malignant crosstalk of mechanical and biochemical signals of lung as well as preparation method and application of nano preparation

Abstract

The invention discloses a nano preparation for blocking malignant crosstalk of mechanical and biochemical signals of a lung. The preparation is composed of lipid nanoparticles composed of phospholipid, cholesterol and DSPE-PEG2K-NH2, the surfaces of the lipid nanoparticles are grafted with L-arginine and tannic acid, the two drugs are cooperatively loaded, and double intervention on mechanical and biochemical signal channels is achieved. Tannic acid modification can remove active oxygen and enhance lung targeting, and L-arginine is helpful to penetrate through a compact extracellular matrix and promote delivery of drugs to the deep part of pulmonary alveoli. On one hand, by promoting phenotype reversion of myofibroblast-like endothelial cells to endothelium, endothelial-mesenchymal transformation is reduced, and fibrosis progress is inhibited; on the other hand, nuclear translocation of mechanical conduction factors is blocked, and mechanical signal channels related to fibrosis are intervened. The nano preparation can effectively reverse malignant crosstalk of mechanical and biochemical signals in fibrotic lung tissues, and provides an innovative and efficient treatment strategy for pulmonary fibrosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanobiomedicine, in particular to a nano-preparation for blocking the malignant crosstalk of mechanical and biochemical signals in the lung and a preparation method and application thereof. BACKGROUND

[0002] Pulmonary fibrosis is a chronic progressive lung disease caused by various known or unknown factors, and its main pathological feature is the abnormal deposition of extracellular matrix (ECM), which leads to the destruction of lung tissue structure and the continuous impairment of lung function. Idiopathic pulmonary fibrosis is the most common type of pulmonary fibrosis with unknown etiology, and the main population of the disease is people aged 65 and above. The median survival time after the onset of the disease is usually 2 to 5 years. With the intensification of global population aging, the physical, psychological and socio-economic burden caused by pulmonary fibrosis is on the rise, which seriously affects the quality of life of patients and puts a great pressure on the medical system. The core of the intractability of pulmonary fibrosis lies in the unknown etiology, complex mechanism, late diagnosis and the fact that existing drugs can only treat the symptoms but not the root cause.

[0003] The US Food and Drug Administration has approved two oral drugs for the treatment of pulmonary fibrosis, namely pirfenidone and nintedanib. These two drugs mainly exert anti-fibrotic effects by interfering with the biochemical signaling pathways related to fibroblasts, but they can only slow down the decline in lung function (e.g., reduce the forced vital capacity annual decline rate by about 50%), but cannot stop the progression of the disease, nor can they reverse the fibrosis that has already formed. Some patients do not respond: about 30-50% of patients do not respond to existing drug therapy, and the disease continues to worsen. Therefore, existing drugs cannot fundamentally block the continuous deterioration of the mechanical microenvironment in the lung.

[0004] Therefore, there is an urgent need to develop a new treatment strategy to achieve more effective and less side effect precise treatment of pulmonary fibrosis. SUMMARY

[0005] The first object of the present application is to provide a strategy for blocking the malignant crosstalk of mechanical and biochemical signals in the lung by combining drug V and drug B to treat pulmonary fibrosis; the second object of the present application is to provide an aerosol inhalation preparation for removing active oxygen that penetrates the extracellular matrix and reaches the alveoli; and the third object of the present application is to provide a nano-preparation for treating pulmonary fibrosis by blocking the malignant crosstalk of mechanical and biochemical signals.

[0006] Technical solution: The present application provides an application of a nano-preparation for targeted treatment of pulmonary fibrosis by blocking the malignant crosstalk of mechanical and biochemical signals in fibroblasts and endothelial cells in the fibrotic lung.

[0007] Further,

[0008] A nano-preparation for treating pulmonary fibrosis by blocking malignant crosstalk of mechanical and biochemical signals, comprising a nano-carrier and a drug, wherein the drug can include the following two categories:

[0009] The drug V for blocking mechanical signal transduction is verteporfin, dobutamine, CA3, etc.

[0010] The drug B for blocking biochemical signal transduction is berbamine, pirfenidone or galunisertib, etc.

[0011] The nano-carrier includes phospholipids, cholesterol, D-PEG 2K -NH2, and tannic acid and L-arginine grafted on the outer layer of the nano-preparation, wherein the phospholipids are one or more of soybean phospholipids, HSPC, egg yolk lecithin, and synthetic phospholipids; the D material can be selected from DSPE, PLGA, PLA, PGA, PCL, and PS, and different types of D-PEG2K-NH2 can be used to form a stable dispersed nano-film. 2K The above nano-carrier can be prepared by a film dispersion method, an ethanol injection method or a reverse evaporation method, and can be grafted with tannic acid and L-arginine through DSPE-PEG

[0012] Further, the mass ratio of phospholipids to cholesterol is 2:1 to 14:1, the addition amount of L-arginine is 0.574 mM, and the addition amount of tannic acid is 1 mM.

[0013] Preferably, the nano-preparation has good particle size distribution and dispersity, and the polydispersity index (PDI) thereof is between 0.15 and 0.3, and the average particle size of the preparation is 90-150 nm.

[0014] The preparation method of the above nano-preparation for treating pulmonary fibrosis by blocking malignant crosstalk of mechanical and biochemical signals, comprising the following steps:

[0015] (1) preparing nanoparticles by a film dispersion method, an ethanol injection method or a reverse evaporation method, using the drug, phospholipids, cholesterol and D-PEG 2K -NH2 as raw materials;

[0016] (2) dissolving L-arginine in a 5% glucose solution, activating the carboxyl group with EDC and NHS at room temperature, adjusting the pH to about 7.5, then introducing the activated L-arginine into the nanoparticles for modification, and obtaining L-arginine-modified nanoparticles (VB-R nanoparticles) after incubation and impurity removal by ultrafiltration.

[0017] (3) Tannic acid was dissolved in 5% glucose solution, and the pH was adjusted to about 7.5, and was ready for use. The VB-R obtained above was mixed with tannic acid, and after standing and reaction, ultrafiltration was performed to obtain L-arginine nanoparticles modified by tannic acid (VB-RT nanoparticles).

[0018] Further, in the step (1), the mass ratio of the phospholipid and the cholesterol is 2:1-14:1.

[0019] Preferably, the drug loading amount in the anti-fibrosis nano-preparation is between 0.5-1.5%, and the particle size of the nano-preparation is 90-150 nm.

[0020] Further, in the step (1), the mass ratio of the phospholipid and the cholesterol is 2:1-14:1, and more preferably 8:1.

[0021] In the step (2), the input amount of L-arginine is more preferably 0.574 mM.

[0022] In the step (3), the input amount of tannic acid is more preferably 1 mM.

[0023] Further, in the step (3), the loaded drugs are verteporfin and berberine; preferably, the verteporfin encapsulation efficiency is 93-99%, and the drug loading amount is 1.4-1.7%; the berberine encapsulation efficiency is 95-97%, and the drug loading amount is 0.7-1.5%.

[0024] The nano-preparation is used for preparing a drug for treating pulmonary fibrosis.

[0025] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages:

[0026] (1) A large number of studies have shown that transforming growth factor-β (TGF-β) plays a key role in the pathogenesis of pulmonary fibrosis. Fibrosis stimulation can induce cytokines such as TGF-β and tumor necrosis factor-α (TNF-α) to promote the activation, differentiation and survival of myofibroblasts through multiple signaling pathways. The activation of myofibroblasts is accompanied by an increase in the expression of α-smooth muscle actin (α-SMA), leading to cytoskeletal remodeling and changes in cell traction, thereby driving the reconstruction of the pulmonary biomechanical environment. TGF-β is stored in the ECM in the form of latent complex, deposited by forming a complex with latent-associated peptide and latent TGF-β binding protein 1. When the tissue is damaged or stimulated by mechanical force, myofibroblasts perceive and transmit mechanical signals through stress fibers and αν integrins, causing the conformational change of the RGD site of latent-associated peptide, thereby releasing active TGF-β and further activating fibrosis-related signaling pathways. In addition, the release of vascular endothelial growth factor (VEGF) by damaged endothelial cells disrupts the balance between angiogenic regulators, triggering abnormal proliferation and apoptosis of endothelial cells, while the destruction of the basement membrane and the impairment of endothelial barrier function also enhance the sensitivity of the tissue to mechanical stimulation and changes in matrix stiffness.

[0027] However, mechanical signals and biochemical signals stimulate each other in the process of fibrosis, forming a positive feedback loop. The release of TGF-β promotes ECM synthesis and increases tissue stiffness, further activating mechanical signals, leading to the release of more latent TGF-β and accelerating disease progression. In addition, oral drugs often have significant adverse reactions during delivery to the lungs, further limiting their clinical application effect.

[0028] The present application adopts a combination of drug V that blocks mechanical signal conduction and drug B that blocks biochemical signal conduction. Drug B that blocks biochemical signal conduction inhibits the expression of inflammatory factors such as TGF-β in lung fibroblasts and endothelial cells, thereby effectively slowing down the progression of pulmonary fibrosis. Drug V can effectively prevent the nuclear translocation of the mechanically sensitive protein YAP, block the mechanical conduction pathway of fibrosis, and inhibit the force-dependent conformational changes of the cell and extracellular matrix complex, thereby slowing down the polymerization of F-actin and inhibiting the resulting increase in cell contraction. The present application creatively combines drugs targeting mechanical signals and biochemical signals, breaking the vicious crosstalk between mechanical and biochemical signals in the lungs, thereby achieving effective treatment of pulmonary fibrosis, and the two drugs have a synergistic effect.

[0029] Furthermore, the combination drug strategy adopted by the present application exhibits good ability to treat pulmonary fibrosis and significantly improves the mechanical microenvironment of the lungs, while breaking the vicious crosstalk between mechanical and biochemical signals in pathological fibroblasts and endothelial cells, providing a new treatment strategy for reversing the disease.

[0030] (2) The present application is prepared into a nebulized inhalation type nano preparation which can better penetrate into the alveoli and reduce the toxic side effects of drugs. At the same time, the surface of the lipid nanoparticles is modified with tannic acid and L-arginine, wherein the high affinity of tannic acid to collagen promotes the targeted accumulation of nanoparticles in the lung tissue. In addition, L-arginine promotes the production of nitric oxide by bronchial epithelial cells, enhances the uptake of cells by the carrier, activates endogenous matrix metalloproteinases, degrades the dense extracellular matrix layer, promotes the penetration of nanoparticles into the lung interstitium and effectively delivers them to the deep alveoli. The prior art shows that particles with a particle size greater than 5 μm are easily deposited in the upper respiratory tract, while particles smaller than 500 nm can enter the alveoli, but particles that are too small (<10 nm) can be exhaled. Therefore, the present application controls the particle size to be 90-150 nm, which not only helps its effective deposition in the alveolar region, but also reduces the risk of being exhaled and systemically transferred, thereby achieving a more stable and efficient local drug delivery effect in the lungs. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is a schematic diagram of the preparation process of the nano preparation of the present application; Lipo is a lipid nanoparticle, including phospholipid, cholesterol and DSPE-PEG 2K -NH2, V is verteporfin, B is berberine, R is L-arginine, and T is tannic acid, wherein arginine penetrates the collagen barrier and penetrates deeply, and tannic acid can eliminate excess ROS in the lungs;

[0032] Figure 2 It is a particle size distribution and polydispersity index diagram after screening of the drug formula of the nano preparation;

[0033] Figure 3 It is a particle size diagram and Zeta potential diagram of each nano preparation prepared;

[0034] Figure 4 It is the calculation of the grafting rate of L-arginine and tannic acid of the prepared nano preparation, wherein A is the standard curve and grafting rate of L-arginine, and B is the standard curve and grafting rate of tannic acid;

[0035] Figure 5 It is the ultraviolet-visible spectrum of the drug loaded by the carrier;

[0036] Figure 6 It is the evaluation of the reduction of ROS level by the nano preparation, wherein A is the immunofluorescence diagram of ROS in 16HBE cells, and B is the fluorescence quantification diagram;

[0037] Figure 7 It is the evaluation of the production of NO by the nano preparation, wherein A is the fluorescence diagram of the production of NO by 16HBE cells, and B is the fluorescence quantification diagram;

[0038] Figure 8Evaluation of the nanoformulation to block the malignant interaction of mechanical and biochemical signals in fibroblasts, wherein A is the evaluation of TGF-β expression level induced by different nanoformulations, and B is the expression of α-SMA induced by different nanoformulations under soft and hard matrix conditions;

[0039] Figure 9 Evaluation of the nanoformulation to block the malignant interaction of mechanical and biochemical signals in endothelial cells, wherein A is the expression analysis of Vimentin and E-cadherin proteins in fibroblasts after treatment with different nanoformulations; B is a comparison chart of the expression of F-actin and pMLC induced by different nanoformulations in 3D soft and hard matrix;

[0040] Figure 10 Evaluation results of the anti-fibrosis effect of the nanoformulation in vivo, wherein A is the H&E staining and Masson staining of the lung tissue of mice in different treatment groups, B is the body weight change chart of the mice, C is the hydroxyproline content chart of the lung of the mice, and D is the Ashcroft score of the lung of the mice. DETAILED DESCRIPTION

[0041] In order to more clearly illustrate the technical content of the present application, specific embodiments are described in detail in conjunction with the drawings. Obviously, the listed embodiments are only preferred embodiments of the technical solution, and other technical solutions that can be obtained by those skilled in the art based on the disclosed technical content still belong to the protection scope of the present application.

[0042] Example 1 Preparation of VB-RT nanoparticles

[0043] I. Prescription screening of VB nanoparticles

[0044] Soybean phospholipid and cholesterol with a mass ratio of 14:1, 10:1, 8:1, 6:1, and 2:1 were weighed, respectively, and the cholesterol was fixed at 5 mg. Then 5 mg of DSPE-PEG 2K -NH2, 2 mg of verteporfin, and 1 mg of berberine were added, and the mixture was dissolved in 15 mL of dichloromethane. The organic solution was placed in a rotary evaporator to spin dry, forming a uniform transparent film. 5 mL of PBS was added to the bottle, and it was placed under water for 20 minutes. Then the film was washed for 10 minutes under ultrasonic oscillation, and placed in an ice bath for ultrasonic treatment with an ultrasonic cell crusher for 5 minutes. Then, centrifugation was performed at 5000 rpm for 5 minutes to remove free drugs, and the supernatant was collected. Finally, the supernatant was squeezed through a filter membrane with a pore size of 100 nm to obtain VB nanoparticles.

[0045] The average particle size and particle size distribution of the prepared nanoparticles were determined using a Malvern ZetasizerNano ZS.

[0046] Results:

[0047] As shown in Figure 2 , the particle size was measured to be 90-150 nm, and the polydispersity index (PDI) was between 0.15-0.3. According to the comprehensive analysis of the particle size and PDI, the preferred mass ratio of soybean phospholipid to cholesterol was 8:1.

[0048] II. Preparation of VB-RT nanoparticles

[0049] The following experiments were performed with a mass ratio of soybean phospholipid to cholesterol of 8:1.

[0050] 1. The preparation method of VB nanoparticles is as follows:

[0051] Lipid nanoparticles (Lipo-VB) were prepared by thin film dispersion method. Specifically, 40 mg of phospholipid, 5 mg of cholesterol, 5 mg of DSPE-PEG 2K -NH2, 2 mg of verteporfin, and 1 mg of berberine were weighed and dissolved in dichloromethane. After mixing uniformly, the solvent was evaporated under reduced pressure using a rotary evaporator to form a uniform lipid film. Then, 5 mL of PBS was added and hydrated for 20 minutes to form a membrane layer. The probe-type ultrasonic treatment was performed under ice bath conditions for 5 minutes. Subsequently, a centrifuge was used at 5000 rpm for 5 minutes to collect the supernatant without drug loading, and Lipo-VB nanoparticles were obtained. The average particle size, particle size distribution, and Zeta potential of the obtained nanoparticles were measured using a Malvern Zetasizer Nano ZS instrument. Transmission electron microscopy (TEM, JEM-200CX, JEOL, Japan) was used to observe the external morphology.

[0052] Results:

[0053] As shown in Figure 3 , the average particle size of VB nanoparticles was 104.30 ± 1.01 nm, and the Zeta potential was -13.89 ± 0.2646 mV.

[0054] 2. The preparation method of VB-R nanoparticles is as follows:

[0055] L-arginine was dissolved in 5% glucose solution, and 0.25 mol / L EDC and 0.25 mol / L NHS were added to activate the carboxyl group at room temperature for 30 minutes. Then the pH of the solution was adjusted to 7.5. The activated L-arginine was added to the unmodified nanoparticles and incubated at room temperature overnight. Finally, the catalyst and free L-arginine in the reaction system were removed by ultrafiltration to obtain arginine-modified VB-R nanoparticles. The average particle size, particle size distribution, and Zeta potential of the prepared nanoparticles were measured using a Malvern Zetasizer Nano ZS instrument.

[0056] result:

[0057] like Figure 3 As shown, the particle size of VB-R was measured to be 108.72±4.58 nm; due to the introduction of positively charged L-arginine, its Zeta potential increased to -10.7±0.5 mV.

[0058] 3. The preparation method of VB-RT nanoparticles is as follows:

[0059] Tannic acid was dissolved in a 5% glucose solution, and the pH was adjusted to 7.5. VB-R was mixed with the prepared tannic acid solution, and after standing at room temperature for 30 minutes, free tannic acid was removed by ultrafiltration to obtain tannic acid-functionalized VB-RT nanoparticles. The average particle size, particle size distribution, and Zeta potential of the obtained nanoparticles were measured using a Malvern ZetasizerNano ZS instrument.

[0060] result:

[0061] like Figure 3 The results showed that after further modification with tannic acid via electrostatic adsorption, the particle size of the nanoparticles increased to 126.87±3.65 nm, and the Zeta potential decreased to -18.19±0.87 mV, which is more conducive to its penetration of the negatively charged tracheal mucus layer. High-performance liquid chromatography (HPLC) determined that the encapsulation efficiency (EE) of verteporfen in VB nanoparticles was 99.04±0.78%, and the drug loading (LC) was 1.67±0.06%; while the EE of verteporfen in VB-RT was 95.91±1.05%, and the LC was 0.76±0.01%.

[0062] In addition, such as Figure 4 The colorimetric analysis results and Sakaguchi reaction shown indicate that the grafting rate of tannic acid in VB-R nanoparticles is 0.88±0.02%, and the grafting rate of L-arginine in VB-RT nanoparticles is 1.56±0.04%, further confirming the successful modification of nanoparticles by TA and L-arginine, laying the foundation for subsequent experimental studies.

[0063] Example 2: UV-Vis spectroscopy determination of drug-loaded VB-RT nanoparticles

[0064] Using the VB-RT nanoparticles prepared in Example 1 as the research object, appropriate amounts of the drugs verteporfen and berberine were weighed and prepared into solutions of suitable concentrations, with the same solvent used as a blank control. Ultraviolet spectrophotometers were used to perform full-wavelength scanning measurements on each sample within the wavelength range of 200-800 nm.

[0065] result:

[0066] like Figure 5As shown, the results showed that the prepared VB-RT nanoparticles had obvious response at the characteristic absorption peak position, indicating that they could successfully encapsulate the two drugs of verteporfin and berberine.

[0067] Example 3: Determination of ROS scavenging ability of VB-RT nanoparticles

[0068] A549 cells were evenly inoculated in a confocal microscope special culture dish, and were co-incubated with different drug treatment groups (V-RT, B-RT, VB-R, VB-RT) for 12 hours. Subsequently, the active oxygen positive control reagent was added, and the incubation was continued for 4 hours, and was co-incubated with the fluorescence probe DCFH-DA for 30 minutes under light-proof conditions. After the incubation was completed, the cells were washed with PBS and the cell nucleus was stained with DAPI. Image acquisition was performed using a laser confocal microscope (CLSM-800, Zeiss, Germany).

[0069] Results:

[0070] As shown: Figure 6 compared with the blank control group, the 16HBE cells pretreated with H2O2 showed obvious green fluorescence, indicating that the intracellular ROS level was significantly increased; while after treatment with VB-RT nanoparticles, the generation of ROS was significantly inhibited, and the fluorescence intensity was significantly weakened, proving that the VB-RT nanoparticles had good ROS scavenging activity.

[0071] Example 4: Determination of the ability of VB-RT nanoparticles to induce the production of nitric oxide (NO)

[0072] The generation of intracellular nitric oxide was detected using the fluorescence probe DAF-FM DA, as follows: 16HBE cells were inoculated in a confocal microscope special culture dish, and were co-incubated with VB, VB-R, and VB-RT nanoparticles for 4 hours. After the cells were washed with PBS, the fluorescence probe DAF-FM DA was added, and the incubation was performed in a dark room for 20 minutes. Subsequently, the cell nucleus was stained, and the images were collected for analysis.

[0073] Results:

[0074] As shown: Figure 7 the DAF-FM DA results showed that the nanoparticles modified with L-arginine (VB-R and VB-RT nanoparticles) could significantly promote the generation of nitric oxide in 16HBE cells, and exhibited enhanced ability to promote the expression of nitric oxide.

[0075] Example 5: Determination of the synergistic effect of VB-RT nanoparticles

[0076] To evaluate the synergistic effect of the two drugs in the nanoparticle preparation, fibroblasts were inoculated in a 96-well plate at a density of 1 × 10 4The cells were seeded in 96-well plates at a density of 1000 cells / well, and when the cell density reached more than 80% of the bottom of the plate, different concentrations of B-RT, V-RT and VB-RT were added to the plates in the order of 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM and 100 μM. At the same time, blank control groups without cells and negative control groups without drugs were set up, and each group had 6 replicates. After 24 h, the plates were taken out, the drug-containing medium was removed, 120 μL / well of MTT solution (1 mg / mL) was added using a gun, and the plates were incubated in a cell culture incubator for another 4 h. After incubation, the MTT solution was removed, 150 μL / well of DMSO solution was added using a gun, and the plates were shaken to completely dissolve the methylene blue. The absorbance (OD value) of each well was measured at 490 nm using a multifunctional enzyme label instrument, and the synergistic index was calculated according to the following formula based on the inhibition rate of each group of drugs on the cells:

[0077]

[0078] wherein (Dx)1 and (Dx)2 represent the doses of drug 1 and drug 2, respectively, required to produce an x% effect, and D1 and D2 represent the doses of drug 1 and drug 2, respectively, required to produce the same effect when used in combination.

[0079] Results:

[0080] The CI indexes of the two drugs at IC25, IC50, IC75 and IC90 were 0.673, 0.623, 0.659 and 0.538, respectively, and the calculated values were all less than 1, indicating that V-RT and B-RT had synergistic effects, i.e., the effect of VB-RT was greater than the sum of the individual effects when the two drugs were used in combination.

[0081] Example 6 Ability of VB-RT nanoparticles to block the malignant cross-talk of mechanical and biochemical signals of fibroblasts

[0082] The myofibroblasts were seeded in the chamber, washed twice with pre-cooled PBS after 3 days of culture, and then incubated with lysis buffer containing 0.5% sodium deoxycholate, 1 mM PMSF and 10 mM Tris-HCl (pH 8.0) at 4°C for 10 min. After washing, washing buffer containing 2 mM Tris-HCl (pH 8.0) and 1 mM PMSF was added. Then the fibroblasts were co-implanted with PMVEC in the ECM layer and cultured under tension or without tension, while different drug formulations (B-RT, V-RT, VB-R and VB-RT) were added for stimulation. The cells in the chamber after treatment were collected and centrifuged to obtain the supernatant, and the content of TGF-β was determined.

[0083] Results:

[0084] AsFigure 8 As shown in A, mechanical tension significantly promoted the activation of latent TGF-β, and its release was increased by 2.16 times compared with the non-tension group. Each drug treatment group could inhibit the release of active TGF-β, among which the VB-RT group reduced the TGF-β level to near the baseline level, and the inhibitory effect was the most significant.

[0085] Fibroblasts were seeded in the chamber of programmable cell stretching system (Cell&Force, China) and cultured for 24 hours before drug treatment. Then mechanical stretching (mode: SIN F, amplitude: 5, duration: 20 seconds) was applied for 24 hours. After treatment, the expression of a-SMA was detected by immunofluorescence staining, and the images were obtained using a laser confocal microscope (CLSM-800, Zeiss, Germany).

[0086] Results:

[0087] As Figure 8 As shown in B, stretching treatment significantly induced the enhancement of a-SMA expression, suggesting the activation of fibroblasts. Each drug group showed different degrees of inhibition, among which the VB-RT treatment group had the lowest fluorescence intensity, indicating that it had the strongest inhibition on the activation of fibroblasts.

[0088] Example 7 Ability of VB-RT nanoparticles to block the malignant cross-talk of mechanical and biochemical signals in endothelial cells

[0089] Endothelial cells were lysed in lysis buffer, and the supernatant was collected after centrifugation. The protein concentration was determined using a BCA protein assay kit, and an equal amount of protein sample was separated by SDS-PAGE electrophoresis and transferred to a nitrocellulose membrane. After blocking the membrane with 5% skim milk, the primary antibody was incubated at 4°C for 12-16 hours, and the secondary antibody was incubated at room temperature for 2 hours. The protein bands were visualized using a CCD imaging system (Tanon 4200, Shanghai, China). As Figure 8 The WB detection results in the middle show that TGF-β stimulation significantly up-regulates the mesenchymal marker Vimentin and down-regulates the endothelial marker VE-cadherin, suggesting that the cell undergoes endothelial-mesenchymal transition (EndMT). Each drug treatment group can inhibit this process to varying degrees, among which the VB-RT group has the most significant regulatory effect, with decreased Vimentin expression and restored VE-cadherin expression, indicating that it has a significant inhibitory effect on EndMT.

[0090] 1 x 10 6Endothelial cells were suspended in collagen solution to a final suspension volume of one-eighth of the collagen solution volume, and the pH of the mixture was adjusted to 7.4. 200 μL of the collagen-EC mixture was dispensed into untreated TC-treated 48-well plates and incubated at 37°C and 5% CO2 for 60 min to induce gelation. Subsequently, 400 μL of incomplete culture medium was added, and the gel was resuspended by streaking along the well walls with a pipette. After 48 hours of incubation, the culture medium was removed, and the desired stimulation was applied. Finally, immunofluorescence staining for pMLC and F-actin was performed, and fluorescence images were obtained.

[0091] result:

[0092] like Figure 9 As shown, p-MLC expression was significantly increased in hard collagen gel, and clear nuclear localization was observed in the 3D reconstructed images, indicating enhanced cell contraction activity and increased cell diffusion. In contrast, in soft collagen gel, p-MLC was mainly located in the cytoplasm, with less nuclear localization. These results suggest that high matrix stiffness promotes integrin aggregation and stabilizes adhesion sites, and VB-RT can better alleviate cell tension and promote the assembly of complex blood vessels.

[0093] Example 8: Efficacy analysis of VB-RT nanoparticles in treating fibrotic mouse lungs.

[0094] C57 mice were acclimatized for one week and then anesthetized with a single intratracheal injection of 2.0 U / kg bleomycin (BLM). An experimental pulmonary fibrosis model was established on day 14. In the in vivo treatment study, on days 15, 17, 19, 21, 23, and 25 post-modeling, mice were nebulized with saline, B-RT, V-RT, VB-R, and VB-RT nanoparticles (verteporfen: 10 mg / kg; berberine: 8 mg / kg), respectively, and the effects were compared with those of mice treated with PFD (25 mg / kg) via gavage. After treatment, the therapeutic effects of each formulation on pulmonary fibrosis were evaluated using H&E staining and Masson's staining.

[0095] result:

[0096] like Figure 10 As shown in Figure A, H&E staining results revealed that healthy mice exhibited normal alveolar structure with intact alveolar walls and air spaces; while mice in the BLM-treated group showed significant lung tissue thickening, alveolar wall destruction, and perivascular fibrosis. Compared to the saline group, the VB-RT treatment group significantly reduced lung tissue damage, including interstitial edema and alveolar wall thickening, while the PFD group showed no significant improvement. Masson staining further indicated extensive collagen deposition in the lungs of BLM-treated mice, while collagen deposition was significantly reduced in the lungs of VB-RT-treated mice, demonstrating its significant inhibitory effect on pulmonary fibrosis. Furthermore, Figure 10B shows the gradual decline in body weight over time in untreated fibrotic mice, while all treatment groups show varying degrees of recovery. Among them, mice treated with VB-RT recovered to levels comparable to healthy donors. Consistent with histology results, Figure 10 C shows that the hydroxyproline (HYP) content of untreated fibrotic mice is 2.5 times higher than that of healthy donors. VB-RT treatment reduced HYP levels to 2.42 times, while PFD treatment only reduced it to 1.70 times relative to the BLM group. As Figure 10 D shows that pathological scoring shows severe fibrosis in the BLM group. All treatment groups show a decrease in fibrosis score, with the most significant improvement in the lungs of mice in the VB-RT group.

Claims

1. A nanoformulation that blocks the malignant cross-talk of mechanical and biochemical signals in the lung, characterized in that, It comprises a nanocarrier and a drug; wherein the drug can include the following two categories: The drug V for blocking mechanical signal transduction is verteporfin, dobutamine or CA3; The drug B for blocking biochemical signal transduction is berbamine, pirfenidone or galunisertib.

2. The nanoformulation according to claim 1, wherein, The nano-carrier comprises phospholipid, cholesterol, D material-PEG 2K -NH2, tannic acid or L-arginine; the phospholipid is any one or several of soybean phospholipid, HSPC, egg yolk lecithin, synthetic phospholipid; the D material is any one or several of DSPE, PLGA, PLA, PGA, PCL or PS.

3. Nanopreparation according to claim, characterized in that, The mass ratio of phospholipid and cholesterol is 2:1-14:1, the added amount of L-arginine is 0.574 mM, and the added amount of tannic acid is 1 mM.

4. Process for the preparation of a nanoforumla- tion according to any one of claims 1 to 3, characterized in that, It comprises the following steps: (1) Drug, phospholipid, cholesterol and D class material-PEG 2K -NH2, nanoparticles are prepared by film dispersion method, ethanol injection method or reverse evaporation method; (2) Dissolve L-arginine in 5% glucose solution, activate the carboxyl group with EDC and NHS at room temperature, adjust the pH to about 7.5, then introduce the activated L-arginine into the nanoparticles for modification, remove impurities by ultrafiltration after incubation, and obtain L-arginine modified nanoparticles; (3) Dissolve tannic acid in 5% glucose solution, adjust the pH to about 7.5, and mix the above obtained VB-R with tannic acid, then ultrafiltrate after standing reaction, and obtain tannic acid modified L-arginine nanoparticles.

5. The method of claim 4, wherein, In the step (1), the mass ratio of phospholipid and cholesterol is 2:1-14:

1.

6. The method of claim 5, wherein, In the step (1), the mass ratio of phospholipid and cholesterol is 8:1; In the step (2), the input amount of L-arginine is 0.574 mM; In the step (3), the input amount of tannic acid is 1 mM.

7. The method of claim 6, wherein, The drug loading amount in the nanometer preparation is between 0.5-1.5%, and the particle size of the nanometer preparation is 90-150 nm.

8. The use of the nanometer preparation according to any one of claims 1-3 in the preparation of a drug for treating pulmonary fibrosis.

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